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Alicyclobacillus Testing: A Guide to Prevent Alicyclobacillus Contamination

Alicyclobacillus, a genus of thermophilic, acidophilic, spore-forming bacteria, has emerged as a significant threat to the global fruit juice and beverage industry. Although it poses no risk for human consumption, many Alicyclobacillus species can produce off-flavors in beverages, leading to distrust in your brand.

These bacteria can survive the pasteurization processes commonly used in juice production and flourish in the acidic environment of fruit juices and other beverages. The economic impact of Alicyclobacillus contamination is staggering, with estimates suggesting that better control of microbial spoilage could significantly increase profits and reduce waste in the juice industry. In this article, we will discuss the unique characteristics of Alicyclobacillus, the financial implications of contamination, potential contamination sources, and detection and prevention measures.

Why is Alicyclobacillus a problem?

Alicyclobacillus presents unique challenges in detection and control due to its specific characteristics:

  1. Survival in acidic environments: Alicyclobacillus species are acidophilic, meaning they can grow and thrive in low pH environments, such as fruit juices and other acidic beverages. This ability to survive in acidic conditions sets them apart from many other spoilage microorganisms.
  2. Resistance to pasteurization: The spores of Alicyclobacillus,  particularly those of A. acidoterrestris, are highly heat-resistant and can survive the pasteurization processes commonly used in producing fruit juices and beverages. This heat resistance allows the spores to remain dormant in the product until conditions become favorable for germination and growth. 
  3. Difficult to detect by visual inspection: Alicyclobacillus contamination is often tricky until the product reaches the consumer. This is because the bacteria do not typically cause visible changes in the product's appearance, such as gas formation, turbidity or sediment.
  4. Difficult to detect by the slow culture-based tests: Traditional culture-based testing for Alicyclobacillus lacks the sensitivity of a modern PCR-based method and oversees spoilers in low concentrations. To make things worse, they typically require a prolonged incubation period of 5 days, followed by an additional 3 days for confirmation, resulting in 8 days before results are available. This extended testing time can delay the release of products and hinder timely decision-making in quality control processes. Furthermore, some older agars used in culture-based methods may contain toxic components that can harm operators.
  5. Spoilage in low amounts: The primary indicator of Alicyclobacillus spoilage is the development of off-flavors and odors, particularly a medicinal or smoky taste attributed to guaiacol production. Studies have shown that consumers can detect the off-flavors produced by guaiacol at concentrations as low as 2 ppb. It requires the most sensitive PCR-based technology to pick up such low concentrations. These factors make Alicyclobacillus a significant challenge for the beverage industry, highlighting the need for robust prevention and control strategies to mitigate the risk of spoilage and protect product quality.

What is the financial impact of Alicyclobacillus?

Alicyclobacillus contamination has a significant financial impact on the beverage industry, primarily due to the costs associated with product spoilage, recalls, and damage to brand reputation.

  1. Product spoilage and waste: When Alicyclobacillus contamination leads to the development of off-flavors and odors in beverages, the affected products often become unsellable, resulting in direct financial losses for the manufacturer.
  2. Product recalls: In the event of widespread Alicyclobacillus contamination, beverage manufacturers may be forced to initiate product recalls. The costs associated with recalls can be substantial, including expenses related to notification, product retrieval, disposal, and customer compensation. In addition to the direct costs, recalls can lead to indirect costs, such as lost sales and reduced consumer confidence in the brand.
  3. Damage to brand reputation: Alicyclobacillus spoilage incidents can have a lasting impact on brand reputation. Consumers who experience off-flavors or odors in a product may be less likely to purchase that brand in the future, leading to a loss of customer loyalty and long-term sales. Negative publicity surrounding a spoilage incident can further erode brand trust and market share.
  4. Increased storage costs: If product release requires testing for Alicyclobacillus, the finished product will need to be stored until test results are available. Current testing methods, such as culture-based methods, can take a long time to yield results. This extended storage period while awaiting test results leads to increased storage costs for the manufacturer, as the product cannot be released for distribution until it has been cleared.

