Original article
Thermal resistance of Alicyclobacillus acidoterrestris spores in
different heating media
Summary The aim of this work was to study the influence of temperature (85, 90, 95 and 100 C), total soluble solids
(SS: 10 and 20Brix or % by weight of sucrose) and pH (3.5 and 4.0) on decimal reduction time (D-value) of
the Alicyclobacillus acidoterrestris strain DSM2498 spores in apple juice, orange juice and malt extract broth
(MEB). The effects of SS and pH on D-values and z-values in each media were insignificant (P > 0.05). In
apple juice, orange juice and MEB, z-values of A. acidoterrestris for pH 3.5 and pH 4.0 were 12.2 ± 1.3–
14.2 ± 3.2 C, 11.2 ± 0.3–9.4 ± 0.0 C and 11.9 ± 0.8–10.3 ± 0.4 C, respectively. z-values of apple
juice, orange juice and MEB samples with SS = 10Brix and SS = 20Brix were 14.1 ± 3.2–12.2 ± 1.3 C,
10.2 ± 0.7–10.5 ± 1.1 C and 11.3 ± 1.5–10.9 ± 0.2 C, respectively. However, D-values of all samples
were affected by temperature significantly (P < 0.01). Average D-values of apple juice, orange juice and
MEB were 101.2 ± 14.7, 34.4 ± 7.9, 20.3 ± 4.9 and 4.3 ± 1.3 min for 85, 90, 95 and 100 C. This study
demonstrated that A. acidoterrestris spores exhibited high resistance to thermal processing applications. pH
and SS of the media did not affect thermal resistance.
Keywords Alicyclobacillus acidoterrestris, apple juice, D-value, orange juice, thermal resistance, z-value.
doi:10.1111/j.1365-2621.2009.01995.x
2009 The Authors. Journal compilation 2009 Institute of Food Science and Technology
Thermal resistance of A. acidoterrestris spores G. Ceviz et al. 1771
result of the germination and growth of these spores 44 C, after applying heat treatment between 80 and
surviving in juices, taste and aroma defects can be 96 C. In the relevant study, decrease at critical
occured (Jensen, 2000). Therefore, this micro-organism cinnamaldehyde concentration in accordance with pH
has been suggested as the target to be used in the design reduction at medium was reported.
of adequate pasteurisation processes of acidic food The effects of temperature and high pressure on the
products (Murakami et al., 1998; Silva et al., 1999; Silva inactivation of A. acidoterrestris have been both
& Gibbs, 2001, 2004). Indeed, A. acidoterrestris spore separately and jointly investigated. Alpas et al. (2003)
germination and growth to 106 cfu mL)1 under acidic reported that there is a significant increase in the
conditions has also been reported in orange juice stored inactivation of A. acidoterrestris cells in model system
at 44 C for 24 h (Pettipher et al., 1997). Komitopoulou (BAM broth), orange juice, apple juice and tomato
et al. (1999) reported that A. acidoterrestris spores juice. Lee et al. (2002) and Buzrul et al. (2005) explained
germinate and grow in apple, orange and grapefruit that the processing temperature has obvious and signif-
juices stored at 30 C. Pontius et al. (1998) showed that icant effects on the pressure inactivation. For rais-
A. acidoterrestris can survive for 2 min at 88–96 C ing threat of A. acidoterrestris at fruit juice industry,
during the hot filling processes. A. acidoterrestris have Walker & Phillips (2008) evaluated the applicable
been determined to germinate and grow best under the isolation techniques and rapid identification methods,
conditions of pH 4.5 and 43 C (Sinigaglia et al., 2003). spoilages created by these bacteria and the novel
Some preservatives used in the preservation of food- applications for preventing spoilages in their extensive
stuffs reduce the thermal resistance of micro-organisms review.
(Acar & Cemeroglu, 1998). As well as the studies The thermal resistance characteristics of food-spoilage
concerning the temperature variables, some other stud- micro-organisms are quite important determinants of
ies have been conducted on how A. acidoterrestris is the design of the thermal processes used to preserve
affected by several chemicals (Pettipher & Osmundson, various food products. As put forward by many
2000; Yamazaki et al., 2000; Lee et al., 2004). Lee et al. researches, A. acidoterrestris is an important source of
(2004) investigated the reduction of A. acidoterrestris microbial spoiling in fruit juices. Only limited numbers
spores in several culture media and apples with chlorine of studies have been carried out to determine the
dioxide. It was found that increasing the concentration thermal resistance characteristics of this bacterium.
