Characterization Of Fungal Community In Fuzhuan Brick-tea And Its Intestinal Regulation Functions Investigation

Fuzhuan brick-tea is a kind of microbial-fermented tea and is the leading product amongst the Hunan dark teas that provided for the border tea market in China. It is an essential beverage in daily life for the minorities living in northwestern China and is a common folk medicine as well. Nevertheless, several open problems troubling the manufacturers and consumers that were encountered in the Fuzhuan brick-tea product processing and utilization, id est,1) The classical processing of Fuzhuan brick-tea based on natural fermentation that induced the complex diversity of microorganisms existing in the tea product, unstable product quality, slow rate of turnover of fermentation room since longer fermentation periods.2) Although modern uncontaminated Fuzhuan brick-tea processing lines and fermentation rooms were constructed, there had not any applicable fermentation starters and practicable employment techniques for starters. Those became technique bottlenecks of the equipments.3) The effects of Fuzhuan brick-tea products and the functional fermentation fungi on the physiological functions in human body should be studied thoroughly and intensively, since the consumers had some concerns about that. It constrained the product-market development. The perfect solution to those problems was to deeply characterize the fermentation microbial communities and to study the physiological functions of the tea products. Consequently, powerful technical support would be provided for the Fuzhuan brick-tea product industry, and the mysteries of Fuzhuan brick-tea would be disclosed for the consumers.The main objectives of present study include analysis of the fungal communities in the Fuzhuan brick-tea processing and product samples, identification of the predominant fungi, determination of the anthraquinone derivatives in the Fuzhuan brick-tea related samples, the effects of Fuzhuan brick-tea intervention into EHEC O157:H7 infection, and the effects of Eurotium cristatum on colonic microbiota. The results are stated as follows:1. Analysis of the fungal community structure in Fuzhuan brick-teaThe piling tea samples and fermenting tea samples in the fermentation room at 2-day intervals were carried out in a major Fuzhuan brick-tea production factory in Hunan province, China. The samples of raw dark green tea and ready made Fuzhuan brick-tea were collected. The fungal communities in these samples were characterized with classical culture-depended methods and the most recent culture-independed approach such as polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE).Physicochemical analysis showed that the optimal growth conditions for xerophilic fungi were provided during the fermentation processing of Fuzhuan brick-tea. The genus Eurotium is the predominant fungi during the processing of Fuzhuan brick-tea detected with culture-based method.Three sets of universal primers targeting at fungal rDNA, such as NS1//GCfung5, su-0817-GC//ssu-1196 and NL1F-GC//LS2R, were adopted to amplify total DNA extracted from each tea sample. After PCR, the amplicons were separated by DGGE. Aimed at analysis of fungal community structure in samples, a comparison of the resulted three PCR-DGGE profiles showed that the best effect was obtained with the primer set of NL1F-GC//LS2R.In present study, at least eight genera of fungi, Eurotium, Debaryomyces, Aspergillus, Verticillium, Pichia, Pestalotiopsis, Rhizomucor and Beauveria in the Fuzhuan brick-tea related samples were identified by PCR-DGGE analysis. Except for genera Rhizomucor and Beauveria, fingerprints of the remaining six genera were detected in the fermenting tea samples. Fingerprints of Eurotium, Debaryomyces and Aspergillus were consistently visible, with the genus Debaryomyces not previously reported in Fuzhuan brick-tea. Xerophilic fungi such as genera Eurotium and Debaryomyces were predominant fungi during the fermentation processing of Fuzhuan brick-tea, and the genus Aspergillus acted as a subordinate partner under normal conditions.The genera Rhizomucor and Verticillium which were dominant fungi in raw dark green tea had never been detected previously. This is the first report of the genus Beauveria in stored ready made Fuzhuan brick-tea. The presence of genus Beauveria may be related to long-term preservation of the stored Fuzhuan brick-tea. It might be considered to utilize this safe microorganism to help protect tea products from insect spoilage.The PCR-DGGE fingerprints of fungal communities in industrial scale fermentation processing of Fuzhuan brick-tea were achieved in the present study. The results gave directions to screening of functional fungi used as fermentation starters for Fuzhuan brick-tea.2. Identification and phylogenetic analysis of the functional strains isolated from Fuzhuan brick-teaIdentification of the microbial species is the most importance for developing an industrial microorganism. For the isolated strain of Eurotium sp. FZ, the morphological features of colonies growing on media CZA30, CZA200 and M40Y, the microscopic features of sexual reproduction structures, the sub-microscopic features of ascospores under scanning electron microscope, were investigated. The morphological species name of the strain was identified as Eurotium cristatum relied on those typical morphological features.When applying multilocus sequence typing (MLST) approach to identify the strain of Eurotium sp. FZ, we determined the sequences from the four loci, ID region (ITS 1-5.8S rDNA-ITS2 and D1/D2 divergences of 28S rDNA), RPB2 (RNA polymerase II), BT2 (β-tubulin) and CF (calmodulin) and deposited the resulted sequences in GenBank under the accession numbers HQ148160, HQ148161, HQ148162 and HQ148163, respectively. The phylogenetic species name of the strain was identified as Eurotium cristatum based on the MLST approach.The identification results based on the morphological attributes and on the MLST approach were in agreement with each other.The ID region sequence of the yeast strain was determined and submitted in GenBank under the accession number of JF710627. The