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Survival ofMegasphaera cerevisiaeheated in laboratory media, wort and beer
Food Microbiology, 1996, 13, 205–212
ORIGINAL ARTICLE
Survival of Megasphaera cerevisiae heated in laboratory media, wort and beer D. Watier*1, I. Chowdhury1, I. Leguerinel2 and J. P. Hornez1
The heat resistance of two strains of spoilage bacteria (Megasphaera cerevisiae DSM 20461 and Megasphaera cerevisiae DSM 20462) was measured at different temperatures (50–60°C). The values of D50 are lower at pH 5·2 and 6. At pH 4, the heat resistance is 4·2 times higher. With values of 0·55 mn for the D60 in wort and beer, DSM 20461 was the more resistant strain. In MRS medium, ethanol and hop bitter acids decreased the thermal resistance. The results were discussed in relation to factors affecting heat resistance and pasteurization regimes in brewery. For a treatment of 8 min at 60°C and initial population of 1·3× 106 bacterial ml−1, the sterility degree is 2·7×105 i.e. one bottle out of 2·7×105 can be contaminated by Megasphaera. For a flash pasteurization (1 min, 70°C), the sterility degree was of 4·5×102, i.e. one bottle out of 4·5×102 bottles were spoiled. 1996 Academic Press Limited
Introduction The quality of beer is determined by the physicochemical stability and biological stability. The growth of micro-organisms induces unpleasant cloudiness accompanied by offflavor. Beer pasteurization is a common practice which guarantees the biological stability of the product and makes its shelf life longer. The scale of pasteurization usually used enables the destruction of all viable forms of life in beer. In brewing literature, the most resistant organisms are different according to the authors; but in practice, the standard pasteurization corresponds to a treatment between 8 and 30 pasteurization units (PU) (Findlay 1971). One PU is equivalent to a heating at 60°C for 1 min (Del Vecchio et al. 1951). This treatment is based on the heat resistance of *Corresponding author. 0740-0020/96/030205+08 $18.00/0
the most heat resistant spoilage organisms. However, the latter must be limited in order to reduce the creation of off-flavors and energy consumption. In practice, the standards of pasteurization have been established thanks to the z value. The formula used gives a lethality rate (Lt) according to z and to the treatment duration (t). This equation is as follows Received: 28 July 1995
Lt (°C)=1/(log−1((60−t)/z)) The recommended z value by Del Vecchio et al. (1951) was 6·94°C. However, it has been proved (Sokal and Rohlf 1969) that the use of z could bring important mistakes in the calculation of Lt. Ingram in 1969 (Ingram 1969) and Tsang and Ingledew in 1982 had already showed that the use of z was not well adapted to established scales of pasteurization in breweries. These authors have suggested to use the inactivation factor (IF) characterized by the treatment duration t and the DT. The
1
Universite´ des Sciences et Technologies de Lille, Laboratoire de Micobiolgic SN2, 59655 Villeneuve DAscq Cedx, France 2 IUT De´partement de Biologie, 2 Rue de l’Universite´, BP319, 29191 Quimper Cedex, France
1996 Academic Press Limited
206 D. Watier et al.
IF is the factor by which the micro-organism population is divided in a treatment of a duration t. The IF is defined by the following relation IF=10 (t/DT) The main beer spoilage are lactic acid bacteria such as Lactobacillus. Moreover, for the last 20 years, new bacteria such as Megasphaera cerevisiae have been encountered. M. cerevisiae is a strictly anaerobic bacteria which was isolated for the first time in Germany by Weiss et al. in 1979 (Weiss et al. 1979, Back et al. 1979). Since then, the appearance of Megasphaera has been described by Haı¨kara (Haı¨kara 1985, Haı¨kara and Lounatmaa 1987) in Finland. When this bacteria develops in beer, it provokes, a synthesis of volatile fatty acids and especially butyric acid in addition to cloudiness. M. cerevisiae is more sensitive to oxygen than other strictly anaerobic spoilage bacteria, such as Pectinatus. However, Megasphaera can develop in beer (Haı¨kara and Lounatmaa 1987). The oxygen reduction during processing can favor the growth of this micro-organism. Megasphaera was isolated in beer, in the piping and in the surrounding air of a brewery (Soberka et al. 1989). The supposed presence of Megasphaera in wort during the fermentation, and an inappropriate standard of pasteurization can explain the increase of the number of contaminations by this bacteria. The growth can also be favored by the marketing of alcohol-free beers and of beers with a highresidual carbohydrate level in which the heat resistance is generally higher (Gibson 1973). This paper reports on the heat resistance of two strains of M. cerevisiae in different media (MRS, wort and beer), and different environmental and physiological conditions (pH, hop bitter acids and alcohol).
Materials and Methods Micro-organisms and media The two studied strains, M. cerevisiae DSM 20461 and DSM 20462 come from the international collection Deutsche Sammlung von
Mikro-organismen und Zellkulturen (Brounsweig, F.R.G.). Three media were used: a modified MRS medium (Man–Rogosa–Sharpe) (Watier et al. 1995) (2 g l −1: glucose), wort (8·7% Plato from GBM, Roubaix, France) and beer (3·3% v/v of ethanol). The beer was obtained by fermentation of wort during 8 days at 8°C and then 30 days at 2°C. The influence of ethanol and hop on the heat resistance was determined in two distinct media. The medium containing ethanol was prepared by the volumetric addition (3·3% v/v) of absolute ethanol to the MRS medium after sterilization. In the medium containing hop, 0·25 g of dehydrate hop was introduced in 1 l of water. After being boiled for 1 h, this solution was used for the preparation of the modified MRS medium. For every medium used, the initial pH values were adjusted with NaOH 1M or H2SO4 1M at pH 6 for MRS, pH 5·2 for wort and pH 4·2 for beer. The pH influence on heat resistance is tested for pH 4, 5·2, 6 and 6·3. The media (19·5 ml) were dispensed in 25 ml antibiotic flasks flushed continuously with N 2/CO 2 (85/15, v/v). Na2S (0·5 g l−1 final) and resazurin (0·5 ml of a 5% stock solution) were added, just before sealing with butyl rubber stoppers. The different media were sterilized at 105°C for 30 min.
