Weak Acid Preservatives Research Articles

Sorbic acid is an efficient preservative in pea-based meat analogues

The popularity of plant-based alternatives to meat has increased due to advancements in important attributes such as taste. Using adequate processing at elevated temperatures combined with aseptic packaging, especially in combination with storage below the freezing point, microbial hazards in these products are negligible. However, for plant-based alternatives to ham, sausages, spreads, or cheese, for example, on sandwiches, the product must withstand microbial growth in the fridge after breaking the seal of the food packaging, as everything is not typically consumed directly. Currently, there are few preservations methods, but one solution is adding weak acid preservatives, with less inherent health concerns compared to sulfites and nitrite, e.g. carcinogenicity and hypersensitivity. To be fully effective an acidic pH is required. In this study, an approach where sausage-resembling meat analogues with different pHs (4.14–5.56) were prepared without preservatives, with potassium sorbate (2000 mg/kg), or sodium disulfite (200 mg/kg), and thereafter subjected to standardised challenge tests to elucidate the efficacy. Sorbate was the only preservative effective to prevent growth of all tested organisms, Penicillium roqueforti, Pichia fermentans and Listeria monocytogenes. The identified superiority of sorbate compared to other tested preservatives is relevant knowledge when developing preservation strategies for other types of meat analogues.

Trehalose metabolism targeting as a novel strategy to modulate acid tolerance of yeasts and its application in food industry

Some spoilage yeasts are able to develop resistance to commonly used weak-acid preservatives. We studied the trehalose metabolism and its regulation in Saccharomyces cerevisiae in response to propionic acid stress. We show interruption of trehalose synthetic pathway caused the mutant hypersensitive to the acid stress, while its overexpression conferred acid-tolerance to yeast. Interestingly, this acid-tolerance phenotype was largely independent of trehalose but relied on the trehalose synthetic pathway. We demonstrate trehalose metabolism played a vital role in regulation of glycolysis flux and Pi/ATP homeostasis in yeast during acid-adaptation, and the PKA and TOR signaling pathways were involved in regulating trehalose synthesis at transcriptional level. This work confirmed the regulatory function of trehalose metabolism and improved our understanding of molecular mechanism of acid-adaptation of yeast. By exemplifying trehalose metabolism interruption limited the growth of S. cerevisiae exposed to weak acids, and trehalose pathway overexpression conferring acid-resistance to Yarrowia lipolytica enhanced citric acid production, this work provides new insights into the development of efficient preservation strategies and robust organic acid producers.

Efficacy of weak acid preservatives on spoilage fungi of bakery products

Organic acids and their salts are usually the first choice in the bread industry to restrict fungal spoilage, but their efficacy is pH-dependent and spoilage by fungi remains as a common threat. Therefore, this study aimed to evaluate the susceptibility of spoilage fungi of bakery products to acetic, sorbic, and propionic acids at different pH. Penicillium roqueforti, Penicilium paneum, Aspergillus pseudoglaucus, Aspergillus montevidensis and Hyphopichia burtonii strains isolated from spoiled products had their minimum inhibitory concentration (MIC) defined by macrodilution. The concentrations tested were: (i) sorbic acid up to 32 mM; (ii) propionic acid up to 1024 mM and (iii) acetic acid up to 800 mM with pH adjusted in 4.5, 5.0, 5.0 and 6.0 after setting the agent concentration. The lowest MICs for all agents were obtained at pH 4.5, usually doubling with every 0.5 pH increase. P. roqueforti strains isolated from spoiled products were the most resistant to all tested preservatives; while strains of the related species P. paneum, showed similar tolerance to acetic and propionic acids but was double more susceptible to sorbic acid. Strains of A. pseudoglaucus and A. montevidensis were indistinctly susceptible to the preservatives and were the most susceptible species to propionic and acetic acids. H. burtonii strains demonstrated the most variable behaviour in comparison to the other strains being the most susceptible to sorbic acid, were like Aspergillus strains regarding propionic acid, but tolerate well acetic acid. Propionic acid concentrations usually allowed in baked goods are lower than the concentrations required to inhibit the most tolerant isolates tested in this study. The same is true for sorbic acid at higher pH levels. Spoilage species of bakery ware presents a distinct susceptibility profile to the preservatives commonly used in this sector, but the high tolerance observed is a cause of concern.

