Influence of the hot-fill water-spray-cooling process ...

International Journal of Food Microbiology 137 (2010) 295–298

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International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o

Influence of the hot-fill water-spray-cooling process after continuous pasteurization on the number of decimal reductions and on Alicyclobacillus acidoterrestris CRA 7152 growth in orange juice stored at 35 °C Ana Cláudia N.F. Spinelli a, Anderson S. Sant'Ana a,b, Cristiana P. Pacheco-Sanchez a, Pilar R. Massaguer a,c,⁎ a b c

Department of Food Science, Faculty of Food Engineering, State University of Campinas, Rua Monteiro Lobato, 80, Caixa Postal: 6121. 13083-862, Campinas, SP, Brazil Department of Food and Experimental Nutrition, Faculty of Pharmaceuthical Sciences, University of São Paulo. Av. Prof. Lineu Prestes, 580. Caixa Postal: 05508-900. São Paulo, SP, Brazil Department of Chemical Processes, Chemical Engineering Faculty, State University of Campinas, Rua Albert Einstein 500, C.P.6066,CEP 13083-852 Campinas, SP, Brazil

a r t i c l e

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Article history: Received 2 April 2009 Received in revised form 2 November 2009 Accepted 8 November 2009 Keywords: Alicyclobacillus acidoterrestris Orange juice Hot-fill Spoilage Inactivation Growth and predictive microbiology

a b s t r a c t In this study, the influence of the hot-fill water-spray-cooling process after continuous pasteurization on the number of decimal reductions (γ) and growth parameters (lag time; λ, ratio Nf/No; κ, maximum growth rate; μ) of Alicyclobacillus acidoterrestris CRA 7152 in orange juice stored at 35 °C were investigated. Two different inoculum levels of A. acidoterrestris CRA 7152 (102 and 103 spores/mL) in orange juice (110Brix, pH 3.7) and a Microthermics UHT-HTST pilot plant were used to simulate industrial conditions. Results have shown that regardless of the inoculum level (102 or 103 spores/mL), the pasteurization processes were unable to cause even 1 γ. Predictive modeling using the Baranyi model showed that only κ and time to reach 104spores/mL (t104 — time to juice spoilage) were affected by the spore inoculum used (p b 0.05). It has been concluded that A. acidoterrestris was able to survive the hot-fill process and to grow and spoil orange juice in 5–6 days when the final storage temperature was 35 °C. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Since the first report in 1982 of aseptically packed apple juice spoilage in Germany (Cerny et al., 1984) Alicyclobacillus has become a concern in the fruit juice industry. A. acidoterrestris is recognized as the most important Alicyclobacillus species due to frequently isolation from fruit juices and/or involvement in episodes of fruit juice spoilage (Chang and Kang, 2004; Groenewald et al., 2009). It is a spore former thermoacidophilic bacterium, that grows and produces off-flavors (medicinal or phenolic) in fruit juices (Komitopoulou et al., 1999; Yamazaki et al., 1996; Chang and Kang, 2004). Thus, A. acidoterrestris has been suggested as the target microorganism in the design of thermal processes for these products (Bahçeci and Acar, 2007; Tribst et al., 2009). Fruit juice pasteurization can be carried out using hot-fill and hold pasteurization processes. In hot-filling process, juices are heated up to the target temperature (normally between 92 and 105 °C) for 15–30 s, sent to surge tanks where the temperature is decreased to 82–84 °C, and then filled into the bottles and maintained hot for approximately