yeast was identified as Debaryomyces hansenii. This yeast is proposed to grant QPS (Qualified Presumption of Safety) status along the list of units referred to EFSA (European Food Safety Authority). The yeast strain of Debaryomyces hansenii would be a candidate microorganism for developing mixture fermentation starters for production of Fuzhuan brick-tea.3. Analysis of anthraquinone compounds in the Fuzhuan brick-tea related samplesThe presence of three anthraquinone derivatives (emodin, physcion and chrysophanol) in five Fuzhuan brick-tea related samples (Pu’er tea, raw dark green tea, Fuzhuan brick-tea, tea fermented by E. cristatum, biomass of E. cristatum) was detected by HPTLC (High performance thin layer chromatography). Under the experimental conditions for HPTLC, the three standards of emodin, physcion and chrysophanol were separated effectively with the Rf values 0.28,0.43 and 0.51, respectively. The bands’absorption peak and average peak areas were detected at 430 rm. The results indicated that emodin was present in all five samples in descending order of peak areas:raw dark green tea, Pu’er tea, tea fermented by E. cristatum, Fuzhuan brick-tea and biomass of E. cristatum. The physcion contained in the biomass of E. cristatum far exceeded that of tea fermented by E. cristatum, with no physcion found in the remaining three samples. Chrysophanol was not detectable in any of the samples. The emodin was present in all the dark tea samples detected which implied that the anthraquinone compounds (e.g. emodin) existing in dark teas (including Fuzhuan brick-tea) were important sources of dietary anthraquinones for tea consumers.4. Intervention of EHEC O157:H7 infected mice with water extracts from Fuzhuan brick-teaThe EHEC O157:H7 infected murine models were established via intragastric administration with fresh EHEC O157:H7 cells suspension (OD600=1.0) to the mature SPF grade KM mice. The detection of EHEC O157:H7 in their fresh feces was positive. The mice behaved with disease symptoms.Five groups of mice were designed, including normal control, EHEC O157:H7 infected model, intervention with low-dosage water extract (ig.1.0 g/kg daily), middle-dosage (ig.10.0 g/kg daily) and high-dosage (ig.20.0 g/kg daily). The dosages corresponded to normal consumption of Fuzhuan brick-tea (6.6 g/d) for a mature human being (60 kg) and by a factor of 10 and 20, respectively.A comparison analysis was conducted for the five groups of experimental mice in aspects of histopathology, haematology, immunology and intestinal microecology. The results showed that the infected mice model displayed typical symptoms of EHEC O157:H7 infectious disease. The histopathological examination indicated that severe lesions were observed in their gastric, duodenal, ileal, colonic and renal tissue and cell structures. The haematological indices reflected their impaired liver function and renal function. The immunological indices approved their compromised immune system.Low-dosage tea extracts showed rehabilitation-promoting effects on EHEC O157:H7 infected mice to some extent. Moreover, for the middle- and high-dosage groups, the detected indices were improved in comparison with that of infected models, recovered to levels of normal controls and even become superior to that. The results indicated appropriate dosage of water extracts from Fuzhuan brick-tea would exhibit protection and recovery effects on the detriments induced by EHEC O157:H7 infection. The extracts of Fuzhuan brick-tea showed anti-infective effects against EHEC O157:H7 as following:1) maintaining the integration of intestinal epithelium, 2) regulation of the intestinal microbiota,3) improving the body immunity, and 4) protecting liver and kidney against impairment, and ensuring the normal functions of these organs. Considering the dose administered to middle- and high- dosage groups matched with the 10- and 20-times of normal consumption of Fuzhuan brick-tea (6.6 g) for mature human being (60 kg), extrapolating to human, it was suggested that the 10- 20 times concentrated extracts from daily consumed Fuzhuan brick-tea would have therapeutic effect on EHEC 0157:H7 caused infectious diarrhea.The results provided experimental basis and theoretical references for applying Fuzhuan brick-tea to treat EHEC O157:H7 caused infectious diarrhea.5. Effects of Eurotium cristatum on mice colonic microbiotaThe acute toxicity of E. cristatum to SPF grade KM mice was determined. Under the maximal tolerance dose (10000 mg/kg) of suspension of E. cristatum after intragastric administration, zero mortality of the experimental mice was evident. The result indicated that the maximal tolerance dose of the trial (ig), E. cristatum, was greater than 10000 mg/kg. According to the grades of acute toxicity (ig) of chemical compounds, the LD50 range of 5001-15000 mg/kg for mice (ig) indicates the trial belonging in toxicity grade II, that is, an actual non-toxic substance.Four groups of mice were designed, including normal control, intragastric administration with low-dosage water suspension of E. cristatum (50 mg/kg, daily), middle-dosage (200 mg/kg, daily) and high-dosage (1000 mg/kg, daily). The dosages corresponded to daily consumption of E. cristatum 0.33 g,1.32g and 6.6g for a mature human being (60 kg), respectively.The general bacteria, Lactobacillus, Bacteroides and Clostridium cluster IV population structure were detected by PCR-DGGE method. The results showed the colonic bacterial structures in mice were different individually. Low-dosage suspension of E. cristatum increased the population diversity of general bacteria, Lactobacillus and Clostridium cluster IV, but had not obvious effects on Bacteroides population. Considering the health-promoting benefits of Lactobacillus and Clostridium cluster IV, the results implicated that low-dosage suspension of E. cristatum would have up-regulation function to intestinal microbiota. Extrapolating to human, it was implicated that daily consumption of E. cristatum 0.33 g for a mature human being (60 kg) would have up-regulation function to human intestinal microbiota.The results provided experimental basis for developing E. cristatum preparation.