Isolation and Identification of Arisaema Cum Bile Fermentation Strain and Preliminary Investigation on Its Compound Strain Fermentation / 中国实验方剂学杂志

ObjectiveIn order to solve the problem that the quality and stability of Arisaema Cum Bile in the fermentation process with hybrid bacteria were not easy to control， the microorganism in the fermentation process of Arisaema Cum Bile was isolated and identified， the dominant strains were screened and the fermentation process of Arisaema Cum Bile with compound bacteria was investigated. MethodThe submerged culture during the fermentation process of Arisaema Cum Bile was taken out for strain separation and purification. Bacteria and fungi multiphase identification and detection methods and automatic microbial analysis system were used to analyze and compare DNA sequences and identify microorganisms. The isolated and identified strains were respectively inoculated and fermented. After screening the dominant strains， a preliminary exploration of compound strain fermentation were carried out. The contents of index components in Arisaema Cum Bile fermented by compound strain and traditional Arisaema Cum Bile were compared by ultra-performance liquid chromatography-triple quadrupole tandem mass spectrometry （UPLC-QqQ-MS/MS）. Mmobile phase was 0.1% formic acid acetonitrile solution （A）-0.1% formic acid aqueous solution （B） for gradient elution （0-2 min， 35%-45%A； 2-10 min， 45%-48%A； 10-12 min， 48%-100%A； 12-12.01 min， 100%-35%A； 12.01-15 min， 35%-65%A）， the flow rate was set at 0.35 mL·min-1. The mass spectrographic analysis employed electrospray ionization （ESI）， negative ion acquisition mode and multiple reaction monitoring （MRM） scanning mode were adopted to collect information， the collection range was m/z 50-1 000. ResultEight microorganisms were isolated and identified from the submerged culture of Arisaema Cum Bile. Among them， Enterococcus sp. （anaerobic） and E. casseliflavus were selected as the dominant strains in the fermentation process. Compared with the traditional fermentation method， the contents of chenodeoxycholic acid， hyodeoxycholic acid and hyocholic acid in free cholic acid increased by 1.76， 0.06， 0.19 mg·g-1， respectively. In bound cholic acid， glycochenodeoxycholic acid， taurochenodeoxycholic acid， glycohyodeoxycholic acid， taurohyodeoxycholic acid， glycohyocholic acid， taurine porcine cholic acid decreased by 0.63， 0.23， 0.26， 0.16， 0.03， 0.04 mg·g-1， respectively. ConclusionArisaema Cum Bile with compound strain fermentation （Enterococcus sp. and E. casseliflavus） can be fermented more completely， the fermentation cycle can be shortened， and the quality and stability of products can be improved.

Screening of plant growth-promoting rhizobacteria and its effect on seed germination of Polygonum multiflorum / 中国中药杂志

In this study, the rhizobacteria and actinomycetes of Polygonum multiflorum were screened for the strains with indole acetic acid(IAA)-producing capacity by Salkowski method, the siderophore-producing strains by Chrome Azurol S(CAS) assay, and the strains with inorganic phosphorus-solubilizing capacity by PKO inorganic phosphorus medium. The strains were identified by morphological identification, physiological and biochemical characteristics, and 16 S rDNA sequences. Furthermore, the effect of growth-promoting strains on the seed germination and development of P. multiflorum was tested. The results showed that among 196 strains, two strains F17 and F42 were found to be capable of producing IAA and siderophore and solubilizing inorganic phosphorus simulta-neously. For F17 and F42, the results are listed below: 38.65 and 33.64 mg·L~(-1) for IAA production, 0.85 and 0.49 for siderophore-producing capacities(A_s/A_r), and 1.35 and 1.70 for inorganic phosphorus-solubilizing capacities(D/d), respectively. Comprehensive analysis revealed that strains F17 and F42 were identified as Pseudochrobactrum asacharolyticum and Bacillus aryabhattai, respectively, and both could significantly promote the seed germination of P. multiflorum.

