Alicyclobacillus Spoilage in Juices: Testing Methods

Microbial Spoilage of Fruit Juices Caused by Alicyclobacillus Bacteria

Alicyclobacillus is a genus of acidophilic, strictly aerobic, rod-shaped, Gram-variable and spore-forming bacteria. Due to their characteristic slow and late growth, alicyclobacilli pose a challenge for juice manufacturers. The growing demand for natural organic drinks has made the quality control for such acidophilic bacteria even more crucial. Spoilage microorganisms can cause increased turbidity, color changes and unpleasant sensory properties in juices (disinfectant-like off-flavors in products caused by formation of guaiacol). Guaiacol is produced by degradation of phenolic acids (important antioxidants like caffeic, p-coumaric, ferulic, sinapic acids) in fruits and leaves. Off-flavors and odors caused by spoilage typically occur at levels of 10⁴ to 10⁵ cfu Alicyclobacillus/mL. Alicyclobacillus acidoterrestris is recorded as the most important spoilage species within the genus,¹ but in total 19 Alicyclobacillus species have been isolated from spoiled product, including A. acidocaldarius, A. pomorum, and A. herbarius². Soil is the natural habitat of alicyclobacilli, and probably also often the source of beverage contamination by way of the raw materials (syrups, concentrates, fruit purees, etc.) used for beverage production³. About 30% of fruits entering a processing plant are contaminated with Alicyclobacillus. The endospores can typically be found in 15 to 75% of juice concentrates, but not necessarily spoilage causing types.

These microbes do not grow in juice concentrates but remain viable for a long time—their spores can germinate in the diluted (finished) product. Alicyclobacillus spores can also survive a typical pasteurization process. Detection of these organisms, which is traditionally done by incubation on culture medium, takes five to twelve days, or even longer. Most of these acidophilic and thermophilic species grow at a pH range of 2 to 6 and at a temperature range of 20 to 70 °C and are heat resistant (D₉₅ 2 to 15 min, depending on the strain and the fruit juice/concentrate)^1,4.

Division: Firmicutes
Class: Bacilli
Order: Bacillales
Family: Alicyclobacillaceae
Genus: Alicyclobacillus

Alicyclobacillus species:

Not reported to be involved in off-flavor	Reported to be involved in off-flavor	Frequently reported to be involved in off-flavor
acidocaldarius subsp. acidocaldarius	acidiphilus	acidoterrestris
acidocaldarius subsp. rittmannii	acidocaldarius
aeris	cycloheptanicus
contaminans	fastidiosus
disulfidooxidans	herbarius
ferrooxydans	hesperidum
kakegawensis	pomorum
macrosporangiidus
sendaiensis
shizuokensis
tolerans
vulcanalis
pohliae
sacchari

Regulatory Requirements for Alicyclobacillus Testing: IFU Method No. 12

The revised IFU method No 12 (2019)⁶ for Alicyclobacillus spp. testing in juice-containing drinks recommends using 3 different methods according to ISO procedures, as well as BAT broth and BAT agar. The new formulation is less toxic as cobalt and boric acid are not included anymore. All three methods (A, B and C) involve heat shock treatment for 10 min at 80 °C to induce germination of spores. The procedure A is a direct enumeration by pour plate technique and is used for samples which cannot be filtrated because of particles or other matrix properties. Procedure B is a quantitative method used for the direct enumeration by filtration technique. The procedure C is for the detection of spore-forming thermo-acidophilic bacteria (Alicyclobacillus spp.) by enrichment in BAT broth. For confirmation the colonies on BAT agar are transferred onto fresh BAT agar and an agar with neutral pH, for example PCA (plate count agar) for growth comparison. Alicyclobacillus spp. should not grow on PCA due to its neutral pH. Optionally the guaiacol test can also be performed.

Learn more about testing according to IFU Method No 12

Alicyclobacillus acidoterrestris DSM 2498 on BAT agar.

Culture results after 72 ± 4 h incubation at 45 °C	Growth on BAT agar	Growth on culture medium with neutral pH, e.g. PCA
Positive for spore-forming thermo-acidophilic bacteria (Alicyclobacillus spp.)	+	-
Negative: Other bacteria	+	+

Alicyclobacillus Testing Methods

• Culture-based methods:
  BAT broth for enrichment or dilution of samples and BAT agar for detection, enumeration, and confirmation of Alicyclobacillus spp. according IFU Method No. 12:2019.
• Rapid molecular biology method without PCR:
  Alicyclobacillus organisms can be detected and identified using in-situ hybridization. The HybriScan® system provides the accuracy of a genetic test but without the need for PCR. While detecting living cells only, the system is not sensitive to sample matrix and is low in costs, which makes it interesting for the beverage industry. With HybriScan^® I Alicyclobacillus the detection, identification and confirmation are done in a single step, so no further confirmation step is needed.

Method	IFU No. 12:2019 (Procedures A&B)	IFU No. 12:2019 (Procedure C)	HybriScan® I Alicyclobacillus
Enumeration	5 days	-	-
Detection	-	7-8 days	72 ± 4 hours
Confirmation	72 ± 4 hours	72 ± 4 hours	2 hours

References

1. Spinelli ACNF, Sant'Ana AS, Rodrigues-Junior S, Massaguer PR. 2009. Influence of Different Filling, Cooling, and Storage Conditions on the Growth of Alicyclobacillus acidoterrestris CRA7152 in Orange Juice. Appl Environ Microbiol. 75(23):7409-7416. https://doi.org/10.1128/aem.01400-09

2. McKnight I, Eiroa M, Sant?Ana A, Massaguer P. 2010. Alicyclobacillus acidoterrestris in pasteurized exotic Brazilian fruit juices: Isolation, genotypic characterization and heat resistance. Food Microbiology. 27(8):1016-1022. https://doi.org/10.1016/j.fm.2010.06.010

3. Pornpukdeewattana S, Jindaprasert A, Massa S. 2020. Alicyclobacillusspoilage and control - a review. Critical Reviews in Food Science and Nutrition. 60(1):108-122. https://doi.org/10.1080/10408398.2018.1516190

4. PONTIUS AJ, RUSHING JE, FOEGEDING PM. 1998. Heat Resistance of Alicyclobacillus acidoterrestris Spores as Affected by Various pH Values and Organic Acids?. 61(1):41-46. https://doi.org/10.4315/0362-028x-61.1.41

5. IFU International Fruit and Vegetable Juice Association. Method on the detection and enumeration of spore-forming thermo-acidophilic spoilage bacteria (Alicyclobacillus spp.). Method of Analysis No. 12:2019.. [dissertation].