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Jul 15, 2003 - on Alicyclobacillus acidoterrestris during apple juice production. Received: 28 .... taken into account, this reduction was found to be ca. 2.
Eur Food Res Technol (2003) 217:249–252 DOI 10.1007/s00217-003-0750-z

ORIGINAL PAPER

K. Savas¸ Baheci · Vural Gkmen · Arda Serpen · Jale Acar

The effects of different technologies on Alicyclobacillus acidoterrestris during apple juice production Received: 28 January 2003 / Published online: 15 July 2003  Springer-Verlag 2003

Abstract In this study, the effects of various processing steps applied during apple juice processing on Alicyclobacillus acidoterrestris were investigated. Raw apple juice was inoculated with A. acidoterrestris spores at two inoculum levels of 1103 cfu/ml and 1106 cfu/ml. Following enzymatic treatment, the raw juice was processed into clear juice using either conventional clarification or ultrafiltration. The number of A. acidoterrestris spores in the final product was determined to be dependent on both initial contamination level and processing conditions. Increasing temperature to 50 C during depectinization resulted in a higher spore counts at both inoculum levels. Even if the ultrafiltration process was found to be much more convenient for the retention of A. acidoterrestris when compared to conventional clarification, the spores could penetrate the ultrafiltration membranes having both 20 and 50 kDa. Increasing membrane pore size and initial spore counts in the feed solution also increased the number of spores penetrating the membrane during ultrafiltration. Keywords Alicyclobacillus acidoterrestris · Thermoacidophilic · Apple juice · Conventional process · Ultrafiltration

Introduction Until recently, it was believed that bacterial spores did not have the ability to germinate and produce vegetative growth in high acid foods such as many fruit juices. So, pasteurization processes utilizing temperatures of 85– 95 C are often adequate for this class of foods [1, 2]. However, in 1984 Cerny et al. [3] reported an unusual spore-forming bacterium that spoiled aseptically packaged apple juice in Germany. This bacterium was K. S. Baheci ()) · V. Gkmen · A. Serpen · J. Acar Food Engineering Department, Hacettepe University, 06532 Beytepe, Ankara, Turkey e-mail: [email protected]

subsequently named Bacillus acidoterrestris [4] and in 1992, with the creation of the genus Alicyclobacillus, based on the presence of the w-alicyclic acids and on DNA sequence evidence, it was re-identified as Alicyclobacillus acidoterrestris [5]. A. acidoterrestris is a Gram positive, motile, spore forming, rod shaped organism with central, subterminal or terminal spores. Growth has been reported over the pH range 2.5–5.5 [6, 7]. The spores of A. acidoterrestris are highly resistant to heat and can survive during the usual pasteurization regimes used in the juice industry. So they find in the single strength juice a favorable environment for germination and growth that, under certain conditions, can lead to product deterioration [8]. It has been isolated and identified in spoiled commercial fruit products such as apple and orange juice [8, 9, 10] and also in ice tea [11]. The visual detection of spoilage is very difficult because A. acidoterrestris does not produce gas during growth and incipient swelling of containers does not occur. Due to these facts, spoilage during the storage of a retail product can occur without visible changes and produce what is usually described as an off-flavor or odor [12]. The flavor taint compounds have been reported as guaiacol [13] and 2,6-dibromophenol [14]. Today, there are two common technologies for the production of clear apple juice: one is a conventional process using gelatin and bentonite as the clarification aids, and the other is ultrafiltration. Most of the studies on A. acidoterrestris in relation to fruit juices have dealt with detection and enumeration methods, deterioration of fruit juices and surveillance of A. acidoterrestris in commercial fruit juices [9, 15, 16, 17]. There is no information about how the processing variables affect this acidothermophilic, sporeforming bacterium during juice production. Therefore, the aim of this study was to determine the effects of various treatments on A. acidoterrestris during clear apple juice processing.

