Sourdough lactic acid bacteria were selected for antifungal activity by a conidial germination assay. The 10-fold-concentrated culture filtrate of Lactobacillus plantarum 21B grown in wheat flour hydrolysate almost completely inhibited Eurotium repens IBT18000, Eurotium rubrum FTDC3228,Penicillium corylophilum IBT6978, Penicillium roqueforti IBT18687, Penicillium expansum IDM/FS2,Endomyces fibuliger IBT605 and IDM3812, Aspergillus niger FTDC3227 and IDM1, Aspergillus flavus FTDC3226,Monilia sitophila IDM/FS5, and Fusarium graminearum IDM623. The nonconcentrated culture filtrate ofL. plantarum 21B grown in whole wheat flour hydrolysate had similar inhibitory activity. The activity was fungicidal. Calcium propionate at 3 mg ml−1 was not effective under the same assay conditions, while sodium benzoate caused inhibition similar toL. plantarum 21B. After extraction with ethyl acetate, preparative silica gel thin-layer chromatography, and chromatographic and spectroscopic analyses, novel antifungal compounds such as phenyllactic and 4-hydroxy-phenyllactic acids were identified in the culture filtrate of L. plantarum 21B. Phenyllactic acid was contained at the highest concentration in the bacterial culture filtrate and had the highest activity. It inhibited all the fungi tested at a concentration of 50 mg ml−1 except forP. roqueforti IBT18687 and P. corylophilumIBT6978 (inhibitory concentration, 166 mg ml−1). L. plantarum 20B, which showed high antimold activity, was also selected. Preliminary studies showed that phenyllactic and 4-hydroxy-phenyllactic acids were also contained in the bacterial culture filtrate of strain 20B. Growth of A. niger FTDC3227 occurred after 2 days in breads started with Saccharomyces cerevisiae 141 alone or with S. cerevisiae andLactobacillus brevis 1D, an unselected but acidifying lactic acid bacterium, while the onset of fungal growth was delayed for 7 days in bread started with S. cerevisiae and selectedL. plantarum 21B.
Fungal growth is the most frequent cause of spoilage in baked goods. In addition to the great economic losses associated with spoilage, another concern is the possibility that mycotoxins could cause public health problems (20).
Fungal contamination of baked goods is influenced by several factors: the type of product (bread or sweet baked goods), ingredients (type of flour and other dry ingredients), leavening sources (chemical, baker's yeast, or sourdough), size and architecture of the bakery, and conditioning and packaging of the products (slicing, wrapping, and materials used for packaging). Since fungal spores are killed during baking, airborne molds contaminate the baked goods during cooling, slicing, wrapping, and storage operations (20).
The most common spoiling fungi isolated from bakery products belong to the genera Penicillium, Aspergillus,Monilia, Mucor, Endomyces,Cladosporium, Fusarium, and Rhizopus(20, 24). Baked goods can be protected from fungal spoilage by destroying any spores which have contaminated the products (e.g., infrared and microwave radiation), using fungal inhibitors such as ethanol and propionic, sorbic, benzoic, and acetic acids and some of their salts, using modified atmospheres or other packaging techniques, and using sourdough with antifungal activity (20, 25).
The use of sourdough in rye and wheat breadmaking, as well as in other sweet baked goods, increases shelf life due to the presence of lactic acid bacteria (11). To date, only a few studies have reported the antifungal activity of lactic acid bacteria, and many of them have considered food processes other than those used for leavened baked goods. The improved microbial shelf life of sourdough baked products was initially attributed to the organic acids produced by lactic acid bacteria (27, 28). By increasing the amount of sourdough used, the shelf life of bread inoculated with conidia of typical bread molds such as Aspergillus niger,Cladosporium herbarum, and Penicillium verrucosumhas been extended. The fungistatic effect was attributed to lactic and, especially, acetic acids produced by lactic acid bacteria (25). Further studies have confirmed that the acetic acid concentration was strictly related to the antifungal activity and that other bacterial metabolites also have inhibitory activity (3, 13, 22). It has been shown that the antifungal activity of sourdough lactic acid bacteria varies and is found mainly in obligately heterofermentative Lactobacillus spp. Within this group,Lactobacillus sanfranciscensis CB1 (31) had the largest spectrum of antifungal activity due to the production of a mixture of organic acids (3).
