Inactivation of Lactobacillus brevis in Beer Utilizing a Combination of High-Pressure Homogenization and Lysozyme Treatment

M. A. Franchi*, A. A. L. Tribst and M. Cristianini

ABSTRACT

J. Inst. Brew. 117(4), 634638, 2011 Inactivation of Lactobacillus brevis in beer by high-pressure homogenization (HPH) and lysozyme addition was evaluated. The minimum inhibitory concentration of lysozyme against L. brevis was determined and found to be 100 mg.L1. The effects of the homogenization process on lysozyme antimicrobial activity and muramidase activity, and the microbial reduction promoted by HPH, and by HPH associated with lysozyme, were evaluated. A significant reduction in lysozyme muramidase activity was observed under 250 MPa, however, no reduction in antimicrobial activity was observed after homogenization up to 300 MPa. The HPH at 100, 140 and 150 MPa promoted 1, 3 and 6 decimal reductions in L. brevis microbial counts, respectively. The HPH and lysozyme association had an additive effect on microbial inactivation, immediately after homogenization, and the lysozyme remained active during 10 days of storage, increasing the inactivation of L. brevis up to a 6 decimal reduction. Therefore, the application of lysozyme with HPH has the potential to reduce the level of pressure required for beer processing, improving the economic costs when utilizing HPH. Key words: beer, dynamic high pressure, hurdle, shelf life.

INTRODUCTION
Pilsen beer is the most popular and widely accepted type of beer in the Brazilian market. It is characterized as a light-bodied beer, with a light-yellow colour, an alcoholic percentage of around 4.5GL and with good foam stability. Common microbial contaminants that may be found in beer include lactic acid bacteria (Lactobacillus and Pediococcus), acetic bacteria (Acetobacter and Acetomonas), Pectinatus, Zymomonas, Megasphaera, Micrococcus and wild yeasts29. Among these contaminants, L. brevis is the main spoilage microorganism of fermented beverages, being responsible for 40% of total product deterioration10 and capable of producing off-flavours and turbidity in beer1.

Faculty of Food Engineering, Department of Food Technology, University of Campinas, UNICAMP, P.O.Box. 6121, 13083-862, Campinas, S.P. Brazil. * Corresponding author. E-mail: franchi.mark@uol.com.br
Publication no. G-2012-0207-1174 2011 The Institute of Brewing & Distilling

Industrially, beer is often pasteurized in order to guarantee microbiological stability during its expected shelflife31. However, the heat can cause protein denaturation, promoting the formation of new tannin-protein complexes, with consequent turbidity enhancement23. In addition, heat processing promotes the Maillard reaction, resulting in an alteration of beer colour to a reddish hue4 and the formation of undesirable flavours23. These are related to oxidation and staling9, with the development of paper and cardboard flavours (long chain aldehydes)31. Additionally, the heating process of beer results in the isomerisation of hop -amino acids, which increases beer bitterness2. Hence, the pasteurization process promotes intense changes in the sensory quality of the product. On the other hand, consumers are requesting food and beverages similar to unprocessed ones, and this is promoting the development of non-thermal techniques to ensure food preservation25. Following this request, the application of antimicrobials seems to be effective in inactivating or delaying the growth of beer contaminants21,22, increasing the beer shelf-life up to a month with no negative sensory changes22. Lysozyme is an enzyme extracted from egg white and is able to lyse the peptidoglycan from the Gram positive bacterial cell wall at the (1-4)-N-acetylmuramic and N-acetyl-glucosamine linkage, thus causing cellular death of such organisms18. Also with respect to its enzymatic activity (muramidase), it has been related by some authors that lysozyme remained active against microorganisms even after its denaturation12,20. High pressure homogenization (HPH) is a continuous process in which fluid is forced under pressure through a narrow gap, where it is subjected to rapid acceleration (200 m/s at 340 MPa)7, after which it undergoes an extreme drop in pressure7, causing shear, cavitation, turbulence and friction14,24, which can promote microbiological inactivation3,14,2426 and molecular conformational changes mainly in proteins17 and polysaccharides14. Its effect on beer quality has indicated that HPH was able to improve the beverage color, although no improvements in oxidation and haze were observed8. A synergistic effect between HPH and antimicrobials has been previously reported5,13,24,29. Also, an improvement in lysozyme activity was observed after homogenization13,28. Therefore, the aim of the present study was to evaluate the inactivation of L. brevis in a high pressure homogenized beer, supplemented with lysozyme.

