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Loop-mediated Isothermal Amplification to Detect and Identify Beer Spoilage Lactobacillus spp. Bacteria
H. Murakami, T. Fujii, and N. Hayashi

Primers for a loop-mediated isothermal amplification (LAMP) method to specifically identify beer spoilage caused by Lactobacillus spp. including Lactobacillus brevis, Lactobacillus lindneri, Lactobacillus backi and Lactobacillus paracollinoides were developed. These LAMP primer sets were designed from target sequences in the 16S rRNA gene or the spacer region. To identify beer spoilage strains in the species, we also developed a LAMP primer set to detect highly specific genetic markers. The LAMP primer sets in this study distinguished the target species or beer spoilage strains from other lactic acid bacteria in 40-60 min. Moreover, using these primer sets in conjunction with the LAMP method enabled detection of approximately 1 x 102 cfu/ml lactic acid bacteria from suspensions in distilled water, beer and beer that contained large amounts of bottom-fermenting yeast cells. Thus, the LAMP method may be useful for direct detection and identification of Lactobacillus spp. in samples taken during the fermentation process and from final products. Contamination of beer by lactic acid bacteria such as Lactobacillus is a major concern in the brewing industry. However, among the numerous Lactobacillus species, only a few cause spoilage in beer [1]. While isolates belonging to the aforementioned species have been reported as beer-spoilage bacteria, some strains have resistance to hop-compounds and can grow in beer, whereas others do not have the ability to spoil beer [1]. Lactobacillus brevis is one of the most common contaminants in breweries and beer [2, 3]. L. brevis is an obligate heterofermentative bacterium that has beer-spoilage abilities that vary according to its strain and origin. Beer-spoilage strains of L. brevis cause clouding, sedimentation and acidification of beer. Another important beer spoilage Lactobacillus sp. is Lactobacillus lindneri. This organism is highly resistant to bitter compounds from hops, and survives a higher degree of thermal treatment than other lactic acid bacteria found in the brewery environment [4, 5, 6, 7, 8]. Recently, two novel species, Lactobacillus paracollinoides and Lactobacillus backi, were isolated from brewery environments [9,10]. Comparison of the 16S rRNA gene sequences of these organisms has shown that L. paracollinoides is most closely related to Lactobacillus collinoides. However, these strains did not show sufficient DNA-DNA homology to be classified as any of the confirmed Lactobacillus species, even though they exhibited strong beer-spoilage ability and L. collinoides strains are not generally considered to be beer-spoilage bacteria [9]. L. backi strains have been isolated from lager, pils and wheat beers from different breweries in Germany and Italy, and are considered to be obligate beer-spoiling bacteria [10]. L. backi strains have been found to have a striking morphological resemblance to Lactobacillus coryniformis and homofermentative behavior. However, they can be differentiated from L. coryniformis on the basis of physiological and biochemical tests and they have been found to show less than 97 % 16S rDNA sequence similarity to any other Lactobacillus species [10].

Descriptors: Lactic acid bacteria, beer-spoilage bacteria, rapid detection, loop-mediated isothermal amplification, LAMP, DNA amplification

BrewingScience - Monatsschrift fr Brauwissenschaft, 62 (November/December 2009), pp. 172-180