International Journal of Food Microbiology 172 (2014) 1–4
Contents lists available at ScienceDirect
International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Short communication
Efficacy of ozone against Alicyclobacillus acidoterrestris spores in apple juice Emrah Torlak ⁎ Necmettin Erbakan University, Faculty of Science, Department of Molecular Biology and Genetics, 42090 Konya, Turkey
a r t i c l e
i n f o
Article history: Received 31 July 2013 Received in revised form 17 November 2013 Accepted 29 November 2013 Available online 7 December 2013 Keywords: Alicyclobacillus acidoterrestris Apple juice Ozone Total phenolic content
a b s t r a c t Alicyclobacillus acidoterrestris survives during the typical pasteurization process and can cause the spoilage of fruit juices thanks to its spore forming and thermo-acidophilic nature. In recent years, A. acidoterrestris has become a major concern to the fruit juices industry worldwide. This study was undertaken to evaluate ozone for the reducing number of A. acidoterrestris spores in apple juice. Apple juice inoculated with A. acidoterrestris spores was bubbled with continuous stream of two different constant concentrations (2.8 and 5.3 mg/L) of ozone at 4 and 22 °C up to 40 min. Level of A. acidoterrestris spores in juice decreased by 2.2 and 2.8 log after 40 min of ozonation at 4 °C with concentrations of 2.8 and 5.3 mg/L, respectively. Treatments at 22 °C for 40 min with 2.8 and 5.3 mg/L ozone resulted in 1.8 and 2.4 log reductions of spore viability, respectively. At the ozone concentration of 5.3 mg/L, significant (P b 0.05) reductions were observed in total phenolic content of juice at both temperature levels. However, treatments performed at 2.8 mg/L were observed to have no significant (P N 0.05) effect on total phenolic content. The results presented in this study indicate that over the 2 log reduction in the count of A. acidoterrestris spores in apple juice can be achieved by bubbling ozonation at 4 °C without causing a significant decrease in total phenolic content of product. Therefore, it can be suggested that bubbling ozonation is a promising method for the control of A. acidoterrestris in fruit juices. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Alicyclobacillus acidoterrestris, firstly named Bacillus acidoterrestris, is a thermo-acidophilic, non-pathogenic and spore-forming bacterium (Silva and Gibbs, 2001). Members of the genus Alicyclobacillus, characterized by ω-alicyclic fatty acids as the major membrane fatty acid component in their cells walls, are able to grow at low pH (3.0–3.5) and high temperature (50–70 °C) (Bevilacqua et al., 2008). The low pH of acidic foods serves as a natural control measure against spoilage. Therefore, spoilage of fruit juices had previously been attributed primarily to the growth of yeasts, fungi and lactic acid bacteria (Jay et al., 2005). Since the early 1980s, when a large-scale spoilage of aseptically packaged apple juice occurred in Germany, A. acidoterrestris has emerged as a food spoilage organism of major significance to the fruit juice industry (Walker and Phillips, 2005). In a comprehensive survey study conducted in Argentina (Oteiza et al., 2011), the percentages of Alicyclobacillus positive samples for fruit and vegetable juices were ranged from 24% to 100%. A survey conducted by the European Fruit Juice Association showed that 45% of participants from the fruit processing industry experienced Alicyclobacillus related problems in the three years prior to the survey (Steyn et al., 2011). A. acidoterrestris was reported as most frequently identified species among the Alicyclobacillus
⁎ Tel.: +90 544 544 89 98; fax: +90 332 236 21 41. E-mail address: [email protected]. 0168-1605/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijfoodmicro.2013.11.035
strains isolated from fruit juices and fruit juice processing environments (Durak et al., 2010). The spoilage caused by A. acidoterrestris is difficult to detect visually and mainly characterized by medicinal or disinfectant off-odors due to the generation of compounds like 2-methoxyphenol (guaiacol), 2,6-dibromophenol and 2,6-dichlorophenol. The detectable concentration of guaiacol in fruit juices by smell, reported as 2 ppb, (Gocmen et al., 2005) can be produced by about 5 log CFU/mL of A. acidoterrestris cells (Pettipher et al., 1997). Thermal pasteurization is currently considered to be the most common method for processing and preserving of fruit juices (Sokolowska et al., 2013). Pasteurization process of fruit juices typically involves heating the product to 90–95 °C for 15–20 s, followed by package filling while the product cools to 82–84 °C (Solberg et al., 1990). The D95 values of A. acidoterrestris spores in different fruit juices were reported to range from 1.00 to 9.98 min (Smit et al., 2011). These values indicate that the spores of A. acidoterrestris may survive in fruit juices after pasteurization treatment commonly applied in the food industry (Bahçeci and Acar, 2007). Therefore several non-thermal processing alternatives have been proposed in the literature to inhibit spore germination and growth of A. acidoterrestris in fruit juices while simultaneously maintaining nutritional quality (Bevilacqua et al., 2008; Steyn et al., 2011). Interest in potential food applications of ozone has expanded in recent years in response to consumer demands for green technologies (Torlak et al., 2013). Ozone is a powerful antimicrobial substance due to its potential oxidizing capacity (Guzel-Seydim et al., 2004).
2
E. Torlak / International Journal of Food Microbiology 172 (2014) 1–4
Moreover, decomposition of ozone to oxygen and lack of toxic residues make it a favorable environment-friendly sanitizer (Achen and Yousef, 2001). Gaseous ozone was recognized as Generally Recognised as Safe (GRAS) by the Food and Drug Administration for direct application on food products. Also, gaseous ozone used in food processing is recognized as allowable by organic certification and regulatory bodies (Selma et al., 2008). The affirmation as GRAS triggered broad usage of ozone in the food industry (Patil and Bourke, 2011). Antimicrobial action of ozone treatment against a wide range of microorganisms in fruit processing has been reported in several studies. These studies demonstrated that over the 5 log reduction of pathogenic microorganisms could be achieved using ozone in fruit juices (Cullen et al., 2010; Patil and Bourke, 2011). However, the efficacy of ozone for the reduction of A. acidoterrestris spores in fruit juices has not been reported. Thus, the main objective of this study was to determine the efficacy of ozone for the reduction of A. acidoterrestris spores in apple juice. Apple juice is an excellent source of several phenolic compounds such as chlorogenic acid, quercetin glycosides and procyanidins (Kahle et al., 2005; Torres et al., 2011). Ozone would be expected to cause the loss of antioxidant bioactive compounds such as phenolics, because of its strong oxidizing activity (Rawson et al., 2011). Therefore, effect of ozone on the total phenolic content in apple juice was also evaluated.
buffered potassium iodide (KI) solution. When the bubbling was stopped, pH of KI solution was adjusted with sulfuric acid (4.5 M) to pH 2, in order to complete the reaction. Immediately after, the liberated iodine was titrated to a starch endpoint with freshly standardized sodium thiosulfate solution (0.1 M). Ozone concentration was calculated based on ozone/iodine stoichiometry of 1. Ozonation was applied to inoculated juice aliquots under continuous stream of two different constant concentrations of ozone in a 250 mL gas washing bottle equipped with a diffuser. Bubbling ozonation was performed at 4 and 22 °C for four exposure times (10, 20, 30 and 40 min). Prior to the treatments, gas washing bottles containing inoculated juice were placed into a refrigerated water bath (Nüve, Ankara, Turkey) which was set to the respective treatment temperature. During the treatments, temperature of water was checked with K-type thermocouple connected to digital thermometer (Fluke, Everett, WA, USA).
