Controlling Microbes at Crush with Chitosan in Cabernet Franc
Kirsty Harmon
Blenheim Vineyards
Summary
The 2018 vintage in Virginia was marked with heavy rainfall leading to damaged fruit and potentially heavier microbial loads on the fruit. Though many winemakers used ample SO2 at crush, wines produced from this vintage show higher than normal levels of volatile acidity. One option for combatting microbial spoilage during fermentation is chitosan. Chitosan is a naturally occurring molecule that has been used at nearly every stage of winemaking to bind to microbial cell walls and cause disruption, leakage and sedimentation. In this experiment, chitosan was used to treat one half of a lot of Cabernet Franc while the other remained untreated. There were no notable differences in volatile acidity nor microbial load between treatments in the finished wine. However, overall microbial load was lower in 2019 than 2018. Chitosan may be an effective companion to SO2 in high microbe years.
Introduction
In 2018, Blenheim Vineyards noticed surprisingly high levels of microbial counts in finished wine, despite using 50 mg/L SO2 at crush. High microbial load can be the source of volatile acidity and other spoilage during aging. One likely source of microbial diversity are the grapes themselves.
Grapes enter the winery covered in microbes from the vineyard. To date, 52 different species of yeast from 22 different genera have been identified on grapes including Hanseniospora (AKA Klockera), Candida, Pichia, Hansenula, Metschnikowia, Sporoblomyses, Cryptococcus, Thodotorula, and Aurobasidium1. The cast of characters changes as grapes ripen, with the greatest abundance of microbes present in the last few weeks1. Healthy grapes are generally inhospitable environments for any microbe because they are covered in plates of wax that form a cuticle to hold in nutrition and repel water. Microbes cluster around the stomata or next to cracks in the cuticle where seepage from the openings provides both water and nutrients. The overall microbial load on grapes depends on environmental factors such as climate, altitude, variety, age of grapes, disease pressure and vineyard practices. Fog, rain, and fruit damage (like that seen in wet vintages like 2018) quickly transform the microbial desert of grape skins into a microbial oasis. Cells that are present in small numbers quickly multiply when given the chance1. For example, Botrytis infection can increase the overall abundance of microbes by 1000x. Grapes with sour rot have significantly higher microbial diversity and abundance2. Insect pressure will also increase abundance due to increased vectoring from diverse environments3. The overall inoculant of non-Saccharomyces yeast and bacteria coming into the winery from the vineyard on the grapes is often larger than the inoculant of selected Saccharomyces yeast added at the beginning of fermentation1.
Non-Saccharomyces yeast have several impacts on the wine, both positive and negative. Klockera apiculata (aka Hanseniaspora uvarum) is a common member of the non-Saccharomyces yeast community found on grapes1,4. This yeast strain is easily identified under a microscope by its lemon shaped cells. It is tolerant to up to 100 mg/L SO2, can grow at low temperature (such as that found during cold soak), and can produce both acetic acid and ethyl acetate (which smells like nail polish remover) under aerobic conditions3. Other offenders in the non-Saccharomyces yeast community include Pichia guilliemondii, a film forming yeast prevalent in warm conditions when fermentation is delayed. This yeast can form spores that become resident in barrels and produce ethyl acetate and 4 ethyl phenol (which can smell like band-aid, wet dog, horse sweat)1–3.
In addition to yeast, many spoilage bacteria also come into the winery on grapes. Sour rot and Botrytisincrease the prevalence of Acetobacter, Gluconobacter and Gluconacetobacter, all of which produce acetic acid. Several Lactobacillus species (hilgardii, plantarum, casei) and Pediococcus (damnosus) are also residents of mature grapes. These can produce acetic acid, mousy flavor and biogenic amines (which have names like putrescine and cadaverine…). They may also produce polysaccharides that lead to ropy texture1,3,5.
High levels of native flora may also cause nutrient depletion early in fermentations, limiting nutrient availability for Saccharomyces, potentially leading to stuck fermentations6,7. In a study of nutrient depletion by non-Saccharomyces yeast species, Medina et al (2012)6 found that Metchnikowia, a non-Saccharomycesyeast strain present in potentially high numbers on grapes8, consumed YAN quickly in fermentation. Mimicking what may be occurring in fermentations with cold soaking or later inoculation, sequential inoculation with Metchnikowia followed by Saccharomyces led to stuck fermentations that could be resolved with nutrient addition. In the same study, Hanseniaspora, another prevalent member of the grape microbiota, did not show large YAN depletion (90% of the YAN remained 3 days after inoculation with this species), however it did deplete thiamine, an essential vitamin for Saccharomyces. Excessive use of SO2, as would occur in vintages with high microbial load, also leads to reduction in thiamine, further increasing the potential for stuck fermentations.
