The dairy industry still relies on thermal processes to ensure safety and quality of fluid milk. However, there is still a perception that these products have diminished nutritional quality. Thus, there has been a resurgence of interest in the adoption of non-thermal processing methods but it is unlikely that a single method can effectively replace thermal pasteurization.
Ross et al. (Reference Ross, Griffiths, Mittal and Deeth2003) and Raso & Barbosa-Cánovas (Reference Raso and Barbosa-Cánovas2003) have reviewed combinations of existing non-thermal technologies that can be used to achieve microbial inactivation in foods without damaging quality. Among these technologies, pulsed electric fields (PEF) have been used to reduce microbial loads in milk (Michalac et al. Reference Michalac, Álvarez, Ji and Zhang2003; Fernández-Molina et al. Reference Fernández-Molina, Bermudez-Aguirre, Altunakar, Swanson and Barbosa-Cánovas2006).
Using a hurdle processing approach, PEF has been combined with moderate heat and antimicrobials (Smith et al. Reference Smith, Mittal and Griffiths2002), thermosonication (Noci et al. Reference Noci, Walkling-Ribeiro, Cronin, Morgan and Lyng2009), and with heat in sequence (Walkling-Ribeiro et al. Reference Walkling-Ribeiro, Noci, Cronin, Lyng and Morgan2009). As PEF is more effective at moderate than low temperatures (Toepfl et al. Reference Toepfl, Mathys, Heinz and Knorr2006; Walkling-Ribeiro et al. Reference Walkling-Ribeiro, Noci, Cronin, Lyng and Morgan2010), a process called high electric field short time (HEST) was proposed by Sampedro et al. (Reference Sampedro, Rivas, Rodrigo, Martínez and Rodrigo2007).
Reviews by Sampedro et al. (Reference Sampedro, Rodrigo, Martínez, Rodrigo and Barbosa-Cánovas2005), and Sobrino-Lόpez & Martín-Belloso (Reference Sobrino-Lόpez and Martín-Belloso2010) suggest that different approaches are necessary to analyse the effect of milk nutrients on PEF effectiveness, with processing conditions, microbial analysis, and the food system as main factors.
Cross-flow microfiltration (CFMF) consists of the cross-flow of skim milk through a filter (1·4 μm pore size) under uniform trans-membrane pressure (Gesan-Guiziou, Reference Gesan-Guiziou and Griffiths2010). Following microfiltration, bacterial populations in the milk are reduced by up to 99·7% (Pedersen, Reference Pedersen1992). Microfiltration has been incorporated into the Bactocatch system in combination with centrifugation, and an ultra-high-temperature (UHT) treatment (Kessler, Reference Kessler2002).
The objective of this study was to evaluate the effectiveness of PEF processing at different steps of milk manufacture to reduce numbers of the natural microbiota in milk with different fat levels. The impact of CFMF prior to PEF and the effects of cream homogenisation were investigated as well as the correlation between the fat and solids content and electrical conductivity.
Materials and Methods
Conventional and alternative milk processing
Milk was obtained from the Elora-Ponsonby Dairy Research Station, University of Guelph. Prior to fat separation (Westfalia LWA 205, Centrico Inc., San Francisco, CA, USA), pre-heating (50°C) was conducted in a dual stage heat exchanger (UHT/HTSTLab-25EDH, Micro-Thermics, Raleigh, NC, USA).
Following the conventional processing sequence shown in Fig. 1, milk was standardised to three fat contents (11, 20 and 31 g/kg). Homogenisation (NS2006H, GEA NiroSoavi S.p.A, Hudson, WI, USA) was conducted at 20 MPa (first stage) and 5 MPa (second stage), 15 Hz and a minimum supply pressure of 0·4 MPa. The heat exchanger was combined with the homogeniser to process milk at 50°C, and subsequently cool it to 12°C.
In the alternative processing sequence (Fig. 1), skim milk (5 g fat/kg) was passed through a CFMF pilot plant system (MFS-1, TetraPak, Aarhus, Denmark) with a membrane pore size of 1·4 μm and inlet and outlet pressures of 90, and 20 kPa, respectively. Creams were homogenised under the same conditions described above.
