If you could learn which yeasts actually ferment your natural fermentation, would you?

The Australian Wine Research Institute (AWRI) has launched a new winery service. Winemakers can now submit samples from natural/wild/native/non-inoculated ferments (or, conceivably, from an inoculated one if they wanted to) and, courtesy of “next-generation DNA sequencing*,” receive a profile of the yeast species present along with approximate percentages. An additional step can give them more specific strain information, and the AWRI can also isolate, freeze down, and store the main yeasts from your sample as “insurance” if you need them in future vintages.

This sounds like marvelous geekery. Winemakers who don’t inoculate probably wonder about what’s going on in there at least occasionally. What I’m unsure of is whether this yeast profile is a useful management tool beyond a fun way to satisfy your curiosity (or give even more detailed information to very well-heeled consumers). I imagine the following scenarios:

Good natural ferment — You like what’s happening with your uninoculated wine. You’re going to keep making it even if you learn that some generally undesirable bug has part of the action because you know you like the results. Maybe having a yeast profile sets you a benchmark so that if the ferment stops working in some future vintage you can send in a sample for comparison and see if the microbial blend changed, but how does that information then change what you do?

Bad natural ferment – You’ve tried not inoculating a wine and it isn’t working for you for whatever reason. You send in a sample from one that didn’t work (too slow, undesirable flavors, or didn’t finish fermentation). Maybe you’ll learn that some toxic bug is out-competing the yeasts you need to grow, or you’ll identify the source of that high volatile acidity you’ve been combatting. What then? The natural ferment still doesn’t work. Can you tweak pH or how much sulfur dioxide you use or your oxygen management to encourage more favorable microbes against the enemy? I don’t know how likely someone is to successfully adjust or amend a natural ferment to work better at that level — from the perspective of philosophy as well as how likely it is to work — but if you’re going to try these strategies, you’ll likely know to try them without knowing which yeast species are involved.

Planning a natural ferment – Maybe you run a test natural ferment to see whether you like what it does, and you want to “double-check” that it’s okay. The primary information you need stays the same: does it ferment to dryness? does it move fast enough to satisfy your economic needs and peace of mind? does it taste good? These will remain the primary drivers of your decision to move ahead or not, independent of what you learn from that yeast profile.

This service can help answer that perennial question of whether the yeasts in your “wild” ferment are really wild or just commercial yeast strains that have colonized your winery, and to some extent (especially if you go down to that extra strain level sequencing) the degree to which your ferment is different from some other winery’s. But the question — the AU $275 question for running a single sample, or AU $792 for the recommended panel of three samples per ferment — is this: will the extra information change what you do?

Nevertheless, forward-thinking actionability isn’t everything. Even if a winery never tries to replicate a previous successful wine by inoculating with its bespoke mix of strains banked through the system (that seems unlikely to succeed simply given the enormous variability in other parameters affecting wine quality directly and indirectly via influencing yeast growth), a retrospective look at yeast mixes in multiple vintages of the same natural ferment could be interesting. Did a change in viticultural management practices, or source of grapes, or fermenting conditions correlate with a clear change in microbial populations? That prospect makes me hope for two things. First, that a winery (or a dozen or two) will use this as a tool for looking back at the path they’ve taken and not just down at where they’re standing. Second, that when they do, they’ll share.

 

*Next-generation DNA sequencing (this introduction from Nature is dated and technical, but it’s also open-access; the article on Wikipedia is also quite good) is a collection of methods so called because they rely on new strategies for sequencing DNA — not just tweaks of the old traditional way, but really new ways of solving the problem — that let us do things differently, faster, and more efficiently. The most important things to know about next-generation sequencing are that, 1) it’s not a method, but a general term for a whole bunch of methods; 2) the idea has been around since 2008, which doesn’t mean that any of those methods can’t be cutting edge but which does mean that they’ve “trickled down” to the general market by now; and 3) they allow for pulling many sequences from many different organisms out of a single small sample. Old sequencing methods required a relatively large sample of (preferably) a single pure target sequence or else the signals would get so jumbled up that the whole instrument read-out would just look like soup (as I recall well from my first sequencing experiments as an undergrad in 2002-3). Now, we can sequence even single stretches of DNA, and even many different single stretches all hanging out in the same sample from some real-life microbe-rich setting: soil, seawater, or an active ferment. And we can do it quickly and inexpensively enough, now, to offer it as a commercial service. Remember those predictions about what genomics would bring us back around 2000, when sequencing genomes really hit the news? We’re getting there.