Screening and identification of fibrinolytic enzyme-producing microbes in fermentation process of Sojae Semen Praeparatum / 中草药

Objective: To screen and identify the dominant strains which produce fibrinolytic enzyme during the processing of Sojae Semen Praeparatum (SSP, Dandouchi in Chinese). Methods: SSP was prepared according to the Chinese Pharmacopoeia (2020 edition), and samples were taken at different time points during the fermenting process of SSP.The casein plate method and fibrin plate method were used to screen the fibrinolytic enzyme-producing microorganisms in samples at different time points. The fibrinolytic enzyme-producing microorganisms were inoculated in the designated liquid medium to obtain single strain fermentation broth, and fibrin plate method was used to measure the fibrinolytic activity of the fermentation broth. The DNA sequences of fibrinolytic enzyme-producing bacteria and fungi were amplified using 16S rDNA and 18S rDNA universal primer by PCR respectively.The amplified products were sequenced, and the sequencing results were identified through NCBI homology comparison. Molecular biological identification was done by phylogenetic tree constructed by MEGA 4.1 software. Results: Three types of fibrinolytic enzyme-producing bacteria were screened out and identified in this study. They were Bacillus subtilis, Stenotrophomonas maltophilia and Micrococcus, respectively. The result of fibrin plate method showed that the fermentation broth of S. maltophilia had the highest fibrinolytic activity, reaching 527.49 IU/mL. Conclusion: There are fibrinolytic enzyme-producing dominant microorganisms existing in the fermenting process of SSP and the thrombolytic effect of SSP is worthy of further study. This study lays the foundation for revealing the formation mechanism of fibrinolytic enzyme in the fermentation process of SSP.

Screening and identification of GABA-producing microbes in fermentation process of Sojae Semen Praeparatum / 中国中药杂志

A high-content GABA was found in Sojae Semen Praeparatum(SSP), which is a famous traditional Chinese medicine and officially listed in Chinese Pharmacopoeia. To screen out and identify GABA-producing microbes from samples at different time points during the fermenting process of SSP, traditional microbiological methods combined with molecular biological methods were used to study the predominant GABA-producing microorganisms existing in the fermenting process of SSP. This study would lay a foundation for further studying the processing mechanism of SSP. The fermenting process of SSP was based on Chinese Pharmacopoeia(2010 edition), and samples were taken at different time points during the fermenting process of SSP. The bacteria and fungi from samples at different time points in the fermenting process of SSP were cultured, isolated and purified by selective medium, and dominant strains were selected. The dominant bacteria were cultured in the designated liquid medium to prepare the fermentation broths, and GABA in the fermentation broth was qualitatively screened out by thin-layer chromatography. The microbial fermentation broth with GABA spots in the primary screening was quantitatively detected by online pre-column derivatization and high performance liquid chromatography established in our laboratory. GABA-producing microorganisms were screened out from predominant strains, and their GABA contents in fermentation broth were determined. The DNA sequences of GABA-producing bacteria and fungi were amplified using 16S rDNA and 18S rDNA sequences by PCR respectively. The amplified products were sequenced, and the sequencing results were identified through NCBI homology comparison. Molecular biological identification was made by phylogenetic tree constructed by MEGA 7.0 software. Through the homology comparison of NCBI and the construction of phylogenetic tree by MEGA 7.0 software, nine GABA-producing microorganisms were screened out and identified in this study. They were Bacillus subtilis, Enterococcus faecium, E. avium, Aspergillus tamarii, A. flavus, A. niger, Cladosporium tenuissimum, Penicillium citrinum and Phanerochaete sordida respectively. For the first time, nine GABA-producing microorganisms were screened out and identified in the samples at different time points during the fermenting process of SSP in this study. The results indicated that multiple predominant GABA-producing microorganisms exist in the fermenting process of SSP and may play an important role in the formation of GABA.