Despite the risks, there are also some benefits to having a rich microbial community early in fermentation. Several non-Saccharomyces yeast species have been shown to produce positive compounds that add complexity to wine aroma such as esters, higher alcohols, glycerol, succinic acid and thiols. Proteases produced by non-Saccharomyces yeast have been shown to break down cells and add nutrients, ultimately making a more protein stable wine. Some produce glycosidases that help unmask aromas compounds that are bound to sugar molecules. Others produce enzymes to break down polysaccharides that would otherwise inhibit clarification and filtration. Lachanacea thermotolerans has been shown to consume acetic acid, reducing volatile acidity1,8,9. It is likely these are some of the mechanisms that occasionally lead winemakers to employ ambient fermentations.
Many of our winemaking decisions affect the abundance and diversity of the microbial community present at the beginning of fermentation. Mechanical harvesting and long transport times, especially at warm temperatures, can lead to high microbial load8,10. As soon as the grapes are crushed, nutrients are released to feed the organisms that are present. Klockera (Hanseniaspora) is often the most abundant species on the grapes , and remains prevalent until alcohol levels rise above 4-7% and oxygen is used up3,10. The low pH environment of the juice, rising alcohol, rising temperatures, and presence of phenolics also tends to inhibit spoilage organisms in early fermentation. Harvesting of wet grapes, prolonged cold soak, cool fermentation conditions, low inoculant of yeast, and lack of clarification (for white wines) can all lead to higher counts of yeast and bacteria in the fermentation3,11
In wet vintages such as 2018, the prevalence of damaged berries and wet grapes likely increased the inoculant of non-Saccharomyces microbes in fermentation and may have contributed to overall higher volatile acidity in wines that year. One approach to microbial management is to use higher than normal levels of SO2. Though SO2 has efficacy against some microbial spoilage, many of these microbes (such as Hanseniaspora) have high tolerance to it. Much of the SO2 added at crush is lost as it binds to grape solids that are prevalent in red wine fermentations, making it less effective. High SO2 additions can also bind thiamine and slow down or halt fermentation, and may even select for SO2 tolerant microbes that will cause spoilage during aging12,13. Still, fermentations that have some SO2 added at crush do tend to have faster onset of fermentation and steadier kinetics (Egli et al 1998).
When SO2 isn’t enough, or when you want to limit SO2 for other reasons, another option for combatting microbial spoilage during fermentation is chitosan. Chitosan is a naturally occurring molecule that can also be produced by the de-acetylation of chitin using NaOH or chitinase enzymes14. Chitin is the second-most common polymer found in nature (after cellulose), making up the cell walls of fungi and shells of crustaceans and thus readily available as a renewable resource5. In winemaking applications, chitin from Aspergillus nigeris used as the source for chitosan. The effectiveness of any given formulation of chitosan depends on its molecular weight, deacetylation degree and the pH of the medium15. Lower molecular weight, higher degree of deacetylation is the favored formulation for antimicrobial purposes16. At juice and wine pH, chitosan is very positively charged, which increases efficacy16.
Different microbes bind chitosan to different degrees. Chitosan binding to cell walls is driven by chemical properties of the cell wall itself, with high degree of correlation to the hydrophilicity of the wall17. Gram negative bacteria are more susceptible to binding than gram positive17. Chitosan has been shown to have some efficacy against a wide range of grapevine and wine microbes including downy mildew18, powdery mildew19, Phomopsis20, Lactobacillus, Oenococcus, and Brettanomyces17. Due to its versatility as an antimicrobial agent, chitosan in various forms has been used worldwide at nearly every stage of wine production including vineyard applications, on grapes during transport and storage, at crush, after fermentation and during the aging of wine16,21,22.
Many different mechanisms of antimicrobial action have been proposed for chitosan in wine. Chitosan has been shown to physically bind to the cell walls of microbes15,17. Binding may aid in sedimentation, leading to reduction in overall cell number with racking. Binding of the positively charged molecular of chitosan may also disrupt cell membranes, leading to leakage of ATP, potassium, and proteins, all essential components of cell function15,17. Leakage may therefore result in semi-viable cells or cell death. Other proposed mechanisms include the physical blocking of cell permeability by chitosan binding, chelation of survival factors such as copper, penetration of the cell membrane and binding to DNA15.
