Modeling yeast spoilage in cold-filled ready-to-drink beverages with Saccharomyces cerevisiae, Zygosaccharomyces bailii, and Candida lipolytica

Mathematical models were developed to predict the probability of yeast spoilage of cold-filled ready-to-drink beverages as a function of beverage formulation. A Box-Behnken experimental design included five variables, each at three levels: pH (2.8, 3.3, and 3.8), titratable acidity (0.20, 0.40, and 0.60%), sugar content (8.0, 12.0, and 16.0 degrees Brix), sodium benzoate concentration (100, 225, and 350 ppm), and potassium sorbate concentration (100, 225, and 350 ppm). Duplicate samples were inoculated with a yeast cocktail (100 microl/50 ml) consisting of equal proportions of Saccharomyces cerevisiae, Zygosaccharomyces bailii, and Candida lipolytica (approximately 5.0 x 10(4) CFU/ml each). The inoculated samples were plated on malt extract agar after 0, 1, 2, 4, 6, and 8 weeks. Logistic regression was used to create the predictive models. The pH and sodium benzoate and potassium sorbate concentrations were found to be significant factors controlling the probability of yeast growth. Interaction terms for pH and each preservative were also significant in the predictive model. Neither the titratable acidity nor the sugar content of the model beverages was a significant predictor of yeast growth in the ranges tested.     

Effectiveness of various food preservatives in controlling the outgrowth of Byssochlamys nivea ascospores

Potassium sorbate, sodium benzoate, sulfur dioxide, and diethylpyrocarbonate (DEPC) were tested for their effectiveness in preventing the outgrowth ofByssochlamys nivea Westling ascospores. Sulfur dioxide was the most inhibitory of the test antimycotics, complete inhibition of colony formation occurring in acidified (pH 3.5) potato dextrose agar containing 50 ppm of the preservative. Complete inhibition ofB. nivea ascospore outgrowth in grape juice stored for 60 days was noted in the presence of 300 ppm sulfur dioxide, 400 ppm potassium sorbate, and 600 ppm DEPC. Growth was observed in grape juice containing 1000 ppm sodium benzoate. The presence of up to 100 ppm potassium sorbate in grape juice during heat activation appears to have a stimulatory effect on breaking dormancy, while the other test preservatives at this concentration decrease the heat resistance ofB. nivea ascospores. The time elapsed between heat shock and exposure to DEPC or sodium benzoate is critical with respect to the sensitivity of ascospores to these preservatives.     

Antimicrobial Activity of Aroma Compounds against Saccharomyces cerevisiae and Improvement of Microbiological Stability of Soft Drinks as Assessed by Logistic Regression

The combined effects of a mild heat treatment (55°C) and the presence of three aroma compounds [citron essential oil, citral, and (E)-2-hexenal] on the spoilage of noncarbonated beverages inoculated with different amounts of a Saccharomyces cerevisiae strain were evaluated. The results, expressed as growth/no growth, were elaborated using a logistic regression in order to assess the probability of beverage spoilage as a function of thermal treatment length, concentration of flavoring agents, and yeast inoculum. The logit models obtained for the three substances were extremely precise. The thermal treatment alone, even if prolonged for 20 min, was not able to prevent yeast growth. However, the presence of increasing concentrations of aroma compounds improved the stability of the products. The inhibiting effect of the compounds was enhanced by a prolonged thermal treatment. In fact, it influenced the vapor pressure of the molecules, which can easily interact within microbial membranes when they are in gaseous form. (E)-2-Hexenal showed a threshold level, related to initial inoculum and thermal treatment length, over which yeast growth was rapidly inhibited. Concentrations over 100 ppm of citral and thermal treatment longer than 16 min allowed a 90% probability of stability for bottles inoculated with 10⁵ CFU/bottle. Citron gave the most interesting responses: beverages with 500 ppm of essential oil needed only 3 min of treatment to prevent yeast growth. In this framework, the logistic regression proved to be an important tool to study alternative hurdle strategies for the stabilization of noncarbonated beverages.     