Anti-Yeast Synergistic Effects and Mode of Action of Australian Native Plant Essential Oils

Yeasts are the most common group of microorganisms responsible for spoilage of soft drinks and fruit juices due to their ability to withstand juice acidity and pasteurization temperatures and resist the action of weak-acid preservatives. Food industries are interested in the application of natural antimicrobial compounds as an alternative solution to the spoilage problem. This study attempts to investigate the effectiveness of three Australian native plant essential oils (EOs) Tasmanian pepper leaf (TPL), lemon myrtle (LM) and anise myrtle (AM) against weak-acid resistant yeasts, to identify their major bioactive compounds and to elucidate their anti-yeast mode of action. The minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC) and minimum bactericidal concentration (MBC) were assessed for EOs against weak-acid resistant yeasts (Candida albicans, Candida krusei, Dekkera anomala, Dekkera bruxellensis, Rhodotorula mucilaginosa, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Zygosaccharomyces bailii and Zygosaccharomyces rouxii) and bacteria (Staphylococcus aureus and Escherichia coli). The EOs showed anti-yeast and antibacterial activity at concentrations ranging from 0.03–0.07 mg/mL and 0.22–0.42 mg/mL for TPL and 0.07–0.31 mg/mL and 0.83–1.67 mg/mL for LM, respectively. The EOs main bioactive compounds were identified as polygodial in TPL, citral (neral and geranial) in LM and anethole in AM. No changes in the MICs of the EOs were observed in the sorbitol osmotic protection assay but were found to be increased in the ergosterol binding assay after the addition of exogenous ergosterol. Damaging of the yeast cell membrane, channel formation, cell organelles and ion leakage could be identified as the mode of action of TPL and LM EOs. The studied Australian native plant EOs showed potential as natural antimicrobials that could be used in the beverage and food industry against the spoilage causing yeasts.

The Preservative Sorbic Acid Targets Respiration, Explaining the Resistance of Fermentative Spoilage Yeast Species

A small number (10 to 20) of yeast species cause major spoilage in foods. Spoilage yeasts of soft drinks are resistant to preservatives like sorbic acid, and they are highly fermentative, generating large amounts of carbon dioxide gas. Conversely, many yeast species derive energy from respiration only, and most of these are sorbic acid sensitive and so prevented from causing spoilage. This led us to hypothesize that sorbic acid may specifically inhibit respiration. Tests with respirofermentative yeasts showed that sorbic acid was more inhibitory to both Saccharomyces cerevisiae and Zygosaccharomyces bailii during respiration (of glycerol) than during fermentation (of glucose). The respiration-only species Rhodotorula glutinis was equally sensitive when growing on either carbon source, suggesting that ability to ferment glucose specifically enables sorbic acid-resistant growth. Sorbic acid inhibited the respiration process more strongly than fermentation. We present a data set supporting a correlation between the level of fermentation and sorbic acid resistance across 191 yeast species. Other weak acids, C2 to C8, inhibited respiration in accordance with their partition coefficients, suggesting that effects on mitochondrial respiration were related to membrane localization rather than cytosolic acidification. Supporting this, we present evidence that sorbic acid causes production of reactive oxygen species, the formation of petite (mitochondrion-defective) cells, and Fe-S cluster defects. This work rationalizes why yeasts that can grow in sorbic acid-preserved foods tend to be fermentative in nature. This may inform more-targeted approaches for tackling these spoilage organisms, particularly as the industry migrates to lower-sugar drinks, which could favor respiration over fermentation in many spoilage yeasts.IMPORTANCE Spoilage by yeasts and molds is a major contributor to food and drink waste, which undermines food security. Weak acid preservatives like sorbic acid help to stop spoilage, but some yeasts, commonly associated with spoilage, are resistant to sorbic acid. Different yeasts generate energy for growth by the processes of respiration and/or fermentation. Here, we show that sorbic acid targets the process of respiration, so fermenting yeasts are more resistant. Fermentative yeasts are also those usually found in spoilage incidents. This insight helps to explain the spoilage of sorbic acid-preserved foods by yeasts and can inform new strategies for effective control. This is timely as the sugar content of products like soft drinks is being lowered, which may favor respiration over fermentation in key spoilage yeasts.

