The increased interest in biopreservation of food systems has recently led to the development of new natural antimicrobial compounds having different origins. A variety of systems to prevent food spoilage have been investigated; these include animal-derived systems (lysozyme, lactiferrin, magainins, etc.), plant-derived products (phytoalexins, herbs, spices), and microbial metabolites, including bacteriocins, hydrogen peroxide, and organic acids (12
). Few of the major food preservation techniques (e.g., low temperature, low water activity, acidification, etc.) act by inactivating the spoilers, while most of the newer or emerging techniques (irradiation, electroporation, high hydrostatic pressure, etc.) act by directly inactivating microorganisms. Nowadays, in the case of bread and bakery products, which can be contaminated by a variety of molds (mainly Aspergillus
species), contamination can be prevented by irradiating a product with infrared rays or microwaves, by using a modified atmosphere, or by adding fungal inhibitors, such as ethanol, propionic acid, sorbic acid, and acetic acid (17
). In particular, propionic acid and its salts are commonly used to extend the shelf lives of bakery products. Recently, the levels of chemical preservatives permitted in bakery products in Europe have been reduced due to application of EU Directive 95/2/CE (1
), which allows concentrations of propionate and sorbate salts of 0.3 and 0.2% (wt/wt), respectively, for packaged sliced breads, although the latter is rarely used because of its secondary effects on bread volume (17
). When 0.3% (wt/vol) calcium propionate was tested in a conidial germination assay with several molds contaminating bakery products, fungal growth still occurred, suggesting that the use of suboptimal salt concentrations might not assure preservation (16
Among the natural preserving systems, sourdough has long been known to improve the shelf lives of bread and bakery products. Rocken (25
) observed that sourdough antifungal activity was strictly related to acetic acid production. More recently, the use of sourdough lactic acid bacteria to inhibit mold growth was studied, which led to the identification of a strain of Lactobacillus plantarum
21B whose culture filtrate showed an important antifungal activity. Phenyllactic acid (PLA) was shown to be one of the major compounds occurring in the culture, together with lactic acid and acetic acid (16
). Dieleveux et al. (8
) isolated PLA from a culture filtrate of Geotrichum candidum
and characterized it as the main compound responsible for the anti-Listeria
activity shown by the fungal culture. These authors obtained relevant inhibition of pathogen growth in an agar diffusion well assay by using dl
-PLA, while d
-3-PLA inhibited the growth of Listeria monocytogenes
cultured in liquid medium or in ultrahigh-temperature whole milk and the growth of several strains of Staphylococcus aureus
, Escherichia coli
, and Aeromonas hydrophila
on solid medium (6
). PLA has been reported to be one of the most abundant aromatic acids to which antibacterial properties have been attributed and to occur in several honeys with different geographical origins (28
In this study the antifungal activity of PLA against a variety of fungal species isolated from bakery products and flours and two ochratoxin A-producing strains isolated from cereals was evaluated. For each strain, the minimal fungicidal or inhibitory PLA concentration was determined together with the behavior at pH conditions more similar to those in real food systems with respect to the ability to inhibit and delay mold growth. The effect of PLA in combination with the main organic acids produced in culture by L. plantarum 21B was also investigated.
MATERIALS AND METHODS
dl-β-PLA was supplied by Sigma-Aldrich Division (Milan, Italy), l-(−)-β-PLA and d-(−)-β-PLA were supplied by Fluka (Sigma-Aldrich Division, Milan, Italy), and lactic and acetic acids were supplied by Carlo Erba (Milan, Italy).
