Heat resistance and the effects of continuous pasteurization on the inactivation of Byssochlamys fulva ascospores in clarified apple juice

Authors


Anderson de Souza Sant′Ana, Department of Food and Experimental Nutrition, Faculty of Pharmaceuthical Sciences, University of São Paulo, Av. Prof. Lineu Prestes, 580, Bloco 14, CEP: 05508-900, Butantã, São Paulo, SP, Brazil. E-mail: assantana@usp.br

Abstract

Aims:  To determine thermal resistance, the effect of pasteurization temperature variations (c. 2°C) in a continuous system in the number of decimal reductions (n) of a Byssochlamys strain in clarified apple juice (CAJ).

Methods and Results:  Thermal destruction kinetics of Byssochlamys fulva IOC 4518 in thermal death tubes were determined at 85°, 90°, 92° and 95°C by using Weibull distribution frequency model. Three processes with different heating and holding temperatures (A: 94°, 92°C; B: 95°, 93°C; C: 96°, 94°C, respectively) were performed in a continuous system. Process time was 30 s. δ (time of first decimal reduction) values were: 42·98, 8·10, 3·62 and 1·81 min. Variable n ranged from 0·16 to >4·78 for process B (equivalent to industrial). Variable n (0·95–2·66 log CFU ml−1) were obtained in CAJ bottles processed under condition B, while process A resulted in total heat-resistant mould (HRM) survival and process C in total HRM destruction.

Conclusions:  This study demonstrates that small variations in temperature during the CAJ pasteurization could result in elimination or survival of HRM due to its nonlogarithmic behaviour.

Significance and Impact of the Study:  This was the first study to use Weibull frequency method to model inactivation of HRM in fruit juices. Temperature variations could culminate in the presence of HRM in pasteurized juices even when low counts (<10 spores per 100 ml) were present in the raw materials.

Introduction

Heat resistant moulds are among the micro-organisms of importance in the spoilage of heat-processed fruit juices. The species most frequently isolated and/or associated with food deterioration belong to the following genera: Byssochlamys, Neosartorya, Talaromyces, Eupenicillium (Hocking and Pitt 2001) and Paecilomyces (Piecková and Samson 2000; Peña et al. 2004).

The main concerns with the presence of heat-resistant moulds in fruit juices, in addition to their elevated heat resistance, is the capacity of some genera, such as Byssochlamys spp. of growing under low oxygen tensions (Hocking and Pitt 1984), deteriorating packaged products. Besides, some species are able to produce mycotoxins such as patulin, byssochlamic acid, byssotoxin A, assymetrin, variotin, fumitremorgins A and C, verruculogen, fischerin and eupenifeldin (Tournas 1994). Soil represents the main reservoir of these micro-organisms and fruits having more contact with soil or dust are more susceptible to contamination by heat-resistant moulds. Although low counts of these micro-organisms have been reported in the soil (101–102 CFU per 10 g; Jesenskáet al. 1992), they are not higher than 101 CFU per 100 g in the fruits before processing of the juice.

Despite the low heat-resistant mould counts in the fruits before juice processing, various control measures have been studied aiming at avoiding or reducing the contamination and deterioration of fruit juices by heat-resistant moulds, as avoiding contact of the fruits with soil or dust during transport from orchard to storage (Jesenskáet al. 1992; fruit washing and selection stages at the factory (Ito et al. 1972) and filtration of the juice through diatomaceous earth (King et al. 1969). Nevertheless, the occurrence of deterioration episodes (Olliver and Rendle 1934; Obeta and Ugwuanyi 1995; Rajashekhara et al. 1996; Suresh et al. 1996; Kotzekidou 1997; Baglioni et al. 1999) indicates that the stage to which microbiological stability and safety of the fruit juices (pasteurization) is attributed, may not, on its own, be efficient in reducing/eliminating even low loads of these contaminants. This can be due to the elevated heat resistance of mould ascospores (Tournas 1994) or to log-linear kinetics behaviour followed by some micro-organisms. The low z values (5–7°C) presented by the heat-resistant moulds show the great dependence on the velocity of the heat destruction reaction on the temperature. In industrial conditions, where variations in temperature (c. 2°C) can easily occur during fruit juice continuous pasteurization, small variations in z values among different heat-resistant fungi strains could affect the design of the thermal process treatment and its efficiency in inactivating fungi spores. Thus, using the most resistant Byssochlamys spp. strain found in clarified apple juice (CAJ), the present study aimed to determine: (i) the kinetic parameters of the heat inactivation of the micro-organism at 85°, 90°, 92° and 95°C in thermal death tubes (TDT) and (ii) the effect of pasteurization in a continuous system and of the temperature variations that can occur in industrial heat exchangers (c. 2°C) on the heat inactivation of this micro-organism.

Materials and Methods

Apple juice

Frozen (−20°C), concentrated CAJ (70°Brix; pH = 3·88; 1·30% acidity as malic acid), was diluted to 11°Brix with sterile distilled water and used in the experiments with the continuous pasteurization system. In order to select the most heat-resistant B. fulva strain and determine its heat resistance, the concentrated juice was previously diluted (11°Brix) with distilled water and pasteurized in an autoclave at 105°C for 10 min. A previous determination of the heat-resistant mould count (Beuchat and Pitt 2001) in samples of the concentrated juice indicated the absence of these micro-organisms in 100 ml of the juice.

