The case for spontaneous fermentation of wine grapes

Saccharomyces cerevisiae (SC) is the yeast species which completes the alcoholic fermentation process in both inoculated and spontaneous fermentations but the role of non-Saccharomyces (non-S) yeasts in the process should not be discounted. Spontaneous fermented wines carry a higher risk of spoilage but bring along the benefits of increased complexity, improved mouthfeel, and a higher degree of flavor integration (Jolly, et al.). This post, drawing heavily on the work of Jolly, et al., takes a closer look into the functioning of non-S yeasts in alcoholic fermentation.

Absent an inoculation, all yeasts found in grape must and wine will originate from one or more of the following sources: vineyard, grapes, or winery processing equipment (E.J. Bartowsky). Wine-associated yeasts are identified in the table below.

The general consensus was that all non-S yeasts died shortly after the beginning of alcoholic fermentation but, according to Jolly, et al., that is not borne out by more recent reasearch. Rather, the progression, they say, is as follows:

• H. uvarum is usually present in the highest numbers initially, followed by various Candida spp.
• The majority of the non-S yeasts disappear during the early stages of a vigorous fermentation
• May be a result of :
• Slow growth
• Inhibition of the combined effects of SO2, low pH, high ethanol, oxygen deficiency, nutrient limitation, and size or dominance of SC inoculants
• Non-S yeasts that do survive and are present till the end of the fermentation (Z. bailii, Pichia spp.)may have a higher tolerance to ethanol.

Jolly, et al., have conducted a literature survey which has identified a number of benefits that accrue to the practitioners of spontaneous fermentation:

• Lower ethanol yields -- as the authors point out, these yields "are sometimes the result of wines with higher residual sugar"
• A range of metabolic products to include terpenoids, esters, higher alcohols, glycerol, acetaldehyde, acetic acid, and succinic acid
• Hydrolization of glycosolated flavorless precursors by the enzyme ß-glucosidase to form free volatiles that can improve the flavor and aroma of wine 
• Several flavor and aroma compounds are present in the grapes as glycosolated flavorless precursors
• Enzyme ß-glucosidase not encoded by the SC genome
• Several of the non-S yeasts possess varying degrees of the enzyme
• Contribution to flavor production -- Non-S yeasts can be divided into neutral and flavor-producing yeasts. P. anomala, K. apiculata, and Candida pulcherima are flavor-producing non-S species, with the latter being known as a high producer of esters.
• Some non-S yeasts can consistently produce high glycerol concentrations during fermentation
• Glycerol important for regulating cell redox potential during fermentation
• Glycerol also contributes to smoothness, sweetness, and complexity of wine
• Glycerol production can also be associated with increased acetic acid production.

In summary, there are risks associated with spontaneous fermentation but those risks seem to be more than offset by the benefits that accrue to the user. And the number of great wines in France and Italy that utilize this approach attest to the fact that most of the leading producers have arrived at this conclusion.

Bibliography
E.J. Bartowsky, Bacterial spoilage of wine and approaches to minimize it, Letters in Applied Microbiology.
Neil P. Jolly, et al., Not your ordinary yeast; non-Saccharomyces yeasts in wine production uncovered, FEMS Yeast Research, 14 (2).
Loureiro and Malfeito-Ferreira (Spoilage yeasts in the wine industry, International Journal of Food Microbiology 86, 2003).

©Wine -- Mise en abyme

Sulfur taint in wine production: Genesis and exodus

A winemaker is continuously on guard to ensure the earliest possible detection of issues that could have potentially negative effects on the quality of in-process or finished wine. One issue that is treated with the utmost respect, and attended to with some alacrity, is sulfur taint, the primarily olfactory manifestation of sulfur compounds in the wine. Sulfur taint is the bane of the winemaker because ( Lansing, Wine defects during fermentation, Wine Business Monthly, April 2011):

• It is generally associated with negative aromas
• It has a low threshold for sensory detection
• It has high chemical reactivity
• It is difficult to mask and/or remove.

The sulfur compounds associated with sulfur taint, and the population of odors associated therewith, are illustrated in the figure below.

In this post I will examine the origins of sulfur in must, the creation of sulfur compounds during winemaking, and strategies for minimizing the incidence and/or removing these compounds from the medium.

Sulfur taint has its origins in either the vineyard, the cellar, or both. In the vineyard, elemental sulfur is sprayed on the vines to combat the potential effects of powdery mildew. If this spraying is conducted too close to harvest, portions of the sulfur will remain on the grapes and make its way into the fermentation process. An example of sulfur-like off odors created in the cellar is the case of hydrogen sulfide production by the yeast to synthesize the sulfur-containing amino acids methionine and cysteine. This process is facilitated by the reduction of sulfates via the sulfur-reduction pathway. A lack of intracellular nitrogen will not curtail the process and the excess hydrogen thus created cannot be incorporated into the amino acid. Rather, it is secreted into the medium (Kennedy and Reid, Yeast nutrient management in winemaking, The Australian and New Zealand Grapegrower and Winemaker, 537, October 2008).

A listing of the sources of sulfur-like off odors is presented in Table 1.

Table 1: Sources of sulfur taint in wine production.

Environment	Source	Action	Impact
Vineyard	Elemental sulfur used as fungicide	Reduction during fermentation
	Sulfur-containing pesticides	do.
	Excess of metal ions
	Vine stress
	Unsound fruit
Cellar	Cold soaking	Growth of yeasts such as Kloeckera	Depletion of amino acids and micronutrients
	Native Yeasts	High hydrogen sulfide production	Compete against other yeasts for dominance of fermentation
	Excess hydrogen sulfide from sulfate reduction	Hydrogen sulfide used to synthesize	Absence of nitrogen causes produced hydrogen sulfide  to be secreted into the medium
	High levels of sulphur dioxide added to must at crush	Allows sulphur dioxide to bypass the sulfate reduction system	Sulfur dioxide enters the yeast cell directly
	Vitamin shortage in high YAN musts
	Nitrogen limitation	Produces sulfur-like off odors	Production begins 30 minutes after ammonia starvation initiates

Source: Compiled from Lansing and Kennedy and Reid)

The timing of the production of sulfur-like off odors is shown in Table 2 below.