Heat stress experiments In all cases, the medium used as an inoculum was identical to the one used for the experiment. An inoculum of each strain was prepared by growing the organisms during 3 days (stationary phase) (Watier et al. 1993) at 32°C, and subsequently, used for heat test. The cell concentration in inoculum was adjusted at 109 cells ml−1. Uninoculated flasks were submerged in a circulating water-bath equipped with a thermoregulator (±0·1°C) (Julobo EM) at temperatures of 45, 50, 55, 60°C in MRS and wort, 48, 52, 56, 60°C in beer. After 20 min of equilibration, the flasks were inoculated with 0·5 ml of inoculum. The flasks were continually shaked to prevent flocculation. The tubes (8×160 mm) containing MRS agar (4·5 ml) were used to determine the formation of col-
Heat resistance of Megasphaera cerevisiae
ony-forming units (cfu). Samples (0·5 ml) were taken and immediately transferred in a tube equilibrated to 45°C. After serial dilutions (1/10) from one tube to another, these tubes were immediately water-cooled and then were incubated at 32°C for 5 days. The experiments at different temperatures were replicated in beer medium for the two strains.
using the sterility degree expression i.e. sterility degree=IF/(average number of organisms by litre), experimentally determined values for IF and the micro-organism concentration found in the fermentation trial. The number of organisms per litre used was 1·3× 106 bacteria ml−1.
Results
Treatment of data The DT-values (i.e. the decimal reduction time) is the necessary time to reduce the number of surviving bacteria by 10 at determined temperature. The DT is independent from the number of initial organisms. Consequently, it is possible to represent the survival curve according to the percentage of cfu ml−1. The z-value is the necessary rise of temperature to reduce the DT-values by 10. The DT-values were calculated by linear regression from the survival curves for the different strains (t=D lg (NO/N)). The same method was used to determine the z-values (T=z lg (D O/D)). The IF-values (inactivation factor) were calculated with the following equation: IF=10 (t/DT). To calculate the IF in case of classical pasteurization, the treatment duration value used was 8 min with D60-value of 0·55 min (DSM 20461) and 0·19 min (DSM 20462). In case of flash pasteurization, the treatment duration value used was 1 min with D70-value of 0·085 min (DSM 20461) and 0·024 min (DSM 20462). The sterility degree was calculated
Whatever the medium used, the M. cerevisiae survival curves heated at different temperatures follow general laws of vegetative cell thermal destruction (see Fig. 1 for example). The DT-values of two Megasphaera strains in three media were shown in Table 1. The method does not enable the determination of the D70 values because of lack of experimental points. Therefore, we have had to extrapolate the D70 -values. Through a calculation from the D70-values with the equation: T=z lg (DO/D) For most of the cases, the correlation coefficients for DT and z were higher than 0·97. The DSM 20461 seems slightly more resistant than the DSM 20462 at 60°C (Fig. 2). With a value of 0·55 mn for the D60 in wort and beer media, M. cerevisiae DSM 20461 was the most resistant strain. For DSM 20461, the DT-values were similar in wort
2 1 –1
A fermentor (Setric set 2M, SGI, France) containing 1·5 l of sterile wort, was prepared. The wort was kept at 8°C and saturated by oxygen during 20 min. The wort was inoculated with Saccharomyces (107 cells ml−1) and Megasphaera (104 cells ml−1). After a fermentation at 8°C during 8 days, viable cells of Megasphaera were counted using the deep agar counting method using MRS agar (12 g l−1 agar, 50 mg l−1 of cycloheximide) incubated at 32°C for 5 days.
log (% of cfu ml )
Simulation of contamination
0 –1 –2 –3 –4 –5 –6 –7
0
Figure
2
4
6 8 10 12 Heating time (min)