Weak Acid Resistance A (WarA), a Novel Transcription Factor Required for Regulation of Weak-Acid Resistance and Spore-Spore Heterogeneity in Aspergillus niger.

Propionic, sorbic, and benzoic acids are organic weak acids that are widely used as food preservatives, where they play a critical role in preventing microbial growth. In this study, we uncovered new mechanisms of weak-acid resistance in molds. By screening a library of 401 transcription factor deletion strains in Aspergillus fumigatus for sorbic acid hypersensitivity, a previously uncharacterized transcription factor was identified and named weak acid resistance A (WarA). The orthologous gene in the spoilage mold Aspergillus niger was identified and deleted. WarA was required for resistance to a range of weak acids, including sorbic, propionic, and benzoic acids. A transcriptomic analysis was performed to characterize genes regulated by WarA during sorbic acid treatment in A. niger Several genes were significantly upregulated in the wild type compared with a ΔwarA mutant, including genes encoding putative weak-acid detoxification enzymes and transporter proteins. Among these was An14g03570, a putative ABC-type transporter which we found to be required for weak-acid resistance in A. niger We also show that An14g03570 is a functional homologue of the Saccharomyces cerevisiae protein Pdr12p and we therefore name it PdrA. Last, resistance to sorbic acid was found to be highly heterogeneous within genetically uniform populations of ungerminated A. niger conidia, and we demonstrate that pdrA is a determinant of this heteroresistance. This study has identified novel mechanisms of weak-acid resistance in A. niger which could help inform and improve future food spoilage prevention strategies.IMPORTANCE Weak acids are widely used as food preservatives, as they are very effective at preventing the growth of most species of bacteria and fungi. However, some species of molds can survive and grow in the concentrations of weak acid employed in food and drink products, thereby causing spoilage with resultant risks for food security and health. Current knowledge of weak-acid resistance mechanisms in these fungi is limited, especially in comparison to that in yeasts. We characterized gene functions in the spoilage mold species Aspergillus niger which are important for survival and growth in the presence of weak-acid preservatives. Such identification of weak-acid resistance mechanisms in spoilage molds will help in the design of new strategies to reduce food spoilage in the future.

Quantifying the effect of sorbic acid, heat and combination of both on germination and outgrowth of Bacillus subtilis spores at single cell resolution

Bacillus subtilis spores are a problem for the food industry as they are able to survive preservation processes. The spores often reside in food products, where their inherent protection against various stress treatments causes food spoilage. Sorbic acid is widely used as a weak acid preservative in the food industry. Its effect on spore germination and outgrowth in a combined, ‘hurdle’, preservation setting has gained limited attention. Therefore, the effects of mild sorbic acid (3 mM), heat-treatment (85 °C for 10 min) and a combination of both mild stresses on germination and outgrowth of B. subtilis 1A700 spores were analysed at single spore level. The heat-treatment of the spore population resulted in a germination efficiency of 46.8% and an outgrowth efficiency of 32.9%. In the presence of sorbic acid (3 mM), the germination and outgrowth efficiency was 93.3% and 80.4% respectively whereas the combined heat and sorbic acid stress led to germination and outgrowth efficiencies of 52.7% and 27.0% respectively. The heat treatment clearly primarily affected the germination process, while sorbic acid affected the outgrowth and generation time. In addition a new ‘burst’ time-point was defined as the time-point at which the spore coat visibly breaks and/or is shed. The combined stresses had a synergistic effect on the time of the end of germination to the burst time-point, increasing both the mean and its variation more than either of the single stresses did.