Table 2. Production timing of sulfur taint by sulfur class.

Sulfur Class	Production Timing	Source
Hydrogen Sulfide	Early in fermentation (2 - 4 days)	Nitrogen/vitamin deficiency
	Fermentation end	Degradation of sulfur-containing compounds
	Sur lie aging	Autolysis
	In bottle	Generally under screw cap
Higher Sulfides	Late in fermentation/Sur lie aging	Release of compounds by metabolically active yeasts Degradation of sulfur-containing amino acids Degradation of cell compounds during autolysis

Source: Compiled from Lansing

There are a number of precautionary steps that can be taken to minimize the potential for sulfur taint (Lansing; Kennedy and Reid):

• Minimize the use of sulfur in the vineyards and cellar
• In the vineyard, ensure adequate time spacing between application and harvest
• Press stressed fruit separately
• Provide adequate nutrition to support the yeast during alcoholic fermentation
• The less assimilable nitrogen in the must, the greater the production of hydrogen sulfide
• Keep yeast cells suspended in the tank during fermentation
• Allows an even distribution of fermentation
• Allows the yeast full access to distributed nutrients
• Manage fermentation temperatures
• Hydrogen sulfide tends to form more commonly in hot, fast fermentations
• Mix the tank contents to prevent stratification
• An especial risk in tall, narrow-diameter tanks
• Remove wine from lees at the first hint of trouble
• Smell, smell, smell.

In the cases where the odors are manifested in the wine, remedies include (i) blowing it off through volatility; (ii) inert gas sparging; (iii) precipitating with copper additions; and (iv) fining.


©Wine -- Mise en abyme

Non-Nitrogen yeast nutrition requirements during alcoholic fermentation

In addition to their utilization of nitrogen as a nutrient during alcoholic fermentation (AF), yeasts synthesize and/or assimilate a number of factors (growth, co, and survival) that help them compete and survive in the hostile environment that is the active fermentation vessel.

Vitamins are growth factors involved in yeast metabolism with some of the needed species synthesized while others are obtained from the juice/must. The most important vitamins in the AF process are indicated in the table below.

Vitamin	Characteristics
Biotin	Plays an important role in sugar, nitrogen, and fatty acid metabolism Yeasts cannot grow without biotin (low concentrations will support growth but 10 microgram/L optimal) Supplementation of musts with biotin increases the viable yeast population and increases fermentation rate
Thiamine	Deficiency leads to accumulation of various products including pyruvic acid which is responsible for much of the non-acetaldehyde sulfite pool
Pantothenate	Involved in the biosynthesis of the sulfur-containing amino acids cysteine and methionine Involved in the formation of acetyl Co-A (acetate donor in esterification) Deficiency results in increased hydrogen sulfide production as well as higher concentrations of acetic acid and glycerol (the latter two producing negative results when coupled)
Pyridoxine	Involved in the biosynthesis of the sulfur-containing amino acids cysteine and methionine Deficiency results in increased hydrogen sulfide production

Source: Kennedy and Reid

Adequate levels of vitamins are normally available to the yeasts but, in some cases (mold infestation, excessive diammonium phosphate addition), a deficiency is recorded and supplements are required. Inactivated yeasts are an excellent source of vitamins.

Yeast cell membrane integrity will be affected by the toxicity of ethanol and the increase in permeability associated with higher ethanol levels will have a negative impact on sugar and amino acid uptake. So-called survival factors, comprised primarily of sterols and long chain fatty acids, are responsible for cell membrane integrity and fending off the ethanol effects until fermentation is complete. Survival factors are formed only in the presence of oxygen and grape musts normally contain enough dissolved oxygen which, when combined with the use of active dry yeast, allows the synthesis of adequate amounts of these factors.

If ascorbic acid is added to the must for any reason, no additional survival factors will be synthesized. Naturally occurring sterols and fatty acids will be depleted by excessive must clarification. In the case of a deficiency of these survival factors, the addition of inactivated yeast cells or yeast hulls will provide a rich source of sterols and fatty acids. Such additions should be adde at the beginning of fermentation and should utilize fresh material to avoid the negative effects associated with lipid oxidation.

Minerals are used as co-factors in enzymatic reactions in the yeast cell with the most important ones being magnesium, potassium, manganese, zinc, iron, and copper. The grape must normally contains adequate levels of these minerals to support AF to its conclusion.

©Wine -- Mise en abyme

Nitrogen as a yeast nutrient in alcoholic fermentation

Wine is a result of using yeasts in an anaerobic (oxygen-free) environment to convert sugars from pressed grape juice into ethanol in the two-step process illustrated in the figure below. The first step -- glycolysis -- results in the 6-carbon glucose being split into two 3-carbon pyruvate molecules. In the next step --- alcoholic fermentation (AF) - four atoms of oxygen and two atoms of carbon leave the pyruvate, resulting in acetaldehyde, which is subsequently converted into ethanol.

Alcoholic fermentation (Source: http://alcoholicfermentation.net/)

According to Fugelsang (Overview of yeast selection and malolactic fermentation on aroma, flavor and phenols), the yeasts (i) extract compounds from the solids in the must/juice in order to form the "characteristic metabolites of fermentation (alcohols, esters, fatty acids, carbonyls, etc.) and (ii) cleave cysteine-containing precursors such that volatile thiols (aroma component of several varieties) can be released. The yeast that receives most of the credit -- and does most of the work -- in alcoholic fermentations is a species called Saccharomyces cerevisiae (SC) which is "specialized in metabolizing media with high sugar content and small quantities of nitrogenous compounds" (Suárez-Lepe and A. Marota, New trends in yeast selection for winemaking, Trends in Food Science and Technology 23 (2012), 39-50.). Yeasts require nutritive support to allow the performance of the above functions in the hostile environment (ethanol-rich, acidic) of the fermentation tank. It is the nitrogenous aspect of that support that is the focus of this blog post.

Proteins are used by the yeast as (i) enzymes for the glycolytic pathway (indicated above), (ii) permeases in the cell membrane responsible for the transportation of compounds into the cells, (iii) cellular constituents (Kennedy and Reid, Yeast nutrient management in winemaking, The Australian and New Zealand Grapegrower and Winemaker, 537, October 2008). These proteins are synthesized by the yeast and nitrogen (N) is a key component in that process. According to Kennedy and Reid, "Efficient protein synthesis is needed for efficient sugar transport and overall yeast metabolism."