Strains

Three strains of Byssochlamys spp. from different origins were used: B. fulva IOC 4518 (Brazil – isolated from apple), Byssochlamys nivea ATCC 24008 (United States – isolated surface of mechanical grape harvester) and B. nivea FRR 4421 (Brazil – isolated from strawberries). These strains were chosen as they had been isolated from fruits or from surfaces that had been in contact with fruits. Byssochlamys fulva was isolated by Salomão et al. (2002) and deposited at Instituto Oswaldo Cruz Culture Collection (IOC), Brazil. The identities of Byssochlamys strains were confirmed from the observation of their micro- and macroscopic characteristics. Microscopic characteristics considered were: observation of absence of cleistothecia, gymnosthecia or bodies bearing the asci during developing and maturation and ascospores and conidial structures (shape, size and ornamentation). Macroscopic characteristics considered were: colour, size, colony appearance, colour of reverse of the colony and presence of exudates and pigments. These characteristics were evaluated after fungi growth in selected culture media (CYA or Czapek yeast extract agar and MEA or malt extract agar) at different temperatures (25° and 30°C; Tournas 1994; Pitt and Hocking 1999).

Preparation of the spore suspensions

Suspensions of the ascospores were prepared after growth for 30 days at 30°C in Roux bottles containing 200 ml of sterile formulated MEA (20 g malt extract; 20 g glucose; 20 g agar-agar and 1 g bacteriological peptone). The ascospores were collected after scraping the growth of fungus on each Roux bottle using 20 ml sterile distilled water and a sterile glass rod with a rounded tip. To clean the suspension and remove hyphal fragments, each suspension was filtered through three layers of sterile gauze and collected in sterile flasks. They were then homogenized and centrifuged at 11 962·6 g and 4°C for 15 min. Sonication between 0 and 4°C was then applied for up to 4 min to separate the ascospore clusters (Splittstoesser and Splittstoesser 1977). The final suspensions were transferred to glass bottles containing sterile glass beads and maintained at 2 ± 0·2°C up to 6 months. The ascospore counts in the suspensions were carried out in formulated MEA after application of heat shocks at 75°C (optimal activation temperature) for 5 min for B. fulva IOC 4518 and 20 min for B. nivea ATCC 24008 and B. nivea FRR 4421. These conditions were determined in the preliminary experiments. These time/temperature binomials were subsequently used for all the experiments performed, and all the suspensions were standardized at a concentration of 107 ascospores ml−1.

Selection of the most heat-resistant Byssochlamys spp. strain

The most heat-resistant Byssochlamys strain was selected using thermal death tubes (TDT; 8 mm external diameter, 6 mm internal diameter and 1 mm wall thickness) containing 1·8 ml of CAJ (heating medium) and 0·2 ml of spore suspension adjusted to 107 spores ml−1 (final concentration: 106 spores ml−1). Heat shocks (80°C for 20 min, 85°C for 15 min, 90°C for 5 min, 90°C for 10 min, 95°C for 5 min, 95°C for 10 min, 100°C for 5 min, 105°C for 5 min and 110°C for 3 min) were based by both literature knowledge and experimental information. The lower time/temperature condition (80°C for 20 min) was based on heat shock temperature applied for detecting and enumerating heat-resistant fungi in foods, according to Beuchat and Pitt (2001). The higher temperature condition (110°C for 5 min) was chosen based on the last condition in which survivors were observed. After this condition, three higher conditions were applied in order to assure complete fungi inactivation. Before applying the heat shocks, the TDT tubes were sealed using a blowtorch (O2/liquified gas of petroleum – LGP). The heat shocks were applied in a thermostatically controlled water bath (Polystat®; Poly Science, lL, USA, with ± 0·1°C of precision), the lag time being considered. After heat shock and cooling in a water bath, the total volume of the tubes (2 ml) was transferred to sterile petri dishes and aseptically pour-plated with formulated MEA and incubated at 30°C for 10 days. Growth (mycelial) of the fungus after this period was considered indicative of their capacity to survive the time/temperature binomial applied. Previously, it was determined that this time was enough for assuring fungal spore growth. In spite of this, the plates were also stored for 30 days in order to verify the presence of any injured spores. The strain confirmed to be the most heat-resistant among the three evaluated had its inactivation kinetics parameters determined. Besides, this strain was used in the experiments in the continuous pasteurization of apple juice. The heating time lag was determined in TDT tubes containing 1·8 ml of juice and 0·2 ml of distilled water, by a flexible copper/constantan thermocouple (TC; TT-T-36 type; Ômega, CT, USA) maintained in the position representing one-third of the volume occupied by the sample and connected to a temperature recorder (Ômega; model CL526). The tubes were placed in a thermostatically controlled water bath adjusted to the treatment temperature, and the time taken to reach the desired temperature was registered.

