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

Non-Saccharomyces yeasts as antidotes to climate change: Fundamentals

In my post on the role of alcohol in wine balance I indicated that, in addition to unbalancing the wine, an excess of alcohol: makes the wine appear hot; will lead to a reduced perception of wine aroma; and can impart a sense of intoxication to the taster.  The winemaker may address the issue of excess alcohol by (i) reducing the concentration of sugar present in grapes or (ii) by removing "excess" alcohol from the wine.

Reducing grape sugar requires harvesting the grapes earlier than normal but this risks affecting wine composition and quality due to fewer aroma flavors, less color intensity, non-attainment of phenolic ripeness, and increased acidity. I have dealt with removing alcohol from the wine in a previous post and technologies/approaches employed include: (i) reverse osmosis, (ii) the spinning cone, and (iii) adding water to the wine. Reverse osmosis and the spinning cone are authorized in the U.S by wine regulation 27 CFR 24.248 Processes Authorized for the Treatment of Wine, Juice, and Distilling Materials.  Under this regulation the processes must be conducted at a Distilled Spirits Plant (DSP) or at a bonded winery that is authorized to alternate between a DSP and a bonded winery.

An additional alcohol-reducing approach has come to my attention by way of a recent Elin McCoy article which discusses how some Oregon producers were turning to non-Saccharomyces yeasts to aid in countering potential climate effects on the region's Pinot Noir wines. According to the article, wines made with the Pinot Noir grape, a famously finnicky variety, could become increasingly unbalanced in the future as the effects of climate change bring riper grapes (with higher potential alcohol) into the cellar.

Source: wineeconomist.com

According to Elin, Chapter 24, an Oregon-based producer was partnering with a professor at MIT to study the potential of non-Saccharomyces yeasts to reduce the alcohol content during fermentation without negatively affecting the aroma and flavor profile of the wines. I explore the science behind this effort in this series but begin with some background material on fermentation, Saccharomyces cerevisiae(SC), and non-Saccharomyces (non-SC) yeasts.

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 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 dominant population.

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).

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.

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.

©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

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