Biodiversity, dynamics and ecology of bacterial community during grape marc storage for the production of grappa

International Journal of Food Microbiology 162 (2013) 143–151

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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro

Biodiversity, dynamics and ecology of bacterial community during grape marc storage for the production of grappa Petros A. Maragkoudakis a, 1, 2, Tiziana Nardi b, 1, 3, Barbara Bovo a, Maura D'Andrea b, Kate S. Howell c, Alessio Giacomini a, b,⁎, Viviana Corich a, b a b c

DAFNAE — Department of Agronomy, Food, Natural Resources, Animals and Environment, Viale dell'Università 16, Legnaro (PD), Italy Centro di Ricerca Interdipartimentale per la Viticoltura e l'Enologia, Università di Padova, via XXVIII Aprile 14, Conegliano (TV), Italy Melbourne School of Land and Environment, University of Melbourne, 3010 Parkville, Australia

a r t i c l e

i n f o

Article history: Received 27 April 2012 Received in revised form 21 December 2012 Accepted 2 January 2013 Available online 11 January 2013 Keywords: Grape marc Lactobacillus plantarum Antimicrobial activity Biofilm Genotyping

a b s t r a c t The Italian spirit obtained from grape marc, grappa, is produced by an extended storage of the marc which allows alcoholic fermentation. Bacterial populations can develop and are associated with off-flavour production. Grape marc acidification before storage is a common practice in distilleries to control bacterial proliferation. Few studies have been published on the microbial biodiversity in grape marc and no information exists about microbiology of acidified marcs and physiological properties needed for colonizing such an environment. The aim of this study was to investigate the composition and dynamics of grape marc bacterial populations during the long-period storage by microbiological analyses of acidified and untreated marcs. Eight bacterial species were identified by ARDRA — 16s rRNA sequencing at the beginning of the fermentation. Among them the bacterial species of Tatumella terrea, Acetobacter ghanensis and Tatumella ptyseos were identified for the first time in a wine environment. In later stages Oenococcus oeni and members of the Lactobacillus plantarum group became dominant in acidified and non-acidified grape marc, respectively. Further molecular typing of L. plantarum isolates yielded 39 strains. To explain the prevalence of L. plantarum in untreated samples, all strains were tested for potential antimicrobial activity and for biofilm formation ability. Although no antimicrobial activity was found, many strains exhibited the ability to form a biofilm, which may confer an ecological advantage to these strains and their dominance during marc storage. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Grape marc consists of the solid parts of grapes, containing grape skins, seeds and sometimes stalks which remain after juice extraction, and represents the main solid waste of the wine-making process. This type of by-product has been traditionally utilised in southern Europe, and spirits obtained from grape marc distillation are produced in almost all the Mediterranean countries (Gerogiannaki-Christopoulou et al., 2006). In addition, this material can be used for animal feed or recycled as soil conditioner due to its organic and nutrient contents. Moreover, grape marcs are also burnt after distillation for heat production (Lo Curto and Tripodo, 2001).

⁎ Corresponding author at: DAFNAE — Department of Agronomy, Food, Natural Resources, Animals and Environment, Viale dell'Università 16, Legnaro (PD), Italy. Tel.: +39 049 8272924; fax: +39 049 8272929. E-mail address: [email protected] (A. Giacomini). 1 These authors contributed equally to this work. 2 Present address: European Commission, Joint Research Institute, via E. Fermi 2749, 21027 Ispra (VA), Italy. 3 Present address: Lallemand Italia, via Rossini 14/B, 37060 Castel d'Azzano (VR), Italy. 0168-1605/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijfoodmicro.2013.01.005

Grappa is a traditional alcoholic beverage produced in Italy from steam distillation of grape marc (EEC, 1989). During the manufacturing process of white grape varieties, marc is separated from grape juice before must fermentation (De Rosa and Castagner, 1994). The marc is sent to the distillery where they are stored, generally in sealed plastic tunnels, for a period ranging from a few days to several weeks. During this period sugars present at 5 to 15% (w/w) in grape marc, are converted into ethanol to reach a final concentration of 4–10% (w/w). Generally, microbial starter cultures are not added and thus many undefined biochemical reactions take place due to the activity of the natural microflora. Although yeasts are essential to the process of alcoholic fermentation, they can also be responsible for the production of spoilage compounds. In particular yeasts often produce excess of higher alcohols that are then concentrated into the distillate (Bovo et al., 2010; Cortés et al., 2010; De Pina and Hogg, 1999; Nykanen, 1986; Weinberg et al., 1988). Bacteria are responsible for the most frequent off-flavours present in the distillate via the production of 2-butanol and ethyl lactate (Bae et al., 2006; Davis et al., 1985; Manitto et al., 1994; Pozo-Bayon et al., 2005; Versini and Margheri, 1979). For these reasons a well-controlled storage process that prevents the growth of spoilage microflora is considered essential. To control