2.4. Enumeration of A. acidoterrestris
2. Materials and methods
Enumeration was performed by the plate count technique on YGS agar. Totally 1 mL of apple juice and additional ten-fold dilutions were surface plated on three YGS agar plates. Inoculated plates were incubated at 45 °C for 5 days. After incubation colonies grown on YGS agar plates were counted and A. acidoterrestris counts were calculated as log CFU/mL.
2.1. Microorganism and preparation of spore suspension
2.5. Determination of total phenolic content
Freeze dried culture of A. acidoterrestris (ATCC 49025) was supplied from Microbiologics Inc. (Saint Cloud, MN, USA). For sporulation, a fresh culture of A. acidoterrestris in B. acidoterrestris (BAT) broth (pH 4, Himedia, Mumbai, India) was spread-plated onto yeast glucose starch (YGS) agar (pH 4, Himedia) and incubated at 45 °C for 5 days. Sporulation was confirmed by microscopy following staining with malachite green. After reaching more than 80% of sporulation, spores were removed by a mild agitation of plates using a glass spreader after adding 5 ml of distilled water. The pool of spores collected from different plates was centrifuged at 3600 g for 10 min at 5 °C (Hettich, Tuttlingen, Germany) and washed three times with sterile distilled water. The final cell pellet was resuspended in sterile distilled water. The concentration of spores in the final suspension, determined by plating 0.1 mL of appropriate dilutions onto YGS agar, was adjusted to 107 spores/mL with sterile distilled water. The suspension was stored at 4 °C until used and heated at 80 °C for 10 min to activate spores before inoculation into apple juice.
Colorimetric assay based on the procedure of Singleton and Rossi (1965) was used to quantitate the total phenolic content. This assay involves the reduction of Folin–Ciocalteu reagent by phenolic compounds, with a concomitant formation of a blue complex. Briefly, 0.1 mL of the juice sample was mixed with 0.9 mL of distilled water and 5 mL of 0.2 N Folin–Ciocalteu reagent (Sigma-Aldrich, Milwaukee, WI, USA). After 3 min, 4 mL of saturated sodium carbonate solution (75 mg/mL) was added to the mixture and then shaken. After 2 h, the absorbance of blue coloration was measured at 765 nm against a blank sample using a spectrophotometer (Thermo Scientific, Waltham, MA, USA). The measurements were compared to a calibration curve constructed with solutions of gallic acid (Sigma-Aldrich). The results were expressed in mg gallic acid equivalent/L (mg GAE/L).
The commercial pasteurized apple juice sample (11.8° Brix, pH 3.8), kindly provided by the Enka Food Company (Konya, Turkey), was determined as Alicyclobacillus-negative by the enrichment method (IFU, 2007). Aliquots (200 mL) of apple juice were transferred into sterile stomacher bags and inoculated with 1 mL of spore suspension. Inoculation level of juice aliquots was determined immediately after inoculation as described in the Section 2.5. 2.3. Ozone treatment Ozone was produced directly from atmospheric oxygen by two ozone generators (Opal, Ankara, Turkey) with different ozone output levels. Ozone flow rate was adjusted to 0.4 L/min using a flow regulator (Dywer Instruments, Michigan City, IN, USA). The ozone concentrations in the air flow produced by the generators were determined as 2.8 and 5.3 mg/L by the iodometric titration method (IOA, 1996) using a washing bottle equipped with a diffuser. Iodometric method was carried out by bubbling of ozone in 200 mL
Three independent trials were conducted. Results were analyzed by one-way analysis of variance (ANOVA) using statistical software (SPSS Inc., Chicago, IL, USA). Mean values were compared using the Duncan grouping test at P b 0.05.
Treatment time (min) 0.0
0
10
20
30
40
-0.5
log (N/N0)
2.2. Inoculation of apple juice
2.6. Statistical analyses
-1.0 -1.5 -2.0 -2.5 -3.0
Fig. 1. Reduction of A. acidoterrestris spores in apple juice during ozonation at 4 °C (N: number of surviving spores, CFU/mL; N0: initial inoculation level, CFU/mL): -▲2.8 mg/L, - ■ - 5.3 mg/L.
E. Torlak / International Journal of Food Microbiology 172 (2014) 1–4
Treatment time (min) 0.0
0
10
20
30
40
log (N/N0)
-0.5 -1.0 -1.5 -2.0 -2.5 -3.0 Fig. 2. Reduction of A. acidoterrestris spores in apple juice during ozonation at 22 °C (N: number of surviving spores, CFU/mL; N0: initial inoculation level, CFU/mL): -▲2.8 mg/L, - ■ - 5.3 mg/L.
3. Results and discussion The reductions in the level of A. acidoterrestris spores in ozonated apple juice at two different constant concentrations (2.8 and 5.3 mg/L) during 40 min of treatment at 4 and 22 °C are shown in Figs. 1 and 2, respectively. Statistically significant (P b 0.05) reductions in the number of spores were obtained after 10 min of treatment with both ozone concentrations at 4 °C, whereas treatments for 10 min at 22 °C did not yield a significant reduction in spore counts. The initial level of A. acidoterrestris in juice, determined as 4.9 log CFU/mL, decreased by 1.8 and 2.4 log during 40 min of treatment at 22 °C with ozone concentrations of 2.8 and 5.3 mg/L, respectively. Treatments at 4 °C with ozone concentrations of 2.8 and 5.3 mg/L for 40 min resulted in 2.2 and 2.8 log reductions of spore viability, respectively. In accordance with results of present study, several studies reported that substantial microbial reductions in fruit juices treated with bubbling ozone. In study of Patil et al. (2009), orange juice samples inoculated with Escherichia coli were treated with ozone at concentration of 75 mg/L. This treatment reduced the E. coli population by 5 log between 1 and 18 min, depending on the pulp content of the samples. In another study (Patil et al., 2010), a 5 log reduction in the counts of E. coli in low pH apple juice was achieved within 4 min in bubble column by ozonation performed at concentration of 48 mg/L. Williams et al. (2004) reported that 15 min of ozone treatment (0.9 g ozone/h) at 50 °C caused greater than a 6 log reduction in the counts of Salmonella in apple cider. The bactericidal effects of ozone have been documented on a wide variety of microorganisms, including spores. Ozone treatments have been successfully utilized for inactivation of bacterial spores in aqueous solutions and various food matrices (Guzel-Seydim et al., 2004). Khadre and Yousef (2001) compared the sporicidal action of ozone and hydrogen peroxide against spores of eight Bacillus species in spore suspension. Results demonstrated that 10% (v/v) concentration of hydrogen peroxide was less effective than ozone at 11 mg/L. Sporicidal activity of ozone can be explained by degradation of outer spore components and inner membrane damages. An alternative to ozone killing spores by damaging the spore coat and inner membrane is that ozone may enter the spore core and inactivate one or more critical core enzymes (Young and Setlow, 2003, 2004). In addition to intrinsic factors such as organic matter content and pH, effectiveness of ozone against microorganisms present in liquid foods depends on extrinsic factors, primarily temperature (Patil et al., 2009; Tiwari and Muthukumarappan, 2012). Enumeration results obtained after 40 min of ozonation indicated that reductions of A. acidoterrestris spores in apple juice at 4 °C were considerably higher compared to those at 22 °C. The effect of temperature on antibacterial effectiveness of ozone in liquids is mainly attributed to the fact that the solubility and decomposition rate of ozone changes substantially with changes in temperature (Tiwari and Muthukumarappan, 2012). As temperature decreases ozone becomes more soluble and more stable
3