PEF treatment
Exponential decay pulses of 1·5 μs width were formed with a generator unit (PPS 30, University of Waterloo, Waterloo, ON, Canada) and applied in a three electrode co-axial treatment chamber, which has been described by Walkling-Ribeiro et al. (Reference Walkling-Ribeiro, Rodríguez-González, Jayaram and Griffiths2011a, Reference Walkling-Ribeiro, Rodríguez-González, Jayaram and Griffithsb).
Skim milk was pumped (Masterflex pump drive 7524-40 and pump head 77201-60, Cole Parmer Instrument Co., Vernon Hills, IL, USA) through silicone tubing to the PEF chamber. The product was either pre-cooled in a stainless steel coil submerged in ice, or preheated in the same coil submerged in a water bath (Isotemp 10L, Fisher Scientific, Hampton, NH, USA) as in Table 1. The milk at the chamber outlet following PEF processing was cooled to 12°C in a coil submerged in a refrigerated water bath (NESLAB RTE-7, Thermo Scientific, Newington, NH, USA).
To measure temperature in the tubing, thermocouples were connected to stainless steel flow-through chambers and monitored using a wireless temperature data logger (OM-SQ2020-2F8, Omega, Stamford, CT, USA). Pulses were recorded with a two-channel digital storage oscilloscope using a bandwidth of 100 MHz and a sample rate of 1 GS/s (TDS1012B, Tektronix Inc., Beaverton, OR, USA).
Based on the PEF treatment conditions (Table 1), specific energy density (w PEF) was calculated according to Zhang et al. (Reference Zhang, Barbosa-Cánovas and Swanson1995) considering chamber resistance (Ω), and product electrical conductivity. For total energy calculation, the heating and cooling energy were calculated based on specific heat capacity (3·9 kJ/kg °C) and temperature change as described by Sepúlveda et al. (Reference Sepúlveda, Gongora-Nieto, Guerrero and Barbosa-Cánovas2009).
Heat treatment of milk
High temperature short time (HTST) treatment was conducted in the aforementioned dual stage heat exchanger using a pre-heating temperature of 50°C in the first stage. The heating and cooling energy required by the HTST treatment was calculated as explained above.
Microbiological Analysis
The raw milk initial counts were below 2,500 CFU/ml for mesophiles, 300 CFU/ml for coliforms and the psychrotrophic count was below 300 CFU/ml. To achieve a reliable estimate of the inactivation rates accomplished by the treatments, a portion of the milk (100 ml/l) was incubated at 18°C for 24 h. This inoculation rate produced milks with populations of about 7·0 log CFU/ml for mesophiles, and 6·0 log CFU/ml for both coliforms and psychrotrophs. The upstream processing conditions (white boxes in Fig. 1) decreased the initial microbial population by less than 1·0 log CFU/ml.
Milk (1·5 ml) was collected before and after each processing stage, and cooled on ice prior to serial dilution in Ringers solution (Oxoid, Thermo Fisher Scientific, Basingstoke, UK), and spread-plating on media. Mesophile counts were performed on plate count agar (BD Difco, Sparks MD, USA) after incubation for 48 h at 32°C (Laird et al. Reference Laird, Gambrel-Lenarz, Scher, Graham, Reddy, Wehr and Frank2004), coliforms were enumerated on MacConkey agar (Remel, Thermo Fisher Scientific, Lenexa, KS, USA) after incubation for 24 h at 35°C (Henning et al. Reference Henning, Flowers, Reiser, Ryser, Wehr and Frank2004), and psychrotroph counts were carried out on plate count agar (BD Difco, Sparks, MD, USA) after incubation for 10 days at 7°C (Frank & Yousef, Reference Frank, Yousef, Wehr and Frank2004). Recovered microorganisms were counted and log reductions were evaluated subsequently. Reductions in microbial population were calculated by subtracting the microbial counts of treated samples from the counts of non-treated samples. The detection limit for these counts was 2·4 log CFU/ml.
