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Understanding Difficult Malolactic Fermentations

By Dr. Molly Kelly, Enology Extension Educator, Department of Food Science

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As harvest comes to a close we have planned which wines will be going through malolactic fermentation (MLF). This article provides some information to assist you in dealing with a potentially difficult MLF.

Malolactic fermentation (MLF) is a process of chemical change in wine in which L-malic acid is converted to L-lactic acid and carbon dioxide. This process is normally conducted by lactic acid bacteria (LAB) including Oenococcus oeni, Lactobacillus spp. and Pediococcus spp. O.oeni is the organism typically used to conduct MLF due to its tolerance to low pH, high ethanol and SO2. Most commercial strains are designed to produce favorable flavor profiles.

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Although inoculation with a commercial starter is recommended, MLF may occur spontaneously. The lag phase associated with spontaneous MLF may increase the risk of spoilage organisms as well as the production of volatile acidity. Inoculation with a LAB culture can help avoid these problems by providing the cell population needed to successfully conduct MLF (more than 2×106 cells/mL). The compatibility of yeast and LAB should be taken into account since failed MLF may be due to incompatibility between these two organisms.

The key to a successful MLF is to manage the process and to monitor the progress. Although there has been extensive research on the MLF process, it may still be difficult to initiate at times. The possible causes of difficult MLF have been studied less extensively than those of stuck/sluggish alcoholic fermentation. In this article, factors that may influence the start and successful completion of MLF will be discussed.

The main chemical properties that influence MLF are well known: pH, temperature, ethanol and SO2 concentration. A study by Vaillant et al (1995) investigating the effects of 11 physico-chemical parameters, identified ethanol, pH and SO2 as having the greatest inhibitory effect on the growth of LAB in wine.

pH

Generally, LAB prefer increased pH’s and usually, minimal growth occurs at pH 3.0. Under winemaking conditions, pH’s above 3.2 are advised. The pH will determine the dominant species of LAB in the must or wine.  At a low pH (3.2 to 3.4) O. oeni is the most abundant LAB species, while at higher pH (3.5 to 4.0), Lactobacillus and Pediococcus will out-number Oenococcus.

Temperature

MLF is generally inhibited by low temperatures. Research demonstrates that MLF occurs faster at temperatures of 200 C (68˚F) and above versus 150C (59˚F) and below. In the absence of SO2 the optimum temperature range for MLF is 23-250C (73.4˚F-77˚F) with maximum malic acid conversion taking place at 20-250C (68˚F-77˚F). However, with increasing SO2 levels, these temperatures decrease and 200C (68˚F) may be more acceptable.

Ethanol

LAB are ethanol-sensitive with slow or no growth occurring at approximately 13.5%. Commercial O. oeni strains are preferred starter cultures due to tolerance to ethanol.  The fatty acid composition of the cell membrane of LAB can be impacted by ethanol content.

Sulfur dioxide

LAB may be inhibited by the SO2 produced by yeast during alcoholic fermentation. A total SO2 concentration of more than 50 ppm generally limits LAB growth, especially at lower pH where a larger portion of SO2 is in the antimicrobial form. Generally, it is not recommended to add SO2 after alcoholic fermentation if MLF is desired.

Some of the lesser known factors impacting MLF are discussed below.

Fatty Acids

MLF can be inhibited by medium chain fatty acids (octanoic and decanoic acids) produced by yeast. It is difficult to finish MLF when octanoic acid content is over 25 mg/L and/or decanoic acid is over 5 mg/L. Bacterial strains that tolerate high concentrations of octanoic and decanoic acids may be important in successful MLF. It is important to check your supplier regarding strain specifications. Yeast hulls may be added before the bacteria are inoculated (0.2g/L) to bind fatty acids. Yeast hulls may also supply unsaturated fatty acids, amino acids and assist with CO2 release.

Fungicide residues

Some fungicide and pesticide residues may negatively impact malolactic bacteria. Residues of systemic pesticides used in humid years to control botrytis can be most detrimental. Care should be taken in harvest years with high incidence of botrytis. Winegrowers should be familiar with sprays used on incoming fruit and also adhere to pre-harvest intervals.

Lees compaction

Lees found at the bottom of a tank can become compacted due to hydrostatic pressure, resulting in yeast, bacteria and nutrients being confined to the point that they cannot function properly. Larger tank sizes may contribute to increased delays in the start of MLF. This inhibition of the start of MLF can be remedied by pumping over either on the day of inoculation or on the second day after inoculation of the bacteria.

Alternatively, contact with yeast lees can have a stimulating effect on MLF. Yeast autolysis releases amino acids and vitamins which may serve as nutrients for LAB. Yeast polysaccharides may also detoxify the medium by adsorbing inhibitory compounds. A general recommendation is to stir lees at least weekly to keep LAB and nutrients in suspension.

Residual lysozyme

Residual levels of lysozyme may impact MLF. Follow the supplier’s recommendations regarding the required time delay between lysozyme additions and the inoculation of the commercial MLF culture. Strains of O. oeni are more sensitive to the effects of lysozyme compared to strains of Lactobacillus or Pediococcus.

Malic acid concentration

Malic acid concentrations vary between grape cultivars and may also differ from year to year in the same grape cultivar. MLF becomes increasingly difficult in wines with levels of malic acid below 0.8g/L. In this case a ML starter culture with high malate permease activity or a short activation protocol is recommended. Check with your supplier to ensure that the chosen strain has these attributes if needed.

Wines with levels above 5 g/L malic acid may start MLF, but may not go to completion. This may be due to inhibition of the bacteria by increasing concentrations of L-lactic acid derived from the MLF itself.

Nutrients

Difficult MLF can result from insufficient nutrients necessary for LAB growth. Since yeast can reduce available nutrients for LAB, time of inoculation is important to avoid competition for nutrients. The addition of nutrients when inoculating for MLF is especially important if the must and wine has low nutrient status or if yeast strains with high nutritional requirements are used. The addition of bacterial nutrients can help ensure a rapid start and successful completion of MLF.

Research demonstrates that the longer it takes to initiate MLF, there is a greater risk for Brettanomyces growth. Some inoculate during alcoholic fermentation (AF) to avoid this problem. Co-inoculation involves adding malolactic starter 24 hours after AF starts. By controlling microbial populations, the growth of spoilage organisms such as Brettanomyces may be inhibited.

Note that inorganic nitrogen (diammonium phosphate) cannot be used by LAB. Check with your supplier for the optimum nutrient product for your particular MLF needs.

Oxygen

Malolactic bacteria are sensitive to excessive amounts of oxygen. The bacteria should not be exposed to large amounts of oxygen after AF is complete. Micro-oxygenation may have a positive impact on the completion of MLF. This impact may be due to the gentle stirring associated with micro-oxygenation that keeps LAB and nutrients in suspension rather than the exposure to oxygen itself.

Tannins

Some red grape cultivars may have difficulty completing a successful MLF. Some varieties that may experience increased MLF problems include Merlot, Tannat and Zinfandel. This may be related to certain grape tannins negatively impacting the growth and survival of LAB.

Polyphenols can have either stimulatory or inhibitory effects on the growth of wine LAB. This effect depends on the type and concentration of polyphenols as well as on the LAB strain. The tannin fraction of wine tends to complex with other compounds, minimizing their inhibitory effects on MLF. However, in wines that contain a large amount of condensed tannins only, LAB are increasingly inhibited.

MLF nutrients containing polysaccharides have been shown to minimize this effect. This may be due to interactions between the polysaccharides and tannins.

Conclusions

MLF difficulties are usually due to a combination of factors. A stuck or sluggish MLF is usually not the result of one factor alone. It is important, therefore, to both understand and manage the MLF process at each step of the winemaking process. Proper measurement of the process is also vital to be aware when MLF is not proceeding as desired.

 

References

Bousbouras, G.E. & Kunkee, R.E., 1971. Effect of pH on malolactic fermentation in wine. Am. J. Enol. Vitic. 22, 121-126.

Britz, T.J. & Tracey, R.P., 1990. The combination effect of pH, SO2, ethanol and temperature on the growth of Leuconostoc oenos. J. Appl. Bacteriol. 68, 23-3 1.

Costello, P.J., Morrison, R.H., Lee, R.H. & Fleet, G.H., 1983. Numbers and species of lactic acid bacteria in wines during vinification. Food Technol. Aust. 35, 14-18.

Davis, C.R., Wibowo, D., Eschenbruch, R., Lee, T.H. & Fleet, G.H., 1985. Practical implications of malolactic fermentation: a review. Am. J. Enol. Vitic. 36, 290-301.

Henick-Kling, T. & Park, Y.H., 1994. Considerations for the use of yeast and bacterial starter cultures: SO2 and timing of inoculation. Am. J. Enol. Vitic. 45, 464-469.

