Monitoring of the bacterial and fungal biodiversity and dynamics during Massa Medicata Fermentata fermentation

The microbial community dynamics play an important role during Massa Medicata Fermentata (MMF) fermentation. In this study, bacterial and fungal communities were investigated based on the culture-dependent method and polymerase chain reaction-denaturing gradient gel electrophoresis analysis. Meanwhile the dynamic changes of digestive enzyme activities were also examined. Plating results showed that MMF fermentation comprised two stages: pre-fermentation stage (0–4 days) was dominated by bacterial community and post-fermentation stage (5–9 days) was dominated by fungal community. The amount of bacteria reached the highest copy number 1.2 × 1010 CFU/g at day 2, but the fungi counts reached 6.3 × 105 CFU/g at day 9. A total of 170 isolates were closely related to genera Enterobacter, Klebsiella, Acinetobacter, Pseudomonas, Mucor, Saccharomyces, Rhodotorula, and Amylomyces. DGGE analysis showed a clear reduction of bacterial and fungal diversity during fermentation, and the dominant microbes belonged to genera Enterobacter, Pediococcus, Pseudomonas, Mucor, and Saccharomyces. Digestive enzyme assay showed filter paper activity; the activities of amylase, carboxymethyl cellulase, and lipase reached a peak at day 4; and the protease activity constantly increased until the end of the fermentation. In this study, we carried out a detailed and comprehensive analysis of microbial communities as well as four digestive enzymes' activities during MMF fermentation process. The monitoring of bacterial and fungal biodiversity and dynamics during MMF fermentation has significant potential for controlling the fermentation process.

[1]  H. M. Abdel-Mageed,et al.  Characterization of Mucor racemosus lipase with potential application for the treatment of cellulite , 2011 .

[2]  Wenyuan Gao,et al.  [Identification of microorganism from traditional Chinese medicine Massa Medicata Fermentata]. , 2012, Zhongguo Zhong yao za zhi = Zhongguo zhongyao zazhi = China journal of Chinese materia medica.

[3]  Vita Kiriliauskait Properties and Immobilization of Enterobacter aerogenes 13 Lipase , 2011 .

[4]  D Schomburg,et al.  Extracellular lipase of Pseudomonas sp. strain ATCC 21808: purification, characterization, crystallization, and preliminary X-ray diffraction data , 1991, Journal of bacteriology.

[5]  Tian-xiang Wu,et al.  Advances in the study on microbial fermentation and transformation of traditional Chinese medicine , 2013 .

[6]  R. Facklam,et al.  Phenotypic and Genotypic Characterization ofPediococcus Strains Isolated from Human Clinical Sources , 2001, Journal of Clinical Microbiology.

[7]  Sang Hyun Lee,et al.  Identification and Functional Characterization of an α-Amylase with Broad Temperature and pH Stability from Paenibacillus sp. , 2013, Applied Biochemistry and Biotechnology.

[8]  Qing Fan,et al.  Characterization and comparison of microbial community of different typical Chinese liquor Daqus by PCR–DGGE , 2011, Letters in applied microbiology.

[9]  Haiyang Wang,et al.  [Review of traditional Chinese medicine processed by fermentation]. , 2012, Zhongguo Zhong yao za zhi = Zhongguo zhongyao zazhi = China journal of Chinese materia medica.

[10]  Narendra Kumar,et al.  Production and purification of α-amylase from hydrogen producing Enterobacter cloacae IIT-BT 08 , 2000 .

[11]  T. Abe,et al.  Microflorae of aquatic moss pillars in a freshwater lake, East Antarctica, based on fatty acid and 16S rRNA gene analyses , 2012, Polar Biology.

[12]  G. L. Miller Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar , 1959 .

[13]  Charles S Henry,et al.  Screening for physical stability of a Pseudomonas amylase using self-interaction chromatography. , 2006, Analytical biochemistry.

[14]  G. Muyzer,et al.  Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology , 2004, Antonie van Leeuwenhoek.

[15]  K. Okada,et al.  [Detection of extracellular protease in Mucor species]. , 2005, Revista iberoamericana de micologia.

[16]  Wenyuan Gao,et al.  [Influence of different processing techniques of massa medicata fermentata on their amylase activity]. , 2012, Zhongguo Zhong yao za zhi = Zhongguo zhongyao zazhi = China journal of Chinese materia medica.

[17]  B. Smiley,et al.  Comparative denaturing gradient gel electrophoresis analysis of fungal communities associated with whole plant corn silage. , 2001, Canadian journal of microbiology.

[18]  J. Coca,et al.  Expression and characterization of lipase produced by mucor griseocyanus @ , 2006 .

[19]  Philip Hugenholtz,et al.  Impact of Culture-Independent Studies on the Emerging Phylogenetic View of Bacterial Diversity , 1998, Journal of bacteriology.

[20]  T. K. Ghose Measurement of cellulase activities , 1987 .

[21]  A. Uitterlinden,et al.  Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA , 1993, Applied and environmental microbiology.

[22]  A. Endo,et al.  Monitoring the lactic acid bacterial diversity during shochu fermentation by PCR-denaturing gradient gel electrophoresis. , 2005, Journal of bioscience and bioengineering.

[23]  P. Teixeira,et al.  Characterization of microbial population of ‘Alheira’ (a traditional Portuguese fermented sausage) by PCR‐DGGE and traditional cultural microbiological methods , 2008, Journal of applied microbiology.

[24]  S. Seseña,et al.  Bacterial biodiversity and dynamics during malolactic fermentation of Tempranillo wines as determined by a culture-independent method (PCR-DGGE) , 2010, Applied Microbiology and Biotechnology.

[25]  I. Haq,et al.  Isolation and screening of fungi for the biosynthesis of alpha amylase , 2002 .

[26]  Q. Shen,et al.  Dynamics of bacterial communities during solid-state fermentation using agro-industrial wastes to produce poly-γ-glutamic acid, revealed by real-time PCR and denaturing gradient gel electrophoresis (DGGE) , 2011, Applied Microbiology and Biotechnology.

[27]  G. Díaz-Godínez,et al.  CHARACTERIZATION OF AN ASPARTIC PROTEASE PRODUCED BY Amylomyces rouxii , 2011 .

[28]  P. Cocconcelli,et al.  Surface microbiota analysis of Taleggio, Gorgonzola, Casera, Scimudin and Formaggio di Fossa Italian cheeses. , 2010, International journal of food microbiology.

[29]  B. Bisping,et al.  Vitamin B12 production by Citrobacter freundii or Klebsiella pneumoniae during tempeh fermentation and proof of enterotoxin absence by PCR , 1994, Applied and environmental microbiology.

[30]  J. Tiedje,et al.  DNA recovery from soils of diverse composition , 1996, Applied and environmental microbiology.

[31]  E. W. Jones,et al.  Protease B of Saccharomyces cerevisiae: isolation and regulation of the PRB1 structural gene. , 1987, Genetics.

[32]  U. Banerjee,et al.  Characterization of a fungal amylase from Mucor sp. associated with the marine sponge Spirastrella sp. , 1998 .

[33]  K. Okada,et al.  Detección de proteasas extracelulares en especies de Mucor , 2005 .