Factors Affecting Death of Yeast by Sulfur Dioxide

Abstract

The interrelated factors that influence the effectiveness of so2 as a preservative against yeast were studied and the correct quantitative relationships of so2 molecular species determined. Widely differing pK values for S0 2 were found in the literature, compared with experimental data, and one set of values was selected. Undissociated H 2 S0 3 is the only effective form of S0 2 against yeast and can be calculated from measurement of free S0 2 and pH, and the correct dissociation constants. Duration of contact, pH, concentration of S0 2 and yeast, and binding of S0 2 all influence the preservative action of S0 2 • Lower total S0 2 concentrations can be used for food preservation by optimum control of these factors. Numerous investigations have been made of one or more factors involved in killing of microorganisms by S0 2 , but it appears that no attempts have been made to consider all the interrelated parameters, or the state of the art was not sufficiently developed for this. Furthermore, those factors which have been investigated have led to widely varying results. This could be because of the influence of the uninvestigated factors, but also because of errors in the chemistry of sulfurous acid and its salts, introduced over SO years ago and perpetuated in the literature. We have attempted to clarif'y the situation by investigating the important factors affecting so2 action and their interrelationships, as well as correcting the errors in published pK values of S0 2 • We have also examined the combined effects of these factors to determine how to control yeast fermentations. Since we have been concerned with fruit preservation, we have limited our work mostly to a pH range of 2.5 to 4.5. This eliminates most vegetable preservation, with its considerably different S0 2 chemistry at higher pH values, and also narrows the types of spoilage organisms to yeasts. Bacteria do not grow significantly in the fruit pH range and molds do not grow if containers are closed and oxygen is restricted. Thus most of our work has been with a standard freeze-dried wine yeast, Saccharomyces cerevisiae var. ellipsoideus (Montrachet, U.C. 522), one of the most SOrresistant yeasts (J). Some conftrmatory 'Reforence to a company and/or product named by the Department is only for purposes of information and does not imply approval or /'!?Commendation of the product to the exclusion qf others which may also be suitable. work was also done with the spoilage yeast Hansenula anomala. The following factors affecting the action of S0 2 on yeast were investigated: (a) pH-effect on yeast and on dissociation ofH 2 S0 3 , (b) effectiveness of each molecular species of H 2 SO 3 against yeasts, (c) extent of combination of carbonyl-containing compounds with so2, (d) effectiveness of these combined SOrcarbonyl compounds against yeast, (e) concentration of so2 and time of contact with yeast and (j) concentration of yeast. Other factors involved are temperature, oxygen concentration and osmotic effect of dissolved substances. The first two of these were held constant. Soluble solids in our experiments were too dilute to cause a significant osmotic effect. EXPERIMENTAL Determination qf S0 2 The method of Panting and Johnson {)1} was used to measure both "free" and "bound" S0 2 • Free S0 2 is defined as all the molecular species of H 2 S0 3 in a solution which are titratable by iodine in acid solution. Free S0 2 S0 2 + H 2 S0 3 + HSOi + gas in solution undissociated sulfurous acid bisulfite ion SOj. sulfite ion Bound S0 2 is obtained by subtracting free S0 2 from total S0 2 • which is obtained by titrating a sample after freeing the bound S0 2 with an alkaline treatment. then acidifYing. Bindin~ ofS0 2 The extent of binding 300 ppm of S0 2 by glucose was measured by equilibrating S0 2 solutions (at various pH values) with 3 and 30o/o glucose (3 days at room temperature, approximately 25 C). A citrate· phosphate buffer (0.1 M) was used to establish pH values. Effect qfpH and r;lucose on .r;;rowth In this and other experiments with yeast, cultures were prepared from a single lot of freeze-dried yeast which had been stored frozen in 5-g aluminum foil packages. The contents of a package were suspended in 100 ml of sterile deionized water containing 0.1% Difco neopeptone, 0.1 o/o Difco yeast extract and 0.1% glucose. This preparation contained approximately 1 x 10 7 viable cells/mi. Usually l ml of yeast suspension was added to 100 ml of the same medium containing one or more test substances, but in some experiments the dilution factor was changed. The mixture was then incubated at room temperature for the desired time, followed by plating at several dilutions on Difco Potato Dextrose Agar. pH 5.6, for counting viable cells. Yeast growth was followed for 48 h at 29 C in glucose concentrations ofO.l M (1.8%) and 1.0 M (18%), over a pH range of l.S-4.5. The initial yeast concentration was approximately l x 10'' viable cells/ml in this experiment.