Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Application of PCR-DGGE for the identification of lactic acid bacteria in acitve dry wine yeasts

Application of PCR-DGGE for the identification of lactic acid bacteria in acitve dry wine yeasts Annals of Microbiology, 57 (1) 137-141 (2007) Application of PCR-DGGE for the identification of lactic acid bacteria in active dry wine yeasts 1 2 1 1 Cristina GIUSTO *, Dagmara MEDRALA , Giuseppe COMI , Marisa MANZANO 1 2 Dipartimento di Scienze degli Alimenti, Universita’ degli Studi di Udine, Via Marangoni 97, 33100 Udine, Italy; Department of Cell and Molecular Biology/Microbiology, Goteborg University, S-40430 Goteborg, Sweden Received 5 September 2006 / Accepted 31 January 2007 Abstract - In this work a Polymerase Chain Reaction (PCR)-Denaturing Gradient Gel Electrophoresis (DGGE) protocol was used to iden- tify the Lactic Acid Bacteria (LAB) contaminants in enological active dry yeasts routinely used in the wine production. The method is based on the PCR amplification of a DNA fragment from the region V1 of 16S rDNA gene followed by a DGGE technique. The main con- taminant was Lactobacillus spp. and Pediococcus spp. Key words: wine spoilage, 16S rDNA, Pediococcus, Lactobacillus, PCR-DGGE. INTRODUCTION tion of fructose (Lonvaud-Funel, 1999). The MLF is of great economic importance to the wine making industries. MLF The sensory profile of a wine is the result of complex inter- causes a rise in pH of the wine and is considered beneficial actions, which include for example the impact of character- in the acid wines because it decreases the acidity and pro- istics and the quality of the yeast strains used as starters. duces traces of compounds which affects the flavour (succi- The contamination of the yeast starter strains with bacteria nate, acetate, acetoin, lactate, diacethyl) (Jackson, 1994; may contribute negatively or positively to the properties and Nielsen et al., 1996); on the contrary in low-acid wines, this wine sensory characteristics. There are numerous oenolog- process is detrimental and must be controlled. Leuconostoc ical characteristics of both yeast and lactic acid bacteria that spp. in general, and Oenococcus oeni in particular are con- are considered important for vinification (Majdak et al., cerned with the malolactic fermentation (Van Vuuren and 2002). Such characteristics are quantitatively and qualita- Dicks, 1993). LAB is a highly heterogeneous group of Gram- tively conserved and widely distributed among strains of positive bacteria: Lactobacillus, Leuconostoc, Pediococcus, Saccharomyces cerevisiae (Querol et al., 1992; Josepa et Streptococcus, Lactococcus, Enterococcus, and Oenococcus al., 2000). Lactic Acid Bacteria (LAB) and acetic acid bacte- expressing a wide range of morphological, metabolic and ria are commonly present on grapes, and in must and wine. physiological characteristics. In the last decade it was shown LAB have the ability to tolerate the stresses of the wine that classical microbiological techniques do not accurately environment, namely, low pH, presence of ethanol and sul- detect microbial diversity, while the Denaturing Gradient Gel phur dioxide, low temperature, and diluted concentration of Electrophoresis (DGGE) method has the potential to provide nutrients, so that they are capable of abundant growth in information about variations in target genes within bacteria the grape juice and, if the yeast growth is delayed, they population because it allows separation of DNA molecules could grow and spoil the juice or cause stuck alcoholic fer- that differ by single bases (Muyzer and Smalla, 1998; mentation. Although LAB are responsible for biological de- Walter et al., 2001; Meroth et al., 2003). The PCR-DGGE acidification of wine performed through a conversion of method has the potential to characterise the LAB flora faster dicarboxylic malic acid into monocarboxylic lactic acid dur- then classical methods routinely used in oenology. ing malolactic fermentation (MLF), they may also trigger The aims of this work were to evaluate the LAB contam- wine spoilage (Dicks and Van Vuuren, 1988; Edwards et al. ination level of the commercial oenological active dry yeasts 1998). Effects of lactobacilli and Oenococcus oeni in wine strains and to identify the LAB contaminating species using are due to the production of diacetyl from citric acid, a PCR-DGGE technique. flavourful in small quantities, but can cause spoilage if pres- ent in excess by the formation of mannitol from fermenta- MATERIAL AND METHODS Samples. A total of 62 dry oenological yeasts in sterile vac- uum packages were tested. Yeasts were stored at room * Corresponding author. Phone: +39 432590746; Fax: +39 432590719; E-mail: giusto.cristina@libero.it temperature in a dry, cool place until they were analysed. 