Vol. 58, No. 10
0099-2240/92/103355-05$02.00/0 Copyright ©) 1992, American Society for Microbiology
Novel Method To Extract Large Amounts of Bacteriocins from Lactic Acid Bacteriat RONGGUANG YANG, MONTY C. JOHNSON, AND BIBEK RAY* Food Microbiology Laboratory, Animal Science Department, University of Wyoming, Laramie, Wyoming 82071 Received 4 May 1992/Accepted 21 July 1992
Antimicrobial peptides, bacteriocins, produced by lactic acid bacteria were adsorbed on the cells of producing strains and other gram-positive bacteria. pH was a crucial factor in determining the degree of adsorption of these peptides onto cell surfaces. In general, between 93 and 100% of the bacteriocin molecules were adsorbed at pHs near 6.0, and the lowest (c5%) adsorption took place at pH 1.5 to 2.0. On the basis of this property, a novel isolation method was developed for bacteriocins from four genera of lactic acid bacteria. By using this method we made preparations of pediocin AcH, nisin, sakacin A, and leuconocin Lcml that were potent and concentrated. This method produced a higher yield than isolation procedures, which rely on precipitation of the bacteriocins from the cell-free culture liquor. It is simple and can be used to produce large quantities of bacteriocins from lactic acid bacteria to be used as food biopreservatives.
In the search for a food biopreservative, investigations on certain antibacterial proteins (bacteriocins) from lactic acid bacteria have been very popular (11, 18, 25). To study their chemical and antibacterial properties and to determine their effectiveness in food systems, it is necessary to obtain relatively large quantities of these peptides in a pure and concentrated form. Currently, most methods rely on ammonium sulfate precipitation of the bacteriocins from cell-free culture liquor. This method has been used to obtain bacteriocins from Pediococcus spp. (4-6, 13), Lactobacillus spp. (17, 21, 23), Lactococcus spp. (12, 15, 16, 24), and Leuconostoc spp. (10, 14). However, there is general agreement that it does not yield a good product because many other proteins from the medium can also be precipitated and the yield is not very high (5, 9, 12). For further purification of precipitated bacteriocins, especially in the determination of the amino acid composition and sequence, these researchers have used various column chromatography techniques. Commercial nisin preparations are available in highly purified food-grade form. However, the methods used commercially are not known. Mattick and Hirsch (20) used a combination of acid treatment of the culture followed by removal of the cells and then solvent extraction and precipitation to obtain nisin with high potency. Later, other workers used several different methods based on propanolNaCl or butanol-acetic acid extraction from culture supernatant (2, 9) and breaking of cells and extraction with acid (1, 31). Although the nisin preparations had high potency, the methods were laborious and total yields were low. Bhunia et al. (7), while studying the mode of bactericidal action of pediocin AcH from Pediococcus acidilactici H on sensitive cells of Lactobacillus plantarum NCDO 955, observed that adsorption of pediocin AcH to the cells is pH dependent. Maximum adsorption occurred at pH 6.0 to 6.5, but when the pH of the cell suspension was reduced to 2.0 or below, the pediocin AcH was not adsorbed. It is known that, in general, cells of a bacteriocin-producing bacterial strain * Corresponding author. t Wyoming Agricultural Experiment Station journal article num-
adsorb the bacteriocin molecules that they produce (7, 13, 18, 25, 30). We hypothesized that if, after fermentation, the culture broth of a producer strain was adjusted to the pH for maximum adsorption of the bacteriocin onto the cell surfaces, the cells with adsorbed bacteriocin molecules could easily be removed from the culture liquor by...