with a decrease in the decomposition rate (Rice et al., 1981). Therefore, a fall in the temperature increases availability of ozone in the aqueous medium and, consequently, its efficacy (Pascual et al., 2007). Zuma et al. (2009) suggested that the free radicals formed by the decomposition of ozone are less effective for microbial inactivation than molecular ozone. Williams et al. (2004) evaluated the inactivation of E. coli O157: H7 and Salmonella in apple cider and orange juice treated with ozone (0.9 g/h) at various temperatures. They found that reductions in the levels of pathogens at 4 °C were considerably higher than those at 22 °C. However this finding is apparently in contrast with another study (Steenstrup and Floros, 2004) that reported that D values of E. coli O157:H7 in ozonated apple cider tended to increase with decreasing temperature. These controversial results indicate that ozone efficacy can vary with experimental conditions, making it difficult to predict the influence of temperature on a particular application (Pascual et al., 2007). It has been well documented that inactivation of microorganisms by ozone in the aqueous medium is much faster at low pH than at higher pH values (Zuma et al., 2009). Therefore, acidic environment of apple juice can be considered suitable matrix for ozonation in terms of antibacterial effectiveness. Patil et al. (2010) investigated the effect of pH on the inactivation rate of E. coli in apple juice. They demonstrated that ozone inactivation of E. coli was much faster at low pH than at higher pH values. The effect of pH on antibacterial effectiveness of ozone, similar to the temperature, is mainly attributed to the fact that the ozone decomposition occurs faster in higher-pH aqueous solutions (EPA, 1999; Patil et al., 2010). The health benefits of apple juice are primarily related to its phenolic content (Kahle et al., 2005). Changes in the total phenolic content of apple juice treated with bubbling ozone up to 40 min at two different concentrations and temperatures are given in Table 1. At the ozone concentration of 5.3 mg/L, initial total phenolic content of apple juice, determined as 571 mg GAE/L, decreased significantly (P b 0.05) to 375 and 358 mg GAE/L after 40 min of treatment at 4 and 22 °C, respectively. However, the reductions in the level of total phenolic content observed at the ozone concentration of 2.8 mg/L were not significant (P N 0.05). It was reported that phenolic compounds in fruit juices are prone to oxidation during the ozonation due to the strong oxidation potential of ozone (Karaca and Velioglu, 2007). Torres et al. (2011) demonstrated that bubbling ozonation at concentration of 69 mg/L (4.8% w/w of oxygen) for 10 min caused significant reductions in the levels of major phenolic compounds and total phenolic content of apple juice. They also reported that color values and rheological properties of apple juice were negatively affected by the increase in processing time and concentration of ozone. Total phenol content of ozonated apple juice did not significantly (P N 0.05) influence by processing temperature. Degradation of phenolic compounds in apple juice during the ozonation can be attributed to direct reactions of molecular ozone and reactions with its free radicals. Ozone attacks OH radicals, preferentially to the double bonds in organic compounds (Tiwari et al., 2009). Free radicals may lead to electrophilic and nucleophilic reactions occurring with aromatic compounds (Cullen et al., 2009). In food industry an agent, providing minimum 2 log in microbial reduction, is accepted to be antimicrobial (Tiwari and Rice, 2012). Results of present study demonstrated that counts of A. acidoterrestris spores in apple juice can be reduced more than 2 log by bubbling ozone with an acceptable change in total phenolic content. Accordingly, these results led us to conclude that ozonation is a promising application for preventing the spoilage caused by A. acidoterrestris in apple juice. A minimum 5 log reduction of spoilage and potentially pathogenic bacteria most commonly associated with fruit juices is considered as a primary performance standard for non-thermal processing methods by FDA (Tiwari and Muthukumarappan, 2012). Reduction values obtained in apple juice after 40 min of ozonation did not meet the FDA's requirement of a 5 log reduction. Nevertheless, the efficacy of ozone
4
E. Torlak / International Journal of Food Microbiology 172 (2014) 1–4
Table 1 Effect of bubbling ozone on the total phenolic content (±standard deviations, mg GAE/L) in apple juice. Treatment temperature
Ozone concentration (mg/L)
Treatment time (min) 0
4 °C 22 °C
2.8 5.3 2.8 5.3
571 571 571 571
10 ± ± ± ±
a
62 62a 62a 62a
557 550 544 527
20 ± ± ± ±
a
54 70a 51a 37a
524 502 492 450
30 ± ± ± ±
a
64 47a 32a 59ab
490 439 464 417
40 ± ± ± ±
a
73 71ab 80a 42b
458 375 429 358
± ± ± ±
68a 75b 83a 87b
ab
Values followed by the same letter in the same row are not significantly different (P N 0.05).
against A. acidoterrestris spores can be increased by increasing concentration and exposure time. However, results of present study also showed that total phenolic content of ozonated apple juice may be significantly influenced by ozone concentration and by processing time. Therefore, treatment of apple juice with gaseous ozone can be suggested as a complementary microbial reduction method for the control of A. acidoterrestris spores. Further validation studies are still needed to determine the critical limits (e.g., ozone concentration and time parameters) for effective treatment in terms of sporicidal activity and bioactive compounds of product. Acknowledgment The author would like to thank Mr. Mehmet Zeki Öçlü, Director of Kemiteks Chemical Company, for the supply of microbiological media and chemicals used. References Achen, M., Yousef, A.E., 2001. Efficacy of ozone against Escherichia coli O157:H7 on apples. J. Food Saf. 66, 1380–1384. Bahçeci, K.S., Acar, J., 2007. Modeling the combined effects of pH, temperature and ascorbic acid concentration on the heat resistance of Alicyclobacillus acidoterrestis. Int. J. Food Microbiol. 120, 266–273. Bevilacqua, A., Sinigaglia, M., Corbo, M.R., 2008. Alicyclobacillus acidoterrestris: new methods for inhibiting spore germination. Int. J. Food Microbiol. 125, 103–110. Cullen, P.J., Tiwari, B.K., O'Donnell, C.P., Muthukumarappan, K., 2009. Modeling approaches to ozone processing of liquid foods. Trends Food Sci. Technol. 20, 125–136. Cullen, P.J., Valdramidis, V.P., Tiwari, B.K., Patil, S., Bourke, P., O'Donnell, C.P., 2010. Ozone processing for food preservation: an overview on fruit juice treatment. Ozone Sci. Eng. J. Int. Ozone Assoc. 32, 166–179. Durak, M.Z., Churey, J.J., Danyluk, M.D., Worobo, R.W., 2010. Identification and haplotype distribution of Alicyclobacillus spp. from different juices and beverages. Int. J. Food Microbiol. 142, 286–291. EPA, Environmental Protection, 1999. Agency guidance manual alternative disinfectants and oxidants. EPA 815-R-99-014. (Washington DC). Gocmen, D., Elston, A., Williams, T., Parish, M., Rouseff, R.L., 2005. Identification of medicinal off-flavours generated by Alicyclobacillus species in orange juice using GC-olfactometry and GC–MS. Lett. Appl. Microbiol. 40, 172–177. Guzel-Seydim, Z.B., Greene, A.K., Seydim, A.C., 2004. Use of ozone in the food industry. Lebensm. Wiss. Technol. 37, 453–460. IFU, International Federation of Fruit Juice Producers, 2007. Method on the detection of taint producing Alicyclobacillus in fruit juices. IFU Method No. 12. (Paris). IOA, International Ozone Association, 1996. Quality Assurance Committee Revised Standardized Procedure 001/96 (Scottsdale). Jay, J.M., Loessner, M.J., Golden, D.A., 2005. Modern Food Microbiology, Seventh ed. Springer Science and Business Media, New York. Kahle, K., Kraus, M., Richling, E., 2005. Polyphenol profiles of apple juices. Mol. Nutr. Food Res. 49, 797–806. Karaca, H., Velioglu, Y.S., 2007. Ozone applications in fruit and vegetable processing. Food Rev. Int. 23, 91–106. Khadre, M.A., Yousef, A.E., 2001. Sporicidal action of ozone and hydrogen peroxide: a comparative study. Int. J. Food Microbiol. 71, 131–138. Oteiza, J.M., Ares, G., Sant'Ana, A.S., Soto, S., Giannuzzi, L., 2011. Use of a multivariate approach to assess the incidence of Alicyclobacillus spp. in concentrate fruit juices marketed in Argentina: results of a 14-year survey. Int. J. Food Microbiol. 151, 229–234.
