Int.J.Curr.Microbiol.App.Sci (2018) 7(5): 850-858 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 7 Number 05 (2018) Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2018.705.104 Screening of Antimicrobial Spectrum of Brevibacillus sp. Isolated from Dairy Environment Sandeep Kumar1*, Ebenezer Jeyakymar2, Rubina Lawrence2, Utkarsh Singh Rathore1 and Monika Mishra1 1Division of Crop Protection, Indian Institute of Pulses Research, Kalyanpur, Kanpur, UP (East), India 2Department of Industrial Microbiology, Sam Higginbottom University of Agriculture, Technology and Sciences, Allahabad, India *Corresponding author ABSTRACT Bacteriocins and Bacteriocin like Inhibitory Substance (BLIS) are ribosomal products exhibiting antimicrobial activity. The present study aimed to screen for production of antimicrobial agents from soil bacteria of dairy environment. Borer was used to dig soil K e yw or ds and collected in sterile plastic bags. Isolation was done on tyrosine agar. To exclude non Brevibacillus brevis, spore formers the soil suspension, was heated at 80ºC. The bacterial isolates were Brevicin, Bacteriocin, identified underpinning biochemical and carbohydrate utilization tests and screened for Antagonistic activity, Tyrosine agar their antibacterial activity against methicillin resistant S. aureus and E. coli by agar well diffusion method. Among forty nine isolates three isolates were identified as Brevibacillus Article Info brevis. Screening of crude brevicin obtained from cell free broth inoculated with B. brevis Accepted: exhibited a strong antagonistic activity against S. aureus but no zone of inhibition was 10 April 2018 recorded against E. coli. The spore forming bacterium, B. brevis produced narrow Available Online: spectrum antibacterial agent. Strains of Brevibacillus are potent source of bacteriocins and 10 May 2018 these antimicrobial peptides can be used as an effective therapeutic agent in pharmaceuticals as well as can also be used as food preservatives in canned or packed food and dairy products, controlling pathogens with natural products instead of synthetic ones. Introduction Fulfilling the consumer demands of ready to eat materials are on hike, placing a challenge Bacteria and their products have been proved for industries to provide quality food. to be beneficial as well as harmful in different Microbes are ubiquitous and very much sectors such as food and dairy, adaptive and evolve new strategies to survive, pharmaceutical, etc. Spoilage of food resulting in altered food safety hazards, WHO materials even after packaging is of great food strategic planning meeting have concern for industries. However, advancement noticeably have shown their concern on the in technology has ameliorated the losses. issue (Food safety strategic planning meeting, 850 Int.J.Curr.Microbiol.App.Sci (2018) 7(5): 850-858 2001). Similarly, rapid spread of antibiotic was isolated from soil samples collected from resistant pathogens due to institutional abuse dairy environment with help of surface of antibiotics is an alarm to find alternative sterilized borer. methods of combating infections. A biological tool of natural origin, which can be used in Materials and Methods both the sectors, is needed. Bacteriocins are ribosomally synthesized peptides produced by The research was conducted in Sam bacteria itself, with ability to kill or inhibit Higginbottom University of Agriculture, microorganisms usually associated, but not Technology and Sciences, Allahabad. Soil always, to the producer strain (Klaenhammer, samples from SHUATS dairy environment for 1988). Recent studies have demonstrated the isolation of bacteriocin producing bacteria was potential use of bacteriocins as natural collected and stored in sterilized zipper plastic preservatives and its therapeutic applications bags. All experiments i.e. isolation, against bacteria with Multiple Drug identification and screening of bacteriocin Resistance (MDR) (Singh et al., 2012; Galvez production was conducted in laboratory of et al., 2007; Bizani and Brandelli, 2002). Department of Industrial Microbiology, Based on their molecular weight bacteriocins SHUATS. have been characterized into three classes; Nisin, one of the broadly studied bacteriocin Isolation and identification of bacterial from LAB fall into Lantibiotics or class I strain bacteriocin due to its small size and granted Generally Recognized as Safe (GRAS) by A total of one hundred soil samples were United States Food and Drug Administration collected from a depth of 5-15 cm at different (USFDA) (Galvez et al., 2007; Klaenhammer, sites. One gram of each soil sample was 1993; Maisnier-Patin, 1992). Among transferred to 10 ml of sterilized Ringer’s bacteriocin producing