According to Schwarcz and Schoeninger (Stable Isotope Analysis in Human Nutrition, Yearbook of Physical Anthropology 34, pp. 293-321), almost 100% of exchangeable nitrogen is found in the atmosphere or dissolved in the world's oceans and is transferred from these environments into the biological system through the processes illustrated in the figure below.  Grape vine plants receive their nitrogen through this terrestrial nitrogen cycle.

Source: http://tolweb.org/notes/?note_id=3920

The nitrogen content of grapes are affected by variety, rootstock, climatic conditions, soil composition, vineyard management practices, fertilization, irrigation, rot incidence, and grape maturity (Kennedy and Reid). The yeast cells extract yeast assimilable nitrogen (YAN) from the grape must in the form of ammonia (preferred source of nitrogen for yeast growth as most easily assimilated) and amino acids and these are stored in the cell walls for later use. This extraction and storage of YAN is front-loaded in the AF process.

Nitrogen is required throughout the fermentation process with larger amounts being utilized during the exponential growth phase of the yesats and small amounts during the stationary phase. In some cases the grape must does not provide adequate amounts of assimilable nitrogen and is supplemented by added nitrogen in the form of diammonium phosphate (DAP). Juice levels of < 25 mg/L ammonia or < 150 mg N/L (measured as YAN) is considered nitrogen-deficient (UCDavis). Insufficient nitrogen can result in sluggish/stuck fermentations or sulfide formation (sulfur-like off-odors, mercaptans, and sulfur-containing acetic esters; the less assimilable nitrogen in the must, the more hydrogen sulfide will be produced). Supplements are best added incrementally and proportional to yeast growth (UCDavis).

Excessive nitrogen in the must can lead to elevated levels of ethyl carbamate (a supposed carcinogen) or urea excretion. The levels of nitrogen required for a successful AF is dependent on (Kennedy and Reid):

• Initial must YAN
• Yeast strain
• Fermentation temperature
• Initial grape sugar
• Other factors.

Amino acids are the building blocks of proteins and, when brought into the yeast cell, can be incorporated as is, transformed into a different amino acid, or broken down as a source of nitrogen or sulfur. The amino acids taken up by the yeasts from the grape must is primarily stored in the vacuole to be used for protein synthesis during yeast growth. Once access to inorganic nitrogen becomes difficult, the yeast begins to break down the stored amino acids to provide nitrogen for protein synthesis. The most important amino acids taken up by the yeasts are shown in the table below.

Amino Acid	Characteristics
Proline	Not metabolized appreciably by yeasts under winemaking conditions One of the predominant amino acids along with Arginine and Glutamine Main amino acid from low-fertilization vineyards
Arginine	One of the predominant amino acids Breakdown results in formation of urea and ammonia (During wine storage, urea can react with ethanol to form ethyl carbamate, a carcinogen). Located mostly in grape skin so processing practices could influence content in juice Main amino acid from low-fertilization vineyards
Glutamine	One of the predominant amino acids Favored by yeasts because it can be broken down to glutamate and ammonia

The less-important amino acids taken up by the yeast cells are alanine, serine, and theronine.

©Wine -- Mise en abyme

Lactic acid bacteria: Pre-, intra-, and post-malolactic-fermentation development

Either concurrent with, or post-alcoholic-fermentation, a number of wines are subjected to a process --malolactic fermentation (MLF) -- wherein the harder malic acid is converted to lactic acid by lactic acid bacteria (LAB). The perceived benefits of this process to the final wine are:

• Deacidification, with a resultant increase in pH
• Increased microbial stability through removal of malic acid as a possible carbon substrate; and
• Modification of the wine's aroma profile.

I described the origin and evolution of malic acid in my most recent post and provide a similar treatment of LAB in the current.

The genera from which the LABs are drawn are shown below. The species associated with wine are (Wibowo et al., AJEV, 36 (4) 1988):

• Oenococcus.oeni -- mainly during MLF
• Pediococcus cerevisiae -- mostly after MLF; predominantly in wines with high pH
• Pediococcus pentosaceous -- mostly after MLF; predominantly in wines with high pH
• Lactobacillus spp. -- mainly after MLF.

Phylogenetic trees of Lactobacillales constructed on the
basis of concatenated alignments of ribosomal proteins
Source: pnas.org

Doctor Murli Dahrmadikari (Lactic Acid Bacteria and Wine Spoilage, extension.iastate.edu) describes LAB thusly:

These organisms are gram positive, catalase negative, nonsporing cocci, coccobacilli or rods. They are microaerophilic (sic) that means that they grow well under conditions of low oxygen content. Since they can grow under low oxygen conditions, they can grow throughout the wine (as opposed to on the surface of the wine) even though the container is kept full. The bacteria can metabolize sugars, acids and other constituents in wine and produce several compounds. Some of these are undesirable and constitute spoilage.

LAB utilize two pathways for the metabolism of glucose and a third for the metabolism of pentose (Lerm et al., Malolactic Fermentation: The ABCs of MLF, S. Afr. J. Enol. Vitic. 31 (2), 2010). One of the glucose pathways (EMP) converts glucose into pyruvate over a number of steps and then into lactic acid. In this process, 1 mole of glucose yields 2 moles of lactic acid plus 2 ATPs. This process is called homolactic fermentation and all Pediococcus species utilize this mechanism. The second glucose pathway (6-PG/PK) yields lactic acid, carbon dioxide, ethanol, acetate, and 1 ATP. The bacteria utilizing this pathway are called heterolactic fermenters and this includes all strains of Leuconstoc, some Lactobacillus strains and Oenococcus.oeni. The pentose pathway combines pentose with a phosphate derivative before coalescing with the later portions of the 6-PG/Pk pathway for completion. The outputs of the pentose pathway are lactic acid, acetic acid, and carbon dioxide.