Regarding other food processes, it has been shown that the culture supernatant of a Lactobacillus spp. mixture from a commercial silage inoculum reduces growth and aflatoxin production byAspergillus flavus subsp. parasiticus(12). The mycostatic activity of a Leuconostoc mesenteroides strain used in cheesemaking has been reported, but the antifungal substances have not been isolated (29). Recently, new antimicrobial compounds in the culture filtrate ofLactobacillus plantarum, which were active againstPantoea agglomerans and also against molds such asFusarium avenaceum, have been identified (22).
In this work, we studied the antifungal activity of several sourdough lactic acid bacteria and selected L. plantarum 21B because it showed a very wide spectrum of inhibitory activity against fungi isolated from bakery products. Novel antifungal compounds have been identified, their production has been characterized, and the selected strain was used in sourdough bread production.
MATERIALS AND METHODS
Microbial species and culture conditions.
Eurotium repens IBT18000, Penicillium corylophilum IBT6978,Penicillium roqueforti IBT18687, Endomyces fibuliger IBT605 (belonging to the Culture Collection of the Technical University of Denmark, Lyngby, Denmark), Aspergillus niger FTDC3227, Aspergillus flavus FTDC3226,Eurotium rubrum FTDC3228 (supplied by the Food Technology Department, University of Lleida, Spain), and Aspergillus niger IDM1, Penicillium expansum IDM/FS2, Monilia sitophila IDM/FS5, Fusarium graminearum IDM623, andE. fibuliger IDM3812 (belonging to the Culture Collection of the Institute of Dairy Microbiology, Agriculture Faculty of Perugia, Italy) were used in this study. All these species are commonly isolated from contaminated baked goods (20, 24).
Fungal cells or conidial suspensions (glycerol-water, 20% [vol/vol]) were routinely stored at −80°C and subsequently grown on malt agar (MA) plates (malt extract; Difco Laboratories, Detroit, Mich.) or potato dextrose agar (PDA; Difco) for 48 h (Aspergillusand Penicillium species) or 72 h at 26°C. OnlyEurotium and Monilia species were grown on Czapek yeast extract S20 (23) agar plates for 72 h at 30°C. To produce conidia for the germination assay, spores were collected from cultures on MA and washed twice with sterile distilled water, and a 50-μl aliquot of conidial suspension was spread on MA with subsequent incubation at 26°C for 72 h.
Lactobacillus alimentarius 1A, 2B, 8D, 5A, 5Q, and 5Z;Lactobacillus brevis 1F, 10R, and 1D; Lactobacillus fermentum 6E, 18C, and 18B; Leuconostoc mesenteroidessubsp. mesenteroides 12K; Weissella confusa 14A (2); Leuconostoc citreum 22A (7) and 10M; Lactobacillus plantarum 20B, 21A, and 21B; andLactococcus lactis subsp. lactis 11M, belonging to the Collection of the Istituto Tossine e Micotossine da Parassiti Vegetali, Consiglio Nazionale delle Ricerche, Bari, Italy, were previously isolated and identified from sourdough breads of the Apulia region of Italy (A. Corsetti, P. Lavermicocca, M. Morea, F. Baruzzi, and M. Gobbetti, submitted for publication). Lactobacillus sanfranciscensis IDM/E20 and IDM/C57 and Lactobacillus acidophilus IDM/A2 were from the Culture Collection of the Institute of Dairy Microbiology, Agriculture Faculty of Perugia, Italy. Strains were grown in sour dough bacteria (SDB) broth (17) at 30 or 37°C for 24 h, and cell suspensions (glycerol-SDB, 20% [vol/vol]) were routinely stored at −80°C.
Production of antifungal compounds by lactic acid bacteria.
Twenty-four-hour-old cells of lactic acid bacteria were used to inoculate 0.2% (vol/vol) wheat flour hydrolysate (WFH) broth (pH 4.8) produced as reported previously (8). WFH was used as the culture medium to select for antifungal activity because its main composition is similar to that of wheat flour. Except for the L. fermentum and L. acidophilus strains, which were incubated at 37°C, all the other strains were incubated at 30°C for 72 h. After incubation, cells were harvested by centrifugation (9,000 × g for 10 min at 4°C), and the supernatants were filter sterilized and freeze-dried. After freeze-drying, the bacterial culture filtrates (BCFs) were concentrated 10-fold with respect to their initial volume and used for the conidial germination assay.