Henick-Kling, T., 1995. Control of malo-lactic fermentation in wine: energetics, flavour modification and methods of starter culture preparation. J. Appl. Bacteriol. Symp. (suppl) 79, 29S-37S.

Henschke, P.A., 1993. An overview of malolactic fermentation research. Wine Ind. J. 2, 69-79.

Ingram, L.O. & Butke, T.M., 1984. Effects of alcohols on micro-organisms. Adv. Microbiol. Physiol. 25, 254-290.

Krieger, 5., 1993. The use of active dry malolactic starter cultures. Austral. New Zealand Wine md. J. 8, 56-62.

Kreiger-Weber, S. and P. Loubser. 2010. Malolactic fermentation in wine. In Winemaking Problems Solved. C.E. Butzke (ed), pp. 88-89.Woodhead Publishing Limited, Cambridge, UK.

Kreiger-Weber, S., A. Silvano and P. Loubser. 2015. Environmental factors affecting malolactic fermentation. In Malolactic Fermentation-Importance of Wine Lactic Acid Bacteria. In Winemaking. R. Morenzoni and K. Specht (eds), pp.131-145. Lallemand Inc., Montreal, Canada.

Kunkee, R.E., 1967. Malo-lactic fermentation. Adv. Appl. Microbiol. 9, 235-279.

Lafon-Lafourcade, S., Carre, E. & Ribereau-Gayon, P., 1983. Occurrence of lactic acid bacteria during the different stages of vinification and conservation of wines. Appl. Environ. Microbiol. 46, 874-880.

Lonvaud-Funel, A. 2001. Interactions between lactic acid bacteria of wine and phenolic compounds. Nutritional aspects II, synergy between yeast and bacteria, Lallemand Technical Meeting, Perugia, Italy.

Loubser, P.A. 2004. Familiarise yourself with malolactic fermentation. Wynboer Technical Yearbook (a Wineland publication). 5:32-33.

Loubser, P., 2005. Bacterial nutrition – essential for successful malolactic fermentation. Wynboer technical yearbook 2005/2006, pp.95-96.

Malherbe, S., F.F. Bauer and M. du Toit. 2007. Understanding problem fermentations-a review. S. Afr. J. Enol. Vitic. 28(2):169-186. Nel, H.A., Moes, C.J. & Dicks, L.M.T., 2001. Sluggish/stuck malolactic fermentation in Chardonnay: possible causes. Wineland Magazine, Wynboer vol. 144, July, pp.1 13-115.

Nielsen, J.C., Pilatte, E. & Prahl, C., 1996. Maitrise de la fermentation malolactique par l’ensemencement direct du yin. Revue Francaise d’Oenologie 160, 12-15.

Nygaard, M. & Prahl, C., 1996. Compatibility between strains of Saccharomyces cerevisiae and Leuconostoc oenos as an important factor for successful malolactic fermentation. Proc. 4 0, Int. Symp. Cool Climate Vitic. Enol., Rochester, NY.

Renouf, V. and M.L. Murat. 2008. L’utilisation de levains malolactiques pour une meilleure maitrise du risqué Brettanomyces. Rev Enol. 126:11-15.

Renouf, V., S. La Guerche, V. Moine and M. Murat. 2009. Techniques for dealing with awkward malolactic fermentations. Wineland Magazine. pp. 82-85.

Vaillant, H., Formisyn, P. & Gerbaux, V., 1995. Malolactic fermentation of wine: study of the influence of some physico-chemical factors by experimental design assays. J. Appl. Bacteriol. 79, 640-650.

Wibowo, D., Eschenbruch, R., Davis, CR., Fleet, G.H. & Lee, T.H., 1985. Occurrence and growth of lactic acid bacteria in wine: a review. Am. J. Enol. Vitic. 36, 301-313.

Zoecklein, B. 2011. Fermentation considerations for the 2011 season. Enology Notes #159. As found on the Wine/Enology Grape Chemistry website

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The Cellar after Harvest’s Dust Settles

By: Denise M. Gardner, Enology Extension Associate

Most likely, all of the wines from the 2016 vintage are happily settling away in tank or barrel at this point.  After such a busy time, his leaves winemakers with that tricky question, “What do I do now?”

Monitoring Malolactic Fermentation (MLF)

Now is a good time to make sure you are monitoring your malolactic fermentations.  Ensure all of your barrels or tanks have been appropriately inoculated, or have started naturally, and get some initial readings on the malic acid concentration.

If you have a spectrophotometer, you can purchase enzymatic kits to measure the concentration of malic acid in your wine over time.  Wines with less than 30 mg/100 mL of malic acid are considered “dry” for MLF or MLF-stable for bottling.

However, winemakers can also monitor malic acid degradation through the use of paper chromatography kits.  These kits are easy enough for home winemakers to use and can also be applied at the commercial level.

MLF paper chromatogram. This image shows the paper after it has dried, where the spots are pertaining to the acid standards and the acid separation for a wine sample. Wine samples above have not completed MLF due to the fact there is a noticeable dot of malic acid in each sample.  Photo by: Denise M. Gardner

MLF paper chromatogram. This image shows the paper after it has dried, where the spots are pertaining to the acid standards and the acid separation for a wine sample. Wine samples above have not completed MLF due to the fact there is a noticeable dot of malic acid in each sample. Photo by: Denise M. Gardner

Paper chromatography works by separating tartaric, malic, and lactic acids from a wine sample (Figure 1).  In addition to blotting small drops of your various wine samples, each paper must also contain 3 standards to show the spots documented by the three acids (tartaric, malic, and lactic).  While paper chromatography is not the best at concentrating how much of each acid remains in the wine, you can get an idea when the bulk of malic acid is converted to lactic acid (i.e., MLF is completed) when the malic acid spot associated with the wine samples disappears.

For a complete protocol on paper chromatography, please see this page: https://www.midwestsupplies.com/media/downloads/421/malolactic_chromatography_kit.pdf

Checking Wines for Off-Flavor Development

It’s also a good time to check wines for hydrogen sulfide (H2S) or sulfur-base off-odor aromas, and volatile acidity (VA), especially for wines that you will want to bottle early in the new year.

Hydrogen sulfide can be treated with copper sulfate.  Penn State Extension offers a great 2-page fact sheet on how to run a copper screen to determine if the wine requires copper sulfate, and a copper bench trial in order for you to assess how much copper is needed to treat the hydrogen sulfide: http://extension.psu.edu/food/enology/wine-production/wine-made-easy-fact-sheets/sulfur-based-off-flavors-in-wine

Now is also a good time to know what the VA is in your wines, especially those that will be seeing some aging.  This is incredibly important to get a baseline value of the VA.  That way, if a problem emerges in the future, you will have an indication how much the volatile acidity has increased.   Penn State Extension also offers a 2-page fact sheet explaining why knowing volatile acidity is important, provides protocols for its analysis, and how to mediate high VA situations: http://extension.psu.edu/food/enology/wine-production/wine-made-easy-fact-sheets/volatile-acidity-in-wine

If you are having problems identifying these key defects in your wine, don’t forget that the annual “Wine Quality Improvement” Short Course is just around the corner in January.  For more information on this workshop and its registration, please go here: 2017 Wine Quality Improvement (WQI) Short Course

Cellar Maintenance

Now is also a good time to clean up any leftover sore spots from the chaotic harvest season:

  • Clean up places in the cellar that have gotten dirty or have become areas that are accumulating materials that should otherwise be put away.
  • Manage all of your harvest records.  Make sure all of the wines have the basic wine chemistries (e.g., pH, TA, residual sugar, alcohol, free and total SO2, malic acid, and volatile acidity) in the record book.  It is easy to forget all of these details as time progresses.
Running basic chemical analysis on your wines and updating records is an essential component of making quality wine. Photo by: Denise M. Gardner

Running basic chemical analysis on your wines and updating records is an essential component of making quality wine. Photo by: Denise M. Gardner

Reflections: Winemaking at Penn State

By: Denise M. Gardner

I can officially say that I have now been involved with 5 harvests here at Penn State, with my first harvest in 2011.  Returning to Pennsylvania from California in 2011 could not have been a greater challenge to an incoming newbie, and I think it will forever be one of the most difficult vintages I have had the experience to deal with to date.  Not only did I manage to lose an entire lot of finished wine down the drain (long story…), but I recognized the need to bring PA-produced research wines to Pennsylvania’s growing wine industry during a daunting season from a weather perspective.  Additionally, I saw an opportunity to educate students on how to make wine while they helped me process fruit from the research vineyards.  In that first year, 5 lucky college seniors helped me process about 8 different varieties from the NE-1020 “multi-state evaluation of wine grape cultivars and clones” project, which was being financially supported by a multi-state SCRI grant.

Looking back today, I now see that the 2011 vintage provided me with a starting point to work with students and a series of winemaking lessons for future vintages that I continue to recall even today.

