ORIGINAL ARTICLE. L. Solieri 1, F. Genova 2, M. De Paola 2 and P. Giudici 1. Abstract

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1 Journal of Applied Microbiology ISSN ORIGINAL ARTICLE Characterization and technological properties of Oenococcus oeni strains from wine spontaneous malolactic fermentations: a framework for selection of new starter cultures L. Solieri, F. Genova, M. De Paola and P. Giudici Department of Agricultural and Food Sciences, University of Modena and Reggio Emilia, Reggio Emilia, Italy AEB-group, S. Polo, Brescia, Italy Keywords 6S ARDRA, malolactic fermentation, Oenococcus oeni, RAPD, starter selection, wine. Correspondence Lisa Solieri, Department of Agricultural and Food Sciences, University of Modena and Reggio Emilia, Via Amendola, 4 Reggio Emilia, Italy. lisa.solieri@unimore.it 9 4: received March 9, revised 9 April 9 and accepted May 9 doi:./j x Abstract Aims: To characterize the genetic and phenotypic diversity of 5 lactic acid bacteria (LAB) strains isolated from Italian wines that undergone spontaneous malolactic fermentation (MLF) and propose a multiphasic selection of new Oenococcus oeni malolactic starters. Methods and Results: One hundred and thirty-five LAB strains were isolated from different wines. On the basis of 6S amplified ribosomal DNA restriction analysis (ARDRA) with three restriction enzymes and 6S rrna gene sequencing, O. oeni strains were identified. M-based RAPD analysis was employed to investigate the molecular diversity of O. oeni population. Technological properties of different O. oeni genotypes were evaluated in synthetic medium at increasing selective pressure, such as low ph (Æ5, Æ and Æ) and high ethanol values (, and % v v). Finally, the malolactic activity of one selected strain was assessed in wine by malolactic trial in winery. Conclusions: The research explores the genomic diversity of wine bacteria in Italian wines and characterizes their malolactic metabolism, providing an efficient strategy to select O. oeni strains with desirable malolactic performances and able to survive in conditions simulating the harsh wine environment. Significance and Impact of the Study: This article contributes to a better understanding of microbial diversity of O. oeni population in Italian wines and reports a framework to select new potentially O. oeni starters from Italian wines during MLF. Introduction Malolactic fermentation (MLF) occurs after completion of the alcoholic fermentation and is conducted by lactic acid bacteria (LAB), mainly Oenococcus oeni. MLF entails the decarboxylation of l())-malic acid to l(+)-lactate and CO, resulting in a decrease in total acidity and an enhancement of both soft mouth feel and microbiological stability in wine (Kunkee 974). In addition, final wine quality is generally improved during MLF by modifying fruit-derived and volatile aromas and producing aromaactive compounds (Amerine and Kunkee 968; Kunkee 99; Maicas et al. 999; Liu ; Pozo-Bayón et al. 5). Spontaneous MLF is often unpredictable and has been described as being capricious. It may occur during, or many months after the completion of alcoholic fermentation (Davis 985; Wibowo et al. 985; Henick-Kling 99; Henschke 99), and it may also fail because of very harsh environmental conditions in the wine for bacterial survival and growth, i.e. low ph, high alcohol content, high SO concentrations and low temperatures Journal compilation ª 9 The Society for Applied Microbiology, Journal of Applied Microbiology 8 ()

2 Selection of new O. oeni starter cultures L. Solieri et al. (Lafon-Lafourcade 98; Wibowo et al. 988). Moreover, there are also undesirable effects of MLF on wine quality, because of the production of off-flavours (Henschke 99; Liu ), reduction in colour (as high as % in red wines) (Liu and Pilone ) and formation of biogenic amines (Davis 985; Henick-Kling 99; Mangani et al. 5). The overall effects of MLF are largely dependent on the species and strains that carry out the process and on the type of wine being manufactured. Several LAB species belonging to Lactobacillus, Pediococcus and Leuconostoc genera can be recovered in grape must, but they progressively disappear during the alcoholic fermentation. Oenococcus oeni is the major bacterial species found in wines during spontaneous MLF, as it is well adapted to the low ph and high ethanol concentration of wine (Wibowo et al. 985). However, O. oeni can be occasionally detected also with other LAB, mainly Lactobacillus spp., Pediococcus parvulus, Pediococcus damnosus, Pediococcus pentosaceus and Leuconostoc mesenteroides (Lafon-Lafourcade et al. 98; Lonvaud-Funel 999; Ribéreau-Gayon et al. 6). In the last four decades, the use of malolactic starter cultures has become widespread to control the MLF process and to prevent off-flavours. Oenococcus oeni is the preferred starter species because of its resistance to the alcohol, ph and SO content of wine. Moreover, O. oeni does not cause any deleterious effects on flavour and aroma, ensures control of the time and the rate of MLF and reduces the potential for spoilage by other bacteria. With the increasing application of O. oeni as starter cultures for vinification, there was also an increasing demand for new starters with defined technological and flavouring abilities. The first O. oeni culture was the strain ML 4 (Kunkee 984), after which a small number of commercial starter cultures have been shown to successfully perform MLF. In this respect, induction of MLF by inoculation with commercial O. oeni strains is not always successful. The starter efficiency depends on its ability to survive and proliferate in wine. Oenococcus oeni is known to be a fastidious bacterium (Kunkee 984) and has been often referred to as a highly heterogeneous species in terms of MLF performance (Garvie 967; Peynaud and