Physico-chemical properties
Electrical conductivity was measured at room temperature with a handheld conductivity meter (CON 11, Oakton Instruments, Vernon Hills, IL), and pH with a pH meter (AB 15 Accumet Basic, Fisher Scientific, Hampton, NH). Compositional analysis of milk was conducted at the Laboratory Services Division, University of Guelph using a MilkoScan (FT 120 type 71200, Foss Analytics, Copenhagen, Denmark).
Statistical analysis
Statistical analyses were conducted using R version 2.10·1 (R Foundation for Statistical Computing, Vienna, Austria) in conjunction with the R Commander (McMaster University, Hamilton, Ontario, Canada) package for each set of data with a minimum of two batches and two repetitions per treatment. The mean values of reductions in microbial populations for each group were compared separately using a multi-way analysis of variance and multiple comparisons of treatments by Tukey's test (HSD) with an alpha value of 0·05. The relationship between electrical conductivity and fat or solids content was determined by a linear regression analysis. Differences between the upstream and downstream products were evaluated by an Analysis of Covariance. Means of pH values were compared for each product and processing step as for the microbial populations.
Results
Microbial reduction in standardised fluid milks
The reductions in microbial counts in fluid milks following PEF are shown in Table 2. Higher reductions of total mesophiles (P<0·05) were observed at lower fat concentrations when a higher electric field and a longer treatment time were applied. A high energy level from a high electric field strength and low pulse frequency proved to be more effective for inactivation of mesophiles than lower electric field strength and higher frequency (Tables 1 & 2). Differences (P⩾0·05) were also found between the minimum and the maximum reductions of coliforms, while numbers of psychrotrophs fell below the detection limit for all treatments and fat concentrations. Higher levels of inactivation were observed for the mesophiles after HTST treatment of all milks, while it resulted in similar coliform count reductions compared with that obtained using the highest electric field strength. The survival of psychrotrophs in milk subjected to HTST was similar to PEF.
A,B,C,a,b,c,d Superscripted letters indicate that means are statistically different within their microbial group, with uppercase indicating the initial population, and lowercase their reduction. SEM=Standard error of the mean. The energy inputs from heat treatment correspond to 530 kJ/L (72°C×15 s), and 632 kJ/L (85 °C×20 s). For detailed PEF treatment conditions see Table 1
Effect of PEF processing temperature on microbial reduction in skim milk
Increasing milk temperature prior to PEF and lowering exposure time affected the survival of the three populations (Table 3). Increasing the temperature of milk from about 6°C to 34°C had almost the same effect on the inactivation of mesophilic bacteria at the two higher treatment intensities but the effect was achieved in half the time at the higher temperature, confirming that microbial inactivation is more effective at mild temperatures, and that colder products require additional energy (Tables 1 & 3). A reduction of 2·0 log CFU/ml for coliforms was achieved at the lowest PEF treatment conditions, which is a different result from the mild temperature conditions of a 2·7 log reduction (P<0·05) (Table 3). The reduction of the psychrotrophs was higher at the low intensity treatment conditions. The data suggested that the increase in inactivation is related to temperature rise, but the difference between outlet temperatures was not significant (P ⩾0·05).
A,M,a,b,c Superscripted letters indicate that means within the microbial groups are statistically different with uppercase indicating the initial population, and lowercase their reductions. SEM=Standard error of the mean. For detailed PEF treatment conditions see Table 1
Milk processing with a combination of CFMF and PEF
Using CFMF and PEF in a hurdle approach resulted in higher reductions of the skim milk bacterial load yielding reductions of ⩾4·0 log CFU/mL for all three microbial groups when the highest electric field (4·8 kV/mm) was applied (Table 4). Doubling the treatment time at 4·8 kV/mm and 12 Hz led to the same results compared with 459 μs, but the latter resulted in lower total energy use (Tables 1 & 4).