Fermentation of Aqueous Plant Seed Extracts by Lactic Acid Bacteria

The effects of lactic acid bacterial fermentation on chemical and physical changes in aqueous extracts of cowpea (Vigna unguiculata), peanut (Arachis hypogea), soybean (Glycine max), and sorghum (Sorghum vulgare) were studied. The bacteria investigated were Lactobacillus helveticus, L. delbrueckii, L. casei, L. bulgaricus, L. acidophilus, and Streptococcus thermophilus. Organisms were inoculated individually into all of the seed extracts; L. bulgaricus and S. thermophilus were also evaluated together as inocula for fermenting the legume extracts. During fermentation, bacterial population and changes in titratable acidity, pH, viscosity, and color were measured over a 72-h period at 37°C. Maximum bacterial populations, titratable acidity, pH, and viscosity varied depending upon the type of extract and bacterial strain. The maximum population of each organism was influenced by fermentable carbohydrates, which, in turn, influenced acid production and change in pH. Change in viscosity was correlated with the amount of protein and titratable acidity of products. Color was affected by pasteurization treatment and fermentation as well as the source of extract. In the extracts inoculated simultaneously with L. bulgaricus and S. thermophilus, a synergistic effect resulted in increased bacterial populations, titratable acidity, and viscosity, and decreased pH in all the legume extracts when compared to the extracts fermented with either of these organisms individually. Fermented extracts offer potential as substitutes for cultured dairy products.     

The effect of potassium sorbate, NaCl and pH on the growth of food spoilage fungi

In this study, the hurdle technology approach was used to prevent fungal growth of common spoilage fungi in naturally fermented black olives (Alternaria alternata, Aspergillus niger, Fusarium semitectum andPenicillium roqueforti). The factors studied included a combination of different concentrations of potassium sorbate (100 up to 1000 mg/L), a range of pH values (4.5, 5, 5.5, 6, and 6.5) and levels of NaCl (0, 3.5, 5, 7.5, and 10%).Alternaria alternata was the most sensitive fungus whereasP. roqueforti was the most resistant fungi against all hurdle factors. The combination of all hurdles completely inhibitedA. alternata andF. semitectum by lowest inhibitory factors, such as 100 mg/L potassium sorbate with 3.5% NaCl at pH 5. On the other hand, at pH 5, A.niger andP. roqueforti were totally prevented by a combination of 300 mg/L potassium sorbate with 10% NaCl and 400 mg/L potassium sorbate with 7.5% NaCl, respectively. Potassium sorbate and 5–10% NaCl interaction had significant stimulation effect onp. roqueforti andA. niger (p<0.05). This study indicates that potassium sorbate is a suitable preserving agent to inhibit growth of fungi in fermented products of pH near 4.5 regardless levels of NaCl. For products of slightly higher pH, the addition of potassium sorbate is suggested in combination with NaCl.     

Modeling of Combined Processing Steps for Reducing Escherichia coli O157:H7 Populations in Apple Cider

Probabilistic models were used as a systematic approach to describe the response of Escherichia coli O157:H7 populations to combinations of commonly used preservation methods in unpasteurized apple cider. Using a complete factorial experimental design, the effect of pH (3.1 to 4.3), storage temperature and time (5 to 35°C for 0 to 6 h or 12 h), preservatives (0, 0.05, or 0.1% potassium sorbate or sodium benzoate), and freeze-thaw (F-T; −20°C, 48 h and 4°C, 4 h) treatment combinations (a total of 1,600 treatments) on the probability of achieving a 5-log₁₀-unit reduction in a three-strain E. coli O157:H7 mixture in cider was determined. Using logistic regression techniques, pH, temperature, time, and concentration were modeled in separate segments of the data set, resulting in prediction equations for: (i) no preservatives, before F-T; (ii) no preservatives, after F-T; (iii) sorbate, before F-T; (iv) sorbate, after F-T; (v) benzoate, before F-T; and (vi) benzoate, after F-T. Statistical analysis revealed a highly significant (P < 0.0001) effect of all four variables, with cider pH being the most important, followed by temperature and time, and finally by preservative concentration. All models predicted 92 to 99% of the responses correctly. To ensure safety, use of the models is most appropriate at a 0.9 probability level, where the percentage of false positives, i.e., falsely predicting a 5-log₁₀-unit reduction, is the lowest (0 to 4.4%). The present study demonstrates the applicability of logistic regression approaches to describing the effectiveness of multiple treatment combinations in pathogen control in cider making. The resulting models can serve as valuable tools in designing safe apple cider processes.     