I was actually one of those bright-eyed students back in my younger days.  I stumbled upon Penn State Extension and Mark Chien by pestering local Extension educators on how to grow grapevines.  I still recall the many opportunities Mark, specifically, provided for me despite my age or lack of wine knowledge.  Mark taught me how to plant a vineyard, from site selection to digging holes for a trellis, how to monitor vine growth through proper pruning techniques, how to ferment grapes into wine, and the various stages involved in production that went beyond the basic texts on how to make wine.  I connected with industry members and was awarded an experience to intern at Lallemand in Toulouse, France before I reached my freshman year in college.

Figure 1: Extension Enologist, Denise Gardner, developed an interest in wine grape growing and production throughout high school.  Photos, from left to right, include an annual fermentation lesson during a high school agriculture class, building a trellis system at the local high school, grape vines after 2 years of growth at the high school vineyard, and a lesson from past Extension Viticulturist, Mark Chien, on how to properly prune grapevines at a PA vineyard.  Photos provided by: Denise M. Gardner

Figure 1: Extension Enologist, Denise Gardner, developed an interest in wine grape growing and production throughout high school. Photos, from left to right, include an annual fermentation lesson during a high school agriculture class, building a trellis system at the local high school, grape vines after 2 years of growth at the high school vineyard, and a lesson from past Extension Viticulturist, Mark Chien, on how to properly prune grapevines at a PA vineyard. Photos provided by: Denise M. Gardner

When I arrived to Penn State in 2011, I had a memory of the opportunities Extension awarded me and a goal of working with students that may have an interest in wine production.  I still laugh when I recall a number of students that experienced a harvest at a local winery, only to tell me it was “the hardest thing they have ever had to do.”

While many may not make the connection, food science offers an incredible foundation of knowledge that is beneficial for winemakers and those whom wish to go into fermented beverage production.  Students engage in a series of classes to develop a foundation in chemistry, microbiology, and biotechnology.  Additionally, they learn important processing parameters that are affiliated with winemaking: sanitation, quality control practices, safety when processing, proper sampling techniques, and experimental practices to improve food/beverage products.

For that reason, the annual harvest and production of wine with use of undergraduate and graduate students’ support has blossomed into many positive ventures:

  • Since 2010, Penn State Food Science and several Pennsylvania wineries have sponsored student co-ops at wineries during vintage seasons. These experiences educate students in wine production, and specifically provided venues for “real world” experiences in the wine industry.
  • Undergraduate students have embarked on undergraduate research experiences pertaining to wine research within the College of Agricultural Sciences. Several of these projects have benefited the local wine industry.
  • Graduates from Food Science have started to “harvest hop” to the southern hemisphere for winemaking and production experiences internationally. Virginia (Smith) Mitchell, head winemaker at Galer Estate Winery, traveled to Australia in the winter months of 2013 while Allie Miller will travel to New Zealand for the 2016 harvest.  These experiences bring a global perspective and education that facilitate innovative changes to the growing Pennsylvania wine industry.
  • Several students have benefited from permanent placement in the wine and fermented beverage industries upon graduation, and many have committed to Pennsylvania operations. The experience gained through research winemaking here at Penn State is invaluable and leaves them with base knowledge in wine production.
  • Many students volunteer for Extension programming, which gives them the opportunity to present research and educational experiences to industry members as well as network with potential employers (you!).
  • Graduate research has flourished. Both Dr. Ryan Elias and Dr. Michela Centinari have several graduate students that are working on applied research projects which address winemaker and grower needs reflected in previous industry needs assessments.
  • Since 2011, the number of research wines being made has more than quadrupled. Today, we have outgrown the equipment I used in 2011 and outgrown our storage capacity for research wines.  The wines produced at Penn State are annually evaluated at regional Extension events.

With such a positive focus on student development and interaction with Pennsylvania’s grape and wine industry, the 2015 vintage was expected to be our best vintage yet!

The 2015 growing season did not leave much hope for Pennsylvania grape growers and winemakers, and I can recall a series of summer meetings in which winemakers from across the state asked me if I was prepared to deal with a lack of fruit and a bunch of rot in our research winemaking curriculum.  Luckily, as Michela will reflect upon next week, the season shaped up to be one of the best I have experienced in my time here at Penn State.

For 2015, we recruited 10 interested undergraduate students for the 2015 harvest season to assist with the research harvests and wine production.   This is double the quantity of students that typically enroll in an independent study experience associated with enology.

Students participate in regular wine processing operations, which can be seen in Figures 2 – 7: crushing, pressing, monitoring fermentation, and completing wines through malolactic fermentation.  Additionally, at the end of the semester, each enrolled student presents on a wine grape variety of interest.

Many students arrive with a genuine interest in fermentation science, or would like to get more experience in food production.  Many of them leave the fall semester with future undergraduate research opportunities, internships/co-ops at wineries, or develop an expectation to graduate with permanent placement in the fermented beverage industry.

Figure 2: The undergraduate students start each fall with a review of lab analysis techniques to learn how to properly analyze juice and wine, which comes in handy during the harvest season.  Photo by: Denise M. Gardner

Figure 2: The undergraduate students start each fall with a review of lab analysis techniques to learn how to properly analyze juice and wine, which comes in handy during the harvest season. Photo by: Denise M. Gardner

Figure 3: Crush is an essential part of the independent study class and graduate student research.  Careful care is taken by the students to ensure that proper sanitation is taken, accurate yields are measured, and that treatments are adequately separated into replicate fermentations. Photos by: Denise M. Gardner

Figure 3: Crush is an essential part of the independent study class and graduate student research. Care is taken by the students to ensure that proper sanitation is utilized, accurate yields are measured, and that treatments are adequately separated into replicate fermentations. Photos by: Denise M. Gardner

Figure 4: Prepping inoculums for primary and malolactic fermentations is an important part of what the students learn how to do throughout the semester.  Here, Blair and Cara prep hydration nutrient and yeasts for inoculations. Photo by: Denise M. Gardner

Figure 4: Prepping inoculums for primary and malolactic fermentations is an important part of what the students learn how to do throughout the semester. Here, Blair and Cara prep hydration nutrient and yeasts for inoculations. Photo by: Denise M. Gardner

Figure 5: Students also learn how to properly inoculate wines for primary fermentation.  A: Marielle, Stephanie, Joe, Garrett, Gary and Blair inoculate Riesling wines; Photo by: Denise M. Gardner; B: Denise and Gary inoculate Cabernet Sauvignon musts; Photo by: Marlena Sheridan.

Figure 5: Students also learn how to properly inoculate wines for primary fermentation. A: Marielle, Stephanie, Joe, Garrett, Gary and Blair inoculate Riesling wines; Photo by: Denise M. Gardner; B: Denise and Gary inoculate Cabernet Sauvignon musts; Photo by: Marlena Sheridan.

Figure 6: Racking techniques without a pump. [From left to right] Liv, Maria, and Marielle rack Riesling juice into replicate fermentation carboys.  Photo by: Denise M. Gardner

Figure 6: Racking techniques without a pump. [From left to right] Liv, Maria, and Marielle rack Riesling juice into replicate fermentation carboys. Photo by: Denise M. Gardner

Figure 7: Pressing [white/rosé] juice or finished [red] wine is always an experience. A: Gary, George, and Garrett press rosé to prepare for overnight settling, B: Stephanie loads the press with crushed white berries, C: Allie fills a carboy of finished red wine, D: Laura, Marlena, Gary, Garrett, and Blair preparing for red wine pressing, and E: Marielle sits in the splash zone for red wine pressing.  Photos by: Denise M. Gardner

Figure 7: Pressing [white/rosé] juice or finished [red] wine is always an experience. A: Gary, George, and Garrett press rosé to prepare for overnight settling, B: Stephanie loads the press with crushed white berries, C: Allie fills a carboy of finished red wine, D: Laura, Marlena, Gary, Garrett, and Blair preparing for red wine pressing, and E: Marielle sits in the splash zone for red wine pressing. Photos by: Denise M. Gardner

I can’t wait to share some of the 2015 wines with the local industry at the March 2016 PA Wine Marketing & Research Board Symposium or at future Extension Enology events.

As a general reminder, many of these projects are financially supported through the multi-state Grape Wine Quality Eastern U.S. Initiative SCRI grant (which partially funds the NE-1020 variety trial research program), the PA Wine Marketing and Research Board, and the Crouch Fellowship, among other grant agencies.