Determination of heat resistance of Byssochlamys fulva IOC 4518

Sterile TDT tubes filled with 1·0 ml of CAJ (11°Brix) and 1·0 ml of ascospore suspension (final concentration is 107 CFU ml−1– this count was used in order to assure the maximum number of possible points in the survival curve) were sealed with the aid of a blowtorch (O2/LGP). After the TDT were placed in thermostatically controlled water baths (Polystat®; Poly Science with ±0·1°C of precision) previously adjusted to the temperatures of 85°, 90°, 92° and 95°C. These values cover the most common temperature range used for fruit juice pasteurization (between 85° and 95°C). The lag heating time was determined previously at each temperature as described in the item of selection of the most heat resistant. For this, 1·0 ml of CAJ and 1·0 ml of distilled water were added to the TDT tubes. After each heating time, the TDT tubes were quickly cooled in an ice bath and opened asseptically. Successive decimal dilutions were prepared and the counts made in duplicate in MEA, incubating at 30°C for 10 days. The counts were expressed as the number of CFU ml−1 (surviving ascospores per millilitre). Using the survivor numbers and respective heating times, survival curves were drawn by regression of the data of log population against the time.

Modelling Byssochlamys fulva IOC 4518 inactivation kinetics in CAJ

As the inactivation kinetics behaviour observed was not log-linear, the Weibull model described by Mafart et al. (2002) was chosen for modelling B. fulva IOC4518 death in CAJ. First, GInaFiT software (Geeraerd et al. 2005) was used in order to estimate the main inactivation parameters of the Weibull model, i.e. δ (time of first decimal reduction) and p (shape parameter). SD and R2 values were obtained for each condition assessed (85°, 90°, 92° and 95°C). z value is independent of the shapes of inactivation curves; z value was determined by regressing temperature vs log δ.

Determination of the heat treatment efficiency for the CAJ

The F value for pasteurization was calculated [eqn (1); Mafart et al. 2002] considering an initial load of ten B. fulva IOC 4518 ascospores per 1 l apple juice package (one ascospore per 100 ml of juice), according to the contamination load commercially found (Hocking and Pitt 1984; Baglioni et al. 1999; Massaguer 2003) and the defects rate was fixed at one contaminated pack for each 104 manufactured, corresponding to a probability of nonsterile units (PNSU) of 10−4. Thus, the required number of decimal reductions would be equal to 5, which can be achievable in the industries. As the most commonly applied commercial processes correspond to 95°C for 20–30 s, the δ value obtained at this temperature was used to calculate F:

image(1)

Determination of the pasteurization effect in a continuous system on the ascospores of Byssochlamys fulva IOC 4518

The experiments were performed in a Microthermics LAB-25-DH aseptic pilot unit (Microthermics, NC, USA; Fig. 1), equipped with a spiral tubular heat exchanger of the indirect type, fed by water previously heated by saturated vapour.

Figure 1.

 Esquematic representation of Microthermics LAB-25-DH UHT-HTST Pilot Plant. TC, thermocouples 1, 2 or 3.

The CAJ, inoculated with a suspension of B. fulva IOC 4518 containing 107 spores ml−1 (to give an initial N0 of between 103 and 104 spores ml−1 juice) was maintained in an aseptic reservoir and fed at a rate of 1·05 l min−1, passing through the heating, holding (tube diameter of ¼-inch) and cooling sections. The pasteurized juice was filled using an aseptic filling unit (SPO) in a class 100 laminar flow chamber, using previously sterilized (121°C 30 min−1) glass bottles (capacity of c. 170 ml). The caps were placed on the bottlenecks with the aid of a sterile pincer, and closed using a manual sealer. Before this, the metal caps were washed and sanitized (on the day of the experiment) in a 0·01% peracetic acid solution for 30 min, then rinsed in 70% alcohol to remove the excess peracetic acid and dried in the laminar flow chamber (close to the burner flame).

During the experiments, the temperature was monitored and collected every 10 s with calibrated Omega T-type needle TC installed at the exit to each section (heating, holding, cooling and before filling), using the Fluke Hydra model 2625A data logger. In addition, two pressure sensors were installed: the first at the preheater and the second at the cooler. Before each experiment, the Microthermics LAB-25-DH unit was sterilized by maintaining a temperature of 121°C for at least 15 min at the coldest point of the equipment (sterile product outlet) so as to guarantee sterility throughout the system after this procedure. After each processing, the equipment was submitted to a clean-in-place (CIP) sanitizing procedure consisting of the application of an alkaline detergent (MIP 2% v/v; Ecolab®, Brazil), and acid detergent (1·3% w/v Elsolve; Ecolab®, Brazil) and a sanitizer based on peracetic acid and hydrogen peroxide (Divosan Forte 1·5% v/v; Johnson Diversey®, Brazil). In addition, the equipment was rinsed with potable water between each stage of the CIP. To determine the efficiency of the CIP system, before filling the equipment with potable water (final rinse), 12 l of sterile water was circulated throughout the Microthermics unit, followed by the collection of 2 l of this rinse water under aseptic conditions for heat-resistant mould count (Beuchat and Pitt 2001). This volume of water was divided into 8 × 250 ml portions, submitted to heat shock (80°C for 30 min), then cooled in an ice bath and filtered through a membrane (0·45 μm). After inoculation onto MEA, the plates were incubated at 30°C for 30 days. These analyses proved the efficacy of CIP applied, as no fungal growth was observed.

