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s vi . C Sou Received 4 September 2008 Keywords: Anti-yeast assay Lager brewing Yeast quality/vitality st is sub and functioning (Casey and Ingledew, 1983; Stewart et al., 1999). During the beer fermentation process the yeast is exposed to barley malt that may contain factors that can induce stress such as anti- microbial peptides (Broekaert et al., 1997) and proteins (Gorjanovic´ et al., 2007). Since most antimicrobial peptides act on the microbial plasmamembrane, the resistance or susceptibility of yeast to barley and Lohner, 2006), damage to the transport systems embedded in themembranes (Guihard et al., 1993) or total membrane disruption resulting in cell lysis (Bechinger and Lohner, 2006). Vitality of all living cells is dependent on membrane integrity and optimal cell functioning and the latter two may be assessed by, for example, simple cell survival and growth assays (i.e. broth dilution assays), the leakage of specific intracellular enzymes such as adenylate kinase out of the cell (Cameron-Clarke et al., 2003), cell capacitance where an applied radio-frequency permits the build up Contents lists availab cr w. Food Microbiology 26 (2009) 192–196 * Corresponding author. Tel.: þ27 21 8085872; fax: þ27 21 8085863. factors (Heggart et al., 1999) such as osmotic pressure, high ethanol and carbon dioxide concentrations (Hammond, 1999; White et al., 2003) and stress due to yeast handling in the brewery that include mechanical stress (Stafford, 2003), high physical pressure and oxidative stress (Martin et al., 2003). These factors impact on the physiological status of the yeast, decreasing the viability (percentage live cells) and the vitality. Vitality is defined as the cell’s ability to withstand stress and still perform (Axcell and O’Çonnor-Cox, 1996; Mochaba et al., 1998). Stress can result in growth inhibition of the yeast, decreased genetic stability and of particular interest in this case; changing cell membrane stability immune/defence system (Broekaert et al., 1997), which is either constitutive or induced to cope with sufficiently pathogenic microbial infestation (Carr and Klessig, 1989) or other forms of stress such as drought, cold or chemical exposure (Torres-Schu- mann et al., 1992). The most common factors associated with plant defence are proteins (Batalia et al., 1996; Boller, 1993; Van Loon and Van Strien, 1999) and peptides (Broekaert et al., 1997; Florack and Stiekema, 1994). When antimicrobial peptides and detergent-like compounds act on the microbial plasma membrane it can lead to leakage of ions, metabolites and in some cases even proteins and other macromolecules by membrane permeabilisation (Bechinger 1. Introduction During the brewing process, yeast E-mail address: mra@sun.ac.za (M. Rautenbach). 0740-0020/$ – see front matter � 2008 Elsevier Ltd. doi:10.1016/j.fm.2008.09.003 that yeast, obtained directly from a brewery, was much more sensitive towards the malt extracts than the same yeast strain propagated in the laboratory. Sensitivity to the malt extracts increased during the course of a laboratory scale fermentation when inoculated with brewery yeast. As the assay was able to differentiate yeast samples with different histories, it shows promise as a yeast quality assay measuring the yeast’s ability to withstand stress which can be equated to vitality. The assay was also able to differentiate between different lager yeast strains of Saccharomyces cerevisiae propagated in the labo- ratory when challenged with a number of malt extracts of varying anti-yeast activity. The assessment of yeast strains in the presence of malt extracts will lead to the identification of yeast strains with improved quality/vitality that can withstand malt-associated anti-yeast activity during brewery fermentations. � 2008 Elsevier Ltd. All rights reserved. ject to ageing and stress malt antimicrobial extracts containing such peptides would relate to yeast vitality. Plants utilise antimicrobial compounds as part of their innate Available online 24 September 2008 towards barley malt extracts with anti-yeast activity was assessed with an optimised assay. It was found Accepted 5 September 2008 membrane damage which is likely to affect yeast vitality and fermentation performance, parameters which are notoriously difficult to analyse. In this work the sensitivity of lager brewery yeast strains Quality assessment of lager brewery yea barley malt extracts with