1370

Journal of Food Protection, Vol. 74, No. 8, 2011, Pages 1370–1373

doi:10.4315/0362-028X.JFP-11-027
Copyright G, International Association for Food Protection

Research Note

Recovery of Alicyclobacillus from Inhibitory Fruit Juice

Concentrates
CHRISTOPHER J. MCNAMARA,* DEBORAH WIEBE, AND MARGARITA GOMEZ

Ocean Spray Cranberries, Inc., One Ocean Spray Drive, Lakeville-Middleboro, Massachusetts 02349, USA

MS 11-027: Received 20 January 2011/Accepted 1 April 2011

ABSTRACT
Growth of Alicyclobacillus in low-pH fruit juices may result in off-odors and off-flavors due to the production of compounds
such as guaiacol (2-methoxy phenol). An important step in preventing Alicyclobacillus contamination of fruit juices is the
screening of incoming ingredients. Many fruit juice concentrates contain compounds that inhibit Alicyclobacillus growth, but
beverages produced from the concentrates may not contain sufficient amounts of the active component to prevent spoilage.
Therefore, accurate screening of juice concentrates is essential to prevent false-negative test results and product spoilage. The
objective of this study was to evaluate isolation methods for detection of Alicyclobacillus in inhibitory juice concentrates.
Recovery of Alicyclobacillus spores from inoculated and naturally contaminated concentrates was compared by using pour plate,
spread plate, and filtration methods. Pour plates consistently recovered the lowest number of spores from inoculated concentrates.
Spread plating was the most effective method used to recover spores from inoculated apple and pomegranate juice concentrates,
while filtration resulted in the highest recovery from cranberry concentrate. When tested on naturally contaminated concentrates,
the pour plate method failed to detect Alicyclobacillus in many samples. Filtration was much more effective. The filtration
method increased the likelihood of detecting Alicyclobacillus contamination of fruit juice concentrates containing inhibitory
compounds.