138 C. Giusto et al. The work was subdivided into three steps: (i) a quantitative mmol/l MgCl , 1.25 UI Taq Gold Polymerase (Applera Italia, assay: commercially available yeast starters routinely Monza, Italy), 2 μl DNA (50 ng/μl). The samples were employed in the wine industry (Manzano et al., 2006) were processed using the following protocol: 95 °C for 5 min, five cycles of 95 °C for 60 s, 60 °C for 60 s, 72 °C for 150 analysed, by classical microbiological methods (plate s, five cycles of 95 °C for 60 s, 54 °C for 60 s, 72 °C for count), to define the degree of LAB contamination; (ii) a 150 s and 25 cycles of 95 °C for 60 s, 50 °C for 60 s, 72 qualitative assay: the identification of the LAB isolated °C for 150 s, and a final extension at 72 °C for 5 min. The strains performed with a PCR-DGGE protocol using specific primers used were: P1 5’-CGCGCGTGCCTAATACATGC-3’ primers for V1 region of 16S rDNA. with a GC clamp and primer P2 5’-TTCCCCACGGCTTACT- CACC-3’ (Cocolin et al., 2001). PCR fragments were sepa- Bacterial reference strains. Lactobacillus sakei DSM rated by electrophoresis in a 2% agarose gel, stained with 6333, Lactobacillus casei DSM 20011, Lactobacillus curvatus ethidium bromide (0.5 μg/ml), visualized under UV light DSM 20019, Lactobacillus brevis DSM 20054, Lactobacillus and filed using the GeneSnap Syngene Software plantarum DSM 20174, Pediococcus pentosaceus DSM (Cambridge, UK) to verify the size of the amplicons. LAB 20336, Pediococcus parvulus DSM 20332, Pediococcus strains not identified using PCR-DGGE method were identi- damnosus DSM 20331, Oenococcus oeni DSM 20252, fied by sequencing. PCR conditions for sequencing were Leuconostoc cremoris DSM 4196, obtained from Deutsche performed as described by Kljin et al. (1991) using primers Sammlung von Microorganismen und Zellkulturen GmbH P1 and P4 targeting 700 bp of the V1-V3 region of the 16S (Braunshweig, Germany) were used as reference strains. rDNA. After purification, products were sent to a commer- cial facility for sequencing (MWG Biotech, Edersberg, Microbiological analysis. Boxes containing commercially Germany) packaged dry yeasts were opened in sterile conditions and 1 g sample of the content was collected for the analysis. The DGGE analysis. The Dcode universal mutation detection sample was transferred to 9 ml of peptone water (kept at system (Bio-Rad) was used for a DGGE analysis of the PCR -1 30 °C for 1 h before the analysis) to obtain 10 preliminary products obtained from single colonies. Electrophoresis was -1 dilution. To revitalise yeast, the 10 dilution was shaken performed in a 0.8 mm polyacrylamide gel (8%, w/v, acry- four times for 30 s with 5-minutes intervals and subsequent lamide:bisacrylamide, 37.5:1) using two denaturant gradi- -8 serial dilutions (up to 10 ) were prepared. ents from 40% to 60% and from 30% to 50% increasing in To detect and evaluate the presence of LAB, 1 ml of dilu- the direction of the electrophoresis. The gels were subject- -1 -4 tions from 10 to 10 was transferred to plates and poured ed to a constant voltage of 130 V for 3.5 h at 60 °C, and twice using MRS agar (Oxoid, Milan, Italy) supplemented after electrophoresis they were stained for 20 min in a with 25 mg/l of the antifungal/antimould agent Delvocid 1.25X TAE containing 1X (final concentration) SYBR Green Instant DSM (DSM Food Specialities Dairy ingredients, (Molecular Probes, Eugene, Oregon) and visualized under Netherlands). Plates were incubated under microaerophilic UV light and filed using the GeneSnap Syngene Software. conditions at 30 °C for 3 days, according to the instructions of the manufacturer. After counting, for each sample three Sequence analysis. Sequences were aligned in GenBank colonies from MRS plates were randomly selected and using the BLAST program (Altschul et al., 1997) to deter- streaked on to MRS agar, only the catalase-negative, Gram- mine the closest known relatives of the partial 16S rDNA positive rods or cocci were subsequently transferred on to sequences obtained. MRS agar until use. To evaluate the presence of S. cerevisiae strains, 0.1 ml -5 of each dilution starting from 10 and higher was surface- RESULTS plated on WL Nutrient Agar (Oxoid); plates were incubated aerobically at 28 °C for 5 days (Cavazza et al., 1992). After This study determined the LAB contamination of active dry incubation the number of colonies appearing 5 mm in diam- yeasts. Results obtained by MRS plating count are shown in eter and from cream-coloured to light green was counted Table 1. Of the analysed yeasts, 21% showed a LAB con- and, based on the data, the total number of CFU/g was cal- tamination less than 1.0 x 10 CFU/g, 65% ranged from 1.0 culated. 2 5 x 10 to 1.0 x 10 CFU/g, and 14% of yeasts showed a con- tamination level higher than 1.0 x 10 CFU/g. Due to the Extraction of DNA from pure cultures. Isolated bacteria detection limit of the plate count method (10 CFU/g), no were grown for 48-72 h at 30 °C on MRS agar (Oxoid). Then colonies were isolated from samples showing a LAB con- a single colony was selected from the plate, transferred into tamination under that value. a 1.5 ml microcentrifuge tube containing 0.3 g of glass A total of 104 strains of Gram-positive, catalase-nega- beads (0.1 mm diameter) and resuspended in 200 ml of tive, rod-shaped and/or cocci bacteria were amplified after breaking buffer and DNA extraction was performed as isolation with the primer pair P1-P2, which targeted the described by Manzano et al. (2003). DNA concentration was region V1 of 16S rDNA. They were identified using two TM measured using a SmartSpec 3000 spectrophotometer DGGE denaturant gradients. To obtain a high performance (Bio-Rad, Milan, Italy) and standardized at about 50 ng/μl in the DGGE method it was necessary to use two different before being used in the PCR assay