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Materials and methods Apple juice production. Apples (Golden Delicious) were washed to remove any surface dirt and microbial flora. The raw juice obtained by pressing apple mash was inoculated with aliquots of A. acidoterrestris (DSM 2498, Germany) spore suspension to achieve two inoculum levels of 1103 and 1106 cfu/ml raw juice, separately. The raw juice was then divided into two lots and enzymatically treated with 1.0 ml/l of pectolytic enzyme preparation (Novo, Pectinex 100 L) and 0.2 ml/l of amylase (Novo, Amylase AG 200/300 L,) either at 50 C for 2 h for hot depectinization or at 25 C for 24 h for cold depectinization. Depectinized juice was subdivided into two lots and processed into clear juice by a conventional technique using gelatin and bentonite or by ultrafiltration. Enzymatically treated juice was sequentially flocculated with 500 mg/l of gelatin and 2,500 mg/l of bentonite at 50 C for 2 h and filtered for the conventional process, or ultrafiltrated through 20 and 50 kDa cut-off membranes at a gauge pressure of 2 bar in a plate-and-frame dead-end ultrafiltration module (Amicon) for the ultrafiltration process. Clear juice was concentrated in a vacuum rotary evaporator (Buchi) at 80 C to ca. 60Bx. Following reconstitution (12Bx), juices were bottled and pasteurized at 96 C for 20 min. Pasteurized juices were subjected to an incubation test at 46 C for 2 weeks to determine any risk of the growth of A. acidoterrestris. The whole procedure was repeated twice with duplicated enumeration of A. acidoterrestris at certain processing steps as shown in Fig. 1.

Enumeration of A. acidoterrestris. The sample aliquots from certain processing steps (shown in Fig. 1) were heat treated at 80 C for 10 min prior to enumeration of A. acidoterrestris. Heat-treated juice sample (1 ml) was incorporated directly into Bacillus acidocaldarius medium (BAM) by using the pour plate technique and incubated at 46 C for 3 days, with monitoring of the plates for up to 7 days. For the enumeration of A. acidoterrestris in ultrafiltrated juice samples, 100 ml of heat-treated juice was filtered through a 0.45 mm membrane (Millipore) and the membrane was laid on top of the BAM plates and incubated at 46 C for 3 days, with monitoring of the plates for up to 7 days. BAM composed of three solutions following sterilizing was used for the enumeration of A. acidoterrestris [4, 12, 18]. The compositions of these solutions were as follows: -1. Basic BAM: CaCl2.7H2O, 0.25 g; MgSO4.7H2O,0.50 g; (NH4)2SO4, 0.20 g; yeast extract, 2.00 g; glucose, 5.00 g; KH2PO4, 3.00 g; and distilled water, 500 ml. The medium was adjusted to pH 4.0 with 1 N H2SO4 and sterilized at 121 C for 10 min. -2. Trace elements [19]: CaCl2.2H2O, 0.66 mg; ZnSO4.7H2O, 0.18 mg; CuSO4.5H2O, 0.16 mg; MnSO4.4H2O, 0.15 mg; C Cl2.6H2O, 0.18 mg; H3BO3, 0.10 mg; Na2MoO4.2H2O, 0.30 mg; and distilled water, 1.00 ml; The solution was sterilized at 121 C for 15 min and kept in the refrigerator upon use. -3. Agar: agar, 15.0 g; and distilled water, 500 ml. The solution was sterilized at 121 C for 15 min. For confirmatory tests, the basic BAM with 10.0 g of erytritole instead of glucose, and additionally 15.0 mg of bromophenol blue as indicator was used. The initial blue color of this medium changed to green with the growth of A. acidoterrestris [20]. Spore production. The procedure applied was according to Eiroa et al. [10] and Lee et al. [21] with some modifications. Using the strain A. acidoterrestris DSM 2498, spores were produced in slant BAM agar with incubation at 46 C for 5–7 days. After obtaining more than approximately 80% of spores, as determined by microscopic examination, spores were removed by stirring each tube using a Vortex stirrer after adding 5 ml of sterile distilled water (4 C). The spore crop was centrifuged at 5,000 rpm for 15 min. The supernatant was decanted and the pellet was resuspended. Spores were cleaned by alternate centrifugation and washing of the pellets by sterile distilled water five times. Final pellets were resuspended in sterile phosphate buffer (pH 7.0). Suspensions were heated at 80 C for 10 min and stored at 4 C until used.