New World Ingenuity, Interdisciplinarity, and New Zealand’s Sauvignon Blanc Programme

I’ve been reading James Halliday’s and Hugh Johnson’s lovely, rambling The Art and Science of Wine (Firefly, 2007), with something of the feeling that I’m sitting beside the old gentlemen’s fireplace listening to them hold forth. The book is short on citations and uneven on explanations, but full of two careers’ worth of wisdom. They describe without getting bogged down too much in the how or why of things, a good technique for teaching in a hand-waving, appreciative sort of way and for learning without paying too much notice to the reality that you’re being taught.

A theme — mostly tacit, but persistent — that sticks with the first few sections of the book is the difference in marketing and, therefore, winemaking strategies between the Old World (especially the classic French regions) and the New (especially Australia and New Zealand). Old World: make it and they will come. New World: no one knows who we are, so we’d better be distinctive and creative and different (and tasty). The socio-cultural-historical-economic factors driving that difference are too extensive to explain here and, besides, are largely a matter of common sense. And if it’s an oversimplification, it’s still largely true.

New Zealand has had to fight hard for a place in the global wine eye. Not only is the Kiwi wine industry young and from a remote location, their production is tiny. Yet they’ve unquestionably succeeded. We’ve all heard about Marlborough Sauvignon Blanc, and many if not most of us have heard about Marlborough and Central Otago Pinot Noir. New Zealand’s problem (okay, one of New Zealand’s problems) now is that they’ve played the region-grape association game too well. Kiwi Sauvignon Blanc can be too easy to stereotype. The industry seems especially concerned that their wine is too expensive, with cheaper Sauv Blanc from Chile and South Africa and elsewhere on the market, to keep being competitive without some kind of new consumer incentive.

So, the Kiwis are trying to teach their pony every trick in the book (and maybe invent a few new ones) in an effort called, creatively, the New Zealand Sauvignon Blanc Programme.

A partnership between essentially every major wine research institute in the country and a few major producers (notably Pernod Ricard), the Sauvignon Blanc Programme was first funded in 2004 and has a promise of continued funding through 2016. 2010-2016 is it’s second phase: “Sauvignon Blanc 2: ‘novel wine styles for new markets.'” Phase I largely served as information-gathering about Sauvignon Blanc-specific flavors and flavor production; phase II is aimed at optimizing and manipulating those flavors. The goal is to improve the quality of existing wine, but also to carve out new styles from the harmonious-but-homogenous* cat-piss-on-gooseberry  style for which New Zealand in general and Marlborough in particular has become world-famous.

The exemplary thing about this programme, in addition to it’s duration, is its interdisciplinarity. An effort to solve one problem — how to diversify Kiwi Sauvignon Blanc — is bringing together plant cell biologists and viticulturists and wine chemists and yeast geneticists and sensory scientists and cognitive scientists and assorted biotechnologists and industry folk — winemakers and growers and business and marketing people — all with different perspectives on how to solve that one problem. In the process, they’re creating solid new science, funding Masters and PhD students who will be important to the industry in a few years, and fueling market growth: good for research, good for industry.