The cell concentration in the culture filtrate was estimated by plating on SDB agar after incubation at 30°C for 48 h. The concentrations of lactic and acetic acids and ethanol were determined by enzymatic methods (Boehringer-Mannheim, Milan, Italy).
Conidial germination assay.
The antifungal activity of the BCFs was determined using the conidial germination assay described previously (18), with some modifications. Ten microliters of a conidial suspension in 0.05% (vol/vol) Triton containing about 103 conidia of the fungal species per ml was added to 190 μl of (i) a solution of 10-fold-concentrated BCFs (500 mg of dry matter per ml, pH 3.6 to 3.8), (ii) a solution of 10-fold-concentrated WFH (500 mg of dry matter per ml, pH 4.8), or (iii) solutions of WFH containing calcium propionate and sodium benzoate at concentrations of 0.003, 0.03, and 0.3% (wt/vol) and incubated at 25°C for 30 min. When E. fibuliger strains were assayed as indicators, a suspension of 103 spores per ml was used. Chemicals were from Aldrich Chemical Company, Milwaukee, Wis. Aliquots (50 μl) of each mixture were then spread onto the surface of three petri dishes (60-mm diameter) containing 5 ml of PDA. Plates were incubated at 26 or 30°C for 24 h. After incubation, the number of germinated conidia was determined by stereoscopic observations. The assay was repeated three times.
Isolation and assay of the antifungal compounds produced byL. plantarum 21B.
Aliquots (20 ml) of the BCF ofL. plantarum 21B were initially extracted (1:1, vol/vol) with an eluotropic series of organic solvents, namelyn-hexane, chloroform, ethyl acetate (pH 2.0, 3.6, and 10.0), and n-butanol. Ethyl acetate gave the highest recovery of the antifungal activity. BCF (200 ml, pH 3.6) was extracted four times with 200 ml of ethyl acetate, and the combined organic extract of culture filtrate was dried (Na2SO4) and evaporated under reduced pressure to give a crude residue of ca. 338 mg. Thin-layer chromatography (TLC) analysis of the residue was performed on silica gel plates (Merck, Kieselgel, Germany; 60 F254, 0.25 mm). The spots were visualized by exposing the plates to UV radiation and spraying with 10% H2SO4 in CH3OH and then with 5% phosphomolybdic acid in CH3OH, followed by heating at 110°C for 10 min. The residue (100 mg) was partially purified by preparative TLC (Merck; 60 F254, 0.5 mm) to give three fractions, A, B, and C (5.2, 4.5, and 44.0 mg, respectively). The extract and fractions were dissolved with CHCl3-methanol (MeOH) (1:1, vol/vol), and inhibitory activity against E. fibuliger IBT605 and P. roqueforti IBT18687 was determined by the antifungal disk assay. Fifty-microliter aliquots containing crude extract or fractions (corresponding to 20 ml of BCF for crude extract and 60, 60, and 30 ml of BCF for fractions A, B, and C, respectively) or 50-μl aliquots of CHCl3-MeOH (1:1) were added to 6-mm sterile disks (sterile blanks; Difco), allowed to dry, and placed onto PDA. Plates were overlaid with soft agar (2 ml, 0.7%) containing about 103 cells per ml of test fungi. After incubation at 25°C for 24 h, the inhibition areas on and around the disks were recorded.
The antifungal disk assay was used instead of the conidial germination assay since fractions from BCF were extracted in CHCl3-MeOH and palmitic acid was insoluble in aqueous solution.
Identification of the antifungal compounds by GC/MS.
For gas chromatography/mass spectrometry (GC/MS), samples of ethyl acetate extract (7 mg) of the L. plantarum 21B culture filtrate and fractions (0.5 mg) A, B, and C obtained from its preparative TLC were esterified by dissolving in CH3OH and treated with an ether-diazomethane solution for 30 min at 0°C with stirring. The mixtures were evaporated with an N2 stream. A QP5000 GC/MS instrument (Shimadzu, Kyoto, Japan), equipped with a nonpolar capillary column MDN-5 (30 m by 0.25 mm inner diameter; film thickness, 0.25 μm; Supelco, Belleforte, Pa.), was used for the analysis. The GC oven was held at 140°C for 1 min and then increased to 240°C at 4°C per min for 5 min. Helium was used as the carrier gas with a flow of 30 ml/min. The identification of the compounds was based on 90% similarity between the MS spectra of unknown and reference compounds in an MS spectra library.
