30 July 2014

Composition of Grape vs Honey Musts, Part 2

This part will deal with yeast assimilable nitrogen. Now, we are all familiar with the concept that mead musts don't have enough yeast assimilable nitrogen (YAN), or at least they tend not to have enough for us to be comfortable with. Here, we will take a look, not only at the total levels of YAN, but which constituent parts are involved in the differences between grape and honey must.

What is going to be our baseline? That is a very interesting question as the specific amino acid profile of a grape must depends on the variety, specific clone, climate, vintage, soil type, and what vineyard practices occur. There are some basic profiles that can be drawn based on grape varietal, and differenced between red and white, so we will look at a few white varieties and the average for them. The same problem exists with honey: the specific amino acids and their ratios are heavily determined by floral source, but the weather will also play a major role (with drier weather usually meaning more pollen content, and thus more amino acids), as will the region (again having to do with nectar sources).

Just look how different they all are. To better illustrate the point, lets look at the profiles of an average white grape must, honey, and pollen.

Notice how honey and pollen have much higher levels of proline compared to the other amino acids, over half, while grape musts have proline representing about one fourth of the amino acids. Keep in mind that yeast can only use minute traces of proline (only when respiration is dominant compared to fermentation, which is incredibly rare in fermenting musts), and while arginine is utilized by yeast, they far prefer glutamic acid and glutamine, which show higher ratios in pollen (and therefore honey), in comparison to grape musts. 

Here we get to see how two different honey musts compare to average white grape must. The honey must with pollen (30g/L) is closer to the levels of wine must, but it still has a ways to go. This represents a mead must made with 3lbs honey per gallon, but changing the concentration of honey to water changes the amino acid levels to a considerable degree. 

The more honey, the more amino acids, but also a higher starting gravity. Increasing pollen additions also help add amino acids, and if your honey is not filtered you may have anywhere from 1g/L to 5g/L, though this still represents only a small increase in YAN (<20ppm), far from what is considered acceptable.

While pollen additions seem very effective, there can be a bitter flavor that accompanies the pollen, especially if it is of low quality (be warned, the majority of freeze dried pollen sold in the US is from china, even if the guy at the farmers market is selling it).

P.S. See . . . I told you more pictures! Also I'll get a half-a$$ bibliography soon; just a busy drone, even if I am male.

16 July 2014

Carbohydrate Creation and Utilization

This is part two of a series covering yeast's metabolism of sugars. The first part (renamed for more catchy-ness) can be found here.

In this section we will discuss the synthesis and degradation of storage carbohydrates within a cell. For medically minded people, these processes are used to balance blood glucose levels within humans (and most mammals). First we need to familiarize ourselves with the nomenclature.
Maltose and trehalose, both made
 from two glucose molecules:
maltose has an α(1→4) bond,
trehalose has an α(1→1)α bond.

Monosaccharides are the simplest carbohydrates. What's a carbohydrate? Carbo- containing carbon; hydrate - a compound in which water (H2O) is bound to other elements; hence CX(H2O)X, where X > 3 (typically), is the chemical formula for most carbohydrates. Examples include glucose, fructose, galactose, etc..

Disaccharides are two monosaccharides linked together, remember your greek δίς - two. Disaccharides form via dehydration (the removing of a water molecule), and can be linked on any hydroxyl group (-OH). This results in several types of compounds being possible from the same two monosaccharides. 

Polysaccharides would obviously contain many individual monosaccharides linked together.

To form very complex carbohydrates it is necessary to put branches in this ever-growing chain of monosaccharides. 

Ok, now on to the show.

When yeast are first introduced into a new environment, they break up internal glycogen (a very large chain of glucose molecules) reserves to produce their glucose without transporting external glucose across the cell membrane in order to reach a stable, internal osmotic pressure before the onset of full fermentation.

In order to make the glycogen reserves that yeast need, glucose-6-phosphate (the product of the first step of glycolysis) is acted on by the enzyme phosphoglucomutase to form glucose-1-phosphate.

Then, the enzyme uridyl transferase forms UDP-glucose and pyrophosphate (which is broken up into two phosphate groups by the enzyme pyrophosphatase; these Pi groups are used in many metabolic processes).

