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From Vineyard to Wine, Lactic Acid Bacteria and Yeast Identification Using Molecular Methods
UNIVERSITY OF PADOVA UDINE VERONA Magister Degree course in VITICOLTURE, OENOLOGY AND MARKETING Department of Food Science Developed for the achievement of Master of Science (LM - 69) From vineyard to wine, Lactic Acid Bacteria and yeast identification using molecular methods Supervisor: Prof. Marisa Manzano Assistant supervisor: Priya Vizzini Student: Anabella Varela ACADEMIC YEAR: 2014-2015 CONTENTS ABSTRACT p.3 INTRODUCTION p.5 1. Picolit grape……………………………………………………………… ….. p.5 2. Microorganisms from vineyard to wine environment……………………... p.7 2.1. Yeast evolution during fermentation……………………………… ….. p.8 2.2. Lactic Acid Bacteria evolution during fermentation………………….. p.10 2.3. Yeast and Lactic acid bacteria interaction…………………………….. p.13 3. Microorganism identification: Traditional Methods Vs Molecular Methods p.14 4. Polymerase chain reaction…………………………………………………… p.16 5. Theoretical aspects of Denaturant Gradient Gel Electrophoresis (DGGE) p.18 MATERIALS AND METHODS p.20 1. Samples and Sampling procedures…………………………………………. p.20 1.1. Vineyard………………………………………………………………. p.20 1.2. Winery………………………………………………………………… p.20 1.3. Zymaflore ST Active dry yeast……………………………………… p.21 1.4. Must and wine………………………………………………………… p.21 2. Revitalization of vineyard and winery samples……………………………. p.24 3. Morphologic classification and identification……………………………… p.26 3.1. Storage of pure colonies………………………………………………. p.27 4. DNA extraction ……………………………………………………………… p.28 4.1. DNA extraction from isolated colonies……………………………….. p.28 4.2. DNA extraction from must and wine samples………………………… p.29 5. DNA Standardization………………………………………………………… p.29 6. Molecular analysis……………………………………………………………. p.30 6.1. PCR-DGGE protocol for Lactic Acid Bacteria……………………….. p.30 6.2. PCR-DGGE protocol for Saccharomyces sensu stricto strains……….. p.32 1 6.3. Nested PCR-DGGE protocol for non-Saccharomyces yeasts………. -
Dessert Wines 1
Dessert Wines 1 AMERICA 7269 Macari 2002 Block E, North Fork, Dessert Wines Long Island tenth 75.00 1158 Mayacamas 1984 Zinfandel Late Harvest 50.00 (2oz pour) 7218 Robert Mondavi 1998 Sauvignon Blanc 27029 Kendall-Jackson Late Harvest Chardonnay 7.50 Botrytis, Napa tenth 100.00 26685 Château Ste. Michelle Reisling 7257 Robert Mondavi 2014 Moscato D’Oro, Late Harvest Select 8.00 Napa 500ml 35.00 26792 Garagiste, ‘Harry’ Tupelo Honey Mead, 6926 Rosenblum Cellars Désirée Finished with Bern’s Coffee Blend 12.00 Chocolate Dessert Wine tenth 45.00 27328 Ferrari Carano Eldorado Noir Black Muscat 13.00 5194 Silverado Vineyards ‘Limited Reserve’ 26325 Dolce Semillon-Sauvignon Blanc Late Harvest 115.00 by Far Niente, Napa 19.00 7313 Steele 1997 ‘Select’ Chardonnay 27203 Joseph Phelps ‘Delice’ Scheurebe, St Helena 22.50 Late Harvest, Sangiacomo Vineyard tenth 65.00 6925 Tablas Creek 2007 Vin De Paille, Sacerouge, Paso Robles tenth 105.00 - Bottle - 7258 Ca’Togni 2009 Sweet Red Wine 7066 Beringer 1998 Nightingale, Napa tenth 65.00 by Philip Togni, Napa tenth 99.00 7289 Château M 1991 Semillon-Sauvignon Blanc 7090 Ca’Togni 2003 Sweet Red Wine by Monticello, Napa tenth 65.00 by Philip Togni, Napa tenth 150.00 6685 Château Ste. Michelle Reisling 7330 Ca’Togni 2001 Sweet Red Wine Late Harvest Select by Philip Togni, Napa tenth 150.00 7081 Château St. Jean 1988 Johannisberg Riesling, 6944 Ca’Togni 1999 Sweet Red Wine Late Harvest, Alexander Valley tenth 85.00 by Philip Togni, Napa tenth 105.00 7134 Ca’Togni 1995 Sweet Red Wine 6325 Dolce 2013 Semillon-Sauvignon Blanc by Philip Togni, Napa tenth 125.00 by Far Niente, Napa tenth 113.00 27328 Ferrari Carano Eldorado Noir Black Muscat 13.00 7000 Elk Cove Vineyard Ultima Riesling, 15.5% Residual Sugar, Willamette tenth 80.00 6777 Eroica 2000, Single Berry Select Riesling, by Chateau Ste. -
2019 Napa Valley Ribolla Gialla, Matthiasson Vineyard