The compounds identified in the active fraction were purchased commercially (Fluka, Sigma-Aldrich Division, Milan, Italy) and tested by a GC/MS method to compare their retention times and MS spectra with those of the metabolites present in the C fraction.
The antifungal activity of the identified compounds was evaluated individually and in mixture by the antifungal disk assay againstE. fibuliger IBT605 and P. roqueforti IBT18687. Compounds were dissolved with CHCl3-MeOH (1:1) or with MeOH.
Sourdough fermentation with L. plantarum 21B.
The wheat flour contained moisture (12.8%), protein (N × 5.70), 10.6% dry matter (d.m.), fat (1.79% d.m.), and ash (0.60% d.m.). Wheat flour (250 g), 110 ml of tap water, and 40 ml of cellular suspension, containing 107 CFU of S. cerevisiae IDM141 (Culture Collection of the Institute of Dairy Microbiology, Agriculture Faculty of Perugia, Italy) per ml andL. plantarum 21B (109 CFU ml−1),S. cerevisiae IDM141 and L. brevis 1D (109 CFU ml−1), or S. cerevisiaeIDM141 alone were mixed to produce 400 g of dough (dough yield = 160) with a continuous-high-speed mixer (60 × g; optimal dough mixing time, 5 min) (Chopin & Co., Boulogne, Seine, France). Doughs were individually placed in aluminum pans (25 cm by 10 cm by 8 cm high) and incubated at 30°C for 150 min. After fermentation, the three sourdoughs were baked in a batch oven (Mondial Forni, Verona, Italy) at 220°C for 30 min. Sourdough breads were then cooled at room temperature for 90 min, sliced (10 cm by 1 cm by 9 cm high), and 2 ml of conidial suspension of A. niger FTDC3227, prepared as previously reported and containing about 104conidia per ml, was spread by nebulization on the slice surface. The slices were then packed in polyethylene bags, 95-μm thick (Tillmans S.p.a, Milan, Italy), and stored at 20°C for 7 days.
RESULTS
Preliminary screening.
After 72 h of incubation in WFH broth, all the lactic acid bacteria assayed reached a cell number of ca. 109 CFU ml−1 and all the BCFs used in the conidial germination assay had a pH of between 3.6 and 4.0.
Fusarium graminearum IDM623, Endomyces fibuliger IDM3812, Penicillium expansum IDM/FS2,Aspergillus niger IDM1, and Monilia sitophilaIDM/FS5 were initially used as fungal indicators to select the 25 sourdough lactic acid bacteria strains. When the germination of conidial or yeast-like spores (E. fibuliger strains) was inhibited by from 80 to 100%, antifungal activity was considered positive.
Only the BCFs of Lactobacillus plantarum 20B and 21B inhibited all the indicator fungi (data not shown), whileLactobacillus alimentarius 5A, 5Q, and 5Z andLactococcus lactis subsp. lactis 11M inhibitedF. graminearum IDM623, E. fibuliger IDM3812, andP. expanusm IDM/FS2. Lactobacillus fermentum 18B,Leuconostoc citreum 10M, and L. plantarum 21A were active against F. graminearum IDM623 and E. fibuliger IDM3812. The BCFs of Lactobacillus brevis,Leuconostoc mesenteroides subsp. mesenteroides,Weissella confusa, Lactobacillus sanfranciscensisstrains, and those of the other strains belonging to the above-mentioned species showed appreciable antifungal activity only against F. graminearum IDM623. The ten-fold-concentrated WFH did not show inhibition.
Spectrum of antifungal activity of selected lactic acid bacteria.
L. plantarum 20B and 21B, L. alimentarius 5Q,L. lactis subsp. lactis 11M, and L. citreum 10M were selected because of their greater inhibitory spectra, and the fungicidal activity was further characterized by using eight other fungal species or strains as indicators (Table1). F. graminearum was omitted because it was inhibited by all 25 strains of lactic acid bacteria assayed and different strains were used for the species considered as indicators in the preliminary screening. The activity was compared with antifungal chemicals such as calcium propionate and sodium benzoate.
Table 1.
