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Challenging the assumptions around the pasteurisation requirements of beer spoilage bacteria

2018; Wiley; Volume: 124; Issue: 4 Linguagem: Inglês

10.1002/jib.520

2050-0416

Grzegorz Rachon, Christopher J. Rice, Karin Pawlowsky, Christopher P. Raleigh,

Identification and Quantification in Food

Journal of the Institute of BrewingVolume 124, Issue 4 p. 443-449 Research articleFree Access Challenging the assumptions around the pasteurisation requirements of beer spoilage bacteria Grzegorz Rachon, Corresponding Author Grzegorz Rachon grzegorz.rachon@campdenbri.co.uk orcid.org/0000-0003-3936-5392 Campden BRI, Centenary Hall, Coopers Hill Road, Nutfield, Surrey, RH1 4HY UKCorrespondence to: Grzegorz Rachon, Campden BRI, Centenary Hall, Coopers Hill Road, Nutfield, Surrey RH1 4HY, UK. E-mail: grzegorz.rachon@campdenbri.co.ukSearch for more papers by this authorChristopher J. Rice, Christopher J. Rice Campden BRI, Centenary Hall, Coopers Hill Road, Nutfield, Surrey, RH1 4HY UKSearch for more papers by this authorKarin Pawlowsky, Karin Pawlowsky Campden BRI, Centenary Hall, Coopers Hill Road, Nutfield, Surrey, RH1 4HY UKSearch for more papers by this authorChristopher P. Raleigh, Christopher P. Raleigh Campden BRI, Centenary Hall, Coopers Hill Road, Nutfield, Surrey, RH1 4HY UKSearch for more papers by this author Grzegorz Rachon, Corresponding Author Grzegorz Rachon grzegorz.rachon@campdenbri.co.uk orcid.org/0000-0003-3936-5392 Campden BRI, Centenary Hall, Coopers Hill Road, Nutfield, Surrey, RH1 4HY UKCorrespondence to: Grzegorz Rachon, Campden BRI, Centenary Hall, Coopers Hill Road, Nutfield, Surrey RH1 4HY, UK. E-mail: grzegorz.rachon@campdenbri.co.ukSearch for more papers by this authorChristopher J. Rice, Christopher J. Rice Campden BRI, Centenary Hall, Coopers Hill Road, Nutfield, Surrey, RH1 4HY UKSearch for more papers by this authorKarin Pawlowsky, Karin Pawlowsky Campden BRI, Centenary Hall, Coopers Hill Road, Nutfield, Surrey, RH1 4HY UKSearch for more papers by this authorChristopher P. Raleigh, Christopher P. Raleigh Campden BRI, Centenary Hall, Coopers Hill Road, Nutfield, Surrey, RH1 4HY UKSearch for more papers by this author First published: 06 September 2018 https://doi.org/10.1002/jib.520Citations: 9AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract Current recommendations for beer pasteurisation are based on the study in 1951 by Del Vecchio and coworkers. In this work, 14 beer spoilage bacteria were screened for their ability to grow or survive in ale and stout together with the determination of their thermo tolerance at 60°C. Using a capillary tube method, the D-value (decimal reduction time) and z-value (temperature resistance coefficient) of the three thermo tolerant bacteria (Acetobacter pasteurianus, Lactobacillus brevis and Lactobacillus hilgardii) were determined. Validation of pasteurisation at a range of pasteurisation units (PU) in packaged product were performed in a tunnel pasteuriser. This study showed that eight of the 14 microorganisms were able to grow in both beer styles, whilst different thermo tolerances were observed amongst the spoilage bacteria. Effective pasteurisation of the selected microorganisms was achieved at significantly lower PU values than those recommended by the European Brewery Convention Manual of Good Practice. In package pasteurisation conducted at 1.6 PU resulted in greater than an 8-log reduction in viable cell numbers, resulting in 'commercial sterility'. Although this study demonstrated that successful pasteurisation was achieved for vegetative cells at significantly lower PU values than those recommended, further studies are required to demonstrate the optimal level of pasteurisation for spore forming bacteria and for yeast. © 2018 The Institute of Brewing & Distilling Introduction Contamination of beer is an important problem for the brewing industry, necessitating