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marc spoilage, modern distilleries often adopt various techniques such as pH adjustment, temperature control and oxygen depletion in order to limit the growth of spoilage bacteria. In most cases, bacterial overgrowth is hindered by acidification of grape marc during ensilage, by addition of diluted sulphuric acid (Da Porto, 2002). Despite the popularity of grappa in Italy, the specific aspects of bacterial development during grape marc fermentation and the impact that technological treatments may have on it have not been reported. In particular, no molecular characterization of the bacterial microflora has been described. In the limited literature available, lactobacilli and Lactobacillus plantarum was found to be the most abundant species isolated from grape marc (De Pina and Hogg, 1999; Silva and Malcata, 2000). This species is widely used in fermentations of vegetables (Leal-Sánchez et al., 2003; Ruiz-Barba and Jimenez-Diaz, 1994) and occurs during winemaking process (Capozzi et al., 2012; Lerm et al., 2011; Miller et al., 2011; Rodriguez et al., 2009). From an ecological point of view, lactobacilli and lactic acid bacteria (LAB) in general possess various mechanisms that allow them to successfully compete with other microbes in food matrices, including the production of antimicrobial substances, such as organic acids and bacteriocins (Holzapfel et al., 1995), as well as the ability to grow in the low-pH environment of fermented foods (Maragkoudakis et al., 2006). Interestingly, recent findings highlighted the ecological importance of biofilm formation in many bacterial species within the Lactobacillus genus, including L. plantarum (Kawarai et al., 2007) and other LAB associated with grapes including Pediococcus parvulus (Llauberes et al., 1990), Lactobacillus sanfranciscensis (Korakli et al., 2003) and Leuconostoc mesenteroides (Richard et al., 2005). The aim of this study was to investigate the composition, evolution and dynamics of bacteria during storage of grape marc, and to consider the influence of acidification on bacterial diversity. We studied the composition of marc from Prosecco grapes, a variety cultivated in north-eastern Italy. Physiological traits of representative bacteria were assessed in order to evaluate their impact on the longevity of these strains on grape marc during grappa production. The grape marc environment under storage is an important determinant of grappa quality, thus a better understanding of bacterial composition will improve distillate quality.

carefully washed before use and no sulfites were added throughout the process. In the distillery the grape marc was divided into two 120 kg aliquots, one acidified by addition of H2SO4 33% (v/v) to reduce the pH from 3.8 to 3.5, and the other left untreated. Sugar content as measured by the Fehling titration, was 7.5 g/100 g, which is consistent with Prosecco grape marcs from this region. The grape marc was stored into four 30 kg-capacity plastic bags, sealed hermetically, and placed at room temperature (10–18 °C). Grape marc samples were collected the day of processing (0 day) and after 30, 120 and 180 days of storage. At each sampling two 30-kg bags, one acidified and one untreated, were opened and five samples of 250 g were collected from each treatment. The pH of ensilaged grape marc was monitored at each microbiological sampling with a pH metre (Radiometer Copenhagen pHM82, Cecchinato, Italy).

2. Materials and methods

2.3. Grape marc microbiological analysis

2.1. Bacterial and yeast strains and culture conditions All bacterial and yeast strains used in this study are described in Table 1. The LAB were cultured for routine use in de Man, Rogosa and Sharp broth (MRS, Oxoid, UK) for 24 h at 30 °C except for Oenococcus oeni strains which were grown in Tomato Juice Broth (Oxoid) at pH 4.8 for 5 days at 30 °C in anaerobic conditions (Anaerogen, Oxoid, UK). Gluconobacter oxydans and Acetobacter ghanensis were cultured in YPM broth (5.0 g/L yeast extract, 3.0 g/L peptone, and 25.0 g/L mannitol, Oxoid) for 48 h at 30 °C, with shaking. Saccharomyces cerevisiae strains were grown in YPD broth (20.0 g/L peptone, 10.0 g/L yeast extract, 20.0 g/L dextrose, Oxoid) for 24 h at 25 °C. In order to mimic oenological conditions, the synthetic nutrient medium SNM was prepared according to Delfini and Costa (1993) with the following modifications: glucose, 200 g/L; tartaric acid, 3 g/L; malic acid, 2 g/L; (NH4)2SO4, 0.3 g/L; (NH4)2HPO4, 0.3 g/L; KH2PO4, 1 g/L; MgSO4·7H2O, 0.5 g/L; NaCl, 0.1 g/L; CaC12, 0.1 g/L; inositol, 2 mg/L; pH 4.0 reached with KOH. All strains were stored at −80 °C in cryovials with the relevant broth supplemented with 40% (v/v) of glycerol and 5% skim milk (Sigma-Aldrich).

Enumeration of bacterial microflora was carried out using standard serial dilution methods on Plate Count Agar (PCA, Oxoid) solid medium supplemented with 200 μg/mL of cycloheximide (Sigma-Aldrich) to prevent yeast growth. From the 250 g sample, 20 g was used for standard serial dilutions and plate count analysis. Samples were plated and grown to assess the total aerobic bacterial counts after seven days of aerobic incubation at 25 °C and total anaerobic bacterial counts after seven days of anaerobic incubation at 25 °C using the Anaerogen anaerobiosis kit (Oxoid). From both growth conditions, between 30 and 35 colony isolates were collected for genetic analysis from sampling after storage for 0, 30 and 120 days. On acidified marc sampling, the colonies at 30 and 120 days were re-streaked on MRS agar medium (DeMan Rogosa Sharp, Oxoid) and grown under anaerobic conditions. Quantification of yeasts microflora was performed by plating aliquots from serial dilutions on Wallerstein Laboratory (WL) nutrient agar (Green and Gray, 1962) supplemented with 100 μg/mL chloramphenicol to prevent bacterial growth. Counts were made after incubation at 25 °C for 48 h. Identification of these yeasts is described by Bovo et al. (2011).