Heat processes applied

A process equivalent to that applied by a Brazilian apple juice-processing plant (B) was used, corresponding to 94·3°C for 53 s in the Microthermics LAB-25-DH pilot plant. Two additional processes (A and C) were also adopted so as to determine the influence of temperature fluctuations on the lethality of the process and the number of decimal reductions. For the three processes, the mean holding time was 30 s (15 s for the fastest particle). The juice feed and filling temperatures were both 24° ± 1°C. The mean temperatures programmed for the heating and holding sections were respectively, as follows: A (95° and 93°C); B (96° and 94°C); and C (94° and 92°C).

Survivor count and determination of the number of decimal reductions (n)

The spore loads present in the juice before pasteurization (N0) and the final counts (NF) after each processing were confirmed after applying heat shock at the optimum activation temperature of the fungus (75°C for 5 min). Pour plating of the appropriate decimal suspensions was performed in formulated MEA and incubated at 30°C for 30 days. The colonies that grew on the plates were observed with respect to the macroscopic and microscopic characteristics of B. fulva, according to Pitt and Hocking (1999). Twenty bottles of pasteurized juice were analysed after each process (A, B and C), and the number of decimal reductions for each process (n) determined from the ratio between N0 and NF. In parallel, additional 100 bottles (c. 150 ml juice per bottle) for each experiment, were filled and incubated (30°C for 30 days). The objective of this procedure was to allow for the recovery of fungal spores injured by the heating process of the juice, increasing the probability of detecting survivors (one spore per 100 ml – detection limit of the heat-resistant mould count method for one spore per 16 l juice of bottle incubation).

The fluid flow regime was determined from the thermophysical properties of the CAJ that affected its flow (ρ = 105 kg (m3)−1); μ  =  1·4 × 10−3 Pa s; Constenla et al. 1989; Li et al. 2006) and the internal diameters of the tubes in each section of the unit. As the Reynold’s number (Re) was in the transition zone (c. 2400), it was assumed, for questions of safety, that the fluid flowed with a laminar regime, such that the fastest particle was submitted to the minimum pasteurization condition (Toledo 1991). As the flow rate of the process (1·05 l min−1), the distance between the TC and the dimensions (length and internal radius; Table 1) of each section and of the system connections were known, the minimum residence time in each section could be determined using the maximum flow velocity of the juice in the tubes.

Table 1.   Dimensions of each section and of the connections of the Microthermics UHT/HTST unit used to perform the experiments
Process phaseSection and connectionsLength (m)Diameter (m)Internal area of tubes (m2)
HeatingHeating11·580·0070·00003848
Connection1·930·00720·00004072
HoldingConnection1·660·00720·00004072
Extended holding5·040·01040·00008495
Connection1·030·00720·00004072
CoolingConnection1·190·00720·00004072
Cooler11·580·0070·00003848
Connection2·610·00720·00004072

Considering that the fluid (apple juice at 11°Brix) heated up and cooled down in an isothermal way, and knowing the heating/cooling temperatures of the medium (steam or hot/cold water) at each step, the Deindoerfer and Humphrey (1959) equation, re-arranged by Swartzel (1982), for the calculation of the temperature in spiral heat exchangers [eqn (2)] could be used.

image(2)

As from this equation and knowing the lengths of each section and the initial and final fluid temperatures, the product temperature at any time in the step under study could be found. The division of the residence time for each section into 20 equal intervals allowed the estimation of the temperature profile. As from the thermal histories, the lethality (L min at 95°C) of the process [eqn (3)] for B. fulva (IOC 4518) could be calculated for each time interval using the z value.

image(3)

However, as B. fulva TDT inactivation curves in this study did not obey a first-order kinetic, pasteurization values (F) for each section cannot be added. In spite of this, n, for each section remains additive and were determined for each process performed by eqn (4) as shown (Mafart et al. 2002):

image(4)

However, kinetic parameters obtained with GinaFiT were not independent, showing a clear dependence of p with temperature and a strong correlation with δ. Considering this dependence and the fact that heat processes were nonisothermal, fixing an average p for calculating n, as described by Mafart et al. (2002) would not be the most appropriated approach, which could lead to errors on estimates depending on temperature. Thus, a unique p value used in the determination of n for the effects of continuous pasteurization on B. fulva IOC 4518 ascospores was estimated directly from eqn (5) by an iterative procedure using the solver capability of Excel software (Mafart and Leguerinel, personal communication). Concerning the δ values for each kinetic, these values are fitted together with the p value, by linear regression between tp values and logN. The adjustment of the best p value for all temperature conditions was done by minimizing the square error while the z value was obtained by linear regression of log δ vs temperature.

image(5)

Results

Selection of the most heat-resistant Byssochlamys spp. strain

Of the three strains studied, all survived the heat shock of at 80°C for 20 min, confirming their heat resistance. Byssochlamys fulva IOC 4518 was the most heat-resistant strain, surviving a shock at 95°C for 5 min and was only eliminated by a heat treatment of 100°C for 5 min. Byssochlamys nivea FRR 4421 only survived a shock at 80°C for 20 min, being eliminated in treatments with temperature higher than 85°C while B. nivea ATCC 24008 only survived at 90°C for 5 min, being eliminated after exposure at 90°C for 10 min or higher.