14
16
1. The survival of Megasphaera cerevisiae DSM 20461 in beer at 48°C (j), 52°C (h), 56°C (m) and 60°C (n).
207
208 D. Watier et al.
and beer, but decreased in MRS medium. The differences between the DT-values in wort and in MRS medium were not significant. In wort for different pH, the values of D50 are widely variable (Table 2 and Fig. 3). The values of D50 are lower for the pH 5·2 and 6. For pH 4, the heat resistance is 4·2 times higher. Whatever the z-values, the heat resistance of the DSM 20461 strain was superior to the DSM 20462 one. Moreover, the z-values varied according to the medium tested. The simulation of a wort fermentation by Saccharomyces with contamination by Megasphaera shows that the final population can
reach 1·3×106 bacteria ml−1 for a beer at 3·3% v/v of ethanol. Table 3 shows the IF and sterility degree for a spoiled non-filtered beer. In case of classical pasteurization (i.e. 8–20 min at 60°C), the sterility degree is rather high. For a treatment of 8 min at 60°C, the sterility degree is 2·7×105 i.e. one bottle out of 2·7×10 5 may be contaminated by Megasphaera DSM 20461. For a flash pasteurization (1 min at 70°C), the sterility degree is much lower. In this case, the sterility degree was 4·5×102, i.e. one bottle out of 4·5×102 bottles were spoiled. The IF and sterility degree obtained for the DSM 20462 strain were lower, especially for
Table 1. Estimated values of D50 and z for the destruction of Megasphaera cerevisiae at pH 6 in MRS, pH 5·2 in wort and pH 4·1 in beer Temperature (°C)
DT (min)
r2
δ
z (°C)
r2
δ
45 50 55 60
64·18 8·57 2·87 0·36
0·98 0·98 0·99 0·92
6·94 0·54 0·13 0·05
6·82
0·99
0·55
Wort
45 50 55 60
39·22 6·13 1·07 0·55
0·99 0·99 0·99 0·98
2·61 0·47 0·05 0·04
7·62
0·96
1·05
Beer
48 52 56 60 70
10·65
0·97
1·23
Strain and Medium Megasphaera cerevisiae DSM 20461 MRS
Megasphaera cerevisiae DSM 20462 MRS
7·11 2·78 1·06 0·55 0·085*
0·97 0·78 0·99 0·038 0·99 0·020 0·99 0·065 (extrapolation)
45 50 55 60
13·76 3·68 2·15 0·25
0·98 0·99 0·97 0·97
0·97 0·19 0·28 0·029
9·16
0·97
1·35
Wort
45 50 55 60
19·96 2·28 0·69 0·36
0·98 0·99 0·97 0·97
1·58 0·038 0·12 0·08
8·69
0·99
0·51
Beer
48 52 56 60 70
0·99 0·15 0·98 0·11 0·99 0·039 0·97 0·021 (extrapolation)
11·17
0·97
1·52
2·2 1·17 0·48 0·19 0·024*
*Theoretical value calculated with the formula: log (D)=T/z+constant.
Heat resistance of Megasphaera cerevisiae
a pasteurization at 60°C. For a flash pasteurization, the risk was one spoiled bottle out of 3·1×1032.
resistance (i.e. linearity of survival curves), it is not a general rule for all the micro-organisms (Tsang and Ingledew 1982, Barille`re et al. 1985). The main factors that affect the shape of the survival curve are the physicoch-
Discussion 1.60
If the heat resistance of M. cerevisiae is in accordance with the classical laws of heat
1.20
1.3
1.00 log D
1.1 0.9
0.79
0.80
0.77
0.60
0.7 log D
1.48
1.41 1.40
0.40
0.5
0.20
0.3
0.00
0.1
4
5.2
5.97
6.3
pH
–0.1 –0.3
Figure 3. Values of D50 for the destruction of
–0.5 45
60
50 55 Temperature (°C)
Megasphaera cerevisiae DSM 20461 at different pHs in wort.
Figure 2. D-values of Megasphaera cerevisiae
2
DSM 20461 (j) and DSM 20462 (h) in beer at different temperatures.
1
log (% of cfu ml )
0 –1
Table 2. Estimated values of D50 (±δ) for the destruction of Megasphaera cerevisiae DSM 20461 at different pHs in wort and with hop or ethanol in MRS δ
2
Medium
pH
D50 (min)
r
Wort
4 5·2 6·0 6·3
25·89 6·20 5·93 30·11
0·99 0·94 0·95 0·99
MRS
8·13
0·99
MRS+Hop MRS+Ethanol 3·5% v/v
2·76 3·92
0·98 0·98
0·50 2·01 1·05 0·27 0·0079 0·025 0·024
–1 –2 –3 –4 –5 –6 –7
0
10 Heating time (min)
20
Figure 4. The survival of Megasphaera cerevisiae DSM 20461 at 50°C, in MRS (j), with hop (m) or ethanol (3·3% v/v) (d).
Table 3. Inactivation factor and sterility degree of Megasphaera cerevisiae DSM 20461 and DSM 20462 in beer for different conditions of pasteurization Pasteurization (8 min, 60°C)
DSM 20461 DSM 20462
Flash pasteurization (1 min, 70°C)
Inactivation factor
Sterility degree (for 1 litre)
Inactivation factor
Sterility degree (for 1 litre)