sterile filtration or heat treatment to maintain the biological integrity of the product. Approximately 70% of beer spoilage cases result from contamination by species of Lactobacillus and Pediococcus 1, while Lactobacillus brevis has been reported as the most prevalent spoilage organism, regardless of the beer style investigated 2. In addition, there are other aerobic and anaerobic beer and brewery-related spoilage organisms that represent a potential source of contamination in the brewing environment and where beer is served in a draught dispensing system, such as acetic acid producing bacteria (e.g. Acetobacter spp.), Zymomonas spp., Megasphaera spp., Kocuria spp. and Pectinatus spp. 3, 4. A number of yeast species can also spoil beer; however, this study focused on bacteria. It is anticipated that futher work will study the heat resistance of spoilage bacteria in biofilms and yeast ascospores. To combat these contaminants, pasteurisation is widely employed in the brewing industry; however the effectiveness of the process varies depending on the processing time and temperature, product composition and the type of contaminating organisms present 5. Thermo tolerant bacteria and yeast are able to tolerate standard heat treatment regimes. Therefore, selecting the most suitable time–temperature pasteurisation regime for a particular product is not always straightforward. The brewing industry currently bases its pasteurisation regimes on long-established microbiological parameters: D- and z-value, and pasteurisation units (PU) of an organism under certain conditions 6. The D-value is the time required at a specific temperature for a decimal (i.e. 1 log or 90%) reduction in the population of a microorganism; the z-value is defined as the change in temperature required for a 10-fold change in the D-value. The European Brewery Convention (EBC) Manual of Good Practice 6 provides basic recommendations for pasteurisation of a range of beer styles (Table 1), but it is suggested that the stated heat loads are over-estimated, resulting in over processing of the beverages, which may result in damage to aroma and flavour compounds 7. Table 1. Range of pasteurisation units for beers – EBC recommendations 6 Product Typical minimum PU Typical maximum PU Pilsner and lager beer 15 25 Ale and stout 20 35 Low alcohol beer 40 60 Non-alcoholic beer 80 120 These EBC recommendations are based on a historical study, conducted by Del Vecchio et al. in 1951 8. Despite a number of concerns 9-11 associated with using Del Vecchio's z-value (6.94°C), it is still widely used. McCaig et al. 12 reported the failure of a flash pasteurisation at 26.5 PU owing to the presence of an L. brevis strain with a z-value >6.94°C. Similarly, Tsang and Ingledew 13 and Molzahn et al. 14 reported higher z-values for common beer spoilage bacteria. The Del Vecchio study includes several curious aspects that do not reflect the real world beer environment. For example, the authors used a completely fermented beer, supplemented with 5% boiled wort, thereby increasing the overall sugar concentration in the product. In addition, the authors inoculated the product with a cocktail of beer spoilage organisms (bacteria and yeast), basing the death kinetics on the most thermo-tolerant organism, an unidentified (and uncharacterised) 'abnormal yeast'. Crucially, this early work was never intended to provide a catch-all model for pasteurisation. Indeed, the authors themselves reported in the study that 'It should not be concluded that the resistance found for the organisms in these tests are necessarily the maximum existing in the beer industry. Only numerous tests with different types and strains and on different beers and ales can determine this point' 8. However, since its publication, the Del Vecchio study has been widely interpreted as providing an indicator for pasteurisation and its findings have been applied to a variety of