Here are just a few snap shots that depict everything that we are currently working on for the 2015 harvest season:

Figure 8: This year’s NE-1020 variety trial projects include yeast trials, an evaluation of tartaric acid additions to red wine varieties grown in high potassium vineyard sites (A), and pre-fermentation juice treatments in Vidal Blanc wines (B).  Photos by: Denise M. Gardner

Figure 8: This year’s NE-1020 variety trial projects include yeast trials, an evaluation of tartaric acid additions to red wine varieties grown in high potassium vineyard sites (A), and pre-fermentation juice treatments in Vidal Blanc wines (B). Photos by: Denise M. Gardner

Figure 9:  The Crouch Fellowship currently supports a project pertaining to the impact of spray-on frost protection products on grape and wine quality.  A: Graduate student, Maria Smith, gets ready for a full day of processing after a full day of harvest. B: Marielle and Cara monitor the red wine fermentations through daily punch downs, temperature logs, and Brix measurements.  Photos by: Denise M. Gardner

Figure 9: The Crouch Fellowship currently supports a project pertaining to the impact of spray-on frost protection products on grape and wine quality. A: Graduate student, Maria Smith, gets ready for a full day of processing after a full day of harvest. B: Marielle and Cara monitor the red wine fermentations through daily punch downs, temperature logs, and Brix measurements. Photos by: Denise M. Gardner

Figure 10: Graduate student, Marlena Sheridan, takes a photo of a cluster representation for her research project on red wine color stability. Photo by: Denise M. Gardner

Figure 10: Graduate student, Marlena Sheridan, takes a photo of a cluster representation for her research project on red wine color stability. Photo by: Denise M. Gardner

Figure 11: Graduate student, Laura Homich, enjoys time in the Noiret vineyard for her research project that focuses on the effect of canopy management practices on rotundone (black pepper flavor) development in Noiret grapes and wine.

Figure 11: Graduate student, Laura Homich, enjoys time in the Noiret vineyard collecting berry samples for her research project that focuses on the effect of canopy management practices on rotundone (black pepper flavor) development in Noiret grapes and wine. Photo by: Maria Smith

Figure 12: Graduate student, Gal Kreitman, prepares inoculates on Vidal Blanc in relation to a project on the influence of copper on thiol-containing aroma/flavor compounds. Photo by: Denise M. Gardner

Figure 12: Graduate student, Gal Kreitman, prepares inoculates on Vidal Blanc in relation to a project on the influence of copper on thiol-containing aroma/flavor compounds. Photo by: Denise M. Gardner

Figure 13: Another full year of research winemaking at Penn State – vintage 2015.  Photo by: Denise M. Gardner

Figure 13: Another full year of research winemaking at Penn State – vintage 2015. Photo by: Denise M. Gardner

To follow all of our annual research harvest activities, please ‘Like’ us on Facebook: www.facebook.com/PennStateExtensionEnology

 

Technical Information about Pét-Nats (Pétillant Naturels, or Sparkling Wines Produced by Méthode Ancestrale)

By: Denise M. Gardner

Author’s Note: Current technical information regarding the production of pétillant naturels is limited.  The following information is summarized and detailed from a series of text books and personal discussions with Paul Guyard from Enartis, Daniel Granes from the ICV in Languedoc-Rousillion, whose contact comes courtesy of Gordon Specht from Lallemand, and Michael Jones from Lallemand.  The author would like to thank all contributors for the following information.

 

The recent interest in sparkling wine production (http://bit.ly/SparklingWineTechniques) has winemakers and sommeliers talking about another trendy bubbly: Pétillant Naturels, or pét-nats when abbreviated.  These bubblies are consumer friendly: less expensive than traditional Méthode Champenoise-produced wine, usually contain an enhanced fruitiness, are meant to be consumed early (i.e., no long term aging required by the consumer), and are currently trendy amongst wine professionals, bloggers and sommeliers.  A quick search online can lead one to a plethora of articles indicating consumer awareness of pét-nats:

Recent food trends indicate that consumers are searching for “more natural” selections (http://fortune.com/2015/05/21/the-war-on-big-food/), and pét-nats may appear as a less intrusive winemaking approach in the eyes of consumers.  Pét-nats offer a winery marketing potential, as many are highlight as being made with limited technological influence and following more traditional winemaking practices.

The concept of production is rather simple: start fermentation and bottle before it is finished fermenting to retain some residual carbon dioxide, and likely sugar, in the final product.  However, production requires winemaker attention to ensure final wine quality.  As David Lynch quoted one producer in his Bon Appetit article, pét-nats production can seem like “Russian roulette winemaking” from the production perspective.  Although in French, a detailed diagram displaying the steps of the méthode ancestrale production practices associated with pét-nats (follow the column labeled “méthode rurale”) can be found here: http://www.wine-and-bubbles.com/maj/phototheque/photos/schema/ShemaEtTexteCv4_3.jpg

History of Pét-nats

Pét-nats are believed to be the original source of sparkling wine production in France, preceding Champagne production (Robinson and Harding 2006).  It is believed that wines from naturally-cooler regions in France would undergo primary fermentation until the winter when temperatures would naturally drop and inhibit fermentation.  Winemakers, unaware that the wine was not fully fermented, bottled the young wine and found that it re-fermented in the bottle when the ambient temperatures became warmer.  Some of the first sparkling wines produced have been traced back to Gaillac, located in the southwest part of France, north of Toulouse, and Limoux, located in the higher mountains of the Languedoc-Roussillon region (Robinson and Harding 2006).

The term “pétillant” generally describes a sparkling wine with less retention of carbon dioxide compared to a sparkling wine like Champagne (WSET 2001).    The grape variety traditionally used for pét-nat production in Gaillac and Limoux is mauzac (known locally as blanquette in Limoux), which has a distinguishable “dried-apple-skin” flavor (Robinson et al. 2014).  Today, pét-nat production has exceeded the boundaries of their origins, extending through the Loire and various regions around the world.

The production method associated with of Blanquette de Limoux is often referred to as the méthode ancestrale, or as the méthode gaillacoise in Gaillac (Robinson and Harding 2006).  The methods are quite similar in execution, which consists of one primary fermentation that is started in tank and finished in the bottle.  This results in a cloudy wine, typically with varying concentrations of residual sugar, and retention of carbon dioxide.

Thinking of Giving Pét-Nat Production a Try?

While the production of pét-nats may seem appealing, one of the experts suggested trying to bottle condition a wine before attempting the full méthode ancestrale production technique as it involves a lot of winemaker attention.  This may also be a practical alternative when current production facilities are not equipped for full-range temperature control.   Bottle conditioning is typically used by homebrewers, home cider makers, and home winemakers to get carbon dioxide in bottles.  You can read some of the home production literature here if you are unfamiliar with the process:

It is recommended that you use a low alcohol (≤12% alcohol v/v), low pH (<3.50) wine if you are exploring the bottle conditioning technique.  Add enough sugar to generate 3-4 ATM of pressure, maximum, and bottle with a yeast addition based on the suggestions below.  Bottles should be suitable to retain pressure and sealed with a crown cap.

Bottle conditioning a wine should give you a clear indication regarding the finishing technique and style associated with pét-nats.  It also acts as good practice before committing to pét-nat production.

Safety First

Since pét-nats are sparkling wine products that contain a fair amount of pressure, winemakers and cellar staff should proceed with caution during production.  Use common sense: purchase appropriate bottles made to withstand pressure, double check calculations for sugar-to-pressure conversions, and use protective eye glasses.  Accidents can happen, and it is best to be prepared for any hazard associated with any stage of wine production.  Sanitation is a key point through production, and proper protective clothing should be worn at all times when using sanitizing agents of any kind.

Parameters to Look for in the Fruit

Pét-nat production may be applied to any grape variety, and offers a wide opportunity for winemakers to explore the production of new and unique wine products.  Although there are no variety limitations, production experts caution that grapes should lack vegetal flavors in the berries.

Berry sensory analysis may be useful for winemakers to evaluate grape flavor quality and to help determine picking times.  In general, ripe (non-vegetal) flavors should persist in the berry in order to encourage their development in the final wine.  However, grapes should avoid “overly ripe” flavor characteristics as this may be an indication of higher pH and lower acidity values that may cause complications through the winemaking process.

Grapes are often picked with a potential alcohol of 10 – 12% v/v, and at this concentration of natural sugar, the pH should be lower (<3.50).  The pH of the wine will offer microbial protection to the wine through the méthode ancestrale process and offer some protection to wine quality through vulnerable production steps.

Fruit should also be of sound quality (i.e., with limited disease pressure) to avoid detriment to flavor and overall quality of the wine.  Some diseases may contribute secondary byproducts which could cause fermentation complications.  Therefore, the winemaker is encouraged to use sound fruit.  Cellar hygiene, or proper sanitation techniques, will be essential for quality control purposes through production.  Extra sanitary care should be taken if the winemaker wants to remove the lees from bottles by traditional disgorging techniques (refer to a previous post on Sparkling Wine Production Techniques).  A summary of grape parameters required for pét-nat production is shown in Figure 1.