2.2. Grape marc preparation

2.4. ARDRA analysis

Prosecco grapes were mechanically harvested and crushed in a commercial winery. The must was separated from the juice by pressing, and the marc was transported to a nearby distillery. All equipment was

DNA samples were extracted by NaOH-SDS cellular lysis by picking single colonies from agar plates (Corich et al., 2007). Identification was carried out using the 16S-ARDRA method with primers PA-PH

Table 1 Bacterial and yeast strains used in this study. Species

Designation

Origin

Acetobacter ghanensis Gluconobacter oxydans Lactobacillus brevis Lactobacillus casei Lactobacillus hilgardii Lactobacillus paraplantarum Lactobacillus plantarum Lactobacillus pentosus Lactobacillus mesenteroides subsp. mesenteroides Oenococcus kitharae Oenococcus oeni Pediococcus damnosus Saccharomyces cerevisiae S. cerevisiae S. cerevisiae S. cerevisiae S. cerevisiae S. cerevisiae S. cerevisiae Weissella paramesenteroides Weissella viridescens

T0PCP14 T0PCP27 DSMZ 20054T DSMZ 20011T T120PCM20 DSMZ 10667T DSMZ 20174T DSMZ 20314T DSMZ 20343T

Prosecco grape marc Prosecco grape marc International collection International collection Prosecco grape marc International collection International collection International collection International collection

DSMZ 17330 DSMZ 20252T DSMZ 20331T PerdominiFR95 AR1 AR3 AR8 NR14 P225.5 P304.13 DSMZ 20288 DSMZ 20410

International collection International collection International collection Commercial starter Prosecco grape marc Prosecco grape marc Prosecco grape marc Prosecco grape marc Prosecco grape must Prosecco grape must International collection International collection

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as described by Rodas et al. (2003) The amplification reaction was carried out in a 25 μL mixture containing 1U of Taq polymerase (Promega, USA) according to the manufacturer's instructions. MseI (Fermentas International Inc., Canada) restriction enzyme was used for the subsequent DNA digestion. The DNA digestion was performed at 65 °C for 2 h in a 20 μl mixture containing 5 U of MseI. Restriction fragments were run on 2% (w/v) agarose gel containing 0.1 μg/mL of ethidium bromide. Bands were visualised by UV transillumination, digital images were acquired with the EDAS290 capturing system (Kodak, USA) Restriction profiles were analysed using the BioNumerics software (v. 4.6, Applied Maths, Belgium). Reference type strains (Table 1) were also included in the analysis. 2.5. 16S rDNA sequencing PCR products amplified with PA and PH primers (Rodas et al., 2003) were purified using the ExoSap™ Cleanup system (United States Biochemical, Cleveland, USA). When necessary, extraction of DNA bands from agarose gel was performed with QIAquick Gel Extraction Kit (Qiagen) according to the manufacturer's instructions. Sanger sequencing was performed using an ABI protocol for Taq-Dye Terminator Sequencing on an automated ABI377 sequencer. Species identification was done after BLASTN alignment (www.ncbi.nlm.nih.gov/BLAST) with sequences in the GenBank public database.

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(1:1) with the appropriate culture media containing the indicator strain to reach a final concentration of 10 5 cfu/mL. Growth of the indicator strains at the appropriate conditions was monitored by optical density (OD) measurement at 600 nm at hourly intervals for a period of 24–48 h, using a spectrophotometric plate reader (Spectrafluor, TECAN, Switzerland). 2.9. Biofilm formation assay The ability of bacterial strains to form a biofilm was analysed in 96-well microtitre plates using the crystal violet assay as described by O'Toole et al. (1999) with the addition of three preliminary washing steps with PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) before crystal violet staining. After washing, the stained biofilm was solubilised by using 95% ethanol. Biofilm formation was quantified by measuring the OD590 in the wells with a spectrophotometric plate reader. L. plantarum strains were grown in MRS broth or in SNM and S. cerevisiae strains were grown in YPD for 24 h until stationary phase. Cultures were then adjusted by dilution to same OD values before the assay. The biofilm formation assay was also performed in a co-culture system with combinations of selected L. plantarum and S. cerevisiae strains, inoculated in SNM at a ratio of 10:1. The absorbance in the microtitre plates for L. plantarum strains, S. cerevisiae strains and the co-culture system was measured immediately after bacteria and yeasts inoculation and after 24 h at 30 °C.

2.6. Bacterial strain analysis by Rep-PCR 2.10. Statistical analysis The strain diversity of lactobacilli was determined by repetitive PCR (rep-PCR). Here, DNA was extracted as described by Gevers et al. (2001) with the following modifications. After the first centrifugation step the pellet was resuspended in 88 μL of TE buffer (Tris 10 mM, EDTA 1 mM, pH 8.0) with the addition of 10 μL of lysozyme (250 mg/mL, United States Biochemical) and 1 U of mutanolysin (Sigma). The mixture was incubated at 37 °C for 90 min, afterwards samples were centrifuged and the pellet resuspended in 500 μL of TE2 buffer (Tris 50 mM, EDTA 20 mM, pH 8.0). The lysate was then extracted as described, without the phenol/chloroform purification step. Primers BOX, A1R, ERIC2, (GTG)5 (Versalovic et al., 1994) and the primer pair ISRh1out22F and ISRh1out22R designed on bacterial insertion sequences (Muresu et al., 2005) were used. The resulting fingerprints were analysed by the BioNumerics software. The dendrogram was constructed using Pearson's correlation coefficients with the unweighted pair-group method using arithmetic averages (UPGMA) clustering method. 2.7. Bacteriocin assay Fresh cultures of selected potential producers, grown in MRS and SNM for 24 and 48 h, were centrifuged at 15 000 g at 4 °C for 30 min. Supernatants were then collected and adjusted to pH 6.5 with NaOH. The assay was carried out using the well diffusion assay (WDA) as described previously (Zoumpopoulou et al., 2008). Briefly, cell free culture supernatant (50 μL) was added to 5 mm wells drilled in solidified soft agar (1.2% w/v) medium containing approximately 10 5 cfu/mL of the indicator strain. Isolates of A. ghanensis, G. oxydans and Lactobacillus hilgardii were used as indicator strains (Table 1). Plates were checked for the presence of inhibition halos around the wells after 24 and 48 h of incubation at the growth conditions appropriate for each indicator organism. MRS broth and SNM (pH 6.5) without supernatant additions were used as negative controls. 2.8. Growth inhibition assay Growth inhibition was assessed by growth in 96-well microtitre plates (Cellstar, Greiner, Germany). Culture supernatants (adjusted to pH 6.5 with NaOH) from potential producer strains were diluted