beer styles containing a broad range of chemical parameters, without the robust microbiological data required to guarantee the microbial integrity of the product. In response to this uncertainty, many brewers have altered their processes to increase the level of pasteurisation to kill off any contaminants 15, providing microbiological stability but risking damage to flavour compounds 7 and increasing the cost of the process 16. Therefore, optimising pasteurisation for different beer styles and microorganisms is likely to result in reduced costs, lower energy utilisation and decreased water utilisation for breweries. However, to reap these benefits, a detailed study is required to provide evidence backed support for the brewing industry. Existing thermal survival data for these microorganisms is scarce 17, with Lactobacillus spp. the best characterised bacteria 18. From the few published studies, it is clear that a wide range of thermo tolerances exist, even within the same type of organism. For example, Lactobacillus D-values at 60°C (D60) in beer have been reported which range from 0.77 to 3.70 min when performed in laboratory scale pasteurisation experiments 13. However, data from a previously performed study at Campden BRI demonstrated considerably lower D60 values of 0.06–0.10 min for a strain of L. brevis (unpublished observations). Studies conducted by Adams et al. 19 highlighted the impact of the chemical environment on microbial thermo tolerance, particularly the beverage pH and ethanol concentration, with other chemical factors also likely to play a significant role. The present study assessed the growth and thermo tolerance of common beer and brewery related spoilage organisms in two beer styles. Once the lowest calculated PU value to provide microbial commercial sterility was determined, tunnel pasteurisation was performed and commercial sterility confirmed. Methodology Selection of beer To assess the growth and pasteurisation survival post pasteurisation of the microorganisms in beer, two styles were selected: a light coloured beer and a dark beer. It has been demonstrated previously that different beer styles have an impact on bacterial growth and survival 20. The light coloured beer was an ale, produced by Wychwood brewery (Witney, UK) and the dark coloured beer was a stout, produced by Meantime brewery (London, UK). To minimise the effect of alcohol concentration and pH on bacterial survival 21, both beer styles selected for this study had a declared alcohol content of 4.5% ABV. The alcohol content measured by distillation was 4.6 and 4.8% ABV, for the ale and stout. The pH, measured with an AR15 pH meter (Accumet Research, USA) was 3.7 and 3.9 for the ale and stout. The bitterness measured by spectrophotometery was 22.1 and 23.5 IBU (International Bitterness Units), for the ale and stout. Culture selection and inoculum preparation Fourteen microorganisms were selected for this study comprising isolates that are associated with beer spoilage. A full list of the organisms is presented in Table 2. Table 2. Microorganisms selected for this study Microorganism Campden BRI code Source Gluconobacter oxydans BSO395 Brewery isolate Gluconacetobacter saccharivorans BSO545 Brewery isolate Acetobacter pasteurianus BSO547 Fermented beverage Kocuria kristinae BSO428 Culture collection Obesumbacterium proteus BSO456 Isolated from vinegar Enterobacter kobei BSO573 Brewery isolate Bacillus megaterium BSO589 Beer Lactobacillus brevis BSO494 Beer (ale) Lactobacillus paracasei BSO564 Brewery isolate Lactobacillus brevis BSO566 Fermented beverage Lactobacillus hilgardii BSO600 Beer (ale) Pediococcus cerevisiae BSO214 Culture collection Pediococcus pentosaceus BSO328 Brewery isolate Pediococcus damnosus BSO596 Brewery isolate All organisms used in this study were recovered from long-term storage (liquid nitrogen) and grown in liquid media – Wallerstein Nutrient broth (WLN; Oxoid, UK) for