Results and discussion

Fig. 1 Laboratory scale apple juice processing scheme (number shows sampling points for the enumeration of Alicyclobacillus acidoterrestris spores)

Here, the raw apple juice was inoculated with two levels of A. acidoterrestris spores (1.0103 cfu/ml and 1.0106 cfu/ml), and the effects of various treatments on the spore population were determined during processing. One of the most established technologies for the production of clear apple juice is conventional clarification of enzymatically depectinized raw juice using certain clarification aids, such as gelatin and bentonite. The solid residue is then removed by vacuum filtration and stable clear juice obtained. This process generally results in a decrease in common microbial metabolites like patulin and fumaric acid due to retention of molecules by physical mechanisms [22, 23]. However, changing conditions applied during depectinization and subsequent fining treatments may significantly affect the final microbial population at the end of this process.

251 Table 1 Change of Alicyclobacillus acidoterrestris spore counts through conventional clarification of apple juice. nd Not detected, a after hot depectinization, b after cold depectinization. For explanation of the processing steps, see Fig. 1

Processing step

After depectinization After clarification After concentration After pasteurization Positive samples after incubation test

2a 2b 3a 3b 4a 4b 5a 5b 5a 5b

Number of spores cfu/ml 1.0103 cfu/ml inoculum

1.0106 cfu/ml inoculum

2.9102–3.1102 0.8102–1.7102 1.1102–1.3102 5.0101–7.0101 4.0101–7.0101 2.0101–3.0101 nd nd 1 1

2.3105–3.0105 1.9105–2.3105 3.9104–1.3105 8.0103–1.8104 7.1103–1.1104 1.2103–1.4103 nd nd 3 2

Table 2 Change of A. acidoterrestris spore counts after ultrafiltration using 20 and 50 kDa cut-off membranes.nd Not detected, UF ultrafiltration, a after hot depectinization, b after cold depectinization. For explanation of the processing steps, see Fig. 1 Number of spores cfu/ml

UF feed solution (depectinized juice) UF permeate (clear juice)

2a 2b 6a 6b

1103 cfu/ml inoculum

1106 cfu/ml inoculum

20 kDa

50 kDa

20 kDa

50 kDa

2.9102–3.1102 0.8102–1.7102 nd nd

2.9102–3.1102 0.8102–1.7102 1.710-1 nd

2.3105–3.0105 1.9105–2.3105 5.510-1 3.010-2

2.3105–3.0105 1.9105–2.3105 3.0100 7.810-1

Table 1 summarizes the change in A. acidoterrestris spore populations during apple juice production by the conventional process. The spore population after depectinization carried out at 50 C for 2 h (hot, 2a) was found to be higher than that carried out at 25 C for 24 h (cold, 2b). Because of its thermophilic nature, a 25 C temperature was not favorable for the growth of A. acidoterrestris, even if the holding period was extended up to 24 h. Cerny et al. [3] have found that A. acidoterrestris spores in apple juice could not grow during incubation at 20–25 C. Similar results have been reported for A. acidoterrestris spores in orange juice incubated at 4 C and 25 C and monitored for up to 28 days [17]. Flocculation of depectinized juice with gelatin and subsequently with bentonite at 50 C for 2 h resulted in a decrease of approximately 1 log cycle in the A. acidoterrestris spore population. When the processing steps of enzymatic treatment, clarification and concentration were taken into account, this reduction was found to be ca. 2 log cycles for an initial inoculum level of 1.0103 cfu/ml and ca. 3 log cycles for that of 1.0106 cfu/ml. However, all these processing treatments could not completely eliminate A. acidoterrestris spores in the final clear apple juice concentrate (Table 1). The spore population in the final juice concentrate was found to be very much dependent on the initial contamination level and the processing temperature. Apple juice following reconstitution was cold-filled into bottles, hermetically sealed and pasteurized in boiling water (96 C) for 20 min. In the bottle, the juice temperature was recorded to be 87–88 C when the

