Beverage Spoiling Organisms

Simple Detection & Characterization of Beer Spoiling Bacteria

From micro­bi­o­logical perspective, breweries have various critical points where microor­ganisms can negatively affect product quality. Therefore, raw materials, product inter­me­diates, end products, surfaces and special areas are frequently monitored in terms of hygienic control proce­dures. Breweries invest a lot of time, energy and money to ensure micro­bi­o­logical stability of their beloved beer in order to protect their label and reputation.

 

Ensuring excellent product quality is the essential motivation of micro­bi­o­logical quality control in breweries of all sizes. Fast and simple molecular biological methods can be an extremely useful tool in any brewery’s toolbox!”

Beer Spoiling Bacteria – the Bad and the Ugly – Knowing Your Enemies

Beer can be seen as a very special ecological niche and a compar­a­tively hostile environment for bacterial life. It is charac­terized by low oxygen– and high CO2 levels, a low pH, presence of alcohol and hop-​​compounds, as well as a lack of nutrients. But not only Jurassic Park fans know: „Life finds a way“. Of course, there are some bacterial specialists who can grow in this environment and thus affect product stability and quality.

The most frequently detected beer spoiling bacteria belong to the genus Lacto­bacillus and Pedio­coccus (The BAD). These lactic acid bacteria are gram-​​positive, facul­tative anaerobic and non-​​motile. The charac­ter­istic spoilage profile is deter­mined by changes in taste, odor (sour, diacetyl)  or  /​ and texture (cloudiness, sedimen­tation, slime-​​formation). This heterogenous group of potential and obligate spoilers can occur ubiqui­tously in the brewery environment – as primary or secondary conta­m­i­na­tions.

Anyone who has ever drunk a sour beer will not be enthu­si­astic, but it is harmless compared to conta­m­i­nated beers with repre­sen­ta­tives of the genera Pecti­natus or Megas­phaera (The Ugly). In most cases the cloak-​​like odor effec­tively protects customers from drinking conta­m­i­nated beer. This massive product damage is caused by bacterial formation of hydrogen sulfide and methylmer­captane. The typical secondary conta­m­i­nants Megas­phaera and Pecti­natus are strongly connected to biofilms and therefore cause the sporadic conta­m­i­nation especially in filling area of a brewery.

more infor­mation

Overview of the most relevant Beer Spoiling Bacteria
Overview of the most relevant Beer Spoiling Bacteria

Overview of the 4 Milenia GenLine Kits for the Detection of Beer Spoiling Bacteria

Milenia GenLine AssayBenefits of the Test System
Lacto­bacillus /​ Pedio­coccus — ScreeningThe Lacto­bacillus /​ Pedio­coccus screening assay is a partic­u­larly robust and easy-​​to-​​handle test system allowing specific detection of obligate and potential beer spoiling Lacto­bacillus and Pedio­coccus species — An extremely helpful and versatile tool to check colonies from solid media or “hard-​​to-​​microscope” samples, such as semi-​​selective enrichment cultures or yeast containg samples.
Megas­phaera /​ Pecti­natus — ScreeningA simple and robust screening assay to confirm the presence or absence of the most feared bacterial spoilers, Megas­phaera and Pecti­natus. Moreover, the Swab PCR is an innov­ative and unique tool for rapid hygienic surface monitoring, especially in the filling area.
Hop Resis­tance ScreeningThis innov­ative test system is an excellent tool to assess the spoilage potential of an bacterial isolate of concern. This analysis can be compared to time intensive product spoilage assays, in which a bacterial, gram-​​positive isolate is inocu­lated into the existing product. There is a high proba­bility that the spoilage ability corre­lates with the result of the Milenia GenLine Hop Resis­tance Screen.
Lacto­bacillus brevis Identi­fi­cationLacto­bacillus brevis is the most relevant beer spoiling organism in breweries. Therefore, this quick and simple identi­fi­cation tool can be an partic­u­larly helpful addition to the existing Milenia GenLine Screening Assays.

The Workflow of the Milenia GenLine Test System

The general procedure to detect beer spoiling bacteria was simplified as much as possible. Therefore the overall detection procedure has been reduced to three general working steps. The ampli­fi­cation cocktail is prepared by mixing two ready-​​to-​​use reagents. After the addition of the sample the reaction tubes were placed into a simple-​​to-​​handle thermal cycler. After 45 min of cycling, an aliquot of the ampli­fi­cation mix has to be applied to the universal Lateral Flow Device, Milenia HybriDetect. The strips were placed into running buffer and within a few minutes test and control lines appear. Results are inter­pretable with naked eye after 5 minutes. The total analysis time is around 1 hour, with a hands-​​on time of around 15 mintues.

Worfkflow-Milenia-GenLine-Detection-Beer-Spoiling-Bacteria
General Workflow of the Milenia GenLine Assays for the Detection of Beer Spoiling Bacteria — (1) samples were mixed with a ampli­fi­cation cocktail (2). Reactions were placed into a thermal cycler (3). After ampli­fi­cation reaction, amplicons were analyzed with the universal lateral flow device, Milenia HybriDetect (4 & 5)

Modular Character of the Milenia GenLine Tests

The Milenia GenLine tests are dedicated to give breweries of every size the possi­bility to benefit from the power of molecular biology. Due to its easy-​​to-​​handle character, the test system is partic­u­larly designed to work for experi­enced and non-​​experienced users. In addition, the tests are designed to be modular, which allows the user to integrate every Milenia GenLine test easily into the lab routine.

“The central dogma of the Milenia GenLine test devel­opment has always been to make this system as simple as possible.”