The genus Alicyclobacillus consists of nonpathogenic,
thermophilic, acidophilic, spore-forming bacteria that are
common in soil (8). Alicyclobacillus typically enters
processing plants on fruit surfaces, and the spores are able
to survive the common pasteurization of juices (8).
Subsequent growth of some species (e.g., Alicyclobacillus
acidoterrestris) in low-pH fruit juices may result in off-
odors and off-flavors due to the production of compounds
such as guaiacol (2-methoxyphenol) or halophenols (2,6-
dibromophenol and 2,6-dichlorophenol) (2, 3, 8).
A number of studies have compared growth media and
culturing methods for detection of Alicyclobacillus spp. in
beverages. For example, Chang and Kang (7) developed the
growth medium SK agar by optimizing pH and the
concentrations of organic acids, inorganic nutrients, and a
nonionic surfactant. Murray et al. (13) compared growth
media, incubation temperatures, incubation times, and
plating methods. Optimal recovery and growth from
beverages were obtained on Bacillus acidoterrestris ther-
mophilic (BAT) agar incubated at 43uC for 3 days.
Optimization of media and recovery conditions is
typically performed with spores suspended in relatively
simple and benign matrices such as apple juice or buffer (5,
13). However, many natural products contain antimicrobial
compounds (4, 5). Compounds isolated from cranberry with
* Author for correspondence. Tel: 508-946-7897; Fax: 508-946-9227;
E-mail: cmcnamara@oceanspray.com.
antimicrobial activity include phenolics, anthocyanins, and
organic acids (10). Cranberry antimicrobial activity has been
demonstrated against many bacteria, including Escherichia
coli, Listeria monocytogenes, Salmonella enterica serovar
Typhimurium, and Staphylococcus aureus (18), and against
some fungi (17).
Splittstoesser et al. (16) demonstrated that cranberry
beverages inhibited Alicyclobacillus. Research at Ocean
Spray Cranberries (OSC) has also shown that cranberry
juice can inhibit Alicyclobacillus growth (Fig. 1) (unpub-
lished data). In addition to cranberries, many fruits such as
blueberries and pomegranates have antimicrobial properties
(6, 12, 14) and may affect Alicyclobacillus growth in
beverages.
Screening ingredients for Alicyclobacillus contamina-
tion is one hurdle that can be used to reduce the likelihood
of product spoilage. However, for screening to be effective,
proper methods must be used. The presence of inhibitory
compounds in fruit juice concentrates can lead to false-
negative test results if these compounds are introduced into
the test medium at a high level. Inclusion of concentrates in
the final beverage product could dilute inhibitors to an
ineffective level, leading to spoilage by undetected spores.
Therefore, an important step in preventing Alicyclobacillus
contamination of fruit juices is the accurate screening of
incoming ingredients.
The purpose of this study was to compare three culture-
based methods for detection of Alicyclobacillus in fruit juice
J. Food Prot., Vol. 74, No. 8
FIGURE 1. Inhibition of Alicyclobacillus sp. growth by cranber-
ry juice. The top half of the plate contains acidified potato dextrose
agar. The bottom half of the plate contains a mixture of cranberry
juice and agar.
concentrates. Spread plating, pour plating, and filtration
were used to enumerate Alicyclobacillus organisms in
experimentally inoculated fruit juice concentrates and in
naturally contaminated concentrates. Concentrates contain-
ing natural inhibitors and noninhibitory concentrates were
tested.
MATERIALS AND METHODS
Comparison of recovery methods. Four strains of A.
acidoterrestris isolated from fruit juices were used in this study.
Three strains (232, 258, and 259) were isolated in the OSC
microbiology laboratory, and one strain (N-1155) was acquired
from the Grocery Manufacturers’ Association (Washington, DC).
Strains isolated in the OSC laboratory were identified as A.
acidoterrestris based on 16S rDNA sequences as well as
phenotypic characterization (gram-positive to gram-variable bacil-
li, endospore formation, growth on potato dextrose agar acidified
with tartaric acid [PDAzTA] at 43uC, no growth on standard
methods agar, and positive for guaiacol production). The three
strains isolated in the OSC microbiology laboratory differed in 16S
rDNA sequences and in growth characteristics on select media.
To prepare spores, cells of each strain collected from the