Staphylococcus aureus Growth Boundaries: Moving towards Mechanistic Predictive Models Based on Solute-Specific Effects

The formulation of shelf-stable intermediate-moisture products is a critical food safety issue. Therefore, knowing the precise boundary for the growth-no-growth interface of Staphylococcus aureus is necessary for food safety risk assessment. This study was designed to examine the effects of various humectants and to produce growth boundary models as tools for risk assessment. The molecular mobility and the effects of various physical properties of humectants, such as their glass transition temperatures, their membrane permeability, and their ionic and nonionic properties, on S. aureus growth were investigated. The effects of relative humidity (RH; 84 to 95%, adjusted by sucrose plus fructose, glycerol, or NaCl), initial pH (4.5 to 7.0, adjusted by HCl), and potassium sorbate concentration (0 or 1,000 ppm) on the growth of S. aureus were determined. Growth was monitored by turbidity over a 24-week period. Toxin production was determined by enterotoxin assay. The 1,792 data points generated were analyzed by LIFEREG procedures (SAS Institute, Inc., Cary, N.C.), which showed that all parameters studied significantly affected the growth responses of S. aureus. Differences were observed in the growth-no-growth boundary when different humectants were used to achieve the desired RH values in both the absence and the presence of potassium sorbate. Sucrose plus fructose was most inhibitory at neutral pH values, while NaCl was most inhibitory at low pH values. The addition of potassium sorbate greatly increased the no-growth regions, particularly when pH was <6.0. Published kinetic growth and survival models were compared with boundary models developed in this work. The effects of solutes and differences in modeling approaches are discussed.     

Factors determining rock phosphate solubilization by microorganisms isolated from soil

Forty two soil isolates (31 bacteria and 11 fungi) were studied for their ability to solubilize rock phosphate and calcium phosphate in culture medium. Eight bacteria and 8 fungi possessed solubilizing ability. Pseudomonas cepacia and Penicillium purpurogenum showed the highest activity. There was a correlation between final pH value and titratable acidity (r=–0.29 to –0.87) and between titratable acidity and soluble phosphate (r=0.22 to 0.99). Correlation values were functions of insoluble phosphate and of the group of microorganisms considered. A high correlation was observed between final pH and soluble phosphate only for the rock phosphates inoculated with the highest concentration of solubilizing bacteria (r=–0.73 to –0.98).     

Growth and fumitremorgin production by Neosartorya fischeri as affected by food preservatives and organic acids

The effects of potassium sorbate, sodium benzoate, sulphur dioxide and citric, malic and tartaric acids on growth and fumitremorgin production by a heat-resistant mould, Neosartorya fischeri , cultured on Czapek yeast autolysate agar (CYA) were studied over a 32-day incubation period. Colonies were examined, and extracts of agar and mycelia were analyzed for mycotoxin content using high performance liquid chromatography (HPLC). Growth of N. fischeri always resulted in production of the fumitremorgins verruculogen and fumitremorgin A and C. Growth on CYA (pH 3.5) was highly repressed by potassium sorbate and sodium benzoate; 75 mg/1 completely inhibited germination of ascospores. Sulphur dioxide was less inhibitory; growth occurred on CYA containing 100 but not 200 mg/1. Growth of N. fischeri was significantly reduced when the pH of CYA was reduced from 7.0 to 4.5 to 3.5 to 2.5. Citric, malic and tartaric acids promoted growth and fumitremorgin production when supplemented to CYA (pH 2.5). These observations indicate that growth and fumitremorgin production by N. fischeri are influenced by pH and type of acid present and can be controlled by small amounts of preservatives.     

Alcohol fermentation in olive oil extraction effluents

Eight culture-collection yeast strains of various species and five newly isolated strains were tested for both growth in olive oil extraction effluents and fermentation of the sugars in the same media. The culture-collection yeast strains did not grow in an effluent containing 2·86% sugar (w/v), 8 g/litre phenolic substances, 4·58 g/litre titratable acidity and pH 4·96, whereas the newly isolated strains of Torulopsis sp. MK-1, Saccharomyces norbensis MC-1, S. oleaceus MC-2 and S. oleaginosus grew well and fermented the sugars. In the medium mentioned above, they produced alcohol in amounts of 1·63 to 1·38%, respectively. None of the yeasts grew in an olive oil extraction effluent vacuum-concentrated to over 13–14% of dry matter. The strain of T. sp. MK-1 showed a hogher stability.     

Comparison of anti-Vibrio activities of potassium sorbate, sodium benzoate, and glycerol and sucrose esters of fatty acids.

