Quantification of Glycosidase Activity in Selected Strains of Brettanomyces bruxellensis and Oenococcus oeni

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1 Quntifiction of Glycosidse Activity 303 Quntifiction of Glycosidse Activity in Selected Strins of Brettnomyces ruxellensis nd Oenococcus oeni Ann K. Mnsfield, 1 Bruce W. Zoecklein, 2 * nd Roert S. Whiton 3 Brettnomyces ruxellensis nd lctic cid cteri re common microorgnisms cple of modifying wine rom nd flvor. The ctivity of ß-glucosidse ginst p-nitrophenyl-ß-d-glucopyrnoside ws determined in model system for 14 strins of Brettnomyces ruxellensis yest nd 9 strins of lctic cid cteri (Oenococcus oeni). All Brettnomyces strins nd 7 Oenococcus strins exhiited enzymtic ctivity ginst this sustrte. B. ruxellensis ß-glucosidse ctivity ws primrily intrcellulr; O. oeni showed some extrcellulr ctivity. Strins showing ctivity greter thn 1000 nmole ml -1 g dry cell mss hr -1 for Brettnomyces, or 100 nmole ml -1 g dry cell mss hr -1 for Oenococcus, were evluted for their effect on ntive Viognier grpe glycosides. Neither genus ws ctive on Viognier grpe glycosides. Key words: Glycosides, glycosidse, glucosides, glucosidses, Brettnomyces ruxellensis, Oenococcus oeni Wine rom nd flvor re influenced y grpe-derived compounds, which exist s free voltiles nd/or s sugr-ound glycosides [1,2,39]. Products of grpe glycoside hydrolysis include liphtic residues, monoterpenes, sesquiterpenes, norisoprenoids, nd shikimic cid metolites [2]. Glycosides my exist s mono- or discchrides, with sugr moieties occurring s ß-D-glucose, 6-O-α-L-rhmnopyrnosyl-ß-D-glucopyrnose, 6-O-α-L-rinofurnosyl-ß-D-glucopyrnose, nd 6-O-α-Lpiofurnosyl-ß-D-glucopyrnose [36]. ß-D-glucopyrnosides pper to predominte [38]. Grpe glycosides re source of vrietl rom nd flvor [39], nd their hydrolysis my led to incresed wine qulity [2,13]. Glycoside hydrolysis my occur enzymticlly through glucosidses or vi cid hydrolysis [13,20,37]. Enzymtic hydrolysis of discchride glycosides occurs s two-step process. In the cse of monoglucosides, the glucosidse cts directly [12]. Acid hydrolysis cleves glycosides of ctivted lcohols, producing croction cple of cusing rom nd flvor chnges [22,32,33,37]. Enzyme hydrolysis cleves the glycosidic ond without ltering the glycone [33]. Endogenous grpe ß-glucosidses result in some hydrolysis during fruit mturtion, ut show low ctivity [19]. Enzymes from molds nd yests my lso relese glycones [22]. McMhon et l. [29] found glycosidic ctivity in five strins of Brettnomyces ruxellensis. Although often deemed s spoilge orgnism, Brettnomyces my result in enhnced 1 Grdute Student, 2 Associte Professor, nd 3 Reserch Scientist, Enology-Grpe Chemistry Group, Deprtment of Food Science nd Technology, Virgini Tech, Duckpond Dr., Blcksurg, VA *Corresponding uthor [Fx: ; emil: zoeckle@vt.edu] Reserch conducted t the Deprtment of Food Science nd Technology, Virgini Tech. Mnuscript sumitted Novemer 2001; revised My 2002 Copyright 2002 y the Americn Society for Enology nd Viticulture. All rights reserved. rom nd complexity nd my impct red wine color [14]. Mono-glucosylted nthocynins re the primry red pigments in Vitis vinifer grpes [34] nd comprise lrge portion of the totl glycoside concentrtion [24,40]. Hydrolysis of glucose usully results in corresponding nthocynidin, which is converted to the colorless pseudose [23], which my ffect color nd stility. An increse in glucose concentrtion coinciding with mlolctic fermenttion (MLF) hs een documented [5,10] nd my e cused y glycoside hydrolysis [27]. Grimldi et l. [16] demonstrted ß-glucosidse ctivity in 12 strins of Oenococcus oeni. However, it is difficult to link cteril enzyme ctivity with glycoside hydrolysis, s