Domercq 968; Irwin et al. 98; Tracey and Britz 987). Because the resistance to wine conditions is strictly strain dependent, the development of new malolactic starters is a multiphasic approach, whose identification and typing of O. oeni strains naturally occurring in wines that undergone spontaneous MLF are relevant steps. In this work, LAB responsible for spontaneous MLF in Italian wines were characterized to construct a O. oeni strains collection of oenological interest. Moreover, we proposed a selection plan based on RAPD genotyping and technological assays in order to simplify the screening of suitable starter cultures. Materials and methods Reference strains The type strains included in this study were Lactobacillus brevis DSM54 T, Lactobacillus buchneri DSM57 T, Lactobacillus plantarum DSM74 T, Lactobacillus hilgardii DSM76 T, Ped. parvulus DSM T, Ped. pentosaceus DSM6 T, Leuc. mesenteroides ssp. mesenteroides DSM4 T, Leuc. mesenteroides ssp. cremoris DSM46 T and O. oeni DSM5 T. Also, an O. oeni commercial starter (AGF5) was used as control in MLF assays. Type strains were maintained on MRS (de Man et al. 96; Oxoid, UK), while strain AGF5 on AJ-MRS medium [MRS supplemented with apple juice % (v v)]. Samples, strains isolation and growth conditions LAB strains were isolated from Italian wines during spontaneous MLF. Oenological parameters of wines were determined according to Ribéreau-Gayon et al. (6). Wine aliquots of ml from a tenfold dilution series were plated out on MRS, AJ-MRS and LG (Larpent and Larpent-Gourgaud, 985) media, solidified with agar (Æ5% w w) and supplemented with cycloheximide (Æ mgl ) ) to inhibit yeasts. Each dilution was plated out in triplicate and maintained under facultative anaerobic conditions (Gas Generating kit; Oxoid Ltd) for 4 6 days at C. After incubation, the number of colonies was counted and expressed as CFU ml ). For each sample, colonies were randomly picked from the highest dilution plates, and the isolates were collected and labelled, as reported in Table. After purification on the same isolation medium, presumptive LAB were phenotypically characterized by Gram staining, determination of morphology, arrangement and motility by phase-contrast microscopy as well as catalase activity. Purified LAB cultures were maintained at )8 C in the isolation medium supplemented with glycerol 5% (v v). Bacterial DNA extraction, 6S rrna gene amplification and ARDRA analysis Bacterial chromosomal DNA was extracted from LAB overnight cultures according to mechanical lysis protocol previously described (Gala et al. 8). DNA precipitates were resuspended in an appropriate volume of TE solution ( mmol l ) Tris HCl, ph 8Æ; mmol l ) EDTA) and quantified using a Nanodrop Nd spectrophotometer (Nano-drop Technologies, Wilmington, DE, USA). 6S 86 Journal compilation ª 9 The Society for Applied Microbiology, Journal of Applied Microbiology 8 () 85 98

3 L. Solieri et al. Selection of new O. oeni starter cultures rrna gene was PCR amplified using the eubacterial universal pair of primers 7f and 49r (numbering is based on the Escherichia coli 6S rrna gene) (Sato et al. ). PCR amplifications were performed in a 5 ll volume using a Hybaid PCR Express Thermal Cycler (Ashford, Middlesex, UK), according to the cycling parameters previously described (Sato et al. ). Each amplification mixture contained Taq polymerase buffer, lmol l ) deoxynucleotides, lmol l ) of each primer, units of Takara Ex Taq DNA polymerase (Takara, Kyoto, Japan) and 5 ng of DNA template. The amplified 6S rrna gene fragments ( ng) were digested with 5 U of the restriction enzymes HaeIII, BfaI and MseI (MBI Fermentas, Hannover, CA, USA) for Æ5 h at 7 C. The restriction products were separated by electrophoresis on a % (w v) agarose gel in Æ5 TBE buffer (9 mmol l ) Tris borate ph 8, mmol l ) EDTA) containing ethidium bromide. Gels were photographed under UV light using the BioDoc- Analyze gel imaging and analysis system (Biometra, Göttingen, Germany). Estimation of fragment lengths was evaluated by comparison to a -bp DNA ladder marker (GeneRuler bp DNA Ladder Plus; Fermentas, Hanover, MD, USA). 6S rrna gene partial sequencing and data analysis 6S rrna PCR products were purified using DNA Clean and Concentrator kit (Zymo Research, Orange, CA, USA) and sequenced in an automatic sequencer (MWG DNA Biotech Company, Ebersberg, Germany) using the external primers 7F and 49R, as well as two internal primers, WLAB and WLAB (López et al. ). Sequencing data were analysed with DNAstar 4.5 software package (DNASTAR Inc, Madison, WI, USA). The consensus sequences were compared with GenBank sequences using the basic local alignment search tool (blast) (Benson et al. ). 6S rrna partial sequences were deposited in the GenBank under the accession numbers: FM87859, FM87859, FM87859, FM87859, FM878594, FM878595, FM878596, FM878597, FM878598, FM878599, FM8786, FM8786, FM8786 and FM8786. RAPD-PCR typing RAPD-PCR analyses were performed using the single primers M (5 -GAGGGTGGCGGTTCT- ) and OPA (5 -GTTGCGATCC- ), as described by Zapparoli et al. (998) and Zavaleta et al. (997) respectively, with the exception of final concentration of lmol l ) for M primer. A volume of Æ5 ng of bacterial DNA was amplified in a 5 ll final volume using a Bio-Rad thermal cycler (My Cycler; Bio-Rad Laboratories, Hercules, CA) and a cycling program with 5 cycles (94 C for min; 4 C for s; 7 C for min and final extension at 7 C for 5 min) for primer M and 4 cycles (94 C for min; 6 C for min; and 7 C for min, with a final extension step at 7 C for 7 min) for primer OPA. The amplicons were resolved by electrophoresis in Æ5% agarose gels in TAE buffer (4 mmol l ) Trisacetate, mmol l ) EDTA ph 8Æ). The molecular size marker was the EcoRI HindIII digested lambda DNA ladder. The gels were stained with ethidium bromide