surface of agar plates were mixed and suspended in phosphate buffer
(PB; 1.13 g of K2HPO4 and 5.92 g of KH2PO4 per liter), heat
shocked for 10 min at 76uC, and cooled in a room temperature water
bath. The concentration of the spore suspension was determined by
using a hemacytometer. The spore suspension was diluted with PB,
and concentrates were inoculated with 100 spores per ml.
One inhibitory concentrate (cranberry, 50uBx) and one
noninhibitory concentrate (apple, 70uBx) were tested. A third
concentrate, pomegranate (65uBx), was also included. We are not
aware of any published literature indicating that pomegranate
concentrate or juice prevents the growth of Alicyclobacillus.
However, pomegranate was included due to the relatively broad
spectrum of antimicrobial activity of pomegranate extracts (12, 14)
and the ability of other dark red juices such as cranberry and red
RECOVERY OF ALICYCLOBACILLUS FROM FRUIT JUICE 1371
grape to inhibit Alicyclobacillus (16). Additionally, preliminary
tests (data not shown) indicated that pomegranate juice can inhibit
Alicyclobacillus.
Immediately after inoculation, the number of spores in each
concentrate was determined by using three methods: spread
plating, pour plating, and filtration. Spread plates (10 per replicate)
were inoculated with 0.1 ml of concentrate. Pour plates were
inoculated with 1.0 ml of concentrate, and 19.0 ml of agar was
added. For the filtration method, 1.0 ml of each concentrate was
diluted with 10 ml of PB. Samples were filtered onto a 0.45-mm-
pore-size filter (Microcheck II Beverage Monitor, Pall Corporation,
Port Washington, NY), rinsed with 20 to 30 ml of sterile distilled
water, and transferred to the surface of an agar plate. All tests were
run in triplicate, and PDAzTA was used for all methods. Previous
comparisons of recovery of these four isolates on PDAzTA, K
agar, and yeast extract–starch–glucose agar demonstrated that
recovery on PDAzTA was equivalent to, or in some cases
superior to, the other media (data not shown). Plates were
incubated at 43uC in plastic bags, and CFU were enumerated after
7 and 14 days. Means were compared by the general linear model
followed by the Tukey multiple comparison test using Minitab
(version 16.1.1).
Screening naturally contaminated concentrates. Blueberry
(65uBx), Concord grape (68uBx), pomegranate (65uBx), and raisin
(70uBx) concentrates were screened for the presence of Alicyclo-
bacillus by filtration and pour plates. Many of the samples had
very low concentrations of spores, making spread plating
impractical. The methods used were identical to those described
above, but the volumes sampled were larger. For filtration, 10 ml
of concentrate was diluted with 100 ml of PB. Ten pour plates
containing 1.0 ml of concentrate and 19.0 ml of agar were
constructed for each replicate. All tests were run in triplicate using
PDAzTA. Plates were incubated at 43uC in plastic bags, and CFU
were enumerated after 7 and 14 days. Isolates recovered from the
concentrates were identified as Alicyclobacillus based on cellular
morphology, endospore production, growth on PDAzTA at 43uC,
lack of growth on standard methods agar, and guaiacol production.
RESULTS AND DISCUSSION
Comparison of the recovery of A. acidoterrestris spores
from inhibitory and noninhibitory fruit juice concentrates
indicated that the method used can significantly influence
the number of CFU recovered. For all three juice
concentrates, the lowest numbers of CFU were recovered
by pour plating (Fig. 2). The highest numbers of CFU were
recovered from apple and pomegranate concentrates by
spread plating. Filtration was the most effective method
used to culture Alicyclobacillus from cranberry concentrate.
Analysis by the general linear model found significant
differences for the main effects of enumeration method (P ,
0.001) and concentrate type (P , 0.001) and for the method
| concentrate interaction (P , 0.001).
Pour plating is one method recommended for testing
juices, concentrates, and ingredients for Alicyclobacillus
contamination (9) and is commonly used in many
laboratories. The use of pour plating with pomegranate
and cranberry concentrates reduced or eliminated the
recovery of Alicyclobacillus CFU (Fig. 2). It is likely that
despite dilution by the growth medium, inhibitory compo-
nents of the pomegranate and cranberry concentrates
remained at a level sufficient to reduce Alicyclobacillus
1372 MCNAMARA ET AL. J. Food Prot., Vol. 74, No. 8