3·5×1014 1·3×1042
2·7×105 9·8×1033
5·8×1011 4·6×1041
4·5×102 3·1×1032
209
210 D. Watier et al.
emical composition of the medium and the physiological state of cells (Barille`re et al. 1985). To avoid the deviation curve and enable the determination of DT, a pre-culture media similar to the one used in the experiments, as well as an homogenate inoculum taken at the beginning of the stationary phase were used. It is well known that the heat resistance is very sensitive to the variations of the environmental conditions (Schmid 1957). Oxygen (Linton et al. 1992), low pH (Hansen and Riemann 1963, Stumbo 1973), the presence of peroxides (Linton et al. 1992), ethanol (Adams et al. 1989) and of hop bitter acids (Adams et al. 1989) generally decrease the thermal resistance. Moreover, the heat resistance could be affected by the presence of salt (Stumbo 1973), cations and phosphate ions (Sugiyama 1951, Amaha and Ordal 1957), oxygen (Linton et al. 1992) or a higher sugar concentration (water activity) (Gibson 1973). In order to determine pasteurization standard it was essential to determine the DT-values in the same physico-chemical environment as that encountered in breweries. For this reason, we used the wort and beer medium. The MRS medium was used as a reference medium. Na 2S was introduced in media as a reducing agent to eliminate traces of molecular O2. Moreover, the advantage of using Na2S is that it does not lead to the formation of peroxides (Morgan et al. 1986). The method used for heat stress experiments is not the conventional method. This method derives from the one used by Barille`re et al. (1985). Its advantage is that it reproduces better the pasteurization conditions i.e. pasteurization of packaged beer under hot water. Whatever the temperatures lower than 70°C, the DT-values of the DSM 20461 strain are higher than these of the DSM 20462 strain. M. cerevisiae DSM 20461 is the strain that might be the most bothering for the brewer, as far as pasteurization is concerned. For that reason, we have studied that strain more specifically. Generally, the heat resistance of the micro-organisms is lower in acid medium than in neutral pH medium (Hansen and Riemann 1963, Stumbo 1973). For M. cerevisiae the opposite is observed in wort. The initial
pH of wort at the beginning of the fermentation corresponds to the pH for which the heat resistance is the highest. The presence of hop bitter acids which have an antibacterial activity (Simpson 1993) and of ethanol, affects the heat resistance. Adams et al. (1989) suggest, the addition of hops for lower the heat resistance in the alcohol-free beer. Similar results have been obtained with Lactobacillus when hop extracts or ethanol are added to alcohol-free beer (Adams et al. 1989). We can suppose that the opposite effect of hop and pH on the heat resistance have a cancelling effect on one another. This study shows that M. cerevisiae can grow during fermentation. Indeed, the low D60-values in wort show that Megasphaera cannot survive when wort is boiled before fermentation. In these conditions, the contamination can only occur once the wort has been boiled. The optimization of heat treatment in breweries requires the knowledge of D60 of the most heat resistant micro-organisms in beer. Tsang and Ingledew (1982) suggest the D60 of Pediococcus acidilactici as a reference, which is 0·867 min. For M. cerevisiae, the most heat resistant strain is DSM 20461 with D60-value of 0·55 min in beer. The choice of P. acidilactici as micro-organism reference should not be called into question. However, the sterility of a beer directly depends on the number of micro-organisms. Tsang and Ingledew (Tsang and Ingledew 1982) have introduced the sterility degree, which enables estimation of the contamination risks. The sterility degree corresponds to the probable number of packaging units still containing a live cell which could lead to a bacterial development. The simulation of a fermentation with contamination by Megasphaera showed that the spoilage population can be important. For the strain DSM 20461, an important bacterial population and a high DT-value lead to a low sterility degree. In this case, for a pasteurization at 60°C and for a treatment duration superior to 8 min, the treatment enables to avoid all risks of contamination by Megasphaera. The D70-value shows that a flash pasteurization at 70°C during 1 min might not be sufficient to eliminate all risk of
Heat resistance of Megasphaera cerevisiae
contamination by M. cerevisiae DSM 20461 when this bacterium is already existent before pasteurization. We can, therefore, wonder whether the pasteurization standards are well adapted to this new spoilage bacterium in the case of a flash pasteurization. However, in industry the great majority of beers are filtered before conditioning and pasteurization, which reduces much the number of bacteria in beer. In this case, according to the efficiency of the filtration, the contamination risks are even lower. Pasteurization, as well as flash pasteurization, must be perfectly efficient. It would then be necessary to take as a reference the DT of two micro-organisms, the D60 of P. acidilactici (Tsang and Ingledew 1982) for pasteurization at 60°C and the D70 of M. cerevisiae for pasteurization at 70°C.
Acknowledgements This work was supported by a grant of Region Nord-Pas de Calais.
References Adams, M. R., O’Brien, P. J. and Taylor, G. T. (1989) Effect of the ethanol content of beer on the heat resistance of a spoilage Lactobacillus. J. Appl. Bacteriol. 66, 491–495. Amaha, M. and Ordal, Z. J. (1957) Effects of divalent cations in the sporulation medium on the thermal death rate of B. coagulans var thermacidurans. J. Bacteriol. 75, 596–604. Back, W., Weiss, N. and Seidel, H. (1979) Isolierung und systematische Zuordnung bierscha¨dlicher gramnegativer Bakterien. II: Gramnegative anaerobe Sta¨bchen/Anhang: Aus Bier isolierte gramnegative fakuitativ Sta¨bechen. Brauwissenschaft 32, 233–238. Barille`re, J. M., Mimouni, A., Dubois, C. and Bidan, P. (1985). Thermore´sistance de souches de levures isole´es de vins. Sci. Aliments. 5, 365–378. Del Vecchio, H. W., Dayharsh, C. A. and Baselt, F. C. (1951) Thermal death time studies on beer spoilage organisms. Am. Soc. Brew. Chem. Proc. 40–50. Findlay, W. P. K. (1971) Modern Brewery Technology. London, The MacMillan Press. p. 214. Gibson, B. (1973) The effect of high sugar concentrations on the heat resistance of vegetative