and photographed as reported earlier. The pictures were subsequently analysed using the Bionumerics software version.5 (Applied Maths, Kortrijk, Belgium). RAPD-PCR patterns were grouped by means of the Pearson product moment correlation coefficient and the unweighted-pairgroup method using arithmetic averages (UPGMA) cluster analysis. For convenience, the correlation levels were expressed as percentages of similarity. The identity level for strain discrimination between different genotypes was determined using a cut-off of 8% similarity. Reproducibility was assessed obtaining M- and OPA-based patterns from six randomly selected isolates two or three times. The discriminatory power of M- and OPA- based typing methods was determined by calculating the discriminatory index (DI) for a randomly selected group of 45 strains using Simpson s index of diversity, as described by Hunter and Gaston (988). MLF performances assays The first panel of malolactic microvinification assays was performed in synthetic wine (SW) [4 g l ) yeast extract, gl ) glycerol, 6 g l ) d,l-malic acid, (Carreté et al. )] in the presence of % (v v) ethanol at ph Æ5 and Æ respectively. Cells grown at exponential phase on grape juice medium (GJM) at ph 4Æ8 [ g l ) glucose, 5gl ) yeast extract, g l ) peptone, Æ gl ) MgSO 4 7H O, Æ5 g l ) MnSO 4 7H O, g l ) d,l-malic acid, Æ% (v v) Tween 8, 5% (v v) grape juice; ph was adjusted with N NaOH] were inoculated at a final concentration of 6 CFU ml ) ( unit OD 6 =5Æ 8 CFU ml ) ) in the same medium at ph Æ5 andæ respectively. After incubation of 48 h at 8 C, cells were washed with physiological solution and resuspended in SW with the corresponding ph value (Æ5 or Æ respectively) at the final concentration of 6 CFU ml ). During incubation at 4 C for month, samples were taken at different interval times, and l-malic acid concentrations were determined with enzymatic kit (Boehringer Mannheim, Germany) according to the manufacturer s protocol. Viable cell numbers were measured by plating diluted SW aliquots on solid AJ-MRS medium. Selected strains were then submitted to second screening panel in SW medium as described earlier, with the Journal compilation ª 9 The Society for Applied Microbiology, Journal of Applied Microbiology 8 ()

4 Selection of new O. oeni starter cultures L. Solieri et al. following combination of ph and ethanol content: (i) ph Æ5 and % (v v) ethanol, (ii) ph Æ5 and % (v v) ethanol, (iii) ph Æ and % (v v) ethanol, (iv) ph Æ and % (v v) ethanol, (v) ph Æ and % (v v) ethanol. Starter preparation and MLF in winery One preculture (5 l) of the selected strain LB4 was obtained after three days of growth on LG medium at 8 C. Cells were centrifuged, resuspended in 5 ml of fresh LG medium and frozen at ) C. After 8 days from the end of alcoholic fermentation, frozen preparation was revitalized in LG medium (4 h at C) and inoculated both 5 l (oak barrel) and l (steel tank) of Chardonnay wine at the final concentration of 6 CFU ml ). Uninoculated wine in 5 l oak barrel was used as control. The temperature was around C. Physicochemical and microbiological parameters of Chardonnay wine resulted as follows (values were determined as reported earlier as mean of three replicas, SD in brackets): Æ gl ) (±Æ) sugars, 4Æ4 g l ) (±Æ7) l-malic acid, g l ) (±) l-lactic acid, 9Æ9 g l ) (±Æ) total acidity, Æ5 g l ) (±Æ) volatile acidity, ph Æ (±Æ), Æ7% (v v) (±Æ) ethanol, 4Æ ppm (±6Æ5) total SO, Æ67 ppm (±Æ) free SO, indigenous LAB < CFU ml ). At the middle stage of MLF, colonies (five from barrel and six from tank wine respectively) were randomly picked from plates at highest dilution and identified at species level as previously reported. Genotyping was performed using M and OPA-RAPD techniques, as reported earlier. Results Identification of LAB strains LAB were isolated from different wines collected in four Italian wine-producing localities. For this purpose, we used the generic LAB medium MRS, as well as the media AJ-MRS and LG, supplemented with apple juice to improve O. oeni growth because of the presence of growth-promoting factors (Ribéreau-Gayon et al. 6). Chemical and microbiological parameters of the samples are recorded in Table. The ph values of the wines ranged from Æ to Æ78. LAB populations were variable, ranging from 4Æ to Æ5 7 CFU ml ) on AJ-MRS and from Æ7 to Æ 7 CFU ml ) on LG medium. No growth on MRS medium occurred in eight samples. With respect to LAB metabolism and MLF, l-malic content was very low in relation to l-lactic acid content in eight samples, indicating that these wines were undergoing this transformation. A total of 5 Gram-positive and catalase negative rods and coccobacilli were isolated at the middle or the end of the MLF. We applied a genotypic approach comprising 6S ARDRA analysis with three restriction enzymes and 6S rrna gene sequencing to identify LAB isolates. Fingerprinting analysis based on combined HaeIII, BfaI and MseI ARDRA patterns permitted to distinguish all the reference strains (data not shown), with the exception of strains Leuc. mesenteroides spp. mesenteroides DSM4 T and Leuc. mesenteroides spp. cremoris DSM46 T. Moreover, six different groups of wine LAB isolates were identified (Table ). Oenococcus oeni species ( strains) Table Chemical and microbiological parameters of wines samples Chemical determination CFU ml ) Sample Cultivar Localities ph L-malic acid* Lactic acid AJ-MRS LG MRS No. of isolates MZ Marzemino Trentino Alto Adige Æ75 Æ Æ8 6 Æ ( ) PN Black Pinot Trentino Alto Adige Æ57 Æ7 Æ9 4 5 ND ( ) MRL Merlot Trentino Alto Adige Æ78 Æ6 Æ8 Æ5 5 Æ6 5 ND ( ) AM5 Cabernet-sauvignon Trentino Alto Adige Æ45 Æ Æ9 5 7 ND 7(4++) 5 Barbera Piemonte Æ5 Æ Æ9 7Æ 5 Æ7 ND (7 + + ) SOA Soave s Garganega Veneto Æ55 Æ Æ4 5 Æ6 5 ND 6 ( ) 88 Prosecco Veneto Æ Æ4 Æ Æ 5 Æ7 5 ND ( ) 8 Prosecco Veneto Æ5 Æ5 Æ8 5Æ67 5 Æ6 5 ND 9(5+4+) 84 Prosecco Veneto Æ5 Æ Æ 5 Æ6 6 5 ( ) 85 Prosecco Veneto Æ4 Æ Æ 5Æ 5 Æ5 5 ND ( ) 74 Inzolia Sicilia Æ65 Æ Æ Æ ( ) 5 Chardonnay Sicilia Æ49 Æ Æ4 Æ5 7 Æ 7 8 ( ) ND, not detected. *l-malic and l-lactic acids in g l ). In bracket colonies numbers isolated from AJ-MRS, LG and MRS media respectively. 88 Journal compilation ª 9 The Society for Applied Microbiology, Journal of Applied Microbiology 8 () 85 98