Glycogen synthase then assembles smaller glycogen molecules using UDP-glucose and preexisting glycogen molecules or glycoginin (a "seed" protein). Larger glycogen molecules are created by adding branches of smaller glycogen molecules utilizing the enzyme transglycosylase (α(1,4 

To utilize glycogen as an energy source, large glycogen molecules are broken by a different transglycosylase (α(1
→ 4)) enzyme into smaller glycogen molecules. The enzyme glycogen phosphorylase then forms glucose-1-phosphate from the terminal glucose molecules of a chain with the addition of a phosphate group. This process will work down the chain until 4 glucose molecules are left before the branch point, at which point it is joined to the terminal position of another chain. Phosphoglucomutase then transforms these glucose-1-phosphate molecules into glucose-6-phosphate (the opposite reaction of the first step of glycogenesis) that can be used directly in glycolysis.

Trehalose synthesis
D-glucose-6-phosphate can also be directed down a different pathway to produce trehalose. UDP-glucose and D-glucose-6-phosphate are the substrates that trehalose-6-phosphate synthase acts on to produce UDP and trehalose-6-phosphate. Trehalose-6-phosphate phosphatase then cleaves the phosphate group off of T6P  producing a free phosphate group (that can be used in further metabolic processes) and trehalose.

This pathway has several benefits: it consumes ATP which creates a loss of energy, in turn driving glycolysis; it stabilizes cell membrane structures which helps protect the cell from temperature swings; it helps prevent damage to membranes by its ability to prevent phase transition events in lipid bi-layers; it prevents glycolysis from progressing too rapidly by diverting phosphorylated sugars used in glycolysis; it also exerts a restrictive control on the influx of sugar by resticting hexokinase activity. These last two act as flow-valves by limiting the amount of glucose that can enter glycolysis to moderate (healthy) levels preventing stalled metabolism that can occur when glycolysis progresses too quickly.


  • Only small portions of glucose-6-phosphate are diverted to either of these paths
  • The yeast will utilize glycogen during lag phase for its sole carbon source while it trys to reach equilibrium with the external osmotic pressure
  • Trehalose is vital for yeast health, especially when put under external temperature stress or changes in osmotic pressure

The importance of a functional trehalose biosynthetic pathway for the life of yeasts and fungi. 2003
Reserve carbohydrates metabolism in the yeast Saccharmyces Cerevisiae. 2000
The role of trehalose synthesis for the acquisition of thermotolerance in yeast. 1994

(P.S. sorry for this crappy bibliography, but i didn't take great notes on sources, and really don't have the time. Also, sorry about only one picture, it takes time to make them; I promise the next post will be more . . . colorful)

09 July 2014

My Hydrometer Broke! What Now?

OK! Time for a break from the science heavy stuff. Want a quick tip/technique? Try this on for size.

This for those of you who run into a broken hydrometer and find yourself unable to take an original gravity reading for your beer, wine, or mead.
First things first, always have a spare hydrometer. I've got 2 normal hydrometers and 3 fine scale hydrometers, and if any break I have a replacement that covers a similar range and immediately get a replacement. However, if you are not prepared for some reason, this process works perfectly (in fact it's how brewers used to measure their gravities, though they used pounds and barrels for their measures), and if you have very accurate scales and beakers it can actually be more precise than a standard hydrometer.

Things you'll need:
A scale that is precise to a minimum of 4 significant figures (look it up if you don't know what that means, it's important)
A way to measure a given volume (either 100 or 1000mL works best) such as a beaker or a flask
A way to take samples (a wine thief is essential to have period! (I meant exclamation point.))

Make sure that your volume beaker/flask can fit on the scale, and make sure your scale can measure the volume your using (for 1000mL you need a max of 1.3kg or more; for 100mL it needs to be able to weigh at least 130g).
Note: It is far easier to use SI units unless you feel like converting.

Here's the procedure for a 100mL sample:
1. Weigh your beaker/flask dry
2. Take a 100mL sample and put it into your beaker/flask
3. Weigh the filled beaker/flask
4. Subtract your beaker/flask weight from the reading
5. Transpose your decimal point so your number reads 1.XXX(X)
Ta Da!