The diverse role of Pdr12 in resistance to weak organic acids

Resistance to weak organic acids is important relative to both weak organic acid preservatives and the development of inhibitor tolerant yeast as industrial production organisms. The ABC transporter Pdr12 is important for resistance to sorbic and propionic acid, but its role in tolerance to other weak organic acids with industrial relevance is not well established. In this study, yeast strains with altered expression of PDR12 and/or CMK1, a protein kinase associated with post-transcriptional negative regulation of Pdr12, were exposed to seven weak organic acids: acetic, formic, glycolic, lactic, propionic, sorbic and levulinic acid. These are widely used as preservatives, present in lignocellulosic hydrolysates or attractive as chemical precursors. Overexpression of PDR12 increased tolerance to acids with longer chain length, such as sorbic, propionic and levulinic acid, whereas deletion of PDR12 increased tolerance to the shorter acetic and formic acid. The viability of all strains decreased dramatically in acetic or propionic acid, but the Δpdr12 strains recovered more rapidly than other strains in acetic acid. Furthermore, our results indicated that Cmk1 plays a role in weak organic acid tolerance, beyond its role in regulation of Pdr12, since deletion of both Cmk1 and Pdr12 resulted in different responses to exposure to acids than were explained by deletion of Pdr12 alone.

Population heterogeneity and dynamics in starter culture and lag phase adaptation of the spoilage yeast Zygosaccharomyces bailii to weak acid preservatives

The food spoilage yeast Zygosaccharomyces bailii shows great resistance to weak-acid preservatives, including sorbic acid (2, 4-hexadienoic acid). That extreme resistance was shown to be due to population heterogeneity, with a small sub-population of cells resistant to a variety of weak acids, probably caused by a lower internal pH reducing the uptake of all weak acids. In the present paper, it was found that resistant cells were extremely rare in exponential cultures, but increased by up to 8000-fold in stationary phase. Inoculation of media containing sorbic acid with a population of Z. bailii cells gave rise to what appeared to be a prolonged lag phase, suggesting adaptation to the conditions before the cells entered the period of exponential growth. However, the apparent lag phase caused by sorbic acid was largely due to the time required for the resistant sub-population to grow to detectable levels. The slow growth rate of the sub-population was identical to that of the final total population. The non-resistant bulk population remained viable for 3days but had lost viability by 6days and, during that time, there was no indication of any development of resistance in the bulk population. The sub-population growing in sorbic acid showed very high population diversity in colony size and internal pH. After removal of sorbic acid, the population rapidly reverted back to the normal, largely non-resistant, population distribution. The data presented suggest that a reevaluation of the lag phase in microbial batch culture is required, at least for the resistance of Z. bailii to sorbic acid. Furthermore, the significance of phenotypic diversity and heterogeneity in microbial populations is discussed more broadly with potential relevance to bacterial “persisters”, natural selection and evolution.

Hormetic Concentrations of Hydrogen Peroxide but Not Ethanol Induce Cross-Adaptation to Different Stresses in Budding Yeast

The biphasic-dose response of microorganisms to hydrogen peroxide is a phenomenon of particular interest in hormesis research. In different animal models, the dose-response curve for ethanol is also nonlinear showing an inhibitory effect at high doses but a stimulatory effect at low doses. In this study, we observed the hormetic-dose response to ethanol in budding yeast S. cerevisiae. Cross-protection is a phenomenon in which exposure to mild stress results in the acquisition of cellular resistance to lethal stress induced by different factors. Since both hydrogen peroxide and ethanol at low concentrations were found to stimulate yeast colony growth, we evaluated the role of one substance in cell cross-adaptation to the other substance as well as some weak organic acid preservatives. This study demonstrates that, unlike ethanol, hydrogen peroxide at hormetic concentrations causes cross-resistance of S. cerevisiae to different stresses. The regulatory protein Yap1 plays an important role in the hormetic effects by low concentrations of either hydrogen peroxide or ethanol, and it is involved in the yeast cross-adaptation by low sublethal doses of hydrogen peroxide.