2 min. Afterwards, the bottles are cooled in a cooling tunnel. Hotfilling pasteurization is normally applied when the fruit juice is filled into glass or plastic bottles instead of carton packages, or when a nonaseptic system is used. Several studies have shown the thermal inactivation parameters for A. acidoterrestris in fruit juices (Murakami et al, 1998; Komitopoulou et al., 1999; Eiroa et al., 1999; Silva et al., 1999; Vieira et al., 2002; Bahçeci and Acar, 2007), however, none have investigated the effect of pasteurization on the γ of this bacterium, when exposed to conditions similar to those applied in industries. Thus, this research aimed to analyze the behavior of two A. acidoterrestris CRA 7152 inoculum levels (102 and 103 spores/mL of orange juice) on the number of decimal reductions (γ) after a hot-fill-water spray-cooling to 35 °C/30 min process. Furthermore, the predictive growth parameters including lag time (λ, days), maximum growth rate (μ, log (CFU/mL)/h) and maximum population ratio (κ, [log10 Nf/No]) of this bacterium were determined.

2. Materials and methods ⁎ Corresponding author. LabTermo. Fundação André Tosello. Rua Latino Coelho, 1301. Parque Taquaral. CEP: 13087-010. Campinas, SP - Brazil. Tel./fax: +55 19 3213 1501. E-mail address: [email protected] (P.R. Massaguer). 0168-1605/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2009.11.003

2.1. Orange juice Frozen concentrated orange juice (FCOJ) (pH 3.8 and 66.17°Brix) was reconstituted to 11°Brix, pH 3.5 with sterile distilled water.

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2.2. Culture and spore suspension A. acidoterrestris strain CRA 7152 (recognized as producing guaiacol in three days) was provided by Danisco Cultor. A. acidoterrestris spore suspension was prepared as described in Spinelli et al. (2009). After preparation the spore suspension was stored at 4 °C until used. The concentration of viable spores in the suspension was 9.4 × 108 spores/mL using Yeast Extract Soluble Starch Agar (YSG). 2.3. Equipment, inoculum level and thermal process All hot fill processes were conducted in a Microthermics LAB-25-DH aseptic pilot unit (Microthermics, NC, USA), equipped with a spiral tubular indirect heat exchanger, heated with water previously heated by saturated steam. The reconstituted orange juice was aseptically inoculated with one of two spore inoculums (102 or 103 spores/mL) in an aseptic reservoir before feeding into the HTST system. The flow rate applied was 1.7 L/min. The inoculum levels were set considering that the usual contaminant loads of Alicyclobacillus spp. found in orange juice were in the range from 100 to 102 spores/mL (Pinhatti et al., 1997). A higher level was tested (103 spores/mL) in order to investigate the behavior when spore counts were uncommonly high in the raw juice. Industrial conditions were simulated by processing juice at 92 °C for 10 s followed by filling at 85 °C, cap twisting, inversion of the bottles for 20 s (disinfection of bottle tops), and holding at 85 °C for 150 s in a previously adjusted water bath to simulate surge tank holding. After the holding time, the juice was filled into disinfected PET bottles (500 mL volume) using a sterile product outlet (SPO) in a laminar cabinet (class 100). The bottles were then cooled to 35 °C by water spraying in about 30 min. During the experiments, the temperature was registered every 10s with a calibrated Omega T-type needle thermocouples installed at the exit to each Microthermics section used (heating, holding and before filling), using the Fluke Hydra model 2625A data logger. Filling and cooling of orange juice were monitored using two PT1000 sensors (TMI-ORION — NanoVACQ-L/1Tc), inserted into one of two different bottles (one in the beginning of the processes and the other in the ending). Three processes were carried out in duplicate in order to verify the number of A. acidoterrestris CRA 7152 decimal reductions (γ) under the following conditions: hot filling without holding at 85 °C/150 s (103spores/mL), hot filling with holding at 85 °C/150 s (103spores/mL) and hot filling with holding at 85 °C/150 s (102spores/mL). Further information on operating and monitoring conditions during the experiments as well as clean-in-place conditions can be found in Sant'Ana et al. (2009) and Spinelli et al. (2009). 2.4. Number of decimal reductions of A. acidoterrestris CRA 7152 in hot-filled orange juice The initial population count (N0) of A. acidoterrestris CRA 7152 spores in the orange juice was determined using YSG agar (incubation at 45 °C/3–5 days), after heat shock at 70 °C/20 min. For the survival examination (Nf), counts were made immediately after filling for both spores and vegetative cells, by adequate decimal dilutions, plating and incubating as described earlier. Under the latter condition, heat shock was not applied. Thus, the number of decimal reductions (γ) was calculated according to the following equation. γ = logðNf Þ logðN0 Þ

