Lisa Van de Water “The Bad Wine Lady” Vinotec Napa 607 Cabot Way, Napa, CA Technical Fax Direct

“When Wild Flora Go Bad” “Debaryomyces does Davis” 2

HOW TO WORK WITH YEASTS AND OTHER MICROBES?  NEVER TRUST A MICROBE  PAY ATTENTION  REACT RIGHT AWAY IF SOMETHING GOES WRONG  KNOW WHEN TO GET TESTS DONE AND WHEN NOT TO  HAVE A PLAN FOR RESPONDING TO EACH POTENTIAL PROBLEM 3

Wine microbiology involves competition among life forms for a food source, and it is all about risk management. The same microbes are found all over the world (though not necessarily the same strains). The same microbes behave differently in different wines, and different wines respond differently to them.

Assessing Microbial Hazards  Start with grapes and proceed through bottling, examining each step of the winemaking procedure for each product.  Consider which microbes could be involved at each point, and what levels are acceptable.  Determine the relative risk and severity of potential problems.  Decide how to detect and monitor the microbes’ presence and effects.  Write procedures to follow if microbes exceed set levels.

To download a newsletter including a discussion of HACCP by Dr. Bruce Zoecklein ALSO ask for ENOLOGY NOTES (FREE!!)

Some recommended texts  Wine Analysis & Production (Zoecklein et al.)  Winery Technology (Margalit)  Handbook of Enology 1 & 2 (Ribereau-Gayon, et al.)  Principles and Practices of Winemaking (Boulton)  Techniques & Concepts: Monitoring the Winemaking Process (Iland)  Knowing and Making Wine (Peynaud)  Winemaking Problems Solved (Butzke, ed.) S 7

Where do yeasts in juice come from? 1. Vineyard Grapes, leaves, dust, and other vineyard sources: almost all non-Saccharomyces yeast 2. Winery Juice processing equipment, containers, and winery surfaces: mostly Saccharomyces, some non-Sacch. surface film yeasts (and sometimes Brett) 3. Winemaker Cultures or starters added: Saccharomyces or mixtures of Saccharomyces and selected non-Sacch. 8

Sources of Yeasts: 1. Vineyard  Kloeckera apiculata (Hanseniaspora uvarum)  Similar microscopically in size and shape to Brettanomyces  Candida, Pichia, Debaryomyces, Hansenula, Metschnikowia, Schizosaccharomyces, Rhodotorula, Cryptococcus, etc.  Many other species of yeasts not very tolerant to alcohol  VERY FEW Saccharomyces yeasts  (Also: Molds, bacteria)  Brettanomyces/Dekkera absent or extremely rare 9

Danger signals in the vineyard for microbial problems in the wine Vineyard history of stuck ferments or other problems Stressed or struggling vines Vines with trunk diseases, deficiencies, pests High heat spells during ripening Damaged, rotten, or moldy grapes

When do vineyard yeasts grow?  On grapes before or after picking if mould/rot/damage is present  On grapes before crushing if any skins are broken  In juice before fermentation  During first part of fermentation  Note: Adverse effects (especially stuck ferments) may NOT be noticed until late in fermentation, even though growth occurred MUCH earlier!

What to call yeasts that were not added?  Natural? No! All yeasts that have not been genetically modified are natural. Cultured yeasts are not “chemical’ or “artificial”. They were isolated from natural fermentations.  Wild or Feral? No. There are many non-Saccharomyces yeast species present in the vineyard which are truly ‘wild’, and there are large populations ofSaccharomyces yeasts present (‘wild’) in every winery. This is a VERY important difference! Calling any yeast that is not added ‘wild’ muddles up this distinction. 12

What to call yeasts that were not added?  Indigenous or native? Maybe. But indigenous/native to where? The vineyard? The cellar?  Spontaneous? Yes. This term correctly indicates that the yeasts, of whatever species or origin, arose without a deliberate addition.  Uninoculated? Yes. This simply means that there were no yeasts added by the winemaker, whether dry yeast or starter tank. 13

Common Misconceptions About Yeast in Vineyards  The best yeast for fermenting grapes come in from the vineyard.  There are a lot of Saccharomyces yeasts on grapes.  Studies in the past two decades have shown that there are not. There are a few… but not very many. An Italian study tested more than 1000 samples of grapes before they found even one Saccharomyces cell.  Spreading pomace in the vineyard may or may not increase the Saccharomyces population. In a recent study, Saccharomyces sprayed on the grapes did not remain as part of the grapes’ microflora. 14

What is the problem with vineyard yeast? Aren’t they all natural and desirable? Natural, yes. Desirable, often NO.