Since the first reported case of massive apple juice spoilage in Germany in 1984 involving Alicyclobacillus species, the incidence of spoilage events caused by these bacteria has increased significantly. According to a report by Kang (2006), Alicyclobacillus spoilage may cause annual economic losses of at least $400,000 in Washington state, USA, alone. In a survey by Lee et al. (2002), 60% of the 57 companies surveyed experienced spoilage events.


What products are most affected by Alicyclobacillus?

Alicyclobacillus species have been isolated from fruit juices, juice concentrates, and acidic beverages. The products most affected by Alicyclobacillus spoilage include:

  1. Apple juice and apple juice concentrates: Several studies have reported high levels of Alicyclobacillus contamination in apple juice and concentrates. In a survey by Borlinghaus and Engel (1997), 30.5% to 75% of apple juice concentrate samples contained Alicyclobacillus.
  2. Orange juice and orange juice concentrates: Alicyclobacillus has been frequently isolated from orange juice and concentrates. Groenewald et al. (2009) reported that 82.1% of orange juice concentrate samples were contaminated.
  3. Other fruit juices and concentrates: Alicyclobacillus has been detected in various other fruit juices and concentrates, including tomato, white grape, pear, mango, pineapple, apricot, peach and coconut.
  4. Fruit juice blends: Alicyclobacillus spoilage has been reported in mixed fruit juice beverages.
  5. Shelf-stable iced tea: Alicyclobacillus has been isolated from shelf-stable iced tea products, leading to spoilage incidents.

The risk of contamination varies depending on the type of juice product. Müller and Schulz-Schroeder (2009) reported that 17% of 53 shelf-stable juices tested contained Alicyclobacillus. Jensen (2005) estimated that 20% to 30% of orange and apple concentrates used in Australia are likely to contain Alicyclobacilli. These findings emphasize the need for effective control measures to prevent spoilage and ensure product quality.

What are the sources of contamination for Alicyclobacillus?

Understanding the sources of contamination is crucial for preventing spoilage and maintaining product quality. The primary sources of Alicyclobacillus species that contaminate fruit juices include:

  1. Soil: Alicyclobacillus naturally inhabits soil, making it the primary source of contamination. The bacterium can contaminate fruits during harvesting.
  2. Unwashed or poorly washed raw fruits: Using unwashed or inadequately washed fruits during juice processing can introduce Alicyclobacillus contamination.
  3. Contaminated raw materials: Alicyclobacillus endospores can be found in juice concentrates, syrups, fruit purees, and other raw materials used for beverage production. Using contaminated ingredients can lead to spoilage issues in the final product.
  4. Contamination during harvesting and processing: Alicyclobacillus contamination can occur at several steps during fruit juice harvesting, supply chain, and processing. Dusty environments, insects, birds, rain, and flooding can also contribute to contamination.
  5. Food processing equipment: Alicyclobacillus, especially A. acidoterrestris, can colonize and form biofilms on various food processing equipment surfaces, such as stainless steel, glass, PVC, etc. These biofilms are difficult to eliminate and can serve as a persistent source of contamination.

How can Alicyclobacillus be detected?

Traditional culture-based testing for Alicyclobacillus typically requires a period of over a week to identify the species and confirm guaiacol production. Furthermore, these methods often struggle to detect low levels of the bacteria in the product. However, rapid methods have been developed to expedite the detection process.

The foodproof® Alicyclobacillus Detection LyoKit is intended for rapid screening for the presence of guaiacol-producing strains in preparations from juices, juice concentrates and other beverages that can potentially be contaminated with Alicyclobacillus. In addition to the detection of DNA of Alicyclobacillus spp. in channel HEX, guaiacol-producing Alicyclobacillus strains are detected in channel FAM.

