Fermentation of lignocellulosic hydrolysates. II: inhibitors and mechanisms of inhibition.

Aims: The minimum inhibitory concentration (MIC) of oregano essential oil (OEO) and two of its principle components, i.e. thymol and carvacrol, against Pseudomonas aeruginosa and Staphylococcus aureus was assessed by using an innovative technique. The mechanism of action of the above substances was also investigated. Methods and Results: The applied technique uses 100-well microtitre plate and collects turbidimetric growth data. To produce the inhibition profiles, a wide range of concentrations were tested for each of the three compounds, as well as for carvacrol‐thymol mixtures. Following a specific mathematical analysis of the observed inhibition profiles from all compounds, it was suggested that mixtures of carvacrol and thymol gave an additive effect and that the overall inhibition by OEO can be attributed mainly to the additive antimicrobial action of these two compounds. Addition of low amounts of each additive: (a) increased permeability of cells to the nuclear stain EB, (b) dissipated pH gradients as indicated by the CFDA-SE fluorescent probe irrespective of glucose availability and (c) caused leakage of inorganic ions. Conclusions: Mixing carvacrol and thymol at proper amounts may exert the total inhibition that is evident by oregano essential oil. Such inhibition is due to damage in membrane integrity, which further affects pH homeostasis and equilibrium of inorganic ions. Significance and Impact of the Study: The knowledge of extent and mode of inhibition of specific compounds, which are present in plant extracts, may contribute to the successful application of such natural preservatives in foods, since certain combinations of carvacrol‐ thymol provide as high inhibition as oregano essential oil with a smaller flavour impact.

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A study of the minimum inhibitory concentration and mode of action of oregano essential oil, thymol and carvacrol.

Fermentation of lignocellulosic hydrolysates. I: inhibition and detoxification

Preservative agents in foods. Mode of action and microbial resistance mechanisms.

The application of physical stress to microorganisms is the most widely used method to induce cell inactivation and promote food stability. To survive, microorganisms have evolved both physiological and genetic mechanisms to tolerate some extreme physical conditions. This is clearly of significance to the food industry in relation to survival of pathogens or spoilage organisms in food. In some microorganisms, the “cold shock response” has been observed in response to abrupt changes to lower temperatures. This results in the production of specific sets of proteins (cold shock proteins), the continued synthesis of proteins involved in transcription and translation, and the repression of heat shock proteins. The addition of weak acid preservatives (for example, sorbates, benzoates) also induces a specific pattern of gene expression (for example, ‘Acid Tolerance Response’), which is likely to be required for optimal adaptation of bacteria to weak acid preservatives and low pH. The primary mode of the antimicrobial action of low pH is to reduce the internal cell pH (pHi) below the normal physiological range tolerated by the cell, leading to growth inhibition. Survival mechanisms involve maintaining pH homeostasis, and this is achieved by a combination of passive and active mechanisms. Microorganisms adapt to osmotic stress by accumulating non-ionic or compatible solutes such as trehalose, glycerol, sucrose, and mannitol. These compatible solutes help balance the osmotic pressure and help preserve protein function inside the cells. By understanding and controlling such mechanisms of adaptation, it may be possible to prevent growth of key microorganisms in food products.

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Adaptation of Microorganisms to Cold Temperatures, Weak Acid Preservatives, Low pH, and Osmotic Stress: A Review

The weak acid sorbic acid transiently inhibited the growth of Saccharomyces cerevisiae in media at low pH. During a lag period, the length of which depended on the severity of this weak-acid stress, yeast cells appeared to adapt to this stress, eventually recovering and growing normally. This adaptation to weak-acid stress was not due to metabolism and removal of the sorbic acid. A pma1-205 mutant, with about half the normal membrane H+-ATPase activity, was shown to be more sensitive to sorbic acid than its parent. Sorbic acid appeared to stimulate plasma membrane H+-ATPase activity in both PMA1 and pma1-205. Consistent with this, cellular ATP levels showed drastic reductions, the extent of which depended on the severity of weak-acid stress. The weak acid did not appear to affect the synthesis of ATP because CO2 production and O2 consumption were not affected significantly in PMA1 and pma1-205 cells. However, a glycolytic mutant, with about one-third the normal pyruvate kinase and phosphofructokinase activity and hence a reduced capacity to generate ATP, was more sensitive to sorbic acid than its isogenic parent. These data are consistent with the idea that adaptation by yeast cells to sorbic acid is dependent on (i) the restoration of internal pH via the export of protons by the membrane H+-ATPase in an energy-demanding process and (ii) the generation of sufficient ATP to drive this process and still allow growth.

Activity of the plasma membrane H(+)-ATPase and optimal glycolytic flux are required for rapid adaptation and growth of Saccharomyces cerevisiae in the presence of the weak-acid preservative sorbic acid.

M. Stratford

Papers

Activity of the plasma membrane H(+)-ATPase and optimal glycolytic flux are required for rapid adaptation and growth of Saccharomyces cerevisiae in the presence of the weak-acid preservative sorbic acid.