- Research Bulletin -
Effect of Fluoride on Fermentation Rate and Population Density in Fourteen Strains of Saccharomyces sp.
by
V.L. Wahlstrom, K.C. Fugelsang and C.J. Muller
CATI Publication #960102
© Copyright January 1996, all rights reserved
INTRODUCTION
Fluoride-containing compounds such as the natural mineral cryolite or proprietary formulations such as Kryocide, have been shown to be unsurpassed in terms of efficacy and benefit/cost ratio for the control of serious vineyard pests such as grape leaf skeletonizer (Harrisina brillians) and omnivorous leaf roller (Platynota stultana).
Grape growing in the central San Joaquin Valley of California requires an effective method for controlling the above mentioned pests. In worst cases, depredation can be so severe and the quality of grapes produced without control so poor, that it may not be economically feasible to harvest a vineyard so affected. Thus, grape growers faced with this problem use one form of control or another; most use fluoride-containing compounds. Although alternatives exist for pest control, such as Dipel (Bacillus thuringiensis) and other methods, their cost is substantially greater.
Previously unpublished (6) and anecdotal information suggest that high levels of fluoride adversely affect fermentation rate. At low concentrations, it is known to be an effective inhibitor of glycolytic pathway enzymes including phosphoglucomutase and enolase (phosphopyruvate hydratase).
Furthermore, fluoride (at levels greater than 1 mg/L [50 uM] ) also adversely affects the efficacy of acid urease, (4, 5) an enzyme which is used to bring about hydrolysis of urea (the identified precursor of ethyl carbamate) prior to bottling.
This report summarizes a two-year study which was initially undertaken to survey several commonly available commercial yeasts with respect to fluoride sensitivity.
MATERIALS AND METHODS
Grape juice: French colombard grapes were harvested from Fresno-area vineyards which had not received fluoride treatment for at least two years. Juice chemistry is presented below:
At harvest, fruit was refrigerated at 7°C for 24 hours prior to crush. Crushing, dejuicing (without enzyme addition), and pressing followed standard winemaking protocol. Sulfur dioxide was added to combine free-run and press juice at 15 mg/L. One hundred eighty nine liters (189-L) of collected juice was cold-clarified (24 hrs at 7°C) without bentonite addition. After clarification, 3-Liter volumes of juice were transferred to previously sterilized 4-L glass fermenters. Fluoride was measured by ion selective electrode as described by De Baenst et. al., and Durst (1, 2).
Sample preparation: In 1992, five commercial active dry yeast strains (S. cervisiae) were compared. Using NaF, each lot was adjusted from baseline (control) of 0.17 mg/L (9.0 uM) to 2.5 (130 uM), 3.0 (160 uM), the international action level, and 3.5 mg/L (180 uM) fluoride. In 1993, a second group of yeasts was analyzed. In this case, however, fermentation rate was followed only at 3.0 mg/L (160 uM) fluoride versus the control lots (baseline fluoride concentration 0.20 mg/L [11 uM] ). The decision to use 3.0 mg/L as a comparative was based upon its acceptance as the U.S. drinking water standard as well as the OIV variance for U.S. wineries exporting into common market countries.
In each case, the control and all treated lots were prepared in triplicate. Juice was sterilized using dimethyldicarbonate (DMDC) at 350 mg/L (21° C). After 12 hr. (allowing for complete decomposition of excess DMDC), each lot was equilibrated to fermentation temperature (15° C) prior to yeast inoculation. A freshly rehydrated inoculum of each was added to juice at the supplier's recommended equivalent of 2.2 lbs. per 1,000 gallons. Fermentation rate was monitored by daily analysis of reducing sugar.
Cell counting method: Total cell titer in the juice was determined microscopically using a Neubauer cell-counting slide as described in Zeocklein et al. (7). Appropriately diluted aliquots were subsequently plated on YM-agar for determination of viable cell number. Viable cell inoculum ranged from 10-15 x 106 CFU/mL in each lot and its replicates. Reducing sugar analysis: During the course of fermentation, 5-mL aliquots of each lot and replicates thereof were collected daily and immediately frozen for later analysis of reducing sugar. Subsequently, analysis of reducing sugar for each sample was carried out using Lane-Eynon titration as described in Zoecklein, et al. (7).