Table 1. Inhibition of germination of conidia or yeast-like spores by chemicals and culture filtrates of selected lactic acid bacteriaa
Fungal species
Mean % inhibition ± SD
Chemicals
Lactic acid bacterial strains
Calcium propionate (3 mg ml−1)
Sodium benzoate (3 mg ml−1)
L. alimentarius 5Q
L. lactis subsp. lactis 11M
L. citreum 10M
L. plantarum 20B
L. plantarum 21B
Aspergillus nigerFTDC3227
0
100
28.8 ± 3
31.1 ± 5.2
97.5 ± 1.9
69 ± 7.3
98.6 ± 0.8
Aspergillus flavusFTDC3226
15 ± 7
100
17 ± 3.5
18.6 ± 4.5
35 ± 6.7
55 ± 5.5
86.5 ± 5.5
Eurotium rubrumFTDC3228
0
70 ± 9.1
97 ± 1.6
97 ± 0.4
98.3 ± 0.9
99.5 ± 0.6
99.7 ± 0.56
Eurotium repensIBT18000
22.6 ± 2.3
75 ± 2.9
52.4 ± 9.8
15.9 ± 2.9
25 ± 7.6
100
100
Endomyces fibuligerIBT605
13.2 ± 0.9
36.6 ± 5
77.5 ± 4.5
26.8 ± 9.2
60 ± 6.4
100
100
Penicillium corylophilumIBT6978
15.6 ± 5.3
68 ± 4.5
26.2 ± 3
20 ± 3.4
15 ± 4.7
100
100
Penicillium roquefortiIBT18687
0
99 ± 0.4
0
0
0
80 ± 5.5
86.3 ± 3.5
Monilia sitophilaIDM/FS5
23.5 ± 6.6
100
97.6 ± 1.7
87 ± 2.9
100
100
100
a
Percent inhibition was calculated using WFH medium (500 mg ml−1) as a negative control. Each value is the mean percentage of three experiments ± standard deviation.
The selected strains produced comparable amounts of d- andl-lactic acid, which ranged from 9.0 to 7.6 mmol/liter, and ethanol production was only notable in heterofermentative L. citreum culture (5.5 mmol/liter). All the strains produced only traces of acetic acid.
All the fungal species were inhibited markedly by BCFs of L. plantarum 21B and 20B; the former showed more than 86% inhibition (Table 1). P. roqueforti IBT18687 was most resistant to the BCFs of sourdough lactic acid bacteria and was inhibited markedly only by L. plantarum 21B and 20B. Except for E. rubrumFTDC3228 and M. sitophila IDM/FS5, L. alimentarius 5Q, L. lactis subsp. lactis11M, and L. citreum 10M showed variable antifungal activities which were markedly lower than those of the L. plantarum strains. Calcium propionate used at the highest concentration (3 mg ml−1) inhibited the fungal species very little. Compared to the activity of BCF by L. plantarum21B, sodium benzoate inhibition at a concentration of 3 mg ml−1 was slightly higher for A. flavus FTDC3226 and P. roqueforti IBT18687, lower for E. rubrum FTDC3228, E. repens IBT18000, E. fibuliger IBT605, and P. corylophilum IBT6978, and similar for the other fungal species. At the lowest concentration tested (0.3 mg ml−1), the inhibitory activity of sodium benzoate decreased greatly.
When the BCF of L. plantarum 21B was concentrated 2-fold instead of 10-fold, ca. 50% inhibitory activity was obtained. The same activity level was found when cell growth in WFH broth was increased to ca. 1010 CFU ml−1 due to a higher inoculum size and the inhibitory activity of the nonconcentrated culture filtrate of L. plantarum 21B grown in whole wheat flour hydrolysate was ca. 90% of that of the 10-fold-concentrated BCF (data not shown).
The activity of L. plantarum 21B was found to be fungicidal, since after treatment of A. niger FTDC3227 conidia orE. fibuliger IBT605 yeast-like spores with BCF and further washing, they were not able to germinate even after prolonged incubation (5 days) in PDA medium (data not shown).
Further experiments were conducted by using E. fibuligerIBT605 and P. roqueforti IBT18687 as indicator fungi, since the former had a sensitivity similar to that of the other fungal species and the latter was the most resistant to the BCF ofL. plantarum 21B.
Characterization of the antifungal activity of L. plantarum 21B.