Experimental data were analysed with the StatGraphics Centurion XV software (StatPoint Inc., USA) using the ANOVA multiple sample comparison. Statistically significant effects (P ≤ 0.05) were further analysed and means were compared by the Tukey's HSD test. Log cfu/g values in the text and graphs are presented in the form of mean ± standard deviation (SD). 3. Results 3.1. Acidification of grape marc and pH determination The pH value of grape marc was reduced from pH 3.8 to 3.5 after addition of sulfuric acid. The pH of the marc changed during storage, and after 30 days the pH values of the marc increased to 4.0 for untreated and 3.7 for acidified marc, and maintained the same values until 120 days (4.0 and 3.7 respectively). At the end of the ensilage period (180 days) pH value of acidified marc significantly (P ≤ 0.05) increased to 4.3 while untreated marc maintained the same value (3.7). At each sampling time, pH value differences between treated and control marc were statistically significant (P ≤ 0.05). 3.2. Grape marc microbiological analyses The microbiological analysis of grape marc from untreated and acidified samples is reported in Fig. 1. As regards yeast population (Fig. 1a), a statistically significant decrease (P ≤ 0.05) was observed during the first 30 days of ensilage in both untreated (6.5 ± 0.38 log cfu/g) and acidified marc (6.2 ± 0.08 log cfu/g). Population remained stable after 120 days but at the end of the storage a significant increase (P ≤ 0.05), almost to the initial numbers, occurred in both untreated and acidified marc (7.4 ± 0.05 and 7.2 ± 0.20 log cfu/g respectively). In the untreated samples, both total aerobic and anaerobic bacterial populations (Fig. 1b, c) exhibited a rapid, statistically significant (P ≤ 0.05), increase (1.4 log and 2.4 log cfu/g, respectively) within the first 30 days of ensilage. Population levels remained then stable between 30 and 180 days. In the acidified samples, total aerobic bacteria remained stable up to 120 days and increased to 6.8 log cfu/g

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a

8,5 8,0 7,5

log cfu/g

7,0 6,5 6,0 5,5 5,0 4,5 4,0 0

30

60

90

120

150

180

Days

b

8,5 8,0 7,5

log cfu/g

7,0 6,5 6,0 5,5 5,0 4,5 4,0 0

30

60

90

120

150

180

120

150

180

Days

c

8,5 8,0 7,5

log cfu/g

7,0 6,5 6,0 5,5 5,0 4,5 4,0 0

30

60

90

Days Fig. 1. Yeast (a), total aerobic (b) and anaerobic (c) bacteria population in acidified (○) and non-acidified (●) grape pomace at 0, 30, 120 and 180 days of storage. Values of each time point are the means of five replicates ± SD (error bars).

after 180 days. Total anaerobic flora of acidified samples increased during the first four months (from 5.7±0.08 log cfu/g at the beginning to 7.0 ±0.14 log cfu/g after 120 days, P ≤0.05), and then remained stable. 3.3. Species identification For each sampling time-point bacterial isolates were re-streaked on PCA medium. However, bacteria isolated from the acidified marc

