The effect of food preservatives on pH homeostasis in Escherichia coli

The effects of cinnamic, propionic, benzoic and sorbic acids on the growth and intracellular pH of Escherichia coli were investigated. The data suggest that the potency of weak acids as food preservatives is related to their capacity to reduce specifically the intracellular pH. The data also suggest that although both the undissociated forms of the acid cause the intracellular pH to fall, growth inhibition is due predominantly to the undissociated acid.     

Microbial spoilage of pre-cooked potato-topped pies

The ecological succession of bacteria which developed in pre-cooked potato-topped pies stored at two different temperatures was examined. Bacillus, Streptococcus and Staphylococcus-Micrococcus spp. were the predominant organisms isolated from freshly prepared pies and those stored at 4°C and 37°C. None of these groups of bacteria caused significant biodeterioration of pies held at 4°C, but all groups grew well in pies stored at 37°C and achieved counts of ca 10⁸/g of sample. Bacillus spp. were the first group to grow, followed by Streptococcus and Staphylococcus- Micrococcus spp. Growth which occurred at 37°C did so at the expense of glucose, lactate accumulated and the pH of pie components decreased. Amylase activity detected in all pie components during storage was associated with the growth of Bacillus spp. and probably supplemented glucose already present in pies, by hydrolytic cleavage of potato, flour or binder starches. Spoilage caused by growth and activity of the bacteria isolated was not associated with visual signs of bio-deterioration, nor production of 'off' odour usually associated with spoilage of meats. These results suggest that pre-cooked potato-topped pies held at inappropriate temperatures represent a potential public health risk.     

Microbial spoilage of pre-cooked potato-topped pies

The ecological succession of bacteria which developed in pre-cooked potato-topped pies stored at two different temperatures was examined. Bacillus, Streptococcus and Staphylococcus-Micrococcus spp. were the predominant organisms isolated from freshly prepared pies and those stored at 4 degrees and 37 degrees C. None of these groups of bacteria caused significant biodeterioration of pies held at 4 degrees C, but all groups grew well in pies stored at 37 degrees C and achieved counts of ca 10(8)/g of sample. Bacillus spp. were the first group to grow, followed by Streptococcus and Staphylococcus-Micrococcus spp. Growth which occurred at 37 degrees C did so at the expense of glucose, lactate accumulated and the pH of pie components decreased. Amylase activity detected in all pie components during storage was associated with the growth of Bacillus spp. and probably supplemented glucose already present in pies, by hydrolytic cleavage of potato, flour or binder starches. Spoilage caused by growth and activity of the bacteria isolated was not associated with visual signs of biodeterioration, nor production of 'off' odour usually associated with spoilage of meats. These results suggest that pre-cooked potato-topped pies held at inappropriate temperatures represent a potential public health risk.     

Inhibition of type A and type B (proteolytic) Clostridium botulinum by sorbic acid

The effect of sorbic acid in the pH range 4.9 to 7.0 on the probability P of growth of a single vegetative bacterium of proteolytic strains of Clostridium botulinum has been determined by comparison of the most probable number count of the bacteria in media at pH 4.9 to 7.0 containing a series of concentrations of potassium sorbate and in a nutrient medium at pH 6.8 to 7.0. The media were maintained under strictly anaerobic conditions at a redox potential equivalent to lower than -350 mV at pH 7. In medium adjusted to the required pH with HCl, P for strain ZK3 (type A) at pH 5.1 or 5.5 after 2 days at 30 degrees C was similar to that at pH 6.8 to 7.0 but was slightly lower at pH 4.9. Potassium sorbate inhibited growth, the inhibition being a function of the concentration of undissociated sorbic acid. A calculated undissociated sorbic acid concentration of 156 mg/liter delayed growth of strain ZK3 (type A) but did not result in a significant decrease in P after an incubation time of 14 days. Higher concentrations of undissociated sorbic acid caused longer delays before maximum most probable number counts developed, and a calculated undissociated sorbic acid concentration of 282 mg/liter decreased log P for strain ZK3 after an incubation time of 14 days by a factor of 5.5 to 7.5. Four additional type A strains and five type B strains were inhibited to an extent comparable to inhibition of strain ZK3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bacteriological Survey of the Frozen Prepared Foods Industry: I. Frozen Cream-Type Pies

During Food and Drug Administration inspections of 12 imitation cream pie producers, 453 finished product samples and 350 line samples were collected and analyzed bacteriologically. Sanitary conditions in the plants varied from good to poor and, in general, were reflected in the bacteriological results. The survey revealed that, in the great majority of cases, frozen imitation-cream pies produced in plants operating under good conditions of sanitation had the following bacteriological content: (i) a most probable number (MPN) of fewer than three Escherichia coli cells per gram (i.e., absent from all tubes in the methodology employed), (ii) an average MPN of fewer than 50 coliforms per gram (10 or more pies), (iii) the absence of coagulase-positive staphylococci in 0.1-g portions, and (iv) an aerobic plate count of fewer than 25,000 per gram (geometric mean of 10 or more pies).     