2019 Napa Valley Ribolla Gialla, Matthiasson Vineyard Ribolla Gialla originates in the Friuli region of Italy and across the border in Slovenia. An ancient grape variety, its earliest known mention was by the Pope in 1296. Ribolla Gialla makes an unparalleled food wine, filling the gap in food pairing between white wines and red wines—it can be amazing with pork and stewed pome fruit, fatty raw seafood, cured meats, and intense cheeses. We have been making “orange wine” from this fruit since 2008, fermenting the wine whole cluster, with the skins and stems, until dryness. The wine is 100 percent Ribolla gialla from our home vineyard. The wine was fermented whole cluster with native yeast in an open-top tank using punchdowns. After two weeks the must was pressed, and the wine was aged for 18 months in neutral barrels. The wine aged sur lees and it was never racked. No SO2 was used until a tiny amount at bottling. The alcohol is 13.0%, the pH is 3.9 and the TA is 5.35. 253 cases produced. The Ribolla gialla tastes like hazelnuts, baking spices, meyer lemon curd, Bartlett pear, but most of all it has a stony minerality that is incredibly distinctive. There is supple tannic structure and fresh balanced acidity, with great weight and length given the moderate alcohol. The Vineyard The Ribolla gialla represents seven rows in our home (“Matthiasson”) vineyard in the Western Oak Knoll area of Napa Valley. It was grafted onto 15 year old Merlot vines. George Vare was given a few sticks of the budwood from Josko Gravner in 2001 and grafted it into his own vineyard, which Steve was managing. -
CONTENTS ABSTRACT P.3 INTRODUCTION P.5 1. Picolit Grape……………………………………………………………
CONTENTS ABSTRACT p.3 INTRODUCTION p.5 1. Picolit grape……………………………………………………………… ….. p.5 2. Microorganisms from vineyard to wine environment……………………... p.7 2.1. Yeast evolution during fermentation……………………………… ….. p.8 2.2. Lactic Acid Bacteria evolution during fermentation………………….. p.10 2.3. Yeast and Lactic acid bacteria interaction…………………………….. p.13 3. Microorganism identification: Traditional Methods Vs Molecular Methods p.14 4. Polymerase chain reaction…………………………………………………… p.16 5. Theoretical aspects of Denaturant Gradient Gel Electrophoresis (DGGE) p.18 MATERIALS AND METHODS p.20 1. Samples and Sampling procedures…………………………………………. p.20 1.1. Vineyard………………………………………………………………. p.20 1.2. Winery………………………………………………………………… p.20 1.3. Zymaflore ST Active dry yeast……………………………………… p.21 1.4. Must and wine………………………………………………………… p.21 2. Revitalization of vineyard and winery samples……………………………. p.24 3. Morphologic classification and identification……………………………… p.26 3.1. Storage of pure colonies………………………………………………. p.27 4. DNA extraction ……………………………………………………………… p.28 4.1. DNA extraction from isolated colonies……………………………….. p.28 4.2. DNA extraction from must and wine samples………………………… p.29 5. DNA Standardization………………………………………………………… p.29 6. Molecular analysis……………………………………………………………. p.30 6.1. PCR-DGGE protocol for Lactic Acid Bacteria……………………….. p.30 6.2. PCR-DGGE protocol for Saccharomyces sensu stricto strains……….. p.32 1 6.3. Nested PCR-DGGE protocol for non-Saccharomyces yeasts……….. p.34 7. Sequencing…………………………………………………………………… p.37 7.1. Issuing of the samples………………………………………………… p.38 RESULTS p.39 1. Bacteria differentiation…………………………………………………….. p.39 2. Saccharomyces differentiation …………………………………………….. p.43 3. Non Saccharomyces differentiation ……………………………………….. p.45 CONCLUSIONS p.49 ACKNOWLEDGEMENTS p.50 REFERENCES p.51 2 ABSTRACT Usually all attempts to characterize the microbial diversity in wine fermentations have employed standard methods of enrichment and isolation to cultivate various microbial constituents before taxonomic identification. -
Guerra Albano | Ribolla Gialla Friuli Colli Orientali
Ribolla Gialla Friuli Colli Orientali DOC Records of Ribolla Gialla go all the way back to the 13th century in Colli Orientali (before that it came from the Greek island of Kefalonia, where it's still known as Robola) and today it is synonymous with Friuli's most prestigious DOCs. In order to Winemaker: Dario Guerra create a balanced wine from this tricky varietal, savvy producers know you always Generation: 3rd have to harvest by hand: clusters are irregular and unpredictable, and the size of grapes can vary widely, even on a single bunch. As with all of his whites, Dario's small batch Ribolla Gialla benefits not only from meticulous, season-long pruning, but also from rich calcareous soils and 40+ year-old vines. WINEMAKER BIOGRAPHY Dario Guerra is a 3rd generation producer (his father’s name was Albano) who farms 10 hilltop hectares in Friuli’s esteemed Colli Orientali DOC. While it’s normal for producers to have 2 or 3 green