incresed glucose concentrtions my e the result of residul grpe or yest hydrolytic enzymes [10]. ß-Glucosidses cn e inhiited y ph, temperture, sugrs, ethnol, nd phenols [17,22]. The degree of inhiition of production nd/or ctivity is dictted y the orgnism nd strin [3,11,16,30]. The cidic conditions in wine my result in denturtion nd inhiition of enzymtic hydrolysis [11]. However, one strin of Oenococcus oeni ws found to retin 78% of mximum ß-glucosidse ctivity t ph 3.5 [5]. The optimum temperture for yest ß-glucosidses hs een found to e 45 to 50 C [11]. At ethnol concentrtions of 10% (v/v), glucosidses of Aspergillus niger, Scchromyces cerevisie, nd Cndid wickerhmii showed no loss of ctivity [22]. Thus, numer of fctors my limit the production nd/or ctivity of ß-glucosidses in wine. However, limited ctivity could hve n influence on wine qulity nd stility. This reserch determined the ility of 14 strins of Brettnomyces ruxellensis nd 9 strins of Oenococcus oeni to hydrolyze model glycoside. Selected strins were ssyed for the site of ß-D-glycosidse ctivity nd the ility to hydrolyze ntive Viognier glycosides. 303

2 304 Mnsfield et l. Mterils nd Methods Cultures. Yest nd cteril strins used in this reserch were provided y Lllemnd, Inc. (Montrel, Cnd) nd re listed in Tles 1 nd 2. Pure cultures of yest strins were isolted nd mintined on Yest Mold Agr (YMA; Difco, Detroit, MI) pltes, ph 5. Bcteril cultures were isolted nd mintined on Tomto Rogos Agr s descried y Fugelsng [15]. Growth nlysis. B. ruxellensis cells were grown in 200 ml Yest Nitrogen Bse (YNB; 7 g/l YNB, 5 g/l rutin) (Difco, Detroit, MI) nd O. oeni cells in 200 ml Tomto Rogos Broth (TRB) [15] t 30 C. Beginning t 24 hr postinocultion, cultures were gitted every 12 hr for 5 min on Thermolyne RotoMix (Brnsted/Thermolyne, Duuque, IA). Enzymtic ctivity. The procedure of Blondin et l. [4] s modified y Chroenchi et l. [8] ws used to determine hydrolytic enzyme ctivities on ß-D-glucoside. B. ruxellensis ws cultured s descried y McMhon et l. [29]. O. oeni cells were grown in 10 ml liquid medium consisting of TRB [15] nd 5 g/l rutin (Sigm, St. Louis, MO), ph 5.0. Cultures were inoculted t 24, 48, or 72 hr nd incuted t 30 C until ech culture reched log phse. Cultures were centrifuged (5000 x g, 10 min, 4 C), wshed with cold sterile sline (0.7% NCl), nd recentrifuged. Pellets were then resuspended in 10 ml filter-sterilized growth medium contining 6.7 g/l YNB (for B. ruxellensis) or TRB (for O. oeni) nd 1 mm of the sustrte p-nitrophenyl-ß-d-glucopyrnoside (Sigm, St. Louis, MO). The medium ws uffered to ph 3.5 with 0.9 g/l trtric cid nd 0.1 g/l K 2 HPO 4 to pproximte wine ph. Rection tues were incuted for 48 hr t 30 C. The superntnt ws ssyed for lierted p-nitrophenol (pnp): 1.0 ml ws mixed with 2.0 ml sodium cronte uffer (0.2 M, ph 10.2) nd mesured spectrophotometriclly (Genesys 5, Spectronic Instruments Inc., Rochester, NY) t 400 nm. A series of stndrds ws prepred tht contined 0 to 200 nm pnp. A sustrte lnk (uffer Tle 1 Enzyme ctivities for Brettnomyces ruxellensis strins (expressed s nmole of hydrolyzed ß-glucoside /ml ssy medium /grm dry cell mss). Vlues re verges of triplicte replictions. nd sustrte) nd smple lnks (cell preprtion nd uffer) were prepred nd sutrcted from experimentl sornce redings. All ssys were performed in triplicte. Enzyme ctivity loction. Strins tht demonstrted enzyme ctivity ginst p-nitrophenyl-ß-d-glucopyrnoside were further nlyzed to determine loction of ctivity (whole cells, permeilized cells, nd superntnt) s descried y Rosi et l. [31]. A loopful of culture ws trnsferred from stock slnts to 10 ml of liquid medium (YNB 6.7 g/l, rutin 5 g/l, ph 5.0 for yest; TRB, ph 5.0, for cteri) nd incuted t 30 