Regardless of the mechanism, chitosan has been shown to be quite effective in treating microbial infection of wine. When treating wine already inoculated with Brettanomyces bruxellensis, Taillander et al (2014) found that 85% of the population of Brettanomyces cells were dead within 20 hours of treatment. There was a dose effect in the rate of cell death, with 0.4 g/L treatment killing cells faster than 0.04 g/L. They also found differences in susceptibility based on the strain of Brettanomyces used, presumably due to genetic differences in cell wall components. Many cells initially compromised by chitosan treatment recovered, as evidenced by growth in the Brettanomyces population after 7 days. Other researchers tested the effect of 0.04 g/L chitosan on aging wine and found that, even at low dose, aging of wine on chitosan helped prevent infection by Brettanomyces23.
Most chitosan products are recommended for use in finished wine. Stab Micro M (Enartis) is a chitosan-based product specially formulated for use on juice and must to reduce the activity of a wide range of microbes, leading to reduction in volatile acidity, sulfide defects, volatile phenols and production of other off-flavors. It can be used in conjunction with SO2 or as a replacement, depending on the condition of grapes and preference of the winemaker16. In this experiment, a single harvest lot of Cabernet Franc was split so that half received treatment with chitosan while the other half did not. Both bins received SO2. Resulting chemistry and microbial load were assessed post fermentation and after aging.
Methods
At the time of harvest, fruit was divided evenly and randomly among four TBins (two each received treatment and control). Cabernet Franc was made according to the winery’s standard protocol. Grapes were picked, chilled overnight, sorted, destemmed, and loaded into TBins with 50 mg/L SO2. There were two TBins per treatment. All bins received SO2. Stab Micro M was sprinkled in layers into the treatment at the de-stemmer at alternative times with SO2. Tbins were moved to a temperature-controlled warehouse and inoculated at a rate of 15g/hL of EC1118 yeast the following day. Tartaric acid (1.5 g/L) was added after the end of lag phase (on day 4 after destemming). Fermentations were monitored for Brix and temperature and punched down twice daily throughout the fermentation until Brix were below -1.5. Both Tbins per treatment were pressed together 10 days after processing, after the completion of alcoholic fermentation, with free run and press fractions combined. Pressed wine was allowed to settle, racked, inoculated with 1 g/hL ML One (Enartis), then transferred to barrel. Malic acid depletion was checked after three weeks and confirmed dry with enzymatic analysis. Tartaric acid (2 g/L) and SO2 (75 ppm) were added without racking. Two TBins of each treatment were pressed together. Each treatment had three finished barrels of the same cooperage, age, and forest after pressing, which were maintained separately until the time of sampling.
Results
Fruit was harvested at 22.9°Bx with pH of 4.07. Fermentation proceeded normally with no noticeable differences among TBins (data not shown). After completion of primary fermentation, there were no differences in pH or malic acid (Table 1). After completion of malolactic fermentation, all malic acid was <0.10 g/L and glucose/fructose was <1.0 g/L. General chemistry was similar for all barrels in both lots, with no difference in volatile acidity (Table 2), color (Figure 1) or microbiology (Table 3).
Preliminary Conclusions
This experiment was initiated to address higher than acceptable microbial loads found in 2018 wines. The rationale was that SO2 may not be enough to control microbial growth and additional measures were needed. The overall microbial load on grapes varies with vintage, variety, and region, an effect seen here.
When compared with overall microbial loads from 2018, both treatment and control lots in 2019 had notably lower levels of spoilage organisms (Table 3). Lower levels of acetic acid bacteria, Brettanomyces, and lactic acid bacteria can significantly reduce risk of spoilage during longer aging. In 2019, the winery’s normal dose of SO2 (50 ppm at crush) was sufficient to control populations of spoilage organisms. However, in 2018 it was not. Though this experiment showed few differences in 2019, chitosan may still be a valuable tool to augment SO2 in wet or otherwise compromised vintages.
Table 1: Wine chemistry following primary fermentation for two treatments of Cabernet Franc (in-house lab)
Table 2: Wine chemistry after malolactic fermentation and aging for two treatments of Cabernet Franc (ICV labs)
Figure 1: Color intensity for two treatments of Cabernet Franc (ICV Labs)
Table 3: Microbiology after aging for two treatments of Cabernet Franc (ETS Labs)
References
(1) Jackson, R. S. Wine Science: Principles and Applications, 4 edition.; Academic Press: Amsterdam, 2014.
(2) Barata, A.; Malfeito-Ferreira, M.; Loureiro, V. The Microbial Ecology of Wine Grape Berries. International Journal of Food Microbiology 2012, 153 (3), 243–259.
(3) Wine Microbiology. UC Davis Viticulture and Enology.
(4) Diversity of Wine Yeasts | Viticulture and Enology https://wineserver.ucdavis.edu/industry-info/enology/wine-microbiology/yeast-mold/diversity-wine-yeasts (accessed 2018 -11 -13).