micro-organisms. J. Appl. Bacteriol. 36, 365–376. Haı¨kara, A. (1985) Detection of anaerobic, Gramnegative bacteria in beer. Monatsschrift fu¨r Brauwissenschaft. 6, 239–243. Haı¨kara, A. and Lounatmaa, K. (1987) Characterisation of Megasphaera sp., a new anaerobic beer spoilage coccus. EBC Congress. 21st, 473–480. Hansen, N. H. and Riemann, H. (1963) Factors affecting the heat resistance of nonsporing organisms. J. Appl. Bacteriol. 26, 314–333. Ingram, M. (1969) Sporeformers as food spoilage organisms. In The Bacterial Spore (Eds Gould, G. W. and Hurst, A.), pp. 549. New York, Academic Press. Linton, R. H., Webster, J. B., Pierson, M. D., Bishop, J. R. and Hackney, C. R. (1992) The effect of sublethal heat shock and growth atmosphere on the heat resistance of Listeria monocytogenes. Scott A. J. Food Protect. 55, 84–87. Morgan, R. W., Christman, M. F., Jacobson, F. S., Storz, G., and Ames, B. N. (1986) Hydrogen peroxide-inducible proteinsin Salmonella typhimurium overlap with heat shock and other stress proteins. Proc. Nat. Aca. Sci. 83, 8059–8063. Schmid, C. F. (1957) Thermal resistance of microorganisms. In Antiseptics, Disinfectants, Fungicides and Sterilization, (2nd ed) (Eds Reddish, G. F.) Philadelphia, Lea and Febiger Simpson, W. J. (1993) Studies on the sensitivity of lactic acid bacteria to hop bitter acids. J. Inst. Brew. 99, 405–411. Soberka, R., Sciazko, D. and Warzecha, A. (1989) Pectinatus, nouvelle bacte´rie pouvant affecter la stabilite´ biologique du mouˆt et de bie`re. Biosynthesis 20, 51–59. Sokal, R. R. and Rohlf, F. J. (1969) Biometry, the principles and practice of statistics. In Biological Research. (Eds Freeman W. H. and Co.) San Francisco. Stumbo, C. R. (1973) Thermobacteriology. In Food Processing. (2nd ed). New York, Academic Press. Sugiyama, H. (1951) Studies on factors affecting the heat resistance of spores of Cl. Botulinum. J. Bacteriol. 62, 81–96. Tsang, E. W. T. and Ingledew, W. M. (1982) Studies on the heat resistance of wild yeasts and bacteria in beer. Am. Soc. Brew. Chem. 40, 1–8. Watier, D., Leguerinel, I., Hornez, J. P. and Dubourguier, H. C. (1993) Influence de facteurs physico-chimiques sur les cinetiques de croissance de Pectinatus cerevisiiphilus et de Megasphaera cerevisiae. Sci. Aliments 13, 297–304. Watier, D., Leguerinel, I., Hornez, J. P., Chowdhury, I. and Dubourguier, H. C. (1995) Heat resistance of Pectinatus sp., a beer spoil-
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age anaerobic bacterium. J. Appl. Bacteriol. 78, 164–168. Weiss, N., Seidel, H. and Baack, W. (1979) Isolierund und systematische Zuordung bierscha¨d-
licher gramnegativer bakterien. I: Gramnegative strikt anaerobe Kokken. Monatsschrift fu¨r Brauwissenschaft. 32, 189–194.
ORIGINAL ARTICLE
Survival of Megasphaera cerevisiae heated in laboratory media, wort and beer D. Watier*1, I. Chowdhury1, I. Leguerinel2 and J. P. Hornez1
The heat resistance of two strains of spoilage bacteria (Megasphaera cerevisiae DSM 20461 and Megasphaera cerevisiae DSM 20462) was measured at different temperatures (50–60°C). The values of D50 are lower at pH 5·2 and 6. At pH 4, the heat resistance is 4·2 times higher. With values of 0·55 mn for the D60 in wort and beer, DSM 20461 was the more resistant strain. In MRS medium, ethanol and hop bitter acids decreased the thermal resistance. The results were discussed in relation to factors affecting heat resistance and pasteurization regimes in brewery. For a treatment of 8 min at 60°C and initial population of 1·3× 106 bacterial ml−1, the sterility degree is 2·7×105 i.e. one bottle out of 2·7×105 can be contaminated by Megasphaera. For a flash pasteurization (1 min, 70°C), the sterility degree was of 4·5×102, i.e. one bottle out of 4·5×102 bottles were spoiled. 1996 Academic Press Limited
Introduction The quality of beer is determined by the physicochemical stability and biological stability. The growth of micro-organisms induces unpleasant cloudiness accompanied by offflavor. Beer pasteurization is a common practice which guarantees the biological stability of the product and makes its shelf life longer. The scale of pasteurization usually used enables the destruction of all viable forms of life in beer. In brewing literature, the most resistant organisms are different according to the authors; but in practice, the standard pasteurization corresponds to a treatment between 8 and 30 pasteurization units (PU) (Findlay 1971). One PU is equivalent to a heating at 60°C for 1 min (Del Vecchio et al. 1951). This treatment is based on the heat resistance of *Corresponding author. 0740-0020/96/030205+08 $18.00/0
the most heat resistant spoilage organisms. However, the latter must be limited in order to reduce the creation of off-flavors and energy consumption. In practice, the standards of pasteurization have been established thanks to the z value. The formula used gives a lethality rate (Lt) according to z and to the treatment duration (t). This equation is as follows Received: 28 July 1995
Lt (°C)=1/(log−1((60−t)/z)) The recommended z value by Del Vecchio et al. (1951) was 6·94°C. However, it has been proved (Sokal and Rohlf 1969) that the use of z could bring important mistakes in the calculation of Lt. Ingram in 1969 (Ingram 1969) and Tsang and Ingledew in 1982 had already showed that the use of z was not well adapted to established scales of pasteurization in breweries. These authors have suggested to use the inactivation factor (IF) characterized by the treatment duration t and the DT. The
1
Universite´ des Sciences et Technologies de Lille, Laboratoire de Micobiolgic SN2, 59655 Villeneuve DAscq Cedx, France 2 IUT De´partement de Biologie, 2 Rue de l’Universite´, BP319, 29191 Quimper Cedex, France