The LAB in wine originate from grape and grape leaves and are brought into the winery at harvest (Dharmadhikari; Lerm et al.; Wibowo et al.). The LAB diversity and population density are limited by  grape maturity and sanitary conditions and levels are generally on the order of 100 cells/gm (Wibowo et al.) The species that are present at this time include Pediococcus and Leuconstoc (Lerm et al.).

Once the grapes are brought into the winery, the LAB population increases dramatically, implicating the winery environment in this proliferation. According to Lerm et al., the population rises to 10³ - 10⁴ cells per ml shortly after crushing and prior to alcoholic fermentation (AF). In a study of the evolution of LAB, Lafon-Lafourcade et al. (Occurrence of Lactic Acid Bacteria During the Different Stages of Vinification and Conservation of Wines, Appl. Environ. Microbiol. 1983, 46 (4)) show populations of 10⁴ cells/ml at 14℃ and 19℃ if (i) unsulfited and (ii) sulfited at 50 mg/l. At 100 mg/l of SO2, however, the LAB population declines 10-fold. The LAB species present at this stage are L. plantarium, L. casei, Leuconstoc mesenteries, P. damnosus, and, to a lesser extent, O. oeni (Lerm et al.).

During the course of AF, the LAB population levels fall precipitously with only 200 cells/ml surviving the process (Wibowo et al.; Lafon-Lafourcade et al.). Wibowo et al., attribute this decline to ethanol sensitivity. The only LAB strain that survives AF is O. Oeni.

AF is followed by a lag phase which is, in turn, followed by rapid proliferation of LAB with levels rising to between 10⁶ - 10¹⁰ prior to MLF. Lafon-Lafourcade et al., posit that this growth originates from winery equipment which serve as incubators for the LAB that would perform a natural inoculation of MLF. O. oeni is the main species that develops here but Lactobacillus and Pediococcus spp. may proliferate and conduct the MLF if the wine pH approaches 4.0.

There are a number of factors that affect the development of LAB and, as a result, the activation and effectiveness of MLF. For example, wine pH affects (Wibowo et al.):

• LAB growth rate
• The LAB species that proliferate
• The metabolic behavior of the species that grow
• The survival of LAB.

A listing of the factors beyond pH that affect LAB growth and development are provided in Tables 1 and 2 below.

TABLE 1. The influence of different winemaking practices on LAB growth

Practice	Influence
Degree of must clarification	Significant impact on bacterial growth. Yeast produce more medium chain fatty acids in highly clarified must
Skin contact prior to AF	Direct effect on extraction of nitrogenous and other macromolecules stimulate LAB growth and malolactic activity
Choice of yeast strain	Inhibitory and stimulatory effects differ between strains
Aging of wine on yeast lees	Yeast autolysis release nutrients that stimulate LAB growth and malolactic activity

Source: Lerm et al., TABLE 2

TABLE 2. Yeast activity inhibiting LAB via the production of yeast metabolites

Yeast Metabolite	Effect on LAB and/or MLF
Ethanol	Affects growth ability
SO2	AF with SO2 producing yeast strain results in wine inhibitory to MLF
Medium chain fatty acids	Affect LAB growth and reduce ability to metabolise malic acid. Combination of fatty acids (hexanoic, octanoic and decanoic acid) cause greater inhibition than individual compounds.
Metabolites of protein nature	Peptide produced by S. cerevisiae during AF: inhibit O. oeni by disruption of cell membrane; inhibition dependent on SO2

Source: Lerm et al., TABLE 3

According to Wibowo et al., additions of sulfur dioxide, and storing the wine at higher temperatures, leads to the progressive loss of viability of any LAB surviving MLF. According to Lafon-Lafourcade et al., under standard conditions, the surviving LAB remained viable post-MLF, exhibiting, initially, a tendency for further growth and then showing a slow, progressive decline over a 200-day storage period. At temperatures over 20℃, a rapid decline in viability was recorded and at 26℃, no LAB activity was recorded after 80 days. Lafon-Lafourcade et al. found that the decline in LAB viability was accelerated by increasing temperature, lowering the wine pH, and increasing the alcohol concentration. These actions, it seems, combine to provide a toxic environment for the bacteria. Addition of sulfur dioxide, they agree, does result in a rapid loss in cell viability but growth recommences at a later date.

At the end of barrel aging, the wine microbial population is stabilized but its population prior to bottling is 103 - 104 cells/ml, if unfiltered, with microbes such as Acetobacter, S. cerevisiae, and O. oeni predominant (Renouf et al., Survival of Wine Microorganisms in the Bottle during Storage, AJEV 58(3), 2007). Filtering with a 0.4 micron filter sheet will eliminate all bacteria from the wine.

Now that the two most important players in the MLF drama have been described, I will cover the MLF process itself when I revisit this topic.

©Wine -- Mise en abyme

Inclement weather and lengthy fermentation time risks in natural yeast fermentations

I have previously characterized the risks associated with natural-yeast fermentations thusly: (i) stuck fermentations; (ii) yeasts washed off grapes during inclement weather; (iii) spoilage yeast contamination; (iv) lengthy fermentation times; and (v) persistence of negative characteristics.  I have begun a process of exploring these perceived risks in greater detail beginning with a post on spoilage yeast contamination and continuing with a subsequent post on the risks of sluggish and stuck fermentations.  In this post I will examine the risks associated with (i) inclement weather and (ii) lengthy fermentation times.

Yeasts and Inclement Weather

Regardless of the source (bird droppings, stoamch of bees, etc.), Saccharomyces yeasts are present in very small quantities on the grapes exiting the vineyard at harvest; according to Bisson and Butzke (Diagnosis and Rectification of Stuck and Sluggish Fermentations, American Journal of Enology and Viticulture 51(2), 2000), as low as 100 viable cell/ml.  With this low initial level of Saccharomyces, it is understandable that there would be some concern that rain could separate the grape from its precious cargo and, conceptually, leave the must laying around in the tanks pining for  a long lost suitor. There are two issues with this scenario.