C for 24 to 72 hr. Severl uthors hve demonstrted mximum enzyme production t ph of 5.0 [4,16]. Therefore, n elevted ph ws selected for loction ssys. Once log phse ws reched, 0.2 ml of the inoculum ws dded to 200 ml of fresh liquid medium in 500-mL medi ottles. Cultures were incuted t 30 C for 48 hr. Whole cells. Cells were hrvested from 1 ml of culture (centrifuged t 5000 x g, 10 min, 4 C) nd wshed twice with cold distilled wter. The pellet ws resuspended in 0.2 ml citrtephosphte uffer (100 mm, ph 5.0) nd ssyed for ctivity. Permeilized cells. The procedure of Rosi et l. [30] ws used. The culture (5 ml) ws centrifuged (5000 x g, 10 min, 4 C), nd the pellet ws wshed with 5 ml of cold distilled wter. The pellet ws resuspended in 1 ml imidzole uffer (75 nm, ph 7.5), nd 50 µl 0.3 M glutthion, 10 µl 10% Triton X-100, nd 50 µl toluene/ethnol (1:4 v/v) were dded. The suspension ws plced on mechnicl shker for 5 min nd then centrifuged. The pellet ws suspended in 5 ml of cold distilled wter, nd 1 ml of this suspension ws centrifuged nd the pellet ws wshed with cold distilled wter. The pellet ws resuspended in 0.2 ml citrte-phosphte uffer (100 mm, ph 5.0). Therefore, the permeilized frction consisted of wshed cells with the cell wll compromised. Superntnt. The superntnt frction ws comprised of 0.2 ml of the unconcentrted growth medium. Superntnt fluid, whole, or permeilized cells (0.2 ml ech) were mixed with 0.2 ml of 5 mm pnp glucopyrnoside in 100 mm citrtephosphte uffer (ph 5.0). The rection mixture ws incuted t 30 C for 1 hr. Addition of 1.2 ml cronte uffer (0.2 M, B. ruxellensis strin Glucopyrnoside (nm/ml) cde jk hijk cdef ghj fghij Brux 1231 Souche Ave 1476 Souche O 2501 m Souche M 878 cdefg Vin fghi Vin defgh Vin Vin Limit of detection: 3 nmole/ml. Different letters indicte significnce t p <0.05. Tle 2 Enzyme ctivities for Oenococcus oeni strins (expressed s nmole of hydrolyzed ß-glucoside /ml ssy medium /grm dry cell mss). Vlues re verges of triplicte replictions. O. oeni Glucopyrnoside (nm/ml) 508 <LOD <LOD Limit of detection (LOD): 3 nmole/ml. Different letters indicte significnce t p <0.05.

3 Quntifiction of Glycosidse Activity 305 ph 10.2) stopped enzyme ctivity, nd then the rection mixture ws centrifuged t 10,000 x g for 2.5 min. Lierted pnp ws mesured spectrophotometriclly s descried previously. A series of stndrds ws prepred contining 0 to 200 nm pnp. All ssys were performed in triplicte. Isoltion of grpe glycosides. Viognier (Vitis vinifer L.) grpes grown in northwestern Virgini were pressed nd the juice prtilly fermented (to 7% lcohol). Viognier glycosides were isolted using Wters (Milford, MA) C-18 reverse-phse Sep-Pk columns, ctivted with 10 ml methnol followed y 10 ml deionized wter. One hundred ml of undiluted juice ws dded, nd the columns were wshed three times with 15 ml deionized wter. Glycosides were eluted with 15 ml methnol. Ten elutions were comined per liquot, concentrted to dryness, nd stored t -20 C. Glycoside concentrtion ws estimted y the nlysis of glycosyl-glucose [25,38]. Hydrolysis of glycosides. Culture (50 ml) grown in YNB plus rutin (yest) or TRB (cteri) ws centrifuged nd the pellet resuspended in 50 ml of 100 mm citrte-phosphte uffer (ph 5.0) nd dded to n liquot of Viognier glycosides (80 mm glycosyl-glucose). Pectinolytic enzyme (0.03 g/l) (AR2000, Gist-Brocdes, Inc., Seclin, Frnce) in citrte-phosphte uffer ws used to verify the potentil for glycoside hydrolysis. After 48 hr incution t 30 C, lierted glycosyl-glucose ws mesured spectrophotometriclly t 340 nm, using n enzymtic glucose ssy (Roche, Mnnheim, Germny). Dry weight Dry cell weight ws determined y filtering 50 ml of culture on preweighed memrne filter (0.45 µm; Pll Gelmn