aerobic bacteria, and de Man, Rogosa and Sharpe (MRS; Oxoid, UK) broth for anaerobic bacteria. All cultures were grown for 5 days at 25 ± 1°C. The working stock cultures were prepared and broths with the addition of sterile glycerol (10% v/v) were stored at −70°C until required. Prior to inoculation, all strains were adapted to the beer environment. Aliquots of 100 μL stock culture were added to 50:50 solutions of broth (WLN or MRS) and beer (ale or stout) and incubated for 5 days at 25 ± 1°C. After incubation, cells were washed by centrifugation at 3500 g and the resulting pellet was resuspended in either ale or stout. Thermo tolerance in ale and stout The thermo-tolerance of a range of common beer spoilage organisms was determined at 60°C in ale and stout. Individual strains were recovered from the working stock cultures and grown in the appropriate adaptation broth, as described above. Thereafter, cells were washed by centrifugation at 3500 g for 15 min. The pellets were re-suspended in 10 mL of ale or stout and the thermo resistance at 0.5 PU (60°C for 30 s) was tested using capillary tube method 22. Briefly, 50 μL of solution containing between 107 and 108 CFU/mL was introduced into the soda glass capillary tubes G119/0,2 (Fisher Scientific, UK); the tube ends were heat sealed and processed within 15 min. As shown by Bradshaw et al. 23, sealing the capillary tubes did not affect the testing solution. The sealed capillary tubes were then submerged in a water bath at the test temperatures (54, 56, 58 or 60°C) and held for a required pre-established time. Although the ramp time was not measured in this study, Jordan et al. 18 and Basaran-Akgul 24 showed that this period in glass capillary tubes was short ( 106 CFU/mL) was required. Thus bacteria were grown in larger volumes (first broth then broth + beer adaptation medium). Following the microbial cocktail preparation and the adaptation step, three 500 mL bottles of ale and three 500 mL bottles of stout were opened and inoculated with an aliquot of microbiological cocktail. Immediately after inoculation, bottles were capped and the content mixed for 2 min by inversion. The level of inoculation was then enumerated by spread plating 100 μL of the appropriate decimal dilutions onto RR agar for enumerating Lactobacillus spp. and onto WLN for the enumeration of A. pasteurianus. The bottles were re-capped, placed into the tunnel pasteuriser and the pasteurisation process started. Three pasteurisation trials were conducted: one for which a kill of less than 6 logs was expected so that a small number of bacteria should be recovered, for trials two and three all inoculated bacteria were expected to be inactivated, resulting in a high log reduction (>6 logs). Following pasteurisation, the number of viable bacteria was enumerated using two techniques. First, the samples were analysed by the spread plate technique where 100 μL of adequate dilutions were spread onto WLN and RR agar. In addition, 1 mL (2 × 0.5 mL) of undiluted sample was also spread plated (limit of enumeration <1 CFU/mL). Secondly, 10 mL of sample was filtered through a 0.45 μm filter (MF – membrane filtration) and viable cells recovered on WLN and RR agar (limit of enumeration 6 logs of inoculated microorganisms during the pasteurisation process. The determined PUs (PUtot) and the lethality of the process (LTProc – logarithmic reduction of inoculated microorganisms achieved by the process) are shown in Table 4. The PUtot and LTProc values were different when using the experimentally determined z-values or the Del Vecchio z-value. Lower PUtot and LTProc values were determined when the z-value used for the calculation was lower than Del Vecchio's and higher values of PUtot and LTProc were determined when the z-value used for the calculation was higher than Del Vecchio's. For example, such a difference can be seen for A. pasteurianus in the ale at 52°C. Using the z-value