Pascual, A., Llorca, I., Canut, A., 2007. Use of ozone in food industries for reducing the environmental impact of cleaning and disinfection activities. Trends Food Sci. Technol. 18, S29–S35. Patil, S., Bourke, P., 2011. Ozone processing of fluid foods. In: Cullen, B.J., Tiwari, B., Valdramidis, V. (Eds.), Novel Thermal and Non-Thermal Technologies for Fluid Foods. Academic Press, London. Patil, S., Bourke, P., Frias, J.M., Tiwari, B.K., Cullen, P.J., 2009. Inactivation of Escherichia coli in orange juice using ozone. Innov. Food Sci. Emerg. Technol. 10, 551–557. Patil, S., Valdramidis, V.P., Cullen, P.J., Frias, J., Bourke, P., 2010. Inactivation of Escherichia coli by ozone treatment of apple juice at different pH levels. Food Microbiol. 27, 835–840. Pettipher, G.L., Osmundson, M.E., Murphy, J.M., 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. Rawson, A., Patras, A., Tiwari, B.K., Noci, F., Koutchma, T., Brunton, N., 2011. Effect of thermal and non thermal processing technologies on the bioactive content of exotic fruits and their products: review of recent advances. Food Res. Int. 44, 1875–1887. Rice, R.G., Robson, C.M., Miller, G.W., Hill, A.G., 1981. Uses of ozone in drinking-water treatment. J. Am. Water Works Assoc. 73, 44–57. Selma, M.V., Ibanez, A.M., Cantwell, M., Suslow, T., 2008. Reduction by gaseous ozone of Salmonella and microbial flora associated with fresh-cut cantaloupe. Food Microbiol. 25, 558–565. Silva, F.V.M., Gibbs, P., 2001. Alicyclobacillus acidoterrestris spores in fruit products and design of pasteurization processes. Trends Food Sci. Technol. 12, 68–74. Singleton, V., Rossi, J., 1965. Colorimetry of total phenolics with phosphomolybdic phosphotungstic acid reagents. Am. J. Enol. Vitic. 16, 144–158. Smit, Y., Cameron, M., Venter, P., Witthuhn, R.C., 2011. Alicyclobacillus spoilage and isolation—a review. Food Microbiol. 28, 331–349. Sokolowska, B., Skąpska, S., Fonberg-Broczek, M., Niezgoda, J., Chotkiewicz, M., Dekowska, A., Rzoska, S.J., 2013. Factors influencing the inactivation of Alicyclobacillus acidoterrestris spores exposed to high hydrostatic pressure in apple juice. High Press. Res. Int. J. 331, 73–82. Solberg, P., Castberg, H.B., Osmundsen, J.I., 1990. Packaging systems for fruit juices and non-carbonated beverages, In: Hicks, D. (Ed.), Production and Packaging of Nonacarbonated Fruit Juices and Fruit Beverages, First ed. Blackie and Son Ltd., London. Steenstrup, L.D., Floros, J.D., 2004. Inactivation of E. coli 0157:H7 in apple cider by ozone at various temperatures and concentrations. J. Food Process. Preserv. 28, 103–116. Steyn, C.E., Cameron, M., Witthuhn, R.C., 2011. Occurrence of Alicyclobacillus in the fruit processing environment—a review. Int. J. Food Microbiol. 147, 1–11. Tiwari, B.K., Muthukumarappan, K., 2012. Ozone in fruit and vegetable processing. In: O'Donnell, C., Tiwari, B.K., Cullen, P.J., Rice, C.R.G. (Eds.), Ozone in Food Processing. A John Wiley & Sons Ltd., Oxford. Tiwari, B.K., Rice, C.R.G., 2012. Regulatory and legislative issues. In: O'Donnell, C., Tiwari, B.K., Cullen, P.J., Rice, C.R.G. (Eds.), Ozone in Food Processing. A John Wiley & Sons Ltd., Oxford. Tiwari, B.K., O'Donnell, C.P., Patras, A., Brunton, N., Cullen, P.J., 2009. Anthocyanins and color degradation in ozonated grape juice. Food Chem. Toxicol. 47, 2824–2829. Torlak, E., Durmuş, S., Ulca, P., 2013. Efficacy of gaseous ozone against Salmonella and microbial population on dried oregano. Int. J. Food Microbiol. 165, 276–280. Torres, B., Tiwari, B.K., Patras, A., Wijngaard, H.H., Brunton, N., Cullen, P.J., O'Donnell, C.P., 2011. Effect of ozone processing on the colour, rheological properties and phenolic content of apple juice. Food Chem. 124, 721–726. Walker, M., Phillips, C.A., 2005. The effect of intermittent shaking, headspace and temperature on the growth of Alicyclobacillus acidoterrestris in stored apple juice. Int. J. Food Sci. Technol. 40, 557–562. Williams, R.C., Sumner, S.S., Golden, D.A., 2004. Survival of Escherichia coli O157:H7 and Salmonella in apple cider and orange juice as affected by ozone and treatment temperature. J. Food Prot. 67, 2381–2386. Young, S.B., Setlow, P., 2003. Mechanisms of killing of Bacillus subtilis spores by hypochlorite and chlorine dioxide. J. Appl. Microbiol. 95, 54–67. Young, S.B., Setlow, P., 2004. Mechanisms of Bacillus subtilis spore resistance to and killing by aqueous ozone. J. Appl. Microbiol. 96, 1133–1142. Zuma, F., Lin, J., Jonnalagadda, S.B., 2009. Ozone-initiated disinfection kinetics of Escherichia coli in water. J. Environ. Sci. Health A Tox./Hazard. Subst. Environ. Eng. 44, 48–56.