harvests, Dario – rather amazingly – treats every vine as an individual, constantly sculpting them all season long! ENOLOGIST Dario Guerra TASTING NOTES Color Straw yellow with golden highlights Nose Layers of apple, pineapple, citrus, and peach Palate High-toned structure and acidity, with soft, enveloping tannins Finish Long, persistent, mineral-driven VINEYARD & VINIFICATION Vineyard Location Friuli Colli Orientali DOC, Friuli-Venezia Giulia Vineyard Size 10 ha Varietals List 100% Ribolla Gialla Farming Practices Grapes organically farmed (non-certified), dry farmed, and picked by hand. Elevation 220 m Soils Ponca (flysch) and calcareous clay Maturation Summary Bottled for 6 months Alcohol 12.5 % Acidity 6.3 g/liter Residual Sugar 3 g/liter REGION FRIULI-VENEZIA GIULIA Situated in the NE corner of Italy, Friuli-Venezia Giulia borders Veneto to the west, the Austrian Alps to the north, Slovenia to the east, and to the south has a short coastline along the Adriatic Sea. -
Grower Profile
MIANI nzo Pontoni works quietly and tirelessly in his tiny Calvari, 100% Refosco dal Peduncolo Rosso aged in 100% plots totalling 15 hectares of vineyards nestled in new oak barrels. Out of respect to his predecessors many of the hills near the small towns of Buttrio and the wines are named after the families who tended the Rosazzo in the Colli Orientali del Friuli region of vineyards, Filip, Banel, Buri, and Saurint. EItaly. Some might even call Pontoni a recluse. Some describe him as a true Frulian, a little rough on the edges. Enzo is “Uncompromisingly taut, minerally precise, wonderful notoriously fastidious in the tending of his vineyards. freshness, flamboyant richness, detail, textural elegance, Nothing but the best fruit makes its way to his bare bones extreme concentration, textured and lavish palate, mineral- little cellar in the town of Buttrio. The cellar is reminiscent imbued acidity and striking finesse, power with irresistible of a large garage with Enzo’s mother living above the tiny drinkability, muscle and velvet with elegance” are all facility. Here Pontoni produces his miraculous wines named descriptors various wine writers have used to describe Miani after his mother’s maiden name, Miani. wines. Need we say more? Due to the extremely low yields, (1.2 tons/acre) and rigorous selection of grapes, output is miniscule, roughly 800 cases spread between nine different wines. Few have tasted Miani wines, and far fewer still have ever met the man himself. Yet, from his earliest of releases in the early ‘80s, Enzo Pontoni has been known as a legendary winemaker to the primo cognoscenti of the wine world. -
Picolit D.O.C
PICOLIT D.O.C. COLLI ORIENTALI DEL FRIULI − Controlled and Guaranteed Designation of Origin Sweet white wine prized of a straw yellow, delicate scent reminiscent of honey and wildflowers, taste warm, harmonious, sweet with a pleasantly bitter aftertaste. GRAPES: 100% Picolit VINE: Indigenous variety from Friuli, undoubtedly ancient, already cultivated in the Roman imperial period, had the honor to delight the palates of popes and emperors. Among the admirers of Picolit, it also includes Carlo Gol doni, who defines this wine the brightest gem of Friuli wine. Vine arrived today thanks to the work of Count Fabio Asquini of Fagagna who in 1700 saved this precious vine f rom certain death. It was the same Asquini that made him known to the "foreigners" in high−prestige markets su ch as London, Paris, Genoa, Milan, Ancona. The Picolit was appreciated and consumed not only in Italy, but he found among his great admirers even the Court of France, the Papal Court and even the King of Sardinia, the E mperor of Austria and the Czar of Russia. The Picolit is characterized by very limited production due to a peculiarity in the development of the grapes that undergo a partial floral abortion, leaving the loose bunch with smaller and sweeter grapes. Today it is cultivated in the hills of Friuli Venezia Giulia, in the provinces of Udine and Gorizia. The 2006 vintage is the first as Denomi nation of controlled and guaranteed origin and thanks to his particular value, Picolit is the second denomination of controlled and guaranteed origin of the whole Friuli Venezia Giulia. -
RONCHI DI CIALLA Friuli Venezia-Giulia, Italy
