|The fungistatic action of oleic acid|
W. Sheba Davidson, R. K. Saxena* and
Department of Microbiology, University of Delhi, South Campus, Benito Juarez Road, New Delhi 110 021, India
Oleic acid (C 18:1) has been found to be fungistatic against a wide spectrum of saprophytic moulds and yeasts. The fatty acid causes a delay of 68 h in the germination of fungal spores and is very effective at a low concentration of 0.7% (v/v). The application of this property of oleic acid finds great use in preserving foodstuffs including bakery products like cakes and pastries with sweet soft cream as a topping which are prone to quick spoilage under conditions of non-refrigeration.FATTY acids of varying chain lengths are known for their antimicrobial action primarily against Gram-positive bacteria and yeasts at low pH16. The observed inhibition is explained as a consequence of the uptake of undissociated fatty acids which dissipate the transmembrane proton gradient and thereby affect
tations of grape must2,4. In the present communication, we report the wider ability of oleic acid to inhibit a variety of moulds and yeasts. The fungistasis is effected at the germination stage of the spores. This observation can find use in the preservation of foodstuffs, as oleic acid can be metabolized easily in vivo in the same manner as fatty acids normally found in food.
A number of saprophytic yeasts and moulds (as listed
in Table 1) were grown and maintained on slants of potato dextrose agar (PDA) medium10
at 30 ± 1°C or 37 ± 1°C. Following incubation for five days, the
cultures were then stored at 4 ± 1°C till use. The organisms were subcultured
once in every fifteen days and the
purity of the cultures was checked regularly under microscope.
The inhibition tests were carried out in two stages. (i) The plate method; where oleic acid (0.2 ml) was loaded in wells (8 mm diameter) on PDA plates containing a luxuriant lawn of the different yeasts and moulds; (ii) Candida albicans and Aspergillus sp. (showing largest zones of inhibition) were grown in potato dextrose broth containing 0.12% (v/v) oleic acid at 30 ± 1°C and 37 ± 1°C, respectively in a controlled environment, using new Brunswick shaker (G25-KC) at 250 rpm. Biomass in each case was measured using predried (80°C for 48 h) and preweighed discs of Whatman filter paper no.1 or aluminium foil cups.
For the study of inhibition of aerial microflora, PDA plates containing 1%, 2%, and 3% (w/v) of dextrose were exposed to air for 1 h. A high concentration of 3% dextrose was tried, as bakery products often contain a high concentration of sugar and this supports the growth of osmophilic yeasts and moulds. The air-exposed plates were incubated at 37 ± 1°C and observed for microbial growth after 24, 48 and 72 h.
For determination of the exact stage of growth at which oleic acid inhibited the fungi, spores of Aspergillus sp. were observed in cavity slides containing 3% dextrose solution with oleic acid. Control slides were devoid of the fatty acid. The slides were observed under a Nikon microscope (Type 102) at regular intervals of 30 min.
Oleic acid and all other chemicals used in the present investigation were of analytical grade and purchased locally from E. Merck, India.
The addition of oleic acid to the cultures of C. albicans (Figure 1) and Aspergillus sp. resulted in a deviation from the growth curves, normally obtained in a defined medium. The effects observed can be generally described as a decrease in the specific growth rate and an increase in the length of the lag phase, which were particularly well marked in the presence of higher concentrations of oleic acid. The specific growth rate dropped to a low of 0.030.04 h1, and the observed lag period was of 78 h in case of C. albicans.
It has earlier been reported that the effects of sublethal concentrations of organic acids are the expressions of a complex interaction between the acid molecule and the microorganism. The toxicity level of the compound is associated with the concentration of the unionized form of the acid and the size of the carbon chain. The resistance of the microorganism to acid toxicity depends on the effectiveness of its tolerance mechanism, and varies from one species to another. Acid stress results in changes at all levels of cell physiology leading to a modification of the microbial growth profile11.
When a variety of yeasts and moulds were grown on plates containing a central well loaded with 0.2 ml of oleic acid, zones of inhibition ranging from 19 mm were observed after 48 h (Table 1; Figure 2). This finding clearly showed that oleic acid has antimicrobial effect over a wide spectrum of yeasts, a good number of aspergillii, penicillii, and many other moulds.Figure 1. Effect of oleic acid (0.7% v/v) on growth of Candida albicans under shake culture conditions.
Figure 2. Zone of inhibition caused by oleic acid against yeasts and moulds after 48 h in (a) Cryptococcus neoformans; (b) Candida albicans; (c) Aspergillus sp. and (d) Curvularia lunata.
Figure 3. Potato dextrose agar plates exposed to air for 1 h and incubated at 37 ± 1°C for 48 h. (a) control (without oleic acid); and (b) test (with oleic acid).
Extending this experiment further, PDA plates containing 1, 2, and 3% (w/v) dextrose and 0.7% (v/v) oleic acid incorporated in the medium were prepared. In addition, PDA plates with the same concentrations of dextrose after solidification were overlaid with a film of oleic acid. These sets of plates were exposed to air for 1 h with a view to trap the aerial microflora.
Figure 3 clearly shows that oleic acid was extremely effective in preventing/reducing the growth of aerial microflora. In particular, the plates with oleic acid incorporated in them showed better results. This might be due to the inability of oleic acid to form a uniform film
Figure 4. Fungistatic effect of different concentrations of oleic acid on Candida albicans after 8 h.Figure 5. Inhibition of germination of spores of Aspergillus sp. by oleic acid (0.7% v/v). (a) control (without oleic acid); and (b) test (with oleic acid).
on the surface of the PDA plates owing to its hydrophobic nature. Our study showed that while lower concentrations of oleic acid were effective when incorporated in the medium, higher concentrations were required for the spread plate method. Similar results were also obtained using milk cream.
Oleic acid acts in a concentration-dependent manner with 0.7% (v/v) being the least concentration which is most effective (Figure 4). At high concentrations, growth is largely inhibited.
It was observed that the time of addition of oleic acid was an important factor. Oleic acid added at the time of incubation (0 h) was very effective in causing inhibition of growth. Addition of the fatty acid later during incubation of the organisms was not determined to be a useful measure. Thus, the fungistatic nature of oleic acid was identified. In order to confirm this property of oleic acid, germination of spores of Aspergillus sp. was observed in cavity slides. Figure 5 clearly shows the delay in the germination of the fungal spores by 8 h in presence of oleic acid. This clearly established the fungistatic nature of oleic acid.
It can hence be concluded that oleic acid can be used in combination with other compounds routinely in the
preservation of cakes and pastries which are easily prone to spoilage, particularly during power breakdowns in developing countries12. Oleic acid is not highly reactive, and is also easily miscible with any lipophilic preparation. In addition, it causes no interference with the preservative effect of other chemicals. Oleic acid can be metabolized easily in vivo in the same manner as fatty acids normally found in food and thus the use of oleic acid as a preservative tool for processed foods is advocated.
ACKNOWLEDGEMENTS. We thank Ms Rekha Kohli for her help in preparation of this manuscript. Ms W. Sheba Davidson acknowledges grant of Senior Research Fellowship from the University Grants Commission.
Received 30 October 1998; revised accepted 16 January 1999