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Effects of Lysozyme on the Microbiological Stability and Organoleptic Properties of Unpasteurized Beer

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Effects of Lysozyme on the Microbiological Stability and Organoleptic Properties of Unpasteurized Beer Effects of Lysozyme on the Microbiological Stability and Organoleptic Properties of Unpasteurized Beer T. Silvetti1, M. Brasca1,*, R. Lodi1, L. Vanoni1, F. Chiolerio2, M. de Groot2 and A. Bravi3 ABSTRACT The so-called beer spoilage microorganisms belong to both Gram-positive and Gram-negative bacteria, as well J. Inst. Brew. 116(1), 33–40, 2010 as to wild yeasts. Gram positive beer spoilage bacteria Lysozyme is an antimicrobial enzyme that could be applied to include several species of lactic acid bacteria (LAB), counteract those bacterial species which, due to their own meta- which are reported as the most hazardous bacteria for bolic activity, possess notable beer spoilage ability and lead to breweries as they are responsible for approximately 70% loss of beer quality. Experiments were carried out to assess ly- 3,31 sozyme potential to prevent the growth of beer spoilage bacteria, of all microbial spoilage incidents . Indeed, LAB can be and to verify the effect of lysozyme on the microbiological sta- found in brewing materials at almost every stage of the bility and sensory characteristics of unpasteurized beer. Eight malting and brewing process, from the standing barley replicates, all from the same lot of Italian beer, were treated with crop to the finished beverage13,17,28,33. These bacteria occur 0 and 100 ppm lysozyme. Microbiological analyses were con- as part of the natural barley micro-biota, and persist dur- ducted bimonthly to investigate the presence of spoilage bacte- ing malting and mashing29,35 due to their aero tolerant na- ria. Sensory analyses were performed to determine whether ture and tolerance of low pH and high ethanol levels. there were any significant differences in sensory impressions The genera Lactobacillus and Pediococcus are the pre- between beers produced with and without lysozyme. Lysozyme dominant beer spoilage LAB species20. Among them the exerted a strong inhibitory action on the lactic acid bacteria most important spoilage organisms are, according to (LAB) present in the beer and was very stable throughout the brewing literature, L. brevis, L. lindneri, L. paracolli- shelf life. Sensory tests revealed no unfavourable influence on 2,30,32,34 beer flavour when using lysozyme. Indeed, the shelf life of beer noides and P. damnosus . In addition, L. backi and P. with added lysozyme proved to be extended. Even as late as 1 inopinatus have been reported as potential beer spoil- month after the expiry date it still met with the panellists’ ap- ers7,22,33. A recently characterised strain, P. clausseni, has proval. Lysozyme may be regarded as an effective agent for been shown to spoil beer12. The strictly anaerobic Pectina- preventing microbiological contamination and prolonging the tus and Megasphaera, showing characteristics of both stability of unpasteurized beer. Gram-negative and Gram-positive organisms, are com- Key words: lactic acid bacteria (LAB), lysozyme, shelf life, monly isolated from unpasteurized, packaged beer and unpasteurized beer. brewery environments15,16,25,27. Important Gram-negative contaminants in the context of beer brewing belong to various Enterobacteriaceae, acetic acid bacteria, Seleno- INTRODUCTION monas, and the strictly anaerobic bacteria Zymomonas, Although beer shows notable microbiological stability and Zymophilus23,31. due to its physiochemical properties, certain bacterial Lysozyme (muramidase, EC 3.2.1.17) is an antimicro- species have been recognized as possessing potential beer bial enzyme that can be applied against microbial growth spoilage ability. These contaminants can emerge during in food and beverages19. It occurs in several mammalian the malting and brewing processes, and consequently secretions (milk, saliva, tears) and also in hen egg white, spoil the beer by their own metabolic activity, which leads which represents the raw material of choice for the pro- to turbidity, acidity and undesirable off-flavours such as duction of lysozyme on an industrial scale. Lysozyme is a diacetyl or hydrogen sulphide. These changes have a neg- fully natural product and no solvents are used to extract ative impact on beer quality, with detrimental financial the protein from the albumen. This low molecular weight consequences for the brewing industry. enzyme (14,400 Dalton), which consists of 129 amino acids cross-linked by four disulphide bridges, shows hy- drolytic activity against the β(1→4) glycosidic bond be- N N 1 Institute of Science of Food Production, National Research Coun- tween -acetyl-D-glucosamine and -acetylmuramic acid cil, Milan, Italy. in the cell wall of bacterial species, particularly Gram- 18 2 Fordras