at 30 and 120 days were small, indicating non-optimal growth on PCA. These colonies were re-streaked onto MRS medium in anaerobic conditions, to achieve better growth. This behaviour supports preliminary identification as LAB. MseI restriction analysis of 16S RNA sequences was performed on bacterial isolates collected from grape marc at 0, 30 and 120 days and grown in aerobic and anaerobic conditions. A total of 300 isolates from both natural and acidified grape marc were analysed. For species identification 16S-ARDRA analysis was used as described by Rodas et al. (2003). The restriction enzyme MseI was chosen for the analysis since it can resolve most of lactic acid bacteria associated with wine. In addition to the species analysed by Rodas, the discriminatory power of MseI was tested on Oenococcus kitharae, Weissella paramesenteroides and Weissella viridescens, three recently described species genetically close to the enological Leuconostoc and Oenococcus species (Table 1). In all the cases a specific profile was obtained for each species, except for the L. plantarum phylogenetic subgroup, which is composed of three species (L. plantarum, Lactobacillus pentosus and Lactobacillus paraplantarum). These species gave identical genetic profiles. Recently two more species were added to this group, Lactobacillus fabifermentans which shares 98.6% of 16S rDNA nucleotide sequence with L. plantarum type strain (De Bruyne et al., 2009) and Lactobacillus xiangfangensis which shows 98.8% sequence similarity of 16S rDNA gene with L. plantarum subsp. plantarum type strain (Gu et al., 2012). Performing an in silico analysis of the 16S rDNA MseI digestion, L. fabifermentans gave the same profile of L. plantarum, L. pentosus and L. paraplantarum, while L. xiangfangensis showed a different profile (see supplementary material S1). Cluster analysis was performed to evaluate differences at the species level. Based on a computerized numerical analysis of ARDRA electrophoretic patterns, the isolates were grouped into 11 clusters. At least one sample for each cluster was sequenced and BLAST search was used to indicate the most probable species identification. Alignment against the GenBank database was carried out using filters, thus only sequences proofread by NCBI (REFSEQ) or relative to type strains were included. Similarity percentage threshold was set at 98%. Species identification results, along with related NCBI accession numbers, are shown in Table 2. Eleven species were identified, with O. oeni being the most representative of the isolates (41% of all colonies) and also the unique species found in acidified grape marc from 30 days onwards. The most frequent pattern rescued in both acidified and untreated marcs at the start of the experiment and in non-acidified marcs after 30 and 120 days belonged to the L. plantarum group (38%). The 16S-rDNA sequence of the isolates was identical to the L. plantarum type strain sequence. Therefore, according to ARDRA results and in silico 16S rDNA analysis, the cluster was ascribed to the taxonomic group including L. plantarum, L. pentosus, L. paraplantarum and L. fabifermentans (see supplementary materials S1 and S2). This result confirms that species belonging to the L. plantarum group are the most representative bacteria during marc ensilage without any technological treatment, as described in a previous study (De Pina and Hogg, 1999). G. oxydans and P. parvulus represented the 23% and 17% of the total isolates, with the latter found only in non-acidified grape marcs. The other species were detected at values lower than 10%. 3.4. L. plantarum strain typing Among the primers tested in this study for L. plantarum strain typing, the (GTG)5 oligomer was selected as it showed higher reproducibility and pattern complexity, with patterns containing 15–20 bands ranging from 300 to 4000 bp (data not shown). A similarity value of 95% among electrophoretic profiles, obtained with the BioNumerics software from the comparison of three completely independent analyses of the same isolate (data not shown), was set as the threshold. This similarity value is consistent with previous works using the same primer (Gevers et al., 2001; Kostinek et al., 2008).

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Table 2 Evolution of bacterial species during grape marc fermentation. ARDRA profile

16S seq Genbank accession no.

Species

B C D E I L N O P Q R

HM562982 HM562983 HM562984 HM562985 HM562989 HM562990 HM562996 HM562993 HM562995 HM562998 HM562994

Lactobacillus hilgardii Pediococcus parvulus Acetobacter ghanensis Lactobacillus paracollinoides Oenococcus oeni Lactobacillus plantarum groupa Gluconobacter oxydans Tatumella terrea Acetobacter cerevisiae Gluconobacter cerinus Tatumella ptyseos Total colonies %

Total

%

Non-acidified T0 (%)

7 17 5 3 123 114 23 3 1 1 3 300 —

2.3 5.7 1.7 1.0 41.0 38.0 7.7 1.0 0.3 0.3 1.0 – 100

T30 (%)

Acidified T120 (%)

T30 (%)

T120 (%)

Aer

Microaer

Aer

Microaer

Aer

Micro aer

Aer

Micro aer

Aer

Microaer

– – 10 – – – 78 3 3 3 3 30 100

– – 7 – 10 70 – 7 – – 6 30 100

– – – – – 100 – – – – – 30 100

– 17 – – – 83 – – – – – 30 100

21 14 – 7 – 58 – – – – – 29 100

3 26 – 3 – 68 – – – – – 31 100

– – – – 100 – – – – – – 30 100

– – – – 100 – – – – – – 30 100

– – – – 100 – – – – – – 30 100

– – – – 100 – – – – – – 30 100

Aer: aerobiosis; microaer: micro-aerophilia. a (L. plantarum/Lactobacillus pentosus/Lactobacillus paraplantarum/Lactobacillus fabifermentans).

A total of 62 isolates belonging to the L. plantarum group randomly selected among those recovered from non-acidified marc at 0, 30 and 120 days of storage were screened and the results reported in a dendrogram (Fig. 2). The isolate names contain the day of isolation (T0, T30 and T120) followed by the isolate number. A total of 39 clusters were formed, each one including isolates with a genetic similarity percentage higher than the threshold. All isolates were clearly separated from L. pentosus, L. paraplantarum and O. oeni type strains. Most of the isolates grouped together with L. plantarum type strain but 22 isolates clustered separately. These 22 isolates were further identified as L. fabifermentans by 16S rDNA sequencing (data not shown). Only three fingerprinting patterns contained isolates present at the beginning of the ensilage and also after 30 or 120 days, whereas all the others were distinctive of one storage period. This finding indicates that an assemblage of three strains persisted throughout the storage process. The 39 strains identified as belonging to the L. plantarum subgroup, as well as the reference type strain of L. plantarum (Fig. 3a), were selected for further studies. Additionally, three more isolates were included. According to our typing, isolates T0-14 and T30-38, T30-32 and T120-16, T0-17 and T120-41 share profiles 10, 13 and 24 respectively, but were isolated at different sampling times (Fig. 3a). These strains were further studied to assess the physiological properties of bacteriocin production and/or biofilm-forming ability that may allow persistence throughout the grape marc fermentation.

