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Title: Inducer bacteria, unique signal peptides and low nutrient media stimulate in-vitro bacteriocin production by Lactobacillus spp. and Enterococcus spp. strains

item Svetoch, Edward
item Eruslanov, Boris
item Perelygin, Vladimir
item Levchuk, Vladimir
item Seal, Bruce
item Stern, Norman

Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/15/2010
Publication Date: 9/1/2010
Citation: Svetoch, E.A., Eruslanov, B.V., Perelygin, V.V., Levchuk, V.P., Seal, B.S., Stern, N.J. 2010. Inducer bacteria, unique signal peptides and low nutrient media stimulate in-vitro bacteriocin production by Lactobacillus spp. and Enterococcus spp. strains. Journal of Agricultural and Food Chemistry. Volume 58 pages 6033-6038.

Interpretive Summary: Applications of bacteriocins (BCN) portend a valuable option to the use of traditional therapeutic antibiotic treatments. These antimicrobial peptides appear to promise an important alternative for controlling bacterial infections in human and veterinary medical applications as well as to control spoilage and pathogenic bacteria in foods and feeds. To enable commercial application and further scientific evaluations of BCN economically feasible methods to optimize production are needed. To reproducibly enhance the fermentation/purification output of our three previously published BCN we provided specified quantities of three isolated and sequenced signal peptides, which shared a unique signature amino acid sequence, together with specified BCN inducer bacteria and, the previously unreported use of a low nutrient medium. We observed that 100% brucella broth provided yields of 25 to 32 mg BCN/L while 10% brucella broth in the presence of inducer bacteria and SP provided yields of 225 to 510 mg BCN/L, an increase of more than 9 fold the quantities of BCN when compared to those levels previously produced. These BCN can now be efficiently produced for application to control numerous bacterial associated problems.

Technical Abstract: Bacteriocins (BCN) provide promising potential to control bacterial infections in a variety of applications. We previously reported three Type IIa BCN produced by Lactobacillus salivarius B-30514 (OR-7), Enterococcus durans/faecium/hirae B-30745 (E 760) and Enterococcus faecium B-30746 (E 50-52). In vitro, these BCN are notably effective against a wide array of pathogenic bacteria. In an infected adult chicken model, milligram quantities of BCN reduced six logs of Campylobacter jejuni colonization. To commercially use such BCN, adequate quantities must be produced and harvested. We determined that a combination of endogenously synthesized signal peptides (SP) in the presence of both producer and inducer bacteria grown in a dilute fermentation medium enabled marked increase in BCN output. Each SP shared the unique terminal carboxyl sequence of VKGLT. The inducer bacterial isolates cultured with the BCN producer fermentations were Lactobacillus acidophilus B-30510 and Lactobacillus crispatus B-30884. We demonstrated enhanced BCN production using a nutrient-limited medium of 10% brucella broth to increase output. Using the combination of these above three parameters enabled us to reproducibly harvest at least 200 mg BCN per liter of the spent fermentation broths for each of the three BCN studied. These three-Type IIa BCN can now be reproducibly and efficiently produced in commercial quantities.