The lactic acid bacteria metabolite phenyllactic acid inhibits both radial growth and sporulation of filamentous fungi

BackgroundFood spoilage caused by molds is a severe problem. In food and feed, e.g. dairy products, sourdough bread and silage, lactic acid bacteria are used as starter cultures. Besides lactic and acetic acid, some strains produce other low molecular weight compounds with antifungal activities. One of these metabolites is phenyllactic acid (PLA), well known for its antifungal effect. The inhibitory effect of PLA has only partially been investigated, and the objective of this study was to elucidate in detail the antifungal properties of PLA.ResultsWe investigated the outgrowth of individual conidia from Aspergillus niger, Cladosporium cladosporioides and Penicillium roqueforti, and observed the morphologies of resulting colonies on solid media using different acid concentrations. We found that PLA inhibits molds similar to weak acid preservatives. Furthermore, it has an additional activity: at sub-inhibitory concentrations, fungal colonies displayed slower radial growth and inhibited sporulation. The L isoform of PLA is a more potent inhibitor than the D form. Increased expression of phiA was observed during PLA treatment. This gene was initially identified as being induced by Streptomyces-produced macrolide antibiotics, and is shown to be a structural protein in developed cells. This suggests that PhiA may act as a general stress protectant in fungi.ConclusionFrom a food protection perspective, the results of this study support the usage of lactic acid bacteria strains synthesizing PLA as starter cultures in food and feed. Such starter cultures could inhibit spore synthesis, which would be beneficial as many food borne fungi are spread by airborne spores.

Extreme resistance to weak-acid preservatives in the spoilage yeast Zygosaccharomyces bailii

Weak-acid preservatives, such as sorbic acid and acetic acid, are used in many low pH foods to prevent spoilage by fungi. The spoilage yeast Zygosaccharomyces bailii is notorious for its extreme resistance to preservatives and ability to grow in excess of legally-permitted concentrations of preservatives. Extreme resistance was confirmed in 38 strains of Z. bailii to several weak-acid preservatives. Using the brewing yeast Saccharomyces cerevisiae as a control, tests showed that Z. bailii was ~3-fold more resistant to a variety of weak-acids but was not more resistant to alcohols, aldehydes, esters, ethers, ketones, or hydrophilic chelating acids. The weak acids were chemically very diverse in structure, making it improbable that the universal resistance was caused by degradation or metabolism. Examination of Z. bailii cell populations showed that extreme resistance to sorbic acid, benzoic acid and acetic acid was limited to a few cells within the population, numbers decreasing with concentration of weak acid to <1 in 1000. Re-inoculation of resistant sub-populations into weak-acid-containing media showed that all cells now possessed extreme resistance. Resistant sub-populations grown in any weak-acid preservative also showed ~100% cross-resistance to other weak-acid preservatives. Tests using 14C-acetic acid showed that weak-acid accumulation was much lower in the resistant sub-populations. Acid accumulation is caused by acid dissociation in the higher pH of the cytoplasm. Tests on intracellular pH (pHi) in the resistant sub-population showed that the pH was much lower, ~ pH5.6, than in the sensitive bulk population. The hypothesis is proposed that extreme resistance to weak-acid preservatives in Z. bailii is due to population heterogeneity, with a small proportion of cells having a lower intracellular pH. This reduces the level of accumulation of any weak acid in the cytoplasm, thus conferring resistance to all weak acids, but not to other inhibitors.