anti-yeast acti Sandra N.E. van Nierop a,b, Barry C. Axcell a,c, Ian C aGroup Brewing Research, SABMiller plc, PO Box 782178, Sandton 2146, South Africa bDepartment of Biochemistry, Stellenbosch University, Private Bag X1, Matieland 7602, cDepartment of Microbiology, Stellenbosch University, South Africa a r t i c l e i n f o Article history: a b s t r a c t Membrane active anti-yea Food Mi journal homepage: ww All rights reserved. t samples and strains using ty antrell a, Marina Rautenbach b,* th Africa compounds, such as antimicrobial peptides and proteins, cause yeast le at ScienceDirect obiology elsevier .com/locate/ fm of charge due to the dielectric nature of the cell’s (intact)membrane, and M, were propagated as described previously (Van Nierop et al., 2008). All yeast propagation was performed in MYGP broth (3 g each of malt and yeast extract, 5 g peptone and 10 g glucose dis- solved in 1 L water, autoclaved 15 min, at 121 �C). For the anti-yeast assays each strain was harvested at approximately mid-log phase (growth times ranged between 14.5 and 20 h) and diluted to a cell count of 3.6�106 cells/mL. All yeast cell counts were performed microscopically in a Hawksley–Cristallite Haemocytometer with improved Neubauer ruling (Boeco, Hamberg, Germany). To deter- mine the yeast quality, IC50 values were calculated from dose– response data according to Du Toit and Rautenbach (2000). The IC50 value was taken as the concentration of extracted malt (mg malt extracted per 100 mL assay volume) that causes 50% inhibition of yeast growth (also refer to Van Nierop et al., 2008). Brewery production yeast samples (strain S only) were used directly in the assay after aseptic collection and storing on ice for less than 24 h, using the same yeast counts (3.6�106�10% cells/ mL) as for the laboratory propagated yeast but taking the viability (percent alive) into account. Yeast viability was performed Microbiology 26 (2009) 192–196 193 the active uptake of vital stains by cells with functioning membranes only (Heggart et al., 1999; Smart et al., 1999), and the acidification power test (Iserentant et al., 1996) and internal pH measurements (Imai et al., 1994) which both measure the cells ability tomaintain the pH gradient by actively pumping out protons. The assessment of yeast quality and prediction of fermentation performance are notoriously difficult as variations in yeast vitality are very subtle and there is a continued need to develop more accurate means of measuring yeast quality (Heggart et al., 1999; Iserentant et al., 1996; Lodolo and Cantrell, 2007). In order to study yeast quality, the influence of barley malt antimicrobial compounds in malt extracts on yeast growth was assessed with a microbroth dilution anti-yeast assay, a cell survival/growth assay adapted for execution in microtiter plates (Van Nierop et al., 2008). We report here the utilisation of the optimised anti-yeast microbroth dilution assay as a yeast vitality assay to compare the susceptibility of different lager brewery yeast samples and strains of Saccharomyces cerevisiae (renamed Saccharomyces pastorianus by Barnett, 2004 and Rainieri et al., 2006) to malt antimicrobial factors. 2. Experimental 2.1. Preparation and quality control of malt extracts The malt extracts were prepared as described previously by Van Nierop et al. (2008). In brief: selected commercial lager-type two row barley malts were milled to a flour consistency and duplicate 5 g aliquots were extracted using 30 mL of 0.5 M sulphuric acid (3 h at 0 �C) (method of Okada et al., 1970 with slight modifications by Van Nierop et al., 2008). The protein/peptide containing superna- tants were collected by centrifugation (4000 g, 15 min) and dia- lysed at 4 �C against distilled water (1 kDa cut-off dialysis tubing, preblocked with casamino acid). The dialysed samples were centrifuged again (6500 g, 10 min, 4 �C) and filtered through a 0.45 mm acetate syringe filter (Osmonics, Warren, Indiana, U.S.A.). Aliquots (containing extract from 0.50 g malt) were freeze-dried and stored at 20 �C until use. For the anti-yeast assays the samples were re-suspended in 1.0 mL acetonitrile/water (25:75 v/v) (acetonitrile – HiPerSolv�, BDH, Poole, England) and centrifuged at 6500 g for 15 min before addition to the assay. The same extraction procedure was applied to 0.05 M sulphuric acid, without the malt, as a negative reagent control. The lytic activity of selected extracts was confirmed by an ade- nylate kinase