boiling point of the pasteurization medium was reached. No A. acidoterrestris spores could be detected in any of apple juices just after pasteurization. However, 7 bottles of pasteurized juices of 24 gave positive results after enrichment by incubation at 46 C for 2 weeks (Table 1). D values of A. acidoterrestris spores were reported as 16–23 min at 90 C and 2.4–2.8 min at 95 C by Splittstoesser et al. [1]. This data suggesting that spores can survive the typical juice pasteurization at 85–95 C for 2–20 min compares very well with our pasteurization and incubation test results. However, it should be noted that the prolonged pasteurization applied in our study is not identical to those mostly applied in commercial practices. A more common procedure is a high temperature short-time treatment using a plate or tubular heat exchanger followed by hot-filling into bottles and sealing under “clean” conditions, or by aseptic processing [24]. Table 2 summarizes the effects of ultrafiltration on A. acidoterrestris spores using membranes having cut-off values of 20 and 50 kDa. The results have clearly shown us that both the initial spore population and the membrane cut-off value significantly affect the presence and population of A. acidoterrestris spores in ultrafiltrated juice. Interestingly, A. acidoterrestris spores could penetrate through the ultrafiltration membrane having both 20 and 50 kDa cut-offs, although a significant proportion of spores present in the depectinized apple juice used as feed were retained by the membrane. In ultrafiltration trials, increasing the initial spore population and membrane cutoff also increased the number of spores detected in the ultrafiltration permeate stream, so-called clear juice. The

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usual enumeration procedure applied in this study was unable to detect A. acidoterrestris spores in ultrafiltrated juices. Therefore, 100 ml of ultrafiltrated apple juice was filtered through a 0.45 m membrane for enrichment and the membrane was laid on top of the BAM plates prior to incubation. The spores were detected in all clear juice samples after ultrafiltration when the raw juice was inoculated with 1106 cfu/ml of A. acidoterrestris spores. When the inoculum level was decreased to 1103 cfu/ml, only the clear juice sample which had been hot-depectinized and ultrafiltrated through a 50 kDa cut-off membrane contained A. acidoterrestris spores, while others were free of them (Table 2). Nissen and Junker [25] have also claimed that Alicyclobacillus spores can penetrate the micro and ultrafiltration membrane. Even though mold spores and yeast cells are removed, retention of bacteria is not absolute during the ultrafiltration process [26]. Both the dimensions of the microorganism and membrane pore size are expected to affect the passage of bacteria and their spores through the ultrafiltration membrane. The size of A. acidoterrestris cells range from 2.9 to 4.3 m in length, and from 0.6 to 0.8 m in width [4, 27], but there is no information about the size of their spores. In conclusion, the avoidance of microbial spoilage of beverages is the focus of attention of many treatments, and is realized through the application of different technologies. Here, two common technologies for the production of clear apple juice were studied in view of their effects on A. acidoterrestris spores. As expected, the ultrafiltration process was found to be much more effective for the retention of spores from the final product to a greater extent when compared to the conventional clarification process using gelatin and bentonite. However, it is a fact that A. acidoterrestris spores were detected in the finished product after membrane filtration. A limited number of spores penetrated the ultrafiltration membranes having both 20 and 50 kDa cut-offs and thus entered the permeate (product) side of the system which is supposed to be sterile. The presence and population of A. acidoterrestris spore in the final product was found to be dependent on initial contamination levels and processing conditions, especially temperature. The temperature optimum for the growth of A. acidoterrestris is 45–50 C, which is also the temperature preferred for the enzymatic treatment of raw juice and subsequent flocculation with clarification aids to produce a good quality clear juice in an acceptable duration. These processing conditions may result in an increase in A. acidoterrestris population if extended.

Since the pasteurization norms cannot guarantee a complete inactivation of A. acidoterrestris spores, the final product should be kept at temperatures below 25 C to prevent their growth, and thus any risk of spoilage. Acknowledgements Authors would like to thank Hacettepe University Scientific Researches Unit (Project no. 01.001.006) for financial support.

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