Determination of the heat resistance of Byssochlamys fulva IOC 4518

Figure 2 shows the survivor curves experimental and calculated by GInaFiT for B. fulva IOC 4518 in CAJ (11°Brix) at temperatures of 85°, 90°, 92° and 95°C. As can be seen, all the curves were characterized by a nonlog-linear behaviour, with the presence of a shoulder followed by a fall in the survivor counts. Low SD were obtained, showing reproducibility of the experiments performed.

Figure 2.

 Nonlog-linear survivor curves experimental and calculated with GInaFiT for Byssochlamys fulva IOC 4518 in clarified apple juice (11°Brix) at 85°, 90°, 92° and 95°C.

Table 2 shows the parameters δ and p found for B. fulva IOC 4518 in CAJ by using the GInaFiT software (Geeraerd et al. 2005). It can be observed that the higher the heating temperature, the lower was the δ value. A demonstration of dependence between temperature and p values can be observed. Target F value found for the pasteurization of the CAJ at 95°C was 2·90 min, considering an N0 equal to 101 spores per package and PNSU of 10−4 spores ml−1.

Table 2.   ‘δ’ and ‘p values determined by GinaFiT at temperatures of 85°, 90°, 92°C and 95° and z value (°C) for Byssochlamys fulva IOC 4518 in clarified apple juice
Temperature (°C)δ ± SD (min) ± SDR2
  1. SD, standard deviation.

8542·98 ± 16·802·06 ± 0·960·89
908·10 ± 1·332·05 ± 0·360·98
923·62 ± 0·353·36 ± 0·740·97
951·81 ± 0·713·43 ± 3·360·78
z (°C)7·1

The effect of pasteurization in a continuous system on Byssochlamys fulva IOC 4518 ascospores

A mean fall of 1·78°, 2·5° and 2·82°C in product temperature between the end of heating and holding was observed for processes A, B and C, respectively. This fall in temperature is plausible of ocurring in industrial heat exchangers making our results realistic. Figure 3 shows the thermal histories of the processes (A, B and C) obtained from the temperature data measured at the start and end of each step, focusing on the heating and holding regions (main responsibility for the lethality of the heating process).

Figure 3.

 Thermal histories of the processes applied to pasteurize clarified apple juice. (inline image, Process A; inline image, Process B; inline image, Process C)

The adjustment of Weibull frequency model by the iterative procedure with Solver yielded a p value of 4·68 and a minimum square error of 6·7. z value found in this case was 6·3°C (R2 0·99). These values were then applied to the determination of n values for the three heat processes applied. Table 3 shows the decimal reduction values calculated (nc) and experimentally observed (ne).

Table 3.   Numbers of calculated and experimentally obtained reductions of Byssochlamys fulva IOC 4518 ascospores during the clarified apple juice pasteurization processes
Processes (heating/holding)N0 ± SD (log CFU ml−1)*NF ± SD (log CFU ml−1)*Decimal reductions experimentally obtained (ne) ± SD (log CFU ml−1) Decimal reduction ratio calculated (nc)Percentage of spoiled samples
  1. *Mean counts from 20 bottles.

  2. †Survivals were absent after counting in 100 ml of the samples.

  3. SD, standard deviation.

A (94°C/92°C)4·68 ± 0·214·52 ± 0·190·16 ± 0·195·0 × 10−3100
B (95°C/93°C)3·36 ± 0·011·66 ± 0·491·70 ± 0·491·6 × 10−2100
C (96°C/94°C)4·78 ± 0·10<10−2>4·786·2 × 10−20

According to the calculations, processes A, B and C would be responsible for causing a total of 5·0 × 10−3, 1·6 × 10−2 and 6·2 × 10−2 decimal reductions (nc), respectively, in the populations of B. fulva IOC 4518, considering a value for δ95°C = 1·88 min. As can be seen, of the three processes performed, process C showed the greatest decimal reduction ratio, followed by processes B and A, respectively. As process B was the equivalent to industrial conditions, the variation in temperature (Table 4) between it and process C was responsible for a 74% increase in the decimal reduction value. Between processes B and A, the decimal reduction value was reduced by 31% (Table 3).

Table 4.   Mean temperature values at the entrance and exit sections of Microthermics pilot plant and J values during clarified apple juice pasteurization on Byssochlamys fulva IOC 4518 ascospores
ProcessSectionMean temperature (ºC)J (s−1)
Entrance ± SDExit ± SD
  1. *Entrance apple juice temperature was adjusted before feeding.