pairs T0-14 and T30-38 with paired strains T30-32 and T120-16, biofilm formation improved during storage, when grown in both in MRS and SNM (Fig. 3a). Strains T0-17 and T120-41 showed a constant very high biofilm formation capability throughout the storage period both in MRS and in SNM. The S. cerevisiae strains used were recovered during a previous study on yeast populations from acidified (AR1 and AR3) and non-acidified (NR8 and NR14) grape marcs (Bovo et al., 2011). All of these S. cerevisiae strains isolated from grape marc storage have the ability to form biofilms in both SNM and YPD media (Fig. 3b). 3.7. Biofilm formation in co-culture system Based on the results of the biofilm formation assay on single cultures, representative L. plantarum strains were chosen to be used in co-culture systems with S. cerevisiae. The L. plantarum strains used included isolates with lower (T30-01 and T30-07) and higher (T0-49) biofilm formation ability, as well as the three L. plantarum strain pairs, containing isolates sharing the same profile but collected at different sampling times, described above. Results of biofilm assay using co-cultures are reported in Table 3. Five bacteria out of 9 tested showed increased biofilm values in the presence of any S. cerevisiae tested. Two S. cerevisiae strains (AR1 and AR3) showed a higher ability to form a biofilm when co-cultured with any L. plantarum strain tested. The exception was for co-culture of S. cerevisiae AR1 and L. plantararum T30-38, where the biofilm formation was lower.

3.5. Determination of antimicrobial activity 4. Discussion Antimicrobial activity was not observed when neutralised 24-h culture supernatants of the 42 L. plantarum isolates, grown both in MRS and SNM, were used against the indicator strains of A. ghanensis, G. oxydans and L. hilgardii (Table 1) using the well diffusion and microtitre plate assays (data not shown). 3.6. Biofilm formation in a single culture system The biofilm formation ability of the 42 examined L. plantarum isolates and the S. cerevisiae strains used in this study, measured as absorbance at OD590 is reported in Fig. 3a and b, respectively. A ratio of 1 or less (not shown on graph) is interpreted as limited or no biofilm formation (no increase in absorbance after 24 h of incubation). In order to define biofilm formation, a ratio of 1:5 has been arbitrarily set and represents a 50% increase in absorbance at 590 nm. Thus, 33% and 49% of the L. plantarum strains identified in this study, grown in SNM and MRS respectively, can form biofilms. For some strains, namely typing

Prosecco grape marc used for grappa production was examined for the bacterial profiles during the storage period that precedes distillation. A common practice in distilleries to reduce fermentation off-flavours via bacterial growth is to acidify the marc. From our results, the yeast populations were only slightly influenced by this treatment, confirming that S. cerevisiae, the main yeast present during the active phase of grape marc fermentation (Bovo et al., 2009, 2011, 2012), is tolerant to low pH (Kalathenos et al., 1995; Kudo et al., 1998). However, a reduction of bacterial population was observed in acidified marc, particularly during the first 30 days of storage. This result could explain the positive effect on the distillate quality observed when acidification is used in distillery. A previous study demonstrated that this treatment can positively modify the distillate composition in terms of lowering specific off-flavours such as ethyl lactate and 2-butanol (Da Porto, 2002). As the increase in ethyl lactate concentration in wine is linked to lactic acid bacteria growth (Davis

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100

95

90

85

80

75

70

65

60

55

50

45

40

35

30

25

MIX x GTG 20

15

10

MIX x GTG

T0

46

GTG profile 1

T0

47

GTG profile 1

T0

49

GTG profile 2

T120

10

GTG profile 3

T120

15

GTG profile 3

T120

14

GTG profile 3

T120

12

GTG profile 4

T120

36

GTG profile 6

T30

18

GTG profile 7

T30

19

GTG profile 7

T30

29

GTG profile 8

T30

17

GTG profile 9

T30

38

GTG profile 10

T0

14

GTG profile 10

T30

02

GTG profile 12

T30

10

GTG profile 12

T30

11

GTG profile 12

T30

33

GTG profile 11

T30

34

GTG profile 11

T30

37

GTG profile 11

T120

16

GTG profile 13

T30

32

GTG profile 13

T120

09

GTG profile 14

T30

31

GTG profile 15

T0

18

GTG profile 5

T0

32

GTG profile 5

T0

21

GTG profile 18

T0

23

GTG profile 18

T120

17

GTG profile 16

T120

35

GTG profile 17

T0

22

GTG profile 19

T30

09

GTG profile 20

T0

37

GTG profile 21

T30

25

GTG profile 22

T30

26

GTG profile 22

T30

23

GTG profile 22

T30

30

GTG profile 23

T120

41

GTG profile 24

T0

17

GTG profile 24

T0

26

GTG profile 24

L. plantar.

20174

type

L. hilgardii 20176

type

T120

30

GTG profile 25

T120

37

GTG profile 25

T30

05

GTG profile 26

T0

48

GTG profile 27

T120

03

GTG profile 28

T120

05

GTG profile 28

T120

08

GTG profile 28

T120

04

GTG profile 29

T120

19

GTG profile 30

T120

38

GTG profile 30

T30

03

GTG profile 31

T30

08

GTG profile 31

T30

13

GTG profile 32

T120

39

GTG profile 33

T120

40

GTG profile 33

T30

21

GTG profile 34

T30

22

GTG profile 34

T30

16

GTG profile 35

T120

06

GTG profile 36

T30

01

GTG profile 37

T30

06

GTG profile 38

T30

07

GTG profile 39

L. parapla.

10667

type

L. pentosus 20314

type

O.oeni

type

Fig. 2. Cluster analysis of GTG5 rep-PCR fingerprints of L. plantarum group isolates. Lactobacillus hilgardii, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus plantarum and Oenococcus oeni type strains are included. The vertical line indicates the threshold value (95% similarity) used for strain designation. Isolates with same profiles collected at different sampling times are boxed. The isolate names contain the day of isolation (T0, T30 and T120) followed by the isolate number.