All fungal strains used in this study were isolated from bakery products, wheat flour, or cereals (Table 1
). In particular, Aspergillus flavus
ITEM5134 and ITEM5135, Aspergillus niger
ITEM5132, Aspergillus terreus
ITEM5136, Penicillium brevicompactum
sp. strains ITEM5147 and ITEM5148 (species morphologically related to P. brevicompactum
but not yet characterized), Penicillium chrysogenum
ITEM5151 and ITEM5152, Penicillium citrinum
ITEM5144, ITEM5145, and ITEM5146, Penicillium commune
ITEM5150, Penicillium polonicum
ITEM5142, ITEM5143, and ITEM5141, Penicillium solitum
ITEM5149, and Fusarium
sp. strain ITEM5153 were isolated in Apulia, Italy, identified and confirmed by different morphological procedures (14
), and deposited in the ITEM Culture Collection of the CNR Institute of Sciences of Food Production, Bari, Italy. A. flavus
FTDC3226 and A. niger
FTDC3227 were obtained from the Culture Collection of the Food Technology Department, University of Lleida, Lleida, Spain; Penicillium roqueforti
IBT18687 and two strains of ochratoxin A producers, Aspergillus ochraceus
FR21991 and Penicillium verrucosum
FR22625, were obtained from the Culture Collection of the Technical University of Denmark, Lyngby, Denmark. For some strains, a high-performance liquid chromatography analysis of culture extracts obtained by microscale extraction (27
) was performed to determine the chromatographic metabolite profiles. In particular, production of citrinin was ascertained for the three strains of P. citrinum
, while the three A. flavus
strains did not produce aflatoxins when they were grown on the solid media yeast extract sucrose agar (26
) and Czapek yeast extract agar (23
Preparation of inoculum suspension.
Fungal conidia were collected from 7-day-old cultures on potato dextrose agar (PDA) (Difco Laboratories, Detroit, Mich.) and washed twice with distilled water, and a 50-μl aliquot of each conidial suspension was spread on PDA plates and incubated at 26°C for 72 h. Conidia were collected by using 0.05% (vol/vol) Triton X-100, and 10 μl of the conidial suspension, containing about 5 × 104 conidia, was used as the inoculum.
Preliminary experiments showed that there were not significant differences (P > 0.05) in the inhibitory activities against A. niger FTDC3227 between the racemic and d and l isomers of PLA at a concentration of 20 mg ml−1; therefore, dl-β-PLA was used in all experiments.
(i) MIC and MFC evaluation.
To determine the 90% MIC (MIC90
) and the minimal fungicidal concentration (MFC), a PLA stock solution (pH 2.6) containing 20 mg of PLA ml−1
in wheat flour hydrolysate (WFH) broth (11
) was serially diluted to obtain concentrations of 15, 10, 7.5, 5, 3.75, 2.5, and 1.87 mg ml−1
, and the preparations were filter sterilized. A WFH solution was used as a control.
(ii) Influence of pH on PLA activity.
In order to test PLA at pH values closer to those in real food systems, the influence of pH on PLA activity was assayed by using A. niger FTDC3227. pH values of 2.6, 4.0, 4.5, 5.0, and 5.5 were obtained by dissolving PLA (20 mg ml−1) in WFH diluted 1:1 (vol/vol) with water and phosphate buffer containing 0.18, 0.22, 0.25, and 0.3 mol of KH2PO4-K2HPO4 liter−1, respectively. WFH diluted 1:1 with water was used as a control solution.
(iii) Antifungal activity of PLA at pH 4.
Antifungal activity at pH 4 was evaluated by using all of the fungal strains and PLA solutions containing 20, 15, 10, 7.5, 5, and 3.75 mg of PLA ml−1 in WFH diluted 1:1 (vol/vol) with phosphate buffer at different concentrations (0.18, 0.12, 0.08, 0.06, 0.04, and 0.03 mol of KH2PO4-K2HPO4 liter−1, respectively). WFH diluted 1:1 with water was used as a control solution.
(iv) Inhibitory effect of PLA in the presence of other organic acids.