Industry dollars are a main source of funding for wine research everywhere, but rarely is the collaboration this diverse or long-standing. The scope of SB1 and SB2 are fueling research far beyond just bringing a new and improved white wine to market. Here, the Kiwi homogeneity is serving them well: even if not everyone makes Sauvignon Blanc, the industry as a whole obviously rides on it. I wonder what would happen if other winemaking regions could identify one massive problem relevant to more or less everyone, focus their resources, and sponsor all of the region’s top researchers to help solve it.

I suspect that multiple different Sauvignon Blanc flavor profiles are going to be a hard sell to all but the most esoterically sophisticated Americans, though perhaps the more important UK market will be better educated enough to pay attention. We’ve already seen scientific publications from this project; we may just have to wait to judge the success of the wine.

Market pressures don’t always make for good science. But, sometimes, they do.

Can Occam’s razor slice through a Scorpion?

Occam’s Razor: use the simplest means possible to accomplish your goal.

Scorpion: 1) An arachnid; 2) a genetic method, patented by ETS Labs, for detecting bacteria and yeasts in wine (or grape juice, or beer) samples based on real-time fluorescence PCR (poymerase chain reaction.)

Can Occam’s Razor slice through a Scorpion?

“Plurality should not be posited without necessity” or, in the words of William of Ockham, “Pluralitas non est ponenda sine neccesitate.” According to The Skeptic’s Dictionary, the eponym was awarded to the monk from Ockham because he used the argument so often, even though it was already a common tenent of Medieval logic. Philosophers refer to the Razor in arguing over the existence of God, but most of us translate the phrase as “Don’t make it more complicated than necessary (stupid.)”

If Ockham’s monastery pew became a time machine one day and he was transported to 2010, the philosopher might be curious about the many incredible scientific advances we’ve made in the past 800 years.

In addition to being a cousin of the tarantula, Scorpion is ETS Labs’ patented name for a genetic method to detect common spoilage yeast and bacteria in wine samples. Send ETS Labs 60 mL of your wine and they will send back a report listing which, if any organisms are swimming around in your tank or barrel or bottle. Scorpion analysis relies on differences between the genomes of different organisms. Probes designed to bind to DNA sequences that uniquely identify a species are labeled (“tagged”) with fluorescent markers. Toss probes specific to many different organisms into a wine sample, and the tags show which probes are bound and, therefore, which organisms are in the sample. (This is a gross oversimplification, but I’m trying to avoid a detailed discussion of RT-PCR here. For a little more detail, see ETS Labs’ website.)

The first assignment for my wine microbiology lab this semester is to identify the bacteria and/or yeast contaminating an unidentified wine sample. The professor will give each group two wines — one spiked with nasties — and ask us to give him a report on what we found in the wine and how we found it. The first part of the assignment is to propose a method for attacking the problem: when we have the wine, how will we analyze it?

Oooohhh…There are lots of ways to analyze wine, and I could show my prof that I know about them by including all sorts of nifty things in my report. Scorpion analysis is outside my budget, but I could always run my own genetic tests if I can find out where to buy the right genetic probes.

Or I could smell it. “The nose knows” may be cheesy (why cheesy? Why not yogurty, or cucumbery? There’s a whole ‘nother kettle of fish…) but such aphorisms arise because they are true. Looking at my lab manual and the list of microorganisms that could be the unknown contaminant, each has a peculiar smell. Brettanomyces bruxellensis is probably the most famous — many wine lovers can identify “Brett” — but Pediococcus parvulus, Acetobacter, and Lactobacillus species have distinctive aromas, too, as I know from culturing them in the lab.

Oenococcus oeni, a bacteria very often responsible for malolactic fermentation, is a little trickier to identify based on smell alone, so I might need to move up to the next level of complexity (by the way, we aren’t allowed to use taste as part of our analysis; some of my classmates are underage.) If my nose isn’t quite sure, I can drip a few milliliters of wine onto a Petri dish and see what grows. We make Petri dishes full of growth media for yeast and bacteria by combining sugar, some protein and a few other basic nutrients, and adding agar — a gelatin-like substance from seaweed — to make it solidify. Culture media in a dish is essentially Jello (mmmm….yeast extract-flavored Jello!) Any bacteria and yeast in my wine will grown and reproduce on this media and, after a few days in a nice warm incubator, each little microbe will have grown into a colony of identical offspring microbes that I can see with my naked eye. Different bacteria and different yeasts have different colony morphologies; they look different; even within the same species, different strains can have different morphologies. One of my favorite strains of Brettanomyces bruxellensis looks like this.

