Detection of Beer Spoilage Microorganisms

Spoilage Microorganisms in Beer

Rapid detection of pathogens and spoilage organisms is a critical step in microbiology and the brewing industry as microorganisms can negatively affect product quality. The popularity of beer remains high, but the quality of beer has to be very high to survive in a competitive market. Therefore, raw materials, product intermediates, end products, surfaces, and special areas are frequently monitored through hygienic control procedures.

Lactobacilli (beer spoilage organisms)

The common beer spoilage microorganisms are either lactic acid bacteria belonging to the genus Lactobacillus and Pediococcus or the obligate anaerobes of the genus Pectinatus and Megasphaera (Table 1). Among the Lactobacilli species that are known to cause spoilage of beer, only certain strains can grow in the beer and are responsible for spoiling (however, all strains of L. lindneri cause spoilage). L. brevis is the most common beer spoilage bacterium followed by L. lindneri.¹

Additionally many wild yeasts are responsible for beer spoilage such as Saccharomyces cerevisiae and Candida pelliculosa.² One of the biggest problems is biofilm formation in beer plants, which makes it very difficult to remove spoilage organisms. Highly skilled lab staff perform microbiological analysis in specific quality control laboratories. Most laboratories still use conventional standard-based cultivation methods, which are very time-consuming and take 3 to 5 days for beer to be released to the market.

HYBRISCAN™ System - An Innovative Screening Method for Beer Spoilage Microorganisms

HybriScan™ Beer kit is a rapid test system developed in collaboration with Scanbec GmbH. This system is an alternative to the conventional method to detect beer spoilage microorganisms at a faster rate, with reliable results in as little as two hours (pre-enrichment for 24 h, if necessary) for a faster product release.

Species of beer-spoilage microorganisms that can be detected with the HybriScan™ D Beer–kit (Product No. 62533 , 96 assays)

Genus Lactobacillus

Lactobacillus acidophilus
Lactobacillus brevis
Lactobacillus brevisimilis
Lactobacillus buchneri
Lactobacillus casei
Lactobacillus collinoides
Lactobacillus coryniformis
Lactobacillus curvatus
Lactobacillus fermentum
Lactobacillus fructivorans
Lactobacillus lindneri
Lactobacillus malefermentans
Lactobacillus parabuchneri (frigidus)
Lactobacillus paracasei
Lactobacillus paraplantarum
Lactobacillus plantarum
Lactobacillus rhamnosus

Genus Pediococcus

Pediococcus acidilactici
Pediococcus claussenii
Pediococcus damnosus
Pediococcus inopinatus
Pediococcus parvulus
Pediococcus pentosaceus

Genus Pectinatus

Pectinatus cerevisiiphilus
Pectinatus fringensis

Genus Megasphaera

Megasphaera cerevisiae

Beer Spoilage Monitoring Based on the Detection of rRNA

HybriScan™ molecular screening system uses sandwich hybridization to provide fast and accurate detection, identification, and quantification of spoilage and pathogenic microorganisms in beer. It is ideal for the comprehensive and reliable routine control of raw materials and concentrates on all production steps up to the quality check of finished goods. The test is based on the detection of rRNA on an economical microtitre plate that takes less than 2 hours. This low-cost molecular biology method uses a photometric readout.

A variety of applications have been developed for HybriScan™ system, including the detection of bacteria and yeast in non-alcoholic beverages. The robustness of the HybriScan™ assay enables it, in contrast to other rapid test systems, to detect bacterial contamination in brewer’s yeast and leads to efficient use of this valuable resource. Furthermore, the HybriScan™ test system is a perfect tool for microbiological control of dispensing equipment. The legal standard for sterility control of dispensing equipment is 100,000 cfu/mL; a fast, direct determination of beer-spoiling bacteria is possible without a pre-enrichment procedure delivering results within two hours.

HYBRISCAN™ Molecular Screening System Vs. Other Rapid Test Systems: A Comparison

Performing quality control by using the standard culture-based method is time-consuming. In recent years many companies have developed rapid test systems to hasten this procedure. For quality control of beer and beverages, three main technologies are available:

• HybriScan™ system (sandwich hybridization)
• PCR (polymerase chain reaction)
• VIT (vermicon identification technology)

The benefits of using HybriScan™ over PCR or VIT are given below.

• Fast and cost-efficient analysis: Comparison of the HybriScan™ test with a cultivation-based analytical method (MRS agar) displays the equivalent results within the limits of microbiological sample variability at a faster rate.
• Accurate: Using two different probes for the detection of microbial RNA reduces false-positive results
• Inexpensive read-out technology
• High sensitivity and specificity

Equivalent results within the limits of microbiological sample variability are displayed for comparison tests between HybriScan™ system and a cultivation-based analytical method (MRS agar). A comparison of these different technologies is given in Table 1.

Table 1. Comparison of different technologies for the detection of beer spoilage bacteria.

	Cultivation based method	PCR	VIT	HybriScan™ system
Method	cultivation based method with optical or microscopic readout	PCR/real-time PCR	fluorescence microscopy	sandwich hybridization and photometrical signal readout
Detection spectrum	detection and identification of all beer spoilage microorganisms	identification of all relevant Beer spoilage microorganisms possible	Lactobacillus sp. and Pediococcus damnosus, Lactobacillus sp. + L. brevis, Pectinatus + Megasphaera cerevisiae	identification of all relevant beer spoilage microorganisms possible
Sample preparation	selective pre-enrichment	enrichment and lysis of bacteria, if necessary, pre-enrichment	selective pre-enrichment	enrichment and lysis of bacteria, if necessary, pre-enrichment
Time	3 to 7 days	3 hours to 2 days	2 days	3 hours to 2 days
Costs per test	ca. 1 €	12 €	15 €	3 €
Detection limit (cfu)	1	1-5 x 105	1 x 103	1-5 x 103
Devices	None	PCR cycler	fluorescence microscopy	microplate reader
Advantages	high sensitivity, relatively cheap	high sensitivity, quantitative analysis	simple detection technology set up, detects only living cells (RNA)	rapid and sensitive, qualitative, and quantitative detection of living cells, cost-efficient analysis
Disadvantages	time-consuming, no detection of non-culturable microbes, labor expensive	expensive devices needed, no discrimination between live and dead cells, not officially accepted	time-consuming, low sample throughput, expensive, not automatable, difficult data analysis, not officially accepted	no differentiation of serotypes or subspecies, limited probe design (rRNA target), not officially accepted

References

1. Storåards E, Suiiiko M, Pot B, Vanhonacker K, Janssins D, Broomfield PLE, Banks JG. 1998. DETECTION AND IDENTIFICATION OFLACTOBACILLUS LINDNERIFROM BREWERY ENVIRONMENTS. 104(1):46-54. https://doi.org/10.1002/j.2050-0416.1998.tb00974.x

2. Timke M, Wang-Lieu NQ, Altendorf K, Lipski A. 2008. Identity, beer spoiling and biofilm forming potential of yeasts from beer bottling plant associated biofilms. Antonie van Leeuwenhoek. 93(1-2):151-161. https://doi.org/10.1007/s10482-007-9189-8