5 L. Solieri et al. Selection of new O. oeni starter cultures Table 6S partial gene ARDRA analysis with three endonucleases of the wine lactic acid bacteria isolates 6S ARDRA analysis Sample Medium Strainà HaeIII BfaI MseI Attribution MZ LG LB, LB, LB, LB4, LB5 59,,, 9, 9 5, 5, 9, 5, 6, 5,, 4, 5 Oenococcus oeni AJ-MRS LA, LA LA, LA5 59,,, 9, 9 5, 5, 9, 5, 6, 5,, 4, 5 O. oeni LA4 59, 4,, 9 85, 8, 9, 8 4, 6, 5, 5, 4, 9 Profile A PN MRS LZ, LZ, LZ, LZ4, LZ5 59,,, 9, 9 5, 5, 9, 5, 6, 5,, 4, 5 O. oeni LG LB, LB, LB, LB4, LB5 59,,, 9, 9 5, 5, 9, 5, 6, 5,, 4, 5 O. oeni AJ-MRS LA, LA4 59,,, 9, 9 5, 5, 9, 5, 6, 5,, 4, 5 O. oeni LA, LA 59, 4,, 4 58,, 9, 9, 8 45, 6, 5,, 8, 4, 9 Lactobacillus casei LA5 59, 4,, 9 99,, 8 4, 6, 5,, 4,, 9 Lactobacillus hilgardii MRL LG LB, LB, LB, LB4, LB5 59,,, 9, 9 5, 5, 9, 5, 6, 5,, 4, 5 O. oeni AJ-MRS LA, LA, LA, LA4, LA5 59,,, 9, 9 5, 5, 9, 5, 6, 5,, 4, 5 O. oeni AM5 LG LB4, LB5, LB6, LB 59,,, 9, 9 5, 5, 9, 5, 6, 5,, 4, 5 O. oeni AJ-MRS LA, LA 59,,, 9, 9 5, 5, 9, 5, 6, 5,, 4, 5 O. oeni LA 59, 4,, 4 85,,,, 8 4, 6, 5, 5, 4, 9 Pediococcus parvulus 5 LG LB, LB, LB, LB4, LB6, LB8, LB 59,,, 9, 9 5, 5, 9, 5, 6, 5,, 4, 5 O. oeni AJ-MRS LA, LA6, LA8 59,,, 9, 9 5, 5, 9, 5, 6, 5,, 4, 5 O. oeni SOA LG LB, LB, LB4, LB5, 59,,, 9, 9 5, 5, 9, 5, 6, 5,, 4, 5 O. oeni LB6, LB9, LB LB 59, 4,, 4 58,, 9, 9, 8 45, 6, 5,, 8, 4, 9 Lact. casei AJ-MRS LA, LA4, LA6, LA7, 59,,, 9, 9 5, 5, 9, 5, 6, 5,, 4, 5 O. oeni LA9, LA,LA, LA 88 LG LB4, LB4, LB4, LB44, LB45 59,,, 9, 9 5, 5, 9, 5, 6, 5,, 4, 5 O. oeni AJ-MRS LA4, LA4, LA4, LA44, LA45 59,,, 9, 9 5, 5, 9, 5, 6, 5,, 4, 5 O. oeni 8 LG LB5, LB5, LB5, LB54 59,,, 9, 9 5, 5, 9, 5, 6, 5,, 4, 5 O. oeni AJ-MRS LA5, LA5, LA5, LA54, LA55 59,,, 9, 9 5, 5, 9, 5, 6, 5,, 4, 5 O. oeni 84 LG LB6, LB6, LB6, LB64, LB65 59,,, 9, 9 5, 5, 9, 5, 6, 5,, 4, 5 O. oeni AJ-MRS LA6 59,,, 9, 9 5, 5, 9, 5, 6, 5,, 4, 5 O. oeni LA6, LA65 LA6, LA64* 59, 4,, 9,, 8 4, 6, 5,, 4,, 9 Lact. hilgardii MRS LZ6, LZ6, LZ64 59, 4,, 4 85,,,, 8 4, 6, 5, 5, 4, 9 Ped. parvulus LZ6, LZ65 59, 4,, 9,, 8 4, 6, 5,, 4,, 9 Lact. hilgardii 85 LG LB7, LB7, LB7, LB74, LB75 59,,, 9, 9 5, 5, 9, 5, 6, 5,, 4, 5 O. oeni AJ-MRS LA7, LA7, LA7 LA74, LA75 59,,, 9, 9 5, 5, 9, 5, 6, 5,, 4, 5 O. oeni 74 LG LB, LB, LB, LB4, LB5 59,,, 9, 9 5, 5, 9, 5, 6, 5,, 4, 5 O. oeni AJ-MRS LA, LA, LA, LA4, LA5 59,,, 9, 9 5, 5, 9, 5, 6, 5,, 4, 5 O. oeni MRS LZ, LZ, LZ5 59,,, 9, 9 5, 5, 9, 5, 6, 5,, 4, 5 O. oeni 5 LG LB, LB, LB, LB4, LB5 59,,, 9, 9 5, 5, 9, 5, 6, 5,, 4, 5 O. oeni AJ-MRS LA, LA, LA, LA4, LA5 59,,, 9, 9 5, 5, 9, 5, 6, 5,, 4, 5 O. oeni *Underlined strains were submitted to malolactic activity assays Profiles did not consider restriction fragments under 7 bp. àstrains in bold were subjected to 6S rrna gene sequencing. Journal compilation ª 9 The Society for Applied Microbiology, Journal of Applied Microbiology 8 ()

6 Selection of new O. oeni starter cultures L. Solieri et al. predominated in all the samples, whereas obligately heterofermentative Lact. hilgardii (six strains), obligately homofermentative Ped. parvulus (four strains) and facultative heterofermentative Lactobacillus casei (three strains) appeared sporadically. Only the strain LA4 showed restriction profiles, which did not matched with any reference strains used in this work (profile A). To confirm the differences in the rrna gene tandem repeat, 6S rrna encoding genes of 4 LAB strains representative of each restriction profile were partially sequenced. Phylogenetic analysis confirmed the conspecificity for strains belonging to O. oeni, Lact. hilgardii, Ped. parvulus and Lact. casei species. 6S sequence analysis of strain LA4 (profile A) revealed high homology with the Lactobacillus mali 6S rrna gene sequence. The majority of wines showed O. oeni as the only isolated species, suggesting that O. oeni is one of the most representative LAB species harbouring in wines collected in this study. Five samples showed more than one species, and their LAB species distribution was reported in Fig.. Diversity of Oenococcus oeni strains We used RAPD molecular markers to assess the genomic variability and to determine possible clonal relationships among the O. oeni strains. For this purpose, preliminary evaluation of two RAPD-PCR primers (M and OPA) was carried out using a subset of 45 randomly selected strains. Amplification with primer M generated 6 7 fragments, ranging from 9 to 5 bp, and primer OPA generated 7 fragments of 4 55 bp. Isolates that showed very similar RAPD patterns (similarity coefficient >8%) were considered as belonging to the same LAB species (%) % 8% 6% 4% % % MZ PN AM5 Wines Figure Distribution of lactic acid bacteria species in five wine samples (MZ, PN, AM5, SOA and 84): () Oenococcus oeni; () profile A; () Lactobacillus hilgardii; (4) Lactobacillus casei and (5) Pediococcus parvulus. SOA 84 strain (and thus to the same genotype). The DI values were of Æ88 and Æ86 for M-RAPD and OPA-RAPD assays respectively (data not shown). Therefore, RAPD analysis with M primer was chosen for our study. All the O. oeni isolates generated DNA fingerprints that were clustered in a unique dendrogram. On the basis of a computerized numerical analysis of M-RAPD patterns, most strains clustered within the range of 6 % similarity. As showed in Fig., ten very well-defined groups (from A to L) at a similarity level below 5% and 4 subclusters (from C to C4) at a similarity level of 8% were discerned. The