1996 Academic Press Limited
206 D. Watier et al.
IF is the factor by which the micro-organism population is divided in a treatment of a duration t. The IF is defined by the following relation IF=10 (t/DT) The main beer spoilage are lactic acid bacteria such as Lactobacillus. Moreover, for the last 20 years, new bacteria such as Megasphaera cerevisiae have been encountered. M. cerevisiae is a strictly anaerobic bacteria which was isolated for the first time in Germany by Weiss et al. in 1979 (Weiss et al. 1979, Back et al. 1979). Since then, the appearance of Megasphaera has been described by Haı¨kara (Haı¨kara 1985, Haı¨kara and Lounatmaa 1987) in Finland. When this bacteria develops in beer, it provokes, a synthesis of volatile fatty acids and especially butyric acid in addition to cloudiness. M. cerevisiae is more sensitive to oxygen than other strictly anaerobic spoilage bacteria, such as Pectinatus. However, Megasphaera can develop in beer (Haı¨kara and Lounatmaa 1987). The oxygen reduction during processing can favor the growth of this micro-organism. Megasphaera was isolated in beer, in the piping and in the surrounding air of a brewery (Soberka et al. 1989). The supposed presence of Megasphaera in wort during the fermentation, and an inappropriate standard of pasteurization can explain the increase of the number of contaminations by this bacteria. The growth can also be favored by the marketing of alcohol-free beers and of beers with a highresidual carbohydrate level in which the heat resistance is generally higher (Gibson 1973). This paper reports on the heat resistance of two strains of M. cerevisiae in different media (MRS, wort and beer), and different environmental and physiological conditions (pH, hop bitter acids and alcohol).
Materials and Methods Micro-organisms and media The two studied strains, M. cerevisiae DSM 20461 and DSM 20462 come from the international collection Deutsche Sammlung von
Mikro-organismen und Zellkulturen (Brounsweig, F.R.G.). Three media were used: a modified MRS medium (Man–Rogosa–Sharpe) (Watier et al. 1995) (2 g l −1: glucose), wort (8·7% Plato from GBM, Roubaix, France) and beer (3·3% v/v of ethanol). The beer was obtained by fermentation of wort during 8 days at 8°C and then 30 days at 2°C. The influence of ethanol and hop on the heat resistance was determined in two distinct media. The medium containing ethanol was prepared by the volumetric addition (3·3% v/v) of absolute ethanol to the MRS medium after sterilization. In the medium containing hop, 0·25 g of dehydrate hop was introduced in 1 l of water. After being boiled for 1 h, this solution was used for the preparation of the modified MRS medium. For every medium used, the initial pH values were adjusted with NaOH 1M or H2SO4 1M at pH 6 for MRS, pH 5·2 for wort and pH 4·2 for beer. The pH influence on heat resistance is tested for pH 4, 5·2, 6 and 6·3. The media (19·5 ml) were dispensed in 25 ml antibiotic flasks flushed continuously with N 2/CO 2 (85/15, v/v). Na2S (0·5 g l−1 final) and resazurin (0·5 ml of a 5% stock solution) were added, just before sealing with butyl rubber stoppers. The different media were sterilized at 105°C for 30 min.
Heat stress experiments In all cases, the medium used as an inoculum was identical to the one used for the experiment. An inoculum of each strain was prepared by growing the organisms during 3 days (stationary phase) (Watier et al. 1993) at 32°C, and subsequently, used for heat test. The cell concentration in inoculum was adjusted at 109 cells ml−1. Uninoculated flasks were submerged in a circulating water-bath equipped with a thermoregulator (±0·1°C) (Julobo EM) at temperatures of 45, 50, 55, 60°C in MRS and wort, 48, 52, 56, 60°C in beer. After 20 min of equilibration, the flasks were inoculated with 0·5 ml of inoculum. The flasks were continually shaked to prevent flocculation. The tubes (8×160 mm) containing MRS agar (4·5 ml) were used to determine the formation of col-
Heat resistance of Megasphaera cerevisiae
ony-forming units (cfu). Samples (0·5 ml) were taken and immediately transferred in a tube equilibrated to 45°C. After serial dilutions (1/10) from one tube to another, these tubes were immediately water-cooled and then were incubated at 32°C for 5 days. The experiments at different temperatures were replicated in beer medium for the two strains.
using the sterility degree expression i.e. sterility degree=IF/(average number of organisms by litre), experimentally determined values for IF and the micro-organism concentration found in the fermentation trial. The number of organisms per litre used was 1·3× 106 bacteria ml−1.
Results
Treatment of data The DT-values (i.e. the decimal reduction time) is the necessary time to reduce the number of surviving bacteria by 10 at determined temperature. The DT is independent from the number of initial organisms. Consequently, it is possible to represent the survival curve according to the percentage of cfu ml−1. The z-value is the necessary rise of temperature to reduce the DT-values by 10. The DT-values were calculated by linear regression from the survival curves for the different strains (t=D lg (NO/N)). The same method was used to determine the z-values (T=z lg (D O/D)). The IF-values (inactivation factor) were calculated with the following equation: IF=10 (t/DT). To calculate the IF in case of classical pasteurization, the treatment duration value used was 8 min with D60-value of 0·55 min (DSM 20461) and 0·19 min (DSM 20462). In case of flash pasteurization, the treatment duration value used was 1 min with D70-value of 0·085 min (DSM 20461) and 0·024 min (DSM 20462). The sterility degree was calculated
Whatever the medium used, the M. cerevisiae survival curves heated at different temperatures follow general laws of vegetative cell thermal destruction (see Fig. 1 for example). The DT-values of two Megasphaera strains in three media were shown in Table 1. The method does not enable the determination of the D70 values because of lack of experimental points. Therefore, we have had to extrapolate the D70 -values. Through a calculation from the D70-values with the equation: T=z lg (DO/D) For most of the cases, the correlation coefficients for DT and z were higher than 0·97. The DSM 20461 seems slightly more resistant than the DSM 20462 at 60°C (Fig. 2). With a value of 0·55 mn for the D60 in wort and beer media, M. cerevisiae DSM 20461 was the most resistant strain. For DSM 20461, the DT-values were similar in wort
2 1 –1
A fermentor (Setric set 2M, SGI, France) containing 1·5 l of sterile wort, was prepared. The wort was kept at 8°C and saturated by oxygen during 20 min. The wort was inoculated with Saccharomyces (107 cells ml−1) and Megasphaera (104 cells ml−1). After a fermentation at 8°C during 8 days, viable cells of Megasphaera were counted using the deep agar counting method using MRS agar (12 g l−1 agar, 50 mg l−1 of cycloheximide) incubated at 32°C for 5 days.
