“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!”
Simple Detection of Alicyclobacillus & Their Capability to Produce Guajacol
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.
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.
Milenia GenLine Alicyclobacillus tests are two kits, which are can be used in combination with each other
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.
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.
Performance Data of Milenia GenLine Alicyclobacillus Tests
|Test System||Alicyclobacillus spp.||A. acidoterrestris & Guajacol Formation|
|Sensitivity||approx. 1000 cfu / mL||approx. 1000 cfu / mL|
|Time to Result||120 minutes||120 minutes|
|Sample Type||filterable and non-filterable juice, juice concentrate, puree, smoothie||filterable and non-filterable juice, juice concentrate, puree, smoothie|
|Results||qualitative, visual reading||qualitative, visual reading|
|Specificity||Alicyclobacillus 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 Screening||A. acidoterrestris Identification||Guajacol producing ACB|
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:
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