determined in this study gave only a 2.3 log reduction in cell numbers for this process, whereas using Del Vecchio's z-value, an over-estimated 7.9 log reduction was calculated. Table 4. Calculated PUtot and LTProc of the validation process Microorganism Temperature (°C) Beer Campden BRI trial – various z-values Del Vecchio trial – z = 6.94 PUtot LTProc PUtot LTProc Acetobacter pasteurianus (zale = 5.17, zstout = 6.71) 52 Ale 0.2 2.3 0.7 7.9 Stout 0.6 4.5 0.7 5.1 54 Ale 0.6 6.9 1.6 17.6 Stout 1.4 10.2 1.6 11.3 56 Ale 1.5 16.2 3.0 32.9 Stout 2.8 19.6 3.0 21.2 Lactobacillus brevis (zale = 9.48, zstout = 8.68) 52 Ale 2.0 10.0 0.7 3.6 Stout 1.5 10.2 0.7 4.8 54 Ale 3.5 17.5 1.6 7.9 Stout 2.9 19.0 1.6 10.6 56 Ale 5.5 27.5 3.0 14.8 Stout 4.7 31.1 3.0 19.7 Lactobacillus hilgardii (zale = 7.72, zstout = 8.47) 52 Ale 1.1 8.1 0.7 5.5 Stout 1.4 8.9 0.7 4.5 54 Ale 2.1 16.3 1.6 12.2 Stout 2.7 16.8 1.6 9.9 56 Ale 3.7 28.6 3.0 22.8 Stout 4.5 27.9 3.0 18.5 Three validation pasteurisation trials were performed. The first trial (T1) was conducted at 0.7 PU, the second (T2) at 1.6 PU and the third (T3) at 3.0 PU (calculated using the Del Vecchio z-value). For each trial, one inoculated bottle of ale, one inoculated bottle of stout and one non-inoculated bottle filled with tap water, containing the temperature probe (for temperature profiling), were placed in the middle of the pasteuriser and the trial performed. Preliminary temperature profiling trials showed that a pasteuriser setup of 45 min ramp time, 1 min holding time at 52°C and cooling to 35°C for 25 min would achieve ~0.75 PU. A 45 min ramp time, 1 min holding time at 54°C and cooling to 35°C for 25 min would achieve ~1.6 PU and a 45 min ramp time, 1 min holding time at 56°C and cooling to 35°C for 25 min would achieve ~3 PU. For each trial the temperature in the un-inoculated bottle was measured and logged using a WiFi-TP – Temperature Data Logger. The temperature profiles of the three pasteurisation runs are presented in Fig. 3. Although the required temperature was not reached, the calculated cumulative PU values were within the expected assumptions. For the first trial, the temperature reached 50.8°C and the calculated cumulative PU value was 0.7; for the second trial the temperature reached 53.4°C and the cumulative PU value was 1.6; and for the third trial, the temperature reached 55.3°C and the cumulative PU value was 3. Figure 3Open in figure viewerPowerPoint Temperature profiles for the three tunnel pasteurisation validation trials. The level of inoculated bacteria before and after pasteurisation was determined (Table 5). The results from Trial 1 showed that the inoculated microorganisms were not completely inactivated. Only 4.8 and 7.5 log reductions were achieved for A. pasteurianus in ale and stout, respectively, but the two Lactobacillus spp. were completely inactivated. The calculated PU for this trial (Trial 1) was 0.72. In trials 2 and 3 (T2 and T3) all inoculated microorganisms were inactivated and achieved log reductions of over 8.7 and 8.8 for ale and stout respectively. Table 5. Pasteurisation validation results Trial no. – maximum temperature PU Beer Test times Lactobacillus (BSO566 and BSO600) Acetobacter pasteurianus (BSO547) CFU/mL Log10 (CFU/mL) δLog CFU/mL Log10 (CFU/mL) δLog T1, Tmax = 50.8°C 0.7 Ale Start (SP) 6.2 × 107 7.8 >8.8 5.0 × 107 7.7 4.8 End (SP) <1.0 × 100 <0.0 8.3 × 102 2.9 End (MF) <1.0 × 10−1 <−1.0 >3.0 × 101 >1.5 Stout Start (SP) 4.8 × 107 7.7 >8.7 5.2 × 107 7.7 7.5 End (SP) <1.0 × 100 <0.0 1.0 × 100 0.0 End (MF) <1.0 × 10−1 <−1.0 1.8 × 100 0.3 T2, Tmax = 53.4°C 1.6 Ale Start (SP) 4.9 × 107 7.7 >8.7 4.7 × 107 7.7 >8.7 End (SP) <1.0 × 100 <0.0 <1.0 × 100 <0.0 End (MF) <1.0 × 10−1 <−1.0 <1.0 × 10−1 <−1.0 Stout Start (SP) 4.1 × 107 7.6 >8.6 5.2 × 107 7.7 >8.7 End (SP) <1.0 × 100 <0.0 <1.0 × 100 <0.0 End (MF) <1.0 × 10−1 <−1.0 <1.0 × 10−1 <−1.0 T3, Tmax = 55.3°C 3.0 Ale Start (SP) 4.9 × 107 7.7 >8.7 