ð1Þ

2.5. Determination of the A. acidoterrestris CRA 7152 growth parameters in orange juice after hot-fill processing Two orange juice hot-fill processes similar to those used in an industry were carried out in duplicate to estimate the growth parameters of A. acidoterrestris CRA 7152 using predictive modeling.

The processes consisted of holding at 92 °C for 10s, followed by hotfilling at 85 °C, inversion of the bottles for 20 s (bottle cap disinfection), with holding at 85 °C/150 s, cooling down to 35 °C/30 min, holding at 25 °C/48 h and final storage at 35 °C. Two A. acidoterrestris CRA 7152 inoculum levels were applied (102 and 103spores/mL). The growth curves were accompanied up to the stationary phase by sampling the juice every 8 h. Appropriate dilution and plate counting were carried out, plating on YSG agar (incubation at 45 °C/3–5 days) (Goto et al., 2002). For predictive modeling, data were analyzed using DMFit program (version 2.1), fitting the Baranyi model (Baranyi and Roberts, 1995). The following bacterial growth kinetic parameters were estimated: lagtime λ (days), maximum growth rate μ (log (CFU/mL)/h) and maximum population ratio κ (log10 Nf /No). By analyzing each growth curve, it was determined that an A. acidoterrestris population of 104 CFU/mL was required to produce enough guaiacol to be detected by the Kirin Kit (Niwa and Kuriyama, 2003). Therefore, the time required to reach 104 CFU/mL was determined and identified as “t104” under every storage condition. This value was considered critical for evidence of hot-filled orange juice spoilage. 2.6. Statistical analysis Descriptive statistical calculations were applied to obtain the data required to determine the mean and standard deviation. The Analysis of Variance, Tukey test was applied at 5% significance to check for significant statistical differences among the treatments, using the Statistica 7.0 (Statsoft, USA). 3. Results and discussion This is the first study that clearly proves the hot fill process for orange juice with hot-holding and slow cooling is unable to reduce/ eliminate A. acidoterrestris contamination. Cerny et al. (1984) reported the contamination of hot fill apple juice by A. acidoterrestris. Other researchers have also reported the ability of this microorganism to survive fruit juice thermal processes (Walls and Chuyate, 2000; Eiroa et al., 1999), but they did not carry out processing simulating industrial conditions, nor report a specific response by A. acidoterrestris to withstand fruit juice pasteurization. Furthermore, a predictive modeling approach was used throughout this study to show the behavior of an Alicyclobacillus spoilage strain surviving and germinating in such a product. Table 1 presents the number of decimal reductions obtained for A. acidoterrestris CRA 7152 in orange juice after the hot-fill-water spraycooling processes. Although the inactivation without hot-holding was still rather minor (γ = 0.3 log CFU/mL), it was significantly higher (P b 0.05) than with hot-holding (γ = 0.03log CFU/mL). Despite this, due to uncertainty of plate count methods, this difference may not be totally attributed to the effects of the different conditions. Therefore, further studies should be done to investigate if such conditions result in damages in A. acidoterrestris' spores. The mean temperature values registered during the different thermal pasteurization processes for each section of the Microthermics pilot plant were not significantly different (p b 0.05) (see Supplementary material). Thus, it was concluded that variation in the process and temperature was not responsible for the difference observed between the processes with or without holding at 85 °C/150 s for a load of 103 A. acidoterrestris CRA 7152 spores/mL juice. The data found in the Microthermics pilot plant experiments (0.03 to 0.3 γ) were very close to the 0.1 γ obtained when the efficiency of heating process was calculated using previously published data on A. acidoterrestris inactivation kinetics (Eiroa et al., 1999). This determination considered a mean D95°C = 4.4 min and z = 8.4 °C values for A. acidoterrestris strains using in orange juice as heating substrate and pasteurization conditions applied during Microthermics experiments (92 °C/10 s at the holding section). In this case, since the γ value