remaining 6 subclusters contained only one isolate. The number of RAPD profiles (and thus of different genotypes) identified with a cut-off value of 8% similarity was 4 out of isolates, according to a type variation of Æ%. Considering the subclusters from C to C4, we observed that strains isolated from different wines were grouped in different clusters, with the exception of subclusters C and C, which consisted of isolates from two different wines, such as sample pairs 8 88 and AM5 5 respectively. Distribution of RAPD types for sample showed from two to six O. oeni RAPD types growing in the same sample with the exception of sample 88, from which only one type was isolated (Table ). Malolactic microvinifications Twenty-four strains representative of different M-RAPD genotypes and one commercial O. oeni starter (AGF5) were submitted to comparative assay of malolactic performance at laboratory scale (microvinifications) using a synthetic medium similar to wine (SW) with an ethanol concentration of % (v v). In particular, we studied the inhibitory effect of ph (Æ5 and Æ) on cell growth of O. oeni. A previous adaptation of strains in GJM-rich medium at the corresponding ph value was carried out to improve the survival of O. oeni cells (Guzzo et al. 994; Beltramo et al. 6). All the strains were able to consume completely l-malic acid in SW at ph Æ5 after 7 or 4 days, with the exception of strains LB and LB. Representative malic consumption curves are showed in Fig.. Compared to ph Æ5, SW at ph Æ was more selective for the majority of strains, as the complete consumption of l-malic acid was generally delayed. Three strains that did not show growth in precultivation medium at ph Æ were eliminated, resulting in strains tested. For each strain, viable cells counts confirmed the maintenance of the inoculation level (about 6 CFU ml ) ) during the first month of bioconversion, and then a decrease was observed (data not shown). l-malic acid consumption curves differed strongly among the strains (Fig. 4). Five 9 Journal compilation ª 9 The Society for Applied Microbiology, Journal of Applied Microbiology 8 () 85 98

7 L. Solieri et al. Selection of new O. oeni starter cultures A 5% similarity 5 5 B C 45 8 D E % similarity C C C LA65 LB64 C4 LB5 C5 C6 C7 C8 LA LA C9 LA6 C C Sample 85 Sample 84 Sample 74 Samples 8 and F 5 G H I 5 4 L C LA LB C C4 C5 C6 C7 C8 LA LB5 C9 C C LB4 LB LB LB4 LB C C LA8 C4 Sample SOA Samples MRL,PN,MZ Samples AM5 and 5 Figure Dendrogram based on the UPGMA clustering with Pearson coefficient M-RAPD of patterns of Oenococcus oeni strains isolated from spontaneous malolactic fermentations. The vertical line indicates the 8% similarity value for delineating subclusters (from C to C4). The dotted vertical line indicates the 5% similarity value for delineating main clusters (from A to L). of strains were unable to completely consume l-malic acid, whose concentration was about Æ5 gl ) after 5 days of microvinifications (Fig. 4a). Reduction of l-malic acid was very slow for eight strains and for the commercial starter, which completely decarboxylated malate after 5 days (Fig. 4b). Eight strains were fast Journal compilation ª 9 The Society for Applied Microbiology, Journal of Applied Microbiology 8 ()

8 Selection of new O. oeni starter cultures L. Solieri et al. Table Number of different Oenococcus oeni types obtained by M-RAPD-based analyses Samples RAPD types MZ 5 (4)* PN 6 (7) MRL 4 () AM5 (6) 5 () SOA (5) 88 () 8 (9) 84 4 () 85 () 74 4 () 5 5 () TOT types 4 () *In bracket, total number of O. oeni isolates in each sample. L-malic acid (g l ) Figure Evolution of L-malic acid concentrations in synthetic wine (ph Æ5 and % v v ethanol) inoculated with the following Oenococcus oeni strains: ( ) LA; ( ) LA; ( ) LB; ( ) LB; ( ) LA; ( ) LB4; ( ) LB; ( ) LA; ( ) LA; ( ) LA; ( ) LB4; ( )LB4; ( ) LB5; ( ) LB; ( ) LB and ( ) AGF5. Values reported are mean of two independent replicas. consuming, seven of which showed a malate decarboxylation kinetic of 5 days and strain LB74 of 7 days respectively (Fig. 4c). Four strains (LA, LB4, LB4 and LB74) showing the best malolactic activity in previous steps were submitted to a second panel of microvinification assays with five combinations of ph and ethanol as described earlier. Independently from ethanol content (% or % v v), ph value of Æ5 was found to be the best survival condition for the strains tested, permitting the l-malic acid consumption for three (Fig. 5a) and two (Fig. 5b) of four strains considered. In particular, strains LA and LB74 showed a more rapid decarboxylation of l-malic acid to l-lactic acid (a) 5 L-malic acid (g l ) L-malic acid (g l ) L-malic acid (g l ) 5 (b) 5 5 (c) Figure 4 Evolution of L-malic acid concentrations in synthetic wine (ph Æ and % v v ethanol). (a) Strains unable to consume L-malic acid: ( ) LA4; ( ) LB5; ( ) LB; ( ) LA and ( ) LB. (b) Strains with slow-consuming L-malic acid: ( ) LA; ( ) LA; ( ) LB; ( ) LA; ( ) LB; ( ) LA; ( ) LB4; ( ) AGF5 and ( ) LB75. (c) Strains fastconsuming L-malic acid: ( ) LB74; ( ) LA; ( ) LB4; ( ) LB; ( ) LB6; ( ) LB; ( ) LA6 and ( ) LA5. Values reported are mean of two independent replicas. than commercial starter AGF5, which is used as control. At ph Æ, the lowest ethanol content (% v v) was the only condition suitable for survival of O. oeni strains (Fig. 5c,d). Moreover, no strain was found to be able to consume l-malic acid at ph value of Æ (Fig. 5e). 9 Journal compilation ª 9 The Society for Applied Microbiology, Journal of Applied Microbiology 8 () 85 98