First, if the rain is heavy and persistent enough, the greater risk is for the development of rot and the mold and bacteria which accompany it.  These molds and bacteria can make their way into the must if care is not exercised and proliferate during the lag phase with an associated wine-spoilage risk.  Secondly, there is a much greater yeast population in the winery than there ever was on the grape at any time during its residence in the vineyard. According to Bisson (Introduction to Wine Production, Viticulture and Enology, Section 3, Lecture 11, enologyaccess.org), yeast cell population in the winery is 102 cells/ml early in the vintage and 106 cells/ml late in the harvest as cells build up on the winery equipment.  These cells can more than make up for any cells washed off the grapes during a rainstorm.

Lengthy Fermentation Times

Longer fermentation times can result from (Bisson): (i) long lag before the onset of fermentation; (ii) normal start but a slowdown during fermentation; (iii) sluggishness throughout the process; and (iv) arrested fermentation.  Natural fermentations do have longer lag times because of the growth requirements placed on the yeast populations.  For example, the maximal yeast density during fermentation is 108 cells/ml while most inoculations are 106 cells/ml.  It requires seven generations (and 24 to 35 hours) to bridge the gap (Bisson).  To this we must add 12 to 24 hours for the yeasts to adjust to the must environment.  In the case of a natural ferment, the growth requirements are more intense.  To get from 100 cells/ml to 106 cells/ml will require 13 generations and to this must be added the time to maximal yeast population.  Even if the must is colonized by winery-resident yeasts, the growth curve is still steep.

It is true that lengthier fermentation times are associated with natural yeast fermentations but the overall lengthening of the wine production process might be viewed by the natural-yeast practitioner as an essential characteristic and requirement in realizing a more complex end product.


©Wine -- Mise en abyme

The risks of sluggish and stuck fermentations when utilizing natural yeasts

I have previously characterized the risks associated with natural-yeast fermentations thusly: (i) stuck fermentations; (ii) yeasts washed off grapes during inclement weather; (iii) spoilage yeast contamination; (iv) lengthy fermentation times; and (v) persistence of negative characteristics.  I have begun a process of exploring these perceived risks in greater detail -- beginning with a recent post on spoilage yeast contamination -- and will continue on that path with the current post on the risks of sluggish and stuck fermentations.

Slow or sluggish fermentation is characterized by low sugar utilization by the attendant yeasts while incomplete (stuck) fermentations occur when a higher-than-desired level of residual sugar remains at the conclusion of alcoholic fermentation (Klaus A. Sutterlin, Fructophilic yeasts to cure stuck fermentations in alcoholic beverages, PhD dissertation, Stellenbosch University, March 2010).  Sluggish or stuck fermentations are the second most significant enological problem faced by winemakers (2003 American Vineyard Foundation survey and 1996 Association for the Development of Wine Biotechnology survey, both cited in Sutterlin 2010) and, with more than 60% of respondents  admitting to having experienced one or both of these problems, the economic costs are perceived as being enormous.

According to Bisson and Butzke (Diagnosis and Rectification of Stuck and Sluggish Fermentations, American Journal of Enology and Viticulture 51(2), 2000), there are four types of fermentations which deviate from the "normal" fermentation profile: (i) sluggish initiation with the rate eventually becoming normal; (ii) normal initiation becoming sluggish; (iii) sluggish throughout the entire process; and (iv) abrupt arrest late in the fermentation.

Bisson and Butzke report that slow fermentation initiation (Type (i) in the foregoing) can occur in both natural and inoculated fermentations and, in the case of natural fermentations, "the sluggish start may simply be due to low numbers of yeasts in the must and not reflect any particular problem other than an initial low biomass."  Based on their research, the authors aver that fermentation will go to completion, depending on juice conditions and the presence of other organisms, with as little as 100 viable Saccharomyces cells/mL present at initiation.

The risk of deficient (< 100 viable cells/mL) Saccharomyces populations leading to a problem fermentation is highest in the earliest portions of the crush.  As crush is prolonged, Saccharomyces bacteria will colonize the winery equipment such that juice and must passing through said equipment will have their levels of Saccharomyces elevated.

In the cases where there are low initial levels of Saccharomyces, holding juice at low temperatures is risky as it (i) encourages the growth of Kloeckera apiculata and (ii) is injurious to the existing Saccharomyces yeasts.  If the Saccharomyces yeasts are able to dominate, the fermentation will proceed to completion; if not, the fermentation will arrest.  It should be noted that the lower the level of the initial Saccharomyces population, the greater the growth requirements of the juice.  Bisson and Butzke estimate that it will take 13 generations to get from an initial level of 100 cells/mL to a typical innoculum level of  10⁶ cells/mL.

According to Malherbe, stuck fermentations can be caused by glucose/fructose ratio imbalance, nutritional limitations of the must (nitrogen deficiency, oxygen deficiency, mineral deficiency, vitamin deficiency), inhibitory substances (ethanol, toxic acids, the effects of sulphites, killer toxins, fungicide/pesticide residues), and a number of physical factors (excessive must clarification, temperature extremes, excessive use of Sulphur Dioxide).  When compared to this range of potential stuck-fermentation causative factors, the risk of low initial yeast population in natural fermentations does not seem that stark.  Bisson and Butzke has shown that fermentation can conclude successfully even beginning with yeast levls as low as 100 cells/mL and if the fermentation continues in a sluggish manner, or gets stuck, it is a biomass rather than a starter problem.  Further, the risk of sluggish initiation is not restricted to natural yeast fermentations.  According to Bisson and Butzke, poor starter culture can lead to sluggish initiation in the case of inoculated fermentations.

To conclude then, sluggish/stuck fermentations is an issue that a winemaker always has to be cognizant off and has to constantly monitor against.  There are many opportunities for this curse to be visited upon the winemaker and one of those cases is at the initiation of the fermentation where it will register as a sluggish start.  Such a manifestation could be apparent whether the fermentation is natural or inoculated.  All things being equal in the biomass, the natural yeast fermentation should right itself and proceed to completion.  There does not seem to be an outsized and determinative risk of stuck fermentations if one practices natural yeast fermentation.  There is an obvious lag phase however, as the yeast levels build up from cellar-entry levels to standard starter inoculate levels.

©Wine -- Mise en abyme

The risk of spoilage yeast contamination during natural yeast fermentations

In a comment on my post on indigenous- versus inoculated-yeast fermentations, reader Ben opined that three of the five disadvantages of natural fermentation that I had listed were "discountable" but that the remaining two provided the basis for an "interesting debate."  Ben's comment has prompted me to provide readers with a fuller discussion of the mentioned disadvantages beginning with the current post on spoilage yeasts as a risk in indigenous fermentations.