concentrations of denaturant. The 40 to 60% denaturant gradient allowed a good differentiation of all the PCR conditions. Amplification was performed in a DNA Lactobacillus reference strains used (Fig. 1A) and the 30 to TM Engine DYAD SYSTEM (CELBIO, Milan, Italy) in a final 50% denaturant gradient (Fig. 1B) was useful for the differ- volume of 50 μl containing 10 mmol/l Tris-HCl, 50 mmol/l entiation of Pediococcus damnosus, P. pentosaceus, P. KCl, 200 μmol/l each dNTP, 0.2 μmol/l each primer, 2.5 parvulus, Oenococcus oeni and Leuconostoc cremoris. The Ann. Microbiol., 57 (1), 137-141 (2007) 139 TABLE 1 - Yeast cells number and Lactic Acid Bacteria (LAB) contamination of the active dry yeasts tested Dry yeast Yeasts LAB Dry yeast Yeasts LAB sample (CFU/g dry yeast) (CFU/g dry yeast) sample (CFU/g dry yeast) (CFU/g dry yeast) 9 2 9 2 01 4.5 x 10 3.0 x 10 32 4.8 x 10 2.8 x 10 9 3 9 2 02 3.5 x 10 3.5 x 10 33 9.2 x 10 7.8 x 10 10 4 9 4 03 1.1 x 10 8.5 x 10 34 5 x 10 5.5 x 10 9 2 9 2 04 8.2 x 10 <10 35 6.1 x 10 <10 9 3 10 2 05 9.8 x 10 3.0 x 10 36 1.8 x 10 <10 10 2 9 3 06 1.2 x 10 3.5 x 10 37 4.8 x 10 1.5 x 10 9 3 9 5 07 4 x 10 1.8 x 10 38 0.2 x 10 4.3 x 10 10 4 9 2 08 1.0 x 10 4.8 x 10 39 5.4 x 10 6.5 x 10 9 2 9 2 09 2 x 10 <10 40 2.0 x 10 <10 9 2 9 2 10 6.8 x 10 9.5 x 10 41 2.2 x 10 1.5 x 10 9 4 10 2 11 6.1 x 10 2.8 x 10 42 1.0 x 10 9.5 x 10 10 2 9 4 12 1.5 x 10 3.5 x 10 43 5.8 x 10 9.6 x 10 10 6 9 3 13 1.5 x 10 1.8 x 10 44 6.4 x 10 7.1 x10 9 2 10 2 14 5 x 10 6.6 x 10 45 1.0 x 10 <10 9 3 10 2 15 5.5 x 10 2.8 x 10 46 6.3 x 10 2.8 x 10 9 3 10 6 16 6.8 x 10 4.4 x 10 47 4.0 x 10 8.0 x 10 10 4 9 2 17 1.0 x 10 3.4 x 10 48 3.2 x 10 5.5 x 10 9 4 10 3 18 7.5 x 10 8.0 x 10 49 1.5 x 10 1.2 x 10 10 5 9 2 19 1.7 x 10 1.3 x 10 50 5.1 x 10 <10 9 3 9 2 20 5.3 x 10 2.0 x 10 51 3.8 x 10 <10 10 6 9 2 21 1.0 x 10 8.0 x 10 52 4.4 x 10 7.5 x 10 10 5 9 2 22 2.0 x 10 1.2 x 10 53 0.1 x 10 <10 10 5 9 5 23 1.5 x 10 8.0 x 10 54 3.3 x 10 1.5 x 10 8 3 10 4 24 6.0 x 10 1.0 x 10 55 1.0 x 10 1.4 x 10 9 3 9 5 25 1.9 x 10 1.0 x 10 56 4 x 10 8.5 x 10 9 3 9 2 26 3.3 x 10 2.4 x 10 57 4 x 10 3.5 x 10 10 4 9 2 27 1.5 x 10 3.7 x 10 58 4 x 10 <10 9 3 9 2 28 7.2 x 10 3.7 x 10 59 1.2 x 10 <10 10 2 9 4 29 1.1 x 10 5.0 x 10 60 4.5 x 10 2.0 x 10 10 2 9 4 30 3.3 x 10 <10 61 3.3 x 10 9.7 x 10 9 2 10 4 31 5.8 x 10 <10 62 1.0 x 10 4.4 x 10 48 rod-shaped bacteria (46% of LAB isolated) gave a total Figure 2 shows the percentages of the different LAB of six different profiles belonging to the Lactobacillus spp. strains isolated from the oenological active dry yeasts test- The strains were identified as L. plantarum (13 strains), L. ed. Lactobacillus brevis emerged as the predominant sakei (7 strains), L. curvatus (20 strains), L. brevis (3 species whereas the Oenococcus oeni the less diffused. A lot strains), L. casei (4 strains) and L. paracasei (1 strains). The of Pedioccocus strains, in particular P. pentosaceus and P. 28 spherical cells isolated (27%) were identified as: parvulus, were isolated from all the analysed yeasts boxes, Pediococcus damnosus (8 strains), P. pentosaceus (18 then Leuconostoc cremoris was the most numerous strain strains) and P. parvulus (2 strains). The last 13 strains were isolated from the others. identified as Oenococcus oeni (5 strains) corresponding to the 5% and Leuconostoc cremoris (8 strains) corresponding to 7%. For 15 cocci strains (14%) it was necessary to use DISCUSSION the sequencing method followed by comparison with DNA sequences retrieved in GeneBank to obtain identification as Lactobacillus spp. turned out to be the most common con- no profiles corresponding to the reference strains used were taminants of analysed samples (Fig. 2), in fact they occur in obtained. For these bacteria a PCR product using primers P1 many types of wine (Dicks and Van Vuuren, 1988), despite and P4 specifics for regions V1 and V3 of 16S rDNA was the high level of ethanol, the low pH (3.2-3.8) and the addi- made. The strains identified by sequencing were: tional SO content present. Oenococcus oeni strains tolerate Enterococcus hirae (1 strain), Enterococcus durans (2 low pH better than Leuconostoc, Pediococcus and strains), Enterococcus pseudoavium (2 strains), Weissella Lactobacillus strains and generally predominate in wines cibaria (2 strains), Weissella kimkii (3 strains), Pediococcus with pH 3.0 to 3.5. Wines with pH values exceeding 3.5 acidilactici (4 strains) and Staphyloccocus caseolyticus (2 might have a mixed microflora, consisting of O. oeni and strain). various species of Pediococcus and Lactobacillus which tol- 140 C. Giusto et al. 5 -1 wine quality. Presence of 10 CFU g of LAB in must is a 1 2 3 4 5 6 1 2 345 crucial microbial concentration for the development of unde- sired MLF during or after alcoholic fermentation. The occur- rence of LAB in the tested pool of starters differed signifi- cantly as reported in Table 1. In conclusion, 31% of the strains were shown to have a level of contamination in 4 -1 excess of 10 CFU g . This is a critical level of contamina- tion considering that there is already a concentration 3 4 -1 between 10 and 10 CFU ml of natural bacterial flora present in the must. Producers recommend adding 20 g h 1 -1 L of dry yeast to the must (Fleet, 2003) which can cause LAB contamination to exceed values of 10 CFU/ml, which is enough to start MLF. REFERENCES Altschul S.F., Madden T.L., Shaffer A.A, Zhang J., Zhang Z., Miller W, Lipman D.J. (1997). Gapped BLAST and PSI-BLAST: a new A B generation of protein database search programs. Nucleic Acids Research, 25: 3389-3402. Cavazza A., Grando M.S., Zini C. (1992). Rivelazione della flora FIG. 1 - A: DGGE profiles obtained for PCR products in the 40- microbica dei mosti e dei vini. Vignevini, 9: 17-20. 