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MATERIALS AND METHODS

Beer and lysozyme Pasteurized lager beer (pH 4.5, 4.7GL, 2.5B) was obtained from a Brazilian brewery. The beer was degassed and heat treated at 121C/15 min. Lysozyme was obtained from the commercial formula (Novagard) from DANISCO S.A.S. (Cotia, Brazil). Bacteria and growth medium Lactobacillus brevis (CCT 3745) was obtained as a donation from the Tropical Culture Collection/Tropical Research and Technology Foundation (Campinas, Brazil). L. brevis was grown in OXOID (Cambridge, UK) lactobacillus media (MRS - De Man, Rogosa, Sharpe - agar and broth) and preserved in litmus milk (DIFCO - Lawrence, USA). High pressure homogenizer The high-pressure treatments were performed in a Stansted homogenizer, model FPG 7400H:350 (STANSTED Fluid Power LTD. Essex, UK) at pressures from 0 to 300 MPa, with a flow rate of approximately 270 mL.min1. A heat exchanger (SPIREC) for cooling was connected to the homogenizer to reduce the temperature of the fluid exiting the homogenizer valve. The heat exchanger outlet was connected to an aseptic collection system. Lysozyme minimum inhibitory concentration (MIC) The MIC determination was performed as previously described24. It was tested using lysozyme concentrations ranging from 0.5 to 1,000 mg.L1. The MIC value was the lowest concentration of lysozyme that showed inhibition of bacterial growth in 24 h. The tubes were inoculated for 48 h to evaluate if lysozyme inhibition was permanent or transitory. Residual activity of lysozyme after HPH Beer containing 50 mg.L1 of lysozyme was homogenized at 100, 150, 200, 250 and 300 MPa. The residual activity of lysozyme (muramidase) was measured by the turbidity decrease of Micrococcus lisodeikticus24. The residual activity was calculated following equation 1. The tests were performed in triplicate. % Residual activity = (activity of homogenized lysozyme/activity of unprocessed lysozyme) 100 (1) The antimicrobial activity of unprocessed and homogenized lysozyme at 150 and 300 MPa was measured using the L. brevis growth method24, with beer as growth media. A control assay was performed by inoculation of a target microorganism in beer with no lysozyme. The tests were performed in triplicate. Effect of HPH on target microorganism A suspension of L. brevis was inoculated in beer at 106 CFU.mL1 and then homogenized at 60, 100, 140, and 150 MPa. Following these treatments, the samples were plated and incubated at 30C/48 h. The initial (control)

microorganism count was determined by direct plating of the inoculated beer. Results were evaluated by the number of decimal reduction (NDR), of organisms following equation 2. NDR= log (initial count) log (survivors count) (2) Effect of lysozyme and HPH on the target microorganism The combination of lysozyme (50 mg.L1) and HPH was evaluated using homogenization pressures of 100 and 140 MPa, which were pressures not able to reduce the total microbial load. L. brevis suspension was inoculated at 106 CFU.mL1 in three litres of beer previously dosed with 50 mg.L1 of lysozyme. The samples were kept at 25C for 1 h to allow lysozyme action. Samples were then homogenized and collected in a horizontal laminar flow cabinet and packaged in 355 mL dark glass bottles, previously flushed with CO2, and capped with metal lids. Then, bottles were stored 10 days at room temperature (25C). The L. brevis counts were performed after beer inoculation, after one hour of lysozyme action, after homogenization and after the storage. NRD of each step was calculated following equation 2. Assays were performed in triplicate.

RESULTS AND DISCUSSION

The MIC of lysozyme against L. brevis was determined as 50 mg.L1 after 24 h. When samples were re-incubated for an additional 24 h, it was seen that at this lysozyme concentration, L. brevis was able to grow, showing that the initial 24 h inhibition was only transitory. A permanent L. brevis inhibition was however observed at 100 mg.L1 of lysozyme concentration. Previous results evaluated the effect of lysozyme in controlling beer spoilage, showing that the antimicrobial action was not only capable of slowing19 or inhibiting22 the growth of beer contaminants, but was also able to inactivate microorganisms22, thus increasing the shelf life of non-pasteurized beer over one month with no sensory changes22. These data highlight the potential application of the use of lysozyme in beer. Previous results indicated that HPH can promote activation, stabilization15,16,17,27 or inactivation14,30 of enzymes, with the effects being specific for each enzyme present and the level of applied pressure15,16. The enzymatic activity (muramidase) after lysozyme homogenization in beer is shown in Table I. The results showed that homogenization negatively affected the enzymatic activity of lysozyme at pressures above 200 MPa, reducing to around 50% the muramidase activity at 300 MPa. Similar results were previously obTable I. Enzymatic activity of lysozyme after homogenization in beer. Pressure (MPa) 0 100 150 200 250 300 Residual activity (%) 100 1.7 90.2 12.2 87.8 16.0 93.0 16.1 84.8 4.7 52.9 11.2

