Progress 07/01/01 to 06/30/04
Outputs
A comprehensive and systematic research approach has been applied to minimize problem alcoholic fermentations. Specifically, the laboratory is investigating how changes in availability of one nutrient important for yeast growth (e.g., assimilable nitrogen) affects the cells need for other nutrients such as vitamins. To do this, a chemically defined fermentation medium was developed based on the amino acid composition of Cabernet Sauvignon grape musts from Washington. The project initially focused on two vitamins, biotin and pantothenic acid, given their roles in the metabolism of Saccharomyces. Factorial designs (2 x 3) were employed with the concentrations of yeast assimilable nitrogen or YAN (60 and 250 mg/l) and biotin (0, 1, or 10 ug/l) or pantothenic acid (10, 50, and 250 ug/l) as variables. Yeast growth and H2S production were significantly influenced by biotin, pantothenic acid, and nitrogen, synthesis of other molecules important for odor and flavor (esters,
alcohols, etc.) were also altered. Alcoholic fermentations were induced in a synthetic grape juice using different yeast strains. During primary fermentation, samples were periodically removed, sterile filtered, and inoculated with O. oeni to induce malolactic fermentation (MLF). Prior to inoculation with O. oeni, some samples were treated with a protease or a concentrated solution of nutrients. S. cerevisiae strain V1116 produced the highest levels of SO2 during fermentation and these media were highly inhibitory to MLF. The addition of nutrients did not lessen the inhibition; however, MLF was enhanced in protease-treated samples removed two days after the start of alcoholic fermentation. Addition of the protease did not affect MLF in media fermented by other yeast strains, namely Saint Georges or EC1118. Despite similar amounts of total SO2 produced, MLF was highly inhibited in media fermented by RubyFERM but not EC1118. The addition of nutrients to the RubyFERM fermented media did
not decrease bacterial inhibition.
Impacts
The yeast nutrient study has important implications for the winemaking industry where a better understanding of the nutritional requirements of Saccharomyces is necessary to reduce fermentation problems and to improve final product quality. One important finding of the research was that H2S was increased with an increase of available nitreogen under conditions of pantothenic acid deficiency (10 to 250 ug/L). This finding may help explain why some wineries experience an increase in the amount of H2S evolution when extra nitrogen is added. Future studies will focus on the role of temperature towards H2S formation. Regarding interactions between Saccharomyces and Oenococcus, the findings suggest that the inhibition of MLF by S. cerevisiae is caused by a number of mechanisms including the synthesis of SO2 and/or antibacterial peptides/proteins. Isolation and characterization of these antibacterial peptides/proteins will potentially allow natural inhibition of MLF when
undesired. Alternatively, yeasts with better compatibilities with malolactic bacteria can be found so as to allow simultaneous alcoholic and malolactic fermentations.
Publications
- Larsen, J.T., J.C. Nielsen, B. Kramp, M. Richelieu, M.J. Riisager, N. Arneborg, and C.G. Edwards. 2003. The inhibitory effect of Saccharomyces cerevisiae on malolactic fermentation by Oenococcus oeni. Am. J. Enol. Vatic. 54:246-251.
- Lu, G., J.K. Fellman, C.G. Edwards, D.S. Mattinson and J. Navazio. 2003. Biosynthetic origin of geosmin in red beets (Beta Vulgaris L.). J. Agric. Food Chem. 51:126-129.
- Lu, G., J.K. Fellman, C.G. Edwards, D.S. Mattinson, and J. Navazio. 2003. Quantitative determination of geosmin in red beets (Beta vulgaris L.) using headspace solid-phase microextraction. . J. Agric. Food Chem. 51:1021-1025.
- Wang, X.D., J.C. Bohlscheid and C.G. Edwards. 2003. Fermentative activity and production of volatile compounds by Saccharomyces grown in synthetic grape juice media deficient in assimilable nitrogen and/or pantothenic acid. J. Appl. Microbiol. 94:1-11.