P.A. Maragkoudakis et al. / International Journal of Food Microbiology 162 (2013) 143–151

a

Medium Type strain T120-41 T120-39 T120-36 T120-35 T120-30 T120-19 T120-17 T120-16 T120-13 T120-12 T120-10 T120-09 T120-06 T120-04 T120-03 T30-38 T30-33 T30-32 T30-31 T30-30 T30-29 T30-25 T30-21 T30-18 T30-17 T30-16 T30-13 T30-11 T30-09 T30-08 T30-07 T30-06 T30-05 T30-02 T30-01 T0-49 T0-48 T0-46 T0-37 T0-32 T0-22 T0-21 T0-17 T0-14

149

3

2

1 2

b medium P304.13 P225.5 NR14 NR8 AR3

3

AR1

1 1,0

1,5

2,0

2,5

3,0

3,5

4,0

OD590 t24/t0 ratio

4,5

5,0

1,0

2,0

3,0

4,0

5,0

6,0

OD590 t24/t0 ratio

Fig. 3. Biofilm formation values of (a) 42 selected L. plantarum group strains and (b) 6 S. cerevisae strains used in this study. Biofilm formation data are presented as the ratio of absorbance value (OD590) after 24 h and at the beginning of incubation, in synthetic must SNM ( ) and MRS or YPD (□). Absorbance values are means of two repetitions with triplicate measurements. Non-inoculated growth media were used as negative controls. 1,2,3 Isolate pairs including bacteria with the same profile and collected at different sampling times.

et al., 1985), acidification treatment, lowering the bacterial concentration, could improve the distillate quality. Molecular analyses on isolates from acidified and untreated marcs allowed identification of 11 microbial groups, indicating that grape marc is a selective environment for bacteria. Most of the species recovered belong to genera observed in oenological environments (De Pina and Hogg, 1999; Torija et al., 2010). Interestingly, species that are reported here for the first time in grape marc, such as Tatumella terrea, A. ghanensis and Tatumella ptyseos have been previously described in cocoa and coffee bean fermentations (Cleenwerck et al., 2007; Garcia-Armisen et al., 2010; Silva et al., 2008). The highest number of species was observed at the beginning of the storage period, where 10 species were identified. After 30 days just three species were recovered that raised to five after 120 days, all identified as LAB (Table 2). In the untreated marc, species belonging to the L. plantarum group appeared to be dominant throughout

the whole ensilage period, confirming results described by De Pina and Hogg (1999). On the contrary, the acidification treatment of grape marc, in addition to a numerical reduction of the bacterial population, caused a strong selection of species, as suggested from the colonies growth on primary isolation medium (PCA). In fact at 30 and 120 days samplings mainly small colonies were observed. Only when they were re-streaked on the MRS medium we obtained a stronger growth, suggesting the presence of a group of LAB. The ARDRA analysis revealed that the dominant species was O. oeni (Fig. 2). O. oeni was present in low numbers at the beginning of storage, yet increased during after 30 and 120 days of storage. This bacterium is characterised by particular nutritional requirements not satisfied by PCA, and tolerates low pH (Terrade and Mira de Orduña, 2009; Zarazaga et al., 2004). It is possible that the poor growth observed on the PCA medium was allowed by transfer of essential nutrients from the marc.

150

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Table 3 Biofilm formation of 9 Lactobacillus plantarum strains and 4 Saccharomyces cerevisiae strains, in single and co-culture assays. Biofilm formation is presented as the ratio of the OD590 readings taken after 24 and 0 h of incubation (t24/t0). Original OD590 values (not shown) are means of two repetitions with triplicate measurements. L. plantarum single cultures

S. cerevisiae single cultures AR1

AR3

NR8

NR14

1.5

1.9

4.1

5.2

6.8c,d 3.3c 7.7c,d 2.5c 4.0 3.8c 5.5d 5.5 3.8

2.6b,c 4.1c 2.5b 1.3b 3.1 2.8 3.1 5.5 2.5b

Co-culture combination T30-01 T30-07 T0-49 T0-141 T30-381 T30-322 T120-162 T0-173 T120-413

1.2 0.7 2.2 1.3 3.5 1.9 3.0 4.2 3.7

2.3c,d 4.7c,d 3.0d 2.3c,d 3.2d 3.4c,d 2.7d 2.6c 2.1

4.5c,d 3.4c,d 6.0c,d 4.5c,d 1.7a 3.9c,d 4.7c,d 4.5d 4.4d

1,2,3 Isolates with the same superscript number have the same molecular typing profile, as shown in Fig. 2. a Biofilm formation reduced in co-culture at ≤50% of the single culture of L. plantarum. b Biofilm formation reduced in co-culture at ≤50% of the single culture of S. cerevisiae. c Biofilm formation increased in co-culture at ≥150% of the single culture of L. plantarum. d Biofilm formation increased in co-culture at ≥150% of the single culture of S. cerevisiae.