60% denaturant gel. Lane 1: Lactobacillus plantarum Cocolin L., Manzano M., Cantoni C., Comi G. (2001). Denaturing DSM 20174; Lane 2: Lactobacillus sakei DSM 63333; Gradient Gel Electrophoresis analysis of the 16S rRNA gene Lane 3: Lactobacillus curvatus DSM 20019; Lane 4: V1 region to monitor dynamic changes in the bacterial popu- Lactobacillus brevis DSM 20054; Lane 5: Lactobacillus lation during fermentation of Italian sausages. Applied and Environmental Microbiology, 67: 5113-5121. casei DSM 20011; Lane 6: Lactobacillus paracasei. B: DGGE profiles obtained for PCR products in the 30- Dicks L.M.T., Van Vuuren H.J.J. (1988). Identification and physi- 50% denaturant gel. Lane 1: Pediococcus damnosus ological characteristics of heterofermentative strains of DSM 20331; Lane 2: Pediococcus pentosaceus DSM Lactobacillus from South African red wines. Journal of Applied Bacteriology, 64: 505-513. 20336; Lane 3: Pediococcus parvulus DSM 20332, Lane 4: Oenococcus oeni DSM 20252, Lane 5: Leuconostoc Edwards C.G., Haag K.M., Collins M.D. (1998). Identification of cremoris DSM 4196. some lactic acid bacteria associated with sluggish/stuck fer- mentations. American Journal of Enology and Viticulture, 49: 445-448. Fleet G.H. (2003). Yeast interactions and wine flavour. International Journal of Food Microbiology, 86: 11-22. erate higher concentrations of sulphur dioxide better than Jackson R.S. (1994). Wine Science: Principles and Applictions, O. oeni and are more likely to occur in wines with higher San Diego Academic Press. amounts of this substance. Considering the importance of a Josepa S., Guillamon J.M., Cano J. (2000). PCR differentiation of pure alcoholic fermentation, one of the main requirements Saccharomyces cerevisiae from Saccharomyces is to limit the level of bacteria contamination of dry yeast bayanus/Saccharomyces pastorianus using specific primers. starters to the lowest possible level, especially during wine FEMS Microbiology Letters, 193: 255-259. maturing and ageing. Depending on the species, strains or Kljin N., Weerkamp A.H., deVos W.M. (1991). Identification of even growth stage, LAB may be beneficial or detrimental to mesophilic lactic acid bacteria by using polymerase chain reaction-amplified variable regions of 16S rRNA and specific DNA probes. Applied and Environmental Microbiology, 57: 3390-3393. Lonvaud-Funel A. (1999). Lactic acid bacteria in the quality 50% improvement and depreciation of wine. Antonie Van 45% Leeuwenhoek, 76: 317-331. 40% Majdak A., Heriavec S., Orlic S., Redzepovic S., Mirosevic N. 35% (2002). Comparison of wine aroma compounds produces by Saccharomyces paradoxus and Saccharomyces cerevisiae 30% strains. Food Technology and Biotechnology, 40: 103-109. 25% Manzano M., Giusto C., Iacumin L., Cantoni C., Comi G. (2003). 20% A molecular method to detect Bacillus cereus from a coffee 15% concentrate sample used in industrial preparations. Journal of Applied Microbiology, 95: 1361-1366. 10% C D F 5% Manzano M., Medrala D., Giusto C., Bartolomeoli I. Urso R., Comi G. (2006). Classical and molecular analyses to characterize 0% commercial dry yeast used in wine fermentation, Journal of Applied Microbiology, 100: 599-607. FIG. 2 - Percentage of Lactic Acid Bacteria isolated from enologi- Meroth C.B., Walter J., Hertel C.B., Brandt M.J., Hammes W.P. cal dry yeast starters. A: Lactobacillus spp., B: (2003). Monitoring the bacterial population dynamics in sour- Pediococcus spp., C: Oenococcus oeni, D: Enterococcus dough fermentation process by using PCR- Denaturing Gradient Gel Electrophoresis. Applied and Environmental spp., E: Leuconostoc cremoris, F: Weissella spp., G: Microbiology, 69: 475-482. Staphyloccocus caseolyticus. Ann. Microbiol., 57 (1), 137-141 (2007) 141 Muyzer G., Smalla K. (1998). Application of denaturing gradient Van Vuuren H.J.J., Dicks L.M.T. (1993). Leuconostoc oenos: A gel electrophoresis (DGGE) and temperature gradient gel review. American Journal of Enology and Viticulture, 44: 99- electrophoresis (TGGE) in microbial ecology. Antonie Van 112. Leeuwenhoek, 73: 127-141. Walter J., Hertel C., Tannock G.W., Lis C.M., Munro K., Hammes Nielsen J.C., Praahl C., Lonvaud-Funel A. (1996). Malolactic fer- W.P. (2001). Detection of Lactobacillus, Pediococcus, mentation in wine by direct inocultion with freeze-dried Leuconostoc and Weissella species in human feces by using Leuconostoc oenos cultures. American Journal of Enology and group-specific PCR primers and denaturing gradient gel elec- Viticulture, 47: 42-48 trophoresis. Applied and Environmental Microbiology, 67: 2578-2585. Querol A., Huerta T., Barrio E., Ramon D. (1992). Dry yeast strain for use in fermentation of Alicante wines: selection and DNA patterns. Journal of Food Science, 57: 183-185. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Application of PCR-DGGE for the identification of lactic acid bacteria in acitve dry wine yeasts

Loading next page...