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Progress 01/01/03 to 12/31/03
Outputs
A comprehensive and systematic research approach has been applied to minimize problem alcoholic fermentations. Specifically, the laboratory is investigating how changes in availability of one nutrient important for yeast growth (e.g., assimilable nitrogen) affects the cells need for other nutrients such as vitamins. To do this, a chemically defined fermentation medium was developed based on the amino acid composition of Cabernet Sauvignon grape musts from Washington. The project initially focused on two vitamins, biotin and pantothenic acid, given their roles in the metabolism of Saccharomyces. Factorial designs (2 x 3) were employed with the concentrations of "yeast assimilable nitrogen" or YAN (60 and 250 mg/l) and biotin (0, 1, or 10 ug/l) or pantothenic acid (10, 50, and 250 ug/l) as variables. Yeast growth and H2S production were significantly influenced by biotin, pantothenic acid, and nitrogen, synthesis of other molecules important for odor and flavor (esters,
alcohols, etc.) were also altered. Alcoholic fermentations were induced in a synthetic grape juice using different yeast strains. During primary fermentation, samples were periodically removed, sterile filtered, and inoculated with O. oeni to induce malolactic fermentation (MLF). Prior to inoculation with O. oeni, some samples were treated with a protease or a concentrated solution of nutrients. S. cerevisiae strain V1116 produced the highest levels of SO2 during fermentation and these media were highly inhibitory to MLF. The addition of nutrients did not lessen the inhibition; however, MLF was enhanced in protease-treated samples removed two days after the start of alcoholic fermentation. Addition of the protease did not affect MLF in media fermented by other yeast strains, namely Saint Georges or EC1118. Despite similar amounts of total SO2 produced, MLF was highly inhibited in media fermented by RubyFERM but not EC1118. The addition of nutrients to the RubyFERM fermented media did
not decrease bacterial inhibition.
Impacts
The yeast nutrient study has important implications for the winemaking industry where a better understanding of the nutritional requirements of Saccharomyces is necessary to reduce fermentation problems and to improve final product quality. One important finding of the research was that H2S was increased with an increase of available nitreogen under conditions of pantothenic acid deficiency (10 to 250 ug/L). This finding may help explain why some wineries experience an increase in the amount of H2S evolution when extra nitrogen is added. Future studies will focus on the role of temperature towards H2S formation. Regarding interactions between Saccharomyces and Oenococcus, the findings suggest that the inhibition of MLF by S. cerevisiae is caused by a number of mechanisms including the synthesis of SO2 and/or antibacterial peptides/proteins. Isolation and characterization of these antibacterial peptides/proteins will potentially allow natural inhibition of MLF when
undesired. Alternatively, yeasts with better compatibilities with malolactic bacteria can be found so as to allow simultaneous alcoholic and malolactic fermentations.
Publications
- Edwards, C.G. and L. Petersen. 2003. Controlling malolactic fermentation. Vinarsky obzor (Czech Republic), 96(9): 409-410.
- Larsen, J.T., J.C. Nielsen, B. Kramp, M. Richelieu, M.J. Riisager, N. Arneborg, and C.G. Edwards. 2003. The inhibitory effect of Saccharomyces cerevisiae on malolactic fermentation by Oenococcus oeni. Am. J. Enol. Vitic. 54: 246-251.
- Lu, G., C.G. Edwards, J.K. Fellman, D.S. Mattinson, and J. Navazio. 2003. Biosynthetic origin of geosmin in red beets (Beta vulgaris L.). J. Agric. Food Chem. 51: 1026-1029.
- Lu, G., J.K. Fellman, C.G. Edwards, D.S. Mattinson, and J. Navazio. 2003. Quantitative determination of geosmin in red beets (Beta vulgaris L.) using headspace solid-phase microextraction. J. Agric. Food Chem. 51: 1021-1025.
- Wang, X.D., J.C. Bohlscheid, and C.G. Edwards. Fermentative activity and production of volatile compounds by Saccharomyces grown in synthetic grape juice media deficient in assimilable nitrogen and/or pantothenic acid. J. Appl. Microbiol. 94: 1-11 (2003).
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Progress 01/01/02 to 12/31/02
Outputs
The impact of various nutrients on growth of Saccharomyces under vinification conditions is being studied. This medium is being used to determine if there is a relationship between the yeasts' need for key vitamins and available nitrogen. It is well known that musts from WA are deficient in nitrogen and possibly other nutrients. Conditions that favor H2S production are currently being investigated as well as its biochemical source during fermentation. A new part to the project has been initiated analyzing different grape cultivars to determine concentrations of biotin and pantothenic acid present in musts. Samples from commercial grape vineyards have been obtained and analysis of biotin and pantothenic acid have been initiated. Additional research is being conducted involving malolactic fermentation of wines to attempt to identify to anti-bacterial factor produced by some yeasts that inhibits this important secondary fermentation. While yeasts produce various
quantities of SO2, the antibacterial form of SO2 (free SO2) was only present in minor amounts. Results indicate that SO2 is probably involved in the inhibition of Oenococcus by yeast but that other factors are present.