Lortory, Ann Aror, MI). Filters were plced in tred luminum pns, dried overnight t 100 C, nd reweighed. Sttisticl nlysis. All dt were sttisticlly nlyzed using the Tukey-Krmer HSD method in JMP, revision 4 (SAS Institute, Cry, NC). Results All 14 of the Brettnomyces ruxellensis strins displyed ß-glucosidse ctivity ginst p-nitrophenyl-ß-d-glucopyrnoside, rnging from 418 to 2501 nmole ml -1 g dry cell mss hr -1 (Tle 1). With the exceptions of Vin 1 nd Vin 4, ll B. ruxellensis strins exhiited greter intrcellulr (permeilized) enzymtic ctivity thn tht ssocited with the whole cells. Limited extrcellulr or superntnt ctivity ws noted (Tle 3). Seven of the nine Oenococcus oeni strins showed ß-glucosidse ctivity, rnging from 21 to 178 nmole ml -1 g dry cell mss hr -1 (Tle 2). All strins exmined hd lower ß-glucosidse ctivity thn the B. ruxellensis strins. No extr- or intrcellulr ß-glucosidse ctivity ws found for the O. oeni strins. Strins 528, 531, 566, 648, nd 649 displyed moderte whole cell or prietl enzyme ctivity. Viognier glycosides were used to determine enzyme hydrolysis of nturl sustrte in B. ruxellensis strins Brux, Souche Ave, Souche O, Vin 4 nd Vin 5, nd O. oeni strins 528, 566, 648, 649, nd 655. No ctivity on ntive glycoside ws detected (dt not shown). Tle 3 ß-Glucosidse ctivities y loction of enzyme ctivity for Brettnomyces ruxellensis strins. Vlues re verges of triplicte replictions. Enzyme ctivity B. ruxellensis strin Whole cell Permeilized Superntnt c c 142 e 11 cd d cd c 105 f 14 cd c 110 f 6 cd 215 <LOD d 74 g 11 cd Brux 26 c 182 d 11 cd Souche Ave 14 c 138 e 7 cd Souche O <LOD <LOD 4 d Souche M d 9 cd Vin 1 32 c 14 g <LOD Vin 3 22 c 232 c 9 cd Vin g 4 d Vin 5 4 d 21 g 11 c Activity is expressed s nmole p-nitrophenol (pnp) per mg cells (dry weight). Activity is expressed s nmole pnp per ml. c Different letters within columns indicte significnce t p <0.05. Mens with the sme letter re not significntly different. d Limit of detection (LOD): 3 nmol/ml. Discussion McMhon et l. [29] found whole cell ctivity to e higher thn intrcellulr in severl Brettnomyces strins; here, the opposite ws shown. The differences etween the two studies my e function of strin nd cell growth stge. In the current study, cultures were ssyed t log phse. The lck of ctivity in superntnt frctions of Brettnomyces in this study my e the result of young cultures, with limited incidence of utolysis. Yests re known to utolyze upon cell deth, relesing intrcellulr compounds [26]. ß-Glucosidse is locted in the periplsmic spce of S. cerevisie cells nd is relesed upon cell deth [9]. During lcoholic fermenttion, cell cultures rech mximum utolytic enzyme ctivity more quickly thn in nonfermenting cell cultures [12]. The permeiliztion technique tests endocellulr ctivity y rpidly mesuring the intrcellulr enzyme ctivity in smll mss of cells [30]. It is elieved tht the enzymes re functioning under conditions ner in vitro concentrtions nd with mcromolecules present [30]. This study suggests tht differences in hydrolytic enzyme ctivity my e function of strin nd my e influenced y cell wll vrition. In the enzyme ctivity procedure used [30], strins forming less cohesive cell pellets my lose some cells during the rinsing. Depending on the extent of the loss, ctivity dt would e rtificilly lowered. Differences etween strins in cell wll mnnn content cn result in vrying degrees of cell ggregtion [7]. In this study, the Brettnomyces strin Souche O exhiited the highest totl ß-glucosidse ctivity (2501 nmole ml -1 g dry cell mss hr -1 ), ut ctivity ws elow the limit of detection in extrcellulr nd prietl whole cell frctions. Empiricl oservtion during the study confirmed