  2. SD, standard deviation.

A (heating at 94°C/holding at 92°C)Heating24·00 ± 0·5*94·20 ± 0·540·225
Holding94·20 ± 0·5492·42 ± 0·470·041
Cooling92·42 ± 0·4724·23 ± 0·250·145
Filling24·23 ± 0·2524·40 ± 0·22
B (heating at 95°C/holding at 93°C)Heating24·00 ± 0·5*95·23 ± 0·190·226
Holding95·23 ± 0·1992·73 ± 0·200·053
Cooling92·73 ± 0·2024·09 ± 0·170·146
Filling24·09 ± 0·1724·40 ± 0·17
C (heating at 96°C/holding at 94°C)Heating24·00 ± 0·5*96·14 ± 0·110·227
Holding96·14 ± 0·1193·32 ± 0·180·057
Cooling93·32 ± 0·1824·52 ± 0·120·142
Filling24·52 ± 0·1224·40 ± 0·17

The effect of temperature variations greater than 0·5% (Table 4) can be visualized in the present study from the number of decimal reductions obtained experimentally (ne) in the processes evaluated (Table 3). The variation in temperature in the exit of holding section between processes B and C was 0·64% (0·59°C), while in the exit of the heating section it was 0·96% (0·91°C). On the other hand, between processes B and A, this difference was 0·33% (0·31°C) in the exit of holding section and 1·08% (1·03°C) in the exit of the heating section. Thus, ne (log CFU ml−1) varied from 1·70 ± 0·49 to >4·78 for processes B and C, respectively, while for processes B and A, the ne varied between 1·70 ± 0·49 and 0·16 ± 0·19 (log CFU ml−1), respectively. In addition to the variation in the number of decimal reductions occurring when small variations in temperature were tested, the number of decimal reductions of the B. fulva IOC 4518 population also varied in a single heat processing. In process B (equivalent to the industrial process), the number of decimal reductions obtained experimentally (ne) (log CFU ml−1) varied from 0·95 to 2·66, with the majority of the bottles presenting survivor counts indicating from one to two decimal reductions (70%) with a mean of 1·70 ± 0·49 decimal log reductions (log CFU ml−1). A smaller percentage of the bottles presented less than one decimal reduction (5%), while 25% of the bottles presented more than two decimal reductions in the B. fulva IOC 4518 population.

Discussion

Selection of the most heat-resistant Byssochlamys spp. strain

As from an initial concentration of 106 ascospores ml−1, it can be seen that the Byssochlamys strains presented variable capacities to resist the time/temperature bionomials applied. The application of successive heat shocks is an interesting strategy that can be used to select the most heat-resistant microbial strain among various isolates. In this case, the most heat-resistant micro-organism should be used as the target for heat processing and for subsequent determination of thermal death kinetics parameters. In this study, B. fulva IOC 4518 surviving at 95°C for 5 min was used in the heat resistance experiment and to determine the effect of pasteurization in continuous system. Considering that the same inoculum level was used for the three Byssochlamys strains studied (B. fulva IOC 4518, B. nivea ATCC 24008 and B. nivea FRR 4421), in industrial conditions and with the same ascospore level, B. fulva IOC 4518 would be the strain able to survive apple juice pasteurization. In a study with samples collected at different points of apple juice processing (fruits before entering the plant processing, before and after the first pasteurization, after concentrating – apple juice with 42°Brix), after cooling, before and after second pasteurization and the final product (juice with 11°Brix), Salomão et al. (2008) have isolated 11 strains of fungi from different processing lines. These strains were submitted to heating for different time/temperature conditions (80°C for 20 min to 97°C for 15 min) and six were not confirmed (survivors have not been recovered after heating at 80°C for 20 min) as heat-resistant strains. Six heat-resistant strains isolated were: two Neosartorya fischeri (isolated from fruits before entering the plant processing and product after concentrator – able to survive after heating at 92°C for 10 min and 95°C for 20 min, respectively), B. fulva (isolated from fruits before entering the plant processing – able to survive after heating at 95°C for 20 min) and Eupenicillium spp. (product after first pasteurization – able to survive after heating at 85°C for 15 min). This study confirms the importance of fruit quality before entering the plant processing and the failure of well-designed pasteurization processes to inactivate heat-resistant fungi during apple juice processing, as heat-resistant species were isolated after these two points.

Determination of the heat resistance of Byssochlamys fulva IOC 4518

This is the first report on thermal inactivation data of heat-resistant moulds by using Weibull frequency distribution model. In the literature, there are several reports on heat resistance of Byssochlamys spp. (Bayne and Michener 1979; Aragão and Massaguer 1990; Gressoni and Massaguer 2003; Hoffmann 2004; Houbraken et al. 2006), however as the majority of these studies have used the linearization method of Alderton and Snell (1970) for setting inactivation data, the parameters obtained by them cannot be judged in the same way as the approach adopted in the present study. By using Weibull frequency distribution approach reported by Mafart et al. (2002), thermal inactivation parameters of B. fulva IOC 4518 indicated that δ values followed the same dependence on temperature as conventional D values, with higher values being observed for the lower temperatures. However, the shape parameter p showed dependence with temperature and δ.

