Colloids and Surfaces B' Biointerfaces . 2 (19941511-5150927-7765/94/$07 . 00

© 1994-Elsevier Science B .N . All rights reserved,

Brief Note

Relationship between foaming and flocculence inSaccharomyces cerevisiae wine yeasts'

Patrizia Romano*, Giovanna Suzzi, Lucia VanniniDipartimento di Protezione e Valorizzazione Agroalimentare, Sezione di Chimica e Tecnologia degli Alimenti,Universitd di Bologna, Via San Giacomo 7, 40126 Bologna, Italy

(Received 18 March 1993; accepted 26 October 1993)

Abstract

The cell surface hydrophobicity (CSH) was studied in foaming and flocculent strains of Saccharornvces cererisiae .Microbial adhesion to hydrocarbons showed a high CSH both in foaming and flocculent strains . When treatedwith EDTA the foaming strains responded differently from flocculent ones, with the CSH decreasing in the formerand increasing in the latter. The treatment of cells with promise, proteinase K . trypsin and chymotrypsin abolishedthe CSH activity in all the strains . When sugars (glucose . fructose, sucrose, galactose . mannose) were present, the strainhydrophobicity did not vary . Tlie results indicate that foaming and flocculation are independentphenomena .

Kev words Cell surface hydrophohicity ; Flocculent ; Foaming : Hydrocarbons; Hydrophohicity, Microbial adhesion to hydrocarbons ;Pro teases; Saccharo,nyces cereri .siae

Introduction

Numerous naturally occurring microorganismsinteract with the air-water interface to form a frothhead or a film on the surface of the fermentationmedia [1] . Among these microorganisms, film-forming yeasts cause formation of a pelliclc on thesurface of wine [2]; sake and wine yeasts abun-dantly produce a froth head at the early stages offermentation both in sake mash and grape must[3,4] .

Froth flotation and film formation are bothrelated to cell surface hydrophobicity [2,5] . Asregards froth formation, its mechanism in yeast isnot clearly understood, as it involves both hydro-

^Corresponding author,'Presented at the second Bioadhesion conference, Louvan-la-Neuve, Belgium, May 23 27 . 1993 .

SSDI 0927-7765(93)01101-V

511

phobic and electrostatic interactions [6] . Foamfrom yeasts is considered to be caused by hydro-phobic yeast-surface proteins [3,7,8] .The presence of two dominant genes for foam

formation, FRO] and FRO2, has been demon-strated by Thornton [9] .

The capacity to form stable and high foam is afrequent characteristic in wine yeasts [4] ; it isassociated with the growth of dispersed cells andis not described in flocculent strains . A relationshipbetween flocculation and foaming has been hypoth-esized in top-fermenting brewery yeasts, whichpossess both properties, whereas Stratford [10]suggests that these phenomena are independent,involving different surface proteins .

In this paper we studied cell surface hydrophobi-city activity in foaming wine yeasts compared withflocculent ones.

512

Experimental

Organisms

Six strains of Saccharomyces ceretisiae wineyeasts were used in this study. Ba53, Ba92 andBa224 are foaming strains ; G11, G17 and G31 areflocculent strains . The yeast cells were maintainedat 4`C on wort agar slopes .

Experimental conditions

Yeasts were cultivated at 25'C in 100 ml cotton-plugged Erlenmeyer flasks containing 50 ml ofSabouraud medium (DIFCO). Cells were har-vested by centrifugation after 48 h and wereimmediately used .

Deternn ion of cell surface hydrophobicity

The cell surface hydrophobicity (CSH) of yeastcells was determined by microbial adhesion tohydrocarbons (MATH) according to limura et al .[2] and Rosenberg et al . [11] .

The distribution ratio of yeast cells in a two-phase system, a buffer solution and an organicsolvent, was measured . Two volumes of yeast cellssuspended in 50 mM sodium acetate buffer (pH4.5) to an A 620 value of 2.5 were mixed for 20swith 1 volume of hexadecane or heptane . After30 mm at room temperature, the optical densitiesof the initial and residual buffer solutions weremeasured . The degree of CSH (H .D .) was calcu-lated from the equation

H.D.(%) = 100( 1 - R/l)

where l and R are the optical densities of the initialand the residual buffer solutions respectively .

Changes in CSH after protease treatment and sugaraddil ion

Protease treatments were performed accordingto the method of Hodgson et al . [12] . Yeast cells,suspended in flocculation buffer, were washed twice

P. Romaao el al ./Colloids S u'%aces B Bmmterfacea 2 (1994) 511-515

in 50 mM sodium phosphate. 50 mM EDTA, pH7 .5 in order to disperse flocs, and twice in water .The resulting suspension was used for treatmentwith the proteinases or with the sugars . For treat-ment with the protcinases EDTA/H,O-washedcells were resuspended in l ml enzyme solutionand incubated at 30'C for 90 mitr. After incuba-tion. CSH activity, foam and flocculation abilitywere evaluated .

Protcolytic enzymes were suspended in 50 mMsodium phosphate, 50 mM EDTA, pH 7.5, inthe following concentrations : proteinase K .200 pg ml ' ; pronase, 200 µg ml- ' ; chymotrypsin ..100 Itg ml'; trypsin, 100 µg ml - ' .

In order to study the influence of added sugars,EDTA/II,O-washed cells were resuspended in I Msugar solution (mannose, fructose, galactose,sucrose, glucose), 50mM acetate buffer (pH 4 .5)and after 90 min were tested for CSH .

Controls were made in the appropriate buffers .

Flocculation test

Yeast cells were harvested, washed twice withwater and resuspended in 50 ml sodium ace-tate 0.1% CaCl, flocculation buffer (pH 4 .5) andstrain ability to flocculate was estimated by eye,as described by Hodgson et al . [12] .

