Api 50 chb

All chemicals used were of analytical grade. Potassium chromate, 0.1 N Sodium hydroxide, silver nitrate, methyl alcohol, ethyl alcohol, and sodium chloride were obtained from Duksan Pure Chemicals (Ansan, Gyeonggi-do, Korea). Sodium hydroxide, sodium hydrogen carbonate, and ammonium hydroxide were purchased from Junsei Chemicals (Tokyo, Japan). Formalin solution, standard methanol, ethanol, pentanol, propanol, and butanol were purchased from Sigma-Aldrich (St. Louis, MO, USA). Plate count agar, nutrient agar, potato dextrose agar (PDA), and potato dextrose broth were purchased from Difco (Becton, Dickinson and Company, Sparks, MD, USA). Mannitol egg yolk polymyxin agar (MYP), egg yolk emulsion, and polymyxin B supplement were purchased from Oxoid LTD (Basingstoke, Hampshire, UK). 3M Yeast and Mold Petrifilm was purchased from 3M Health Care (St. Paul, MN, USA). API 50CHB and API 20E were obtained from bioMerieux (Marcy I’Etoile, France).

Each colony that grew on nutrient agar media was collected and isolated. The isolates were streaked in four quadrants on nutrient agar media (Oxoid) to obtain pure cultures. The isolates were incubated at 28°C for 24 h. Bacterial identification was conducted using the biochemical analytic profile index (API) 50 CH, API 50 CHB, and API 20E kit (BioMérieux, France). Tests were conducted in accordance with the manufacturer’s instructions. Analysis of the test results was carried out on the software https://apiweb.biomerieux.com/login.

Experiments using API 50CHB (Bio Mérieux, Lyon, France) and Gen Ⅲ MicroStation (Biolog, Hayward, CA, USA) were carried out to evaluate the physiological and biochemical characteristics, in accordance with the manufacturers’ protocols. All experiments were conducted in LB broth at 30 °C for 24 h.

The optimal growth conditions for strains J780^T and J316 were detected in nutrient broth at different temperatures (4, 16, 28, 30, 35, 37, and 42°C), salinities [0.5–10% (w/v) NaCl at intervals of 0.5%], and pH [(3–12) at intervals of 1.0 pH unit]. Cell morphology, Gram-staining, and colony appearance were observed using isolated bacterial stains grown on NA plates at 28°C for 24 h. Cell morphology was observed by a scanning electron microscope (SU8010, Hitachi). Gram-staining was performed using a Gram-staining kit (Baso) (Austrian, 1960 (link)). With E. aphidicola DSM 19347^T, E. persicina GDMCC 1.331^T, and E. rhapontici ATCC 29283^T as positive controls for the motility assay, cell motility was tested by growing in semi-solid agar at 22°C for 48 h (Xu et al., 2013 (link)). The catalytic activity of catalase was detected using 3% (v/v) H₂O₂. Enzymatic activity and acid production from various carbohydrates of the novel strains were determined by API ZYM, API 50 CHB, API 20E, and API 20NE strips (bioMérieux), and strains E. aphidicola DSM 19347^T, E. persicina GDMCC1.331^T, and E. rhapontici ATCC 29283^T were used as the parallel references.

Morphological characterisation, temperature optimum, salt tolerance, API gallery tests, and fatty acids cellular composition profiles were carried out by DSMZ Services, Leibniz Institut DSMZ—Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany.
The fatty acids profile was analysed by gas chromatography of fatty methyl esters (GC-FAME), using minor modifications of the method of Miller^{62 (link)} and Kuykendall et al.⁶³ .
Biochemical characterisation, including the analysis of different substrate utilisation and enzyme production, was performed through API 50 CHB, API 20E, API 20NE, and API ZYM systems (Biomérieux) according to the manufacturer’s instructions.

Bacterial strains were isolated from samples of puba that were obtained from different batches of the same producer. The samples were obtained in the District of Saco da Raiz in Estância, a town of Sergipe State, located in northeast of Brazil. The strains were selected among Gram-positive rods obtained from puba samples, where five isolates were presumptively identified as Bacillus species based on standardized methods including observation of cell morphology Gram-staining, phase-contrast microscopy for detection of parasporal crystal proteins formation, and catalase activity (Food and Drug Administration-Bacteriological Analytical Manual [FDA-BAM], 2012 ). Additional tests were conducted using the identification kits API 50 CHB and API 20E (BioMérieux SA, Marcy-l’Étoile, France). The results were analyzed by the API LAB Plus software for strain identification (BioMérieux SA). Following, the antimicrobial potential of these strains was tested against varied bacteria, yeasts and filamentous fungi (data not show). The strains C3 and P5, which presented the most promising results, were selected for the subsequent tests.