As shown in Figure 3, R2 values obtained were between 0·97 and 0·98 for 90° and 92°C, while at 85° and 95°C lower R2 values were obtained, 0·89 and 0·78, respectively for Weibull model adjusted by GInaFiT. It can be observed that for 85° and 95°C inactivation curves, the model describes well the shoulder region but not the sharp decrease that is the actual thermal inactivation (Fig. 3). This can be considered a limitation of this model in the current application. By using the iterative procedure, a best fit of the model to the experimental data was found with R2 of 0·99 and 0·93 for 95° and 85°C, respectively, however a high and unique p value could be obtained in this case.

Casella et al. (1990), found a tendency for linearization of the survivor curve for B. fulva in culture media with increases in the heating temperature, especially in the range near 90°C. In the present study, despite the size reduction of the shoulder with increase in temperature (Fig. 3), the tendency of linearization was not clear even at 95°C (δ of 1·81 min). This fact demonstrates that most of the fungal population was composed of ascospores with elevated heat resistance.

In a preliminary study, the inactivation kinetic parameters were determined by using Alderton and Snell’s (1970) linearization procedure and a z value of 7·4°C was found that is not so different from the z value (7·1°C) calculated by the Weibull model GInaFiT. It can be considered that both approaches determine this parameter by linear regression of either log 1/k vsT (Alderton and Snell 1970) or logδ vsT (Weibull distribution model). It should be clear that the z value is not dependent on the shapes of curves, but it is a characteristic of the relative resistance of the micro-organism studied and both approaches could be used to determine it. In fact, z values found by both the methods were in the range of those reported in the literature, which varied between 4° and 7°C for heat-resistant moulds (mean value of 5°C; Tournas 1994).

Concerning F value calculation, the results of this study indicate that the process which has a time more than 2·90 min would result in the desired PNSU. This process, however, can be considered impracticable, owing to the elevated time required, leading to nutritional and mainly sensory alterations of the product.

F value was estimated by using eqn (1) as proposed by Mafart et al. (2002), which uses inactivation parameters obtained by the Weibull model (p and δ). Thus (no additive), F values of nonlog–linear microbial inactivation kinetics that do not obey Weibull distribution model should be developed in future.

The effect of pasteurization in a continuous system on Byssochlamys fulva IOC 4518 ascospores

Considering only nc, there would be less than one decimal reduction of the target micro-organism population, and thus it seems that a more severe heat treatment would be necessary, compromising the sensory and nutritional qualities of the apple juice. Nevertheless, the survivor counts of the heat processes showed that, depending on the variation in temperature in the heating and holding sections, up to 4·78 decimal reductions (process C; log CFU ml−1) of B. fulva IOC 4518 could be obtained. As the value for δ95°C was used to calculate the number of decimal reductions (nc) of the continuous processes and was different from the number of decimal reductions obtained experimentally (ne), it can be affirmed that there are differences in the inactivation behaviour of the ascospores of this fungus between the static (isothermal) and continuous (nonisothermal) systems. These differences could be related to the shearing forces acting exclusively on the ascospores inside the heat exchanger tubing, most notorious in spiral type heat exchangers owing to the spiral bendings. In practice, this would mean that the shoulder observed before the linear heat inactivation of the fungus (Fig. 2) would be eliminated by the sharp de-aggregation of the ascospores, leading to a quicker death and justifying the differences observed between the calculated and experimental decimal reductions. Another explanation for the results obtained could be the effect of the faster heating rates to which the ascospores are submitted in continuous systems, leading to quicker activation and inactivation. Similar results were obtained by Juneja and Marks (2003) with Salmonella strains when exposed to different heating rates in sous-vide cooked beef. The induced heat resistance phenomenon has also been discussed by Valdramidis et al. (2007). Our results are in agreement with other published works (Fairchild et al. 1994; Etoa and Adegoke 1995; Wescott et al. 1995; Stabel et al. 1997; Tamega and Massaguer 2003; Pacheco and Massaguer 2004). It should also be taking into account that nc was the minimal n value estimated by the fastest particle inside the Microthermics unit, while ne considered all the effects on B. fulva ascospores inside the equipment. Thus, the use of continuous systems to determine the destruction kinetics of these moulds might be a good alternative as compared with the static systems, permitting the simulation of more realistic conditions, closer to those occurring in industries, and taking into account phenomena associated with the de-aggregation, activation and inactivation of the heat-resistant moulds by the heat treatment. Their use would allow for the juice to be submitted to conditions that assure the elimination of the micro-organism targeted by the heat process, without, nevertheless, compromising the sensory and nutritional parameters of the product.

The incubation of the bottles filled with the pasteurized CAJ showed that no survivor could be recovered after process C, as none of the 100 bottles showed signs of fungal growth after 30 days of incubation at 30°C. On the other hand, for the 100 bottles each from processes B and A, all those incubated at 30°C showed signs of fungal growth (mycelium) only 3 days after processing. It could be seen that the growth of B. fulva IOC 4518 occurred preferentially at the bottom of the bottles, later disseminating throughout the bottle, reflecting their ability to multiply under low oxygen tension (Taniwaki et al. 2001).