Modular Character of the Milenia GenLine Tests

The Detection Mechanism of the Milenia GenLine Tests?

Our assays for the detection of beer spoiling bacteria rely on our own lateral flow devel­opment platform, Milenia HybriDetect. These universal lateral flow devices are partic­u­larly suitable for method­ological combi­nation with very sensitive and precise molecular biological DNA ampli­fi­cation techniques. And that’s exactly what we did. We use the power of the polymerase chain reaction to enable specific, robust and sensitive analysis. The special compo­sition of our tests allows the direct analysis of biological material directly from product, swabs or enrichment cultures. The tests are versatile and can be used individ­u­alized.

  • simple: easy-​​to-​​handle and –inter­prete
  • short hands-​​on-​​time (10 minutes)
  • time to result: approx. 1h
  • modular character
  • not restricted to a certain number of tests
  • allows direct analysis from enrichment cultures
  • no DNA-​​extraction needed
  • Controls included: internal ampli­fi­cation control and immunoassay control

Why should I use the Hop Resis­tance Screening Test?

Hop has evolved to one of the key compo­nents of beer in the history of brewing. The addition of hop was origi­nally used to protect the beer from micro­bi­o­logical spoilage. Especially, hop related iso-​​alpha-​​acids are very effective antimi­crobial compounds against a broad spectrum of bacterial growth. Never­theless, some bacterial species developed mechanism(s) to bypass this „growth-​​barrier“. Today it is known that the survival of gram positive bacteria in beer is strongly connected to the individual genetic „equipment“ of these organisms.

Bacillus (related) species in particular are charac­terized by great genetic variability. In adaptation to the respective environ­mental condi­tions, these organisms can acquire genetic infor­mation that enables them to survive in very special ecological niches. Therefore, genetic markers connected to beer spoiling potential of Lacto­bacillus spp. and Pedio­coccus spp. have been identified in the early 2000’s. Especially, the plasmid encoded genes horA and horC show great potential to predict the ability of a Lacto­bacillus /​ Pedio­coccus isolate to grow in hop-​​containing beers.

The Milenia GenLine Hop Resis­tance Screening Assay gives quick infor­mation about the presence of horA or /​ and horC. As a result, this simple screening test can be an innov­ative and helpful addition to the micro­bi­o­logical quality control of a brewery. The detection of at least one these lifestyle markers is a very strong indication for the presence of an obligate beer spoiling isolate.

HorA- and HorC- mediated Hop Resistance Mechanisms
HorA– and HorC– mediated Hop Resis­tance Mecha­nisms

Hygienic Surface Monitoring – Milenia GenLine Swab PCR

From a micro­bi­o­logical perspective, the area around the filling machine is one the most relevant areas of a brewery, because the product is presented to its environment which allows potential secondary conta­m­i­nation. Therefore, hygienic monitoring of the filling area is an absolutely essential part of the micro­bi­o­logical quality control. Biofilm formation is an exciting topic from a scien­tific point of view, but dealing with biofilm-​​associated secondary conta­m­i­na­tions can be extremly challenging for brewers, especially when temper­a­tures increase.

The Milenia SwabPCR is an innov­ative tool which allows rapid detection of beer spoiling bacteria directly from swab samples. The test system is a valuable addition to time consuming enrichment strategies especially of anaerobic obligate spoilers. It is designed and developed with brewers for brewers. In particular, the detection of obligate anaerobic beer spoiling bacteria from swab samples can sometimes be very time-​​consuming and thus can take days to weeks. The Milenia GenLine Swab PCR was explicitly developed to allow counter­mea­sures at the day of the analysis.

more infor­mation

Insen­si­tivity beside high sensi­tivity – a calcu­lation example

Many unexpi­enced users see PCR-​​based test systems as a „super-​​sensitive rocket-​​science technology“ that detects every trace of bacterial conta­m­i­nation. Basically, they are not so wrong. Extremely sensitive PCR-​​based assays are able to detect the one cell. But how beneficial is this sensi­tivity if only a very tiny volume can be analyzed?

The calcu­lation example of a perfect test system: Let’s assume that the PCR-​​based assay is able to detect 1 cell and we can analyze 5 µL of a sample with this method. Now we see a positive result with 1 cell in our test. We are very lucky to have such a sensitive tool, but now the calcu­lation begins…

1 cell in 5 µL corre­sponds to 200 cells in 1 mL. As a result, there would be a bacterial conta­m­i­nation of at least 100.000 cells per in 0,5 L beer. And that in turn doesn’t sound that sensitive. Just a few vital cells per container can be enough to cause massive damage to the product in a matter of weeks.

Small Sample Volume in a PCR can lead to “insen­sitive” Analysis

This simple calcu­lation is intended to explain why the Milenia GenLine tests (and other DNA ampli­fi­cation techniques) are suitable for direct analysis to a limited extent. In most cases, it is essential to benefit from the impact of an enrichment culture in order to detect any bacterial conta­m­i­nation as sensitive as possible.

Possible Appli­cation of the Milenia GenLine Tests?

  • should be combined with selective /​ semi-​​selectiv pre enrichment culture
  • not recom­mended for the direct detection of trace conta­m­i­na­tions
  • can be used for direct analysis of colonies from solid media
  • can be used for confir­mation of a suspicion (liquid enrichment culture, colonies from solid media)
  • perfectly compatible with with the NBB portfolio from Döhler Gmbh (Darmstadt)
  • can be used for hygienic control of surfaces (e.g. filling area)
  • tolerates compar­a­tively high loads of background yeast
  • enrichment cultures containing yeast (e.g. NBB-​​C + yeast beer)
  • perfect for „difficult-​​to-​​microscope“ samples