A,B,a,b Superscripted letters indicate means within the microbial groups that are statistically different with uppercase indicating the initial population, and lowercase their reductions. †The standard CFMF conditions were inlet and outlet pressures of 90 (at 120 L/h), and 20 kPa (at 12 L/h) at 35°C with a membrane pore size of 1·4 μm, with an estimated energy consumption of 37 kJ/L. SEM=Standard error of the mean. The energy inputs from heat treatment correspond to 530 kJ/L (72°C×15 s), and 632 kJ/L (85°C×20 s). For detailed PEF treatment conditions see Table 1
Effect of cream homogenisation on the inactivation of native bacteria
The reduction in mesophiles was similar between the non-homogenized and homogenised creams, with significant increments (P<0·05) in reduction with treatment time and electric field (Table 5). The results indicate that there was a greater inactivation (P ⩾0·05) of the coliforms and psychrotrophs in homogenised cream when treatment time was doubled at 4·0 kV/mm; whereas similar high levels of inactivation for all microbial populations in both homogenised and non-homogenised cream were achieved at 5·6 kV/mm.
A,B,a,b,c Superscripted letters indicate means within the microbial group that are statistically different with uppercase indicating the initial population, and lowercase their reductions. SEM=Standard error of the mean, NON-HOMO=non-homogenised, HOMO=homogenised. Homogenisation was conducted at two pressure stages of 20 MPa and 5 MPa respectively. For detailed PEF treatment conditions see Table 1
Physicochemical properties of milk and conductivity
The non-homogenised products showed the highest correlation between conductivity and fat content (Adj-R2 of 0·970) (Fig. 2A). When all the products were included, the correlation decreased slightly (Adj-R2 of 0·824). This decrease in correlation may have been due to physicochemical variations caused by homogenisation and heating. A similar trend was observed for the relation between solids content and electrical conductivity for CFMF products, which resulted in a high Adjusted R2 (0·706 to 0·966). When combined with skim milk, all the products gave a higher correlation (0·921) as shown in Fig. 2B.
From the slopes it can be estimated that for each 100 g/kg increase in fat the conductivity of the non-homogenised products decreased by 0·07 S/m in the range of 0·5 and 500 g fat/kg (Fig. 2A), while the conductivity increased 0·05 S/m for each 10 g/kg increase of solids content (Fig. 2B). These electrical conductivity measurements can be also related to temperature rise (Table 1). For example, applying 2·4 kV/mm at 12 Hz in skim milk (6·3°C) resulted in a temperature rise to 56·1°C compared with standardised cream (5·7°C), which reached a maximum temperature of 50·4°C, and homogenised cream (5·0°C), the temperature of which was raised to 50·5°C.
The pH of all milk products investigated ranged from 6·71 to 6·98 with no differences (P⩾0·05) between products.
Discussion
Microbial reduction in milks
The low impact of the fat content on the PEF effectiveness in fluid milks was confirmed (Table 2). Various reports describing the effect of milk fat content on microbial inactivation by PEF reach different conclusions (Sobrino-Lόpez & Martín-Belloso, Reference Sobrino-Lόpez and Martín-Belloso2010). Grahl & Märkl (Reference Grahl and Märkl1996) treated milks containing Escherichia coli (15 and 35 g fat/kg) and estimated regression coefficients between survivors and electrical field (B E) or treatment time (B t). Differences were found for the effect of these parameters on the two milks; suggesting that milk fat protected Esch. coli from inactivation during PEF treatment. Sobrino-Lόpez et al. (Reference Sobrino-Lόpez, Raybaudi-Massilia and Martín-Belloso2006) inactivated Staphylococcus aureus in milks (0 and 30 g fat/kg) and applied a response surface quadratic model considering fat content, electric field, and pulse number, width, and type. They concluded that fat did not affect microbial inactivation. Reina et al. (Reference Reina, Jin, Zhang and Yousef1998) treated Listeria monocytogenes in milks (2 to 35 g fat/kg) and reported no differences in survival. Walkling-Ribeiro et al. (Reference Walkling-Ribeiro, Noci, Cronin, Lyng and Morgan2009) suggested that for the protective effect of fat to be observed a threshold level must be attained. Therefore, the application of mathematical models like surface response analysis or tertiary models considering composition could aid the optimization of milk processing.
The sensitivity of the microbial groups to PEF inactivation agrees with previous reports in that mesophilic microorganisms are more resistant than coliforms and psychrotrophs. Sepúlveda et al. (Reference Sepúlveda, Gongora-Nieto, Guerrero and Barbosa-Cánovas2009) PEF treated whole milk and subsequently obtained high populations of mesophiles, followed by coliforms and psychrotrophs. The same trend has also been reported by Sepúlveda et al. (Reference Sepúlveda, Gongora-Nieto, Guerrero and Barbosa-Cánovas2005).
