Characterization of Bacteriocin-Producing Lactic Acid Bacteria Isolated from Native Fruits of Ecuadorian Amazon

Abstract

Tropical, wild-type fruits are considered biodiverse "hotspots" of microorganisms with possible functional characteristics to be investigated. In this study, several native lactic acid bacteria (LAB) of Ecuadorian Amazon showing highly inhibitory potential were identified and characterized. Based on carbohydrate fermentation profile and 16S rRNA gene sequencing, seven strains were assigned as Lactobacillus plantarum and one strain as Weissella confusa. Using agar-well diffusion method the active synthetized components released in the neutralized and hydroxide peroxide eliminated cell-free supernatant were inhibited by proteolytic enzymes, while the activity was maintained stable after the treatment with catalase, lysozyme, α-amylase and lipase suggesting their proteinaceous nature. The inhibitory activity was stimulated by acidic conditions, upon exposure to high heat and maintained stable at different ranges of sodium chloride (4-10%). The DNA sequencing analysis confirmed the presence of plw structural gene encoding for plantacirin W in the selected L. plantarum strains. Moreover, we showed that the active peptides of Cys5-4 strains contrast effectively, in a bactericidal manner, the growth of food borne E. coli UTNEc1 and Salmonella UTNSm2, with about tree fold reduction of viable counts at the early stage of the target cell growth. The results indicated that the bacteriocin produced by selected native lactic acid bacteria strains has elevated capacity to suppress several pathogenic microorganisms implying their potential as antimicrobial agents or food preservatives.

Inhibitory Substances Produced by NativeLactobacillus plantarumUTNCys5-4 Control Microbial Population Growth in Meat

“…No significant difference (p > 0.05) was observed between the Cys5-4, LP and nitrite upon 9 days of storage. ese results were in agreement with our previous findings that Cys5-4 inhibited in vitro the growth of E. coli and Salmonella at both vegetative and exponential target growth [13]. ese results are similar to the results obtained using the crude extract of Lactobacillus LAC231 on the microbiota presented in the meat packed in controlled atmospheres [19].…”

“…is observation is consistent with our previous findings about the bactericidal mode of action of the bacteriocin Cys5-4 [13]. In a recent paper, the molecular weight of partial purified bacteriocin from L. plantarum ATCC8014 was estimated at 68 kDa [30], while a previous study indicated that the protein was larger than 122 kDa [31].…”

“…e PP were recovered in 25 mM ammonium acetate (pH 6.5), desalted by using a midi dialysis kit (cat # PURD10005-1KT, Sigma-Aldrich, USA) pre-equilibrated with phosphate buffer (pH 7.0) and stored at (− ) 20°C before use in antimicrobial assays. e antimicrobial activity in vitro of CFS and peptides was evaluated as previously reported [13].…”

“…e meat filets were divided in nine portions (150 g meat/portion/each treatment) and treated as following: (a) portions 1 and 2 were immersed in pure cell culture suspensions of Cys5-4 and LP independently at the final concentration of 8.97 and 9.03 log CFU/mL, respectively; (b) portions 3 and 4 were immersed in CFS of Cys5-4 and LP suspension at the final concentration of 6400 AU/mL (arbitrary units were determined as previously described by Garzón et al [13]); (c) portions 5 and 6 were immersed in Cys5-4 and LP peptides, independently, at the final concentration of 6400 AU/mL; (d) portion 7 was immersed in nitrite at 200 mg/kg according with the INEN Normative for meat preservation [6]; (e) portion 8 was immersed in commercial nisin from Lactococcus lactis 2.5% (balance sodium chloride) at the final concentration 200 mg/mg; and (e) portion 9 was immersed in sterile water (control). Briefly, the immersed samples were maintained for 10-15 min in individual sterile trays containing the inhibitory substances as described above, followed by 30 min under the laminar bench to avoid any crosscontamination and stored in the refrigeration for 9 days in polystyrene food delivery boxes wrapped with sterile plastic bag (Ziploc).…”

“…In general, the e cacy of bacteriocins to control pathogen growth depends on the producer strain's capacity to compete with other bacteria in the same microenvironment; therefore, the selection of strains with wide-ranging antimicrobial activity is of interest. We previously reported the antimicrobial potential of several LAB species originating from extreme microenvironments such as tropical wild-type fruits of the Amazon [13]. Among them, a bacteriocinogenic Lactobacillus plantarum UTNCys5-4 producing plantaricin W showed elevated potential to inhibit E. coli and Salmonella in orange juice when applied as cell-free supernatant [14].…”