Fungal Isolates from a Pu‐Erh Type Tea Fermentation and Their Ability to Convert Tea Polyphenols to Theabrownins

The natural microbiota involved in the fermentation influence the quality and taste of fully postfermented teas such as China's Pu‐erh tea. Ten microbial isolates representing 6 species were recovered from a solid‐state fermentation of a Pu‐erh type tea. The isolates were Aspergillus tubingensis, Aspergillus marvanovae, Rhizomucor pusillus, Rhizomucor tauricus, Aspergillus fumigatus, and Candida mogii. With the exception of A. marvanovae and C. mogii, all these microorganisms have been previously reported in solid‐state fermentations of native Pu‐erh tea. The ability of the isolates for converting the tea polyphenols to bioactive theabrownins in infusions of sun‐dried green tea leaves in a submerged fermentation process was subsequently investigated. All isolates except C. mogii TISTR 5938 effectively produced theabrownins in a 4‐d fermentation in shake flasks at 40 °C, 250 rpm. A. tubingensis TISTR 3646, A. tubingensis TISTR 3647, A. marvanovae TISTR 3648, and A. fumigatus TISTR 3654 produced theabrownins at particularly high levels of 6.5, 12.4, 11.1, and 8.4 g/L, respectively.     

Identification and quantification of fungi and mycotoxins from Pu-erh tea

Pu-erh tea originates from the province of Yunnan in south-western China. As this tea is produced by so called Aspergillus post-fermentation the question arises which molds and mycotoxins may be found in this tea. In total 36 samples of Pu-erh tea were investigated for their content of filamentous fungi and the mycotoxins aflatoxins B₁, B₂, G₁, and G₂, fumonisins B₁, B₂, and B₃, and ochratoxin A. Fungi were isolated from all samples in a concentration of 1.0 × 10¹ to 2.6 × 10⁶ colony forming units (cfu)/g tea, all together 19 fungal genera and 31 species were identified. The most prevalent species were Aspergillus acidus and Aspergillus fumigatus, followed by Zygomycetes and Penicillium species. Aflatoxins and fumonisins were not found in the samples investigated, ochratoxin A was detected in 4 of 36 teas (11.1%).     

发酵方法对普洱茶茶褐素样品组成的影响

采用水提醇沉法从塔宾曲霉液态发酵普洱茶粉和传统固态发酵工艺所产普洱熟茶中提取茶褐素。应用居里点热裂解气相色谱-质谱联用技术及比色法研究液态发酵茶褐素样品和固态发酵茶褐素样品的化学组分。结果显示,液态发酵样品中的茶褐素、总多酚、总黄酮、总儿茶素和氨基酸显著高于固态发酵样品（P＜0.05）;液态发酵样品中的总糖、多糖、茶黄素显著低于固态发酵样品（P＜0.05）;液态发酵样品与固态发酵样品的蛋白质含量差异不显著（P＞0.05）。在不同居里点热裂解温度下,固态发酵样品与液态发酵样品的裂解产物分别有12?种（380?℃）和9?种共同成分（600?℃）,两者在不同温度下的裂解产物中,酚类种类最多,含量最高的化合物是咖啡因。     

Production of Agaricus brasiliensis mycelium from food industry residues as a source of antioxidants and essential fatty acids

Agaricus brasiliensis is a medicinal mushroom traditionally employed in prevention and treatment of cancer, used as immunostimulant and as a source of antioxidants. We investigated the chemical composition of the mycelium produced by submerged (SF) and solid‐state fermentation (SSF), using residues from food industry as substrates. After fermentation, antiradical activity and levels of antioxidants were enhanced, indicating that the micro‐organism produces these metabolites. For myceliated substrate (mushroom mycelia grown around and into the substrate particles) obtained by SSF, phenolics ranged from 18.57 to 70.46 mg g^?1 and flavonoids from 0.83 to 4.51 mg g^?1. For myceliated substrate obtained by SF, the variation was 27.19 to 66.99 mg g^?1 and 0.75 to 5.34 mg g^?1 for phenolics and flavonoids, respectively. The fatty acid profile determined by FT‐ICR MS and UPLC‐MS showed predominance of palmitic, linoleic, linolenic and oleic acids. Our findings indicated that mycelium has nutraceutical potential and can be incorporated in food supplements.     