Contents lists available at ScienceDirect
International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Short communication
Efficacy of ozone against Alicyclobacillus acidoterrestris spores in apple juice Emrah Torlak ⁎ Necmettin Erbakan University, Faculty of Science, Department of Molecular Biology and Genetics, 42090 Konya, Turkey
a r t i c l e
i n f o
Article history: Received 31 July 2013 Received in revised form 17 November 2013 Accepted 29 November 2013 Available online 7 December 2013 Keywords: Alicyclobacillus acidoterrestris Apple juice Ozone Total phenolic content
a b s t r a c t Alicyclobacillus acidoterrestris survives during the typical pasteurization process and can cause the spoilage of fruit juices thanks to its spore forming and thermo-acidophilic nature. In recent years, A. acidoterrestris has become a major concern to the fruit juices industry worldwide. This study was undertaken to evaluate ozone for the reducing number of A. acidoterrestris spores in apple juice. Apple juice inoculated with A. acidoterrestris spores was bubbled with continuous stream of two different constant concentrations (2.8 and 5.3 mg/L) of ozone at 4 and 22 °C up to 40 min. Level of A. acidoterrestris spores in juice decreased by 2.2 and 2.8 log after 40 min of ozonation at 4 °C with concentrations of 2.8 and 5.3 mg/L, respectively. Treatments at 22 °C for 40 min with 2.8 and 5.3 mg/L ozone resulted in 1.8 and 2.4 log reductions of spore viability, respectively. At the ozone concentration of 5.3 mg/L, significant (P b 0.05) reductions were observed in total phenolic content of juice at both temperature levels. However, treatments performed at 2.8 mg/L were observed to have no significant (P N 0.05) effect on total phenolic content. The results presented in this study indicate that over the 2 log reduction in the count of A. acidoterrestris spores in apple juice can be achieved by bubbling ozonation at 4 °C without causing a significant decrease in total phenolic content of product. Therefore, it can be suggested that bubbling ozonation is a promising method for the control of A. acidoterrestris in fruit juices. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Alicyclobacillus acidoterrestris, firstly named Bacillus acidoterrestris, is a thermo-acidophilic, non-pathogenic and spore-forming bacterium (Silva and Gibbs, 2001). Members of the genus Alicyclobacillus, characterized by ω-alicyclic fatty acids as the major membrane fatty acid component in their cells walls, are able to grow at low pH (3.0–3.5) and high temperature (50–70 °C) (Bevilacqua et al., 2008). The low pH of acidic foods serves as a natural control measure against spoilage. Therefore, spoilage of fruit juices had previously been attributed primarily to the growth of yeasts, fungi and lactic acid bacteria (Jay et al., 2005). Since the early 1980s, when a large-scale spoilage of aseptically packaged apple juice occurred in Germany, A. acidoterrestris has emerged as a food spoilage organism of major significance to the fruit juice industry (Walker and Phillips, 2005). In a comprehensive survey study conducted in Argentina (Oteiza et al., 2011), the percentages of Alicyclobacillus positive samples for fruit and vegetable juices were ranged from 24% to 100%. A survey conducted by the European Fruit Juice Association showed that 45% of participants from the fruit processing industry experienced Alicyclobacillus related problems in the three years prior to the survey (Steyn et al., 2011). A. acidoterrestris was reported as most frequently identified species among the Alicyclobacillus
⁎ Tel.: +90 544 544 89 98; fax: +90 332 236 21 41. E-mail address: [email protected]. 0168-1605/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijfoodmicro.2013.11.035
strains isolated from fruit juices and fruit juice processing environments (Durak et al., 2010). The spoilage caused by A. acidoterrestris is difficult to detect visually and mainly characterized by medicinal or disinfectant off-odors due to the generation of compounds like 2-methoxyphenol (guaiacol), 2,6-dibromophenol and 2,6-dichlorophenol. The detectable concentration of guaiacol in fruit juices by smell, reported as 2 ppb, (Gocmen et al., 2005) can be produced by about 5 log CFU/mL of A. acidoterrestris cells (Pettipher et al., 1997). Thermal pasteurization is currently considered to be the most common method for processing and preserving of fruit juices (Sokolowska et al., 2013). Pasteurization process of fruit juices typically involves heating the product to 90–95 °C for 15–20 s, followed by package filling while the product cools to 82–84 °C (Solberg et al., 1990). The D95 values of A. acidoterrestris spores in different fruit juices were reported to range from 1.00 to 9.98 min (Smit et al., 2011). These values indicate that the spores of A. acidoterrestris may survive in fruit juices after pasteurization treatment commonly applied in the food industry (Bahçeci and Acar, 2007). Therefore several non-thermal processing alternatives have been proposed in the literature to inhibit spore germination and growth of A. acidoterrestris in fruit juices while simultaneously maintaining nutritional quality (Bevilacqua et al., 2008; Steyn et al., 2011). Interest in potential food applications of ozone has expanded in recent years in response to consumer demands for green technologies (Torlak et al., 2013). Ozone is a powerful antimicrobial substance due to its potential oxidizing capacity (Guzel-Seydim et al., 2004).
2
E. Torlak / International Journal of Food Microbiology 172 (2014) 1–4
Moreover, decomposition of ozone to oxygen and lack of toxic residues make it a favorable environment-friendly sanitizer (Achen and Yousef, 2001). Gaseous ozone was recognized as Generally Recognised as Safe (GRAS) by the Food and Drug Administration for direct application on food products. Also, gaseous ozone used in food processing is recognized as allowable by organic certification and regulatory bodies (Selma et al., 2008). The affirmation as GRAS triggered broad usage of ozone in the food industry (Patil and Bourke, 2011). Antimicrobial action of ozone treatment against a wide range of microorganisms in fruit processing has been reported in several studies. These studies demonstrated that over the 5 log reduction of pathogenic microorganisms could be achieved using ozone in fruit juices (Cullen et al., 2010; Patil and Bourke, 2011). However, the efficacy of ozone for the reduction of A. acidoterrestris spores in fruit juices has not been reported. Thus, the main objective of this study was to determine the efficacy of ozone for the reduction of A. acidoterrestris spores in apple juice. Apple juice is an excellent source of several phenolic compounds such as chlorogenic acid, quercetin glycosides and procyanidins (Kahle et al., 2005; Torres et al., 2011). Ozone would be expected to cause the loss of antioxidant bioactive compounds such as phenolics, because of its strong oxidizing activity (Rawson et al., 2011). Therefore, effect of ozone on the total phenolic content in apple juice was also evaluated.
buffered potassium iodide (KI) solution. When the bubbling was stopped, pH of KI solution was adjusted with sulfuric acid (4.5 M) to pH 2, in order to complete the reaction. Immediately after, the liberated iodine was titrated to a starch endpoint with freshly standardized sodium thiosulfate solution (0.1 M). Ozone concentration was calculated based on ozone/iodine stoichiometry of 1. Ozonation was applied to inoculated juice aliquots under continuous stream of two different constant concentrations of ozone in a 250 mL gas washing bottle equipped with a diffuser. Bubbling ozonation was performed at 4 and 22 °C for four exposure times (10, 20, 30 and 40 min). Prior to the treatments, gas washing bottles containing inoculated juice were placed into a refrigerated water bath (Nüve, Ankara, Turkey) which was set to the respective treatment temperature. During the treatments, temperature of water was checked with K-type thermocouple connected to digital thermometer (Fluke, Everett, WA, USA).