Population Dynamics: Over the two-year study, five of the 14 yeast strains examined exhibited signs of protracted and/or stuck fermentations in the presence fluoride levels at 3.0 mg/L (160 uM). The two cultures that clearly stuck were not examined further. The three that exhibited some degree of fermentative stress were further studied with respect to changes in populations in the presence of increasing concentrations of fluoride. Montrachet was included in this study as a comparison.
This phase of the study was carried out in model juice solutions prepared using yeast extract (0.5% w/v, KH2PO4 (0.5% w/v). Hydrogen ion concentration (pH) was adjusted to 3.40 using a mixture (1:1) of tartaric and malic acids.
One Liter (1-L) volumes of model juice (prepared in duplicate) were transferred to 1,500-ml sterile Erlenmeyer flasks and fluoride (as NaF) added at levels of 1.0 (53 uM) and 3.0 mg/L (160 uM). Controls with no added fluoride were also prepared. Subsequently, DMDC was added to each lot at 350 mg/L.
Twenty-four hours later, active yeast starters were prepared according to protocol described above and inoculated accordingly. Initial cell titer was determined using the method described above and ranged from 15-20 x 106 cells/mL for each lot and its replicate. These were subsequently verified by plating diluted aliquots on YM agar. Fermentation was carried out at 13°C. Visual cell counts were made daily (as described above) until stationary phase growth was observed.
RESULTS AND DISCUSSION
Results suggest substantial variation among the 14 strains of wine yeasts with respect to fluoride sensitivity during fermentation (see Table 1). Further, among those sensitive strains studied, responses to fluoride varied. This is reflected both in fermentation rate and corresponding studies of population changes when challenged with fluoride. From the vintner's point of view, this sensitivity translates into longer fermentation times and, in some instances, stuck fermentations.
As would be expected, differences in fermentation rate upon fluoride challenge are reflected in relative yeast population densities. Even though fermentation rates may not appear to substantially differ, final population densities, in the presence of increased concentrations of fluoride, were observed to be lower than controls.
This study has demonstrated the negative impact of fluoride (at both 1 and 3 mg/L) on fermentation rates and final reducing sugar as well as on yeast population. If fermentation progress is followed by measuring reducing sugar, the effect of fluoride on some yeasts (e.g., Montrachet) is not obviously apparent. However, when fermentation progress is measured by monitoring population densities, significant inhibition is evidenced at both levels of fluoride (1 and 3 mg/L) as compared with the control. Other yeasts may exhibit similar behavior. Although not addressed in this study, results presented herein suggest that perhaps other known inhibitors of fermentation may act synergistically with fluoride to exacerbate fermentation problems. The interactive effects of fluoride and such inhibitors merits further study.
ACKNOWLEDGMENTS
The authors wish to thank the California Agricultural Technology Institute (CATI) and Lallemand for their generous support of this project.
REFERENCES
1. De Baenst, G., J. Martens, P. Van den Winkel and D.L. Massart. Determination De L'ion Fluorure Dans Le Vine Par Potentiometry. J. Pharm. Belg. 28:188-194. (1973).
2. Durst, R.A. Ion Selective Electrodes. Chapt. 11. Natl. Bur. Stand. Spec. Publ. 314. (1969).
4. Ough, C.S., and G. Tioloi. Urea Removal from Wine by an Acid Urease. Am. J. Enol. Vitic. 39:303-307 (1988).
5. Tegmo-Larsson, I.M., and T. Henrick-Kling. Ethyl Carbamate Precursors in Grape Juice and the Efficiency of Acid Urease on the Removal. Am. J. Enol. Vitic. 41:189-192 (1990).
6. Wahlstrom, V.L., J.S. Burr, K.C. Fugelsang and C.J. Muller. Effect of fluoride on Fermentation. Presented at the 42nd Annual Meeting of the American Society for Enology and Viticulture, Seattle, WA (1991).
7. Zoecklein, B.W., K.C. Fugelsang, B.H. Gump and F.S. Nury. Production Wine Analysis. Chapman-Hall, N.Y. (1990).
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Copyright © 1996. All rights reserved.
CALIFORNIA AGRICULTURAL TECHNOLOGY INSTITUTE - CATI
College of Agricultural Sciences and Technology
California State University, Fresno