普洱茶发酵过程中的雪黄散囊菌研究初探

目的揭示普洱茶发酵过程中的微生物群落结构及其与普洱茶品质形成影响关系。方法采用CTAB法提取普洱茶发酵阶段样的微生物总DNA,并进行基因扩增,基于illumina Miseq技术测序平台进行双末端测序分析,研究普洱茶发酵过程中不同层间的微生物群落结构。结果首次在普洱茶发酵过程中发现了雪黄散囊菌,且各发酵层间和各发酵阶段均有存在,但由于发酵堆温和其他优势菌群的影响,雪黄散囊菌生长随发酵进行受到了抑制。结论在普洱茶生产过程中控制发酵条件,使其有利于雪黄散囊菌的生长,并在普洱茶发酵过程中处于优势地位,从而产生特色新风味的普洱茶。     

2013—2015年广州市市售普洱茶真菌毒素污染调查

目的 了解广州市市售普洱茶真菌毒素污染状况及评估健康风险。方法 2013—2015年,在广州市批发市场、零售店、超市、餐饮单位、网店和茶博会6类场所采集不同生产工艺、包装形态、生产年份、原料产地、价格的普洱茶样品432份,使用多功能净化柱净化-柱后衍生高效液相色谱法测定黄曲霉毒素(B₁、B₂、G₁、G₂),高效液相色谱-质谱法测定雪腐镰刀菌烯醇、脱氧雪腐镰刀菌烯醇和玉米赤霉烯酮。结果 432份普洱茶样品黄曲霉毒素(B₁、B₂、G₁、G₂)、雪腐镰刀菌烯醇、脱氧雪腐镰刀菌烯醇、玉米赤霉烯酮7个项目检测结果均低于所用检测方法的检出限。结论 本次对广州市市售普洱茶的调查研究,未检出真菌毒素。     

Nutritional and medicinal improvement of black tea by yeast fermentation

Black tea fermentation with Dabaryomyces hansenii for 10 days resulted in accumulation of major vitamins, such as A, B₁, B₂, B₁₂ and C in sufficient quantities to fulfil the recommended dietary allowances (RDA). Fermentation of tea by yeast resulted in reduction of caffeine and excess tannins in significant amounts. After fermentation, the amount of theophylline was increased to make fermented tea a potent bronchodilator. Tea fermentation with D. hansenii improved its nutritional and medicinal values. Our observations suggest that intake of fermented tea is advantageous over black tea in terms of its nutritive and therapeutic values.     

Effect of bioaugmentation on biochemical characterisation and microbial communities in Daqu using Bacillus,Saccharomycopsis and Absidia

Daqu, a saccharifying and fermenting agent for the production of Chinese vinegar and liquor, is manufactured through a spontaneous solid-state fermentation process (SSF). To investigate the influence of bioaugmentation with native microorganisms on Daqu SSF process, physicochemical properties, microbial biomass, volatile compounds and microbial communities were analysed in laboratory-scale Daqu. The results showed that the amylase activity of Daqu enhanced 6.35% ± 0.74% by inoculating Bacillus amyloliquefaciens, Saccharomycopsis fibuligera and Absidia corymbifera. Obviously higher microbial richness was found in the Daqu with bioaugmentation, although the microbial community structure remained relatively stable. Moreover, a total of twenty-two volatile compounds were detected in the mature Daqu. Slightly increase in the content of alcohols was found in the enhanced group by fungal inoculation, including ethanol (46.58 ± 1.36 mg kg⁻¹), isobutanol (0.19 ± 0.04 mg kg⁻¹) and isoamyl alcohol (1.55 ± 0.13 mg kg⁻¹). This study demonstrated that bioaugmentation had a positive effect on the amylase activity, main volatile compounds and microbial community richness.     

