Induction of flocculation in brewing yeasts by change in ph value

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1 FEMS Microbiology Letters 136 (1996) Induction of flocculation in brewing yeasts by change in ph value Abstract Malcolm Stratford * Institute of Food Research, Norwich Research Park, Coinq. Norwich NR4 7UA, UK Received 30 October 1995; revised 13 November 1995; accepted I3 November 1995 Most brewing strains of Succharomyces cereuisiae flocculate following growth in beer wart. However, many do not flocculate in laboratory culture media, because their initial ph and buffering capacity do not correspond to the ph range within which these yeasts flocculate. Many, though not all, NewFlo phenotype brewing yeasts flocculate within a narrow ph range only; this is indicative of the existence of more than one NewFlo flocculation phenotype. Such strains may be flocculated by small alterations of ph to within the flocculation range. Induction of flocculation by ph change may be used to separate cells from media at any stage during fermentation. Keywords: 1. Introduction Flocculation; Saccharomyces cerecisiae; Growth media ph; NewFlo phenotype Yeast flocculation is usually associated with the brewing industry where, after fermentation is complete, suspensions of single-celled yeasts gather into clumps, and sediment rapidly from the beer. This has traditionally benefited the brewer in two ways; firstly, the product is clear and largely yeast-free, and secondly, the yeast crop is neatly separated for repitching into later fermentations. In brewing, the timing of flocculation onset is not regulated, flocculation usually occurring in the stationary phase of growth after fermentation has ceased. More recently, there has been considerable interest in flocculation as a cheap and natural method of cell separation in * Corresponding author. Present address: Microbiology Section, Unilever Research, Colworth House, Shambrook, Bedford MK44 ILQ, UK. Tel.: +44 (1234) ; Fax: +44 (1234) other industrial fermentations. Cell separation technology and yeast flocculation have been recently reviewed [1,2]. The mechanism by which yeasts flocculate involves surface proteins on flocculent yeast cells binding to carbohydrate receptors on nearby cells [3]. Sugar-binding proteins, lectins [4], and flocculation share the characteristics of inhibition by specific sugars and a requirement for calcium ions. Calcium ions maintain lectins in active conformation and Ca*+ removal by EDTA chelation causes loss of activity [5]. This mechanism for yeast flocculation is termed the lectin hypothesis. Examination of brewing strains of Saccharumyces cereuisiue has revealed at least two distinct forms of flocculation. The characteristics of Flol phenotype flocculation suggested a mannose-specific lectin bonding, whereas WewFlo phenotype characteristics indicated broader specificity, glucose- and mannosespecific lectins [6]. A further mannose insensitive /96/$ Federation of European Microbiological Societies. All rights reserved SSDI (95)

2 14 M. Stratford / FEMS Microbiology Letters 136 f 1996) mechanism of flocculation has been reported in other strains of S. cerevisiue [7]. Flol phenotype flocculation is relatively insensitive to extreme ph, high salt concentration or protease attack and is usually expressed constitutively. Almost all Flol phenotype strains flocculate throughout growth, in all media. These yeasts are easy to flocculate and in consequence, almost all of the molecular biology and genetic studies of flocculation have involved Flol phenotype flocculation. Flol phenotype flocculation is found in strains containing either FLOI, FLO5, FLO8, tupl or cyc8 genes, but is not typical of brewing yeasts [6]. The majority of brewery ale strains show NewFlo phenotype flocculation [6]. These yeasts grow and ferment in wort as single cells and flocculate late in growth, usually in stationary phase; they will grow but not flocculate in chemically defined Yeast Nitrogen Base medium, and flocculate poorly, if at all, in the other commonly used yeast growth medium, YEPD. In this paper, the reasons for lack of flocculation of these strains in laboratory media are investigated. 2. Materials and methods 2.1. Yeast strains All the yeasts used in this study were recognised brewery ale strains of S. cerevisiae: NCYC 1101; NCYC 1190; NCYC 1224; ARCl. NCYC strains were from the National Collection of Yeast Cultures, Institute of Food Research, Norwich NR4 7UA, U.K. and ARC1 was from Adnams Brewery, Southwold, Suffolk, U.K.. Duplicate experiments were carried out using S. cereuisiae NCYC 1026; NCYC 1028; NCYC 1154; NCYC 1181; NCYC 1183; NCYC 1198; NCYC 1214; NCYC Growth media Yeasts were maintained at 4 C on YEPD agar slopes. YEPD contained, per litre of water, 20 g glucose, 10 g yeast extract and 20 g bacteriological peptone. Starter cultures were grown aerobically in 40 ml YEPD broth in 100 ml conical flasks, for 24 h at 25 C on an orbital shaker, at 150 rpm. Experimen- tal cultures consisted of 1 1 portions of media in 2 1 flasks, inoculated with 5 mg dry weight of yeast/l. Growth was monitored by optical absorbance at 600 nm, and converted to dry weight using calibration curves. Other growth media used were brewer s wort (sweet wort, Adnams Brewery, original gravity 1.035) and Difco Yeast Nitrogen Base (without amino acids and ammonium sulfate) plus 0.5 g NH,SO, and 20 g glucose per litre (YB). Previous studies had shown flocculation to be suppressed by excess ammonium ions [8,9]. The ammonium concentration used here in YB medium is 10% of that recommended by Difco but was sufficient for normal growth Flocculation measurement Flocculation in growth media was assessed by stopping the shaker for 30 s to allow floes to settle and removing samples, 0.2 ml, from just below the meniscus. Samples were diluted in 250 mm EDTA, and cell concentrations were determined by measuring optical density. Larger samples, ml, were removed for assessment of flocculation in buffer. Yeasts were harvested by centrifugation, washed in water and resuspended in a buffer of 50 mm sodium succinate (ph 4.0) containing 10 mm calcium chloride. Suspensions were agitated at 120 rpm on an orbital shaker. After 4-6 h, samples of supernatant fluid were removed, dispersed in EDTA, and cell concentrations were measured by spectrophotometer. The proportion of flocculated cells was determined from the total cell concentration and the non-flocculated fraction Flocculation ph range The ph range over which each yeast flocculated was assessed by resuspending flocculent yeasts in a composite buffer with a very wide buffering range. This buffer contained 50 n-&i Tris, 50 mm succinic acid, 100 mm KOH, and 10 mm CaCl,. Solutions were adjusted to ph with 5 M HCl Buffering capacity 100 ml aliquots of media were adjusted to ph 9.0 with 10 M NaOH and then back-titrated with a series