9 L. Solieri et al. Selection of new O. oeni starter cultures (a) 5 L-malic acid (g l ) 5 (c) 5 L-malic acid (g l ) (b) L-malic acid (g l ) L-malic acid (g l ) (d) (e) L-malic acid (g l ) Figure 5 Evolution of L-malic acid concentration in synthetic wine inoculated with strains LA ( ); LB4 ( ); LB4 ( ); LB74 ( ) and AGF5 ( ). (a) ph Æ5 and % (v v) ethanol content; (b) ph Æ5 and % (v v) ethanol content; (c) ph Æ and % (v v) ethanol content; (d) ph Æ and % (v v) ethanol content; (e) ph Æ and % (v v) ethanol content. Values reported are mean of two independent replicas. Starter preparation and MLF in winery Finally, we evaluated the malolactic performance of a promising strain, LB4, in Chardonnay wine at winery scale. Two different assays were carried out in traditional barrel and steel tank. For this purpose, a frozen preparation of strain LB4 was obtained from LG medium culture. The recovery of viable cells after freezing was close to % (data not shown). Concerning the LAB population, a lag period of 5 days was observed both in traditional barrel and steel tank, during which the LAB population decreased from 6 (inoculum) to CFU ml ). After month from the MLF, an increase in viable cells up to about 6 CFU ml ) was observed in both cases (data not shown). On the contrary, the LAB population was maintained at low level (about CFU ml ) ) in uninoculated wine during the complete vinification period. The kinetics of consumption of l-malic acid and production of and l-lactic acid are reported in Fig. 6. Depletion of l-malic acid occurred in wines inoculated with strain LB4, but not in uninoculated wine, where the indigenous LAB took 6 days to begin MLF. A higher rate of l-malic acid consumption was observed for inoculated wine stored in steel tanks with regard to barrels. This difference could be dependent on important physicochemical parameters, such as redox potential and oxygen concentration, which could be changed between steel tanks and barrels. Microbiological control of LAB population in inoculated wines was performed by 6S ARDRA analysis and RAPD typing on randomly selected colonies from plates at the highest dilution collected in the middle Journal compilation ª 9 The Society for Applied Microbiology, Journal of Applied Microbiology 8 ()

10 Selection of new O. oeni starter cultures L. Solieri et al. L-malic acid (g l ) Figure 6 Evolution of malolactic fermentation in wine inoculated with selected strain LB4 at winery scale. L-malic acid consumption in inoculated ( ) barrel and ( ) steel tank and uninoculated wines ( ) as well as l-lactic acid production in inoculated ( ) barrel and ( ) steel tank and in uninoculated wines ( ) were reported. The dotted vertical line indicates the time of sample collection for microbiological control. phase of MLF (Table 4). All the isolates belonged to O. oeni species, according to their 6S ARDRA profiles. Strain typing carried out using M primer showed that all the isolates, both those from barrel (five colonies) and tank (six colonies), had the same M-RAPD profile than strain LB4, suggesting a good implantation of starter on indigenous LAB population. The same isolates were submitted also to OPA-RAPD analysis. Only two colonies isolated from steel tank showed a RAPD profile similar to that of strain LB4, while the remaining colonies showed a different OPA profile because of a single about -bp long band (data not shown). Regarding barrel MLF, all the isolates had a unique OPA profile, which was different from that of inoculated strain. Table 4 6S partial gene ARDRA analysis and M OPA RAPD typing of lactic acid bacteria strains isolated from wines inoculated with selected strain LB4 during malolactic fermentation in barrel and tank at winery scale Molecular monitoring Barrel Tank 6S ARDRA Oenococcus oeni + + Other species ) ) M-RAPD LB4 profile 5 (5)* 6 (6) Other profiles ) ) OPA-RAPD LB4 ) (6) Other profiles 5 (5) 4 (6) + and ) indicate presence or absence, respectively. *In bracket, total number of O. oeni isolates. L-lactic acid (g l ) Discussion Selection of new starter culture for food and beverages fermentation entails a rational plan consisting of the constitution of microbial genetic pool (clonal selection), followed by a wide functional screening based on trialand-error method (Giudici et al. 5). Given the economic importance of MLF, development of O. oeni starter is an interesting aim in oenology. Recently, advances in molecular biology have provided new opportunities to overtake the time-consuming step of clonal selection by genetic improvement of the industrially useful strain. However, the constitution of biodiversity pool remains a very important step in the case of O. oeni, as effective DNA recombinant tools have been only recently set up for this species (Zuniga et al. ; Beltramo et al. 4; Assad-García et al. 8). In this work, we proposed a selection plan of new O. oeni starter including four phases: (i) strains collection constitution; (ii) O. oeni population genotyping; (iii) bottle-neck screening of different O. oeni genotypes on the basis of their technological properties; (iv) MLF test in winery. Recovery and molecular characterization of a high number of LAB strains is the first stage to establish a strains collection of oenological interest. PCR-based methods have been already successfully used to identify LAB species in different wines (Rodas et al. ; du Plessis et al. 4; López et al. 7; Cappello et al. 8). Here, we found O. oeni isolates out of 5 strains collected from different wine samples that undergone spontaneous MLF, confirming that O. oeni was the bestadapted wine species. The development of the lactobacilli and pediococci in wine samples was linked to the decrease in the wine quality (Ribéreau-Gayon et al. 6). In this respect, comparison of our results with those of similar studies on the biodiversity of wine LAB species indicated that, with the exception of few isolates belonging to Lact. casei, Lact. mali, Lact. hilgardii and Ped. parvulus, many wine LAB species were not recovered in wines considered in this study, despite using three different isolation media. Genotyping of O. oeni isolates is another important stage in development of nonredundant strain collection. Many studies using different genotyping techniques have indicated that O. oeni is a homogeneous species (Kelly et al. 99; Viti et al. 996; Zavaleta et al. 996, 997; Le Jeune and Lonvaud-Funel 997; Zapparoli et al. ; Sato et al. ; Guerrini et al. ; Bartowsky et al. ; Malacrinò et al. ). These data were discordant with phenotypical observation that O. oeni strains diverge in their tolerance towards environmental determinants, such as ethanol concentration, ph and SO, as well as in their growth and carbohydrate fermentation behaviour. 94 Journal compilation ª 9 The Society for Applied Microbiology, Journal of Applied Microbiology 8 () 85 98