Food additives and plant components control growth and aflatoxin production by toxigenic aspergilli: A review

Growth and aflatoxin production by toxigenic aspergilli are partially or completely inhibited by the undissociated form of acetic, benzoic, citric, lactic, propionic and sorbic acids. Salts such as sodium chloride, potassium chloride and sodium nitrate, at low concentrations, can enhance aflatoxin production. At higher concentrations they become inhibitory, but marked inhibition requires amounts of the salts greater than are commonly used in foods. Phenolic antioxidants, sometimes added to foods to prevent oxidative deterioration, also are inhibitory to toxigenic aspergilli. Other inhibitory agents include certain insecticides, methylxanthines (caffeine and theophyllin), and components of some herbs, spices and other plants.     

Effect of cadmium on fungi and on interactions between fungi and bacteria in soil: influence of clay minerals and pH.

Fungi (Rhizopus stolonifer, Trichoderma viride, Fusarium oxysporum f. sp. conglutinans, Cunninghamella echinulata, and several species of Aspergillus and Penicillium) tolerated higher concentrations of cadmium (Cd) when grown in soil than when grown on laboratory media, indicating that soil mitigated the toxic effects of Cd. In soil amended with clay minerals, montmorillonite provided partial or total protection against fungistatic effects of Cd, whereas additions of kaolinite provided little or no protection. Growth rates of Aspergillus niger were inhibited to a greater extent by 100 or 250 mug of Cd per g in soil adjusted to pH 7.2 than in the same soil at its natural pH of 5.1. However, there were no differences in the growth rates of Aspergillus fischeri with 100 or 250 mug of Cd per g in the same soil, whether at pH 5.1 or adjusted to pH 7.2. Growth of A. niger and A. fischeri in a soil contaminated with a low concentration of Cd (i.e., 28 mug/g), obtained from a site near a Japanese smelter, did not differ significantly from growth in a soil collected some distance away and containing 4 mug of Cd per g. Growth of A. niger in sterile soil amended with 100 mug of Cd per g and inoculated with Bacillus cereus or Agrobacterium tumefaciens was reduced to a greater extent than in the same soil containing 100 mug of Cd per g but no bacteria. The inhibitory effects of Agrobacterium radiobacter to A. niger were slightly reduced in the presence of 100 mug of Cd per g, whereas the inhibitory effects of Serratia marcescens were enhanced.     

Inhibition of type A and type B (proteolytic) Clostridium botulinum by sorbic acid.

The effect of sorbic acid in the pH range 4.9 to 7.0 on the probability P of growth of a single vegetative bacterium of proteolytic strains of Clostridium botulinum has been determined by comparison of the most probable number count of the bacteria in media at pH 4.9 to 7.0 containing a series of concentrations of potassium sorbate and in a nutrient medium at pH 6.8 to 7.0. The media were maintained under strictly anaerobic conditions at a redox potential equivalent to lower than -350 mV at pH 7. In medium adjusted to the required pH with HCl, P for strain ZK3 (type A) at pH 5.1 or 5.5 after 2 days at 30 degrees C was similar to that at pH 6.8 to 7.0 but was slightly lower at pH 4.9. Potassium sorbate inhibited growth, the inhibition being a function of the concentration of undissociated sorbic acid. A calculated undissociated sorbic acid concentration of 156 mg/liter delayed growth of strain ZK3 (type A) but did not result in a significant decrease in P after an incubation time of 14 days. Higher concentrations of undissociated sorbic acid caused longer delays before maximum most probable number counts developed, and a calculated undissociated sorbic acid concentration of 282 mg/liter decreased log P for strain ZK3 after an incubation time of 14 days by a factor of 5.5 to 7.5. Four additional type A strains and five type B strains were inhibited to an extent comparable to inhibition of strain ZK3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple modes of inhibition of spore germination and outgrowth by reduced pH and sorbate

Germination and outgrowth of three strains of Clostridium botulinum in PYEG medium were measured by phase contrast microscopy. Reduction in pH from 7 to 5·5 completely inhibited germination of strain 12885A, reduced the extent of germination of strain 62A and had no effect on the extent of germination of strain 53B. At pH 5·5, 225 mg/1 of undissociated sorbic acid had no effect on the germination of strain 53B, while at pH 6·5, 225 mg/1 of undissociated sorbic acid completely inhibited germination of strains 62A and 12885A. Outgrowth of germinated spores of strains 62A and 53B was not inhibited at pH 5·5, but the addition of sorbate (225 mg/1 undissociated sorbic acid) completely inhibited outgrowth. Sorbate inhibited germination of Cl. botulinum and Bacillus cereus spores triggered to germinate by amino acids. Inhibition occurred after germinant binding, as measured by commitment to germinate.     