Foaming test

Yeast cells were harvested, washed twice withwater and resuspended in acetate buffer (pH 4.2) .To evaluate strain ability to produce foam thefroth flotation test was used, as described byKamada and Murata [13] .

Results

Relation between CSH and foam formation

The foaming strains proved to be highly hydro-phobic and the cell affinity to CO, bubbles isshown in Fig . 1 .The MATH test carried out on foaming and

flocculent yeasts (Table 1) showed that the cells

P. Romano et al ./Colloids Surfaces B . Biointerfaces 2 (1994) 511-515

513

Fig . 1 . Micrographs showing the cell affinity of foamingSaccharomrces cererrsiae Ba53 to CO, bubbles,

involved in foaming and flocculation were gen-erally highly hydrophobic . Different behaviour wasexhibited by EDTA-treated cells . In fact, EDTAlowered CSH activity in foaming strains, even ifby differing degrees. In particular, two strainsshowed a direct relationship between the substan-tial decrease in CSH activity and the loss offoaming ability. The flocculent strains exhibited aninverse correlation: EDTA causing the loss offlocculation ability, while increasing CSH activity .Protease treatment abolished almost completelythe CSH activity in both foaming and flocculentstrains . Nevertheless the foaming strains lost theability to foam under all conditions, whereas theflocculent ones lost the ability to flocculate onlyin some cases .

The addition of sugars (Table 2) did not affectCSH activity in either foaming or flocculent strains .However, foaming strains retained their ability tofoam in the presence of all sugars, whereas floc-culcntstrains deflocculated only in the presence ofmannose .

CSII activity as afunctron of growth stage

During growth the CSH varied in both foamingand flocculent strains (Fig . 2). In foaming strainsa general but small decrease in CSH was observed :in Ba92 on the second day and in Ba53 after 96 h

Table 1Cell surface hydrophobicity after EDTA addition and protcmase treatment in foaming and flocculent strains of Saccharomyces cerevisiae

'Hydrophobic degree (H .D.1 expresses the percentage of the yeast cells transferred from buffer to heptane .b A, flocculation or foam ability ; F, floc formation ; NF. no floc formation; S, foam formation : NS, no foam formation .

Strain Control EDTA Protemase K Pronase Trypsin ChymotrypsinH.D.I%1'

H.D. I%1' A' H.D. A° H.D . (got H D. (%1° .A° H.D . (%r A °

FoamingBa3 89 66 S 0 S 0 S 3 S 2 SBa92 64 12 NS 0 S 0 S 4 S SBa244 50 22 NS 9 S I S 5 S S

FlocculentGl1 56 75 NF 0 F 0 NF 0 F 0 FGl7 49 61 NF 4 F 2 F 0 NF 3 FG31 35 60 NF 6 F 6 F 4 F 13 F

For definitions see Table 1 .

24

48

hours72

38

Fig, 2 Change in cell surface hydrophobicny during 96 h ofgrowth . •, foaming strain Ba53, ∎, foaming strain Ba92; * .flocculent strain GI I : A, flocculcnt strain (331

of growth . Flocculent strains behaved differently :CII showed an increasing CSH during growthfrom low to high values, whereas G31 exhibited adramatic decrease in CSH activity during the first3 days and then tended to increase.

Discussion

Both flocculent and foaming wine yeasts have ahigh CSH, the degree of which is a characteristicof the strain .

Both phenomena involve surface proteins thatare more resistant to protease treatments in wineflocculent strains than in wine foaming ones . Theloss of CSH activity in foaming strains treatedwith EDTA leads us to suppose that calcium ions

play an important role in maintaining the elevateddegree of CSH in these strains . Besides. floccula-tion, well known to be promoted by calcium ions[14], is abolished by EDTA treatment, whereasCSF1 activity is unaffected, showing instead a ten-dency to increase .

Taking into account that coordinated bindingof calcium is necessary for the active conformationof some proteins [15,16] . it is possible to hypothe-size that calcium functions as a stcreochcmicalstabilizer for CSH expression .

It is important to note that the treatment offoaming and flocculent cells with sugars had noeffects on either CSH, foam formation or floccula-tion ability, with the exception of mannose, which,as expected, inhibited flocculation expression .

The theory of foaming being independent offlocculation is also supported by the different pat-terns of CSH evolution during the growth offoaming and flocculcnt strains. In wine yeasts, thefact that CSH does not depend on whether thestrain is foaming or flocculent would suggest thatdifferent surface proteins could be involved in thetwo phenomena .

Acknowledgement

This research was supported by the NationalResearch Council of Italy, special project RAISA .subproject 4, paper no . 1272 .

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P. Romano et a7,%Colloids Surfaces B . Bimnterfuces 2 (1994) 511 5)5

Table 2Cell surface hydrophohicrty after sugar addition in foaming and flocculent wine strains of Snccharoinree .s cererfsiae

Strain

Control Mannose Fructose Galactose Sucrose GlucoseH.D . (%i

A H 1) . (%) H.D . I%) A H.D (%) A H.D. (0,) AH I). (%1

FoamingBa53

71) 81 S 81 S 84 S 8'- SBa92

87 73 S 78 85 S 76 S 83 SBa224

57 49 S 41 49 S 37 S 43 S

FlocculentG11

77 72 NF 79 t 76 F 84 F 82 FG17

65 80 NF 65 F 70 F 46 F 59 FG31

70 61 NF 82 F 76 F 79 F 79 F

P. Romano et al ;/Colloids Surfaces B- Biointerfaces 2 (1994) 511-515 515

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