The foodproof Alicyclobacillus Detection LyoKit is designed to work seamlessly with Hygiena's foodproof StarPrep Two Kit for quick and easy extraction of bacterial DNA from enrichment according to IFU No.12 by heat-induced lysis. The detection kit is also compatible with many real-time PCR instruments, including Hygiena's Dualo 32®, a robust 32-well instrument ideal for small to medium throughput facilities.

How can Alicyclobacillus contamination be prevented?

Preventing Alicyclobacillus contamination in fruit juices requires a multi-faceted approach that includes good agricultural practices, adequate sanitation, and regular monitoring. Here's how Hygiena's products and services can be incorporated into a comprehensive prevention strategy:

  1. Environmental Monitoring: Regular environmental monitoring is crucial for identifying potential contamination sources and verifying the effectiveness of sanitation procedures. Hygiena's EnSURE® Touch is a versatile monitoring system used with various tests, including SuperSnap® and UltraSnap®, for reliable cleaning verification. Manufacturers can quickly identify and address contamination risks by monitoring surfaces, equipment, and the processing environment.
  2. Water Monitoring: Water used in fruit juice production, including ingredients such as water and water used for cleaning and sanitation, can be a potential source of Alicyclobacillus contamination. Hygiena's AquaSnap® is a rapid and simple test for detecting ATP in water samples, indicating the overall microbiological quality of the water. Regular water monitoring can help ensure that the water used in the facility meets quality standards and does not contribute to contamination.
  3. Data Analysis: Collecting and analyzing environmental monitoring data is essential for identifying trends, setting appropriate limits, and making informed decisions. Hygiena's SureTrend® software is a powerful tool for managing and analyzing data from multiple monitoring devices, including the EnSURE Touch. By using SureTrend, manufacturers can easily track and trend their monitoring results, identify problem areas, and demonstrate compliance with food safety regulations.
  4. Implementing Good Agricultural Practices (GAPs): Since soil is a primary source of Alicyclobacillus contamination, implementing GAPs can help minimize the risk of contamination at the farm level. This includes minimizing soil contact during harvesting, using clean and sanitized harvesting equipment, and properly storing and transporting fruits.
  5. Raw Material Testing: Testing raw materials, such as juice concentrates, purees, and flavorings, for the presence of Alicyclobacillus can help prevent contamination in the production process. Hygiena's foodproof Alicyclobacillus Detection LyoKit can rapidly and accurately detect Alicyclobacillus in these raw materials.

By incorporating Hygiena's products and services into a comprehensive prevention strategy, fruit juice manufacturers can effectively monitor and control Alicyclobacillus contamination, ensuring the quality and safety of their products.



Alicyclobacillus has emerged as a significant threat to the global fruit juice and beverage industry, causing substantial economic losses due to product spoilage, recalls, and damage to brand reputation. These bacteria's unique characteristics, including their survival in acidic environments, resistance to pasteurization, and difficulty in detection, make them a formidable challenge for manufacturers.

To combat Alicyclobacillus contamination effectively, a comprehensive approach that encompasses good agricultural practices, adequate sanitation, and regular monitoring is essential. Hygiena offers a range of products and services that can be integrated into a robust prevention strategy, including the foodproof Alicyclobacillus Detection LyoKit for rapid and accurate detection, EnSURE Touch for environmental monitoring, AquaSnap for water quality testing and SureTrend software for data analysis and trend identification.

By understanding the sources of contamination, implementing appropriate control measures, and leveraging advanced detection and monitoring tools, fruit juice manufacturers can proactively mitigate the risk of Alicyclobacillus spoilage. This proactive approach helps ensure product quality and safety and protects brand reputation and consumer trust.

As the fruit juice and beverage industry continues to evolve, it is crucial for manufacturers to stay informed about emerging threats like Alicyclobacillus and to adopt innovative solutions to address these challenges. By partnering with trusted experts like Hygiena and implementing a comprehensive food safety strategy, manufacturers can safeguard their products, protect their bottom line, and deliver the high-quality beverages consumers demand.

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