Impacts
Better understanding of the nutritional needs of yeast during fermentaton can yield alternative methods to improve rate of fermentation and overall quality of wines from Washington state. Successful inducement of malolactic fermentation is also important to regional vintners.
Publications
- Abonyi, B.I., Feng, H., Tang, J., Edwards, C.G., Chew, B.P., Mattinson, D.S., Fellman, J.K. 2002. Quality retention in strawberries and carrots dried with refractance window system, J. Food Science 67(2):1051-1056.
- Connell, L., H. Stender, and C.G. Edwards. 2002. Rapid detection of Brettanomyces from winery air samples based on peptide nucleic acid analysis. Am. J. Enol. Vitic. 53(4): 322-324.
- Reynolds, A.G., C.G. Edwards, M.A. Cliff, J.H. Thorngate III, and J.C. Marr. 2002. Evaluation of yeast srains during fermentation of Riesling and Chenin blanc musts. Am. J. Enol. Vitic. 54: 336-344.
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Progress 01/01/01 to 12/31/01
Outputs
Using a synthetic grape juice medium that mimics nutritional conditions found in WA musts, growth of Saccharomyces under different conditions is being studied. This medium will be used to determine if there is a relationship between the yeasts' need for biotin or pantothenic acid (key vitamins) and available nitrogen. It is well known that musts from WA are deficient in nitrogen and possibly other nutrients. Research has indicated that wine yeast (Saccharomyces) can not synthesize these key nutrients. Using the synthetic grape juice, the rate of fermentation and yeast growth seem more dependent on available nitrogen rather than biotin or pantothenic acid. However, the presence of these vitamins greatly impacts production of H2S. H2S was greatly reduced if panthenic acid and nitrogen were in abundance. Conditions that favor H2S production are currently being investigated as well as its biochemical source during fermentation. A new part to the project has been initiated
analyzing different grape cultivars to determine concentrations of biotin and pantothenic acid present in musts. Additional research is being conducted involving malolactic fermentation of wines to attempt to identify to anti-bacterial factor produced by some yeasts (and not others) that inhibits this important secondary fermentation. The composition of the synthetic grape juice medium is being altered.
Impacts
Better understanding of the nutritional needs of yeast during fermentation can yield alternative methods to improve rate of fermentation and overall quality of wines from Washington state. Successful inducement of malolactic fermentation is also important to regional vinters.
Publications
- Folwell, R.J., T.A. Bales, and C.G. Edwards. 2001. Cost economics and economic impacts of pricing and product mix decisions in premium table wine wineries. J. Wine Res. 12(2): 111-124.
- Semon, M.J., C.G. Edwards, D. Forsyth, and C. Dinn. 2001. Inducing malolactic fermentation in Chardonnay musts/wines from Washington state using different strains of Oenococcus oeni. Aust. J. Grape Wine Res. 7: 52-59.
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Progress 01/01/00 to 12/31/00
Outputs
Fermentations have been induced in a synthetic grape juice medium developed last year that mimics nutritional conditions found in WA musts. This medium will be used to determine if there is a relationship between the yeast's need for biotin or pantothenic acid (key vitamins) and available nitrogen. It is well known that musts from WA are deficient in nitrogen and possibly other nutrients such as these vitamins. Research has indicated that wine yeast (Saccharomyces) can not synthesize these key nutrients. Using the synthetic grape juice, the rate of fermentation and yeast growth seem more dependent on available nitrogen. However, the presence of these vitamins does greatly influence production of H2S, a highly undesirable volatile produced by yeast. Conditions that favor H2S production are currently being investigated as well as its biochemical source during fermentation. Additional research is being conducted involving malolactic fermentation of wines to attempt to
identify to anti-bacterial factor produced by some yeasts (and not others) that inhibits this important secondary fermentation.
Impacts
A better understanding of the nutritional needs of yeast during fermentation can yield alternative methods to improve rate of fermentation and overall quality of wines from Washington state. Research to improve the inducement of the secondary fermentation of wines involving bacteria ("malolactic bacteria") is very important because most wineries in the Pacific Northwest rely on this fermentation to improve wine quality.
Publications
- Bohlscheid, J.C., C.G. Edwards, J.K. Fellman, and D. Ansen. 2000. Interactions between biotin and nitrogen source on yeast growth and metabolism. Presented at the American Society for Enology and Viticulture Annual Meeting, Seattle, WA June 20-23 (2000). Abstract.