Distinct Effects of Sorbic Acid and Acetic Acid on the Electrophysiology and Metabolism of Bacillus subtilis

Sorbic acid and acetic acid are among the weak organic acid preservatives most commonly used to improve the microbiological stability of foods. They have similar pK_a values, but sorbic acid is a far more potent preservative. Weak organic acids are most effective at low pH. Under these circumstances, they are assumed to diffuse across the membrane as neutral undissociated acids. We show here that the level of initial intracellular acidification depends on the concentration of undissociated acid and less on the nature of the acid. Recovery of the internal pH depends on the presence of an energy source, but acidification of the cytosol causes a decrease in glucose flux. Furthermore, sorbic acid is a more potent uncoupler of the membrane potential than acetic acid. Together these effects may also slow the rate of ATP synthesis significantly and may thus (partially) explain sorbic acid''s effectiveness.     

Multiple modes of inhibition of spore germination and outgrowth by reduced pH and sorbate

Germination and outgrowth of three strains of Clostridium botulinum in PYEG medium were measured by phase contrast microscopy. Reduction in pH from 7 to 5.5 completely inhibited germination of strain 12885A, reduced the extent of germination of strain 62A and had no effect on the extent of germination of strain 53B. At pH 5.5, 225 mg/l of undissociated sorbic acid had no effect on the germination of strain 53B, while at pH 6.5, 225 mg/l of undissociated sorbic acid completely inhibited germination of strains 62A and 12885A. Outgrowth of germinated spores of strains 62A and 53B was not inhibited at pH 5.5, but the addition of sorbate (225 mg/l undissociated sorbic acid) completely inhibited outgrowth. Sorbate inhibited germination of Cl. botulinum and Bacillus cereus spores triggered to germinate by amino acids. Inhibition occurred after germinant binding, as measured by commitment to germinate.     

Growth characteristics of Geotrichum ingens on propionic acid and acetic acid in batch and continuous culture

Growth of Geotrichum ingens in batch cultures was completely inhibited by 47 g acetic acid/l or 33 g propionic acid/I. With mixtures of acetic and propionic acids, however, growth only ceased at 55 g/l. Acetic acid inhibited growth linearly, whereas propionic acid inhibited growth non-linearly. In continuous culture, two steady states at each dilution rate were observed at high dilution rates for acetic acid and propionic acid. The highest yield coefficient (0.69 g cells/g substrate) was achieved with propionic acid as substrate. On both substrates and their mixtures, the protein content of the biomass increased when the dilution rate was increased.The authors are with the Department of Microbiology and Biochemistry, University of the Orange Free State, P.O. Box 339, Bloemfontein 9300, South Africa     

Propionic Acid Production by a Propionic Acid-Tolerant Strain of Propionibacterium acidipropionici in Batch and Semicontinuous Fermentation

A propionic acid-tolerant derivative of Propionibacterium acidipropionici P9 was obtained by serially transferring strain P9 through broth that contained increasing amounts of propionic acid. After 1 year of repeated transfers, a strain (designed P200910) capable of growth at higher propionic acid concentrations than P9 was obtained. An increase in the proportion of cellular straight-chain fatty acids and uncoupling of propionic acid production from growth were observed for strain P200910. Growth rate, sugar utilization, and acid production were monitored during batch and semicontinuous fermentations of semidefined medium and during batch fermentation of whey permeate for both strain P200910 and strain P9. The highest propionic acid concentration (47 g/liter) was produced by P200910 in a semicontinuous fermentation. Strain P200910 produced a higher ratio of propionic acid to acetic acid, utilized sugar more efficiently, and produced more propionic acid per gram of biomass than did its parent in all fermentations.     