 
/lp/springer-journals/application-of-pcr-dgge-for-the-identification-of-lactic-acid-bacteria-wNGIx37km0

References (22)

Publisher
Springer Journals
Copyright
Copyright © 2007 by University of Milan and Springer
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Fungus Genetics; Medical Microbiology; Applied Microbiology
ISSN
1590-4261
eISSN
1869-2044
DOI
10.1007/BF03175063
Publisher site
See Article on Publisher Site

Abstract

Annals of Microbiology, 57 (1) 137-141 (2007) Application of PCR-DGGE for the identification of lactic acid bacteria in active dry wine yeasts 1 2 1 1 Cristina GIUSTO *, Dagmara MEDRALA , Giuseppe COMI , Marisa MANZANO 1 2 Dipartimento di Scienze degli Alimenti, Universita’ degli Studi di Udine, Via Marangoni 97, 33100 Udine, Italy; Department of Cell and Molecular Biology/Microbiology, Goteborg University, S-40430 Goteborg, Sweden Received 5 September 2006 / Accepted 31 January 2007 Abstract - In this work a Polymerase Chain Reaction (PCR)-Denaturing Gradient Gel Electrophoresis (DGGE) protocol was used to iden- tify the Lactic Acid Bacteria (LAB) contaminants in enological active dry yeasts routinely used in the wine production. The method is based on the PCR amplification of a DNA fragment from the region V1 of 16S rDNA gene followed by a DGGE technique. The main con- taminant was Lactobacillus spp. and Pediococcus spp. Key words: wine spoilage, 16S rDNA, Pediococcus, Lactobacillus, PCR-DGGE. INTRODUCTION tion of fructose (Lonvaud-Funel, 1999). The MLF is of great economic importance to the wine making industries. MLF The sensory profile of a wine is the result of complex inter- causes a rise in pH of the wine and is considered beneficial actions, which include for example the impact of character- in the acid wines because it decreases the acidity and pro- istics and the quality of the yeast strains used as starters. duces traces of compounds which affects the flavour (succi- The contamination of the yeast starter strains with bacteria nate, acetate, acetoin, lactate, diacethyl) (Jackson, 1994; may contribute negatively or positively to the properties and Nielsen et al., 1996); on the contrary in low-acid wines, this wine sensory characteristics. There are numerous oenolog- process is detrimental and must be controlled. Leuconostoc ical characteristics of both yeast and lactic acid bacteria that spp. in general, and Oenococcus oeni in particular are con- are considered important for vinification (Majdak et al., cerned with the malolactic fermentation (Van Vuuren and 2002). Such characteristics are quantitatively and qualita- Dicks, 1993). LAB is a highly heterogeneous group of Gram- tively conserved and widely distributed among strains of positive bacteria: Lactobacillus, Leuconostoc, Pediococcus, Saccharomyces cerevisiae (Querol et al., 1992; Josepa et Streptococcus, Lactococcus, Enterococcus, and Oenococcus al., 2000). Lactic Acid Bacteria (LAB) and acetic acid bacte- expressing a wide range of morphological, metabolic and ria are commonly present on grapes, and in must and wine. physiological characteristics. In the last decade it was shown LAB have the ability to tolerate the stresses of the wine that classical microbiological techniques do not accurately environment, namely, low pH, presence of ethanol and sul- detect microbial diversity, while the Denaturing Gradient Gel phur dioxide, low temperature, and diluted concentration of Electrophoresis (DGGE) method has the potential to provide nutrients, so that they are capable of abundant growth in information about variations in target genes within bacteria the grape juice and, if the yeast growth is delayed, they population because it allows separation of DNA molecules could grow and spoil the juice or cause stuck alcoholic fer- that differ by single bases (Muyzer and Smalla, 1998; mentation. Although LAB are responsible for biological de- Walter et al., 2001; Meroth et al., 2003). The PCR-DGGE acidification of wine performed through a conversion of method has the potential to characterise the LAB flora faster dicarboxylic malic acid into monocarboxylic lactic acid dur- then classical methods routinely used in oenology. ing malolactic fermentation (MLF), they may also trigger The aims of this work were to evaluate the LAB contam- wine spoilage (Dicks and Van Vuuren, 1988; Edwards et al. ination level of the commercial oenological active dry yeasts 1998). Effects of lactobacilli and Oenococcus oeni in wine strains and to identify the LAB contaminating species using are due to the production of diacetyl from citric acid, a PCR-DGGE technique. flavourful in small quantities, but can cause spoilage if pres- ent in excess by the formation of mannitol from fermenta- MATERIAL AND METHODS Samples. A total of 62 dry oenological yeasts in sterile vac- uum packages were tested. Yeasts were stored at room * Corresponding author. Phone: +39 432590746; Fax: +39 432590719; E-mail: giusto.cristina@libero.it temperature in a dry, cool place until they were analysed. 