7.5.1 Emergence as Spoilage Organisms
Food manufacturers continually investigate innovations in product development, processing aids, and processing equipment to meet changing consumer demands in order to maintain adequate profit margins. Changes in products and manufacturing procedures frequently result in unique quality and stability challenges. For example, in the 1980s and 1990s the use of oxygen barrier films in gable-top cartons allowed orange juice packers to increase refrigerated shelf life of orange juice from 35 to 60+ days. This increased shelf life provided enough time for fungal propagules within the paperboard matrix of the carton to germinate and outgrow into the product thereby increasing consumer complaints of mold spoilage and leading to use of a different package design.
Between the mid-1980s and 1990s juice and beverage manufacturers became aware of spoilage in low pH, shelf-stable products by spore-forming bacteria. Processors of shelf-stable apple juices noticed occasional development of strong off-aromas in finished product after a few weeks or months of storage. Also, a few citrus juice processors reported the slow growth of a spore-forming bacterium in aseptic juice samples thermally abused at warm temperatures during quality control testing. Since the final product was marketed as a refrigerated product, these spore-formers did not result in spoilage of the finished product.
The earliest documented fruit juice spoilage event related to alicyclo-bacillus was reported by Cerny et al.  and involved an antiseptic off-aroma in apple juice. The causative organism was described as "related to'' Bacillus acidocaldarius but would likely be classified today as A. acidoterrestris. Splittstoesser et al.  reported growth characteristics of two thermoaciduric spore-forming isolates that resulted in strong off-aromas in finished apple juice products. Other reports also documented the isolation of thermoacido-philes from juices, other low pH beverages, soil, fruit surfaces, and recycled water [39—46]. Based on reports such as these, investigations were conducted into the type of spoilage observed and the necessary conditions for such spoilage to become apparent.
Prior to the recognition of alicyclobacillus as a spoilage agent of juices and beverages, microbial stability problems were limited to yeasts, lactic acid bacteria, acetic acid bacteria, or heat-resistant molds. Typical juice spoilage involves production of CO2 and off-flavors by fermentative organisms resulting in bulging or exploding containers. Spoilage events caused by the alicyclobacilli are different. Since there is no CO2 to indicate microbial growth, the powerful antiseptic and medicinal aromas may not be detected until consumers open the package. The only visual clue to the presence of this organism is the possible presence of a slight haze in clear liquids such as clarified apple juice. Therefore, food manufacturers must implement new quality assurance measures to detect the presence of these spoilage organisms.
The alicyclobacilli produce at least three odiferous phenolic compounds with low detection thresholds. It was established in early research that guaiacol is the principal off-aroma produced by the alicyclobacilli although 2,6-dibromophenol (DBP) and 2,6-dichlorophenol (DCP) were also isolated from products with large populations of Alicyclobacillus spp. Pettipher et al.  found detectable levels of guaiacol in fruit juices, including orange and apple juices. Baumgart et al.  and Borlinghaus and Engel  reported the presence of DBP in tea and juice products. Orr et al.  reported that guaiacol content in apple juice was not correlated with numbers of cells, and that the best estimate threshold for guaiacol added to apple juice was 2.23 ppb. Jensen and Whitfield  described the production of DCP by the alicyclobacilli. Gocmen et al.  reported production of guaiacol, DBP, and DCP by five strains of alicyclobacilli in orange juice.
For some manufacturers it is not feasible to consider the addition of preservatives, such as nisin discussed above, to juice and beverage products as a microbial control measure due to regulatory reasons. In these cases, processors must reduce alicyclobacillus in the product through intensive sanitation of the processing environment, and through strict specifications and current good manufacturing practices for the product ingredients. Greater emphasis on appropriate use of cleaners and sanitizers to control these organisms on food contact surfaces should reduce the potential for product contamination.
It is also important for processors to examine carefully all potential sources of contamination since these organisms are widespread in nature and are common inhabitants of soil. As thermoacidophiles, alicyclobacilli would be expected to proliferate under warm acidic conditions in a manufacturing facility. These conditions can occur in juice/beverage processing plants, especially during the warm summer months. (Empirical evidence of this exists in the large amount of spoilage observed in Europe during the unusually hot summers of 1994 and 1995.) Some facilities that produce concentrated fruit juices reclaim water that is released from the juice during the evaporation process, and utilize it for cleaning purposes within the plant. Since the recovered water is warm to hot and slightly acidic, it provides an appropriate environment for proliferation of the alicyclobacilli.
Wisse and Parish  and Eguchi et al.  reported the presence of significant alicyclobacillus populations in "condensate water'' recovered for use in citrus concentrate facilities. Not only was the water used to wash equipment and incoming fruit, in some cases pulp extracted from the juice was washed with this water to recover sugar solids. This wash water was then diverted to the evaporator and mixed with juice just prior to the concentration process thereby guaranteeing a constant source of contamination in juice concentrates produced by those facilities. Since that discovery, efforts to address the alicy-clobacillus issue at citrus processing facilities have emphasized the cleanliness of the condensate water recovery system and treatment of water used in the facility.
Considering the substantial economic losses sustained due to growth of the alicyclobacilli in juices and other low pH beverages, there are few refereed publications discussing cleaning and sanitation requirements to control these organisms. Orr and Beuchat  exposed five strains of A. acidoterrestris spores to sodium hypochlorite, acidified sodium chlorite, trisodium phosphate, hydrogen peroxide, and Tsunami® sanitizer (Ecolab Inc., St. Paul, MN), for 10 minutes at 23°C. A 5 log reduction in spore population was observed after treatment with 1000 ppm sodium hypochlorite or 4% hydrogen peroxide. At lower concentrations, significant (P < 0.05) reductions of 2 log, 0.4 log, and 0.1 log were observed with 200 ppm hypochlorite, 500 ppm acidified sodium chlorite, and 0.2% hydrogen peroxide, respectively. Based on these results, these researchers continued with practical experiments to determine chemical effectiveness against A. acidoterrestris on apple surfaces. Reductions in spore populations after 1 minute exposure to 500 ppm hypochlorite or 1200 ppm acidified sodium chlorite were statistically significant (P < 0.05) but did not inactivate spores more than 1 log as compared to 5 and 2.5 log in the earlier direct challenge experiments.
Methods for accurate and sensitive detection, isolation, identification, and quantification of the alicyclobacilli in foods have developed slowly and remain somewhat controversial. In general, detection of these organisms in juices and beverages has relied upon their thermoacidophilic character and their ability to produce odiferous phenolic compounds. No standard method detection/recovery protocols have yet been developed that are universally accepted.
Research has been conducted on the use of DNA and PCR-based technologies to detect the alicyclobacilli in food samples. Specific primers for detection have been developed  and a real-time PCR-based detection method has been developed . Rapid test kits for detection or identification of Alicyclobacillus spp. are commercially available from Vermicon AG, MicroBio Corporation, and BioSys. As with any rapid test methods, customers should verify and validate these products for their usefulness in the specific commodity or environmental sample of interest. Customers should further question the manufacturers for information on false negative and false positive tests in order to make an informed decision on the usefulness of such products for a particular application.
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