Growth and membrane fluidity of food-borne pathogen Listeria monocytogenes in the presence of weak acid preservatives and hydrochloric acid.

This study addresses a major issue in microbial food safety, the elucidation of correlations between acid stress and changes in membrane fluidity of the pathogen Listeria monocytogenes. In order to assess the possible role that membrane fluidity changes play in L. monocytogenes tolerance to antimicrobial acids (acetic, lactic, hydrochloric acid at low pH or benzoic acid at neutral pH), the growth of the bacterium and the gel-to-liquid crystalline transition temperature point (Tm) of cellular lipids of each adapted culture was measured and compared with unexposed cells. The Tm of extracted lipids was measured by differential scanning calorimetry. A trend of increasing Tm values but not of equal extent was observed upon acid tolerance for all samples and this increase is not directly proportional to each acid antibacterial action. The smallest increase in Tm value was observed in the presence of lactic acid, which presented the highest antibacterial action. In the presence of acids with high antibacterial action such as acetic, hydrochloric acid or low antibacterial action such as benzoic acid, increased Tm values were measured. The Tm changes of lipids were also correlated with our previous data about fatty acid changes to acid adaptation. The results imply that the fatty acid changes are not the sole adaptation mechanism for decreased membrane fluidity (increased Tm). Therefore, this study indicates the importance of conducting an in-depth structural study on how acids commonly used in food systems affect the composition of individual cellular membrane lipid molecules.

Weak-acid preservatives: pH and proton movements in the yeast Saccharomyces cerevisiae

Weak-acid preservatives commonly used to prevent fungal spoilage of low pH foods include sorbic and acetic acids. The “classical weak-acid theory” proposes that weak acids inhibit spoilage organisms by diffusion of undissociated acids through the membrane, dissociation within the cell to protons and anions, and consequent acidification of the cytoplasm. Results from 25 strains of Saccharomyces cerevisiae confirmed inhibition by acetic acid at a molar concentration 42 times higher than sorbic acid, in contradiction of the weak-acid theory where all acids of equal pKa should inhibit at equimolar concentrations. Flow cytometry showed that the intracellular pH fell to pH4.7 at the growth-inhibitory concentration of acetic acid, whereas at the inhibitory concentration of sorbic acid, the pH only fell to pH6.3. The plasma membrane H+-ATPase proton pump (Pma1p) was strongly inhibited by sorbic acid at the growth-inhibitory concentration, but was stimulated by acetic acid. The H+-ATPase was also inhibited by lower sorbic acid concentrations, but later showed recovery and elevated activity if the sorbic acid was removed. Levels of PMA1 transcripts increased briefly following sorbic acid addition, but soon returned to normal levels. It was concluded that acetic acid inhibition of S. cerevisiae was due to intracellular acidification, in accord with the “classical weak-acid theory”. Sorbic acid, however, appeared to be a membrane-active antimicrobial compound, with the plasma membrane H+-ATPase proton pump being a primary target of inhibition. Understanding the mechanism of action of sorbic acid will hopefully lead to improved methods of food preservation.

Mapping the structural requirements of inducers and substrates for decarboxylation of weak acid preservatives by the food spoilage mould Aspergillus niger