Development of a new method for the simultaneous determination of 21 mycotoxins in coffee beverages by liquid chromatography tandem mass spectrometry

A new method for the simultaneous detection of 21 mycotoxins (ochratoxin A, aflatoxin B₁, aflatoxin B_2, aflatoxin G₁, aflatoxin G₂, sterigmatocystin, nivalenol, deoxynivalenol, 3-acetyl deoxynivalenol, 15-acetyl deoxynivalenol, diacetoxyscirpenol, neosolaniol, HT-2 toxin, T-2 toxin, fumonisin B₁, fumonisin B₂, enniatin A, enniatin A₁, enniatin B, enniatin B₁, and beauvericin) in coffee beverages was internally validated. The method is based on liquid/liquid extraction with a mixture of ethyl acetate/formic acid (95:5 v/v) and detection using triple quadrupole (QqQ) and ion trap (IT) liquid chromatography tandem mass spectrometry. The limits of detection and quantification were 0.02 to 39.64 μg/kg, respectively, and the correlation coefficients were optimal for all mycotoxins (R² ≥ 0.992). The recovery values ranged from 72% to 97%. The developed method was demonstrated in six real samples of roasted and instant coffee, caffeinated and decaffeinated coffee, and coffee with sugar added. The analyses indicate the presence of the studied mycotoxins in coffee beverages at μg/kg concentrations. Ochratoxin A, a mycotoxin that is regulated in coffee, was detected in two samples under the maximum limit established by a European legislation (CE1881/2006).     

普洱茶后发酵过程中多酚类成分生物转化的研究进展

普洱茶产于云南,属于后发酵茶,是我国黑茶的典型代表之一。微生物参与的后发酵过程使晒青毛茶中的多酚类成分发生了一系列复杂的诸如氧化、聚合、缩合、分解等为主的电化学变化,奠定了普洱茶的化学物质基础,赋予了普洱茶特殊的风味品质和保健功效。近年来的研究表明,普洱茶后发酵过程中,在微生物分泌的胞外酶作用下,糖基化、甲基化以及其他类型取代方式的化学结构修饰也是多酚类成分生物转化的重要途径之一,以此形成了众多多酚类成分的化学结构修饰产物。现有研究表明,这些结构修饰产物对普洱茶的风味品质和生物活性产生重要影响,是普洱茶中潜在的重要品质化学成分和标志性成分,具有重要的研究意义。本文综述了普洱茶中多酚类成分化学结构修饰产物的研究进展。     

Phenolic compounds and the biological effects of Pu-erh teas with long-term storage

Pu-erh tea has gained more and more popularity and attracted much attention for its various biological effects. The objective of this study was to determine the active phenolic compounds and the biological effects of 15 differently aged Pu-erh teas. The results showed that 43 active phenolics, containing 7 flavan-3-ols, 11 organic acids, and esters, 3 proanthocyanidin dimers, 2 benzotropolones, and 20 flavonoid glycosides were identified based on high-performance liquid chromatography coupled to electrospray ionisation and quadrupole time-of-flight tandem mass spectrometry (HPLC-ESI-QTOF-MS/MS). In particular, 2’-O-galloylhyperin and quercetin-3-O-p-coumaroyl-rhamnosyl-arabinosyl-hexoside were identified from constituents of tea for the first time. The 15 Pu-erh teas exhibited strong DPPH and ABTS scavenging activity in a concentration-dependent manner with IC₅₀ 0.99–2.12 and 0.97–2.67 mmol Trolox equivalent antioxidant capacity/g, respectively. All of the Pu-erh teas tested significantly inhibited the proliferation of SMMC-7721 cells, in a dosage-dependent manner. Of the 15 Pu-erh teas examined, the cake tea P6 had the strongest effect on SMMC-7721 cells with IC₅₀ = 65.88 ± 3.53 µg/mL. The others Pu-erh teas had an IC₅₀ = 96 – 509 µg/mL. Our results also indicated that not all the older Pu-erh teas display stronger anti-activities and anti-cancer than the younger ones and the youngest Pu-erh tea did not possess the highest level of active components. This study provides useful information for consumers to deeply understand the chemical constituents and bioactivity of Pu-erh tea preserved for a long time.     