Between smell and colony morphology, I expect excellent odds of correctly identifying the bugs my professor has hidden in my wine. My nose, and Jello in a Petri dish. In terms of levels of complexity, I think that I’m ranking far below genetic testing even if I do need to use the Jello. I could spend several hundred dollars to use the fancier technology, but why bother when the good, old-fashioned, simple method will do? Now, I’m not at all knocking ETS Labs; Scorpion is a potent analysis when you need to know “how much” as well as “what,” for complex microbial problems, and for busy wineries amongst other things. Scorpion analysis definitely has its place, but this isn’t it.

Occam’s razor: 1

Scorpion: 0

The “hows” of sulfites, part I: measurement

What is the aerator-oxidator apparatus, and why am I spending so much time using it?

The short explanation: I’m measuring wine sulfites.

The long explanation: I’m using the aerator-oxidator method to measure free SO_2.

A good day measuring free SO2: no breakage!

The molecular formula for the sulfite ion is SO₃^2-, formed when sulfur dioxide, SO₂, reacts with water to form the bisulfate ion, HSO₃^–, which can then dissociate to SO₃^2- + H⁺. All of these forms are found in wine – wine is an aqueous solution – but SO₂ is what winemakers actually add, usually in the form of potassium metabisulfite or K₂S₂O₅.

For as casually as we toss around the phrase “wine sulfites,” their behavior is remarkably complex. Potassium metabisulfite, sold by the kilo in winemaking supply catalogues, is a salt that releases potassium and molecular sulfur dioxide when dissolved in aqueous solution. We care about molecular SO₂; this is the “species” of sulfur to which we can ascribe antimicrobial properties. Molecular SO₂, however, quickly dissociates into ions which either remain “free” in the wine or become “bound” to other solutes like acetaldehyde, pigments, and sugars.

SO what? Beyond plain old cleanliness and sanitation, SO₂ is probably the most widespread antimicrobial agent used to protect wine, During yeast-driven alcoholic and bacterial-driven malolactic fermentation, (the right kind of) yeast and bacteria in your fresh grape must is a good thing. Thereafter, the presence of most yeast and bacteria in fermented wine means contamination, nasty flavors, or even unsafe wine.

SO₂ diffuses into yeast and bacterial cells and does all sorts of destructive things: damaging DNA, glomming up the structure of important enzymes, and reacting with nutrients so that the microorganism can’t use them for growth (like a game of chemical keep-away.) The end result isn’t always to kill the bug, but the durn thing isn’t likely to keep multiplying. Like humans and most other living things, yeast and bacteria don’t reproduce well when they’re starved and stressed. Microbial susceptibility to SO₂ varies: 20mg/L will suppress some, while others withstand upwards of 40mg/L molecular SO₂.

[By the way, American wineries are allowed to use as much as 350mg of SO₂ per liter, but any wine containing more than 10mg/L must be labeled “contains sulfites.” Wine-dwelling microorganisms can naturally produce as much as 100mg/L in the process of fermentation. Most wineries aim for sulfur dioxide levels around 50-100mg/L, but that’s a big generalization.]

SO back to the original question: what is this great big fragile glass apparatus, and what does it have to do with SO₂? The only name for it seems to be “the aerator-oxidator apparatus” and – go figure – it is used to measure SO₂ levels by the “aeration-oxidation” method. Air bubbles  through a wine sample – “aeration” – carrying volatile “free” bisulfite up and through a condenser and into a vial of hydrogen peroxide, where it reacts with hydrogen peroxide and water to form sulfuric acid. In other letters, SO₂ + H₂O₂ → H₂SO₄, an “oxidation” of SO₂.