log (% of cfu ml )
Simulation of contamination
0 –1 –2 –3 –4 –5 –6 –7
0
Figure
2
4
6 8 10 12 Heating time (min)
14
16
1. The survival of Megasphaera cerevisiae DSM 20461 in beer at 48°C (j), 52°C (h), 56°C (m) and 60°C (n).
207
208 D. Watier et al.
and beer, but decreased in MRS medium. The differences between the DT-values in wort and in MRS medium were not significant. In wort for different pH, the values of D50 are widely variable (Table 2 and Fig. 3). The values of D50 are lower for the pH 5·2 and 6. For pH 4, the heat resistance is 4·2 times higher. Whatever the z-values, the heat resistance of the DSM 20461 strain was superior to the DSM 20462 one. Moreover, the z-values varied according to the medium tested. The simulation of a wort fermentation by Saccharomyces with contamination by Megasphaera shows that the final population can
reach 1·3×106 bacteria ml−1 for a beer at 3·3% v/v of ethanol. Table 3 shows the IF and sterility degree for a spoiled non-filtered beer. In case of classical pasteurization (i.e. 8–20 min at 60°C), the sterility degree is rather high. For a treatment of 8 min at 60°C, the sterility degree is 2·7×105 i.e. one bottle out of 2·7×10 5 may be contaminated by Megasphaera DSM 20461. For a flash pasteurization (1 min at 70°C), the sterility degree is much lower. In this case, the sterility degree was 4·5×102, i.e. one bottle out of 4·5×102 bottles were spoiled. The IF and sterility degree obtained for the DSM 20462 strain were lower, especially for
Table 1. Estimated values of D50 and z for the destruction of Megasphaera cerevisiae at pH 6 in MRS, pH 5·2 in wort and pH 4·1 in beer Temperature (°C)
DT (min)
r2
δ
z (°C)
r2
δ
45 50 55 60
64·18 8·57 2·87 0·36
0·98 0·98 0·99 0·92
6·94 0·54 0·13 0·05
6·82
0·99
0·55
Wort
45 50 55 60
39·22 6·13 1·07 0·55
0·99 0·99 0·99 0·98
2·61 0·47 0·05 0·04
7·62
0·96
1·05
Beer
48 52 56 60 70
10·65
0·97
1·23
Strain and Medium Megasphaera cerevisiae DSM 20461 MRS
Megasphaera cerevisiae DSM 20462 MRS
7·11 2·78 1·06 0·55 0·085*
0·97 0·78 0·99 0·038 0·99 0·020 0·99 0·065 (extrapolation)
45 50 55 60
13·76 3·68 2·15 0·25
0·98 0·99 0·97 0·97
0·97 0·19 0·28 0·029
9·16
0·97
1·35
Wort
45 50 55 60
19·96 2·28 0·69 0·36
0·98 0·99 0·97 0·97
1·58 0·038 0·12 0·08
8·69
0·99
0·51
Beer
48 52 56 60 70
0·99 0·15 0·98 0·11 0·99 0·039 0·97 0·021 (extrapolation)
11·17
0·97
1·52
2·2 1·17 0·48 0·19 0·024*
*Theoretical value calculated with the formula: log (D)=T/z+constant.
Heat resistance of Megasphaera cerevisiae
a pasteurization at 60°C. For a flash pasteurization, the risk was one spoiled bottle out of 3·1×1032.
resistance (i.e. linearity of survival curves), it is not a general rule for all the micro-organisms (Tsang and Ingledew 1982, Barille`re et al. 1985). The main factors that affect the shape of the survival curve are the physicoch-
Discussion 1.60
If the heat resistance of M. cerevisiae is in accordance with the classical laws of heat
1.20
1.3
1.00 log D
1.1 0.9
0.79
0.80
0.77
0.60
0.7 log D
1.48
1.41 1.40
0.40
0.5
0.20
0.3
0.00
0.1
4
5.2
5.97
6.3
pH
–0.1 –0.3
Figure 3. Values of D50 for the destruction of
–0.5 45
60
50 55 Temperature (°C)
Megasphaera cerevisiae DSM 20461 at different pHs in wort.
Figure 2. D-values of Megasphaera cerevisiae
2
DSM 20461 (j) and DSM 20462 (h) in beer at different temperatures.
1
log (% of cfu ml )
0 –1
Table 2. Estimated values of D50 (±δ) for the destruction of Megasphaera cerevisiae DSM 20461 at different pHs in wort and with hop or ethanol in MRS δ
2
Medium
pH
D50 (min)
r
Wort
4 5·2 6·0 6·3
25·89 6·20 5·93 30·11
0·99 0·94 0·95 0·99
MRS
8·13
0·99
MRS+Hop MRS+Ethanol 3·5% v/v
2·76 3·92
0·98 0·98
0·50 2·01 1·05 0·27 0·0079 0·025 0·024
–1 –2 –3 –4 –5 –6 –7
0
10 Heating time (min)
20
Figure 4. The survival of Megasphaera cerevisiae DSM 20461 at 50°C, in MRS (j), with hop (m) or ethanol (3·3% v/v) (d).