Figure 1: Grape Specifications Recommended for Pét-Nat Production

Figure 1: Grape Specifications Recommended for Pét-Nat Production

Base Wine Production

The production method associated with pét-nats (Figure 2) is alluded to rather simply in the wine literature: the primary fermentation is started in tank, arrested before primary fermentation is completed, bottled, and finished in the bottle.  The consumer can expect a slightly sweet (i.e., presence of residual sugar), cloudy, lightly bubbled wine (usually below 4 ATM pressure; Amerine et al. 1972).  Winemakers should refer to the TTB for additional tax purposes associated with sparkling wines (http://www.ttb.gov/wine/wine_regs.shtml).

Grapes are crushed/destemmed (if preferred) and pressed.  In France, press cycles and parameters are based on regulation.  Press cycles are set to extract 100 L of juice for every 150 kg of fruit.

Some attention should be given to clarification of the juice, pre-fermentation, in the production process of pét-nats.  It is recommended that juice is clarified to 30 – 80 NTUs with use of centrifugation, flotation or assistance with settling enzymes and/or fining agents.

There is some debate as to whether or not sulfur dioxide should be added to the juice during settling.  In the juice-settling phase, a sulfur addition may help clarify the juice and minimize spoilage yeast and bacteria that could harm the quality of the wine.  However, like with still wine production, sulfur dioxide additions should not be made to excess as too much could hinder primary fermentation.  (Note: For those looking to produce a “more natural” wine, or to appeal to the “no-sulfur-added” market, it would be prudent to skip sulfur dioxide additions at this step.)

Following clarification, the juice should be racked and prepared for inoculation.

Starting Fermentation

Yeast selections (Table 1) should be based on the winemaker’s preference, but there are some tips that have been provided by wine supply companies:

  • Use low-sulfur dioxide-producing yeast strains
  • Select yeasts for secondary aroma potential
  • Supply yeasts with proper hydration and fermentation nutrient additions
  • Use yeasts that grow optimally in cool temperatures, 14-16°C (~57-60°F)
  • If the winemaker is going to remove lees (e., disgorging) before selling the product, and is only going to undergo one fermentation without a second inoculation, choose a yeast strain that is recommended for Méthode Champenoise sparkling wine production
Table 1: Yeast Recommendations from Lallemand and Enartis Vinquiry for Pét-Nat Production (Note: Other suppliers may have additional yeast recommendations. Please consult your regular supplier for further suggestions.)

Table 1: Yeast Recommendations from Lallemand and Enartis Vinquiry for Pét-Nat Production (Note: Other suppliers may have additional yeast recommendations. Please consult your regular supplier for further suggestions.)

Use a hydration nutrient (e.g., GoFerm Protect Evolution, Enartis Ferm Arom Plus) properly at inoculation.  Depending on the winemaker’s preferred techniques or the perceived difficulty of alcoholic fermentation, oxygen additions can be made to activate the fermentation.  Some winemakers choose oxygen ingress through the use of micro-ox, and base dosage rates [of oxygen] on sensorial perceptions.

Use of temperature control is essential for producing pét-nats.  If you need more information and suggestions regarding how to integrate temperature control into your winery operation, please visit this report here: http://bit.ly/LowTempFerm.

Fermentation should proceed at 14-16°C (~57-60°F).  At about +/- 3% v/v from the target alcohol, winemakers should chill the wine down to 8°C (~46°F) to hinder the fermentation.  The act of cooling will also clarify the wine and minimize the transfer of lees.  Too much lees transfer will result in a “yeasty” flavored wine, which is not preferred in pét-nat wines.

Finishing the Wine

Once the wine is properly chilled, it will need to be racked to remove most of the lees.  It is not uncommon for winemakers to remove all of the lees by centrifugation or filtration, and later, restart the wine with a fresh culture and hydration nutrient.  From a French winemaking perspective, the addition of nitrogen is usually added at racking in the pump flow (1 L of nitrogen gas for each 20 L of wine).

Winemakers may opt to blend at the racking stage as well.  Blending can help elicit the production of a “house-style” pét-nat, and ensure consistency despite natural vintage year variation.

Malolactic fermentation is optional, and should be inoculated after racking, based on winemaker preference.  For those that are considering malolactic fermentation, it is important to remember that there is a significant quantity of residual sugar in the wine at this stage in the process, which can lead to a series of winemaking problems:

  • Consider the wine’s pH before undergoing malolactic fermentation. Malolactic bacteria have a higher risk of producing more acetic acid during malolactic fermentation if the wine pH is greater than 3.50.  Great attention and care must be given to a pét-nat undergoing malolactic fermentation with a higher pH to avoid extreme spoilage issues.
  • Malolactic bacteria require a warmer temperature for growth, which requires the winemaker to increase the temperature of the wine. Therefore, it is suggested that winemakers sterile filter the wine prior to inoculating for malolactic fermentation to avoid primary fermentation from re-starting and completing before the wine is bottled.
  • The remaining residual sugar puts the wine at risk for other microbial contaminants. Sanitation and monitoring of malolactic fermentation progression is of the utmost importance.

Tartaric acid stabilization, or cold stabilization, can progress at this stage after the wine is racked.  However, it is more common for winemakers to add CMC to avoid crystallization of tartaric acid as opposed to undergoing a cold stabilization process.

At this point, the wine should be prepared to complete primary fermentation.  If the wine were to go to tank and complete fermentation, then the process of completion follows a Partial Fermentation process that is used in the Asti region of Italy to produce Moscato.

To complete the méthode ancestrale technique, the base wine is bottled to complete fermentation.  A second inoculation of yeast is typically required to complete primary fermentation, but it is optional to add more yeast nutrient at inoculation.  Some wineries choose a second edition of a hydration nutrient (prepared during yeast hydration) and a smaller dose of a complex nutrient (e.g., Fermaid K, Nutriferm Advance).  Yeast addition dosage rate is recommended at about 2 million colony forming units (CFU) per mL of living yeast.  Ideally, yeast addition should be less concentrated than a “normal” inoculation to minimize biomass in the bottle and encourage a slow fermentation in the bottle.  Yeast strain should be selected according to winemaker preference (see the above list, Table 1, for suggestions from Lallemand/Scott Labs and Enartis Vinquiry).

Méthode ancestrale does not involve a sugar addition at the second inoculation.  However, a sugar addition to manipulate the final desired concentration of pressure in the bottle is an option for winemakers at the second inoculation of yeast.

Bottle selection is important, and needs to be of high enough quality to retain the internal pressure left over from fermentation.  If the expected pressure is above 4 ATM, ensure that you are using the correct bottles to retain pressure.  Yeast selection should also be altered if the final preferred pressure is greater than 4 ATMs.

Although a slight detour from the méthode ancestrale process, it is possible to remove the lees after fermentation has completed in the bottle.  If the winemaker would like to riddle and disgorge the yeast at the completion of primary fermentation in the bottle, a riddling agent (e.g., Adjuvant MC by Enartis Vinquiry) may be desired.

After the base wine is re-inoculated in the bottle, bottle fermentation should progress in a temperature controlled space, optimally set at 13-15°C (~55-59°F).  For retention of residual sugar, chill the room to 0-2°C (32-36°F) to arrest fermentation in the bottle.  [Note: When the wine is warmed up, it may continue to ferment in the bottle.]

With the minimal yeast population, minimal nutrient availability, increase pressure in the bottle, and low fermentation temperature, fermentation will progress slowly and may stop with residual sugar naturally as all of these factors put stress on the yeast.  It may take several months until an appropriate amount of pressure has built up in the bottle.

Figure 1: Flow Diagram Representing General Production of Pétillant Naturel Sparkling Wines (Méthode Ancestrale)

Figure 2: Flow Diagram Representing General Production of Pétillant Naturel Sparkling Wines (Méthode Ancestrale)

 

Potential Disgorgement

Some winemakers choose to sell a product that is clearer than traditional pét-nats and disgorge the yeast lees using similar techniques that were previously discussed pertaining to the traditional method, Méthode Champenoise, way of making sparkling wine.  Here, the lees are collected, riddled, and disgorged.  If the wine was fermented to dryness, a sugar addition with a dose sulfur dioxide can minimize risk for re-fermentation when the bottle is in the hands of consumers.  Furthermore, disgorgement allows a winemaker to make sensory alterations to the wine with a dosage addition.  Sensory adjustments can be made using Arabic gums, inactivated yeast/polysaccharide products, or tannins that have been added to the dosage.

Traditionally, pét-nats are sealed with a crown cap.

Final Production Thoughts

Large producers of bottle conditioned cider may opt to flash bottle pasteurize hard ciders that retain some residual sugar.  Flash bottle pasteurization will inactive the yeast and ensure an extra line of protection to ensure that fermentation does not continue to progress once the consumer has purchased the product.

However, part of the fun associated with pét-nats is not truly knowing the end residual sugar!

Note: Do NOT add potassium sorbate to the wine at any stage if you are trying to make a pét-nat.  Potassium sorbate will inhibit the yeast from fermenting through any stage of this process.