Why it works?
100mL of distilled water at 4*C (at standard atmospheric pressure) weighs 100g. A hydrometer works by comparing this known density to the density of the unknown sample, usually correcting to 20*C (68*F). To do this adjustment by hand, multiply your result by 1.00177. If the temperature of your sample is not 20*C (68*F), then take the number after multiplying and plug it into an SG temp correction calculator.

Volume: 100mL
Beaker weight: 200.00
Sample temp: 70*F
Weight of total sample: 311.00g
311.00 - 200.00 = 111.00
1.1100 x 1.00177 = 1.1120 (rounded)
Temp corrected and rounded for SG 1.111

Note that this procedure will not work with fermenting must unless it has been fully removed of CO2, which requires some special equipment. However, if your in a rut and need to measure fermentation progress it will work, it just cannot be compared to a standard SG scale.

02 July 2014

Composition of Grape vs Honey Musts, Part 1

This will be a series of articles that compare the constituent compositions of grape musts and honey musts, which is worse than comparing apples and oranges. Although we try to use similar practices on both for the production of wines and meads, there are intrinsic differences that can cause problems; but that also make each unique, even down to the specific grape variety or nectar source. These will not be all encompassing, comparing Russian River Pinot to Burgundy, or Arizona orange blossom to Floridian, but will serve as a guide to how we should treat our musts: what to add, when to add it, and what not to add it.

First we are going to take a look at the minerals in wine and mead. Why? Brewers worry night and day about their water, and for good reason; winemakers almost never consider it because whatever is in the grapes is considered good enough (most of the time); but mead makers don't tend to consider it at all, even though we are adding considerable amounts of it to our honey. As it stand now, no one has done a comparison of different water salt additions to test the organoleptic quality imparted by them, something I hope to do when I bottle some of last years traditionals. Until such time, all we can do is compare the minerals in mead musts to those of wine musts and try to guess what is going on and what needs to be corrected.

So, what specific minerals are we interested in? There are actually two questions there:

  1. What minerals do yeast need for healthy growth? 
  2. What minerals have an impact on the flavor of the product?
The first is readily available thanks to decades of extensive research on yeast metabolism and preferred growth media. The second is very hard in this context: I have yet to find any published papers on the effect of specific minerals on the quality of wine, let alone mead. Aside from obscure remarks about vineyard salt levels and the quality of grapes from them, it seems that no one is putting in any research on the organoleptic side. This is an area where brewers run circles around winemakers, and it is rather strange considering all the hype over the "minerality"of certain wines, although anyone who says they can taste slate is an idiot (go ahead, lick a piece of slate, then granite, and then quartz, they all taste the same; do it blindfolded if you want!).

To the right is a chart comparing the levels of common ions in wine musts, honey, and mead musts, as well as a common synthetic must that is used in experiments. From it, we can see just how different wine must, mead must, and what scientists think wine must is, all are. Wine musts have far greater levels of potassium (K+), magnesium (Mg2+), zinc (Zn2+), sulfate (SO42-), phosphate (PO43-), in fact, all the minerals listed are in higher concentration in wine musts than mead musts. This is just further proof that honey is a very inhospitable environment for most organisms.

So, the question remains: do we need to adjust these levels, or is grape must just in excess? For that, we need to look at several very important ions, and a few ratios, and ask they yeast what they want, what they need, and what they prefer.

Potassium (K+)
In brewing, potassium is not considered in a water profile because malt provides the amounts needed for yeast, but in wine and mead it is a very important consideration. Potassium plays a few vital roles concerning the yeast cell; first and foremost, yeast will uptake potassium ions in exchange for hydrogen ions in order to balance the pH in the cytoplasm. H+ is produced in several steps of glycolysis, and if allowed to remain in the cell, the pH will drop to fast and cause many problems with the glycolytic pathway (and several other pathways). The second role potassium plays is to increase glucose uptake rates via an unknown process that probably occurs with the yeast's hexose transport pathways.