In the case of HTST the microbial reductions obtained are within the range quoted in previous studies. While Odriozola-Serrano et al. (Reference Odriozola-Serrano, Bendicho-Porta and Martín-Belloso2006) obtained a 2 log reduction in counts in whole raw milk at 75°C for 15 s, Walkling-Ribeiro et al. (Reference Walkling-Ribeiro, Noci, Cronin, Lyng and Morgan2009) obtained a 6·7 log CFU/ml reduction at 72°C for 26 s. Higher reductions in count could have been expected at higher temperatures (85°C×20 s), but the presence of thermoduric microorganisms shown to be present in the milk might have caused a high post-pasteurization count.
Effect of the PEF inlet temperature
The results of this study support the conclusion that PEF is more effective at temperatures above 30°C by demonstrating a decrease in almost half the PEF energy applied to reduce microorganisms to the same level achieved in milks treated at lower temperatures (Tables 1 & 3). Reina et al. (Reference Reina, Jin, Zhang and Yousef1998) observed that increasing the initial temperature from 43 to 50°C increased the inactivation of List. monocytogenes. Sampedro et al. (Reference Sampedro, Rivas, Rodrigo, Martínez and Rodrigo2007) reduced the energy required to inactivate Lactobacillus plantarum in an orange juice/milk-based beverage by increasing the product processing temperature from 35 to 55°C in a PEF system with six co-field chambers. Advanced systems for pre-heating milk include the one used by Sepúlveda et al. (Reference Sepúlveda, Gongora-Nieto, Guerrero and Barbosa-Cánovas2005) in which the heat in the outgoing stream of the PEF chamber was recovered and incorporated into the incoming by using a tube-in-tube heat exchanger. Hoogland & de Haan (Reference Hoogland, de Haan, Lelieveld, Notermans and de Haan2007) estimated that PEF could be feasibly applied using pre-heating from 5 to 35°C, and post-cooling from 50 to 5°C, thereby, representing a more eco-friendly alternative for the inclusion in a PEF-based Bactocatch system.
Microbiologically, the synergistic effect of PEF and high temperature is attributed to increased membrane fluidity caused by phase transition of the phospholipids from gel to a liquid-crystalline state (Heinz et al. Reference Heinz, Toepfl and Knorr2003; Jaeger et al. Reference Jaeger, Schulz, Karapetkov and Knorr2009). In milk, the liquefaction of colloidal particles that affect resistivity may add to the synergism. From a physico-chemical perspective a decrease in sample viscosity, and increase in conductivity may increase treatment efficiency and can be a consequence of the upstream operations (Walstra et al. Reference Walstra, Wouters and Geurts2006; Kessler, Reference Kessler2002).
Combining CFMF and PEF
Walkling-Ribeiro et al. (Reference Walkling-Ribeiro, Rodríguez-González, Jayaram and Griffiths2011a) also studied the variation in natural microflora subjected to a combination of CFMF and PEF. A microbial reduction of >4·0 log CFU/ml was obtained using this combination (Table 4). Studies on milk CFMF by Olesen & Jensen (Reference Olesen and Jensen1989) reported a reduction in the total count of almost 4 log CFU/ml. Saboya & Maubois (Reference Saboya and Maubois2000) reviewed milk CFMF and mention an average microbial reduction of 3·5 log CFU/ml. Madec et al. (Reference Madec, Mejean and Maubois1992) observed similar results for mesophiles, and also noted that the microbial reduction could be constant and independent of the initial population level. The reduction in coliforms and psychrotrophs were closer to those observed by Saboya & Maubois (Reference Saboya and Maubois2000). Elwell & Barbano (Reference Elwell and Barbano2006) reported a reduction in total counts of 3·8 log CFU/ml with CFMF, and a total reduction in count of 5·6 log CFU/ml was achieved when CFMF was combined with HTST pasteurization. This process has the potential of increasing efficacy by increasing the processing temperature.