Absent an inoculation, all yeasts found in grape must and wine will originate from one or more of the following sources: vineyard, grapes, or winery processing equipment (E.J. Bartowsky, Bacterial spoilage of wine and approaches to minimize it, Letters in Applied Microbiology).  According to Loureiro and Malfeito-Ferreira (Spoilage yeasts in the wine industry, International Journal of Food Microbiology 86, 2003), mature, healthy grapes harbor microbial populations (yeasts, lactic and acetic acid bacteria, filamentous molds) at levels of 10³ - 10⁵ CFU/g (colony forming unit -- a measure used in microbiology that indicates the number of micro-organisms present in a water sample (www.legionella.com/cfu)), levels that vary based on environmental conditions (rainfall. temperature, grape variety, the application of chemicals in the vineyard).  Yeasts resident on grape berries tend to congregate in areas where juice might escape (Loureiro and Malfeito-Ferreira).  Wine-associated yeasts are identified in the table below.

As seen from the foregoing, yeasts exist in the vineyard environment and on healthy grapes but, for our purposes, the intersection of yeasts and damaged grapes is of significance.  Grapes can be damaged in any number of ways (hail, birds, etc.) but it is the damage caused by phytopathogenic molds that is of greatest interest.  The effects of these molds on grapes and wine are spelt out in the table below.

Loureiro and Malfeito-Ferreira found a number of ascomycetous yeasts proliferating on grapes damaged by sour rot as well as Zygosaccharomyces spp and other spoilage yeasts such as Dekkera bruxellensis.  In a separate study on sour rot, Berata et al., isolated 17 ascomycetous species from sour-rot-damaged samples and only five from sound grapes.  The most significant find was the presence of Zygosaccharomyces bailii, a species which the authors describe as "acidophilic" and "one of the most dangerous wine spoilage yeasts."  Once introduced, this species was recovered from all of the alcoholic fermentation steps.  The authors conclude that "yeast species from sour rot grapes are an important contamination source of wineries and wines."  Loureiro and Malfeito-Ferreira make much the same argument: "For many of the most important wine spoilage species (Dekkera/Brettanomyces), the main entry to the winery is grapes affected by sour rot."  The consensus, then, is that sour rot is the primary vehicle for wine spoilage yeasts transiting from the vineyard to the winery.  Let us now turn our eyes to the winery environment.

According to Woolford et al., (Genome Survey Sequencing of the Wine Spoilage Yeast Dekkera (Brettanomyces) bruxellensis, Eukaryotic Cell 6(4), April 2007), Brettanomyces bruxellensis is a major microbial cause of wine spoilage worldwide and results in significant economic loss.  The yeast makes "the winery itself a primary habitat surviving in the walls ... interior surfaces of presses and fermentation tanks, or on the wood of barrels."  From these positions the microbe is well situated to "colonize the fermenting must or maturing wine."  Wines infected with Brettanomyces will exhibit aromas of mousiness, wet wool, burnt plastic, horse sweat, or barnyard.  According to the same authors, the severity and frequency of Brettanomyces has been on the uptick as winemaking has trended towads wines with higher levels of residual sugar and that are unsulfited, unfiltered, and aged on lees.

Brettanomyces is exceptionally dangerous because it has all of the characteristics of Saccharomyces cerevisiae (ethanol-tolerant, facultatively anaeorobic, can exist without mitochondrial DNA, ferments preferentially in the presence of high glucose under aerobic conditions) but extend beyond it in that, while slower growing, "it can assimilate a wider variety of carbon choices."  These conditions lead to the following progression in a Brettanomyces-contaminated alcohol fermentation (Woolford et al.):

• Saccharomyces cerevisiae dominates throughout primary fermentation then is replaced by D. bruxellensis during the maturation phase when ethanol concentration is high and minimal amounts of sugar remain.

It was long thought that Brettanomyces contamination was a result of poor hygiene in wineries but contamination persists even in the face of intensive hygiene efforts on their parts (Renouf et al., Interactions between Brettanomyces and other yeast species during the initial stages of winemaking, Journal of Applied Microbiology 100 (6), June 2006).  The research cited in this post seems to indicate that Brettanomyces can enter the winery through sour rot  and can then take up residence within the facility and contaminate batches of wine essentially at will.  Intensive hygiene efforts can clean up an infected location but re-contamination is potentially just another sour-rot affected batch away.  Vigilance in screening for sour rot would seem to be in the best interest of the winery.

Grape must is nutrient-rich and ethanol poor, a candy store for most micro-organisms.  The longer  those conditions exist, the more non-Saccharomyces yeasts will thrive. With the exception of Brettanomyces, most wine-spoilage yeasts are ethanol-intolerant so the quicker that high ethanol levels are obtained, the less opportuntiy for these micro-organisms to proliferate.  The issue with natural fermentations is that they start slowly and take longer to get up to levels in which S. cerevisiae thrives.  And it is in that period that the fermented wine becomes susceptible to the travails of wine-spoilage yeasts.

The remaining disadvantages of natural wine fermentation will be coverd in a number of upcoming posts.

©Wine -- Mise en abyme

Indigenous- versus inoculated-yeast fermentation: The pros and the cons

There is an ongoing battle between natural-wine proponents and pragmatists as to the types of yeast strains that provide the "best" results in the alcoholic fermentation of wine grapes, a battle, according to Isak Pretorius (The Power of Yeast, TONG #12) that is far from new.  According to Pretorius, once Louis Pasteur was able to show that some wild yeasts could spoil wine, the debate began as to whether pasteurization or the addition of sulphur dioxide should be utilized to kill off the spoilage agents or whether inoculated ferments should should be used in lieu of indigenous ferments.  This post looks at both sides of this continuing argument.