Non-Saccharomyces Yeasts in Fermentation  May grow before or at the beginning of fermentation  Often are more cold-tolerant than Saccharomyces  Some are SO2-sensitive, others as resistant as Sacch.  Can make floating clumps and fluffy lees  Use up nutrients, especially vitamins, needed by Saccharomyces  May also produce inhibitors to Saccharomyces  Die before full alcohol potential is reached  Can cause stuck ferments!  Can lead to sensory defects  Sulfides  Esters like ethyl acetate (nail polish) and amyl acetate (banana)  Acetic acid  Etc

Avoiding encouraging unwanted microbes Sort grapes to remove diseased, sunburned, or raisined berries Do not “cold”-soak above 10 C (50 F) Warm quickly after cold-settle-rack or cold-soak Add at least ppm SO2 Keep pH below 3.6 Cover open-top fermentors with plastic sheet Store and handle dry yeast according to directions Add enough yeast if inoculating, and do not wait to inoculate once must is at 60+ F Test YAN, supplement appropriately If unwanted yeasts grow, delay nutrient supplementation until Saccharomyces takes over; add extra vitamins (will be depleted)

(scroll all the way down) pantothenate, biotin, pyridoxine, myo-inositol are TTB-approved as of

Danger Signals: Yeast  What you should see: Saccharomyces  Active fermentation once it begins  Clean, yeasty aroma  Regular Brix drop, continuing to dryness  What you should NOT see: Bad vineyard yeasts  Sluggish or stop-start pattern at the beginning  Odor of ethyl acetate or sulfides  Floating clumps or scum  Brix drop tapering off before dryness  Fluffy lees

20 Are you growing a beautiful ‘garden’ of yeast? (with a ‘weed’ or 2 maybe) Or are you just growing a big crop of ‘weeds’?

HOW DO YOU TELL THE DIFFERENCE (1)?  LOOK, SMELL, TASTE, TEST  Especially with uninoculated fermentations, but with any fermentation, smell them AT LEAST TWICE A DAY BEFORE FERMENTATION STARTS, AND THEN EVERY DAY!  Taste any fermentation that seems a little odd  Look for floating clumps  Test whatever you need to test to track the fermentation progress and to be sure it is not slowing down 21

HOW DO YOU TELL THE DIFFERENCE (2)?  CHECK UNDER PHASE-CONTRAST MICROSCOPE  The phase-contrast microscope is the most powerful tool a winery has to detect unwanted yeasts  Culturing takes too long; by the time they grow, it is too late (though culturing may be used to corroborate observations)  Of the vineyard yeasts, PCR at present detects only Kloeckera (which is always present, and excessive levels can be easily and quickly (and cheaply!) seen under a phase-contrast microscope. Other non-Saccharomyces vineyard yeasts which can cause problems are not detected by PCR at this time. 22

Direct phase-contrast microscopic exam  EXTREMELY IMPORTANT MONITORING TOOL ESPECIALLY DURING FERMENTATION  Possible only if there are > cells/ml, difficult unless > 5000/ml  ‘Uninvited’ yeasts are often morphologically different though some may not be.  Check microscopically for Lactobacilli, which often do not grow in culture even if viable.

Direct Exam: Advantages/Disadvantages  Advantages  IMMEDIATE results  Microbes do not need to be stained  Detects any cells present, alive or dead  Problems can be diagnosed a long time after they happened  Disadvantages  Requires HIGH-QUALITY phase-contrast microscope (US$3000+)  Requires training and skill  Requires a large population of cells  Sometimes can tell the yeast genus, cannot tell the species.

Microscopic Exam of Musts  Examine reds under microscope for non-Saccharomyces yeasts and Lactobacillus especially:  After cold-soak, before yeast inoculation  Every day until active fermentation begins  Every day after that if odd yeasts or any bacteria are seen  If no odd yeasts or any bacteria, every other day during fermentation  Examine whites for non-Saccharomyces yeasts if fermentation does not start quickly

Left: WRONG Right: CORRECT

Yeast Sources: 2. Winery surfaces, containers, equipment  Saccharomyces  If yeast is not added, DNA studies show that the Saccharomyces yeasts present in the winery ferment the wine, if they can out-compete non-Saccharomyces vineyard species.  Candida, Pichia, and other surface film yeasts  A variety of other cellar-resident yeasts  Brettanomyces/Dekkera, especially in barrels Winemaker: “Where do wine yeast come from if you don’t add any?” Dr. Ralph Kunkee: “The winery.” 27