A comparison of the number of decimal reductions obtained experimentally (ne) in processes A, B and C (0·16 to >4·78 log CFU ml−1) demonstrated the effect of varying the temperature during pasteurization, on the inactivation of B. fulva in a continuous system. Considering that temperature is known to be the main factor affecting the heat resistance in micro-organisms, Akterian et al. (1999) reported a value of 0·5% tolerance for variation in this parameter in studies on thermo-bacteriology. In industrial practice, this would signify that a reduction in temperature greater than 0·4°C in the holding section of a heat process set to 95°C, would not deliver the lethality for which it was designed. Burton (1988) proposed a linear variation between the lethality of a heat process and the temperature in the holding tube, and reported that a variation of 1°C in the holding temperature led to an increase in the sterilizing effect of a process targeted for Geobacillus stearothermophillus (previously Bacillus stearothermophillus) from 8D to 10D. Although more attention is given to the holding tube, the come-up time and the rate of heating should also be taken into account, as they can contribute 40–51% of the cumulative lethality observed at the exit to the holding tube (Awuah et al. 2004). Thus, it can be affirmed that the interaction between the variations in temperature between the two sections (heating and holding) was responsible for the variation in the number of decimal reductions obtained between the three processes. Considering that such variations in temperature (c. 2°C) commonly occur in industrial heat exchangers, this could result in packages contaminated with heat-resistant moulds within a single processing, even when raw materials carry very low levels of heat-resistant moulds, such as <101 ascospore per 100 ml−1. This is supported by our findings in which a small range of temperature variation (c. 2°C), the number of decimal reductions varied from 0·16 to >4·78. Hatcher et al. (1979) reported that the control of temperature in heat processes becomes critical when the products being processed contain heat-resistant micro-organisms characterized by low z values, such as heat-resistant fungi (5–7°C). According to this author, as from the data obtained in an isothermal heating system (TDT), 1°C temperature fluctuations would increase the time required to destroy 104 spores ml−1 of B. fulva G-5 from 14 to 20 min.

The results of the distribution of the number of decimal reduction for bottles of pasteurized CAJ and sequentially subjected to heat-resistant mould count could have been originated from differences with respect to the response to heat activation and subsequent inactivation, the natural formation of B. fulva ascospore clusters or exposition to the different temperature profiles to which each ascospore in particular was submitted during the continuous process. Another factor that could affect the efficiency of pasteurization is the fact of not knowing the predominant forms of heat-resistant moulds in the juice (asci or ascospores), which should be taken into account, as extra energy could be required to break the asci, liberate the ascospores and heat inactivate them, in the case of the first being the dominant population.

In conclusion, the results presented here could explain the isolation of heat-resistant moulds from heat-processed fruit juice packs, even when their heat processing was adequately designed (e.g. to cause five decimal reductions of the most heat-resistant target micro-organism) and raw material with a low degree of contamination (<101 CFU per 100 ml) was used. This survival would be a consequence of the temperature variation in the heating and holding sections of the systems. In addition, the limited extent of deterioration (number of packs) caused by heat-resistant moulds in fruit juices, could be because of a combination of the low incidence of these micro-organisms (nonhomogenous contamination) in the product in the step immediately before pasteurization and of small populations (which could contain highly heat-resistant ascospores), rather than widely disseminated incidence, the majority of which was eliminated by the heat treatment. In order to guarantee the efficiency of the heat processes applied in the fruit juice industry and a reduction in the incidence, spoilage and potential production of mycotoxins by heat-resistant moulds, the following suggestions have been made: guarantee a strict control of the process temperature, improve the heat exchange system, use and control temperatures on average 2°C higher than those established and validated in the laboratory. This last suggestion takes into consideration the variability in temperature occurring in industrial plants that could compromise the efficiency (lethality) of an adequately designed process. In addition, the determination of the inactivation kinetics of the heat-resistant moulds in continuous systems might be more efficient in optimizing the microbiological, sensory and nutritional parameters of the juices manufactured.

Acknowledgements

The authors thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundo de Apoio ao Ensaio, à Pesquisa e à Extensão (Faepex) (Processes 282/06 and 129/07) and Prodetab (Embrapa) for providing funds for this research (Process 042-01/01). They also thank Dr P. Mafart and I. Leguerinel for providing information on Weibull distribution model calculation.

Appendix

Appendix 1

Nomenclature

A = heat transfer surface area (m2);
cp specific heat (J (kg K)−1);
D = time in minutes to cause a one logarithmic cycle reduction in a microbial population at a determined temperature (min);
F = sterilization value, based on the target (Tref= 95°C & ztarget) (min);
J = (UtA/cpωp) (s−1);
L = lethality;
N0= initial population (CPU ml−1);
Nf = final population (CPU ml−1).
R2 = coefficient of determination;
t = time (min or s);
T = temperature (°C);
Tref = reference temperature (95°C);
z = variation in the temperature required for a reduction of 1 logarithmic cycle in the D value (°C);
n = decimal reduction ratio;
p = shape parameter;
δ = time of first decimal reduction (min);
δ* = time of first decimal reduction at reference temperature (95°C in this case) (min);
Ut = overall heat transfer coefficient (W (m2 K)−1);
μ = viscosity (Pa s);
= density (kg (m3)−1);
ωp = mass of flowing medium in heat exchanger (kg).

Ancillary