How much potassium is needed? It depends on the amount of H+ ions within the medium. It has been shown that yeast need a molar ratio of 25-30:1 K:H in order to successfully complete a fermentation. If the ratio falls below this level, the fermentation is prone to sticking early, leaving a considerable amount of residual sugar. The amount of potassium needed can easily be calculated utilizing the pH of the must as seen to the right, but this calculation can be complicated when concerning mead due to the low pH buffering of meads. I'd suggest estimating how much you need based on the pH when the must is first created (before any additions are made), and just use that. To simplify matters, an addition of 0.25g/L (~1g/gal) potassium carbonate will yield 141ppm K, adding this value to the average 237ppm K in a SG 1.108 honey must gives 378ppm K, enough for a pH above 3.45. 1g/L cream of tartar  yields 208ppm K, and 0.25g/L potassium carbonate and 1g/L creamo of tartar added together will give 349ppm K, enough to supplement even the lowest K levels found in honey.

It is also important to note that supplying K later in the fermentation does not correct an initial deficit, so there is no point in waiting to add potassium.

Calcium (Ca2+)
Considered important by brewers because it stimulates yeast flocculation (and helps control mash pH), calcium is not terribly important in wine or mead making as the bulk aging time will easily clear yeast from the product. In fact, calcium acts antagonistically to magnesium and zinc uptake, and while small amounts (~1ppm) are needed for cell wall maintenance, it's use should be limited to low levels if any is added at all (be aware that almost all nutrient blends contain calcium as a cation attached to certain vitamins, and they usually contain magnesium to help balance this out). Also note that bentonite additions will add calcium to the product if used as a fining agent.

Magnesium (Mg2+)
As seen in the glycolytic pathway, yeast need magnesium for almost all cellular function, especially glycolysis. This is another mineral that brewers don't have to worry about because malt provides a ton of it; and winemakers tend to have enough of it, they just need to worry about if they have a proper amount compared to their calcium content. What is the proper amount? Something higher than 1:1 Mg:Ca. Many studies have been done showing that yeast function better with higher Mg:Ca ratios (2-4:1), but this may be excessive. I think a 1.5-2:1 ratio is right about where we should be to optimize yeast performance and not risk too rapid growth, zinc interference, or off flavors.

Sodium (Na+)
Have you ever noticed that Australian wines tend to be really savory, almost salty? This mineral is responsible for a lot of that flavor. Yeast don't need it, but it is always amazing how it can bring that cup of broth up to the next level, if used carefully. Brewers concern themselves with the chloride ion to give round, mouth-filling, savoriness to a beer, and some add a lot of salt (NaCl); others would never touch it and just use calcium chloride; but there is a difference between adjusting chloride levels, and adding sodium, and in moderation sodium just gives things that extra bit of something. How very scientific of me!

Chloride (Cl-)
Chloride, just like sodium, has no important function in yeast metabolism, and is an unknown as far as flavor contributions to mead. The levels found in grape musts are obviously higher than those in mead, but most commercial nutrient blend provide small amounts in the form of thiamine hydrochloride and other vitamin or amino acid based salts.

Sulfate (SO42-)
Sulfates are very important for yeast health as yeast need two sulfur containing amino acids (cysteine and methionine) which are often synthesized from other amino acid skeletons and sulfate via the sulfate reduction pathway. It is very important to note that this pathway relies on sulfate (SO42-) and not sulfite (SO32-) or elemental sulfur (S), of which the latter can lead to increased hydrogen sulfide (H2S) production.

Sulfate is generally added as an anion attached to another mineral that we choose to add (ie. MgSO4, magnesium sulfate). The addition of sulfate may have an affect on the flavor profile of wine and mead, though it is unclear what the impact is.

Phosphate (PO43-)
Phosphates are very important for yeast metabolism (see how many times they occur just in glycolysis), allowing the construction of nucleic acids, energy transferring compounds (ATP), cell membranes and other internal structures, and are vital for the transport of extracellular compounds into the cell. Simply put, yeast needs phosphate, and honey does not have enough! The levels found in wine are on average 20-30 times those found in mead musts. Luckily, the P in DAP (diammonium phosphate) is there to help out; this compound is put in all non organic nutrient blends to provide both easy nitrogen, in the form of ammonium, and phosphates. A simple 0.25g/L addition of DAP will give 180ppm phosphate which is more than enough for yeast health. Note, however, that the 53ppm yeast assimilable nitrogen (YAN) provided by this same addition is normally not enough for mead musts; in order to get 212ppm YAN form just DAP, an addition of 1g/L is needed, which results in 719ppm phosphate (almost double the levels found in the highest testing wine samples, and above the legal limit of some countries).