11 L. Solieri et al. Selection of new O. oeni starter cultures Recently, de las Rivas et al. (4) demonstrated a high level of allelic diversity in O. oeni, which has been correlated to the lack of mismatch repair (MMR) genes system (Makarova et al. 6; Marcobal et al. 8). According to these findings, our M-RARD results indicated a high genotypic heterogeneity of O. oeni analysed strains. RAPD-PCR is considered to be a suitable method for typing O. oeni strains in winemaking (Zapparoli et al. ). Our estimation on DI value of M marker (Æ88) showed that M-RAPD can be a useful and fast O. oeni strain differentiation technique, different from that reported by Reguant and Bordons (). The degree of polymorphism was relatively high (type variation of Æ%) and comparable to those reported for other LAB species (Torriani et al. 999; Rodas et al. 5). A good correlation between RAPD profiles and wine samples was found, with only two cases where isolates coming from different samples showed identical RAPD pattern. Interestingly, a combination of different M-RAPD patterns were recovered in the most of wines samples, suggesting that there was a high variability of isolated strains and confirming that several strains can occur in a single spontaneous MLF (López et al. 7; Lechiancole et al. 6; Cappello et al. 8; Larisika et al. 8). A nonredundant biodiversity pool of different O. oeni genotypes reduces the number of strains to screen and simplifies the following evaluation of malolactic performances at increasing stressful conditions. The first assay of malate degradation activity showed that low ph is a crucial parameter to limit O. oeni growth in wine. Wide strain-specific differences were observed, mainly at ph Æ, but no correlation between the different genotypes and their ability to metabolize malic acid was found. Also Guerrini et al. () and Cappello et al. (8) found only a partial correspondence between genotypic data (obtained by PFGE and AFLP analyses respectively) and metabolic performances. Comparing data from two different ph values tested, several strains showed the loss of survival and malate activity on more acidic environment, which is in agreement with the general observation that strains isolated from wines and then cultivated in laboratory media can lose their vitality when re-inoculated in wine (Krieger et al. 99; Ribéreau-Gayon et al. 6). Marcobal et al. (8) suggested that the loss of MMR generated beneficial mutations during adaptation to a restrictive environment, but, at the same time, it could determine a decreased fitness when O. oeni is transferred to a second, less restrictive environment, because of the accumulation of mutations deleterious to growth on the new medium. According to bottle-neck plan, the second screening was carried out at more selective environmental conditions on a selected group of four strains showing the best malolactic performance in the previous stage. Using combinations of low ph values and high ethanol content, we showed that low ph values are the main negative feature affecting the malolactic activity. Independently from ethanol percentage, ph condition of Æ5 permitted the MLF accomplishment by three out of four strains analysed. However, ph values of Æ and Æ [combined with ethanol content of % and % (v v) respectively] were prohibitive conditions for all the strains. The malolactic performance of a selected strain in wine at winery conditions was the last step of selection. Our work evidences that degradation of l-malic acid accompanied by the expected increment in l-lactic acid concentration was successfully completed in wines inoculated with the selected strain, but not in uninoculated wine. However, the time of complete malate consumption is very slow in wine compared to SW, mainly because of the presence of inhibitory compounds and the low fermentation temperature. Analysis of LAB population driving the MLF in inoculated wines confirmed the dominance of O. oeni species over lactobacilli and pediococci, but typing of isolates by OPA-RAPD technique provided discordant results on isolates identity. Regarding the capacity of a selected strain to take over spontaneous LAB population, several works have evidenced that the dominance of the starter is not always guaranteed (Henick-Kling 995; Maicas et al. ; Arnink and Henick-Kling 5). The growth of indigenous LAB and many technological factors can significantly affect the implantation capacity of oenococcal starter (Wibowo et al. 985; Henick-Kling et al. 989; Reguant and Bordons ; Carreté et al. 6). However, elucidating all of these parameters is difficult because of the wine complexity and a trial-and-error panel is often the only procedure available to test O. oeni starter in real winery conditions. Although further investigations including a greater number of isolates are necessary, l-malic acid consumption occurring in inoculated wines but not in control batch suggests that our starter positively affects and stimulates the accomplishment of MLF. In conclusion, in this study, a high number of O. oeni strains from four Italian wine-producing areas were characterized from genetic and technological points of view. Moreover, we indicated an integrated approach based on genotyping and simulated microvinifications data to construct a nonredundant strains collection and to individuate O. oeni strains destined for selection of new malolactic starter cultures. As a result, four strains were selected to be developed as malolactic starter cultures. However, vinification in winery scale suggested that many other factors, such SO, low temperature and inhibitory compound, can significantly stress the O. oeni cells activity. An extensive study on the impact of different winemaking parameters on bacterial starters malolactic Journal compilation ª 9 The Society for Applied Microbiology, Journal of Applied Microbiology 8 ()