The effects of fatty acids and their glycerol and sucrose esters, potassium sorbate, and sodium benzoate on growth of Vibrio parahaemolyticus in laboratory media at pH 6.7 were evaluated. The minimum concentrations at which inhibition by esters of glycerol could be detected were lowest for monolaurin (5 microgram/ml) and monocaprin (40 microgram/ml); these concentrations were lower than those observed for inhibition by lauric and capric acids, respectively. Inhibitory action of sucrose caprylate was detected at 40 microgram/ml, whereas sucrose caprate was effective at 100 microgram/ml; sucrose esters of lauric, myristic, and palmitic acids were ineffective at 100 microgram/ml. Potassium sorbate and sodium benzoate inhibited growth at concentrations as low as 30 and 300 microgram/ml, respectively, and enhanced the rate of thermal inactivation of V. parahaemolyticus at slightly higher concentrations. Fatty acid esters of glycerol and sucrose offer potential as perservatives for slightly acid or alkaline low-fat foods which do not lend themselves to the full antimicrobial action of traditional food preservatives such as potassium sorbate and sodium benzoate.     

Evaluation of Inhibitory Effect of Organic and Inorganic Salts Against Ilyonectria liriodendri,The Causal Agent of Root Rot Disease of Kiwifruit

This study evaluated efficacy of 42 organic and inorganic salts as possible alternatives to synthetic fungicides for the control of Ilyonectria root rot of kiwifruit. Preliminary in vitro tests showed that ammonium bicarbonate, ammonium carbonate, potassium benzoate, potassium sorbate, sodium benzoate and sodium metabisulphite at 2% completely inhibited mycelial growth of the fungus. No significant differences were observed among these salts and disodium EDTA (P ≤ 0.05). However, the ED₅₀, minimum inhibition concentration (MIC), and minimum fungicidal concentration (MFC) values indicated that sodium metabisulphite was more toxic to Ilyonectria liriodendri than these other six salts. Soil bioassays showed that sodium metabisulphite, sodium benzoate and potassium sorbate at 0.25% completely inhibited mycelial growth of the fungus, whereas potassium benzoate reduced the mycelial growth of fungus by 90.30%; however, the differences in inhibitory effects were statistically insignificant (P ≤ 0.05). Moreover, there was no significant difference between 0.1% sodium metabisulphite and 0.5% ammonium carbonate, 0.75% ammonium bicarbonate and 1.5–2.0% disodium EDTA (P ≤ 0.05). Unlike disodium EDTA, complete inhibitory was observed with ammonium carbonate and ammonium bicarbonate at higher concentrations. However, in root bioassays, applications of 2% ammonium bicarbonate, 1.5% ammonium carbonate and 2% disodium EDTA were phytotoxic to kiwifruit seedlings, but 0.25% four other salts were neither phytotoxic to kiwifruit seedlings nor did it adversely affect root length, root fresh weight and root dry weight of seedling. This study also showed I. liriodendri to be capable of growth in both acidic and basic environments. However, while the fungus showed uninhibited growth at pH values of 5–11, growth decreased significantly at both higher and lower pH values (P ≤ 0.05) and was completely inhibited at pH 12.     

Effect of Food Additives and Irradiation on Survival of Salmonella in Oysters

Salmonella typhimurium and S. enteritidis were inoculated into blended oysters, both raw and autoclaved. The oysters were also treated with sodium benzoate (0.1%) or potassium sorbate (0.1%), and irradiated (0.1 Mrad). In both non-irradiated and irradiated samples, greater numbers of Salmonella were recovered after storage at 7 C in the presence of sodium benzoate or potassium sorbate. The results of the autoclaved samples and studies in buffer indicated that this effect was not due to the reduction of competition from the natural flora when the additives were present.     

Occurrence and Growth of Yeasts in Yogurts

Yogurts purchased from retail outlets were examined for the presence of yeasts by being plated onto oxytetracycline malt extract agar. Of the 128 samples examined, 45% exhibited yeast counts above 10³ cells per g. A total of 73 yeast strains were isolated and identified as belonging to the genera Torulopsis, Kluyveromyces, Saccharomyces, Candida, Rhodotorula, Pichia, Debaryomyces, and Sporobolomyces. Torulopsis candida and Kluyveromyces fragilis were the most frequently isolated species, followed by Saccharomyces cerevisiae, Rhodotorula rubra, Kluyveromyces lactis, and Torulopsis versatilis. The growth of yeasts in yogurts was related to the ability of the yeasts to grow at refrigeration temperatures, to ferment lactose and sucrose, and to hydrolyze milk casein. Most yeast isolates grew in the presence of 100 μg of sorbate and benzoate preservatives per ml. Higher yeast counts from yogurts were obtained when the yogurts were plated onto oxytetracycline malt extract agar than when they were plated onto acidified malt extract agar.     