Microbial inactivation by PEF in cream
The microbial reductions achieved in cream by PEF processing in our study were higher than those obtained by Mañas et al. (Reference Mañas, Barsotti and Cheftel2001), and Picart et al. (Reference Picart, Dumay and Cheftel2002). Mañas et al. (Reference Mañas, Barsotti and Cheftel2001) conducted their study on cream with 330 g fat/kg (<30°C), and suggested that a higher fat content did not protect Esch. coli against PEF inactivation. Similarly, Picart et al. (Reference Picart, Dumay and Cheftel2002) obtained a maximum reduction of 2·0 log CFU/ml when processing cream (200 g fat/kg) inoculated with a cocktail of three List. innocua strains (<45°C). Toepfl et al. (Reference Toepfl, Heinz and Knorr2007) suggested that agglomeration of microbial cells or between cells and insulating particles might impair the lethal effect of PEF, resulting in a reduction of the PEF effect by 45%. To compare the effect of fat content on the effectiveness of PEF, Otunola et al. (Reference Otunola, El-Hag, Jayaram and Anderson2008) treated Esch. coli and indigenous microorganisms in milk (20 g fat/kg) and cream (180 g fat/kg) and found no differences in rates of inactivation between the two products.
The PEF treatment of cream at temperatures around 5°C (Table 1) resulted in lower microbial reductions than skim milk (Tables 3 & 5), and also in lower final temperatures under comparable PEF conditions (Table 1), which was most likely due to the lower electrical conductivity of creams. In conclusion, PEF treatment of cream may require an increase in the PEF intensity as in the commercial practice of heat pasteurisation, where cream needs to be treated at temperatures 5 to 7°C higher than whole milk (Kessler, Reference Kessler2002).
Effect of composition in electrical conductivity
This study improves our understanding of the effect of milk fat and solids content on electrical conductivity and its implication for microbial inactivation, and product temperature rise during PEF processing. Mabrook & Petty (Reference Mabrook and Petty2003) and Sobrino-Lόpez et al. (Reference Sobrino-Lόpez, Raybaudi-Massilia and Martín-Belloso2006) observed slightly higher conductivity values and a higher decrease in conductivity with increasing fat content compared with this study. In contrast, Ruhlman et al. (Reference Ruhlman, Jin, Zhang, Barbosa-Canovas and Zhang2001) observed a lower conductivity in skim compared with whole milk. These observations may be a result of the variation in the analytical methods. Michalac et al. (Reference Michalac, Álvarez, Ji and Zhang2003) observed no differences in electrical conductivity or pH of raw, UHT or skim milk after PEF, or heat pasteurisation, and Saboya & Maubois (Reference Saboya and Maubois2000) obtained similar pH values for CFMF products. The studies on PEF treatment of cream have not reported variation in pH.
The effect of conductivity on temperature rise during PEF treatment has been reported previously (Zhang et al. Reference Zhang, Barbosa-Cánovas and Swanson1995; Heinz et al. Reference Heinz, Álvarez, Angersbach and Knorr2001; El-Hag et al. Reference El-Hag, Boggs and Jayaram2006), but these earlier studies did not relate electrical conductivity with temperature rise and microbial inactivation.
Conclusions
In the present study the most effective PEF treatment of milk inoculated with a natural microbiota led to lower microbial reductions than those obtained with HTST. Treatment efficacy of PEF against the microorganisms was enhanced when higher electric field strengths were applied. Moreover, a lower milk fat content enabled higher bacterial inactivation by both PEF and HTST. Combining CFMF and PEF in a hurdle strategy increased the antimicrobial effect to a similar level as HTST treatment and consequently, further research on CFMF/PEF processing is recommended. PEF proved to be slightly more effective for homogenised than for standardised cream. The conductivity of the PEF-treated milk was primarily affected by the solids content and secondarily by the fat content, while no variation in the pH of the milk products was observed during processing. These results complemented with the observations in temperature rise, and microbial reduction will aid to design effective PEF treatments for processing milk and milk products.
The authors would like to thank the Dairy Farmers of Ontario and the Natural Sciences and Engineering Research Council for their financial support, to the Guelph Food Technology Centre for facilitating the use of their equipment, and to people in the industry for guiding this project.