(5) García, M. E. R.; Esteve-Zarzoso, B.; Arroyo, T. Non-Saccharomyces Yeasts: Biotechnological Role for Wine Production; 2016.
(6) Medina, K.; Boido, E.; Dellacassa, E.; Carrau, F. Growth of Non-Saccharomyces Yeasts Affects Nutrient Availability for Saccharomyces Cerevisiae during Wine Fermentation.
(7) Bisson, L. F. Stuck and Sluggish Fermentations. American Journal of Enology and Viticulture 1999, 50(1), 107–119.
(8) Jolly, N. P.; Varela, C.; Pretorius, I. S. Not Your Ordinary Yeast: Non- Saccharomyces Yeasts in Wine Production Uncovered. FEMS Yeast Res 2014, 14 (2), 215–237.
(9) Vilela, A. Lachancea Thermotolerans, the Non-Saccharomyces Yeast That Reduces the Volatile Acidity of Wines. Fermentation 2018, 4 (3), 56.
(10) Ciani, M.; Domizio, P. Controlled Mixed Culture Fermentation: A New Perspective on the Use of Non- Saccharomyces Yeasts in Winemaking. FEMS Yeast Research.
(11) Ciani, M.; Comitini, F.; Mannazzu, I.; Domizio, P. Controlled Mixed Culture Fermentation: A New Perspective on the Use of Non-Saccharomyces Yeasts in Winemaking. FEMS Yeast Res. 2010, 10 (2), 123–133.
(12) Ndlovu, T.; Divol, B.; Bauer, F. F. Yeast Cell Wall Chitin Reduces Wine Haze Formation. Appl. Environ. Microbiol. 2018, 84 (13).
(13) Ciani, M.; Comitini, F. Yeast Interactions in Multi-Starter Wine Fermentation. Current Opinion in Food Science 1, 1–6.
(14) O’Kennedy, K. Chitin, Chitinase, Chitosan ... Wineland Magazine, 2019.
(15) Taillandier, P.; Joannis‐Cassan, C.; Jentzer, J.-B.; Gautier, S.; Sieczkowski, N.; Granes, D.; Brandam, C. Effect of a Fungal Chitosan Preparation on Brettanomyces Bruxellensis, a Wine Contaminant. Journal of Applied Microbiology 2015, 118 (1), 123–131.
(16) Barrett, L. Microbial Stability and Control: EnartisStab Micro (Chitosan) Application during Wine Maturation, 2019.
(17) Chung, Y.; Su, Y.; Chen, C.; Jia, G.; Wang, H.; Wu, J. C. G.; Lin, J. Relationship between Antibacterial Activity of Chitosan and Surface Characteristics of Cell Wall. Acta Pharmacol Sin 2004, 5.
(18) Romanazzi, G.; Mancini, V.; Feliziani, E.; Servili, A.; Endeshaw, S.; Neri, D. Impact of Alternative Fungicides on Grape Downy Mildew Control and Vine Growth and Development. Plant Disease 2016, 100 (4), 739–748.
(19) Iriti, M.; Vitalini, S.; Di Tommaso, G.; D’Amico, S.; Borgo, M.; Faoro, F. New Chitosan Formulation Prevents Grapevine Powdery Mildew Infection and Improves Polyphenol Content and Free Radical Scavenging Activity of Grape and Wine: Grape Powdery Mildew Control by Chitosan. Australian Journal of Grape and Wine Research 2011, 17 (2), 263–269.
(20) Nascimento, T.; Rego, C.; Oliveira, H. Potential Use of Chitosan in the Control of Grapevine Trunk Diseases. Phytopathologia Mediterranea 2007, 46 (2), 8.
(21) Singh, R. K.; Martins, V.; Soares, B.; Castro, I.; Falco, V. Chitosan Application in Vineyards (Vitis Vinifera L. Cv. Tinto Cão) Induces Accumulation of Anthocyanins and Other Phenolics in Berries, Mediated by Modifications in the Transcription of Secondary Metabolism Genes. IJMS 2020, 21 (1), 306.
(22) Singh; Soares; Goufo; Castro; Cosme; Pinto-Sintra; Inês; Oliveira; Falco. Chitosan Upregulates the Genes of the ROS Pathway and Enhances the Antioxidant Potential of Grape (Vitis Vinifera L. ‘Touriga Franca’ and ’Tinto Cão’) Tissues. Antioxidants 2019, 8 (11), 525.
(23) Bağder Elmaci, S.; Gülgör, G.; Tokatli, M.; Erten, H.; İşci, A.; Özçelik, F. Effectiveness of Chitosan against Wine-Related Microorganisms. Antonie Van Leeuwenhoek 2015, 107 (3), 675–686.