- Edwards, C.G., M.D. Collins, P.A. Lawson, and A.V. Rodriguez. 2000. Lactobacillus nagelii sp. nov., an organism isolated from a partially fermented wine. Int. J. Sys. Evol. Microbiol. 50(2): 699-702 (2000).
- Holley, S.L., C.G. Edwards, J.H. Thorngate, J.K. Fellman, D.S. Mattinson, E.J. Sorensen, and R.H. Dougherty. 2000. Chemical characterization of different lines of Daucus carota L. roots. J. Food Qual. 23(5): 487-502 (2000).
- Mills, J.M., J.C. Bohlscheid, J. Morrison, S. Andrews, D.S. Mattinson, J.K. Fellman, and C.G. Edwards. 2000. Effect of post-harvest storage methods on fermentation rate and wine quality of Riesling grapes. Presented at the American Society for Enology and Viticulture Annual Meeting, Seattle, WA June 20-23 (2000). Abstract.
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Progress 01/01/99 to 12/31/99
Outputs
A synthetic grape juice medium has been developed that mimics nutritional conditions found in WA musts. This medium will be used to determine if there is a relationship between the yeast's need for biotin (a key vitamin) and available nitrogen. It is well known that musts from WA are deficient in nitrogen and possibly biotin. Preliminary fermentations using this medium indicated differences in the rate of fermentation and the production of volatile compounds such as higher alcohols and esters important for quality. Additional commercial wines undergoing sluggish fermentations were collected 1998-99 for isolation of spoilage bacteria. A total of 90+ strains of lactic acid bacteria were isolated from 14+ wines. Of these strains, five were found to possess antifungal activities and were later identified as being Lb. vermiforme (Lb. hilgardii). Novel species of Lactobacillus isolated last year has been proposed to be named Lb. nagelii, after Dr. Chas W. Nagel (WSU
emeritus professor). Research is continuing to identify the causative species and mechanisms of inhibition, in order to lead to winery procedures that minimize the problem as well as re-start fermentations.
Impacts
A better understanding of the nutritional needs of yeast during fermentation can yield alternative methods to improve the rate of fermentation and overall quality of wines from Washington state. Research involving bacteria like Lactobacillus will develop strategies to limit spoilage, thus improving quality of wines produced.
Publications
- Bohlscheid*, J.C., C.G. Edwards, J.F. Fellman, D.S. Mattinson, and S.E. Spayd. 1999. Relationship between nitrogen and biotin on the fermentation of a model wine system. Presented at the Northwest Chapter of the American Society for Enology and Viticulture, Stevenson, WA July 18-20.
- Edwards, C.G. 1999. Enology research update. Wine and Grape Research News. Washington State University. 10(1): 4-5.
- Edwards, C.G. 1999. Ferocious lactobacilli infections in wines: a research story. Presented to the Department of Food Science and Human Nutrition, Washington State University, Pullman, WA, February 3.
- Edwards, C.G., A.G. Reynolds, A.V. Rodriguez, M. Semon*, and J. Mills*. 1999. Implication of acetic acid in the induction of slow/stuck grape juice fermentation and inhibition of yeast by Lactobacillus sp. Am. J. Enol. Vitic. 50: 204-210.
- Edwards, C.G., A.V. Rodriguez, and J. Mills*. 1999. Slow/stuck alcoholic fermentations in wines from the Pacific Northwest. To be published in: Proceedings of the Northwest Center for Small Fruits Research. Portland, OR.
- Edwards, C.G., K.M. Haag*, M.J. Semon*, A.V. Rodriguez, and J. Mills*. 1999. Evaluation of processing methods to control the growth of Lactobacillus kunkeei, a microorganism implicated in sluggish alcoholic fermentations of grape musts. S. Afr. J. Enol. Vitic. 20(1): 11-19.
- Edwards, C.G. 1999. Basic wine microbiology workshop: Techniques and procedures. 27 pgs. Presented as part of the Basic Wine Microbiology Workshop, Pasco, WA, March 30.
- Folwell, R.J., T.A. Bales, and C.G. Edwards. 1999. Cost of investment and operatin in various sizes of premium table wine wineries in Washington. Submitted for publication, Washington State University, Pullman, WA.
- Semon*, M.J., and C.G. Edwards. 1999. Inducing malolactic fermentation in Chardonnay musts/wines from Washington State using different strains of Oenococcus oeni. Presented at the American Society for Enology and Viticulture Annual Meeting, Reno, NV June 30-July 2.