leakage assay. Malt extract was added to 6 mL yeast in MYGP (3.6�106 cells/mL) and the mixture was shaken for 2 h at room temperature. The mixture was filtered through a 0.45 mm acetate syringe filters and adenylate kinase activity in the filtrate was measured as described by Cameron-Clarke et al. (2003). The presence of proteins and/or peptides as anti-yeast compounds in the extracts was confirmed by treating selected extracts with Pronase� protease cocktail (Roche Diagnostics GmbH, Mannheim, Germany). Pronase� was added to the malt extract (0.14 g per malt extract aliquot) and the mixture was re- suspended in 750 mL water and incubated for 90 min at 40 �C. The mixture was then diluted with 250 mL acetonitrile for use in the anti-yeast assay. The negative control was Pronase�, without malt extract, treated as above. 2.2. Anti-yeast analysis The anti-yeast assay was performed as previously described (Van Nierop et al., 2008). Four different lager yeast strains of S. cerevisiae were supplied in the cryogenically stored form by The South African Breweries Ltd. Fresh batches of cryogenically stored S.N.E. van Nierop et al. / Food yeast were used, because some strains may be more susceptible to long term storage than others. The yeast strains, designated S, T, P according to the ASBC-approved method (Anon., 1992). 2.3. EBC 2L fermentations European Brewing Congress (EBC) fermentations were per- formed according to Van Nierop et al. (2004) using brewery yeast and wort aseptically collected from breweries Y and Z, both located in South Africa. The two different yeasts were fermented in the same wort ex-brewery Z. The brewery wort used was 40% adjunct wort, where 60% of the fermentable sugars come from barley malt and the rest from maize derived maltose syrup. 3. Results and discussion The anti-yeast assay format, as used to screen malts previously (Van Nierop et al., 2008), was applied to different yeast samples and yeast strains, at the same yeast counts (3.6�105 cell/mL) as prescribed for the anti-yeast assay (Van Nierop et al., 2008). Yeast viability (percent alive) was taken into account for brewery production yeast, but not for freshly propagated laboratory yeast where viability was assumed to be 100%. Adenylate kinasewas released when yeast samples were treated with any one of the malt extracts, confirming that the extracts contain membrane active and lytic activities (results not shown). Such membrane active factors will compromise the sensitive yeast A B D E F Pronase 0 10 20 30 40 50 60 70 80 90 100 110 120 Malt extract % Y e a s t g r o w t h ( 5 m g m a l t e x t r a c t e d p e r 1 0 0 µµL ) Malt extract Pronase treated extract Fig. 1. Anti-yeast activity towards yeast strain S of a number of different malt extracts (extract of 5 malt per 100 mL) before and after treatment with a protease cocktail (Pronase). Malt C was not tested because of limited availability. The error bars represent standard error of the mean (SEM) for duplicate experiments. 1 2 3 4 5 6 7 8 9 I C 5 0 ( m g m a l t e x t r a c t e d / 1 0 0 µL ) Brewery X Brewery Y Laboratory propagated yest 10 30 50 70 90 110 % y e a s t g r o w t h laboratory propagated yeast brewery Z yeast brewery Y yeast brewery X yeast S.N.E. van Nierop et al. / Food Microbiology 26 (2009) 192–196194 cell during fermentation. We found that most of the anti-yeast activity was lost when the Pronase� protease cocktail was added to a malt extract, confirming proteins and/or peptides as the major anti-yeast factors in all the tested malt extracts (Fig. 1). The antagonism of the protease cocktail anti-yeast activity (about 25% inhibition) in the presence of the extract also indicated the pres- ence of peptides and proteins in the malt extract. We have posi- tively identified lipid transfer protein 1a (LTP1a) and a number of peptides, one which may be an a-thionin in malt extracts. A more detailed study of the active protein and peptide factors in these malts will be published elsewhere. When comparing brewery yeast to laboratory propagated log phase yeast (all strain S), brewery yeast was found to bemuchmore sensitive to the inhibitory impact of malt extracts (Fig. 2). This trend was observed for all six samples of brewery yeast tested on three -1.25 -1.00 -0.75 -0.50 -0.25 0.00 0.25 0.50 0.75 1.00 1.25 -10 log amount malt D extracted (mg/100µµL) Fig. 2. Anti-yeast assay results (yeast strain S) expressed as dose–response curves comparing laboratory propagated and brewery yeast samples (from breweries X, Y and Z). The error bars represent SEM for duplicate analyses of duplicate experiments (n¼ 4). different malt extracts (A, C and D), a selection of which is shown in Fig. 3. According to the IC50 values, between 3 and 7 fold less malt extract was required to cause the same yeast growth inhibition compared to laboratory propagated log phase yeast for all six brewery yeast tested with malt extract D. The increased sensitivity of the brewery yeast therefore indicated that the vitality of the viable yeast population was lower than that of the equivalent laboratory propagated yeast. 