2.4. Enumeration of A. acidoterrestris
2. Materials and methods
Enumeration was performed by the plate count technique on YGS agar. Totally 1 mL of apple juice and additional ten-fold dilutions were surface plated on three YGS agar plates. Inoculated plates were incubated at 45 °C for 5 days. After incubation colonies grown on YGS agar plates were counted and A. acidoterrestris counts were calculated as log CFU/mL.
2.1. Microorganism and preparation of spore suspension
2.5. Determination of total phenolic content
Freeze dried culture of A. acidoterrestris (ATCC 49025) was supplied from Microbiologics Inc. (Saint Cloud, MN, USA). For sporulation, a fresh culture of A. acidoterrestris in B. acidoterrestris (BAT) broth (pH 4, Himedia, Mumbai, India) was spread-plated onto yeast glucose starch (YGS) agar (pH 4, Himedia) and incubated at 45 °C for 5 days. Sporulation was confirmed by microscopy following staining with malachite green. After reaching more than 80% of sporulation, spores were removed by a mild agitation of plates using a glass spreader after adding 5 ml of distilled water. The pool of spores collected from different plates was centrifuged at 3600 g for 10 min at 5 °C (Hettich, Tuttlingen, Germany) and washed three times with sterile distilled water. The final cell pellet was resuspended in sterile distilled water. The concentration of spores in the final suspension, determined by plating 0.1 mL of appropriate dilutions onto YGS agar, was adjusted to 107 spores/mL with sterile distilled water. The suspension was stored at 4 °C until used and heated at 80 °C for 10 min to activate spores before inoculation into apple juice.
Colorimetric assay based on the procedure of Singleton and Rossi (1965) was used to quantitate the total phenolic content. This assay involves the reduction of Folin–Ciocalteu reagent by phenolic compounds, with a concomitant formation of a blue complex. Briefly, 0.1 mL of the juice sample was mixed with 0.9 mL of distilled water and 5 mL of 0.2 N Folin–Ciocalteu reagent (Sigma-Aldrich, Milwaukee, WI, USA). After 3 min, 4 mL of saturated sodium carbonate solution (75 mg/mL) was added to the mixture and then shaken. After 2 h, the absorbance of blue coloration was measured at 765 nm against a blank sample using a spectrophotometer (Thermo Scientific, Waltham, MA, USA). The measurements were compared to a calibration curve constructed with solutions of gallic acid (Sigma-Aldrich). The results were expressed in mg gallic acid equivalent/L (mg GAE/L).
The commercial pasteurized apple juice sample (11.8° Brix, pH 3.8), kindly provided by the Enka Food Company (Konya, Turkey), was determined as Alicyclobacillus-negative by the enrichment method (IFU, 2007). Aliquots (200 mL) of apple juice were transferred into sterile stomacher bags and inoculated with 1 mL of spore suspension. Inoculation level of juice aliquots was determined immediately after inoculation as described in the Section 2.5. 2.3. Ozone treatment Ozone was produced directly from atmospheric oxygen by two ozone generators (Opal, Ankara, Turkey) with different ozone output levels. Ozone flow rate was adjusted to 0.4 L/min using a flow regulator (Dywer Instruments, Michigan City, IN, USA). The ozone concentrations in the air flow produced by the generators were determined as 2.8 and 5.3 mg/L by the iodometric titration method (IOA, 1996) using a washing bottle equipped with a diffuser. Iodometric method was carried out by bubbling of ozone in 200 mL
Three independent trials were conducted. Results were analyzed by one-way analysis of variance (ANOVA) using statistical software (SPSS Inc., Chicago, IL, USA). Mean values were compared using the Duncan grouping test at P b 0.05.
Treatment time (min) 0.0
0
10
20
30
40
-0.5
log (N/N0)
2.2. Inoculation of apple juice
2.6. Statistical analyses
-1.0 -1.5 -2.0 -2.5 -3.0
Fig. 1. Reduction of A. acidoterrestris spores in apple juice during ozonation at 4 °C (N: number of surviving spores, CFU/mL; N0: initial inoculation level, CFU/mL): -▲2.8 mg/L, - ■ - 5.3 mg/L.
E. Torlak / International Journal of Food Microbiology 172 (2014) 1–4
Treatment time (min) 0.0
0
10
20
30
40
log (N/N0)
-0.5 -1.0 -1.5 -2.0 -2.5 -3.0 Fig. 2. Reduction of A. acidoterrestris spores in apple juice during ozonation at 22 °C (N: number of surviving spores, CFU/mL; N0: initial inoculation level, CFU/mL): -▲2.8 mg/L, - ■ - 5.3 mg/L.
3. Results and discussion The reductions in the level of A. acidoterrestris spores in ozonated apple juice at two different constant concentrations (2.8 and 5.3 mg/L) during 40 min of treatment at 4 and 22 °C are shown in Figs. 1 and 2, respectively. Statistically significant (P b 0.05) reductions in the number of spores were obtained after 10 min of treatment with both ozone concentrations at 4 °C, whereas treatments for 10 min at 22 °C did not yield a significant reduction in spore counts. The initial level of A. acidoterrestris in juice, determined as 4.9 log CFU/mL, decreased by 1.8 and 2.4 log during 40 min of treatment at 22 °C with ozone concentrations of 2.8 and 5.3 mg/L, respectively. Treatments at 4 °C with ozone concentrations of 2.8 and 5.3 mg/L for 40 min resulted in 2.2 and 2.8 log reductions of spore viability, respectively. In accordance with results of present study, several studies reported that substantial microbial reductions in fruit juices treated with bubbling ozone. In study of Patil et al. (2009), orange juice samples inoculated with Escherichia coli were treated with ozone at concentration of 75 mg/L. This treatment reduced the E. coli population by 5 log between 1 and 18 min, depending on the pulp content of the samples. In another study (Patil et al., 2010), a 5 log reduction in the counts of E. coli in low pH apple juice was achieved within 4 min in bubble column by ozonation performed at concentration of 48 mg/L. Williams et al. (2004) reported that 15 min of ozone treatment (0.9 g ozone/h) at 50 °C caused greater than a 6 log reduction in the counts of Salmonella in apple cider. The bactericidal effects of ozone have been documented on a wide variety of microorganisms, including spores. Ozone treatments have been successfully utilized for inactivation of bacterial spores in aqueous solutions and various food matrices (Guzel-Seydim et al., 2004). Khadre and Yousef (2001) compared the sporicidal action of ozone and hydrogen peroxide against spores of eight Bacillus species in spore suspension. Results demonstrated that 10% (v/v) concentration of hydrogen peroxide was less effective than ozone at 11 mg/L. Sporicidal activity of ozone can be explained by degradation of outer spore components and inner membrane damages. An alternative to ozone killing spores by damaging the spore coat and inner membrane is that ozone may enter the spore core and inactivate one or more critical core enzymes (Young and Setlow, 2003, 2004). In addition to intrinsic factors such as organic matter content and pH, effectiveness of ozone against microorganisms present in liquid foods depends on extrinsic factors, primarily temperature (Patil et al., 2009; Tiwari and Muthukumarappan, 2012). Enumeration results obtained after 40 min of ozonation indicated that reductions of A. acidoterrestris spores in apple juice at 4 °C were considerably higher compared to those at 22 °C. The effect of temperature on antibacterial effectiveness of ozone in liquids is mainly attributed to the fact that the solubility and decomposition rate of ozone changes substantially with changes in temperature (Tiwari and Muthukumarappan, 2012). As temperature decreases ozone becomes more soluble and more stable
3