Evidence that sorbic acid does not inhibit yeast as a classic 'weak acid preservative'

Weak-acid preservatives are widely used to maintain microbial stability in foods and beverages. Classical weak-acid theory proposes that undissociated acid molecules pass through the plasma membrane, dissociate in the neutral pH of the cytoplasm, release protons and inhibit growth through acidification of the cytoplasm. Inhibitory concentrations of sorbic acid are shown to liberate fewer protons than other weak-acid preservatives. Sorbic acid shows similar inhibition to other six-carbon acids, alcohols and aldehydes, the latter being unable to act as weak acids. A survey of 22 yeasts showed high correlation between sorbate resistance and ethanol tolerance. Inhibition by short-chain acids or alcohols showed strong correlation with lipophilicity. It is proposed that sorbic acid acts as a membrane-active substance rather than as a weak-acid preservative.     

Chemicals for preventing moulding in damp hay

Fewer than one third of more than 100 chemicals tested prevented moulding when 0.5% of active ingredient was added to hay containing 35% water or, alternatively, when 0.02% of active ingredient was added with 0.2% propionic acid or ammonium propionate. Chemicals which prevented moulding in these tests when used alone included aliphatic acids with 2–6 carbon atoms, sorbic acid, short chain aldehydes and phenols, propionic acid, fully or half-neutralized with ammonia, and 8-quinolinol and its derivatives. Formic acid, aliphatic acids with 7–9 carbon atoms, longer chain aldehydes and some fungicides delayed moulding for one to two weeks. Only formaldehyde, 8-quinolinol and its derivatives, and p-nitrophenol increased the activity of 0.2% propionic acid. Although the effective doses of propionic acid, ammonium propionate, and propionic acid, half-neutralised with ammonia, were similar, there was evidence that the presence of free acid in the formulation aided redistribution to overcome undertreatment of pockets in the hay. Further tests of 2-phenyl phenol, phenyl propionate and 8-quinolinol showed that the latter was the best candidate for development. Fungi of the Aspergillus glaucus group were first to colonise most tests that failed, indicating their high degree of tolerance of a wide range of chemicals, and their value as test organisms for prospective preservatives.     

Listeria contamination at a poultry processing plant

Escherichia coli grown in broth initially at pH 5.0 (pH 5.0-grown organisms) survived exposure to inorganic acid or to acid pH plus organic acid which prevented subsequent growth by pH 7.0-grown organisms. This resistance of pH 5.0-grown organisms to organic acids was observed at acid pH with lactic, propionic, benzoic, sorbic, trans-cinnamic and acetic acids. Such resistance might allow acid-habituated organisms to survive in acid foods or at body sites such as the urinary tract where organic acids are present at acid pH.     

The antimicrobial effect of dissociated and undissociated sorbic acid at different pH levels

The minimum inhibitory concentration of sorbic acid has been determined for Bacillus subtilis, Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans. The inhibition was shown to be due to both undissociated and dissociated acid, and the effect of each has been calculated in accordance with a proposed mathematical model. Although the inhibitory action of undissociated acid was 10-600 times greater than that of dissociated acid, the latter caused more than 50% of the growth inhibition at pH levels above 6 for most of the organisms tested.     

Transport of acetic acid in Zygosaccharomyces bailii: effects of ethanol and their implications on the resistance of the yeast to acidic environments.

Cells of Zygosaccharomyces bailii ISA 1307 grown in a medium with acetic acid, ethanol, or glycerol as the sole carbon and energy source transported acetic acid by a saturable transport system. This system accepted propionic and formic acids but not lactic, sorbic, and benzoic acids. When the carbon source was glucose or fructose, the cells displayed activity of a mediated transport system specific for acetic acid, apparently not being able to recognize other monocarboxylic acids. In both types of cells, ethanol inhibited the transport of labelled acetic acid. The inhibition was noncompetitive, and the dependence of the maximum transport rate on the ethanol concentration was found to be exponential. These results reinforced the belief that, under the referenced growth conditions, the acid entered the cells mainly through a transporter protein. The simple diffusion of the undissociated acid appeared to contribute, with a relatively low weight, to the overall acid uptake. It was concluded that in Z. bailii, ethanol plays a protective role against the possible negative effects of acetic acid by inhibiting its transport and accumulation. Thus, the intracellular concentration of the acid could be maintained at levels lower than those expected if the acid entered the cells only by simple diffusion.     

The antimicrobial effect of dissociated and undissociated sorbic acid at different pH levels

The minimum inhibitory concentration of sorbic acid has been determined for Bacillus subtilis, Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans. The inhibition was shown to be due to both undissociated and dissociated acid, and the effect of each has been calculated in accordance with a proposed mathematical model. Although the inhibitory action of undissociated acid was 10–600 times greater than that of dissociated acid, the latter caused more than 50% of the growth inhibition at pH levels above 6 for most of the organisms tested.