138 C. Giusto et al. The work was subdivided into three steps: (i) a quantitative mmol/l MgCl , 1.25 UI Taq Gold Polymerase (Applera Italia, assay: commercially available yeast starters routinely Monza, Italy), 2 μl DNA (50 ng/μl). The samples were employed in the wine industry (Manzano et al., 2006) were processed using the following protocol: 95 °C for 5 min, five cycles of 95 °C for 60 s, 60 °C for 60 s, 72 °C for 150 analysed, by classical microbiological methods (plate s, five cycles of 95 °C for 60 s, 54 °C for 60 s, 72 °C for count), to define the degree of LAB contamination; (ii) a 150 s and 25 cycles of 95 °C for 60 s, 50 °C for 60 s, 72 qualitative assay: the identification of the LAB isolated °C for 150 s, and a final extension at 72 °C for 5 min. The strains performed with a PCR-DGGE protocol using specific primers used were: P1 5’-CGCGCGTGCCTAATACATGC-3’ primers for V1 region of 16S rDNA. with a GC clamp and primer P2 5’-TTCCCCACGGCTTACT- CACC-3’ (Cocolin et al., 2001). PCR fragments were sepa- Bacterial reference strains. Lactobacillus sakei DSM rated by electrophoresis in a 2% agarose gel, stained with 6333, Lactobacillus casei DSM 20011, Lactobacillus curvatus ethidium bromide (0.5 μg/ml), visualized under UV light DSM 20019, Lactobacillus brevis DSM 20054, Lactobacillus and filed using the GeneSnap Syngene Software plantarum DSM 20174, Pediococcus pentosaceus DSM (Cambridge, UK) to verify the size of the amplicons. LAB 20336, Pediococcus parvulus DSM 20332, Pediococcus strains not identified using PCR-DGGE method were identi- damnosus DSM 20331, Oenococcus oeni DSM 20252, fied by sequencing. PCR conditions for sequencing were Leuconostoc cremoris DSM 4196, obtained from Deutsche performed as described by Kljin et al. (1991) using primers Sammlung von Microorganismen und Zellkulturen GmbH P1 and P4 targeting 700 bp of the V1-V3 region of the 16S (Braunshweig, Germany) were used as reference strains. rDNA. After purification, products were sent to a commer- cial facility for sequencing (MWG Biotech, Edersberg, Microbiological analysis. Boxes containing commercially Germany) packaged dry yeasts were opened in sterile conditions and 1 g sample of the content was collected for the analysis. The DGGE analysis. The Dcode universal mutation detection sample was transferred to 9 ml of peptone water (kept at system (Bio-Rad) was used for a DGGE analysis of the PCR -1 30 °C for 1 h before the analysis) to obtain 10 preliminary products obtained from single colonies. Electrophoresis was -1 dilution. To revitalise yeast, the 10 dilution was shaken performed in a 0.8 mm polyacrylamide gel (8%, w/v, acry- four times for 30 s with 5-minutes intervals and subsequent lamide:bisacrylamide, 37.5:1) using two denaturant gradi- -8 serial dilutions (up to 10 ) were prepared. ents from 40% to 60% and from 30% to 50% increasing in To detect and evaluate the presence of LAB, 1 ml of dilu- the direction of the electrophoresis. The gels were subject- -1 -4 tions from 10 to 10 was transferred to plates and poured ed to a constant voltage of 130 V for 3.5 h at 60 °C, and twice using MRS agar (Oxoid, Milan, Italy) supplemented after electrophoresis they were stained for 20 min in a with 25 mg/l of the antifungal/antimould agent Delvocid 1.25X TAE containing 1X (final concentration) SYBR Green Instant DSM (DSM Food Specialities Dairy ingredients, (Molecular Probes, Eugene, Oregon) and visualized under Netherlands). Plates were incubated under microaerophilic UV light and filed using the GeneSnap Syngene Software. conditions at 30 °C for 3 days, according to the instructions of the manufacturer. After counting, for each sample three Sequence analysis. Sequences were aligned in GenBank colonies from MRS plates were randomly selected and using the BLAST program (Altschul et al., 1997) to deter- streaked on to MRS agar, only the catalase-negative, Gram- mine the closest known relatives of the partial 16S rDNA positive rods or cocci were subsequently transferred on to sequences obtained. MRS agar until use. To evaluate the presence of S. cerevisiae strains, 0.1 ml -5 of each dilution starting from 10 and higher was surface- RESULTS plated on WL Nutrient Agar (Oxoid); plates were incubated aerobically at 28 °C for 5 days (Cavazza et al., 1992). After This study determined the LAB contamination of active dry incubation the number of colonies appearing 5 mm in diam- yeasts. Results obtained by MRS plating count are shown in eter and from cream-coloured to light green was counted Table 1. Of the analysed yeasts, 21% showed a LAB con- and, based on the data, the total number of CFU/g was cal- tamination less than 1.0 x 10 CFU/g, 65% ranged from 1.0 culated. 2 5 x 10 to 1.0 x 10 CFU/g, and 14% of yeasts showed a con- tamination level higher than 1.0 x 10 CFU/g. Due to the Extraction of DNA from pure cultures. Isolated bacteria detection limit of the plate count method (10 CFU/g), no were grown for 48-72 h at 30 °C on MRS agar (Oxoid). Then colonies were isolated from samples showing a LAB con- a single colony was selected from the plate, transferred into tamination under that value. a 1.5 ml microcentrifuge tube containing 0.3 g of glass A total of 104 strains of Gram-positive, catalase-nega- beads (0.1 mm diameter) and resuspended in 200 ml of tive, rod-shaped and/or cocci bacteria were amplified after breaking buffer and DNA extraction was performed as isolation with the primer pair P1-P2, which targeted the described by Manzano et al. (2003). DNA concentration was region V1 of 16S rDNA. They were identified using two TM measured using a SmartSpec 3000 spectrophotometer DGGE denaturant gradients. To obtain a high performance (Bio-Rad, Milan, Italy) and