4 306 Mnsfield et l. tht the pellet ws noncohesive. In contrst, some strins of B. ruxellensis re reported to hve high cohesion [35]. Oenococcus oeni re fstidious nd show decresed growth t ph ove 5.0 [15]. Use of TRB my hve promoted higher titer nd susequent enzyme production thn in n erlier study, which demonstrted n sence of glucosidse ctivity [29]. Additionlly, stggering inocultion times resulted in log phse smpling, potentilly resulting in higher hydrolytic ctivity. Grimldi et l. [16] pproximted erly nd lte log phse in modified MRS medium t 26 nd 40.5 hr, respectively, consistent with the rnge of 24 to 72 hr growth found in this study. Activity ws found to vry widely due to strin nd smpling period, s in this study. Different strins of O. oeni were used, ut ctivity rnges found y Grimldi et l. [16] were consistent with those of the current study. Boido et l. [5] found the extent of reduction in glycosylted compounds vried y strin. In the seven O. oeni strins tested in this study, the ctivity loction ssy indicted no extr- or intrcellulr ctivity nd whole cell ctivity in five strins (528, 531, 566, 648, nd 649, dt not shown). Decreses in internl cell ph, resulting from fermenttion, my cuse decrese in whole cell enzyme ctivity [28]. Alterntively, the lck of ctivity in the permeilized cell frction my hve een due to the relese of enzymes, initilly leding to higher ß-glucosidse concentrtion, ut the enzymes my hve een dentured or lost during pellet wsh. Cells were permeilized using plsmolysis process, which cn led to susequent utolytic relese of enzymes [6]. Grimldi et l. [16] found tht ß-glucosidse ctivity in iomss nd superntnt frctions vried widely due to strin nd phse of growth. Vrinces in the loction of hydrolytic enzyme ctivity reported in Tle 1 versus Tle 3 my hve occurred due to cell wll difference. The lck of ctivity of B. ruxellensis or O. oeni ginst ntive Viognier glycosides my e due to lck of enzymes cple of hydrolyzing discchrides. Discchride hydrolysis occurs in two steps, wherein the terminl sugr is first seprted y hydrolse, nd then glucose from the glycone y ß-glucosidse [21]. Monoscchride glycoside hydrolysis cn occur directly. In exmining yests, McMhon et l. [29] found tht only Aureosidium pullulns ffected ntive glycoside hydrolysis, perhps due to its rinosidse nd rhmnosidse ctivity. Different cultivrs my hve different pools of monond diglucosides [37] nd, therefore, different glycone pools. Glucosidses evince sugr- nd origin-sed selectivity. In ddition, enzymes from single orgnism my hve different ctivities; intrcellulr nd extrcellulr enzymes hve een found to hydrolyze the sme type onds, ut disply selectivity to specific glycones [18]. Conclusions The enzymtic liertion of glycoside hydrolysis products my produce rom, flvor, nd color chnges, influencing wine qulity. All strins of Brettnomyces ruxellensis nd 78% of Oenococcus oeni displyed ß-glucosidse ctivity. B. ruxellensis ctivity ws primrily intrcellulr. O. oeni strins showed some extrcellulr ctivity. No strins studied were le to hydrolyze glycosides isolted from Viognier grpes. It hs een previously demonstrted tht yest strins could produce intrcellulr ut not prietl ß-glucosidse [29]. Enzyme ctivity my e enhnced through the presence of oxygen, reduced ethnol, elevted ph nd temperture, nd the removl of glucose s n end product [30]; fermenttive environments seldom meet these requirements. Some hydrolytic enzyme production is depressed y feedck inhiition through glucose concentrtions less thn 0.5% (v/v) [31]. The results of this study suggest tht while B. ruxellensis nd O. oeni re cple of producing ß-glucosidses in model solutions, the production nd hydrolytic ctivity of these enzymes my e inhiited in wine. 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