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Table 1 Mean number of decimal reductions and growth parameters for A. acidoterrestris CRA 7152 in hot-fill orange juice with and without holding at 85 °C/150 s1. Inoculum level (A. acidoterrestris spores/mL)

Holding at 85 °C/150 s

N0 (spores/mL)

Nf (spores/mL)

Decimal Reductions (γ)

λ (h)

μ (log [CFU mL− 1/h])

κ

t104 (h)

10² 10³ 10³

Yes No2 Yes

2.8 ± 0.1 3.6 ± 0.5 3.8 ± 0.1

2.8 ± 0.1 3.3 ± 0.6 3.7 ± 0.1

0.03 ± 0.1b 0.25 ± 0.1a 0.03 ± 0.1b

114.1 ± 14.5a – 108.2 ± 0.4a

0.05 ± 0.1a – 0.11 ± 0.1a

4.5 ± 0.3a – 2.1 ± 0.2b

147 ± 4.2a – 115 ± 7.1b

Different letters in the same column indicate significant statistical difference according to the Tukey test (p b 0.05). Mean values ± standard deviation. 2Growth parameters are only available for treatments with maintenance at 85 °C/150 s because the condition where holding is not applied is not used by industries. Hot-filling requires this holding time to assure bottles disinfection.

1

calculated were very close to those experimentally obtained, these data could be used in order to determine the efficiency of heat processing. Since the used thermal processes were designed based on industrial practice as applied to orange juices, it can be affirmed that such processes are inefficient against the target microorganism (b1 γ). Considering that the temperature range of A. acidoterrestris growth is about 35–55 °C (Chang and Kang, 2004) the microorganism will be able to grow and spoil the product exposed to such a condition. Fig. 1 shows that A. acidoterrestris CRA 7152 was able to survive and grow in hot-fill orange juice water-spray-cooled after continuous pasteurization. For both spore loads in the raw material studied, the microorganism reached counts of about 106 CFU/mL (with k-values of 4.5 and 2.1, for 102 and 103 spores/mL, respectively). The high R2 values (0.98) for all spore levels indicated a very good fit of the data to the Baranyi model in each of the replicates shown. Table 1 shows that only the values for κ and t104 were significantly different (p b 0.05) considering the different spore inoculum levels studied (102 and 103/mL). The higher the spore inoculum level, the lower the values of the parameters κ and t104. This is due to the fact that κ is estimated as the difference between the maximum (Nf) and the inoculum (No). Then, the higher the inoculum level the smaller the κ value. In addition, the higher the inoculum level the quicker A. acidoterrestris reached the critical population for guaiacol production (104 CFU/mL). Despite the significant differences, the values for t104 were very close (about 5–6 days) under the conditions evaluated. Since spoilage by A. acidoterrestris is imperceptible before the packages are opened, the present results indicated that in almost all the cases the juice would only deteriorate after leaving the factories, reaching the retail center or the consumer home. The control of A. acidoterrestris in orange juice and other fruits is difficult to achieve. During juice pasteurization, time and temperature conditions would have to be increased to assure its elimination, which would result in a juice with reduced quality, thus, other alternatives should be considered. In addition, the control of the juice storage temperature during the

Fig. 1. Growth of A. acidoterrestris CRA 7152 (102 and 103 spores/mL) surviving the orange juice hot-fill water-spray-cooled process after continuous pasteurization.