bacteria, the genus solution aseptically and serially diluted to 10-6. Bacillus is one of the diverse group reported to Each of the dilution tube was heated at 80 ºC produce a variety of antimicrobial peptides or in a preheated water bath for 20 min to BLIS, such as, subtilin from B. subtilis, cerein exclude non-spore formers. After cooling to from B. cereus, megacin from B. megaterium, room temperature, 1 ml of suspension from lichenin from B. licheniformis, B. each tube was put onto the petriplates and stearothermophilus, etc. (Pattnaik et al., 2005; sterilized molten tyrosine agar was poured Hyung et al., 2001). over the suspension. As soon as the agar got solidified the plated were flipped upside down Bacillus brevis, primarily as soil bacterium and incubated at 37 ºC. Observations were was reclassified into the genera Brevibacillus recorded after 48 to 72 hr; colonies with dark in 1996 (Shida et al., 1996) and have been pigment around the colonies were quarantined reported for production of peptide antibiotics on nutrient agar slants after purification with Gramicidin and Tyrocidine. Different strains quadrant streaking. To identify the bacterial of B. brevis have shown antagonistic effect on strain, isolates were carried through series of, various gram positive and gram negative cultural, morphological and physiological organisms, the bacterium have also reported tests, including Grams reaction, spore staining, for extending the shelf life of curd (Anja et al., growth under various conditions, carbohydrate 2014; Appaiah et al., 2012; Gillor et al., utilization, etc. according to “Bergey’s 2008). In consideration of the above Manual of Systematic Bacteriology” (Table background a strain of Brevibacillus brevis 1). 851 Int.J.Curr.Microbiol.App.Sci (2018) 7(5): 850-858 Bacteriocin production and 55 identified as Brevibacillus brevis (6%) and 20% as B. licheniformis, 18% as B. Muller Hinton Broth (MHB) (Hi-media) with megaterium, 6% as B. pumilus, 4% as B. pH at neutrality was used as production coagulans based on morphological and medium, 25 ml broth was prepared in 50 ml biochemical characteristics enlisted in Table 1. Erlenmeyer flasks and sterilized at 121 ºC for 15 min, selected isolates were inoculated as The organisms were found motile, spore soon as the broth came to room temperature bearer, gram positive and strictly aerobic. followed by incubation in shaking incubator at 35 ºC with 100 rpm for 24 and 48 hr. The carbohydrate profile (Table 2) of bacteria under investigation depicts that it could utilize Detection of antimicrobial activity glucose, fructose, glycerol, mannitol, sucrose, trehalose, maltose, ribose and melezitose with A cell free suspension (CFS) was prepared by weak production of acid and little or no gas centrifuging the MHB, at 4500 x g for 20 min production; but could not utilize xylose, and stored at 4 ºC. The antimicrobial activity sorbitol, salicin, rhamnose, raffinose, determined by agar well diffusion method on melebiose, mannose, lactose, inositol and Muller Hinton Agar (MHA) (Hi-media) arabinose. Similar results have been reported against indicator organisms i.e. E. coli by Ghadbane et al., (2013). (MCCB 0017) and methicillin resistant S. aureus (MCCB 0139) provided by Microbial Detection of antimicrobial activity Collection Culture Bank, Department of Industrial Microbiology, SHUATS. A 12-18 All three isolates were examined for their hr old culture broth of indicator organisms antibacterial activity against E. coli and (107 CFUml-1) were swabbed on the MHA methicillin resistant strain of S. aureus. Crude plates and 5 mm wells were cut out, an aliquot brevicin from isolate no. 18 displayed largest of 50 μl of CFS was filled in the wells zone of inhibition among all the three isolates followed by incubation at 37 ºC. The against S. aureus. antagonistic activity in Arbitrary unit/ml (AUml-1) was calculated (Bhaskar et al., 2007) The crude brevicin obtained after 24 hr of as a measure of bacteriocin production. incubation displayed higher activity than that of 48 hr incubation time. The results are summarized in the (Table 3). On calculating the effectiveness in terms of arbitrary unit per AUml-1 = milliliter (AUmL-1) (Bhaskar et al., 2007; Bhuvaneswari et al., 2015) the activity of Results and Discussion crude brevicin was found with upper limit of 440 AUmL-1 for isolate no. 18 against S. Isolation and Identification of bacterial aureus followed by 360 AUmL-1 and 210 strain AUmL-1 for isolate no. 54 and 55 respectively (Fig. 1). In contrast, no activity was observed 49 isolates were quarantined from hundred against E. coli (Fig. 2). soil samples collected form diary environment, on the basis of the method The genus Brevibacillus is mainly