When cultivated in WFH broth, L. plantarum 21B reached the stationary phase (3.0 × 109 CFU ml−1) after 48 h, then cell viability decreased dramatically after 7 days of incubation at 30°C (Fig. 1). The production of lactic acid followed a similar trend, reaching a maximum of 8.8 mmol liter−1 after 48 h, while acetic acid and ethanol productions were very limited. Fungicidal activity against E. fibuliger IBT605 was detected when the cell number was the highest; it increased slightly and then remained constant over 10 days of incubation. When the pH of BCF from L. plantarum 21B was changed from 5.0 to 7.0, the inhibition of E. fibuligerIBT605 decreased markedly, but by readjusting the pH of BCF to the initial value of 3.7, the antifungal activity was restored (data not shown). In sourdough baked goods, pH values of 3.7 to 4.0 are very common (11). The antifungal activity was heat stable to 100°C for 15 min (data not shown).
Fig. 1.
Fig. 1. Kinetics of growth, fermentation end products (lactic and acetic acids and ethanol), and antifungal activity ofLactobacillus plantarum 21B. Antifungal activity is expressed as percent inhibition of spore germination of Endomyces fibuliger IBT605. Each value is the mean from three replicates ± standard deviation.
Isolation and identification of antifungal compounds produced byL. plantarum 21B.
First, BCF from L. plantarum 21B was exhaustively extracted by using ethyl acetate at pH 3.6. This organic extract still showed 100% inhibitory activity against E. fibuliger IBT605. The crude extract was then fractionated by preparative silica gel TLC, and three fractions (A, B, and C) were recovered. The crude extract (5.5 mg/disk) and fractions A, B, and C (0.8, 0.7, and 2.5 mg/disk, respectively) were assayed by the antifungal disk assay using E. fibuliger IBT605 as the indicator. Only the crude extract and fraction C, containing the more polar compounds, caused a 3-mm inhibition halo around the disks. The same results were found when the crude extract and fractions (concentration of 16, 2.4, 2.1, and 7.5 mg/disk) were tested againstP. roqueforti IBT18687. The solvent CHCl3-MeOH did not inhibit any fungal species. The ethyl acetate extract and all three fractions were analyzed by GC/MS after converting the organic acids to the corresponding methyl esters by reaction with diazomethane. The compounds were identified by comparing their electron impact (EI)-MS spectra with those recorded in the mass spectrum library; identification was considered reliable only if there was >90% similarity with the reference spectrum. The compounds identified in the inhibitory fraction C corresponded to phenyllactic acid,p-hydroxyphenyllactic acid, and palmitic acid in the ratio of 4.2:1.5:1.0 (Fig. 2b). These compounds were also included in the GC/MS profile of the crude extract (Fig. 2a), which was more complex due to the presence of other substances subsequently fractionated by preparative TLC and contained in the other two noninhibitory fractions (A and B).
Fig. 2.
Fig. 2. GC profiles of ethyl acetate extract (a) of culture filtrate from Lactobacillus plantarum 21B and (b) of active fraction C obtained from its purification. Metabolites were identified by GC/MS analysis and compared with standard samples.
When commercial phenyllactic acid, p-hydroxyphenyllactic acid, and palmitic acid were analyzed by GC/MS, the same retention time and EI-MS spectra were obtained as for the compounds contained in the inhibitory fraction.
Antifungal activity of the identified organic acids.
The lowest inhibitory dose of phenyllactic andp-hydroxyphenyllactic acids against E. fibuligerIBT605 (ca. 1.5-mm inhibition halos around the disks) corresponded to 2.5 and 6.6 mg/disk, respectively. Phenyllactic acid was also assayed by the conidial germination assay (concentration of 14 mg/ml of WFH), and the activity was confirmed to be as fungicidal, as were those of BCF from L. plantarum 21B (data not shown). Palmitic acid at the highest concentration tested (10 mg/disk) did not inhibit the indicator fungus. When the inhibitory activity of organic acids was tested against P. roqueforti IBT18687, only phenyllactic acid (8.3 mg/disk) caused an inhibition halo of ca. 1 mm around the disk. Palmitic acid and p-hydroxyphenyllactic (10 and 16 mg/disk, respectively) were not inhibitory. When phenyllactic,p-hydroxyphenyllactic, and palmitic acids were used in a mixture according to the ratio found in fraction C (8.3, 3, and 2 mg/disk, respectively), inhibition of E. fibuliger IBT605 (halo of ca. 3 mm) did not increase over that with phenyllactic acid alone (8.3 mg/disk).
Sourdough fermentation with L. plantarum 21B.