山苍子油对霉菌抗菌性及其与黄曲霉产毒关系的研究

采用平板法比较天然增香剂山苍子油与合成食用防腐剂苯甲酸钠、山梨酸钾对8种霉菌的抗菌效力。结果表明,在培养基pH4．5时山苍子油对多数霉菌的最低抑菌浓度为1．77mg／ml,与山梨酸钾的抑菌强度相近,比苯甲酸钠强;但当培养基pH5．5以上时苯甲酸钠对霉菌几乎无效,山梨酸钾的抗菌效力也有减弱,而山苍子油受影响很小,其活性pH范围为4．5～8．5。山苍子油用脂肪酸蔗糖酯乳化,其抗菌效力增强一倍。同时,从山苍子油与黄曲霉产毒关系的实验中发现,山苍子油对黄曲霉产生黄曲霉毒素有较强的抑制作用。     

Metabolic effects of benzoate and sorbate in the yeast Saccharomyces cerevisiae at neutral pH

Preincubation of yeast cells in the presence of benzoate or sorbate at an extracellular pH value of 6.8 elicited a set of metabolic effects on sugar metabolism, which became apparent after the subsequent glucose addition. They can be summarized as follows: a) reduced glucose consumption; b) inhibition of glucose- and fructose-phosphorylating activities; c) supression of glucose-triggered peak of hexoses monophosphates; d) substantial reduction of glucose-triggered peak of fructose 2,6-bisphosphate; e) block of catabolite inactivation of fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase, but not of cytoplamic malate dehydrogenase. On the whole this pattern resulted in prevention of glucose-induced switch of metabolism from a gluconeogenetic to a glycolytic state. Our data also show that, unlike former assumptions, intracellular acidification is not likely to mediate the bulk of metabolic effects of benzoate and sorbate, since under our working conditions intracellular pH kept close to neutrality.     

Use of organic acids and salts to control postharvest diseases of lemon fruits in Egypt

Abstract

Postharvest diseases caused by Geotricum candidum (sour rot), Penicillium digitatum (green mould), and P. italicum (blue mould) are the most important negative factors affecting handling and marketing of citrus fruits in Egypt. The effect of organic acids (ascorbic, benzoic, citric and sorbic) as well as organic salts (potassium sorbate and sodium benzoate) were evaluated on the growth of causal agents and their disease incidence under in vitro and in vivo conditions. Complete inhibition was observed in the linear growth of all tested fungi when exposed to benzoic, citric and sorbic organic acids at concentrations of 4% and 2% of either sodium benzoate or potassium sorbate, respectively. Minimizing the tested concentration of organic acid down to 2%, the tested fungi fluctuated in their response such that only benzoic and sorbic acids could completely inhibit the growth of either P. digitatum or P. italicum only. Different organic acids and salts showed various levels of either protective or therapeutic effect for coated lemon fruits against mould infection whatever the time of their artificial inoculation under in vivo conditions. All treated fruits showed reduction in sour rot and green and blue mould diseases when compared with untreated fruits. Complete inhibition of mould incidence was obtained in coated lemon fruits with 4% of water or wax mixtures of sodium benzoate and potassium sorbate 24 hours before inoculation. Also, high reduction in mould incidence was observed in lemon fruits coated with the same concentration at 48 hours after inoculation under the same conditions. On the other hand, the tested organic acids showed a lesser effect on mould incidence. Moreover, they were more effective against mould incidence when dissolved in water than wax, that only 4% of water mixture of sorbic and benzoic acids showed 100% protection against mould incidence. Furthermore, the severity of infection records followed the same trend. The present findings demonstrate that potassium sorbate and sodium benzoate have potential as environmentally friendly products, nontoxic postharvest fungicides against sour rot, green and blue mould incidence of stored citrus fruits and could be suggested for commercial use in packing-houses in consideration to their wide consumption as safely food preservatives.     