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 A C D Malt extract I C 5 0 ( m g ) Brewery Y Day 1 Day 4 Day 8 Lab prop Fig. 4. Anti-yeast assay results comparing yeast taken at different times from 2L EBC ferme yeast). Yeast was assayed against three different malt extracts (A, C and D). Error bars represe Although brewery yeast was more sensitive, laboratory propa- gated log phase yeast was found to be more discriminating when comparing sensitivity to extracts from three different malts (Fig. 3). These findings support the use of laboratory propagated yeast for the optimised assay (Van Nierop et al., 2008). Here it is evident that all the yeast samples were most sensitive to extract D and least to extract A. The tested brewery yeast samples were in stationary phase, having been collected after flocculation from the bottom of fermentation vessels at the end of fermentation. To determinewhat impact growth phase had on the susceptibility of yeast to malt extracts, brewery yeast samples were re-propagated in MYGP medium as per the method used to propagate yeast for the anti- yeast assay. In all cases the susceptibility decreased upon this re- propagation (exponential phase cultures) in ideal conditions with Malt A Malt C Malt D 0 Extract Fig. 3. Anti-yeast assay results showing relative susceptibilities of yeast from brewery X and Y and laboratory propagated yeast to three different malt extracts (A, C and D). All data for yeast strain S. IC50 expressed in milligram malt extracted. Error bars represent minimum and maximum of duplicate analyses (n¼ 2). no fermentation related stress, but the sensitivity to malt extracts was still far greater than the standard yeast used for the assay (results not shown). However, it is known that stationary phase bacteria and yeast are more robust (‘‘cross-protected’’) and in particular less sensitive to membrane and cell wall perturbing compounds, most probably as a consequence of cell wall changes (Cassone, 1986; McLeod and Spector, 1996; Koo et al., 1996). The stationary phase yeast in our studymay have been already suffering Brewery Z A C D Malt extract 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 I C 5 0 ( m g ) Day 1 Day 4 Day 8 Lab prop ntations of brewery wort inoculated with brewery yeast (ex-brewery Y and Z, strain S nt minimum and maximum of duplicate analyses from duplicate fermentations (n¼ 2). strains S and Mwere most rapid (Fig. 5 insert). Three different malt 0 2 4 6 8 10 12 14 16 18 20 22 24 0 10 20 30 40 50 60 70 80 90 Yeast strains time (hours) m i l l i o n c e l l s p e r m L P M T S 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 Yeast strain G r o w t h r a t e l n ( m i l l i o n c e l l s p e r m L ) / h o u r P M T S Fig. 5. Growth curves of different yeast strains and bar graph (inset) comparing growth rates. Yeast was propagated from cryogenically preserved yeast in MYGP medium. Cell counts were determined microscopically. The dotted lines represent the times at which cells would be taken for the anti-yeast assay. S.N.E. van Nierop et al. / Food Mic from some brewery-induced cell wall damage and/or stress- induced sensitising that were propagated into the daughter cells. Changes in yeast susceptibility with growth phase were exam- ined further by comparing yeast from different stages of the fermentation process. Yeast was sampled on days 1, 4 and 8 from 2L EBC fermentations inoculated with brewery yeast (Fig. 4). All the yeasts at all the stages were more susceptible to inhibition by all three malt extracts than the log propagated yeast (Fig. 4) sup- porting the finding that brewery yeast was more susceptible than laboratory propagated yeast (refer to Figs. 2 and 3). Absolute susceptibility of these yeasts to all the antimicrobial malt extracts changed during the fermentations, although in general the susceptibility trend of A