Computational protocol: Effects of microbial enzymes on starch and hemicellulose degradation in total mixed ration silages

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Protocol publication

[…] The following procedure was employed to screen and isolate amylolytic or hemicellulolytic microorganisms from TMR silages during ensiling. To release the microorganisms in samples, 10 g wet samples were homogenized in 90 mL sterilized distilled water, and serially diluted from 10−1 to 10−5 in sterilized water. Each dilution (50 μL) was evenly spread onto the modified nutrient agar and MRS agar to screen for enzyme-producing aerobic bacteria and LAB, respectively. The modified agar mediums were prepared according to the manufacturer’s (Difco, USA) direction with the exception that beef extract and glucose were replaced with soluble starch or hemicellulose as the sole carbon source to screen for amylolytic or hemicellulolytic microorganisms, respectively []. After cultivation under recommended conditions, strains with a clear halo around the colony were isolated and purified by repeated streaking and checked for homogeneous morphology. The purified isolates were conserved in 20% glycerol at −80°C for further analysis.To identify the species of the purified isolates, polymerase chain reaction (PCR) was carried out to amplify the complete 16S rRNA gene sequence with the forward primer 27f (5′-AG AGTTTGATCCTGGCTCAG-3′) and the reverse primer 1492r (5′-GGTTACCTTGTTACGACTT-3′) []. The PCR procedure was performed as described by Hu et al. []. The PCR products were separated by gel electrophoresis on a 1.0% agarose gel, detected by Gold View (Solarbio, Beijing, China) according to the manufacturer’s instructions and photographed under UV light with a charge-coupled device camera. Sequencing was carried out by Shanghai Sunny Biotechnology Co., Ltd. (Shanghai, China), and then the sequences were analyzed using BLASTN online tool (http:/blast.ncbi.nlm.nih.gov/Blast.cgi). The 16S rRNA gene sequences of isolates and sequences from the type strains held in GenBank were aligned with program CLUSTAL W []. Phylogenetic tree was constructed from the evolutionary distance data that were calculated from Kimura’s two-parameter model [] using the neighbor-joining method []. Bootstrap analyses were performed on 1,000 random resamplings. Evolutionary analyses were conducted in MEGA6 []. The nucleotide sequences for the 16S rRNA gene described in this paper were deposited in the NCBI GenBank data library under accession numbers KU239970 to KU239983. [...] Statistical analysis was performed using the general linear model procedure of IBM SPSS Statistics for Windows (Version 20.0; IBM Co., Armonk, NY, USA). Data on fermentation qualities, microbial counts, chemical compositions, ensiling losses and microbial enzyme activities were subjected to two-way analysis of variance, with the fixed effects of days of ensiling, type of TMR (ATMR vs LTMR), and the interactions between days of ensiling and type of TMR. Significance was defined at a 0.05 probability level. […]

Pipeline specifications

Software tools BLASTN, Clustal W, MEGA, SPSS
Applications Miscellaneous, Phylogenetics
Organisms Glycine max, Zea mays subsp. mays, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus licheniformis, Bacillus subtilis, Enterococcus faecium, Bacillus pumilus
Chemicals Carbohydrates, Lactic Acid