As described in a previous post, wine is the result of applying yeasts to grape berries/must/juice in an anerobic environment in order to convert the resident sugars into alcohol.  The yeast that receives most of the credit -- and does most of the work -- is a species called Saccharomyces cerevisiae (SC) which is "specialized in metabolizing media with high sugar content and small quantities of nitrogenous compounds" (Suárez-Lepe and A. Marota, New trends in yeast selection for winemaking, Trends in Food Science and Technology 23 (2012), 39-50.).  According to Fugelsang (Overview of yeast selection and malolactic fermentation on aroma, flavor and phenols), the yeasts (i) extract compounds from the solids in the must/juice in order to form the "characteristic metabolites of fermentation (alcohols, esters, fatty acids, carbonyls, etc.) and (ii) cleave cysteine-containing precursors such that volatile thiols (aroma component of several varieties) can be released.  SC is the yeast species which completes the alcoholic fermentation process in both inoculated and spontaneous ferments.

Grapes in a vineyard are hosts to what Gourrand (Using non-Saccharomyces yeasts during alcoholic fermentations: taking advantage of yeast biodiversity) calls native microflora -- molds, lactic bacteria, acetic bacteria, Saccharomyces spp, and non-Saccharomyces yeasts (Pichia, Metchnikowia, Kloeckera, Kluyveromyces, Candida, Zygosaccharomyces, Torulaspora, Cryptoccus, Brettanomyces, and Hanseniaspora) -- and it is the yeast element of this microflora that the feral-yeast winemaking adherents seek to exploit.  Wild yeasts accumulate on the grapes from flowering through harvest with the presence of SC being pegged at 1 in 1000 grapes (Robert Mortimer, Vineyard Theory of Wild Yeast, UC Berkeley).  At harvest, SC is the least prevalent of the grape-resident yeast strains.

In the case of indigenous (indigenous, wild, feral, and spontaneous used interchangeably throughout this post) yeast fermentation, the process is kick-started and dominated initially by the "weakly fermentative" -- but numerically dominant -- non-Saccharomyces Kloeckera.  This initiation can take up to a week to begin due to the relatively small amount of wild yeasts present at startup (relative to the amount of yeast used to begin the process in the case of inoculated ferments).  For the first few days of fermentation, the weakly fermentative non-SC population dominates but is then replaced by more adaptive non-SC strains.  As the alcohol level continues to rise, the more alcohol-tolerant SC increases in number at a rapid rate such that at the end of the fermentation it is the only species left standing.

Natural wine adherents assert that the progression from non-SC to SC fermentation in the vessel is an integral part of non-interventionist winemaking and adds complexity to the finished wine (Mortimer; Pretorius).  Critics of the approach see it as akin to Russian roulette because of the inherent risks (Ross; Pretorius): (i) the irregularity of natural fermentation and the associated risk of a stuck fermentation; (ii) in the event of rains around harvest time, the wild yeasts could be washed off the grapes; (iii) spoilage yeasts are often present in grape-derived yeasts; (iv) spontaneous ferments take longer to begin and longer to complete; and (v) while the positive characteristics of natural yeasts are not detectable after 6 or so months of aging, the negative characteristics tend to persist much longer.

For inoculated ferments, a large dose of SC is added to the juice/must in order to initiate fermentation.  The yeast strains utilized have traditionally been selected on the basis of the ability to start the fermentation quickly, the toleration of increasing alcohol levels, low acetic acid production, and resistance to sulfur dioxide (Ross; Suárez-Lepe and A. Marota).

As both Ross and Pretorius point out, the needs of large- and small-production wineries may lead to different emphasis in yeast-strain selection.  For the large producer, effective, efficient production and maintenance of quality is key and a strain that meets that need will be selected.  The smaller producer, on the other hand, is more likely to take advantage of varying yeast strains and temperature regimes as a means of enhancing the wine's aromatic and flavor characteristics.

To gain the benefits associated with both spontaneous and inoculated ferments, some winemakers are employing cocktails of strains hoping to get the "complexity of flavors ... without running the risk of contamination of spoilage yeasts" that comes along with the spontaneity.

According to Fugelsang, the first commercial yeast strain was introduced in 1965 by Red Star Yeast and, since that time, over 100 cultures have been commercially produced.  And winemakers continue to take advantage of these commercial strains in order to improve the capabilities of their wines. According to Suárez-Lepe and A. Marota and Pretorius, winemakers are continually on the lookout for yeast strains that can improve the technological and sensorial properties of their wines.

The advantages that are perceived by "inoculants" are clear: (i) quick, effective, efficient fermentations: (ii) flexibility; (iii) lower risk production process; (iv) the ability to tailor the fermentation; and (v) the ability to take advantage of future advancements in commercially produced strains.  The disadvantage of the use of inoculation is, as perceived by the "naturalists," even more power placed into the hands of the winemaker to manipulate the dickens out of the wine; and the customer loses as a result.

In a future post I will treat the topic of trends in yeast selection.

©Wine -- Mise en abyme

Champagne: The traditional method

Champagne is one of the world's most celebrated wines, the beverage that is, almost universally, tied to adult rite-of-passage events.  The production of Champagne is among the most complex wine production processes and the quality of the product is a testament to the art of blending as practiced by the Chefs de Cave of the prominent Champagne Houses.  This post covers the traditional method for the production of Champagne.  While this method is utilized in the construction of many of the world's sparkling wines, they lack one of the key Champagne critical success factors; grapes grown on the soil of the Champagne region.

The first "blending" decision that is made by a Champagne House is the mix of grapes that will be included in the cuvée or vintage for a specific year.  In my recent post on the terroir and viticulture of Champagne, I pointed out that the Houses only own 10% of all grapes grown in the region.  They have access, however,  to fruit from a broad range of cru vineyards in the major districts and the decision as to which variety to acquire from which cru in which district will have an impact on the style of wine that is produced by that House in that season.

The steps associated with the traditional method follow.

Picking

The starting date for picking the grapes is set by the CIVC (the organization that "coordinates the common interests of wine growers and producers in Champagne") which bases its decision on input from the ripening observation network which was initially established in 1956.  This network allows input variables from 450 control plots to be analyzed and the grapes tracked for ripeness by cru and variety.  Decisions on picking dates, quantities, and alcohol levels are a direct result of this analysis.