Adding a color indicator that flips from royal blue to teal when pH flips from acidic to basic means that sodium hydroxide (a base) can be added to the hydrogen peroxide-plus-sulfuric acid solution to negate the acid. This is just a good old acid-base titration: when the amount of slowly-added sodium hydroxide exceeds the amount of sulfuric acid, pH and color both rapidly change. The final step is calculating sulfuric acid from sodium hydroxide and sulfur dioxide from SO₂. Remember all of those different forms that sulfur dioxide takes in wine? The aerator-oxidator method can be adjusted to measure either free or total SO₂. But wait! I thought that the antimicrobial form was molecular SO₂, and you say that we’re not measuring that? Conveniently, molecular SO₂ can easily be calculated from free SO₂ and the pH of your wine.

To measure free or molecular SO₂, the wine sample is acidified and chilled for the duration of this whole bubbling business. Acidifying the wine – increasing the concentration of H⁺ — forces the equilibrium between SO₂ +H₂O ↔ HSO₃⁼ + H⁺ entirely to the SO₂ side. All of those aforementioned types of SO2are volatile and can make the trip from the wine-containing flask up through the condenser and into the H₂O₂ –containing flask EXCEPT the bisulfate ion that is bound to other compounds in the wine. (Incidentally, chilling the wine sample ensures that heat energy doesn’t break any of those bonds apart.) On the other hand, boiling the wine sample provides enough energy to break (virtually) all of the non-covalent bonds, freeing the bound ion, and allowing the “total” amount of SO₂ in the wine to volatilize. Measuring total SO₂, then, is exactly the same procedure as measuring free/molecular SO₂ save that the wine-containing flask is boiled rather than immersed in an ice bath.

The best part of the whole process? It takes longer to explain and understand than to perform. Yes, I did take general chemistry for a reason!  

Pros of using this method: it’s accurate, easy to read (unless, like my lab-mate, you’re color-blind), inexpensive once you’ve made the set-up, and only takes 10 minutes per sample,

Cons: it takes an entire 10 minutes per sample, all of the SO₂ may not bubble out of the wine, any gaps in the glass-to-glass connections allow SO₂ to escape before reacting with the H₂O₂, and the whole contraption is disturbingly prone to breakage by tired/rushed/frustrated grad students.

By the by, why do some people say that they are allergic to sulfites? A .01%-sized handful of the American population lacks the enzyme sulfite oxidase and literally cannot metabolize sulfites; these folk are also asthmatic and usually have trouble breathing and break out in a rash when they consume sulfites (from wine, dried fruit, lunch meat, ect.) In all great likelihood, you aren’t one of those people.

You know that you’re a food science geek when…

You know that you’re a food science geek when:
 
1. You spend most of Saturday at a food preserving and canning party and think that you’ve had a great weekend.
 
2. You have a book out of the library titled Good Morning, Kim Chi and you’re reading it in the bathroom.
 
3. You routinely take an empty nylon bag on walks so that you can collect wild herbs as you go.
 
4. You wonder how WADY — wine active dry yeast — will make your pizza dough taste.

5. Your friend brings you a can of wine as a present from his road trip.

6. When your friend presents you with the can of wine, a 45-minute conversation ensues about how the can must be lined to prevent aluminum flavors, processing temperatures, oxidative implications of a sealed container versus wine under natural cork, expiration date, ect.

7. Your next big volunteer project is co-teaching a class on non-alcoholic home fermenting.

8. Your counters, the top of your refrigerator, and one of your kitchen cabinets are all full of six different vegetable, dairy, and grain ferments.

9. You’ve been known to take home a petri dish or two to try culturing the organisms fermenting your home-made sauerkraut.

10. When no one is looking, you occasionally taste your experiments.