standardized at about 50 ng/μl in the DGGE method it was necessary to use two different before being used in the PCR assay concentrations of denaturant. The 40 to 60% denaturant gradient allowed a good differentiation of all the PCR conditions. Amplification was performed in a DNA Lactobacillus reference strains used (Fig. 1A) and the 30 to TM Engine DYAD SYSTEM (CELBIO, Milan, Italy) in a final 50% denaturant gradient (Fig. 1B) was useful for the differ- volume of 50 μl containing 10 mmol/l Tris-HCl, 50 mmol/l entiation of Pediococcus damnosus, P. pentosaceus, P. KCl, 200 μmol/l each dNTP, 0.2 μmol/l each primer, 2.5 parvulus, Oenococcus oeni and Leuconostoc cremoris. The Ann. Microbiol., 57 (1), 137-141 (2007) 139 TABLE 1 - Yeast cells number and Lactic Acid Bacteria (LAB) contamination of the active dry yeasts tested Dry yeast Yeasts LAB Dry yeast Yeasts LAB sample (CFU/g dry yeast) (CFU/g dry yeast) sample (CFU/g dry yeast) (CFU/g dry yeast) 9 2 9 2 01 4.5 x 10 3.0 x 10 32 4.8 x 10 2.8 x 10 9 3 9 2 02 3.5 x 10 3.5 x 10 33 9.2 x 10 7.8 x 10 10 4 9 4 03 1.1 x 10 8.5 x 10 34 5 x 10 5.5 x 10 9 2 9 2 04 8.2 x 10 <10 35 6.1 x 10 <10 9 3 10 2 05 9.8 x 10 3.0 x 10 36 1.8 x 10 <10 10 2 9 3 06 1.2 x 10 3.5 x 10 37 4.8 x 10 1.5 x 10 9 3 9 5 07 4 x 10 1.8 x 10 38 0.2 x 10 4.3 x 10 10 4 9 2 08 1.0 x 10 4.8 x 10 39 5.4 x 10 6.5 x 10 9 2 9 2 09 2 x 10 <10 40 2.0 x 10 <10 9 2 9 2 10 6.8 x 10 9.5 x 10 41 2.2 x 10 1.5 x 10 9 4 10 2 11 6.1 x 10 2.8 x 10 42 1.0 x 10 9.5 x 10 10 2 9 4 12 1.5 x 10 3.5 x 10 43 5.8 x 10 9.6 x 10 10 6 9 3 13 1.5 x 10 1.8 x 10 44 6.4 x 10 7.1 x10 9 2 10 2 14 5 x 10 6.6 x 10 45 1.0 x 10 <10 9 3 10 2 15 5.5 x 10 2.8 x 10 46 6.3 x 10 2.8 x 10 9 3 10 6 16 6.8 x 10 4.4 x 10 47 4.0 x 10 8.0 x 10 10 4 9 2 17 1.0 x 10 3.4 x 10 48 3.2 x 10 5.5 x 10 9 4 10 3 18 7.5 x 10 8.0 x 10 49 1.5 x 10 1.2 x 10 10 5 9 2 19 1.7 x 10 1.3 x 10 50 5.1 x 10 <10 9 3 9 2 20 5.3 x 10 2.0 x 10 51 3.8 x 10 <10 10 6 9 2 21 1.0 x 10 8.0 x 10 52 4.4 x 10 7.5 x 10 10 5 9 2 22 2.0 x 10 1.2 x 10 53 0.1 x 10 <10 10 5 9 5 23 1.5 x 10 8.0 x 10 54 3.3 x 10 1.5 x 10 8 3 10 4 24 6.0 x 10 1.0 x 10 55 1.0 x 10 1.4 x 10 9 3 9 5 25 1.9 x 10 1.0 x 10 56 4 x 10 8.5 x 10 9 3 9 2 26 3.3 x 10 2.4 x 10 57 4 x 10 3.5 x 10 10 4 9 2 27 1.5 x 10 3.7 x 10 58 4 x 10 <10 9 3 9 2 28 7.2 x 10 3.7 x 10 59 1.2 x 10 <10 10 2 9 4 29 1.1 x 10 5.0 x 10 60 4.5 x 10 2.0 x 10 10 2 9 4 30 3.3 x 10 <10 61 3.3 x 10 9.7 x 10 9 2 10 4 31 5.8 x 10 <10 62 1.0 x 10 4.4 x 10 48 rod-shaped bacteria (46% of LAB isolated) gave a total Figure 2 shows the percentages of the different LAB of six different profiles belonging to the Lactobacillus spp. strains isolated from the oenological active dry yeasts test- The strains were identified as L. plantarum (13 strains), L. ed. Lactobacillus brevis emerged as the predominant sakei (7 strains), L. curvatus (20 strains), L. brevis (3 species whereas the Oenococcus oeni the less diffused. A lot strains), L. casei (4 strains) and L. paracasei (1 strains). The of Pedioccocus strains, in particular P. pentosaceus and P. 28 spherical cells isolated (27%) were identified as: parvulus, were isolated from all the analysed yeasts boxes, Pediococcus damnosus (8 strains), P. pentosaceus (18 then Leuconostoc cremoris was the most numerous strain strains) and P. parvulus (2 strains). The last 13 strains were isolated from the others. identified as Oenococcus oeni (5 strains) corresponding to the 5% and Leuconostoc cremoris (8 strains) corresponding to 7%. For 15 cocci strains (14%) it was necessary to use DISCUSSION the sequencing method followed by comparison with DNA sequences retrieved in GeneBank to obtain identification as Lactobacillus spp. turned out to be the most common con- no profiles corresponding to the reference strains used were taminants of analysed samples (Fig. 2), in fact they occur in obtained. For these bacteria a PCR product using primers P1 many types of wine (Dicks and Van Vuuren, 1988), despite and P4 specifics for regions V1 and V3 of 16S rDNA was the high level of ethanol, the low pH (3.2-3.8) and the addi- made. The strains identified by sequencing were: tional SO content present. Oenococcus oeni strains tolerate Enterococcus hirae (1 strain), Enterococcus durans (2 low pH better than Leuconostoc, Pediococcus and strains), Enterococcus pseudoavium (2 strains), Weissella Lactobacillus strains and generally predominate in wines cibaria (2 strains), Weissella kimkii (3 strains), Pediococcus with pH 3.0 to 3.5. Wines with pH values exceeding 3.5 acidilactici (4 strains) and Staphyloccocus caseolyticus (2 might have a mixed microflora, consisting of O. oeni and strain). various species of Pediococcus and Lactobacillus which tol- 140 C. Giusto et al. 5 -1 wine quality. Presence of 10 CFU g of LAB in must is a 1 2 3 4 5 6 1 2 345 crucial microbial concentration for the development of unde- sired MLF during or after alcoholic fermentation. The occur- rence of LAB in the tested pool of starters differed signifi- cantly as reported in Table 1. In conclusion, 31% of the strains were shown to have a level of contamination in 4 -1 excess of 10 CFU g . This is a critical level of contamina- tion considering that there is already a concentration 3 4 -1 between 10 and 10 CFU ml of natural bacterial flora present in the must. Producers recommend adding 20 g h 1 -1 L of dry yeast to the must (Fleet, 2003) which can cause LAB contamination to exceed values of 10 CFU/ml, which is enough to start MLF. REFERENCES Altschul S.F., Madden T.L., Shaffer A.A, Zhang J., Zhang Z., Miller W, Lipman D.J. (1997). Gapped BLAST and PSI-BLAST: a new A B generation of protein database search programs. Nucleic Acids Research, 25: 3389-3402. Cavazza A., Grando M.S., Zini C. (1992). Rivelazione della flora FIG. 1 - A: DGGE profiles obtained for PCR products in the 40- microbica dei mosti e dei vini. Vignevini, 9: 17-20. 