Spoilage moulds and aflatoxins from poultry feeds

Aflatoxin producing strains of Aspergillus flayus Link (IMI 280819) and A. oryzae (Ahlb.) Cohn (IMI 280831) were among the eleven spoilage moulds isolated from five types of poultry feeds. The recorded pH and moisture content values of the various feeds are conducive to mould deterioration. All the four principal aflatoxins (B₁, B₂, G₁, G₂) were detected in the analysed feeds though at toxicologically ‘safe’ levels for most farm animals. Significant quantities of aflatoxin B₁ were produced by the two fungal isolates in all the five classes of poultry feeds with A. flavus yielding the larger amounts. Optimum aflatoxin B₁ production and mycelial growth in chick mash infusion medium were recorded for both species at 30 and 35 °C, respectively and similarly on the 8th and 6th day respectively when cultures were incubated at 30 °C.     

Quality Characteristics of a Pickled Tea Processed by Submerged Fermentation

There is a traditional pickled tea fermented under anaerobic condition in many Asian countries, but its quality characteristics are still unclear. The dynamic qualities, chemical components, and volatile components of pickled tea processed by submerged fermentation were investigated. The results showed that the sensory qualities of the pickled tea gradually improved with increasing fermentation time. Sensory qualities decreased gradually after 7 d of fermentation, and the change was extremely significant (p < 0.01). Correspondingly, biochemical indicators (except for soluble extract) decreased continuously and significantly (p < 0.01) within 5–7 d of fermentation, but the decrease was much slower in the later stage. The proper control of fermentation time is a key step for obtaining the desired quality of pickled tea. By submerged fermentation for 7 d, the best sensory quality of pickled tea was obtained: the taste of pickled tea was less bitter and astringent, but certainly acidic; the flavor of pickled tea was sour with sweet and floral tastes; the tea had low caffeine content, high gamma-amino butyric acid content, and high contents of special volatile components, such as hotrienol and 2,6-bis (1,1-dimethylethyl)-4-methyl-phenol. Results indicated that submerged fermentation is potentially a new method of processing pickled tea to obtain the desired quality. Our results contribute to the understanding of the microbial transformation of tea components under anaerobic conditions.     

Production,partial purification and properties of β‐mannanases obtained by solid substrate fermentation of spent soluble coffee wastes and copra paste using Aspergillus oryzae and Aspergillus niger

In order to achieve a higher added value of two galactomannan‐containing wastes, copra paste and spent coffee from the soluble coffee industry (SCW), solid substrate fermentation (SSF) was used. Filamentous fungi Aspergillus oryzae and A niger were used to evaluate the feasibility of producing β‐mannanase by SSF. A 2³ factorial design was used to select the best interaction among the two fungi, the two substrates and two fermentation times. The treatment ‘A niger–copra–2.5 days’ produced a significantly higher (p < 0.05) β‐mannanase activity, having five different isoforms of the enzyme, one of which was partially purified to a specific activity of 764 U mg⁻¹ (U = nmol of mannose released per second from a galactomannan substrate). Copra paste had a higher mannose/galactose ratio (14:1) than SCW (6:1), and low oil content, which led to higher β‐mannanase production from SSF. A β‐mannanase from SSF of copra produced by A oryzae was highly purified using acetone precipitation and cation exchange and size exclusion chromatographies. This enzyme had an MW of 110 kDa, a pI between 3.5 and 4.5 and a specific activity of 1760 U mg⁻¹; purification achieved was 90.7 times. The temperature and pH for optimal activity were 40 °C and 6.0 respectively. The optimal temperature was lower and the optimal pH higher than others previously reported (produced by submerged fermentation), which could be important for viscosity reduction of concentrated coffee extract in instant coffee manufacture. Copra is an interesting alternative for β‐mannanase production, since it is readily available in Mexico; moreover, the residue after SSF has a reduced galactomannan content and may be used for monogastric animal feed. © 2000 Society of Chemical Industry     