Identification of dominant microbes from traditional Chinese medicine Pinelliae Rhizoma Fermentata / 中国中药杂志

To investigate the microbial species, amount changes as well as the isolation and identification of domain strains at different fermentation time points of Pinelliae Rhizoma Fermentata, and provide basis for exploring the mechanism of Pinelliae Rhizoma Fermentata processing. Five samples were chosen at the time points (0, 18, 36, 54, 72 h) of Pinelliae Rhizoma Fermentata processing. Bacteria, mold and yeast from the samples were cultured; their colonies were counted, and the dominant strains were isolated and purified. The dominant bacteria and dominant fungi were identified by 16S rDNA and 26S rDNA sequencing respectively. The results showed that the bacteria count was low with slow and smooth changes in the fermentation process;while mold and yeast grew dramatically after 54 h culturing and reached 1×107 CFU•mL⁻¹ at the end of fermentation. Through the NCBI homology alignment and phylogenetic tree construction, the dominant bacteria were identified as Streptomyces sp., Bacillus pumilus, B. subtilis, B. aryabhattai and other Bacillus sp.; the dominant yeast was identified as Meyerozyma guilliermondii; the dominant mold were identified as Paecilomyces variotii, Byssochlamys spectabilis, and Aspergillus niger in the processing of Pinelliae Rhizoma Fermentata. The results indicated that multiple microbe species, especially yeast and mold, played a role in the fermentation processing of Pinelliae Rhizoma Fermentata. M. guilliermondii, P. variotii, P. variotii and A. niger and Bacillus sp. can be the crucial factors in the processing of Pinelliae Rhizoma Fermentata.

The molecular diversity analysis of Auricularia auricula-judae in China by nuclear ribosomal DNA intergenic spacer

Background: For the crossbreeding of Auricularia auricula-judae, selecting the appropriated parents in hybridization is very important. However, the classification and diversity analysis of A. auricula-judae has been equivocal, due to the similarity of the fruiting body morphology and its susceptibility to environmental influences. For this purpose, the molecular diversity of 32 A. auricula-judae commercial cultivars in China was analyzed by using the nuclear ribosomal DNA intergenic spacer. Results: The complete nuclear rDNA gene complex of A. auricula-judae isolate is 11,210 bp long, and contains the 18S, 5.8S, and 28S rRNA gene as well as the ITS and IGS regions. Based on the sequence data, four more effective primer combinations for the IGS region of A. auricula-judae were designed. Nucleotide sequence variation in the IGS among 32 A. auricula-judae commercial cultivars in China sorted into three strongly supported clades, which is correlated with geographical regions. Most strains originated from the same area were with a narrow genetic basis and could possibly be domesticated from the local wild-type strains. Conclusion: The grouping information obtained in the present work provides significant information for further genetic improvement in A. auricula-judae, and suggested that the IGS region can be used as an excellent tool for identification of genetic variation.

Analysis of pathogen identification from non-mycobacterium pulmonary tuberculosis / 中国感染与化疗杂志

Objective To analyze the nontubercutous mycobaeterium (NTM) identification data of two groups of sputum sam- ples during the periods of 1986 to 1997 and 2000 to 2005 so as to figure out the identification of NTM.Methods A total of 222 strains of non-tuberculosis mycobacteria were included for strain identification and sensitivity test with traditional methods.Re- suits According to Runyon classification, during the period of 1986-1997 there were 15 strains (15.5%) in GroupⅠ, 4 (4.2%) in GroupⅡ, 23 (24.0%)in GroupⅢand 54 (56.3%) in GroupⅣ;during the period of 2000—2005 there were 30 strains (16.1%) in GroupⅠ, 11(5.9%) in GroupⅡ, 51 (27.4%) in GroupⅢand 94 (50.6%) in GroupⅣ.The number of NTM types increased by 133.3%.The absolute number of NTM isolates in the first five years of this century increased by 93.89% compared with the numbers in the 11 years of last century.Conclusions The number of types and absolute number of i- solates of NTM have increased in the first live years of this century compared with the numbers in the 11 years of last century. We should enhance the epidemiological research on pulmonary NTM in order to provide scientific evidence for comprehensive prevention and treatment.