of chlorine dioxide and extending the application time More studies are required to be carried out especially
result in significant increases in the inactivation of on the determination of thermal resistance characteris-
A. acidoterrestris spores. tics of various forms of this bacterium under various
The preservative (sorbic acid, benzoic acid or both) media conditions. Accordingly, the aim of this study is
use in fruit juices is reported to have inhibitive effects on to determine the thermal resistance characteristics of
A. acidoterrestris vegetative cells and spores (Pettipher & A. acidoterrestris spores under different pH, total
Osmundson, 2000). Nisin addition is determined to soluble solids (SS) and temperature values in real food
reduce thermal resistances of A. acidoterrestris spores in media such as apple juice and orange juice as well as
fruit juices and thus being effective on the inhibition of malt extract broth (MEB) model media.
germination and growth of these bacterial spores
(Yamazaki et al., 2000). Bevilacqua et al. (2008)
Materials and methods
searched the antimicrobial effect of sodium benzoate
and some natural compounds like cinnamaldehyde,
Material
eugenol and limonene against outgrowth of A. acidotr-
restris spores (c8 and c4). These researchers determined Alicyclobacillus acidoterrestris strain DSM2498 (Ger-
that limonene compound had no effect on the inhibition man Collection of Micro-organisms and Cell Cultures,
of the relevant spores. However, decreases in between Braunschweig, Germany) spores were used in the study.
57% and 70% at spore outgrowth was observed for The apple juice concentrate and orange juice concentrate
samples stored 13 days at 44 C added with 100 ppm (SS: 69Brix) were obtained from KONFRUT Fruit
cinnamaldehyde or sodium benzoate, whereas com- Juice Factory in Denizli ⁄ Turkey. Malt extract broth
pletely hindered spore outgrowth was established in the (MEB; Merck, Darmstadt, Germany) as the model
presence of 500 ppm eugenol. Separately, it was con- medium and BAT agar (Merck, Darmstadt, Germany)
cluded that cinnamaldehyde was the efficient compound as the counting and sporulation medium were used.
at inhibition of outgrowth, and strain c8 was more
resistant compared with the other strain c4. Similar
Preparation of the spore suspension
researchers, in their other recent study (Bevilacqua
et al., 2009) determined the critical cinnamaldehyde The original A. acidoterrestris in slant agar was taken
concentration for outgrowth inhibition of A. acidoter- from its culture with the help of a sterile loop and put in
restris spores during the 3 and 10 days of storage at the tube filled with sterile physiological water. BAT agar
2009 The Authors. Journal compilation 2009 Institute of Food Science and Technology International Journal of Food Science and Technology 2009
1772 Thermal resistance of A. acidoterrestris spores G. Ceviz et al.
International Journal of Food Science and Technology 2009 2009 The Authors. Journal compilation 2009 Institute of Food Science and Technology
Thermal resistance of A. acidoterrestris spores G. Ceviz et al. 1773
D and z-values
D-value is the thermal processing duration needed for
inhibiting 90% of micro-organisms in the media under
constant temperature. On the other hand, z-value is
defined as the temperature value which needs to be
increased to provide a decimal reduction (90%) in the
D-value (Sahbaz et al., 1996). In order to determine these
values, first of all, the number of bacteria (N) surviving in
the fixed temperatures of each sample and as a result of
the thermal processing carried out on regular intervals
was determined. The logarithms of the number of
surviving bacteria were graphed against the duration of
thermal processing and thus ‘survivor curves’ were
obtained. D-values were calculated from the slope of
the regression line obtained from the linear portion of the
survival curve. z-values were determined from the
regression lines obtained by plotting log D-values against
the corresponding temperatures (85, 90, 95 and 100 C)
(Unluturk & Turantas, 2003; Ozkan & Cemeroglu, 2005).
2009 The Authors. Journal compilation 2009 Institute of Food Science and Technology International Journal of Food Science and Technology 2009