Moulds are able to cause spoilage in preserved foods through degradation of the preservatives using the Pad-decarboxylation system. This causes, for example, decarboxylation of the preservative sorbic acid to 1,3-pentadiene, a volatile compound with a kerosene-like odour. Neither the natural role of this system nor the range of potential substrates has yet been reported. The Pad-decarboxylation system, encoded by a gene cluster in germinating spores of the mould Aspergillus niger, involves activity by two decarboxylases, PadA1 and OhbA1, and a regulator, SdrA, acting pleiotropically on sorbic acid and cinnamic acid. The structural features of compounds important for the induction of Pad-decarboxylation at both transcriptional and functionality levels were investigated by rtPCR and GCMS. Sorbic and cinnamic acids served as transcriptional inducers but ferulic, coumaric and hexanoic acids did not. 2,3,4,5,6-Pentafluorocinnamic acid was a substrate for the enzyme but had no inducer function; it was used to distinguish induction and competence for decarboxylation in combination with the analogue chemicals. The structural requirements for the substrates of the Pad-decarboxylation system were probed using a variety of sorbic and cinnamic acid analogues. High decarboxylation activity, ~100% conversion of 1mM substrates, required a mono-carboxylic acid with an alkenyl double bond in the trans (E)-configuration at position C2, further unsaturation at C4, and an overall molecular length between 6.5Å and 9Å. Polar groups on the phenyl ring of cinnamic acid abolished activity (no conversion). Furthermore, several compounds were shown to block Pad-decarboxylation. These compounds, primarily aldehyde analogues of active substrates, may serve to reduce food spoilage by moulds such as A. niger. The possible ecological role of Pad-decarboxylation of spore self-inhibitors is unlikely and the most probable role for Pad-decarboxylation is to remove cinnamic acid-type inhibitors from plant material and allow uninhibited germination and growth of mould spores.

Study of Weak Acid Preservatives and Modified Atmosphere Packaging (MAP) on Mold Growth in Modal Agar System

Inhibition of spoilage mold of common contaminant of bakery goods in modal agar system by potassium sorbate and calcium propionate in wide range of concentration (0 - 2000 ppm) at different pH (5.5 - 7) was investigated. All Samples were examined daily for mold growth. Results showed that both potassium sorbate and calcium propionate can be used effectively to inhibit mold growth and the effectiveness of these preservatives is enhanced by increasing the concentration and decreasing the pH. The effect of modified atmosphere packaging (MAP) comprised of 60% or 80% CO2 (balance N2) and oxygen absorbents on the growth of mold was also studied on the agar system. The results indicated that mold growth was prevented up to 42 days in packs flushed with 60% or 80% CO2 (balance N2). It was also evident that all samples did not show mold growth for >40 days when packaged in the presence of oxygen scavenger sachet.

The osmotolerant fructophilic yeast Zygosaccharomyces rouxii employs two plasma-membrane fructose uptake systems belonging to a new family of yeast sugar transporters

Owing to its high resistance to weak-acid preservatives and extreme osmotolerance, Zygosaccharomyces rouxii is one of the main spoilage yeasts of sweet foods and beverages. In contrast with Saccharomyces cerevisiae, Z. rouxii is a fructophilic yeast; it consumes fructose faster than glucose. So far, to our knowledge, no specific Z. rouxii proteins responsible for this fructophilic behaviour have been characterized. We have identified two genes encoding putative fructose transporters in the Z. rouxii CBS 732 genome. Heterologous expression of these two Z. rouxii ORFs in a S. cerevisiae strain lacking its own hexose transporters (hxt-null) and subsequent kinetic analysis of sugar transport showed that both proteins are functionally expressed at the plasma membrane: ZrFfz1 is a high-capacity fructose-specific facilitator (K(m)∼400&emsp14;mM and V(max)∼13&emsp14;mmol h(-1) g(-1)) and ZrFfz2 is a facilitator transporting glucose and fructose with similar capacity and affinity (K(m)∼200&emsp14;mM and V(max)∼4&emsp14;mmol h(-1) g(-1)). These two proteins together with the Zygosaccharomyces bailii Ffz1 fructose-specific transporter belong to a new family of sugar transport systems mediating the uptake of hexoses via the facilitated diffusion mechanism, and are more homologous to drug/H(+) antiporters (regarding their primary protein structure) than to other yeast sugar transporters of the Sugar Porter family.

The decarboxylation of the weak-acid preservative, sorbic acid, is encoded by linked genes in Aspergillus spp.