Trace Minerals
Small amounts of manganese (Mn), copper (Cu), iron (Fe), molybdenum (Mo), boron (B), zinc (Zn), cadmium (Cd) and even lead (Pb) are present in grape musts with the table to the left representing an "average" Portuguese white wine must. Manganese tends to run at about 0.5-7.3ppm (mean of 2.7ppm) in most european wines, and zinc is be all over the board from 0.1-10ppm (with some chinese musts registering >12ppm). Typically lead is below 1ppm, as well as copper and iron. Almost all the same trace minerals can be found in honey, but in much lower quantities, with almost no copper or iron.

Fermentation rates seem to be best when there is about 2ppm Zn, 11ppm Mn, and 15ppm Fe, however those concentrations of manganese and iron would clearly impact flavor, and as they are not essential (above trace amounts), there is no need to supplement musts with them. Zinc, however, can have a major impact on the progress of fermentation and should be supplemented to sufficient levels (servomyces works very well at the recommended rate of 8.5ppm (0.32g/gal)).


  • Add potassium, even if you don't have a way to measure pH, your must is probably lacking
  • If you can measure pH, use the chart to guess how much you need, keeping in mind that any addition will raise the pH
  • We don't know what flavor is added or changed, by what ion, yet
  • Don't worry about calcium, maybe try to get it to >50ppm if you want, but make sure you add enough magnesium
  • Add enough magnesium to get to about 100ppm, or about 1.5 times the calcium; remember that nutrient blends usually have it so don't go too crazy, maybe 2g/gal epsom salt
  • Sulfates are needed, but are usually provided by nutrient blends or added epsom salt
  • Phosphates are covered by DAP additions
  • Trace minerals are present, but zinc may need to be added 
  • Lighter honeys have less minerals than dark honeys, but not by a lot

Birch, Rosslyn M., Maurizio Ciani, and Graeme M. Walker. "Magnesium, Calcium and Fermentative Metabolism in Wine Yeasts." Journal of Wine Research 14.1 (2003): 3-15. Taylor & Francis Online. 04 Aug. 2010. Web.
Kudo, Masayoshi, Paola Vagnoli, and Linda F. Bisson. "Imbalance of PH and Potassium Concentration as a Cause of Stuck Fermentations." American Journal of Enology and Viticulture 49.3 (1998): 295-301. Print.
Larcher, Robert, and Giorgio Nicolini. "Elements and Inorganic Anions in Winemaking: Analysis and Applications." Hyphenated Techniques in Grape and Wine Chemistry. Ed. Riccardo Flamini. Chichester, England: John Wiley, 2008. N. pag. Print.
Ribéreau-Gayon, P., Y. Glories, A. Maujean, and Denis Dubourdieu. Handbook of Enology the Chemistry of Wine: Stabilization and Treatments. Chichester: John Wiley, 2006. Print.
Ricardo-da Silva, George/Jane M., H. Mira, P. Leite, and A. S. Curvelo-Garcia. "Metal Reduction in Wine Using PVI-PVP Copolymer and Its Effects on Chemical and Sensory Characters." Vitis -Geilweilerhof 46.3 (2007): 138-47. Print.
Somda, Marius K., Aly Savadogo, Nicolas Barro, Philippe Thomart, and Alfred S. Traore. "Effect of Minerals Salts in Fermentation Process Using Mango Residues as Carbon Source for Bioethanol Production." Asian Journal of Industrial Engineering 3.1 (2011): 29-38. Science Alert. 29 July 2011. Web.
Stobbaerts, R., H. Robberecht, F. Haesen, and H. Deelstra. "Manganese Content of European Wines." International Journal of Vitamin and Nutritional Research 64.3 (1994): 233-36. Print.