Producer Fact Sheet September 2021 RONCHI DI CIALLA Friuli Venezia-Giulia, Italy www.ronchidicialla.it Ronchi di Cialla, a 28 hectare historical Friulian Estate, is a small, family run business. Established in the 1970s, they have established a reputation as one of the finest producers of age-worthy wines in Fruili. ‘Ronchi’, in Fruili’s dialect, means hills cultivated by vines; ‘Cialla’ is a small valley surrounded by woods "The Ronchi di Cialla 2015 with chestnut, oak, and wild cherry trees. Located in the Cialla valley, one of three sub-zones in Colli Schioppettino di Cialla is a tangy, raw and unique wine Orientali del Friuli, it is a picturesque area with perfect growing conditions for the indigenous grapes. with its distinct personality. This The company philosophy has always been to work solely with indigenous varieties, such as, Ribolla, unusual expression certainly Redosco, Schioppettino, Verduzzo, and Picolit. In fact, the Rapuzzi Family restored and revitalized the doles out those dusty, white- Schioppettino grape from near extinction, defying the odds and local wine laws. pepper aromas, and you get them here in large supply. This Their viticultural practices involve minimal treatment with non-polluting products, while vinification is is a mid-weight red that shows carried out naturally. Their continued commitment to agriculture innovation and ecologically-friendly freshness and a light tannic touch. If you are interested in methods have earned them their "biodiversity friendly" certification by the World Biodiversity the indigenous grapes of Italy, Association. This accolade is granted to those with a commitment to progressive increases in biological this wine is well worth your diversity, conservation through the use of natural practices, and minimal interference farming attention." 93 points, Robert Parker, techniques. -
In Friuli, a Newly Proposed DOCG with Roots in Recent History
FORBES.COM Data 11-07-2019 Pagina Foglio 1 / 5 7 views | Jul 11, 2019, 11:26am In Friuli, A Newly Proposed DOCG With Roots In Recent History Susan H. Gordon Contributor Dining & Drinking The what, where, when of wine, with special attention paid to Italy. A view of Oslavia's hills and ponca soils, with the Ribolla Gialla vineyards of local estate winery Primosic PRIMOSIC The move is both a step forward and a return to generations past. Late last year, six winemakers from the Friulian village of Oslavia, today a slip of land at Italy's border with Slovenia and which for most of the 500 years before the 20th century began had been part of east-west crossroads under Austrian rule, proposed a new DOCG for their village’s Ribolla Gialla wines. The six-member group is the Associazione Produttori Ribolla di Oslavia: Fiegl, Il Carpino, La Castellada, Promosic, Princic, Radikon. Of Oslavia’s seven producers, Josko Gravner is missing. Biotype to Slovenia’s Rebula and unrelated to Greece’s Robola, Ribolla Gialla (the color mention is important: 003368 Ribolla Verde which is written about invariably as both clone and unrelated grape is considered lesser either way) has been grown in the hills of the Gorizia province, of which Oslavia is a frazione and home to a few hundred people, for centuries. An “ancient white from the Italy-Slovenia border” reads Nonino FORBES.COM Data 11-07-2019 Pagina Foglio 2 / 5 the subhead in the Jancis Robinson–led Wine Grapes of 2003. White-wine grapes vinified on their skins are traditional to both sides of the national border here, a small area that is the reference point for what outside markets call orange wine. -
Puiatti Metodo Classico Ribolla Gialla
PUIATTI METODO CLASSICO RIBOLLA GIALLA Located in the northeastern corner of Italy in the Friuli region, Puiatti has been a leader in innovative winemaking since 1967. Founder Vittorio Puiatti was a pioneer of current methods in the Friuli region which seek to highlight the uniqueness of grape varieties by emphasizing their purity – no oak is used in production. Puiatti produces a diversity of wines from both indigenous and international grapes including the aromatic varieties for which the region is best known. Bordered by Slovenia, Austria and the Adriatic Sea, Friuli enjoys a combination of continental and maritime climates that result in a unique microclimate which favors northern wine- growing while benefitting from Mediterranean influences. Plentiful sunshine and tangible temperature