Over 100,000 pickers are involved in harvesting the ripe grapes and moving them from the field to one of the 1900 pressing stations that are located throughout the region.  The grapes are picked in clusters and then placed gently into waiting plastic bins.  Picking normally begins in the cool of dawn in order to preserve as much of the grapes' freshness as possible.  Chardonnay is generally picked one or two weeks later than Pinot Noir and Pinot Meunier.

Pressing

The grapes are weighed at the pressing center and relevant information recorded in the pressing logbook.  The grapes are then pressed as whole bunches in a process that is called fractionated winemaking.  In this process, the free-run juice is drawn-off first in three successive pressings.  The product of these pressings is called the cuvée and the middle of the three is called the coeur de  cuvée (heart of the cuvee) and is said to possess an ideal balance of purity and structure.  The maximum amount of juice that can be harvested during the cuvée pressing is 20.5 hl.

The second component of this fractionated winemaking is the heavier press called the taille.  In this stage the juice is harvested in two or three high-force presses that occur subsequent to the removal of the cuvée.  The juice collected at this point is darker due to the impurities extracted from the grape skins.  A total of 5 hl of juice can be legally harvested at this stage.

The cuvée and taille have similar levels of sugar but the cuvée has higher levels of malic and tartaric acids while the taille has higher levels of oxidants, minerals, and pigments.  A total of 25.5 hl of juice can be legally harvested from a 4000 kg marc of fruit.

Addition of Sulphites

The fruit extracted during the press flows into open tanks which are separated by cru, variety, and pressing (If the intent is to make Rosé Champagne via maceration, then the juice stays in contact with destemmed black-skinned grapes  for 24 to 72 hours until the desired color is obtained.).  Sulphites are added to the juice at between 6 and 10 g/hl in order to combat mold and bacteria and reduce the risk of flavor-killing oxidation.

First Racking (Débourbage)

Impurities are removed from the juice through a process called débourbage where the solids fall to the bottom of the tanks while the clear juice is drawn off from the top.

Alcoholic Fermentation

The resulting clear juice is transported to the vat room for alcoholic fermentation.  Today most fermentation is carried out in stainless steel tanks, a change from the prior norm of oak fermentation  (Oak seems to be making a comeback and I will cover its use in Champagne in a future post.).  The juice is chaptalized as necessary to bring it up to 11% potential alcohol after which yeast is added to initiate alcoholic fermentation. Fermentation runs between 10 and 14 days before all the sugar in solution is converted to alcohol and carbon dioxide.  The carbon dioxide produced at this phase is allowed to escape.

Malolactic fermentation is initiated if is a part of the house style.

Cold Stabilization and Clarification

The base wine is cold stabilized to prevent tartrate precipitation later in the life of the wine.  The wine is then racked off the solids and clarified further through fining and/or filtering.

Blending

The next step in the process sets Champagne apart from other sparkling wines and sets the Champagne Houses apart from Grower-Producers.  In order to produce Champagne that aligns with the House style, the Chef de Cave has to memorize and blend wines from a broad array of crus from the current vintage plus wines from the reserve as necessary.  The Grower, on the other hand, is working with a smaller geographic area and a far smaller number of vintages as the base wines for his/her blend.  In the case of a vintage Champagne, the blend can only contain wines sourced from grapes that have been harvested and fermented in the vintage year.

In-Bottle Fermentation

The blended wines are placed into Champagne bottles to which liqueur de tirage (a solution of wine, sugar, and yeast) is added and then the bottle is capped with a crown cork seal.  This addition precipitates a second fermentation, this time in the bottle.  As the bottle is capped, the carbon dioxide created during fermentation cannot escape and the bubbles formed as a result is absorbed into the liquid.  The process by which these bubbles are formed is called prise de mousse and the longer the period, the more refined the bubbles.

Aging

After the sugar has been exhausted, the yeasts die.  The breakdown of the dead yeast cells by enzymes -- autolysis -- adds complexity to the aroma, flavors, and mouthfeel of the Champagne if residency is maintained.  Champagne is legally required to remain on the lees for > 16 months if a non-vintage and > 3 years if designated as vintage.  Quality houses normally age their non-vintage wines for 3 to 4 years and their vintage wines for 7 to 8.

Remuage and Disgorgement

When the house deems that the Champagne has spent enough time on the lees, steps are taken to remove said lees from the bottle.  This is a two-step process with the first step (remuage) designed to move the sediment from the body of the bottle and into the neck and the second step (dégorgement) designed to expel the sediment from the bottle.  In the first step, the bottle is moved slowly from a  horizontal to a vertical, neck-down position, while simultaneously turning it a few degrees at a time to dislodge the sediment from the walls.  This process had historically been done by hand but is now done by a machine (gyropalette) which has resulted in a dramatic reduction in transit time and a marked increase in throughput volume.  Large format bottles are stilled "riddled" by hand.

In the disgorgement phase the bottle is passed neck-down through a freezing brine solution which causes the freezing of the sediment-containing liquid in the neck of the bottle.  Removal of the crown cork seal will cause the pressure in the bottle (6 atmospheres) to forcefully expel the frozen material from the neck.

Liqueur de Dosage

A mixture of base wine and sugar (liqueur de dosage) is added to the Champagne bottle in order to top it up and to attain the desired sweetness level, measured in g/l:

• Zero dosage -- 0 to < 3 g/l
• Extra brut -- 3 to < 6 g/l
• Brut -- 6 to < 12 g/l
• Extra dry -- 12 to 17 g/l
• Sec -- 17 to 32 g/l
• Demi-sec -- 32 - 50 g/l
• Doux -- > 50 g/l

The bottle is plugged with a standard Champagne cork and a steel cage placed around the neck and over the cork to hold it in place.  The bottle is shaken vigorously and left to sit for 6 months to ensure full integration of the liqueur de dosage into the wine.

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The above described method produced 322 million bottles in 2011of which 69% (222 million bottles) were produced by the Champagne Houses with the remaining 31% produced by Growers.  Fifty-six percent of the Champagne produced was consumed in France with the remainder being shipped abroad to the United Kingdom, United States, and Germany among others.  This export market is dominated by the Champagne Houses as only 13% of Grower Champagne is exported.  The Champagne House-Grower split is more evenly balanced within France with 55% of consumption being sourced from the Champagne Houses.

© Wine -- Mise en abyme