Table 3. Inactivation factor and sterility degree of Megasphaera cerevisiae DSM 20461 and DSM 20462 in beer for different conditions of pasteurization Pasteurization (8 min, 60°C)
DSM 20461 DSM 20462
Flash pasteurization (1 min, 70°C)
Inactivation factor
Sterility degree (for 1 litre)
Inactivation factor
Sterility degree (for 1 litre)
3·5×1014 1·3×1042
2·7×105 9·8×1033
5·8×1011 4·6×1041
4·5×102 3·1×1032
209
210 D. Watier et al.
emical composition of the medium and the physiological state of cells (Barille`re et al. 1985). To avoid the deviation curve and enable the determination of DT, a pre-culture media similar to the one used in the experiments, as well as an homogenate inoculum taken at the beginning of the stationary phase were used. It is well known that the heat resistance is very sensitive to the variations of the environmental conditions (Schmid 1957). Oxygen (Linton et al. 1992), low pH (Hansen and Riemann 1963, Stumbo 1973), the presence of peroxides (Linton et al. 1992), ethanol (Adams et al. 1989) and of hop bitter acids (Adams et al. 1989) generally decrease the thermal resistance. Moreover, the heat resistance could be affected by the presence of salt (Stumbo 1973), cations and phosphate ions (Sugiyama 1951, Amaha and Ordal 1957), oxygen (Linton et al. 1992) or a higher sugar concentration (water activity) (Gibson 1973). In order to determine pasteurization standard it was essential to determine the DT-values in the same physico-chemical environment as that encountered in breweries. For this reason, we used the wort and beer medium. The MRS medium was used as a reference medium. Na 2S was introduced in media as a reducing agent to eliminate traces of molecular O2. Moreover, the advantage of using Na2S is that it does not lead to the formation of peroxides (Morgan et al. 1986). The method used for heat stress experiments is not the conventional method. This method derives from the one used by Barille`re et al. (1985). Its advantage is that it reproduces better the pasteurization conditions i.e. pasteurization of packaged beer under hot water. Whatever the temperatures lower than 70°C, the DT-values of the DSM 20461 strain are higher than these of the DSM 20462 strain. M. cerevisiae DSM 20461 is the strain that might be the most bothering for the brewer, as far as pasteurization is concerned. For that reason, we have studied that strain more specifically. Generally, the heat resistance of the micro-organisms is lower in acid medium than in neutral pH medium (Hansen and Riemann 1963, Stumbo 1973). For M. cerevisiae the opposite is observed in wort. The initial
pH of wort at the beginning of the fermentation corresponds to the pH for which the heat resistance is the highest. The presence of hop bitter acids which have an antibacterial activity (Simpson 1993) and of ethanol, affects the heat resistance. Adams et al. (1989) suggest, the addition of hops for lower the heat resistance in the alcohol-free beer. Similar results have been obtained with Lactobacillus when hop extracts or ethanol are added to alcohol-free beer (Adams et al. 1989). We can suppose that the opposite effect of hop and pH on the heat resistance have a cancelling effect on one another. This study shows that M. cerevisiae can grow during fermentation. Indeed, the low D60-values in wort show that Megasphaera cannot survive when wort is boiled before fermentation. In these conditions, the contamination can only occur once the wort has been boiled. The optimization of heat treatment in breweries requires the knowledge of D60 of the most heat resistant micro-organisms in beer. Tsang and Ingledew (1982) suggest the D60 of Pediococcus acidilactici as a reference, which is 0·867 min. For M. cerevisiae, the most heat resistant strain is DSM 20461 with D60-value of 0·55 min in beer. The choice of P. acidilactici as micro-organism reference should not be called into question. However, the sterility of a beer directly depends on the number of micro-organisms. Tsang and Ingledew (Tsang and Ingledew 1982) have introduced the sterility degree, which enables estimation of the contamination risks. The sterility degree corresponds to the probable number of packaging units still containing a live cell which could lead to a bacterial development. The simulation of a fermentation with contamination by Megasphaera showed that the spoilage population can be important. For the strain DSM 20461, an important bacterial population and a high DT-value lead to a low sterility degree. In this case, for a pasteurization at 60°C and for a treatment duration superior to 8 min, the treatment enables to avoid all risks of contamination by Megasphaera. The D70-value shows that a flash pasteurization at 70°C during 1 min might not be sufficient to eliminate all risk of
Heat resistance of Megasphaera cerevisiae
contamination by M. cerevisiae DSM 20461 when this bacterium is already existent before pasteurization. We can, therefore, wonder whether the pasteurization standards are well adapted to this new spoilage bacterium in the case of a flash pasteurization. However, in industry the great majority of beers are filtered before conditioning and pasteurization, which reduces much the number of bacteria in beer. In this case, according to the efficiency of the filtration, the contamination risks are even lower. Pasteurization, as well as flash pasteurization, must be perfectly efficient. It would then be necessary to take as a reference the DT of two micro-organisms, the D60 of P. acidilactici (Tsang and Ingledew 1982) for pasteurization at 60°C and the D70 of M. cerevisiae for pasteurization at 70°C.
Acknowledgements This work was supported by a grant of Region Nord-Pas de Calais.
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