12 Selection of new O. oeni starter cultures L. Solieri et al. metabolism may generate a framework leading to the construction of oenological strains collection. It is a source for the selection of new malolactic starters with increased technological performance for quality wine production. Acknowledgements The authors thank Fabio Licciardello and Chiara Nicoletti for their technical assistance and contribution in microvinification experiments. References Amerine, M.A. and Kunkee, R.E. (968) Microbiology of winemaking. Annu Rev Microbiol, 58. Arnink, K. and Henick-Kling, T. (5) Influence of Saccharomyces cerevisiae and Oenococcus oeni strains on successful malolactic conversion in wine. Am J Enol Vitic 56, 8 7. Assad-García, J.S., Bonnin-Jusserand, M., Garmyn, D., Guzzo, J., Alexandre, H. and Grandvalet, C. (8) An improved protocol for electroporation of Oenococcus oeni ATCC BAA-6 using ethanol as immediate membrane fluidizing agent. Lett Appl Microbiol 47, 8. Bartowsky, E.J., McCarthy, J.M. and Henschke, P. () Differentiation of Australian wine isolates of Oenococcus oeni using random amplified polymorphic DNA (RAPD). Aust J Grape Wine Res 9, 6. Beltramo, C., Oraby, M., Bourel, G., Garmyn, D. and Guzzo, J. (4) A new vector, pgid5, for genetic transfer in Oenococcus oeni. FEMS Microbiol Lett 6, 5 6. Beltramo, C., Desroche, N., Tourdot-Maréchal, R., Grandvalet, C. and Guzzo, J. (6) Real-time PCR for characterizing the stress response of Oenococcus oeni in a wine-like medium. Res Microbiol 57, Benson, D.A., Karsch-Mizrachi, I., Lipman, D.J., Ostell, J., Rapp, B.A. and Wheeler, D.L. () GenBank. Nucleic Acids Res, 7. Cappello, M.S., Stefani, D., Grieco, F., Logrieco, A. and Zapparoli, G. (8) Genotyping by amplified fragment length polymorphism and malate metabolism performances of indigenous Oenococcus oeni strains isolated from Primitivo wine. Int J Food Microbiol 7, Carreté, R., Vidal, M.T., Bordons, A. and Constant, M. () Inhibitory effect of sulfur dioxide and other stress compounds in wine on the ATPase activity of Oenococcus oeni. FEMS Microbiol Lett, Carreté, R., Reguant, C., Rozès, N., Constantí, M. and Bordons, A. (6) Analysis of Oenococcus oeni strains in simulated microvinifications with some stress compounds. Am J Enol Vitic 57, Davis, C.R. (985) Practical implications of malolactic fermentation: a review. Am J Enol Vitic 6, 9. Gala, E., Landi, S., Solieri, L., Nocetti, M., Pulvirenti, A. and Giudici, P. (8) Diversity of lactic acid bacteria population in ripened Parmigiano Reggiano cheese. Int J Food Microbiol 5, Garvie, E. (967) The growth factor and amino acid requirements of species of the genus Leuconostoc, including Leuconostoc paramesenteroides (sp. nov.) and Leuconostoc oenos. J Gen Microbiol 48, Giudici, P., Solieri, L., Pulvirenti, A. and Cassanelli, S. (5) Strategies and perspectives for genetic improvement of wine yeasts. Appl Microbiol Biotechnol 66, Guerrini, S., Bastianini, A., Blaiotta, G., Granchi, L., Moschetti, G., Coppola, S., Romano, P. and Vincenzini, M. () Phenotypic and genotypic characterization of Oenococcus oeni strains isolated from Italian wines. Int J Food Microbiol 8, 4. Guzzo, J., Cavin, J.F. and Diviès, C. (994) Induction of stress protein in Leuconostoc oenos to perform direct inoculation of wine. Biotechnol Lett 6, Henick-Kling, T. (99) Malolactic fermentation. In Wine Microbiology and Biotechnology ed. Fleet, G.H. pp Chur, Switzerland: Harwood Academic Publisher. Henick-Kling, T. (995) Control of malolactic fermentation in wine: energetics, flavour modification and methods of starter culture preparation. J Appl Bacteriol Symp 79(Suppl), 9S 7S. Henick-Kling, T., Sandine, W.E. and Heatherbell, D.A. (989) Evaluation of malolactic bacteria isolated from Oregon wines. Appl Environ Microbiol 55, 6. Henschke, P.A. (99) An overview of malolactic fermentation research. Aus N Z Wine Ind J, Hunter, P.R. and Gaston, M.A. (988) Numerical index of the discriminatory ability of typing systems: an application of Simpson s index of diversity. J Clin Microbiol 6, Irwin, O.R., Subden, R., Lautensach, A. and Cunningham, J.P. (98) Genetic heterogeneity in lactobacilli and leuconostoc of enological significance. Can I Food Sci Tech J 6, Kelly, W.J., Huang, C.M. and Asmundson, R.V. (99) Comparison of Leuconostoc oenos strains by pulsed-field gel electrophoresis. Appl Environ Microbiol 59, Krieger, S.A., Hammes, W.P. and Henick-Kling, T. (99) Effect on medium composition on growth rate, growth yield, and malolactic activity of Leuconostoc oenos LoZH-t7-. Food Microbiol 9,. Kunkee, R.E. (974) Malo-lactic fermentation and winemaking. In Chemistry of Winemaking ed. Webb, A.D. Advances in Chemistry Series 7, pp Washington, DC: American Chemical Society. Kunkee, R.E. (984) Selection and modification of yeasts and lactic acid bacteria for wine fermentation. Food Microbiol, Journal compilation ª 9 The Society for Applied Microbiology, Journal of Applied Microbiology 8 () 85 98

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