shelf life and of the washing treatments should be better explored, applying more effort to improving these stages as good alternatives to avoid microbial spoilage even in these low-cost products considered as commodities. This is particularly important, considering that the growth of A. acidoterrestris does not only occur under thermophilic conditions (55 °C) but also at mesophilic temperatures (35 °C). Thus, juices with greater nutritional and sensory quality, less susceptible to spoilage by A. acidoterrestris could be obtained. Acknowledgments The authors acknowledge the financial support provided by CNPq (The Brazilian Research Council). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ijfoodmicro.2009.11.003. References Bahçeci, K.S., Acar, J., 2007. Modeling the combined effects of pH, temperature and ascorbic acid concentration on the heat resistance of Alicyclobacillus acidoterrestris. International Journal of Food Microbiology 20, 266–273. Baranyi, J., Roberts, T.A., 1995. Mathematics of predictive food microbiology. International Journal of Food Microbiology 26, 199–218. Cerny, Y.G., Hennlich, W., Poralla, K., 1984. Spoilage of fruit juice by bacilli: isolation and characterization of the spoiling microorganism. Zeitschrift für Lebensmitteluntersuchung und -Forschung A (European Food Research and Technology) 179, 224–227. Chang, S., Kang, D., 2004. Alicyclobacillus spp. in the fruit juice industry: history, characteristics, and current isolation/detection procedures. Critical Reviews in Microbiology 30, 55–74. Eiroa, M.N.U., Junqueira, V.C.A., Schmidt, F.L., 1999. Alicyclobacillus in orange juice: occurrence and heat resistance of spores. Journal of Food Protection 62, 883–886. Goto, K., Matsubara, H., Mochida, K., Matsumura, T., Hara, Y., Niwa, M., Yamasato, K., 2002. Alicyclobacillus herbarius sp. Nov., a novel bacterium containing w-cycloheptane fatty acids, isolated from herbal tea. Internationl Journal of Systematic and Evolutionary Microbiology 52, 109–113. Groenewald, W.H., Gouws, P.A., Witthuhn, R.C., 2009. Isolation, identification and typification of Alicyclobacillus acidoterrestris and Alicyclobacillus acidocaldarius strains from orchard soil and the fruit processing environment in South Africa. Food Microbiology 26, 71–76. Komitopoulou, E., Boziaris, I.S., Davies, E.A., Delves-Broughton, J., Adams, M.R., 1999. Alicyclobacillus acidoterrestris in fruit juices and its control by nisin. International Journal of Food Science and Technology 34, 81–85. Murakami, M., Tedzuka, H., Yamazaki, K., 1998. Thermal resistance of Alicyclobacillus acidoterrestris spores in different buffers and pH. Food Microbiology 15, 577–582. Niwa, M., Kuriyama, A., 2003. A. acidoterrestris: rapid detection kit. Fruit Processing 2, 102–107. Pinhatti, M.E.M.C., Variane, S., Eguchi, S.Y., Manfio, G.P., 1997. Detection of acidothermophilic bacilli in industrialized fruit juices. Fruit Processing 9, 350–353. Sant'Ana, A.S., Rosenthal, A., Massaguer, P.R., 2009. Heat resistance and the effects of continuous pasteurization on the inactivation of Byssochlamys fulva ascospores in clarified apple juice. Journal of Applied Microbiology 107, 197–209. Silva, F.M., Gibbs, P., Vieira, M.C., Silva, C.L.M., 1999. Thermal inactivation of Alicyclobacillus acidoterrestris spores under different temperature, soluble solids and pH conditions for the design of fruit processes. International Journal of Food Microbiology 51, 95–103. Spinelli, A.C.N., Sant'Ana, A.S., Rodrigues-Junior, S., Massaguer, P.R., 2009. Influence of different storage temperatures on Alicyclobacillus acidoterrestris CRA7152 growth in hot-filled orange juice. Applied and Environmental Microbiology 75, 7409–7416.

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