Familiarizing Yourself with Pét-Nats

Like with any wine style, it is ideal to have a sensory library of what quality pét-nats taste like using examples from the commercial market.  The practice of tasting multiple examples of a specific wine style creates a benchmark library in the mind of the winemaker, which aids in making processing decisions in relation to an end-goal for the final product.  It also helps define “quality” for that wine style.

While I have not embarked on an exploration to understand pét-nat quality, the following wines have been suggested in the above-mentioned articles or from individuals that have enjoyed pét-nats in today’s market.  I highly suggest that any winemaker aiming to produce pét-nats, obtain various examples to evaluate 1) their individual preference of the product, 2) the potential consumer preference of the product, and 3) the quality parameters that the winemaker will aim for during production of a pét-nat style wine.

References

Amerine, M.A., H.W. Berg, and W.V. Cruess. 1972. Technology of Wine Making, Third Edition.  The AVI Publishing Company: Westport, Connecticut.

Granes, Daniel. 2015. Personal Discussion.

Guyard, Paul. 2015. Personal Discussion.

Jones, Michael. 2015. Personal Discussion.

Robinson, J. and J. Harding. 2006. The Oxford Companion to Wine. ISBN: 978-0198609902

Robinson, J., J. Harding, and J. Vouillamoz. 2014. Wine Grapes.

Wine and Spirit Education Trust (WSET). 2011. Wine and Spirits: Understanding Style and Quality. ISBN: 978-1 905819 15 7.

Winemaking Practices Believed to Affect Red Wine Color Stability

By: Denise M. Gardner

In previous blog posts, we covered an introduction to what anthocyanins (red wine color pigments) are and how they can be stabilized in wine. Additionally, Marlena Sheridan recently discussed current research on acetaldehyde-bridging amongst anthocyanins or anthocyanin and tannin substances in wine.

This post will evaluate several techniques used during red winemaking, and what the scientific literature has found regarding their impact on red wine color stability.

Anthocyanins undergo three main phases during the course of a wine’s life:

  • Development in the grape
  • Extraction during primary fermentation
  • Stabilization through the life of the wine
anthocyanin life

Phases associated with anthocyanins through a wine’s life.

While vineyard development is an important aspect of color stabilization, as starting anthocyanin concentration will ultimately determine potential processing decisions for winemakers, the following processes are reflective of winemaking techniques believed to affect red wine color stabilization. As you siphon through the various techniques, please remember that there is no one fix-all solution to improve red wine color stability. Wine is a complex matrix, and results may vary from one variety to the next, or from one vintage to the next.

Cold Soak

Cold soak is a pre-fermentation process in which grape must is held at low (≤10°C, ≤50°F) temperatures. There are various ways to execute a cold soak step in wine production:

  • Placing a holding vessel (i.e., a macrobin) in a cool, ambient environment
  • Use of tank temperature control
  • Use of dry ice

Cold soaking grape must is believed to increase anthocyanin extraction pre-fermentation. Increasing anthocyanin extraction would make stabilization reactions more favorable, pushing equilibrium to building stable anthocyanin-complexes through and after fermentation.

However, several studies have investigated the use of cold soak on various red wine varieties. In two Pinot Noir studies (Gerbaux 1993, Feuillat 1996, Sacchi et al. 2005*), detrimental effects to color were found on Burgundian wines when cold soak was used pre-fermentation. Similarly, Heatherbell et al. (1996) found no difference in wine color for those wines that were cold soaked versus the non-cold soaked controls.

A study that tested freezing must, however, had a different impact on the finished wine color.  The variety evaluated was Merlot, and musts were frozen with dry ice.  An increase in anthocyanin concentration by 50% and increase in overall tannin concentration by 52% was found in finished wines in which grape must had been frozen pre-fermentation, compared to an untreated grape must, control wines (Couasnon 1999, Sacchi et al. 2005*).  Freezing may have a greater effect on anthocyanin concentration, as freezing physically causes berry cells to burst and release its contents.

Definition of Cold Soak

Definition of Cold Soak

Practical Winemaking Application: Winemakers do not need to utilize a cold soaking step to increase anthocyanin extraction or improve red wine color stability, as most research suggests there is no effect regarding red wine color stability. However, it is important to note that little research has been conducted regarding potential extraction of phenol and/or tannin complexes (including polymeric pigment content) during the cold soak step.

Saignée

Saignée is the French term for “bleed,” and is utilized as a winemaking technique for making rosé wines by “bleeding off” free-run juice from macerated red grapes. While the free-run juice may be used for rosé production, many winemakers utilize the saignée technique to concentrate (increase extraction) of anthocyanins and tannins in the juice that will be made into a finished red wine. Secondary effects may also increase flavor concentration.

In 1972, Singleton found that the use of saignée increased the flavonoid and anothocyanin concentrations in the concentrated red wine, four months post-fermentation. Gerbaux (1993) found slight increases in color (through sensory analysis) and phenolics of young Pinot Noir wines that had been subjected to saignée practices. However, unlike Singleton, Gerbaux’s study did not find increases in anthocyanin concentration. However, Gawel et al. (2001) found initial increases in anthocyanin concentrations at the end of primary fermentation in pressed Syrah wines, but these concentrations were depleted after 6-months post-fermentation. This study did not investigate the fate of monomeric anthocyanins, and depleted concentrations of anthocyanins were likely caused by potential polymerization or adsorption of anthocynanins onto various other wine constituents (e.g. dead yeast cells, tartrates).

saignee

Definition of Saignee

 

Practical Winemaking Application: While the causes of potential increased color stability are unclear, the use of saignée to concentrate red grape must appears to have a positive outcome on the wine’s color stability properties. Currently, there is no evidence that removal of 10% free-run juice is less beneficial than removal of 20%. However, as saignée is a concentration method, if the grapes are of low quality, the winemaker will only concentrate other poor quality components (i.e. off-flavors, excessive tannin, etc.) with use of this technique.

Micro-oxygenation (Micro-ox)

Micro-ox is the “addition of dissolved oxygen at controlled dosage rates at or less than the oxygen uptake rate of wine” (Paul 2002). According to Dykes (2007), typical dosages rates range from 2 – 90 mg of oxygen per liter of wine per month. The utilization of micro-ox is believed to affect the stabilization of red wine color pigments, and therefore requires adequate starting material (i.e. anthocyanins, phenolics/tannins) in order for this method to be effective.

Theoretically, when used between primary fermentation and malolactic fermentation, the integration of dissolved oxygen should provide the chemical constituents needed to initiate polymeric pigment formation of monomeric anthocyanins. Micro-ox is used as a driving force to influence acetaldehyde concentration and influence acetaldehyde-bridged complexes previously described by Marlena Sheridan. One previous study has shown no rise in acetaldehyde contraction by use of micro-ox (Pozo et al. 2010). However, this study did find that wines subjected to micro-ox treatment did have an increased concentration of sulfite-resistant pigments. Many other wine experts have written on the use of micro-ox, and there is a world of scientific literature available regarding its use and outcomes in red wines, with varied guaranteed consensus. Additionally, micro-ox has potential to alter other characteristics of wines, including mouthfeel or flavor, in both positive and negative ways, which is often dependent on the starting wine chemistry. The extent of this summary does not nearly cover the depth of research regarding micro-ox in wine production, and will be tabled for a later date.

Definition of micro-oxygenation (micro-ox)

Definition of micro-oxygenation (micro-ox)

Practical Winemaking Application: The use of micro-oxygenation may be a powerful tool to enhance anothocyanin stabilization. Winemakers are encouraged to work with the unit’s supplier or a consultant with micro-oxygenation experience prior to implementing this strategy into processing procedures. Anecdotally, at Penn State, we have noticed little success of micro-oxygenation to improve red wine color when used on wines that have relatively low anothocyanin concentrations (<200 mg/L GAE).

Thermovinification

Thermovinification is a technique that uses a brief heating step to grape must or juice, and subjecting it temperatures above 60°C (140°F). It is believed that thermovinification enhances both extraction and stabilization of anthocyanins.

Auw et al. (1996) found that the use of thermovinification increased the concentration of free, monomeric anthocyanins in both Cabernet Sauvignon and Chambourcin wines. Additionally, this study also found increased concentrations of phenolics in the Chambourcin wine treated with thermovinification pre-fermentation, and a lower phenolic content in Cabernet Sauvignon wines treated with thermovinification. This emphasizes that solutions to improve color stability may vary depending on the grape variety and its heritage (i.e. native variety vs. hybrid vs. V. vinifera). Additionally, Sacchi et al. (2005) also concluded that it is necessary to have some skin contact time with grape skins after thermovinification treatment in order to enhance extraction of anthocyanins.

Practical Winemaking Application: The use of thermovinification to improve red wine color stability may be a practical tool for red wines, especially those red wines with minimal aging requirement. Thermovinification is not recommended for red wines destined for long term aging. It should be noted that thermovinification may alter flavor of the finished wines. A winery should evaluate the potential sensory impact of thermovinification prior to committing all wines to this process.