FIGURE 2. Comparison of the recovery

of Alicyclobacillus CFU from fruit juice
concentrates by filtration, pour plates, and
spread plates (mean z standard error of
the mean; n ~ 3). Means were compared
by using the general linear model followed
by the Tukey multiple comparison test.
Means that do not share a letter are
significantly different.

growth. While cranberry juice is known to inhibit detected by filtration. Blueberry, Concord grape, and
Alicyclobacillus growth (16), this has not previously been pomegranate juices all exhibit antimicrobial activity (6,
shown for pomegranate juice. 12, 14, 16), and recovery from these juice concentrates was
Even when used to assess noninhibitory products, pour increased at least threefold when filtration was used. In
plating typically results in lower recovery of Alicycloba- contrast, raisin concentrate is not known to exhibit
cillus due to reduced oxygen concentration within the agar antimicrobial activity against Alicyclobacillus, and only a
and difficulty in counting small colonies (13, 15). The low slight increase in recovery by filtration over pour plating
recovery of Alicyclobacillus CFU from apple juice (Fig. 2) was observed.
confirms the results of previous studies and indicates that Filtration increased the likelihood of detecting Alicy-
pour plating should not be used to test for the presence of clobacillus spores in fruit juice concentrates containing
Alicyclobacillus in fruit juice concentrates. inhibitors. For filterable products, use of filtration as
Alicyclobacillus recovery from apple and pomegranate opposed to pour plating reduces the chance of obtaining
concentrate was greatest with spread plating, while the false-negative results when testing ingredients for Alicyclo-
greatest recovery from cranberry concentrate was obtained bacillus contamination and thereby reduces the risk of
by filtration (Fig. 2). Differences among concentrates in product spoilage. For nonfilterable concentrates containing
recovery by spread plating may be related to the compounds that may inhibit Alicyclobacillus growth,
concentration and/or activity of inhibitory compounds. dilution of the concentrate in a large volume of medium
The lower recovery from cranberry concentrate by spread (such as BAT agar, yeast extract–starch–glucose agar, or
plating may indicate a higher concentration or activity of acidified potato dextrose broth) followed by enrichment
inhibitory compounds than is found in pomegranate through incubation at 43 to 45uC is an effective but
concentrate. Presumably, inhibitory compounds in the nonquantitative detection method (1).
pomegranate concentrate were diluted as the material was TABLE 1. Comparison of the number of Alicyclobacillus CFU
absorbed into the agar, allowing colonies to grow on the detected in naturally contaminated concentrates by using the pour
surface. The greater inhibitory activity of cranberry plate and filtration methods
concentrate, compared to that of pomegranate, is also
Avg Alicyclobacillus CFU/10 ml of concentratea
indicated by the pour plate results, in which no organisms
were recovered from the cranberry concentrate. Concentrate Pour plate method Filtration method
More Alicyclobacillus spores were recovered from
apple and pomegranate juice by spread plating than by Blueberry
filtration. This may be the result of some spores either Lot 1 26 81
becoming stuck internally in the membrane matrix or Lot 2 0 4
passing entirely through the membrane. Some loss of spores Lot 3 0 0.4
Lot 4 0 5
through filtration is not unusual. Lee et al. (11) found that
recovery of Alicyclobacillus varied greatly among filter Concord grape
brands and pore sizes. Lot 1 0 75
Detection of Alicyclobacillus in naturally contaminated Pomegranate
fruit juice concentrates was compared using pour plates and Lot 1 0 1
filtration. Spread plating was not feasible due to the low Lot 2 2 7
concentration of spores in many of the concentrates. In all Raisin
cases, more CFU were found by filtration than by pour
Lot 1 94 105
plating (Table 1). Additionally, in many of the concentrates
a
with low levels of contamination, Alicyclobacillus was only Values are the averages of three samples of each lot.
J. Food Prot., Vol. 74, No. 8
ACKNOWLEDGMENT
This research was supported by Ocean Spray Cranberries, Inc.
REFERENCES
1. Anonymous. 2007. Method on the detection of taint producing
Alicyclobacillus in fruit juices. IFU method no. 12. International
Federation of Fruit Juice Producers, Paris.
2. Bahçeci, K., and J. Acar. 2007. Determination of guaiacol produced
by Alicyclobacillus acidoterrestris in apple juice by using HPLC and
spectrophotometric methods, and mathematical modeling of guaiacol
production. Eur. Food Res. Technol. 225:873–878.
3. Bahçeci, K. S., V. Gökmen, and J. Acar. 2005. Formation of guaiacol
from vanillin by Alicyclobacillus acidoterrestris in apple juice: a
model study. Eur. Food Res. Technol. 220:196–199.
4. Bevilacqua, A., M. R. Corbo, and M. Sinigaglia. 2008. Inhibition of
Alicyclobacillus acidoterrestris spores by natural compounds. Int. J.
Food Sci. Technol. 43:1271–1275.
5. Bevilacqua, A., M. Sinigaglia, and M. R. Corbo. 2008. Alicycloba-
cillus acidoterrestris: new methods for inhibiting spore germination.
Int. J. Food Microbiol. 125:103–110.
6. Burdulis, D., A. Sarkinas, I. Jasutiene, E. Stackevicene, L.
Nikolajevas, and V. Janulis. 2009. Comparative study of anthocyanin
composition, antimicrobial and antioxidant activity in bilberry
(Vaccinium myrtillus L.) and blueberry (Vaccinium corymbosum L.)
fruits. Acta Pol. Pharm. 66:399–408.
7. Chang, S., and D.-H. Kang. 2005. Development of novel Alicyclo-
bacillus spp. isolation medium. J. Appl. Microbiol. 99:1051–1060.
8. Chang, S.-S., and D.-H. Kang. 2004. Alicyclobacillus spp. in the fruit
juice industry: history, characteristics, and current isolation/detection
procedures. Crit. Rev. Microbiol. 30:55–74.
9. Evancho, G. M., and I. Walls. 2001. Aciduric flat sour sporeformers,
p. 239–244. In F. P. Downes and K. Ito (ed.), Compendium of
RECOVERY OF ALICYCLOBACILLUS FROM FRUIT JUICE 1373
methods for the microbiological examination of foods. American
Public Health Association, Washington, DC.
10. Lacombe, A., V. C. H. Wu, S. Tyler, and K. Edwards. 2010.
Antimicrobial action of the American cranberry constituents;
phenolics, anthicyanins, and organic acids, against Escherichia coli
O157:H7. Int. J. Food Microbiol. 139:102–107.
11. Lee, S.-Y., S.-S. Chang, J.-H. Shin, and D.-H. Kang. 2007. Membrane
filtration method for enumeration and isolation of Alicyclobacillus spp.
from apple juice. Lett. Appl. Microbiol. 45:540–546.
12. Mathabe, M. C., R. V. Nikolova, N. Lall, and N. Z. Nyazema. 2006.
Antibacterial activities of medicinal plants used for the treatment of
diarrhoea in Limpopo Province, South Africa. J. Ethnopharmacol.
105:286–293.
13. Murray, M. B., J. B. Gurtler, J.-H. Ryu, M. A. Harrison, and L. R.
Beuchat. 2007. Evaluation of direct plating methods to enumerate
Alicyclobacillus in beverages. Int. J. Food Microbiol. 115:59–69.
14. Navarro, V., M. L. Villarreal, G. Rojas, and X. Lozoya. 1996.
Antimicrobial evaluation of some plants used in Mexican traditional
medicine for the treatment of infectious diseases. J. Ethnopharmacol.
53:143–147.
15. Pettipher, G. L., M. E. Osmundson, and J. M. Murphy. 1997.
Methods for the detection and enumeration of Alicyclobacillus
acidoterrestris and investigation of growth and production of taint in
fruit juice and fruit juice-containing drinks. Lett. Appl. Microbiol. 24:
185–189.
16. Splittstoesser, D. F., J. J. Churey, and C. Y. Lee. 1994. Growth
characteristics of aciduric sporeforming bacilli isolated from fruit
juices. J. Food Prot. 57:1080–1083.
17. Swartz, J. H., and T. F. Medrek. 1968. Antifungal properties of
cranberry juice. Appl. Microbiol. 16:1524–1527.
18. Wu, V. C.-H., X. Qiu, A. Bushway, and L. Harper. 2008.
Antibacterial effects of American cranberry (Vaccinium macrocar-
pon) concentrate on foodborne pathogens. LWT Food Sci. Technol.
41:1834–1841.