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served for lysozyme homogenized in phosphate buffer24, indicating that the homogenization effects on lysozyme were similar in different solutions. Some authors11,20,24 have reported that lysozyme antimicrobial effects remained active even after muramidase inactivation. To evaluate if this prerogative was valid for lysozyme added into the beer, the L. brevis lag phase was studied in homogenized beer with previously added lysozyme. The results obtained showed no differences between the lag phase of L. brevis (about 12 h) in beer treated with lysozyme, and homogenized lysozyme at 150 or 300 MPa. In contrast, a lag phase of about 3 h was observed for the control sample with no addition of lysozyme. These results indicate that although HPH inactivates muramidase activity, the lysozyme antimicrobial mechanism continues, corroborating previous results6,24. The results of L. brevis inactivation on beer by high pressure homogenization are shown in Table II. Results showed no significant inactivation of the microorganism at 60 MPa, but partial inactivation at 100 and 140 MPa, with 1 and 3 decimal reduction, respectively; and total load inactivation at 150 MPa (>6 decimal reduction). The temperature reached during HPH at pressures up to 150 MPa was less than 50C, therefore the inactivation of L. brevis cannot be attributed to heating. The results showed a non-linear relationship between the pressure increase and the rate of microbial inactivation. This nonlinear be-

Table II. L. brevis inactivation in beer by high pressure homogenization. Pressure (MPa) 0 60 100 140 150
a NDR

NDRa 0.00 0.00 0.07 0.02 1.01 0.16 2.96 0.29 >6.40

- number of decimal reduction

havior was previously observed by other authors3,24,26 and probably indicates a requirement of a specific pressure homogenization to inactivate each type of microorganism. Considering the results obtained for the inactivation of L. brevis, it was concluded that beer contamination by L. brevis could be controlled by addition of lysozyme at a concentration of 100 mg.L1 or by applying pressures >150 MPa. The combination of lysozyme and HPH might possibility reduce the pressure level required to obtain an effective inactivation of L. brevis. This could be very interesting since homogenizer costs grow exponentially with the linear increment of the pressure operation26. Therefore, the effect on beer microbial stabilization by a combination of 50 mg.L1 of lysozyme and HPH at 100 and 140 MPa was evaluated and results are illustrated in Fig. 1. It was shown that lysozyme promoted around a 0.5 decimal reduction in L. brevis counts and HPH promoted 1 and 4 decimal reductions at 100 and 140 MPa, respectively. Therefore, the addition of lysozyme into beer contaminated with L. brevis did not sensitize the microorganism to the homogenization process, it was an additive effect of lysozyme and HPH. Previous research has reported synergistic effects between these processes, and attributed them to increased cell permeability during pressurization5. This synergistic effect, however, was only observed for E. coli, when homogenized at pressures above 150 MPa5, which perhaps suggests the existence of a minimum pressure required to change cellular permeability. Despite the small inactivation of L. brevis in beer observed just after lysozyme addition, during prolonged storage of lysozyme-treated beer, count reductions of L. brevis were observed, indicating that the lysozyme remained active. In addition, the L. brevis inactivation rate during storage in the samples treated with lysozyme, followed by HPH at 100 MPa, was equal to the inactivation

Fig. 1. Inactivation of L. brevis in beer by a combination of lysozyme (Lz) and HPH treatments.
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rate of this microorganism when submitted only to lysozyme treatment. This suggests that the homogenization at 100 MPa process neither increased nor decreased the antimicrobial activity of lysozyme. Similar results were observed for Listeria monocytogenes in milk13 and for L. monocytogenes and E. coli in skim milk28. For Salmonella enteritidis, however, there was no increase in the inactivation after homogenization. Also, no increase in L. brevis inactivation was observed following a similar process to the one described in this paper, though in phosphate24. Thus, the continued microbial inactivation by lysozyme treatment is dependent on the microorganism species and its natural susceptibility to the antimicrobial, as well as the characteristics of the environment where it was found. Finally, it is possible to conclude that lysozyme alone, at a concentration of 50 mg.L1, may be able to inactivate L. brevis in beer after 10 days of storage, depending on the initial load. On the other hand, to guarantee a 6 decimal reduction, it is possible to apply 100 MPa associated with 50 mg.L1 of antimicrobial.

CONCLUSIONS
Beer spoilage by L. brevis can be controlled by the addition of lysozyme (100 mg.L1) or by applying a homogenization pressure treatment at 150 MPa. To reach a 6 decimal reduction, it was also possible combine both processes, applying lysozyme at concentration of 50 mg.L1, followed by homogenization at 100 MPa. This reduction in the level of pressure required (150 to 100 MPa) will allow for the use of a less expensive homogenizer and easier operation, both of which are needed for the development of cost-effective commercial applications.
ACKNOWLEDGEMENTS

The authors would like to thank the So Paulo Research Foundation (FAPESP) for financial support (project # 2001/06872-2) and the Brazilian National Research Council (CNPq) for providing a fellowship award to MA Franchi.
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(Manuscript accepted for publication January 2012)

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