variations assist the slow maturing of the vines, which yields firm, well- balanced grapes with full flavors and aromas. The Puiatti estate is located in two of Friuli’s most important wine producing areas, 25 acres in Collio and 124 acres in Isonzo. The architecture of the newly constructed winery speaks to the essential philosophy of its wines, sharp, clean and well-balanced. PRODUCTION AREA Country: Italy Region: Friulli From the gravelly terrain of Isonzo,the climate here is mild, rainly and well ventilated. Vines are cultivated using the double-guyot training method with a density of 4700 vines/ha. GRAPE VARIETIES 100% Ribolla Gialla PRODUCTION TECHNIQUES Grapes are harvested by hand in the first week of September. Fermentation takes place off skins in stainless steel and in bottle at a temperature of 68°F. Malolactic fermentation is not carried out. -
Guerra Albano | Ribolla Gialla Brut
Ribolla Gialla Brut With just 10 hectares of vines in the Colli Orientali DOC, Guerra Albano is a discovery we’re exceptionally proud of at Small Vineyards. Here, 40-year-old vines and an average of just 900 grams (less than 1 bottle) per plant help deliver a Winemaker: Dario Guerra sparkling wine with true poise and complexity. With an array of orchard fruit and Generation: 3rd lively minerality, this Brut also exhibits the estate's trademark “marrow” quality that bespeaks of soft minerals and salt. The bubbles are soft and silky, with an elegant, lingering finish. WINEMAKER BIOGRAPHY Dario Guerra is a 3rd generation producer (his father’s name was Albano) who farms 10 hilltop hectares in Friuli’s esteemed Colli Orientali DOC. While it’s normal for producers to have 2 or 3 green harvests, Dario – rather amazingly – treats every vine as an individual, constantly sculpting them all season long! ENOLOGIST Dario Guerra TASTING NOTES Color Sunlight Nose Orchard fruit with floral undertones Palate Lively, mineral-driven, and complex Finish Elegant, lingering VINEYARD & VINIFICATION Vineyard Location Friuli Colli Orientali DOC, Friuli-Venezia Giulia Vineyard Size 10 ha Varietals List 100% Ribolla Gialla Farming Practices Grapes organically farmed (non-certified), dry farmed, and picked by hand. Elevation 220 m Soils Ponca (flysch) and calcareous clay Maturation Summary Bottled for 4 months Alcohol 12.0 % Acidity 5.6 g/liter Residual Sugar 1.3 g/liter Annual Production 10,002 bottles REGION FRIULI-VENEZIA GIULIA Situated in the NE corner of Italy, Friuli-Venezia Giulia borders Veneto to the west, the Austrian Alps to the north, Slovenia to the east, and to the south has a short coastline along the Adriatic Sea. -
Picolit COLLI ORIENTALI DEL FRIULI DOCG
Picolit COLLI ORIENTALI DEL FRIULI DOCG One of the Friulian Enologic prides. In Torre Rosazza we can count on the greatest extension of “Picolit dei Colli Orientali del Friuli” vineyards. This wine possesses a special and symbolic value that underlines a deep connection with the area, that has been established by the estate since its origins. Picolit vineyards are scattered all across the estate, in areas with cultivated in Picolit since 1978. GRAPES Picolit GRAPE YIELD 40 quintals/hectare AREA Colline orientali del Friuli (Manzano Hills) ALTITUDE Terracing Height: 120-180 m amsl. GRAPE TRAINING Sylvoz and guyot PLANTING DENSITY 3.000 trees/hectare AGING at least 24 months in French oak tonneaux ANNATA 2017 FIRST VINTAGE 1986 ANALYSIS DATA Alcohol 14,5% vol., Total acidity 6,3 g/l, Volatile acidity 0,64 g/l Residual sugars 74,3 g/l AGING POTENTIAL 10-15 years Production area Then, the bunches are pressed whole and the must thus obtained carries out a static “Ponca” is the typical element of our soil: decantation. It ferments part in stainless a sedimentary formation integrally made steel tanks and part in oak barrels. This is of clayey marl alternating with layers followed by aging in French oak tonneaux of sandstone. The “Ponca” shows low for at least two years. fertility and good capacity to hold water, gives uniques features to the grapes and the wine such as full-bodied taste and Taste colour, tannins, and minerality. COLOUR: bright gold. The location of the vineyards on BOUQUET: The delicate and captivating terracings, the Ronchi (top of the hills), scent is reminiscent of honey, custard and the perfect exposure allow the full and white peach.