Breads were produced using associations of L. plantarum 21B and S. cerevisiae IDM141, L. brevis 1D andS. cerevisiae IDM141, and S. cerevisiae IDM141 alone. L. brevis 1D is a sourdough strain which did not show appreciable antifungal activity during the preliminary screening but produced about the same amount of lactic acid as L. plantarum 21B in addition to acetic acid. Both breads started with lactic acid bacteria had low pH values (4.4 to 4.6), while those started with the yeast alone had a pH of 5.7. After baking, the breads were sliced, and a conidial suspension of A. niger FTDC3227 was spread by nebulization on the slice surface. The slices were packed in polyethylene bags to maintain constant moisture and then stored at 20°C for 7 days. Figure 3 shows how mold growth occurred mostly after 2 days in the breads started withS. cerevisiae IDM141 alone (the same was found for the association with S. cerevisiae IDM141 and L. brevis 1D), while the selected L. plantarum 21B delayed mold contamination until after 7 days of storage.
Fig. 3.
Fig. 3. Growth of Aspergillus niger FTDC3227 in bread started with S. cerevisiae 141 and Lactobacillus plantarum 21B after 7 days of storage (A) and in bread started with Saccharomyces cerevisiae 141 alone after 2 days of storage (B).
DISCUSSION
Lactic acid bacteria greatly influence the sensory, textural, nutritional, and shelf-life characteristics of sourdough baked goods, especially breads (11). Antifungal activity has to be considered an important tool for selecting sourdough lactic acid bacteria because fungal, rather than bacterial, spoilage is the main cause of substantial economic loss in the baking industry and may also cause public health problems due to the production of mycotoxins (19, 20, 28).
In this study, several sourdough lactic acid bacteria were screened, and novel antifungal compounds from selected Lactobacillus plantarum 21B were purified and characterized. L. plantarum 21B showed a very broad spectrum of activity and inhibited Eurotium repens IBT18000, E. rubrumFTDC3228, Penicillium corylophilum IBT6978, P. roqueforti IBT18687, P. expansum IDM/FS2,Endomyces fibuliger IBT605 and IDM3812, Aspergillus niger FTDC3227 and IDM1, A. flavus FTDC3226,Monilia sitophila IDM/FS5, and Fusarium graminearum IDM623. These fungi represent almost all the species most commonly isolated from contaminated baked goods (20). Phenyllactic acid, its corresponding 4-hydroxy derivative (p-hydroxyphenyllactic acid) and palmitic acid were identified by GC/MS analysis in the active fraction of the BCF fromL. plantarum 21B grown in WFH broth. When the same commercial compounds were used individually, only phenyllactic andp-hydroxyphenyllactic acids showed antifungal activities. A cooperative action has often been reported for antimicrobials produced by microorganisms, because in mixtures such compounds may interact with each other as well as with the test organisms (3, 5, 22). In this study, no synergistic effect of the mixture was found, showing that phenyllactic acid played a key role in inhibiting fungal growth.
Strain L. plantarum 20B, which had broad antimold activity, was also selected. The preliminary purification and characterization of the antifungal compounds produced by strain 20B also led to the identification of phenyllactic and p-hydroxyphenyllactic acids (data not shown).
To our knowledge, this is the first report showing the production of phenyllactic acid and p-hydroxyphenyllactic acid by lactic acid bacteria. Both compounds are involved in phenylalanine metabolism (26). To avoid intracellular accumulation of phenylalanine, this amino acid may be hydroxylated to tyrosine or transaminated to phenylpyruvic acid, which is further metabolized to phenylactic andp-hydroxyphenyllactic acids. Experiments conducted inCandida species with [14C]phenylalanine confirmed that 2-phenyllactic acid was synthesized froml-phenylalanine (21). Based on these findings, it may be supposed that phenyllactic acid was excreted in copious amounts during growth of L. plantarum 21B in WFH to avoid intracellular accumulation of phenylalanine. Neither phenylalanine or tyrosine is an essential or stimulatory amino acid for most of the sourdough L. plantarum strains (9).d-3-Phenyllactic acid was purified and characterized fromGeotrichum candidum, a yeast-like fungus used in the maturation of several cheeses (5). A patent application used mutants of Brevibacterium lactofermentum to produced-3-phenyllactic acid (16). In an extensive study on the interactions among cheese microflora, it was found thatG. candidum produced d-3-phenyllactic acid which inhibited fungi as well as gram-negative and gram-positive bacteria (14). Other authors (4) usedd-3-phenyllactic acid against Listeria monocytogenes grown in culture medium and in milk. They found a bactericidal effect independent of the physiological state of the pathogen and a reduction in the milk population of ca. 4.6 log cycles. A phenyllactic acid concentration ranging from 10 to 20 mg ml−1 was needed to inhibit L. monocytogenes,Staphylococcus aureus, and Enterococcus spp. in the agar well diffusion assay (5, 6). Although different biological tests were applied and the target organism was a fungus, in our conditions, 2.5 mg (which corresponded to 50 mg ml−1) of phenyllactic acid per disk was inhibitory to all the fungi tested except P. roqueforti IBT18687 and P. corylophilumIBT6978 (166 mg ml−1).