Extended Maceration

Extended Maceration is the process of allowing skins and seeds in contact with the finished red wine, post-fermentation, for an extended period of time. Various studies researching the effects of extended maceration on red wines have found increased concentrations of tannins, especially tannins from grape seeds. However, this process has not been shown to increase extraction or concentration of anthocyanins.

Auw et al. (1996) found that a thermovinification treatment was more effective at increasing phenolic composition compared to extended maceration for Chambourcin wines, but that for Cabernet Sauvignon wines, extended maceration was more effective at increasing phenolic concentrations. Watson et al. (1994, 1995) also found an increase in flavanol and polymeric pigment extraction in Pinot Noir wines that underwent extended maceration.

Definition of Extended Maceration

Definition of Extended Maceration

Practical Winemaking Application: Extended maceration will not increase free monomeric anthocyanin concentrations. This practice should only be utilized for winemakers wishing to increase tannin-based extractions.  For a review on why the effects of extended maceration may be focused on phenolic extraction, please see Dr. Anna Katharine Mansfield’s recently published article, “A Few Truths About Phenolics,” in Wines & Vines.

Delayed Malolactic Fermentation (MLF)

The most current scientific literature on delayed MLF is coming out of Dr. James Osborne’s lab in Oregon. This practice is literally as it sounds: winemakers allow a wine to finish primary fermentation and wait (without addition of sulfur dioxide) for a specific period of time until MLF progresses naturally or is inoculated. This is an anthocyanin stabilization processing technique.

On studies in Pinot Noir, James and Osborne (2014) found that a 200 day delay in MLF was required for wines that went through MLF to reach a similar concentration of polymeric pigment, statistically, as the control wine that did not undergo MLF. It is important to remember that in order to maintain stability of wines not treated with sulfur dioxide, adequate temperature control (i.e. maintaining the cellar at 50-55°F or 10-13°C) will cause polymeric pigment formation reactions to progress slowly, which may influence why so much time is needed in delaying MLF. While increase temperature would increase the rate of polymeric pigment formation reactions, the increase in temperature also puts wines at greater risk for microbial spoilage.

Definition of Delayed MLF

Definition of Delayed MLF

Practical Winemaking Application: The jury is not out on this practice! Although some winemakers swear this practice works, it is important to remember than many wineries do not run “control” treatments for various practices. As there is much vintage-to-vintage variation amongst wines, it is challenging to draw solid conclusions from commercial wineries that do not utilize a control treatment. Currently, the literature states a delay in MLF may effectively improve color, but the time required for that delay may not be practical from a commercial operation standpoint.  Additionally, winemakers must maintain effective strategies at monitoring potential spoilage during a vulnerable period of time when the wine is not protected by preservatives (sulfur dioxide).

While this blog post covers several techniques believed to affect red wine color stability, the review article by Sacchi et al. covers several additional topics including: yeast selection, fermentation temperature, the effects of sulfur dioxide, carbonic maceration, the use of pectolytic enzymes, and utilization of pump-overs and punch downs during primary fermentation.  The review of the current scientific understanding of these practices (up to research literature published through 2005) can be found in the American Journal of Enology and Viticulture (AJEV).

Sacchi et al. 2005* The following authors (Couasnon 1999, Feuillat 1996, and Gerbaux 1993) were included in the Sacchi et al. 2005 review article. As initial research article was in another language, information regarding these studies was obtained from Sachhi et al. 2005.

 

Literature Cited

Auw, J.W., V. Blanco, S.F. O’Keefe, and C.A. Sims. 1996. Effect of processing on the phenolics and color of Cabernet Sauvignon, Chambourcin, and Noble wines and juices. Am. J. Enol. Vitic. 47(3):279-286.

Burns, T.R. and J.P. Osborne. 2014. Loss of Pinot Noir wine color and polymeric pigment after malolactic fermentation and potential causes. Am. J. Enol. Vitic.

Couasnon, M.B. 1999. Une nouvelle technique: La maceration préfermentaire à froid-extraction à la neige carbonique. Premiér partie: Résultats oenologiques. Rev. Oenol. 92:26-30.

Dykes, S. 2007. The effect of oxygen dosage rate on chemical and sensory changes occurring during micro-oxygenation of New Zealand red wine. Diss. Food Sci. Univ. of Auckland.

Feuillat, M. 1996. Vinification du Pinot Noir en Bourgogne par maceration préfermentaire à froid. Rev. Oenol. 83:29-31.

Gawel, R., P.G. Iland, P.A. Leske, and C.G. Dunn. 2001. Compositional and sensory differences in Syrah wines following juice run-off prior to fermentation. J. Wine Res. 12(1): 5-18.

Gerbaux, V. 1993. Etude: de quelques conditions de cuvaison susceptibles d’augmenter la composition polyphénolique des vins de Pinot Noir. Rev. Oenol. 69:15-18.

Heatherbell, D., M. Dicey, S. Goldsworthy, and L. Vanhanen. 1996. Effect of cold maceration on the composition, color, and flavor of Pinot Noir wine. In Proceedings of the Fourth International Symposium on Cool Climate Enology and Viticulture. T. Henick-Kling et al. (Eds.), pp. VI: 10-17. New York State Agricultural Experiment Station, Geneva.

Paul, R. 2002. Micro-oxygentation – Where now.” Australian Society of Viticulture and Oenology, Uses of Gases in Winemaking Seminar Proceedings.

Pozo, A.G., I. Arozarena, M.-J. Noriega, M. Navarro, and A. Casp. 2010. Short- and long-term effects of micro-oxygenation treatments on the colour and phenolic composition of a Cabernet Sauvignon wine aged in barrels and/or bottles. Eur. Food Res. Technol. 231: 589-601

Sacchi, K.L., L.F. Bisson, and D.O. Adams. 2005. A review of winemaking techniques on phenolic extraction in red wines. Am. J. Enol. Vitic. 56(3):197-206.

Singleton, V.L. 1972. Effects on red wine quality of removing juice before fermentation to simulate variation in berry size. Am. J. Enol. Vitic. 23:106-113.

Watson, B.T., S.F. Price, H.P. Chen, and M. Valladao. 1994. Pinot Noir processing effects on wine color and phenolics. Abstr. Am. J. Enol. Vitic. 45:471-472.

Watson, B.T., S.F. Price, and M. Valladao. 1995. Effect of fermentation practices on anthocyanin and phenolic composition of Pinot Noir wines. Abstr. Am. J. Enol. Vitic. 46:404.

The Effect of Acetaldehyde on Red Wine Color Stability and Astringency

By: Marlena Sheridan

Dr. Ryan Elias’s Lab

Acetaldehyde is a compound found in wine that has a profound effect on color stability and astringency. Acetaldehyde reacts directly with red wine tannins and anthocyanins to form polymeric pigments and modified tannins. Denise has already provided a great review of polymeric pigment formation (see her post form March 6, 2015). Here, I’ll be focusing on the reactions involving acetaldehyde and research we’ve completed on the topic.

Acetaldehyde reacts with tannins and anthocyanins to form irreversible, covalent bridges. When these reactions are between tannins, they can alter the structure so that its shape and activity are changed. This affects wine astringency, a mechanism based on the interaction of tannins and salivary proteins. Modified tannins, including acetaldehyde-bridged tannins, have been shown to have lower astringency because of their structural changes (Gambuti 2013). A model of these reactions is shown in Figure 1. These changes contribute to the shift from drying or puckering mouthfeel to a more velvety mouthfeel in aged wines.

  Figure 1. Modification of tannins by acetaldehyde bridging reactions.


Figure 1. Modification of tannins by acetaldehyde bridging reactions.

Similar reactions take place between anthocyanins and tannins as well as between anthocyanins themselves. These reactions form polymeric pigments: pyranoanthocyanins and vitisins (see Denise’s previous post; Cheynier 2006). A model for these reactions is shown in Figure 2. As Denise has covered, these polymeric pigments have increased stability to sulfite bleaching and pH changes compared to monomeric anthocyanins.

Figure 2. Polymeric pigment formation by reaction with acetaldehyde.

Figure 2. Polymeric pigment formation by reaction with acetaldehyde.

As has hopefully been made clear, using these reactions of acetaldehyde with tannins and anthocyanins is an important tool for winemakers. Winemakers can use several techniques to get the benefits of acetaldehyde on pigment and tannin structure. These use oxygen incorporation to form acetaldehyde through a series of metal-catalyzed reactions (Danilewicz 2003). Along this pathway, there is the possibility to form detrimental oxidation products instead of the desired acetaldehyde. A simplified version of this mechanism is shown in Figure 3. These other oxidation pathways can lead to some of the risks of oxygen exposure including the loss of desirable aromas, browning, and the formation of off odors.

Figure 3. Formation of acetaldehyde from oxygen.

Figure 3. Formation of acetaldehyde from oxygen.