Enhancement of Fermentation Process in Pu-Erh Tea by Tea-Leaf Extract

ABSTRACT:  Pu-erh tea is known as a fermented tea and longer storage enhances its flavor and taste. Recently,  Aspergillu s,  Blastobotrys , and  Streptomyces  are found to play important roles in nutritional enhancement of Pu-erh tea by fermentation. Since water and temperature affect the microbial growth, we therefore explored the factors that might enhance the Pu-erh tea fermentation. The results showed that the addition of fresh tea-leaf extract (TLE) enhanced the withered tea fermentation (at 37 °C, 80 to 85% RH) as compared with the water only. Contents of statin, GABA, gallic acid, DPPH scavenging and polyphenol oxidase (PPO) activities were increased, whereas polyphenols and caffeine were decreased over 6 mo. TLE dose-dependently enhanced some of the qualities (that is, statin, PPO) of Pu-erh tea significantly as compared with the water only. The effect was related to the increase population of  A. niger  and  A. carbonarius  at 6 mo (from 7.6 ± 1.2 × 10¹ and 3.2 ± 1.3 × 10¹ to 3.1 ± 1.2 × 10⁶ and 2.4 ± 1.1 × 10⁵ colony forming units [CFU]/g, respectively). After drying process (90 °C, 30 min), the total microbial count from these samples returned to background level (3 ± 0.5 × 10² CFU/g). None of ochratoxin and fumonisin, toxins from  Aspergillus , was detected in the final products. The flavor and taste were also enhanced by treatment with TLE. The inoculation with  S. cinereus  Y11 with 2% TLE further enhanced these functional contents (about 2-fold increase of statin level) in the experimental Pu-erh tea. Therefore, this result may add a new process for Pu-erh tea manufacture.     

Effect of substrate and fermentation conditions on pectinase and cellulase production by <Emphasis Type="Italic">Aspergillus niger</Emphasis> NCIM 548 in submerged (SmF) and solid state fermentation (SSF)

The present study deals with the optimization of substrate and fermentation conditions for the production of both pectinase and cellulase by Aspergillus niger NCIM 548 under same fermentation conditions in submerged fermentation (SmF) and solid state fementation (SSF) using a central composite face centered design of response surface methodology (RSM). As per statistical design, the optimum conditions for maximum production of pectinase (1.64 U/mL in SmF and 179.83 U/g in SSF) and cellulase (0.36 U/mL in SmF and 10.81 U/g in SSF) were, time 126 h, pH 4.6, and carbon source concentration 65 g/L in SmF and were time 156 h, pH 4.80, and moisture content 65% in SSF. The response surface modeling was applied effectively to optimize the production of both pectinase and cellulase by A. niger under same fermentation conditions to make the process cost-effective in both submerged and solid state fermentation using agro industrial wastes as substrate.     

The effect of instant green tea on the foaming and rheological properties of egg albumen proteins

The effects of three varieties of instant green tea (from China, Japan and Kenya) on the foaming and thermal properties of 1% (w/v) egg albumen and the gelation properties of 5 and 15% (w/v) egg albumen were investigated. All varieties produced similar effects on the foaming and gelation properties of egg albumen, but to different extents depending on the tea constituents. Mixtures of 1% (w/v) egg albumen and 0.25–0.4% (w/v) instant green teas in distilled water showed the greatest foam expansion (800–1140%) and foam stability (97–100%) at 10 min after whipping compared with 1% (w/v) egg albumen alone (226% for foam expansion and 34% for foam stability). Addition of instant green teas at levels above 0.5% (w/v) decreased foam expansion and stability. Small‐deformation rheology of mixtures of 5% (w/v) egg albumen and 1 or 2% (w/v) instant green teas showed an initial increase in elastic modulus (G′) and viscous modulus (G″) followed by a small, broad peak, indicating that the binding of tea constituents (polyphenols) with proteins may be reversible between 20 and 54 °C; this peak was not seen for 5% (w/v) egg albumen on its own. Large‐deformation rheological tests also indicated increased strength of mixed egg albumen/green tea gels with increasing levels of instant green teas. Differential scanning calorimetry thermograms showed that for the same instant green tea a higher concentration decreased the onset and peak (T_m) temperatures and enthalpy change values of all egg albumen protein peaks. Copyright © 2007 Society of Chemical Industry     

