Beverage Spoiling Organisms

Simple Detection of Alicyclobacillus & Their Capability to Produce Guajacol

Intro­duction 

Alicy­clobacillus (ACB) is a genus of gram-​​positive, thermophilic, acidophilic, spore-​​forming bacteria. These charac­terics make ACB a serious threat for the beverage industry, because they are able to spoil juice, smoothies and soft-​​drinks. Due to their ability to form spores, Alicy­clobacillus is able to survive a pasteur­ization process without any problems. After pasteur­ization spores become activated and start growing. The vegetative cells are able to grow in an environment at low pH and thus spoil the product. The charac­ter­istic picture of Alicy­clobacillus spoilage is the change in taste, smell and texture. Some species are capable to produce phenolic compounds like guajacol, a metabolic product that results in extreme off-​​flavors, even at low concen­tra­tions. The most important species with spoilage capability is A. acidoter­restris.

A reliable detection of these non-​​pathogenic, soilborne bacteria by classical enrichment culture can be very time consuming.

According to the guide­lines of the Inter­na­tional Fruit Juice Union (IFU) a reliable negative result can take up to 15 days. For this reason, there is a high demand for a quick and reliable analytical procedure. DNA-​​amplification methods allow quick, sensitive and precise detection of microor­ganisms and thus show great potential for an improved, accel­erated Alicy­clobacillus analytics.

Manufac­turers of juice need simple, quick and reliable methods for the quality control of their product. Proce­dures allowing to reduce the time to result are strongly needed!”

Milenia GenLine Alicy­clobacillus and Guajacol PCR-​​Tests – As Easy to Handle as a Pregnancy Test

Milenia GenLine Alicyclobacillus-​​tests are dedicated to the sensitive and specific detection of the genus of Alicy­clobacillus. The test system is intended to be used as a sensitive screening tool in combi­nation with a shortened pre enrichment step. In case of a positive result – a second Milenia GenLine test is available for the identi­fi­cation of A. acidoter­restris and other guaiacol producing ACB. This feature allows the evalu­ation of the spoilage potential of frequent and less frequent Alicy­clobacillus species like A. acidoter­restris, A. acidopilus and A. herbarius.

The tests are easy to handle and the technology is basically a combi­nation of a lateral-​​flow test strip, based on the Milenia HybriDetect universal test strip concept, in combi­nation with specific PCR modules. The inter­pre­tation of results is similar to the preganancy test which is available in pharmacy stores. During the product devel­opment, special attention was driven to a simple handling of the test and an easy inter­pre­tation of the results.

Test Procedure for the Detection of Alicyclobacillus with the Milenia GenLine Test System
Test Procedure for the Detection of Alicy­clobacillus with the Milenia GenLine Test System

Milenia GenLine Alicy­clobacillus tests are two kits, which are can be used in combi­nation with each other

Screening-​​Test

First enriched samples are tested for the presence of ACB (Test Strip A). The PCR-​​Control on the test strip confirms the proper perfor­mance of the PCR in the given sample matrix. This creates the basis of valid negative results and excludes false negative results.

Identification-​​Test

In case of a positive Screening-​​Test an identi­fi­cation of A. acidoter­restris and the capability of Guajacol synthesis can be done from the same sample.

Result Interpretation of the Milenia GenLine Alicyclobacillus Test System
Inter­pre­tation of the Milenia GenLine Alicy­clobacillus Test System

Perfor­mance Data of Milenia GenLine Alicy­clobacillus Tests

Test SystemAlicy­clobacillus spp.A. acidoter­restris & Guajacol Formation
Sensi­tivityapprox. 1000 cfu /​ mLapprox. 1000 cfu /​ mL
Time to Result 120 minutes120 minutes
Sample Type filterable and non-​​filterable juice, juice concen­trate, puree, smoothiefilterable and non-​​filterable juice, juice concen­trate, puree, smoothie
Resultsquali­tative, visual readingquali­tative, visual reading
Speci­ficityAlicy­clobacillus spp.A. acidoter­restris /​ Guajacol producing ACB

Results of a Field Study – Comparison of Culture and Milenia GenLine after Enrichment

60 naturally conta­m­i­nated, „real life“ samples were examined in a compre­hensive compar­ative analysis including 30 filterable and 30 non-​​filterable samples. This practi­cally repre­sentive sample set contained several product types like fruit juices, fruit juice concen­trates, smoothies and purees. Therefore, the results of an enrichment procedure according to the reference guideline IFU No.12 has been compared to the Milenia GenLine Tests, which were combined with a universal, semi-​​selective pre enrichment culture.

Of the 60 samples analyzed in the study, 35 turned out to be positive for ACB when tested according to IFU No. 12. After further analysis 22 of these 35 positive samples have been identified as A. acidoter­restris by using 16S rDNA sequencing or MALDI-​​TOF-​​MS. Moreover, 21 guajacol producing ACB isolates could be identified with the help of an enzymatic assay. At the same time, enrichment cultures were analyzed with the Milenia GenLine Alicy­clobacillus tests before incubation (0) and after 2, 3 and 5 days of incubation. The following table shows the results of this compar­ative study.

Enrichment Time Alicy­clobacillus ScreeningA. acidoter­restris Identi­fi­cationGuajacol producing ACB
0 days
2 days
3 days
5 days

Due to the slow growth of individual ACB strains, a direct PCR-​​detection from beverages cannot be recom­mended. Especially if bevarages contain trace conta­m­i­na­tions of spoiling bacteria a positive result even with a very sensitive method like PCR is relatively unlikely. Therefore, an enrichment culture is absolutely necessary to ensure a reliable detection of trace conta­m­i­na­tions by spoiling bacteria.

After 2 – 3 days of enrichment and a strong suspicion or in case of time pressure, Milenia GenLine demon­strated to be a meaningful option. Within this study all 21 ACB strains capable of guajacol synthesis could be identified after 3 days. The recom­mended medium BAT-​​B was especially developed for rapid enrichment of the most relevant spoilage organism Alicy­clobacillus acidoter­restris. As a result, 21 of 22 Alicy­clobacillus acidoter­restris isolates could be identified within 72 hours with the Milenia GenLine test system. In order to achieve reliable negative results, an enrichment period over 5 days seems to be essential. This study clearly showed that especially slow growing ACB need in minimum 4 to 5 days to grow to a detectable level of more than 100 to 1.000 cfu /​ mL.

A timeline of a meaningful test approach with Milenia GenLine PCR test kits is shown in the picture below:

Analysis Regime and possible Timepoints for the Milenia GenLine Alicy­clobacillus Tests

The results of the study have shown that the use of Milenia GenLine Alicy­clobacillus tests could reduce the time to result of 15 days needed by IFU No. 12 can be reduced safely to 5 days after enrichment or even to 3 days if results are needed urgently.

Features and Benefits of Milenia GenLine

  • Sensitive and specific
  • Internal PCR Control on the test strip
  • Positive Control in the kit
  • Robust Test System
  • Simple and quick prepa­ration of the samples
  • Low investment costs in equipment
  • Results in 120 minutes