PLA was also tested at a concentration of 5 mg ml−1
and pH 4 in the presence of lactic and/or acetic acid at the concentrations found in the culture filtrate of L. plantarum
) (0.79 and 0.017 mg ml−1
, respectively) and at the following higher concentrations: 7.9 and 15.8 mg of lactic acid ml−1
; 0.17, 0.34, and 0.67 mg of acetic acid ml−1
; and 15.8 mg of lactic acid ml−1
and 0.67 mg of acetic acid ml−1
Microdilution tests were performed with sterile, disposable, multiwell microdilution plates (96 wells; IWAKI; Scitech Div., Asashi Techno Glass, Tokyo, Japan). Test solutions were dispensed into the wells in 190-μl portions inoculated with 10 μl of a conidial suspension containing about 5 × 104 conidia. Inoculated wells were prepared in quintuplicate, and blanks were prepared in triplicate. All microdilution plates were incubated in a humid chamber at 26°C for 120 h. Fungal growth was observed with a reverse microscope and was measured by determining the optical density at 580 nm every 24 h with a spectrophotometer (Labsystem Multiskan MS, version 3.0, type 352). In each experiment, an uninoculated control (WFH containing antifungal compounds) and an untreated inoculated control were included. The MIC90 was defined as the lowest concentration of PLA that resulted in at least a 90% reduction in growth, as measured by optical density, compared to the growth of an untreated control after 72 h of incubation at 26°C. To quantify the MFC, plate counts of the fungi were determined on PDA by using 10-μl portions from wells containing PLA concentrations higher than the MIC90, the PLA concentration equal to the MIC90, and the PLA concentration that was just below the MIC90. The MFC was defined as the lowest concentration in which no conidial germination was observed after 72 h of incubation at 26°C. Triplicate determinations were performed.
Optical density measurements recorded every 24 h from zero time to 120 h were used to generate growth curves for each fungal strain. The Gompertz model was used as a mathematical means of fitting growth curves to estimate microbial growth kinetics (2
). Three points (optical densities at 72, 96, and 120 h) of the control growth curve were used to calculate with the Gompertz model the additional time (growth delay, expressed in hours) required by PLA-treated suspensions to reach the optical density of the control at that time. The growth delays with respect to the control at these three times (GD72
, and GD120
, respectively) were predicted by the Gompertz model in the cases where the optical density of the control was not reached within the experimental period. The Sigma Plot program (SPSS Science Software Gmb, Erkrath, Germany) was used for graphics and data elaboration.
The findings of the present study indicate that PLA has interesting potential for practical application as an antimicrobial agent in the food industry due to its broad inhibitory activity against a variety of food-borne fungi. The fungal species investigated in this study have been found previously in flours, bakery products, and/or cereals and have the potential to produce bioactive secondary metabolites, including mycotoxins. In particular, A. flavus
has been recovered from flour, bread, and bakery products [9
; F. Valerio, P. Lavermicocca, and A. Visconti, Book Abstr. 5th Congr. Naz. FIMUA (Fed. Naz. Micopatol. Umana Anim.), p. 25, 2000], and the carcinogenic aflatoxins have also been found in bakery products (24
) and rye bread (10
). Even though this fungus is known to produce aflatoxins, the strains evaluated in this study did not show aflatoxin production. Both A. ochraceus
, isolated from flour (3
) and bread (30
), and P. verrucosum
, reported to be a contaminant of cakes (32
) and cereals (19
), produce several mycotoxins, including ochratoxin A. Recently, it has also been established that ochratoxin A is produced by other fungal species, such as A. niger
), which has also been found as a contaminant of bread (32
; Valerio et al., Book Abstr. 5th Congr. Naz. FIMUA).