with a decrease in the decomposition rate (Rice et al., 1981). Therefore, a fall in the temperature increases availability of ozone in the aqueous medium and, consequently, its efficacy (Pascual et al., 2007). Zuma et al. (2009) suggested that the free radicals formed by the decomposition of ozone are less effective for microbial inactivation than molecular ozone. Williams et al. (2004) evaluated the inactivation of E. coli O157: H7 and Salmonella in apple cider and orange juice treated with ozone (0.9 g/h) at various temperatures. They found that reductions in the levels of pathogens at 4 °C were considerably higher than those at 22 °C. However this finding is apparently in contrast with another study (Steenstrup and Floros, 2004) that reported that D values of E. coli O157:H7 in ozonated apple cider tended to increase with decreasing temperature. These controversial results indicate that ozone efficacy can vary with experimental conditions, making it difficult to predict the influence of temperature on a particular application (Pascual et al., 2007). It has been well documented that inactivation of microorganisms by ozone in the aqueous medium is much faster at low pH than at higher pH values (Zuma et al., 2009). Therefore, acidic environment of apple juice can be considered suitable matrix for ozonation in terms of antibacterial effectiveness. Patil et al. (2010) investigated the effect of pH on the inactivation rate of E. coli in apple juice. They demonstrated that ozone inactivation of E. coli was much faster at low pH than at higher pH values. The effect of pH on antibacterial effectiveness of ozone, similar to the temperature, is mainly attributed to the fact that the ozone decomposition occurs faster in higher-pH aqueous solutions (EPA, 1999; Patil et al., 2010). The health benefits of apple juice are primarily related to its phenolic content (Kahle et al., 2005). Changes in the total phenolic content of apple juice treated with bubbling ozone up to 40 min at two different concentrations and temperatures are given in Table 1. At the ozone concentration of 5.3 mg/L, initial total phenolic content of apple juice, determined as 571 mg GAE/L, decreased significantly (P b 0.05) to 375 and 358 mg GAE/L after 40 min of treatment at 4 and 22 °C, respectively. However, the reductions in the level of total phenolic content observed at the ozone concentration of 2.8 mg/L were not significant (P N 0.05). It was reported that phenolic compounds in fruit juices are prone to oxidation during the ozonation due to the strong oxidation potential of ozone (Karaca and Velioglu, 2007). Torres et al. (2011) demonstrated that bubbling ozonation at concentration of 69 mg/L (4.8% w/w of oxygen) for 10 min caused significant reductions in the levels of major phenolic compounds and total phenolic content of apple juice. They also reported that color values and rheological properties of apple juice were negatively affected by the increase in processing time and concentration of ozone. Total phenol content of ozonated apple juice did not significantly (P N 0.05) influence by processing temperature. Degradation of phenolic compounds in apple juice during the ozonation can be attributed to direct reactions of molecular ozone and reactions with its free radicals. Ozone attacks OH radicals, preferentially to the double bonds in organic compounds (Tiwari et al., 2009). Free radicals may lead to electrophilic and nucleophilic reactions occurring with aromatic compounds (Cullen et al., 2009). In food industry an agent, providing minimum 2 log in microbial reduction, is accepted to be antimicrobial (Tiwari and Rice, 2012). Results of present study demonstrated that counts of A. acidoterrestris spores in apple juice can be reduced more than 2 log by bubbling ozone with an acceptable change in total phenolic content. Accordingly, these results led us to conclude that ozonation is a promising application for preventing the spoilage caused by A. acidoterrestris in apple juice. A minimum 5 log reduction of spoilage and potentially pathogenic bacteria most commonly associated with fruit juices is considered as a primary performance standard for non-thermal processing methods by FDA (Tiwari and Muthukumarappan, 2012). Reduction values obtained in apple juice after 40 min of ozonation did not meet the FDA's requirement of a 5 log reduction. Nevertheless, the efficacy of ozone
4
E. Torlak / International Journal of Food Microbiology 172 (2014) 1–4
Table 1 Effect of bubbling ozone on the total phenolic content (±standard deviations, mg GAE/L) in apple juice. Treatment temperature
Ozone concentration (mg/L)
Treatment time (min) 0
4 °C 22 °C
2.8 5.3 2.8 5.3
571 571 571 571
10 ± ± ± ±
a
62 62a 62a 62a
557 550 544 527
20 ± ± ± ±
a
54 70a 51a 37a
524 502 492 450
30 ± ± ± ±
a
64 47a 32a 59ab
490 439 464 417
40 ± ± ± ±
a
73 71ab 80a 42b
458 375 429 358
± ± ± ±
68a 75b 83a 87b
ab
Values followed by the same letter in the same row are not significantly different (P N 0.05).
against A. acidoterrestris spores can be increased by increasing concentration and exposure time. However, results of present study also showed that total phenolic content of ozonated apple juice may be significantly influenced by ozone concentration and by processing time. Therefore, treatment of apple juice with gaseous ozone can be suggested as a complementary microbial reduction method for the control of A. acidoterrestris spores. Further validation studies are still needed to determine the critical limits (e.g., ozone concentration and time parameters) for effective treatment in terms of sporicidal activity and bioactive compounds of product. Acknowledgment The author would like to thank Mr. Mehmet Zeki Öçlü, Director of Kemiteks Chemical Company, for the supply of microbiological media and chemicals used. References Achen, M., Yousef, A.E., 2001. Efficacy of ozone against Escherichia coli O157:H7 on apples. J. Food Saf. 66, 1380–1384. Bahçeci, K.S., Acar, J., 2007. Modeling the combined effects of pH, temperature and ascorbic acid concentration on the heat resistance of Alicyclobacillus acidoterrestis. Int. J. Food Microbiol. 120, 266–273. Bevilacqua, A., Sinigaglia, M., Corbo, M.R., 2008. Alicyclobacillus acidoterrestris: new methods for inhibiting spore germination. Int. J. Food Microbiol. 125, 103–110. Cullen, P.J., Tiwari, B.K., O'Donnell, C.P., Muthukumarappan, K., 2009. Modeling approaches to ozone processing of liquid foods. Trends Food Sci. Technol. 20, 125–136. Cullen, P.J., Valdramidis, V.P., Tiwari, B.K., Patil, S., Bourke, P., O'Donnell, C.P., 2010. Ozone processing for food preservation: an overview on fruit juice treatment. Ozone Sci. Eng. J. Int. Ozone Assoc. 32, 166–179. Durak, M.Z., Churey, J.J., Danyluk, M.D., Worobo, R.W., 2010. Identification and haplotype distribution of Alicyclobacillus spp. from different juices and beverages. Int. J. Food Microbiol. 142, 286–291. EPA, Environmental Protection, 1999. Agency guidance manual alternative disinfectants and oxidants. EPA 815-R-99-014. (Washington DC). Gocmen, D., Elston, A., Williams, T., Parish, M., Rouseff, R.L., 2005. Identification of medicinal off-flavours generated by Alicyclobacillus species in orange juice using GC-olfactometry and GC–MS. Lett. Appl. Microbiol. 40, 172–177. Guzel-Seydim, Z.B., Greene, A.K., Seydim, A.C., 2004. Use of ozone in the food industry. Lebensm. Wiss. Technol. 37, 453–460. IFU, International Federation of Fruit Juice Producers, 2007. Method on the detection of taint producing Alicyclobacillus in fruit juices. IFU Method No. 12. (Paris). IOA, International Ozone Association, 1996. Quality Assurance Committee Revised Standardized Procedure 001/96 (Scottsdale). Jay, J.M., Loessner, M.J., Golden, D.A., 2005. Modern Food Microbiology, Seventh ed. Springer Science and Business Media, New York. Kahle, K., Kraus, M., Richling, E., 2005. Polyphenol profiles of apple juices. Mol. Nutr. Food Res. 49, 797–806. Karaca, H., Velioglu, Y.S., 2007. Ozone applications in fruit and vegetable processing. Food Rev. Int. 23, 91–106. Khadre, M.A., Yousef, A.E., 2001. Sporicidal action of ozone and hydrogen peroxide: a comparative study. Int. J. Food Microbiol. 71, 131–138. Oteiza, J.M., Ares, G., Sant'Ana, A.S., Soto, S., Giannuzzi, L., 2011. Use of a multivariate approach to assess the incidence of Alicyclobacillus spp. in concentrate fruit juices marketed in Argentina: results of a 14-year survey. Int. J. Food Microbiol. 151, 229–234.