3 M. Strat&ord/ FEM.7 Microbiology Letters 136 (1996) I5 of 1 ml additions of 1.O M HCl. Buffering capacities were calculated from the slopes of ph/hci addition graphs. One unit of buffering capacity is the number of millimoles of Hf required to change the ph of 1 1 of media by one ph unit. 3. Results S. cerel,isiae strains NCYC 1101, 1190, 1224 and ARCl, all flocculated strongly after growth in brewer s wort. None of these yeasts flocculated in YB medium. NCYC 1101 and ARC1 flocculated well in YEPD, NCYC 1190 flocculated poorly while NCYC 1224 remained dispersed in this medium. All yeasts grew in all media, albeit somewhat slower in YB medium. While cells of NCYC 1224 remained dispersed throughout growth in YEPD and YB medium, yeast samples resuspended in buffer flocculated strongly. Samples removed after 10 h in YB medium (Fig. 1) and 22 h in YEPD (Fig. 2) were flocculent in buffer. Flocculation in buffer was almost instantaneous, suggesting relief of flocculation inhibition rather than synthesis de novo of a flocculation mechanism. Similarly, cells of NCYC 1190 grown in either YB medium or YEPD flocculated strongly in buffer. Addition of 100 mm calcium chloride to media or substitution of fructose for glucose did not cause NCYC 1224 to flocculate in YEPD or YB medium, Time (II) Fig. I. Growth of S. cerel,isiae NCYC 1224 in YB medium (0) and flocculation of washed yeast cells in buffer, ph 4.0. containing calcium chloride (0). Time (h) Fig. 2. Growth of S. ceret,isiae NCYC 1224 in YEPD (0) and flocculation of washed cells in buffer, ph 4.0, containing calcium chloride (0 ). suggesting that lack of flocculation was not due to calcium shortage or sugar inhibition. The ph range over which different yeasts can flocculate was examined in buffer (Fig. 3). NCYC 1101 and ARC1 were able to flocculate over a broad ph range, , while NCYC 1224 and 1190 only flocculated within a narrow ph window, Measurement of media ph during yeast growth showed that the initial ph of YEPD was 6.2, falling to circa 5.3 by the end of exponential phase. In YB medium, ph fell from 4.2 to 2.3 and in brewer s wort from 5.3 to 3.8 over a similar period. Examination of the buffering capacities of media over this ph range showed YB medium to be poorly buffered (Fig. 41, wort to be moderately buffered and YEPD to be very strongly buffered. These data suggested that in YB medium, the final ph was too low to permit flocculation by any of these yeasts; in YEPD, the final ph was too high for flocculation of NCYC 1190 and The initial ph of wort was somewhat high for flocculation, but following yeast growth, wort ph was optimal for floccualtion in all yeast strains. In order to test this hypothesis, non-flocculating 20 h YEPD-grown NCYC 1224 cultures were acidified to ph 4.0 with HCl. Cells flocculated immediately. Similarly, sodium hydroxide addition to 20 h YB cultures, raising the ph to 4.0, also caused rapid flocculation (Fig. 5).

4 16 M. Strut$ord/ FEMS Microbiology Letten 136 (1996) 13-1X Fig. 3. ph range of flocculation of S. cerecisiue NCYC 1101 (01, ARC1 (0). NCYC 1224 (0) and NCYC 1190 ( 1. Wart-grown yeasts were harvested in the stationary phase, washed, and resuspended in composite buffer containing Tris 50 mm, succinic acid 50 n&l, KOH 100 mm and CaCl, 10 mm. Points are means of duplicate readings. Buffering Capacity (Units) ph value Fig. 4. Buffering capacities of yeast growth media. These were assessed by back titration of media from ph 9.0 with HCI, 1.0 M. One unit of buffering capacity is the amount of H+ in millimoles, required to change the ph of 1 I of medium by one ph unit.