In our experiments the MFC of PLA for A. ochraceus
FR21991 and P. verrucosum
FR22625, both ochratoxin A producers, were 7.5 and 5 mg ml−1
, respectively, while only growth-inhibiting activity against A. niger
ITEM5132 and FTDC3227 was observed. P. citrinum
is known to produce the nephrotoxic mycotoxin citrinin, which has previously been found in bakery products, moldy bread, and rye bread (4
). The three citrinin-producing strains that were tested here were also inhibited by PLA (MIC90
, 7.5 mg ml−1
). PLA showed fungicidal activity against 13 of the 14 species tested, and these 13 species included potential toxigenic organisms, such as A. ochraceus
, A. flavus
, P. roqueforti
, P. verrucosum
, and P. citrinum
. This indicates that application of PLA to reduce fungal mass in food systems has a clear advantage compared with the preservatives now commonly used in bakery products, such as propionic acid and its salts, which act by a fungistatic mechanism (15
) that causes only temporary inhibition of microbial growth. Similar to the activities of other weak acid preservatives (propionic acid, benzoic acid, sorbic acid, etc.) and organic acid acidulants (lactic acid, malic acid, citric acid, acetic acid, etc.), the activity of PLA (pK 3.46) was shown to be pH dependent, indicating that its mode of action is somewhat related to the lipophilic properties which enable the undissociated form to cross microbial membranes (12
). The PLA concentrations showing antifungal activity against molds isolated from bakery products are generally lower than those required for antibacterial activity. In fact, antimicrobial activity of PLA has been reported for Listeria monocytogenes
at a concentration of 13 mg ml−1
and for other human pathogens (S. aureus
, E. coli
, and A. hydrophila
) at a concentration of 20 mg ml−1
A considerable effect of PLA was also observed at pH 4, a pH explored because of its broader application to real food systems; at this pH concentrations lower than 7.5 mg ml−1 were enough to cause both inhibition of more than 50% of fungal growth and a relevant growth delay for all strains tested. In the case of A. flavus (two strains), P. citrinum (two strains), P. commune, and Penicillium sp. (one strain), optical densities equivalent to those of control cultures were never reached by treated fungal suspensions. In conclusion, by using the Gompertz parameters, a PLA concentration of 7.5 mg ml−1 gave unpredictable growth delays (however, the growth delays were always longer than 2 days) for 12 strains, including the common contaminants P. roqueforti and A. flavus and the mycotoxigenic strains of P. verrucosum and P. citrinum.
The growth delays observed in these experiments are of great relevance for extending the shelf lives of food products. In particular, in the case of the acid-tolerant organism P. roqueforti
, application of 5 mg of PLA ml−1
(the MFC) resulted in complete inhibition of the strain even at a low pH (pH 3.0). When used at pH 4, PLA (7.5 mg ml−1
) inhibited fungal development by 52% and delayed growth by an unpredictable time (i.e., the level of contamination observed in the control was not reached during the 5-day experiment). These findings support previous data which showed that the PLA-producing strain L. plantarum
21B, used as a starter in sourdough bread, delayed the growth of A. niger
) and P. roqueforti
IBT18687 (A. Corsetti and P. Lavermicocca, unpublished data) for up to 7 days at room temperature. This delaying effect was not observed in bread started with Lactobacillus brevis
1D, a sourdough strain which in culture produced about the same amounts of lactic acid and acetic acid as L. plantarum
21B (ca. 0.8 and 0.02 mg ml−1
, respectively, for both strains) (16
). This led to the conclusion that PLA was the major factor responsible for the antifungal activity and prolonged shelf life produced by L. plantarum
21B in sourdough bread and the conclusion that these effects were improved by the presence of lactic and acetic acids.
PLA production by food-related bacteria has been reported for a strain of Propionibacterium freudenreichii
and two strains of L. plantarum
). It has been shown that PLA is a product of phenylalanine metabolism; in particular, phenylalanine can be transaminated to phenylpyruvic acid, which is further metabolized to PLA by hydroxy acid dehydrogenase (29
). Therefore, modification of bacterial growth conditions to improve metabolite production may lead to isolation of strains with enhanced PLA production. In addition, screening a relevant number of lactic acid bacteria from several microbiotas, as well as investigations of the inhibitory activities against other microbial contaminants, could widen the potential application of PLA to other food systems. The natural occurrence of relevant amounts of PLA in several honeys from different geographical areas (28
) and the apparent lack of toxicity of PLA for human and animal cell lines (20
) might allow its safe use in foods, even though information about its effect on rheology and flavor should be acquired for each food system. Therefore, PLA, like other antimicrobial substances produced by lactic acid bacteria, represents a promising natural device for controlling contaminants in food systems. An additional advantage compared with some of the other compounds, such as acetic acid, may be the apparent lack of odor of PLA solutions.
Since molds are responsible for contamination of a variety of products destined for both human and animal consumption, such as dairy and meat products, fruit, vegetables, and silages, in fermented foods and feedstuffs PLA-producing strains of lactic acid bacteria may be used for in situ production of the antimicrobial compound, while the conditions for direct application of PLA should be established for other food systems.