Pascual, A., Llorca, I., Canut, A., 2007. Use of ozone in food industries for reducing the environmental impact of cleaning and disinfection activities. Trends Food Sci. Technol. 18, S29–S35. Patil, S., Bourke, P., 2011. Ozone processing of fluid foods. In: Cullen, B.J., Tiwari, B., Valdramidis, V. (Eds.), Novel Thermal and Non-Thermal Technologies for Fluid Foods. Academic Press, London. Patil, S., Bourke, P., Frias, J.M., Tiwari, B.K., Cullen, P.J., 2009. Inactivation of Escherichia coli in orange juice using ozone. Innov. Food Sci. Emerg. Technol. 10, 551–557. Patil, S., Valdramidis, V.P., Cullen, P.J., Frias, J., Bourke, P., 2010. Inactivation of Escherichia coli by ozone treatment of apple juice at different pH levels. Food Microbiol. 27, 835–840. Pettipher, G.L., Osmundson, M.E., Murphy, J.M., 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. Rawson, A., Patras, A., Tiwari, B.K., Noci, F., Koutchma, T., Brunton, N., 2011. Effect of thermal and non thermal processing technologies on the bioactive content of exotic fruits and their products: review of recent advances. Food Res. Int. 44, 1875–1887. Rice, R.G., Robson, C.M., Miller, G.W., Hill, A.G., 1981. Uses of ozone in drinking-water treatment. J. Am. Water Works Assoc. 73, 44–57. Selma, M.V., Ibanez, A.M., Cantwell, M., Suslow, T., 2008. Reduction by gaseous ozone of Salmonella and microbial flora associated with fresh-cut cantaloupe. Food Microbiol. 25, 558–565. Silva, F.V.M., Gibbs, P., 2001. Alicyclobacillus acidoterrestris spores in fruit products and design of pasteurization processes. Trends Food Sci. Technol. 12, 68–74. Singleton, V., Rossi, J., 1965. Colorimetry of total phenolics with phosphomolybdic phosphotungstic acid reagents. Am. J. Enol. Vitic. 16, 144–158. Smit, Y., Cameron, M., Venter, P., Witthuhn, R.C., 2011. Alicyclobacillus spoilage and isolation—a review. Food Microbiol. 28, 331–349. Sokolowska, B., Skąpska, S., Fonberg-Broczek, M., Niezgoda, J., Chotkiewicz, M., Dekowska, A., Rzoska, S.J., 2013. Factors influencing the inactivation of Alicyclobacillus acidoterrestris spores exposed to high hydrostatic pressure in apple juice. High Press. Res. Int. J. 331, 73–82. Solberg, P., Castberg, H.B., Osmundsen, J.I., 1990. Packaging systems for fruit juices and non-carbonated beverages, In: Hicks, D. (Ed.), Production and Packaging of Nonacarbonated Fruit Juices and Fruit Beverages, First ed. Blackie and Son Ltd., London. Steenstrup, L.D., Floros, J.D., 2004. Inactivation of E. coli 0157:H7 in apple cider by ozone at various temperatures and concentrations. J. Food Process. Preserv. 28, 103–116. Steyn, C.E., Cameron, M., Witthuhn, R.C., 2011. Occurrence of Alicyclobacillus in the fruit processing environment—a review. Int. J. Food Microbiol. 147, 1–11. Tiwari, B.K., Muthukumarappan, K., 2012. Ozone in fruit and vegetable processing. In: O'Donnell, C., Tiwari, B.K., Cullen, P.J., Rice, C.R.G. (Eds.), Ozone in Food Processing. A John Wiley & Sons Ltd., Oxford. Tiwari, B.K., Rice, C.R.G., 2012. Regulatory and legislative issues. In: O'Donnell, C., Tiwari, B.K., Cullen, P.J., Rice, C.R.G. (Eds.), Ozone in Food Processing. A John Wiley & Sons Ltd., Oxford. Tiwari, B.K., O'Donnell, C.P., Patras, A., Brunton, N., Cullen, P.J., 2009. Anthocyanins and color degradation in ozonated grape juice. Food Chem. Toxicol. 47, 2824–2829. Torlak, E., Durmuş, S., Ulca, P., 2013. Efficacy of gaseous ozone against Salmonella and microbial population on dried oregano. Int. J. Food Microbiol. 165, 276–280. Torres, B., Tiwari, B.K., Patras, A., Wijngaard, H.H., Brunton, N., Cullen, P.J., O'Donnell, C.P., 2011. Effect of ozone processing on the colour, rheological properties and phenolic content of apple juice. Food Chem. 124, 721–726. Walker, M., Phillips, C.A., 2005. The effect of intermittent shaking, headspace and temperature on the growth of Alicyclobacillus acidoterrestris in stored apple juice. Int. J. Food Sci. Technol. 40, 557–562. Williams, R.C., Sumner, S.S., Golden, D.A., 2004. Survival of Escherichia coli O157:H7 and Salmonella in apple cider and orange juice as affected by ozone and treatment temperature. J. Food Prot. 67, 2381–2386. Young, S.B., Setlow, P., 2003. Mechanisms of killing of Bacillus subtilis spores by hypochlorite and chlorine dioxide. J. Appl. Microbiol. 95, 54–67. Young, S.B., Setlow, P., 2004. Mechanisms of Bacillus subtilis spore resistance to and killing by aqueous ozone. J. Appl. Microbiol. 96, 1133–1142. Zuma, F., Lin, J., Jonnalagadda, S.B., 2009. Ozone-initiated disinfection kinetics of Escherichia coli in water. J. Environ. Sci. Health A Tox./Hazard. Subst. Environ. Eng. 44, 48–56.