Multi-mycotoxins Analysis in Dried Fruit by LC/MS/MS and a Modified QuEChERS Procedure

A sensitive and reliable multi-mycotoxin method was developed for the simultaneous determination of 16 toxicological important mycotoxins, such as aflatoxins B₁, B₂, G₁, and G₂; enniatins A, A₁, B, and B₁; beauvericin; ochratoxin A; fumonisin B₁, B₂, and B₃; diacetoxyscirprenol; HT-2; and T-2 toxin in dried fruits using liquid chromatography combined with electrospray ionization-triple quadrupole tandem-mass spectrometry. Mycotoxins have been extracted from the samples using a modified quick, easy, cheap, effective, rugged, and safe procedure. The method was based on a single extraction with acidified acetonitrile, followed by partitioning with salts, avoiding any further clean-up step. Limits of detections ranged from 0.08 to 15 μg kg^?1 and limits of quantification ranged from 0.2 to 45 μg kg^?1, which were below the legal limit set by the European Union for the legislated mycotoxines. The recoveries in spiked samples ranged from 60 to 135 % except for beauvericin using matrix-matched calibration curves for quantification, with good inter- and intraday repeatability (respective relative standard deviation ≤20 and 9 %). The developed method was applied to 15 commercial dried fruits: raisins, figs, apricots, plums, and dates purchased in local markets from Spain. Among the mycotoxins studied, enniantins and aflatoxins were the most predominant mycotoxins.     

Influence of the addition of lupin sourdough with different lactobacilli on dough properties and bread quality

The aim of this research was to study the effects of solid‐state fermentation (SSF) with Lactobacillus sakei, Pediococcus pentosaceus and P. acidilactici on lupine sourdough parameters and lupine sourdough influence on the physical dough properties and wheat bread quality. The results showed that lactic acid bacteria (LAB) significantly reduced the pH and increased total titratable acidity (TTA) of SSF lupine. The highest protease activity in lupine is excreted by L. sakei (187.1 ± 8.6 PU g^?1), and the highest amylase activity, by P. pentosaceus (155.8 ± 7.5 AU g^?1). Lupine sourdough has a significant effect on the rheological properties of doughs, which affect the baking characteristics of the final product. In conclusion, it can be said that L. sakei, P. pentosaceus and P. acidilactici could be used for lupine SSF, and the addition of up to the 10% SSF lupine products increases the wheat–lupine bread quality.     

Modellversuche zur Aflatoxinbildung in Schmelzk?se

Zusammenfassung Schmelzkäse stellt fürAspergillus favus ein sehr gutes Substrat dar. Nach 16 tägiger Kulturdauer konnten folgende Aflatoxinmengen nachgewiesen werden: 35 ppm Aflatoxin B₁, 66 ppm Aflatoxin G₁, 8,6 ppm Aflatoxin B₂, 5,3 ppm Aflatoxin G₂ und 2,1 ppm Aflatoxin M₁. Eine Erhöhung der Schmelzsalzkonzentration von 3% auf 8% bzw. ein Zusatz von 6% Natriumchlorid reduzierten das Toxinbildungsvermögen vonAspergillus favus deutlich.

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Simulation of natural mould growth in processed cheese

Summary Processed cheese is a very good substrate forAspergillus flavus. After 16 days incubation, a processed cheese inoculated withAspergillus flavus was found to contain 35 ppm aflatoxin B₁, 66 ppm aflatoxin G₁, 8.6 ppm aflatoxin B₂, 5.3 ppm aflatoxin G₂ and 2.1 ppm aflatoxin M₁. An increase in the content of emulsifying salt from 3% to 8% caused a distinct decrease in the contents of aflatoxin B₁ and G₁ in the cheese, as did the addition of 6% sodium chloride.