The ability to resist anti-microbial compounds is of key evolutionary benefit to microorganisms. Aspergillus niger has previously been shown to require the activity of a phenylacrylic acid decarboxylase (encoded by padA1) for the decarboxylation of the weak-acid preservative sorbic acid (2,4-hexadienoic acid) to 1,3-pentadiene. It is now shown that this decarboxylation process also requires the activity of a putative 4-hydroxybenzoic acid (3-octaprenyl-4-hydroxybenzoic acid) decarboxylase, encoded by a gene termed ohbA1, and a putative transcription factor, sorbic acid decarboxylase regulator, encoded by sdrA. The padA1, ohbA1 and sdrA genes are in close proximity to each other on chromosome 6 in the A. niger genome and further bioinformatic analysis revealed conserved synteny at this locus in several Aspergillus species and other ascomycete fungi indicating clustering of metabolic function. This cluster is absent from the genomes of A. fumigatus and A. clavatus and, as a consequence, neither species is capable of decarboxylating sorbic acid.

Inhibition of spoilage mould conidia by acetic acid and sorbic acid involves different modes of action, requiring modification of the classical weak-acid theory

Fungal spoilage of many foods is prevented by weak-acid preservatives such as sorbic acid or acetic acid. We show that sorbic and acetic acids do not both inhibit cells by lowering of internal pH alone and that the “classical weak-acid theory” must be revised. The “classical weak-acid theory” suggests that all lipophilic acids with identical pK a values are equally effective as preservatives, causing inhibition by diffusion of molecular acids into the cell, dissociation, and subsequent acidification of the cytoplasm. Using a number of spoilage fungi from different genera, we have shown that sorbic acid was far more toxic than acetic acid, and no correlation existed between resistance to acetic acid and resistance to sorbic acid. The molar ratio of minimum inhibitory concentrations (MICs) (acetic: sorbic) was 58 for Paecilomyces variotii and 14 for Aspergillus phoenicis. Using flow cytometry on germinating conidia of A spergillus niger, acetic acid at pH 4.0 caused an immediate decline in the mean cytoplasmic pH (pH i) falling from neutrality to ~ pH4.7 at the MIC (80 mM). Sorbic acid also caused a rapid but far smaller drop in pH i, at the MIC (4.5 mM); the pH remained above pH 6.3. Over 0–5 mM, a number of other weak acids caused a similar fall in cytoplasmic pH. It was concluded that while acetic acid inhibition of A. niger conidia was due to cytoplasmic acidification, inhibition by sorbic acid was not. A possible membrane-mediated mode of action of sorbic acid is discussed.

Inhibition ofListeria monocytogenesby acetate, benzoate and sorbate: weak acid tolerance is not influenced by the glutamate decarboxylase system

Weak acids are widely used by the food industry to prevent spoilage and to inhibit the growth of pathogenic micro-organisms. In this study the inhibitory effects of three commonly used weak acids, acetic acid, benzoic acid and sorbic acid, on the growth of Listeria monocytogenes were investigated. In a chemically defined medium at pH 6.4 benzoic acid had the greatest inhibitory effect (50% inhibition of growth at 4 mmol l(-1)), while acetate was the least inhibitory (50% inhibition of growth at 50 mmol l(-1)). Mutants lacking either sigma B (Delta sigB) or two of the glutamate decarboxylase systems (Delta gadAB) were used to investigate the contribution these systems make to weak acid tolerance in L. monocytogenes. The stress-inducible sigma factor sigma B (sigma(B)) was not required for protection against acetate and played only a minor role in tolerating benzoate and sorbate. The glutamate decarboxylase system, which plays an important role in tolerating inorganic acids, played no significant role in the ability of L. monocytogenes to tolerate these weak acids, and neither did the presence of glutamate in the growth medium. These results suggest that the effectiveness of weak acid preservatives in food will not be compromised by the presence of glutamate, at least under mildly acidic conditions.