S.A., Lugano, Switzerland. positive organisms such as LAB . In the cheese industry, 3 Santa Barbara Food S.r.l., Malthus Bier, Como, Italy. its use is authorised as a bio-protectant to prevent butyric * Corresponding author. E-mail: [email protected]. spoilage, which causes the late blowing of semi-hard cheeses by Clostridium tyrobutiricum8,9; in oenology it Publication no. G-2010-0330-1033 represents an alternative way to limit the proliferation of © 2010 The Institute of Brewing & Distilling LAB spoilage bacteria during winemaking5,14. Its antibac- VOL. 116, NO. 1, 2010 33 terial spectrum is similar to what is described for nisin Belgium and one in Italy, and 37 beer samples were inves- and hop acids, and its price is moderate. tigated. Nisin is the best-known and most studied bacteriocin. All the samples were transported to the laboratory and It is produced by strains of Lactococcus lactis subsp. lac- stored at room temperature, until microbiological and tis, and it is the only one approved for food applications. sensorial analysis. Its numerous applications, as a natural food preservative, include dairy products, canned foods and processed Lysozyme effectiveness to prevent or delay cheese. Nisin is an effective bactericidal agent against growth of beer spoilage microorganisms Gram-positive bacteria and spores since it interferes with To assess lysozyme effectiveness against spoilage bac- the cytoplasmic membrane of susceptible species, leading teria in unpasteurized beers of different origin, obtained to pore formation, and dissipates the proton motive through different production processes, six brewers were force24. However many Gram-positive bacteria have been requested to set up the experimental tests. The samples shown to be resistant to it, due their ability to synthesize were taken from the same production lot and were with or an enzyme, nisinase, which can inactivate the bacteriocin; without lysozyme. moreover the cost of nisin addition to all fermentations The lysozyme was added during the brewery bottling, would be prohibitively expensive1,35. and the samples were kept under normal storage condi- Hop acids, mainly iso-α-acids such as iso-humulones, tions (i.e., those applied by the individual producers) and derive from the flowers of the hop plant. They exert bacte- transferred to the laboratory after different periods of stor- riostatic effects on most Gram-positive bacteria as they age (Table I). Once transferred to the laboratory, the sam- act as proton ionophores and dissipate the transmembrane ples were subjected to analysis within 24 h. pH gradient. Several LAB species, such as Lactobacillus The six brands of unpasteurized beers, each one in- spp., have been found to have acquired beer-spoilage abil- cluding one sample with lysozyme (100–300 ppm ly- ity due to hop resistance genes (horA and horC), and con- sozyme) and one or two samples without lysozyme, were sideration must be given to the fact that altering the hop analysed. composition of beer can have a profound impact on the Two samples of yeast were also received in duplicate, organoleptic and physico-chemical properties of the final with and without lysozyme (220 ppm lysozyme and 37.5 product6,21,22,33,35. ppm nisin for the first one, 300 ppm lysozyme for the The aim of this study was to assess lysozyme potential second). to inhibit, or delay, the growth of spoilage LAB in natu- A pH-meter (HI8418, Hanna Instruments, Woonsocket, rally contaminated beer and, consequently, to test the ef- Rl, USA) was used to measure the beer pH immediately fect of lysozyme on the microbiological stability and sen- before microbiological analysis. The pH electrode sory attributes of unpasteurized beer. Of additional (FC200B, Hanna Instruments) was standardised using two interest was the detection, isolation and identification of buffers (pH 4.0 and pH 7.0). The pH was estimated as the spoilage microorganisms commonly present in beer. mean value of two replicates for each sample. For the microbiological control, conventional methods MATERIALS AND METHODS of incubation on culture media were applied. Decimal dilutions of each previously homogenized Sampling sample were prepared in sterile Ringer’s solution (Schar- Samples of unpasteurized beer were collected from lau Microbiology, Barcelona, Spain) and plated in dupli- four European breweries: two in the Netherlands, one in cate onto specific media for viable counts. For each of the Table I. Lysozyme content and pH value of samples used in the study of lysozyme effectiveness on brewing spoilage microorganisms (L = lysozyme added). Added lysozyme Determined lysozyme Sample Geographic origin Source Storage period (days) (ppm) content (ppm) pH A The Netherlands triple fermented ale 206 - <0.25 4.3 AL The Netherlands triple fermented ale 206 100 100 4.4 B The Netherlands triple fermented ale 206 - <0.25 4.5 BL The Netherlands triple
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