Ideally, winemakers would be able to get the benefits of acetaldehyde without the risks of oxygen exposure. By adding acetaldehyde directly, we could avoid the problems of oxidation while simultaneously controlling the beneficial oxidation reactions we’re hoping for. With this in mind, we conducted an experiment where exogenous acetaldehyde was added during red wine fermentation using Cabernet Franc grapes from North East, PA.

The must was separated into three groups – control, low acetaldehyde, and high acetaldehyde. The acetaldehyde groups received four doses during the fermentation, 4×25 mg/L acetaldehyde in the low group and 4×250 mg/L acetaldehyde in the high group. Fermentations were performed in quadruplicate in microfermenters (Figure 4). Wines were fermented to dryness and pressed prior to analysis.

Figure 4. Experimental wines in microfermenters.

Figure 4. Experimental wines in microfermenters.

Wines were analyzed for color stability by the modified Somers assay (Mercurio 2007) with measures of sulfite-resistant pigments, or polymeric pigments, shown here. Astringency was measured using a model protein precipitation, where wines were mixed with bovine serum albumin (BSA) and the amount of tannin precipitated was quantified (Mercurio 2008). Higher tannin precipitation implies higher astringency. Data from this experiment is shown in Figure 5.

Blog_Marlena_Fig5

Figure 5. (A) Tannin content of BSA-tannin precipitates quantified by ferric chloride reaction in (+)-catechin equivalents. (B) Sulfite-resistant pigments in arbitrary units (au). Error bars represent one SD of the mean, and results with different letters (a,b) are significantly different (p < 0.05).

As shown in Figure 5, there was a statistically significant decrease in the measure of astringency and increase in color stability with high acetaldehyde treatment. This data provides evidence that exogenous acetaldehyde can be used in red wines to get beneficial effects on color and astringency without oxygen exposure. A more detailed discussion of the results can be found in the published manuscript (Sheridan 2015).

Based on this work, we are continuing to examine the reaction of acetaldehyde with tannins and anthocyanins. We are currently working with model wine experiments to further understand the chemistry of these reactions including characterizing the final products and the effect of wine composition.

 

Literature Cited:

Cheynier, V. & Dueñas-Paton, M. Structure and properties of wine pigments and tannins. Am. J. Enol. Vitic. 2006, 57, 298–305.

Danilewicz, J. C. Review of Reaction Mechanisms of Oxygen and Proposed Intermediate Reduction Products in Wine : Central Role of Iron and Copper. Am. J. Enol. Vitic. 2003, 54, 73–85.

Gambuti, A., Rinaldi, A., Ugliano, M. & Moio, L. Evolution of phenolic compounds and astringency during aging of red wine: effect of oxygen exposure before and after bottling. J. Agric. Food Chem. 2013, 61, 1618–27.

Mercurio, M. D., Dambergs, R. G., Herderich, M. J. & Smith, P. A. High throughput analysis of red wine and grape phenolics-adaptation and validation of methyl cellulose precipitable tannin assay and modified Somers color assay to a rapid 96 well plate format. J. Agric. Food Chem. 2007, 55, 4651–7.

Mercurio, M. D. & Smith, P. A. Tannin quantification in red grapes and wine: comparison of polysaccharide- and protein-based tannin precipitation techniques and their ability to model wine astringency. J. Agric. Food Chem. 2008, 56, 5528–37.

Sheridan, M.K. & R.J. Elias. 2015. Exogenous acetaldehyde as a tool for modulating wine color and astringency during fermentation. Food Chem. 2015, 177, 17-22.

Questions Pertaining to Malolactic Fermentation in Wine

By: Denise M. Gardner

What is Malolactic Fermentation?

Malolactic fermentation, MLF, is a bacterial fermentation, which converts malic acid to lactic acid. Malic acid, the acid affiliated with apples is perceptibly harsher than lactic acid, which is the primary acid in milk, and perceptibly softer than malic acid.

What is the difference between a native MLF and one that is inoculated?

MLF can occur spontaneously through the native microflora (i.e., lactic acid bacteria) that comes in on the fruit from the vineyard. Most native malolactic bacteria (MLB) strains are in the Lactobacillus genera. These bacterial populations are easily manipulated by pH changes and are sensitive to general environmental changes (e.g. increases in alcohol).

However, due to the fact that native or natural MLB is unpredictable and may contribute to several affiliated off-flavors of the wine, many winemakers choose to inoculate for MLF. The most common bacteria used for commercial inoculation is Oenococcos oeni, which can be purchased through a number of commercial suppliers. Many suppliers offer several different strain selections to accommodate varying wine conditions (e.g. high or low pH, high alcohol environments, use of sulfur dioxide, etc.). Commercial strains are bred to be reliable, consistent, and tolerant of low pH wines. They also contribute less flavor changes to the wine, if specific chains are selected by the winemaker, compared to many native bacterial strains.

One of the biggest problems affiliated with commercial MLF strains is the loss of color in red wines between primary and secondary fermentation. While we will touch briefly on color stability next week, several studies (Gerbaux and Briffox 2003, Morenzoni and Specht 2005, Burns and Osborne 2013) have noted the issues affiliated with red wine color stability since commercial strains have become more prevalent. Earlier this year, José Santos from Enartis Vinquiry discussed various red wine color stability issues during his “Harvest Preparation” workshop. You can read more about his discussion here.

Glass on left contains Chambourcin with deep color intensity while the glass on the right is Chambourcin wine with medium color intensity.

Glass on left contains Chambourcin with deep color intensity while the glass on the right is Chambourcin wine with medium color intensity.

When should wines be inoculated for MLF?

Currently, inoculation and timing of inoculation for MLF is a stylistic decision made by the winemaker. There are several options available for any given wine:

  • Following primary fermentation: Bacteria inoculation occurs after the completion of primary fermentation. This has become the standard way to use MLB.
  • Co-Fermentation of yeast and bacteria: Bacteria inoculation occurs after inoculation of yeast for primary fermentation, but before primary fermentation is complete.
  • Co-Inoculation or simultaneous inoculation: Here, yeast for primary fermentation and bacteria for MLF are inoculated at the same time to the must.

There are some advantages and concerns for co-fermentation or co-inoculation. In terms of advantages, previous research has highlighted how MLF bacteria can utilize remaining primary fermentation macromolecules (Guilloux-Benatier and Feuillat 1991), including mannoproteins (Alexandre et al. 2004) for their own nutritional gain. Beelman and Kunkee (1985) and King and Beelman (1986) found that bacteria acclimate to the changing wine conditions (i.e., increases in ethanol content) while the yeast strain proliferated to complete primary fermentation. Another advantage includes a reduction in total production time from the start of primary fermentation to the end of MLF, which can be beneficial to wineries with space or tank constraints.

In contrast, some research has pointed towards antagonistic relationships between yeast and bacteria, causing an increase in stuck fermentations. Typically, those studies also found an increase in volatile acidity. As mentioned previously, there has been an anecdotal observation of color intensity reduction in red wines, in general, utilizing commercial MLB strains. However, this observation has not yet been linked to timing of inoculation for MLF (Burns and Osborne 2013).

Based on the varying results from past research, it is advised that producers that utilize co-fermentation practices discuss strain selections, of both yeast and bacteria, with their supplier. There is a general review online, authored by Dr. Sibylle Krieger from Lallemand, written in 2007 regarding strain selection and MLF stylistic approaches, which you can read here.

Literature Cited

Alexandre, H. et al. (2004) Saccharomyces cerevisiae – Oenococcus oeni interactions in wine: current knowledge and perspectives. Int. J. Food Micro. 93: 141-154.

Beelman, R.B. and R.E. Kunkee. (1987) Inducing simultaneous malolactic/alcoholic fermentation. Practical Winery & Vineyard Management. July/August 1987. pg. 44-56.

Burns, T.R. and J.P. Osborne. (2013) Impact of Malolactic Fermentation on the Color and Color Stability of Pinot noir and Merlot Wine. Am. J. Enol. Vitic. 64:3, pg. 370-377.

Gerbaux, V. and C. Briffox. (2003) Influence de l’ensemencement en bacteries lactiques sur l’evolution de la couleur des vins de Pinot Noir pendant l’elevage. Revue des Înologues. 103:19-23.

Guilloux-Benatier, M. and M. Feuillat. (1991) Utilisation d’adjuvants d’origine levurienne pour améliorer l’ensemencement des vins en bactéries sélectionnées. Rev. Fr. Oenol. 132:51-55.

King, S.W. and R.B. Beelman. (1986) Metabolic interactions between Saccharomyces cerevisiae and Leuconostoc oenos in a model grape juice/wine system. Am. J. Enol. Vitic. 37(1): 53-60.

Morenzoni, R. and K. Scully Specht (Eds.). (2005) Malolactic Fermentation in Wine: Understanding the Science and the Practice. Lallemand Inc.