Venture index

Venture Center Home Page

Food Science @ Geneva

NYSAES

Food Safety and You: Alicyclobacillus: An Emerging Problem for New York’s Processors of Fruit Juices

D. F. Splittstoesser, R. W. Worobo and J. J. Churey

Until recently it was believed that bacterial spores did not have the ability to germinate and produce vegetative growth in high acid foods such as many fruit juices. As a result, processes in which the juice was filled into containers at temperatures of 185 to 190° F were considered adequate for most beverages.

In 1982 a sporeforming bacillus was isolated from spoiled apple juice that could grow at pH values as low as 2.5 (Cerny et al., 1984). A new genus, Alicyclobacillus, was created for this and similar organisms based on their fatty acid composition and 16S rDNA sequences (Wisotzkey et al., 1992).

We first encountered Alicyclobacillus in 1990 when a commercial apple juice was submitted to us because it exhibited an offensive chemical odor. Our flavor chemist, Dr. T. E. Acree, determined that guaiacol was responsible for the off-odor. When we cultured the bacterium on membranes through which we had filtered over 200 ml of juice, low numbers (about 3 per 10 ml) of acid-tolerant bacterial spores were recovered. We later concluded that the guaiacol must have been generated at an earlier juice processing stage and that most of the bacteria had been removed by subsequent clarification treatments.

Since 1990, we have been involved in additional spoilage outbreaks due to Alicyclobacillus, both in New York as well as in other states. Recognition of the problem appears to be increasing (Walls and Chuyate, 1998).

SUSCEPTIBLE JUICES

Our studies have shown that Alicyclobacillus grows well in apple juice, tomato juice, white grape juice and various blends of juices (Splittstoesser, et al., 1994; Splittstoesser et al., 1998). The bacteria have been isolated from citrus processing lines and are potential spoilage agents of beverages containing citrus juices (Wisse and Parish, 1998).

The organism does not grow in Concord grape juice nor in the juice of the other red grapes that we have tested (Splittstoesser and Churey, 1996). The presence of certain neutral phenolic compounds which may be present in red grape juice have been shown to inhibit growth (Splittstoesser et al., 1994).

Sugar concentrations above 18° Brix are inhibitory as are ethanol levels above 6%.

SOURCE OF CONTAMINATION

The natural habitat of the spores has not been studied thoroughly although soil is considered to be the repository for many sporeformers. Seven of 18 soil samples collected from citrus groves were positive for aciduric sporeformers (Wisse and Parish, 1998). It seems likely, therefore, that fruit in greater contact with soil would be most susceptible to contamination by Alicyclobacillus.

Certain other beverage ingredients are also potential sources of contamination. Our recent unpublished studies revealed the spores to be present in liquid sugar (Worobo and Churey, 1998).

SPOILAGE SIGNS

Only a limited amount of growth occurs in clear beverages such as apple juice and thus spoilage may be manifest by only a slight haze. We believe that oxygen is the limiting growth factor since much heavier growth has been obtained when apple juice was incubated on a rotary shaker after inoculation.

The organism does not produce detectable quantities of carbon dioxide or other gas.

The aroma of guaiacol is quite distinctive and thus many QC personnel have trained their noses to determine whether or not they have a spoilage problem due to Alicyclobacillus.

CULTURAL PROCEDURES

We have used potato dextrose agar (PDA), pH 3.5 as a selective medium for Alicyclobacillus. The juice or other sample is heated at 60°C for 60 minutes prior to culturing to destroy nonheat resistant organisms such as yeasts and to activate dormant spores. The plates are incubated 5 to 7 days at 43° C or at 53° C if the sample also contains spores of heat resistant molds such as Byssochlamys.

Media used by other investigators include K media (Walls and Chuyate, 1998) and ALI agar (Wisse and Parish, 1998).

Because only low populations of the spores may be present, detection often requires the culturing of large volumes of juice. When the juice is filterable, several hundred ml may be passed through 0.45 um membrane filters.

We produce spore crops by inoculating the surface of PDA pH 5.6 plates. The plates are incubated five days at 43° C before the growth is transferred to sterile distilled water. The spores cause a terminal to subterminal swelling of the rods (Splittstoesser, 1993).

HEAT RESISTANCE

Our heat resistance studies have shown that Alicyclobacillus spores can survive the usual hot fill processes that are given to commercial juices (Table 1). The data also show that increasing the Brix makes the spores more resistant to heat. The spores are less resistant as the pH is decreased and the type of organic acid can be important (Pontius et al., 1998). A successful commercial process for Alicyclobacillus control might require a 5 logarithmic spore kill, thus 5 times the D-value at a given temperature.

REFERENCES

Cerny, G., W. Hennlich, K. Poralla. 1984. Spoilage of fruit juice by bacilli: isolation and characterization of the spoilage organism. Z. Lebensm. Unters. Forsch. 179:224-227.

Pontius, A. J., J. E. Rushing, and P. M. Foegeding. 1998. Heat resistance of Alicyclobacillus acidoterrestris spores as affected by various pH values and organic acids. J. Food Protection 61:41-46.

Splittstoesser, D. F. 1993. Recent developments in the microbiology of fruit juices, p. 59-61. In D. L. Downing (ed), Juice Technology Workshop, Report 67, NYS Agric. Expt. Station, Geneva, NY

Splittstoesser, D. F., J. J. Churey, and C. Y. Lee. 1994. Growth characteristics of aciduric sporeforming bacteria isolated from fruit juices. J. Food Protection 57:1080.

Splittstoesser, D. F., and J. J. Churey. 1996. Unique spoilage organisms of musts and wines, p 36-41. In T. Toland and K. C. Fugelsang (ed), Wine spoilage microbiology Conference. California State University, Fresno.

Splittstoesser, D. F., C. Y. Lee and J. J. Churey. 1998. Control of Alicyclobacillus in the juice industry. Dairy Food and Environ. Sanitation 18:585-587.

Walls, I. And R. Chuyate, 1998. Alicyclobacillus — Historical perspective and preliminary characterization study. Dairy Food and Environ. Sanitation 18:499-503.

Wisotzkey, J. D., P. Jurtshuk, Jr., G. E. Fox, G. Deinhard, and K. Poralla. 1992. Comparative sequence analysis on the 16S (rDNA) of Bacillus acidocaldarius, Bacillus acidoterrestris and Bacillus cycloheptanicus and a proposal for creation of a new genus Alicyclobacillus gen. nov. Intl. J. Systematic Bacteriol. 42:263-269

Wisse, C. A., and M. E. Parish. 1998. Isolation and enumeration of sporeforming, thermoacidophilic, rod-shaped bacteria from citrus processing environments. . Dairy Food and Environ. Sanitation 18:504-509.

Worobo, R. W. and J. J. Churey. 1998. Unpublished data.