Effect of potassium sorbate,sodium benzoate,and sodium nitrite on biosynthesis of cyclopiazonic and mycophenolic acids and citrinin by fungi of the <Emphasis Type="Italic">Penicillium</Emphasis> genus

The effect of potassium sorbate, sodium benzoate, and sodium nitrite used as preservatives in the food industry in the production of such mytotoxins as citrinin cyclopiazonic and mycophenolic acids by the contaminating fungi Penicillium citrinum, P. commune, and P. brevicompactum, respectively, was investigated. It was shown that the effect of preservatives used at concentrations relevant to the food industry on the synthesis of mycotoxins depended on the species-specific biochemical and physiological features of the cultures. The growth of P. brevicompactum was inhibited to the highest degree by sodium nitrite and potassium sorbate, and the growth of P. commune was so inhibited by sodium benzoate. It was established that the introduction of 0.015% sodium nitrite into the medium resulted in 1.3- and 1.4-fold reductions of the production of citrinin and mycophenolic acid, respectively, while the production of cyclopiazonic acid did not change in comparison with the control. The introduction of 0.015% sodium benzoate caused a more than 1.5-fold increase of the concentration of citrinin, cyclopiazonic, and mycophenolic acids, and the addition of 0.02% potassium sorbate increased the production of cyclopiazonic and mycophenolic acids by 1.7 and 2.6 times, respectively.     

Concentration effect of Riesling Icewine juice on yeast performance and wine acidity

AIMS: The objective of this study was to determine the effect of increasing juice soluble solids above 40 degrees Brix on wine yeast's ability to grow and ferment the juice, with particular focus on acetic acid production, titratable acidity (TA) changes and the maximum amount of sugar consumed by the yeast. METHODS AND RESULTS: Riesling Icewine juices at 40, 42, 44 and 46 degrees Brix were inoculated with K1- V116 at 0.5 g 1(-1) and fermented at 17 degrees C until sugar consumption ceased. Increasing soluble solids showed strong negative linear correlations with yeast growth, sugar consumption and ethanol production (r = -0.999, -0.997 and 0.984, P < 0.001, respectively). Acetic acid, glycerol and TA production normalized to sugar consumed showed strong positive correlations to the initial juice concentration (r = 0.992, 0.963, and 0.937, P < 0.001 respectively) but no correlation was found for ethanol production. The acetic acid produced as a function of sugar consumed was positively correlated to the glycerol produced (r = 0.970, P < 0.001). The final TA of the wines ranged between 11.8 and 13.7 g 1(-1) tartaric acid, increasing by 2.3-3 g 1(-1) over the starting juice. The increase in TA was positively correlated to the increase in acetic acid produced after normalizing the data to the amount of sugar consumed (r =0.975, P < 0.001). The acid equivalents resulting from the increase in acetic acid accounted for 80-100% of the TA increase when converted to units of tartaric acid. In the final Icewines, acetic acid represented 19-20% of wine TA. CONCLUSIONS: Increasing Icewine juice concentration from 40 to 46 degrees Brix increases the proportion of yeast sugar metabolism towards glycerol and acetic acid production to cope with the increased osmotic stress by decreasing yeast growth, sugar consumption rate, the total amount of sugar consumed and the total amount of ethanol produced. The high proportional contribution of acetic acid to titratable acidity in Riesling Icewine may affect acidity perception. SIGNIFICANCE AND IMPACT OF THE STUDY: We have determined that 10% v/v ethanol would not be achievable with initial juice concentrations above 42 degrees Brix and that Riesling Icewine juice above 52.5 degrees Brix would be theoretically unfermentable. The high proportional contribution of acetic acid to TA may be an important factor in the organoleptic balance of these Icewines.     

Potential for Development of Tolerance by Penicillium digitatum and Penicillium italicum after Repeated Exposure to Potassium Sorbate

Two strains of Penicillium digitatum and one strain of Penicillium italicum were exposed to various levels of sorbic acid and potassium sorbate, and the MICs were determined. Selected strains of the molds were then repeatedly exposed to subinhibitory levels of the compounds to determine whether increased tolerance might develop. The MIC of sorbic acid (pH 4.75) to P. digitatum was between 0.02 and 0.025%. The MIC of sorbate (pH 5.5) to two strains of P. digitatum and P. italicum was found to be between 0.06 and 0.08%. Increasing levels of sorbate resulted in increasing growth suppression of the molds. Populations of P. digitatum were tested for increased tolerance to sorbic acid, and none was found. Individual molds that started from the same parent colony were examined for increased tolerance to potassium sorbate. Two P. digitatum strains developed no observable increased tolerance, but P. italicum developed a slight increase in tolerance to sorbate. When spores of P. italicum and P. digitatum were exposed to higher levels of sorbate for prolonged times, the fungicidal or fungistatic activity of the inhibitor was dependent upon pH, length of exposure time, level of sorbate, and the mold strain.