Isolation and identification of lactic acid bacteria in forage peanut silage
Cultivars of <i>Arachis pintoi</i> species are widespread in tropical and subtropical areas in Brazil. There are a limited number of reports about the use of tropical leguminous for ensiling process mainly with regard to the autochthonous population of lactic acid producing bacteria (LAB). Therefore, the objective of the present study was to isolate and identify the LAB by the partial sequencing of the 16S rDNA gene in silage of <i>Arachis pintoi</i> cv. Belmonte with 0, 3, 7, 14, 28, and 56 days of fermentation. Forage was ensiled in triplicate by vacuum packing the forage in plastic bags and stored at room temperature. Silage samples were mixed with 225 mL of saline solution and serial dilutions were performed. The dilutions were then plated in MRS agar by using Pour plate technique. Plates were incubated at 37 <sup>o</sup>C for 48 h. After incubation period 40 colonies were randomly selected for streak in MRS agar and then incubated at 37 <sup>o</sup>C for 48 h. Selected isolates were tested for catalase, Gram staining and morphology analysis. The isolates characterized as catalase negative and gram positive were selected to be identified by 16S gene rDNA sequencing. The PCR analysis was performed by using GoTaq DNA polymerase kit and a set of primers 1378/P027. The amplified fragment was purified and sequenced. Among the 40 isolates, only 17 isolates had sequences with identity equal to or greater than 97% with sequences already available in GenBank database. From these 17 sequences the <i>Lactobacillus plantarum</i>, <i>Lactobacillus paraplantarum</i>, <i>Pediococcus pentosaceus</i>, and <i>Lactobacillus casei subs. Casei</i> were detected. The majority of the isolates were identified as <i>Pediococcus pentosaceus</i>. The sequence of isolates obtained in the current study was not matched with the sequences of 16S rDNA of <i>L. plantarum</i> or <i>Pediococcus pentosaceus</i> already available at Genbank. The lack of matching among the sequences of the isolates obtained in this study with the sequences available at Genbank does not mean that they are not from the same specie. Microorganisms are susceptible to constant mutations varying according to the environment conditions. The accumulation of these mutations may be the reason of the difference among the sequences of different strain.
Sponsored by FAPEMIG, CNPq and INCT-CA
Keywords: <i>Lactobacillus plantarum</i>, , legume silage, <i>Pediococcus pentosaceus</i>