A Mycological Study on the Candidal Paronychia of the Swine Plant Farmers in Chung-Cheong Area / 대한피부과학회지

BACKGROUND: Candida species are normal flora in human and animals. Also they may cause diseases as pathogen. Bovine mastitis, cutaneous candidiasis in swine, and chicken dermatitis caused by Candida species were reported. Therefore, plant farmers which frequently contact with these animals may be at risk of candidal infection. OBJECTIVE: The purpose of this study is to investigate the possibility of candidal infection from swine to human in candidal paronychia. METHODS: We performed a mycological study in 36 swine plant farmers in Chung-Cheong area between March 1998 and February 2000. To define the species of Candida, culture on Sabouraud's dextrose agar(SDA) with and without chloramphenicol, germ tube test, and API 20C AUX yeast identification system were performed. For identification of strains, pulsed field gel electrophoresis(PFGE) was performed. RESULTS: Among 36 swine plant farmers, we found 15 patients with candidal paronychia and all 15 patients were midwives for swine. The species of Candida isolated from paronychial lesion of the patients and swine which the patients bred were Candida(C.) tropicalis, C. albicans, and C. krusei. When the same species between the patients and their swine were isolated, PFGE was performed for strain identification. The results of PFGE showed that the strains isolated from between the patients and their swine were identical in 6 of 7 cases. CONCLUSION: The results of genotyping analysis suggest that the Candida in swine cause paronychial lesion in human.

IDENTIFICATION OF BACTERIUM D-97 PRODUCING TREHALOSE FROM STARCH OR MALTOOLIGOSACCHRIRIDES / 微生物学通报

A bacterium D 97 whose enducellular enzymes can produce trehalose from starch or maltooligosaccharides was isolated from Datian in Northeast of China The morphological, Cultural and Physiological Characteristics of trehalose prodicing bacterium D 97 were described in this paper There are galactose and lysine in its cell wall but no diaminopimelic acid (DAP) arabinose The content of G+C mol% is 61 3 The full lenth of 16s rDNA (770bp) has been tested and compared with the type strains of all known Arthrobacter sp in Genebank, the results showed the similarity values of 16s rDNA sequence between bacterium D 97 and Arthrobacter nicotinovorus were 97 98%, therefore bacterium D 97 was nameed Arthrobacter nicotinovorus D 97 In this paper we also carried out some comparisons of Physiological Characteristics between Arthrobacter nicotinovorus D 97 and Arthrobacter sp Q36

SCREENING,IDENTIFICATION AND CHARACTERISTIC OF A STRAIN OF FACULTATIVE ANAEROBE METABOLIZING DIESEL / 微生物学通报

The strain of DG7 isolated from the oilwater sample in DAGANG oilfield could produce surfactant ?acids and gas in the culture medium in which the diesel was sole carbon source .The strain was Gram-negative, motive, rod and growed facultatively in the 0 to 18.5% NaCl. Based on its characters, the strain was identified as a member of the genus Aeromonas, but there were some differences between this strain and the described species of this genus in some biochemical features, suggesting that it could be a new species of the genus.

Susceptibility testing and strain identification of Candida species / 重庆医科大学学报

Objective:To identify the strain and to study the in-vitro susceptibilities of Candida species collected from clinical samples of skin and mucosa to Amphotericin B,Ketoconazole,Miconazole,Itraconazole,Fluconazole,5-Flucytosine and Terbinafin. Methods:110 clinical isolates of yeasts were identified by CHROMAGAR and API20C, meanwhile, the susceptibility to six antifungal drugs was examined by microdilution method using Sensititre Yeast One panels. Results:There were 94 C.albicans, 6 C.tropicalis,4 C.krusei,4 C.glabrata,and 2 C.parapsilosis among 110 isolates of yeasts.There were 0.91%,1.81%,18.28%,1.81%,5.45%,3.63% and 94.55% of isolates displaying resistance to Amphotericin B,Ketoconazole,Miconazole,Itraconazole,Fluconazole,5-Flucytosine and Terbinafin, respectively.Conclusions:C.albicans is the main infection strain in our hospital,and candida species display different resistance to six antifungal drugs. It is necessary to test strain identification and susceptibility of candida species to guild the rational clinical drug use.