5 M. Stratford/FEMS Microbiology Letters 136 (IY96) Fig. 5. Induction of flocculation in a 20-h culture of S. cereuisiue NCYC 1224 in YB medium. Flocculation was initiated by NaOH addition (arrow), raising the medium ph to 4.0 (0). Untreated control cultures arc indicated by (0). Points are the means of Examination of other NewFlo phenotype flocculent ale strains showed NCYC 1154 to have a broad ph flocculation range and NCYC 1026, 1028, 1181, 1183, 1198, 1214 and 1245 to flocculate over a narrow range, as exemplified by NCYC Discussion The majority of brewery ale strains flocculate following growth in brewer s wort but show little or no flocculation in media such as YEPD or YB medium [6]. Here we have shown that yeasts removed from laboratory media will flocculate in buffer, and that brewing strains will flocculate in laboratory media if the final ph is suitable. Yeasts are known to acidify their growth media, largely as a result of the activity of the plasma-membrane H+- ATPase [ 101..Given similar yeast growth and AT- Pase activity,, the ph in a growth medium will depend on the initial ph value and the buffering capacity of the medium at that ph value. Initially, YB is at ph 4.5, but having small buffering capacity, the ph falls rapidly during yeast growth to 2.3, where little flocculation occurs. In YEPD, the initial ph of 6.3 and stronger buffering capacity give a final ph barely low enough to permit flocculation. Hence, in order to study flocculation of brewery yeast strains in laboratory media, yeasts may be grown either in YB, buffered to ph 4.5 with citrate or succinate (100 mm) or in YEPD, acidified to ph 4.5. Some of the strains examined here, e.g. NCYC 1224, became flocculent early in growth (Fig. 2). Such strains offer commercial possibilities of a cheap and natural method of separating cells from media at any desired time during fermentation, by a simple change in ph value. In the past, other materials from brewer s wort have been reported necessary for flocculation, notably a proteinacous inducer material [ 1 l]. The data shown suggest that such material is not necessary, at least not for the strains described here. Proteins are generally good buffers, so perhaps the action of this inducer material was to maintain a suitable ph value. Flocculation in 5. cerecisiae is probably associated with several distinct phenotypes [6]. The Flol phenotype shows a very broad tolerance of ph, flocculating between ph 1.5 and IO. The strains used in this study were of the NewFlo phenotype, flocculating in stationary phase and inhibited by mannose and glucose. Evidence has been given here of phenotypic differences between the NewFlo strains: some flocculating over a broad and others within only a narrow ph range. Clearly, it is important to elucidate the genetic and physiological differences between these two groups. Be that as it may, recognition of the ph sensitivity of flocculation in brewing strains will now permit reliable growth and flocculation of these strains under laboratory conditions. Perhaps this will encourage genetic analysis of commercially important NewFlo phenotype strains, an area previously neglected in favour of the more tractable Flol phenotype strains. References [ll Bowden, C.P., Leaver, G., Melling, J., Norton, M.G. and Whittington, P.N. (1987) Recent and novel developments in the recovery of cells from fermentation broths. In: Separations for Biotechnology (Verral, MS. and Hudson, M.J., Eds.), pp Ellis Horwood, Chichester, U.K. [2] Stratford, M. (1992) Yeast flocculation: a new perspective. Adv. Microbial. Physiol. 33, l-7 1. [3] Miki, B.L.A., Poon, N.H., James, A.P. and Seligy, V.L. (1982) Possible mechanism for flocculation interactions governed by the gene FL01 in Saccharomyces cerecisiae. J. Bacterial. 150,

6 18 M. Stratford / FEMS Microbiology Letters 136 f I8 [4] Boyd, W.C. and Shapleigh, E. (1954) Specific precipitating activity of plant agglutinins (Lectins). Science 119, 419. [5] Kalb, A.J. and Levitzki, A. (1968) Metal-binding sites of concanavalin A and their role in the binding of a-methyl D-glucopyranoside. Biochem. J. 109, [6] Stratford, M. and Assinder, S. (1991) Yeast flocculation: Flol and NewFlo phenotypes and receptor structure. Yeast [7] Masy, C.L., Dengis, P., Garsoux, G., Melotte, L., Dufour, J.-P., Mestagh, M.M. and Rouxhet, P.G. (1991) A simple test for screening flocculence of brewer s yeast. Louvain Brew. Lett l-34. [8] Mill, P.J. (1964) The effect of nitrogenous substances on the time of tlocculation of Saccharomyces ceruisiae. J. Cen. Microbial. 35, [9] Stratford, M. (1989) Evidence for two mechanisms of tlocculation in Sacchurotnyces cerwisiue. Yeast 5, S441-S445. [lo] Vallejo, C.C.. and Sermno, R. (1989) Physiology of mutants with reduced expression of plasma membrane Hf-ATPase. Yeast 5, [1 I] Stewart, G.G., Russell, I. and Garrison, I.F. (1975) Some considerations of the flocculation characteristics of ale and lager yeast strains. J. Inst. Brew. 81,