4.7 × 107 7.7 >8.7 End (SP) <1.0 × 100 <0.0 <1.0 × 100 <0.0 End (MF) <1.0 × 10−1 <−1.0 <1.0 × 10−1 <−1.0 Stout Start (SP) 4.1 × 107 7.6 >8.6 5.2 × 107 7.7 >8.7 End (SP) <1.0 × 100 <0.0 <1.0 × 100 <0.0 End (MF) <1.0 × 10−1 <−1.0 <1.0 × 10−1 <−1.0 SP, Spread plate; MF, membrane filtration. Conclusions Predicting the microbial stability and shelf-life of beer is challenging. Factors such as alcohol content, pH and the presence of hop compounds are known to be important in determining microbial growth and beer spoilage 3. Although some guidelines exist, optimising the pasteurisation regime for different beer styles can be time consuming and can often lead to under- or over pasteurised products. This study used a laboratory based method to screen common beer spoilage organisms for their ability to grow in two beer styles and survive thermal treatment. Although the authors are aware that the long term storage of micro-organisms may indeed result in altered characteristics, all reasonable care was taken during the study to ensure that the organisms exhibited phenotypic traits associated with the genus. All organisms were screened by microscopy to ensure normal cell morphology and cultures were streaked onto nutrient agar to ensure a uniform colony morphology. Finally, before the experiments were performed, all strains were examined to ensure their ability to grow in the test beers. Together these physiological checks ensured that the organisms in this study met the basic criteria for spoilage organisms. The results demonstrated the varying abilities of microorganisms to grow in the two beer styles. The heat inactivation trial performed at 0.5 PU showed inactivation levels ranging from 0.5 log for the most resistant microorganism to 7 log reduction for the most heat sensitive microorganism. The three most heat-resistant microorganisms able to grow in the beers were A. pasteurianus, L. brevis and L. hilgardii. The thermo tolerance of these bacteria in ale and stout were similar. Based on the bacteria and beers used in this study, it was shown that the viable cell concentration in ale and stout beers was reduced to achieve 'commercial sterility' at significantly lower PU values than those recommended by the EBC Manual of Good Practice 6. However, it should be borne in mind that the EBC guidelines were compiled >20 years ago and hygiene in breweries has greatly improved since then. Accordingly, it may not be so surprising that lower PUs are now sufficient to achieve stability. The EBC manual recommends using a minimum of 20 PU for ale and stout. The results from the present study indicated that a >8.7 log reduction in the cell numbers of the selected organisms was achieved at just 1.59 PU. It has to be borne in mind that this study only focused on the vegetative forms of bacteria. The most heat resistant morphological forms of bacteria and yeast are spores 26, which were not investigated in this study. Further studies should focus on the heat inactivation of heat resistant yeast ascospores which are potential beer spoilers. This study demonstrated that the z-values of the three most heat resistant bacteria were between 5.17 and 9.48°C and, although the z-value (6.94°C) reported by Del Vecchio et al. 8 was within this range, it was not possible to confirm or refute this value from the findings of this study. However, the calculated lethality of the validation pasteurisation process (LTProc) conducted in this study was correct, and confirmed by the level of recovered microorganisms only when the D- and z-values determined in this study were used. When the Del Vecchio z-value was used, the calculated lethality of the process was not confirmed by the level of recovered microorganisms. Using Del Vecchio's z-value under- or over estimated the lethality of the process. This suggests that Del Vecchio's z-value was not valid for this scenario. 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