are limited and generally not comprehensive in scope. However, there are interesting results to consider. Two studies [30,31] determined that the presence of divalent cations (Ca2+, Mg2+, Ba2+, Mn2+, and Sr2+) in the sporulation medium did not affect the heat resistance of A. acidoterrestris. They also noted that techniques to demineralize and remineralize spores with calcium had no effect on their calcium content or thermal inactivation even though these techniques were capable of decreasing the thermal resistance of Bacillus subtilis spores. Vieira et al.  concluded that extended cold storage of spores at
— 18°C substantially increased z-values. Freshly prepared spores had a z-value of 7.8°C while spores stored at — 18°C for 4 and 8 months had z-values of 22 and 29° C, respectively. Since juice concentrates are typically stored at —5 to
— 10°C in large cold-stored tanks (380,000 L or more per tank) or —18°C in 55 gal drums, establishment of time/temperature processing parameters may require knowledge of the concentrate storage history prior to use by juice and beverage manufacturers.
Of three A. acidoterrestris strains tested by Pontius et al. , one was less heat resistant than the other two at both 91 and 97°C in a model juice system. However, the type of organic acid (citric, malic, or tartaric) in the model system had no significant effect (P > 0.05) on results. These researchers determined that results were significantly (P > 0.05) influenced by pH at 91°C but not at 97°C. Their results were similar to those of Komitopoulou et al.  who showed a decrease in D-values when the pH was changed from 4 to 3 in grapefruit juice. This effect was more apparent at 80°C (32 minutes at pH 3.0 and 52 minutes at pH 4.0) than at 95°C (1.5 minutes at pH 3.0 and 1.7 minutes at pH 4.0).
Under test conditions used by Palop et al. , D-values at 110 to 125° C were not different for spores suspended in orange juice, distilled water, or citrate—phosphate buffers at pH 4 and 7. These researchers noted that a sporulation temperature of 65°C yielded spores with increased heat resistance compared to those produced at 45°C. A linear correlation was noted between sporulation temperature and logD110°C. No differences in z-values were observed in this study among spores in the juice, water, and buffer test matrices.
Further research into processes and relevant parameters for control of the alicyclobacilli is warranted. While it is obvious that the quality of most juice and beverage products would not tolerate typical thermal conditions needed to control these organisms, alternative processes or combined processes may provide a solution.
Processors of 100% juices often choose to rely on thermal treatments as the only hurdle for the production of a safe and stable juice. For those processors selling refrigerated, pasteurized products, there is little cause for concern since the alicyclobacilli do not grow under common refrigeration conditions of 4 to 8°C. These organisms cause problems in shelf-stable, low pH products. In some of these products, sodium benzoate, sorbate or some other preservative may be added to inhibit growth of potential contaminants, but their effects on the alicyclobacilli are not clearly understood. While sorbates have been shown to inhibit germination of spores in some food products , this preservative has not been the subject of public, refereed research in relation to Alicyclobacillus spp.
The addition of the antimicrobial peptide nisin to juice was found to enhance the lethality of thermal treatment [34,37]. These references report use of a commercial nisin compound containing 2.5% active nisin with 106 international units (IU) per gram. Growth of A. acidoterrestris (Z CRA 7182) at 25°C was controlled in apple, grapefruit, and orange juices by as little as 5 IU/ml nisin . In the same study, D80°C of spores in apple juice decreased from 41 minutes without nisin to 24 minutes with 50 IU nisin/ml. As with pH effects, the effect on D-values was more obvious at the lowest (80°C) process temperature tested than at the highest (95°C). Although citrus juices are known to be susceptible to spoilage by the alicyclobacilli, it is interesting to note a greater degree of stability for citrus juice in this study when compared to apple juice. Natural oils in citrus juices are known to have antimicrobial activity, which might partially explain this observation.
When stored at 44°C, growth was observed in apple juice containing 50 IU nisin/ml but was not seen at the 100 IU/ml level over a 6-day period . In contrast, Yamazaki et al.  reported substantial growth of A. acidoterrestris (AB-5) over a 12-day storage trial in a clear apple drink with up to 600 IU nisin/ml. In that study, D90°C values were reduced from 20.8 to 14.8 with the addition of 200 IU nisin/ml of apple drink. Yamazaki et al.  reported that lysozyme increased thermal sensitivity of A. acidoterrestris spores in a citrate buffer (pH 4.0).
Alternative processes other than thermal treatment could provide another avenue of research to investigate alicyclobacillus inactivation. Lee et al.  reported reductions greater than 5 log in spore populations from the application of high-pressure processing (HPP) with thermal treatments. Although HPP potentially reduces the heat resistance of bacterial spores, high pressure alone would not provide adequate kill to prevent spoilage. Other alternative processes, such as pulsed electric field or ultraviolet light treatment, are generally not very effective against bacterial spores. At present, there are no adequate alternatives to stringent sanitation operating procedures coupled with current good manufacturing practices and appropriate raw material specifications for control of alicyclobacilli in beverages. This makes the establishment of specifications difficult. Many customers of juice/beverage ingredients desire to set a zero tolerance for the presence of the alicyclobacilli in concentrates and sweeteners; however, this standard may be unachievable with current processing and sanitizing technology.
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