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Progress 01/01/98 to 12/31/98
Outputs
MLF was induced in a commercially-prepared Chardonnay juice using different strains of Oenococcus oeni. These strains were inoculated during the early, middle or late phases of the alcoholic fermentation. While most strains successfully induced MLF, some strains did not grow well in the starter culture (MT01) or in the inoculated wine (MCW). Experimental results are in agreement with experiments conducted at a commercial winery. Acetic acid was sequentially added to Chardonnay fermentations inoculated with Prise de Mousse. Although fermentations with acetic acid were slowed, yeast viabilities were not different from those fermentations without the acid. These findings do not support the hypothesis that bacterial production of acetic acid is the causative agent for yeast antagonism. Results from agar well assays indicate other mechanisms for yeast inhibition. Additional commercial wines undergoing sluggish fermentations were collected in 1997-98 for isolation of
spoilage bacteria. A total of 30+ strains of lactic acid bacteria were isolated from 16+ wines. One of these strains, LuE10, was characterized for its biochemical traits and 16S rRNA pattern. The results indicate that this microorganism is another novel species of Lactobacillus. Research is continuing to identify the causative species and mechanisms of inhibition in order to lead to winery procedures that minimize the problem as well as re-start fermentations.
Impacts
(N/A)
Publications
- Edwards, C.G., Haag, K.M., Collins, M.D., Hutson, R., and Huang, Y.C. 1998. Lactobacillus kunkeei sp. nov., a spoilage organism associated with grape juice fermentations. J. Appl. Microbiol. 84(5):698-702.
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Progress 01/01/97 to 12/31/97
Outputs
Bacterial strain YH-15, an organism associated with stuck alcoholic fermentations, has been identified as a novel species of Lactobacillus. Addition of acetic acid to fermentations of Riesling grape juice slowed but did not stick, in contrast to the hypothesis that excessive amounts of acetic acid cause stuck fermentations. To better evaluate the influence of acetic acid on yeast, minimum inhibitory concentrations (MIC) of the acid were determined for different yeast strains. Strains differed in their ability to grow in acetic acid with T73, CY3079, 71B, and UCD 522 being most tolerant (MIC=7-7.5 g/L) while strains EC1118, V-1116, UCD 595 and SB1 were most sensitive (MIC=4.5-6.0 g/L). Acetic acid was sequentially added to Chardonnay fermentations inoculated with Prise de Mousse. Although fermentations with acetic acid were slowed, yeast viabilities were not different from those fermentations without the acid. These findings are in agreement with the Riesling
fermentations. Additional commercial wines undergoing slow/sluggish fermentations have been collected for isolation of spoilage bacteria. Since not all species of Lactobacillus are involved in causing slow/sluggish fermentations, it is important to be able to identify the causative species and mechanisms of inhibition. This will lead to winery procedures that minimize the problem as well as re-start fermentations.
Impacts
(N/A)
Publications
- No publications reported this period
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Progress 01/01/96 to 12/30/96
Outputs
Significant effort was made to develop methods for biochemical characterization to identify the Lactobacillus strains (YH-15, YH-24, and YH-37) found to be inhibitory to yeast. Preliminary identification of strains YH-24 and YH-37 indicate that they are strains of Leuconostoc oenos (Oenococcus oeni) while no matches have been found for strain YH-15. Genetic analysis implies that the strain is a new species, although this finding has not been confirmed. Tolerances of the unknown strains of Lactobacillus to pH, temperatures, ethanol, and SO2 were determined to identify possible growh limitations in musts. YH-15, which previously exhibited the strongest inhibition against yeast, did not grow well at low pH, high alcohol, or in the presence of sulfites (>5 ppm total SO2). YH-24 and YH-37 were found to be more tolerant of these conditions. YH-15 produced excessive amounts of acetic acid in stuck fermentations last year, leading to speculation that acetic acid could cause
stuck alcoholic fermentations. However, sequential addition of acetic acid to a fermenting juice at concentrations similar to those produced by YH-15 did not result in a stuck fermentation. In fact, fermentation was only slightly delayed and became dry shortly after the control (without added acetic acid). These data indicate that acetic acid alone is not responsible for stuck fermentations as previously thought and another mechanism(s) of yeast inhibition is occurring. These experiments are being replicated this year.
Impacts
(N/A)
Publications
- HUANG, Y.-C., EDWARDS, C.G., PETERSON, J. C., AND HAAG, K.M. 1996. Relationship between sluggish fermentations of grape juice and the antagonism of yeast by lactic acid bacteria. Am. J. Enol. Vitic. 47:1-10.
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