Genetic Results from Spontaneous Fermentations  Frezier and Dubourdieu [AJEV 43:4, , 2004]  3 years of DNA analysis of yeasts from the grapes, workers’ hands, winery equipment, and fermentations showed that the yeasts doing the fermentations were from the tanks and workers’ hands, not grapes. Wines had from one to 23 strains.  Ciani et al, Italy [Antonie von Leeuwenhoeck 85: , 2004]  58 Saccharomyces isolates from grapes, must, and winery surfaces were tested, and all the strains fermenting the wines came from the winery.  California  Strains in tanks of uninoculated SB were checked against the strains the winery used; the yeasts were all Simi White, which had not been used for10 years. 28

Persistence of Saccharomyces in the Cellar  Peynaud, France: “Imagine the winery surfaces to be lined with yeasts.”  Alessandro Martini (Perugia, Italy): Built a new pilot winery for the student fermentations. Used a ‘labeled’ yeast for the first 2 years, then told the students to kill the yeast. They dismantled everything and steamed every piece of equipment and surface in the cellar. The next fall they did not add yeast, but the labeled strain grew up just as fast as before. 29

What about a new winery with all new equipment?  The very first ferments in a new winery, with no yeast inoculation and no equipment or containers that have ever been in another winery, will begin with non-Saccharomyces yeasts.  If all goes well, the few Saccharomyces yeasts that are on the grapes will grow and will eventually finish the fermentation.  From then on, the cellar is colonized with yeasts and the cellar yeasts will make a major contribution to the fermentations. 30

Yeast Sources: 3. Added by the winemaker  Dry yeast: Almost exclusively Saccharomyces  Many strains available (too many!)  A few blends of Saccharomyces and carefully selected non-Sacch. yeasts  Starter tanks maintained in the winery often contain a mix of vineyard yeasts and Saccharomyces 31

Some factors for success with yeast Storage  As cool as possible (but not frozen): keep opened yeast packs for 6-8 wks at 20C or a year tightly closed in refrigerator Rehydration  Sprinkle yeast into F/38-40 C water  Do not stir for 5 minutes, then stir gently  Wait 20 minutes, no more than 30 Inoculation  Test temperature of must; recommend at least 62 F  If yeast temperature is >10 F higher than must, add small portions of cool must slowly to bring temperature down Do not stress yeasts with temperature shocks!

To add or not to add Why not add yeast  Believed more “Natural”  Long and slow  Complexity Why add yeast  All non-GMO fermentations are natural anyway  Reliability  Predictability  Less spoilage opportunity  Fewer stuck ferments DO NOT ATTEMPT AN UNINOCULATED FERMENTATION UNLESS GRAPES ARE IN PERFECT CONDITION!

Can MOST wineries make successful wines some of the time without adding yeast? YES. Can SOME wineries make successful wines all the time without adding yeast? YES. Can EVERY winery make successful wines all the time without adding yeast? NO. 34

Monitoring fermentations  Graph Brix progress, not just write it down in a list  Test whatever you need to track the fermentation progress and to be sure no unwanted microbes are growing (MLF, VA, pH, etc)  Smell and taste juice (especially if uninoculated!) AT LEAST TWICE A DAY BEFORE FERMENTATION STARTS, AND THEN EVERY DAY, w atch for sulfides and other defects 35

Sulfide origins  Residual spray of elemental sulfur from vineyard  Nitrogen deficiency during fermentation  Deficiency of pantothenate during fermentation  Stressed yeasts near end of fermentation  Others? Competition for nitrogen?

Sulfide descriptors  H2S  Rotten eggs  Toasty  Dirty  “Just not very fruity”  Mercaptans/disulfides  Onion  Garlic  Rubber  Cat pee  Asparagus

Lactobacilli in fermenting wines  Grows quickly  Can grow fully in 2 days to a week  Population increases exponentially  Antagonistic to yeast and other bacteria  Can stop yeast fermentation  May kill other bacterial species including Oenococcus  Produce toxins such as brevicin  Produce acetic acid from sugar: g/L or higher  Do not make ethyl acetate so there is NO SMELL  Musts fermenting at pH 3.6+ are most susceptible “Ferocious Lactobacilli!” - Dr Ralph Kunkee

Preventing Lactobacillus spoilage  Must adjustments  Add ppm SO2 at crushing  Soak on skins no warmer than 15 C /60 F  Lower pH to 3.5 in reds after overnight soak  Provide yeast with proper nutrition  Add ppm lysozyme before fermentation to high-risk wines  During fermentation or in stuck wines  Monitor malic acid depletion  Examine under microscope  Test VA if ferment slows or if Lactobacilli are seen

For workshops and seminars on fermentation and wine microbe identification, and assistance on avoiding or diagnosing wine problems…