1774 Thermal resistance of A. acidoterrestris spores G. Ceviz et al.
Table 2 D and z-values of Alicyclobacillus acidoterrestris strain DSM2498 spores heated in apple juices, orange juices and MEB of different pH and
SS values
D-values (min)*
Apple juice 10 3.5 117.6 ± 9.48a 32.8 ± 1.84b 20.8 ± 1.98b 4.1 ± 0.64c 10.9 ± 0.58b
4.0 105.3 ± 5.52a 36.1 ± 4.53b 27.8 ± 1.70b 11.1 ± 2.55c 16.4 ± 0.81a
20 3.5 103.1 ± 6.43a 52.9 ± 3.96b 41.7 ± 6.51bc 6.2 ± 1.13d 13.3 ± 1.25a
4.0 94.3 ± 4.45a 43.7 ± 2.97b 18.5 ± 2.83c 3.8 ± 0.78d 10.9 ± 0.88a
Orange juice 10 3.5 93.5 ± 5.37a 36.5 ± 2.47b 20.8 ± 3.11c 3.3 ± 0.57d 10.9 ± 0.44a
4.0 120.5 ± 11.46a 30.9 ± 3.61b 20.8 ± 1.27bcd 2.3 ± 0.14d 9.4 ± 0.32a
20 3.5 88.5 ± 8.06a 35.3 ± 3.11b 16.7 ± 2.97c 4.0 ± 0.35dc 11.4 ± 0.30a
4.0 123.5 ± 5.09a 36.1 ± 2.69b 16.7 ± 1.98c 2.7 ± 0.28d 9.4 ± 0.18a
MEB 10 3.5 82.6 ± 5.52a 20.7 ± 2.97b 10.4 ± 2.12bcd 5.2 ± 0.64d 12.8 ± 0.74a
4.0 104.2 ± 3.82a 24.9 ± 1.98b 11.1 ± 2.26c 2.7 ± 0.42d 9.8 ± 0.37a
20 3.5 73.5 ± 4.31a 33.0 ± 2.76b 13.9 ± 0.57c 3.1 ± 0.42d 11.1 ± 0.39a
4.0 107.5 ± 9.48a 30.4 ± 3.18b 23.8 ± 1.70b 2.9 ± 0.21d 10.7 ± 0.13a
values contribute to the increase of thermal resistances. model and fruit juice media, however pH is not effective
As we exceed SS = 20Brix in our study, we do not at 97 C. Similar results were achieved in the study
observe any difference stemming from SS change in carried out by Silva et al. (1999). Researchers put
thermal resistance; and this fact do not contradict to the forward that the increase in pH under model 85 C
literature. increase D-value of A. acidoterrestris (strain, NCIMB
One of the factors having an effect on the thermal 13137) spores, too, in MEB media while D-value is not
resistances of micro-organisms is the pH value of the affected by the increase of pH at 97 C. Similarly, Palop
media. Although there is a general acceptance in et al. (2000) explained that there is no specific effect of
literature that the thermal resistance of micro-organisms pH on the thermal resistance of Alicyclobacillus acido-
decreases as long as they become distanced from neutral caldarius in Mcllvaine citrate–phosphate buffer with pH
pH (Unluturk & Turantas, 2003), no statistical differ- value of 4.0 and 7.0.
ence has been found between D-values obtained for 3.5 Temperature has been found to be the most important
and 4.0 pH in apple juice and orange juice media factor having effects on D-value, which is the main
(P > 0.05). Nevertheless, it has been reported that indicator of the thermal resistance of A. acidoterrestris
D-values obtained for 3.5 and 4 pH and SS = 20Brix spores among SS, pH and temperature values. D-values
under 95 C in MEB media are statistically significantly have been observed to decrease specifically depending on
different (P < 0.05) while D-values for 3.5 and 4 pH the increase of temperature and these values have been
under different temperatures are not statistically differ- found significantly different from each other (P < 0.05).
ent from each other (P > 0.05). The results of the study The decrease in D-value depending on the increase in the
conducted by Murakami et al. (1998) on the effect of temperature is shown in Fig. 2a–c more clearly. Whereas
thermal processing temperature and pH on the thermal D-values show a remarkable decrease in parallel with the
resistance of A. acidoterrestris AB-1 spores support the increase in the temperature, this decrease occurs in greater
results of our study. These researchers determined that rates in 85 C–90 C temperature interval. The decrease
the effect of pH, in general terms, on the thermal in D-values has determined to decrease proportionally in
resistance of A. acidoterrestris AB-1 spores is not 90–100 C interval to 85–90 C interval. In the study
statistically significant (P > 0.05); however they found carried out by Nakauma et al. (2004) on the thermal
that D-values obtained for 5.0 pH and 8.0 pH under resistance of A. acidoterrestris spores, the decrease in the
only 92 C are significantly different from each other number of micro-organisms depending on the tempera-
(P > 0.05) as a result of the study they conducted in ture was examined in two phases. D and z-values were
Mcllvaine buffer, in 3–8 pH interval for six different pH calculated separately for each phase depending on these
values and different temperatures (88, 90, 92 and 95 C). two phases. It was reported in the first logarithmic phase
Pontius et al. (1998) reported that pH at 91 C is that there was a rapid decrease in the number of micro-
effective on the D-values of A. acidoterrestris spores in organisms while in the second phase the resistance of
International Journal of Food Science and Technology 2009 2009 The Authors. Journal compilation 2009 Institute of Food Science and Technology
Thermal resistance of A. acidoterrestris spores G. Ceviz et al. 1775
2009 The Authors. Journal compilation 2009 Institute of Food Science and Technology International Journal of Food Science and Technology 2009
1776 Thermal resistance of A. acidoterrestris spores G. Ceviz et al.
International Journal of Food Science and Technology 2009 2009 The Authors. Journal compilation 2009 Institute of Food Science and Technology
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