60% denaturant gel. Lane 1: Lactobacillus plantarum Cocolin L., Manzano M., Cantoni C., Comi G. (2001). Denaturing DSM 20174; Lane 2: Lactobacillus sakei DSM 63333; Gradient Gel Electrophoresis analysis of the 16S rRNA gene Lane 3: Lactobacillus curvatus DSM 20019; Lane 4: V1 region to monitor dynamic changes in the bacterial popu- Lactobacillus brevis DSM 20054; Lane 5: Lactobacillus lation during fermentation of Italian sausages. Applied and Environmental Microbiology, 67: 5113-5121. casei DSM 20011; Lane 6: Lactobacillus paracasei. B: DGGE profiles obtained for PCR products in the 30- Dicks L.M.T., Van Vuuren H.J.J. (1988). Identification and physi- 50% denaturant gel. Lane 1: Pediococcus damnosus ological characteristics of heterofermentative strains of DSM 20331; Lane 2: Pediococcus pentosaceus DSM Lactobacillus from South African red wines. Journal of Applied Bacteriology, 64: 505-513. 20336; Lane 3: Pediococcus parvulus DSM 20332, Lane 4: Oenococcus oeni DSM 20252, Lane 5: Leuconostoc Edwards C.G., Haag K.M., Collins M.D. (1998). Identification of cremoris DSM 4196. some lactic acid bacteria associated with sluggish/stuck fer- mentations. American Journal of Enology and Viticulture, 49: 445-448. Fleet G.H. (2003). Yeast interactions and wine flavour. International Journal of Food Microbiology, 86: 11-22. erate higher concentrations of sulphur dioxide better than Jackson R.S. (1994). Wine Science: Principles and Applictions, O. oeni and are more likely to occur in wines with higher San Diego Academic Press. amounts of this substance. Considering the importance of a Josepa S., Guillamon J.M., Cano J. (2000). PCR differentiation of pure alcoholic fermentation, one of the main requirements Saccharomyces cerevisiae from Saccharomyces is to limit the level of bacteria contamination of dry yeast bayanus/Saccharomyces pastorianus using specific primers. starters to the lowest possible level, especially during wine FEMS Microbiology Letters, 193: 255-259. maturing and ageing. Depending on the species, strains or Kljin N., Weerkamp A.H., deVos W.M. (1991). Identification of even growth stage, LAB may be beneficial or detrimental to mesophilic lactic acid bacteria by using polymerase chain reaction-amplified variable regions of 16S rRNA and specific DNA probes. Applied and Environmental Microbiology, 57: 3390-3393. Lonvaud-Funel A. (1999). Lactic acid bacteria in the quality 50% improvement and depreciation of wine. Antonie Van 45% Leeuwenhoek, 76: 317-331. 40% Majdak A., Heriavec S., Orlic S., Redzepovic S., Mirosevic N. 35% (2002). Comparison of wine aroma compounds produces by Saccharomyces paradoxus and Saccharomyces cerevisiae 30% strains. Food Technology and Biotechnology, 40: 103-109. 25% Manzano M., Giusto C., Iacumin L., Cantoni C., Comi G. (2003). 20% A molecular method to detect Bacillus cereus from a coffee 15% concentrate sample used in industrial preparations. Journal of Applied Microbiology, 95: 1361-1366. 10% C D F 5% Manzano M., Medrala D., Giusto C., Bartolomeoli I. Urso R., Comi G. (2006). Classical and molecular analyses to characterize 0% commercial dry yeast used in wine fermentation, Journal of Applied Microbiology, 100: 599-607. FIG. 2 - Percentage of Lactic Acid Bacteria isolated from enologi- Meroth C.B., Walter J., Hertel C.B., Brandt M.J., Hammes W.P. cal dry yeast starters. A: Lactobacillus spp., B: (2003). Monitoring the bacterial population dynamics in sour- Pediococcus spp., C: Oenococcus oeni, D: Enterococcus dough fermentation process by using PCR- Denaturing Gradient Gel Electrophoresis. Applied and Environmental spp., E: Leuconostoc cremoris, F: Weissella spp., G: Microbiology, 69: 475-482. Staphyloccocus caseolyticus. Ann. Microbiol., 57 (1), 137-141 (2007) 141 Muyzer G., Smalla K. (1998). Application of denaturing gradient Van Vuuren H.J.J., Dicks L.M.T. (1993). Leuconostoc oenos: A gel electrophoresis (DGGE) and temperature gradient gel review. American Journal of Enology and Viticulture, 44: 99- electrophoresis (TGGE) in microbial ecology. Antonie Van 112. Leeuwenhoek, 73: 127-141. Walter J., Hertel C., Tannock G.W., Lis C.M., Munro K., Hammes Nielsen J.C., Praahl C., Lonvaud-Funel A. (1996). Malolactic fer- W.P. (2001). Detection of Lactobacillus, Pediococcus, mentation in wine by direct inocultion with freeze-dried Leuconostoc and Weissella species in human feces by using Leuconostoc oenos cultures. American Journal of Enology and group-specific PCR primers and denaturing gradient gel elec- Viticulture, 47: 42-48 trophoresis. Applied and Environmental Microbiology, 67: 2578-2585. Querol A., Huerta T., Barrio E., Ramon D. (1992). Dry yeast strain for use in fermentation of Alicante wines: selection and DNA patterns. Journal of Food Science, 57: 183-185.

Journal

Annals of MicrobiologySpringer Journals

Published: Nov 20, 2009

There are no references for this article.