O. oeni and L. plantarum are able to perform malolactic fermentation in grape marc where LAB turn malic acid into lactic acid and often leads to lactic spoilage from production of high concentrations of ethyl lactate (Silva and Malcata, 2000). When added to wine as selective starter cultures, L. plantarum produces a higher concentration of ethyl lactate and ethyl acetate than O. oeni (Pozo-Bayon et al., 2005). Thus, it appears that the acidification treatment of grape marc contributes to the distillate quality by reducing the numerical density of bacteria and favouring the growth of O. oeni, which produces lower concentrations of off-flavours. In order to better characterise the bacterial biodiversity in grape marc, the most represented group in untreated grape marc, L. plantarum, was subjected to further molecular typing. For intra-specific evaluation, PCR amplification of repetitive bacterial DNA elements based on the (GTG)5primer (Rep-PCR) was used to group the isolates and together with 16S DNA sequencing of some representatives from the main clusters allowed to identify in the L. plantarum group the presence of two species, the L. plantarum and the recently described L. fabifermentans (De Bruyne et al., 2009). Among the 62 isolates from the L. plantarum group, it was possible to distinguish 39 different strains many of which were only present at a single sampling time. However, three pairs of isolates were recovered from two different samplings. These couples, along with the 39 strains were chosen for further analyses. To give an explanation for the apparent dominance of L. plantarum, we decided to examine the strains for two characteristics known to confer an environmental advantage to LAB in general, via bacteriocin production and biofilm formation ability (Rojo-Bezares et al., 2007). As L. plantarum is a well-known bacteriocin producers the antimicrobial activity of supernatants from all 39 L. plantarum were tested against bacteria belonging to the genera Acetobacter, Gluconobacter, and the species L. hilgardii. The first two are able to increase acetic acid concentration thus promoting synthesis of ethyl acetate, which can negatively affect the quality of the distillate (Apostolopoulou et al., 2005). No antimicrobial activity was detected from any supernatants of the 42 isolates of L. plantarum against the indicator strains. Therefore, in the current study under these test conditions bacteriocin production does not explain the apparent ecological dominance of L. plantarum. Biofilms may act as extracellular matrices to protect bacterial cells against environmental stresses or to assist them in nutrient trapping (Costerton et al., 1995; Lee and Frank, 1991; Mah and O'Toole, 2001).

Such structures have been described in several vegetables (Filonow et al., 1996; Morris et al., 1997; Rayner et al., 2004) and it has been hypothesised that viticultural practices during grape ripening may also induce biofilm development on grape skin to protect cells against antimicrobial products spread in the vineyard (Renouf et al., 2005; Cabras and Angioni, 2000; Guerra et al., 1999). Thus its formation could confer an ecological advantage to bacteria also in ensilage conditions of a solid matrix such as grape marc. In the present study, many L. plantarum strains revealed biofilm formation ability. Such activity appears to increase during storage, as suggested by biofilm values of isolates with identical Rep-PCR profile collected at different sampling times, and may be due to an adaptative ability to the grape marc matrix. It appears that the co-culturing is beneficial to biofilm formation, since only in three combinations out of 36 the ability decreased for both partners. Moreover, increments reached very high values, frequently higher than 100% and up to 486%. This clearly indicates a general positive effect of microbial population heterogeneity on biofilm formation. The present work describes for the first time the bacterial population dynamics following acidification of marc and during their storage before distillation. The dominance of L. plantarum in non-acidified grape marc during storage could be due to various reasons such as their natural ability to withstand mild-to-low pH values and, as shown in this study, biofilm formation ability. The present study is the first attempt to understand factors that influence microbial interaction within grape marc during storage and provides a valuable microbiological insight that can contribute to a better understanding and control of the grape marc storage, the crucial phase of quality distillate production. Acknowledgements This work has been funded by “Regione Veneto”, “Provincia di Treviso” and in part by POR — FESR 2007/2013 Action 1.1.1. “RISIB” SMUPR n. 4145. “Accademia della grappa e delle acquaviti” and Stefano Soligo are gratefully acknowledged for providing grape marcs. We wish to thank Milena Carlot for skilful technical assistance. Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.ijfoodmicro.2013.01.005. References Apostolopoulou, A.A., Flouros, A.I., Demertzis, P.G., Akrida-Demertzi, K., 2005. Differences in concentration of principal volatile constituents in traditional Greek distillates. Food Control 16, 157–164. Bae, S., Fleet, G.H., Heard, G.M., 2006. Lactic acid bacteria associated with wine grapes from several Australian vineyards. Journal of Applied Microbiology 100, 712–727. Bovo, B., Andrighetto, C., Carlot, M., Corich, V., Lombardi, A., Giacomini, A., 2009. Yeast population dynamics during pilot-scale storage of grape marcs for the production of Grappa, a traditional Italian alcoholic beverage. International Journal of Food Microbiology 129, 221–228. Bovo, B., Fontana, F., Giacomini, A., Corich, V., 2010. Effects of yeast inoculation on volatile compound production by grape marcs. Annals of Microbiology 61, 117–124. Bovo, B., Giacomini, A., Corich, V., 2011. Effects of grape marcs acidification treatment on the evolution of indigenous yeast populations during the production of grappa. Journal of Applied Microbiology 111, 382–388. Bovo, B., Nardi, T., Fontana, F., Carlot, M., Giacomini, A., Corich, V., 2012. Acidification of grape marc for alcoholic beverage production: effects on indigenous microflora and aroma profile after distillation. International Journal of Food Microbiology 152, 100–106. Cabras, P., Angioni, A., 2000. Pesticide residues in grapes, wine, and their processing products. Journal of Agricultural and Food Chemistry 48, 967–973. Capozzi, V., Russo, P., Ladero, V., Fernandez, M., Fiocco, D., Alvarez, M.A., Grieco, F., Spano, G., 2012. Biogenic amines degradation by Lactobacillus plantarum: toward a potential application in wine. Frontiers in Microbiology 3, 122. Cleenwerck, I., Camu, N., Engelbeen, K., De Winter, T., Vandemeulebroecke, K., De Vos, P., De Vuyst, L., 2007. Acetobacter ghanensis sp. nov., a novel acetic acid bacterium

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