Certain Lactobacillus brevis strains are resistant to hop-derived compounds such as isohumulone and are able to grow in beer. In this study, we raised an antiserum against our beer spoilage laboratory strain L. brevis 578 which reacted with 23 of 24 beer spoilers and two of 13 non-spoilers in precipitation reactions using 0.5 M NaOH cell extracts. This specific antigen to the beer spoilage L. brevis strains (SABSL) was demonstrated to be located beneath the S-layer proteins by agglutination reactions using S-layer protein-stripped cells obtained by treatment with 0.1 M NaOH. SABSL was purified using an affinity column coupled with an antibody against SABSL. The purified antigen was hydrolyzed with 2 M HCl and the hydrolyzate was analyzed by thin-layer chromatography and enzymatic analysis. The results showed that SABSL contains glycerol, phosphate, glycerophosphate, D-galactose and D-glucose. D-Galactose and D-glucose accounted for 4.7% and 0.1% of the composition, respectively. Melibiose, but not mannose, inhibited the precipitation reaction. Intense precipitation reactions were obtained with fractions which did not bind to the ConA-column. These results indicate that the immunodominant component of the SABSL is galactose and the SABSL determinant is most probably a galactosylated glycerol teichoic acid. The antiserum raised against the beer spoilage strain L. brevis 578 could distinguish between Pediococcus beer spoilers and non-spoilers in precipitation reactions.

The presence of S-layer proteins in Lactobacillus brevis was examined by SDS-PAGE analysis. Thirty six out of a total of 41 L. brevis strains possessed S-layer proteins of molecular masses ranging from 38 to 55 kDa. Western blot analysis using antisera raised against whole cells of S-layer protein-carrying strains demonstrated the heterogeneity of L. brevis S-layer proteins. No clear relationship was observed between the presence of S-layer proteins or their immunological characteristics and the physiological activity of L. brevis as a beer spoilage organism.

No abstract

An ultrasensitive chemiluminescent enzyme immunoassay (CLEIA) was developed for the rapid detection and quantification of Lactobacillus brevis contaminants in beer and pitching yeast (Saccharomyces cerevisiae slurry collected for reinoculation). L. brevis cells trapped on a 47-mm nucleopore membrane (0.4-m pore size) were reacted with a peroxidase-labelled Lactobacillus group E antibody and then subjected to an enhanced CLEIA analysis with 4-iodophenol as the enhancer. The combination of a nucleopore membrane with low background characteristics that enables the antigen-antibody reaction to proceed through the pores of the membrane and a labelled antibody prepared by the maleimide hinge method with minimal nonspecific binding characteristics was essential to minimize background in the detection of single cells. An ultrahigh sensitive charge-coupled device (CCD) camera equipped with a fiber optics image intensifier permitted the imaging of single cells. A clear correlation existed between the number of luminescent spots observed and the plate count [y (CLEIA) ‫؍‬ 0.990x (plate count) ؉ 15.9, where n ‫؍‬ 7, r ‫؍‬ 0.993, and P < 0.001]. Microscopic observation confirmed that the luminescent spots were produced by single cells. This assay could be used to detect approximately 20 L. brevis cells in 633 ml of beer within 4 h. Our ultrasensitive CLEIA could also be used to detect microcolonies approximately 20 m in diameter which had formed on a membrane after 15 to 18 h of incubation. This method, which we called the microcolony immunoluminescence (MIL) method, increased the signal-to-noise ratio dramatically. The MIL method could be used to detect a 10 0 level of L. brevis contamination in 633 ml of beer and a 1/10 8 level of L. brevis contamination in pitching yeast within 1 day (15 to 18 h to form microcolonies and 2 h for CLEIA).

No abstract