The activity of the BCF of L. plantarum 21B was compared to those of chemicals widely used as preservatives in foods. Conidial germination assays with 0.3 to 3 mg of calcium propionate ml−1 had practically no antifungal activity. Sodium benzoate showed antifungal activity only at 3 mg ml−1which varied slightly in the inhibitory spectrum compared to that ofL. plantarum 21B. Directives of the European Community (1) permit the use of a maximum of 3 mg of calcium propionate ml−1 for packaged sliced breads and rye breads and recommend potassium sorbate at a concentration of 2 mg ml−1, which was not inhibitory against E. fibuliger IBT605, A. niger FTDC3227, or P. roqueforti IBT18687 (data not shown). Potassium sorbate is rarely used because of the secondary effects on bread volume.
The potential of antimicrobials other than bacteriocins produced by lactic acid bacteria is currently being exploited due to the very broad spectrum of activity. After the structural characterization of reuterin by Lactobacillus reuteri (30), pyroglutamic acid, produced by Lactobacillus casei subsp. casei, has also been introduced as an antimicrobial agent (15). Recently, antimicrobial compounds have been identified in the culture filtrate of another strain of L. plantarum (22). They corresponded to benzoic acid, 5-methyl-2,4-imidazolidinedione, tetrahydro-4-hydroxy-4-methyl-2H-pyran-2-one, and 3-(2-methylpropyl)-2,5-piperazinedione and were inhibitory toPantoea agglomerans and also inhibited the fungusFusarium avenaceum to some extent.
For a long time, the improved shelf life of sourdough baked products was attributed to the lactic and acetic acids produced by lactic acid bacteria (27, 28). Further studies (3, 13, 22) have shown that lactic acid is not inhibitory to fungi, while the acetic acid concentration seems to be more strictly related to the antifungal activity (25). The acetic acid concentration in the dough may be increased by adding fructose, which is used as an external electron acceptor by heterofermentative lactic acid bacteria, which consequently increases the growth yield and acetic acid production (10). Very few studies have focused on the antifungal activity of sourdough lactic acid bacteria. A previous screening of sourdough lactic acid bacteria showed that L. sanfranciscensis CB1 (obligately heterofermentative strain) produced a mixture of acetic, caproic, formic, butyric, andn-valeric acids which synergistically inhibited species ofFusarium, Penicillium, Aspergillus, and Monilia. Caproic acid played a key role in inhibiting mold growth (3). It was also shown that sourdough lactic acid bacteria do not have the same antifungal potentialities, which do not depend on the production of lactic and acetic acids. The L. plantarum strains (facultatively heterofermentative) of this study have a very large spectrum of activity based on antifungal compounds which are different from those identified previously (3). It must be emphasized that the association of L. sanfranciscensis and L. plantarum is very often identified in sourdoughs (11).
The antifungal activity of L. plantarum 21B was also found in sourdough bread. Compared to breads started with S. cerevisiae 141 alone or in association with L. brevis1D, the sourdough bread which used L. plantarum 21B in association with S. cerevisiae 141 delayed fungal contamination until after 7 days of storage at room temperature. WhenL. plantarum 21B was grown in a culture medium richer in nutrients, such as the WFH, the inhibitory activity was found to be ca. 90% of that in the 10-fold-concentrated BCF, showing that the antifungal activity may be increased depending on the type of flour used.
To be effective and competitive, the food industry must respond to consumer demands, and recent trends have included the desire for high-quality foods that are not extremely processed and that do not contain chemical preservatives. Because the antimicrobial compounds produced by lactic acid bacteria are considered natural preservatives, the use of L. plantarum 21B to decrease the fungal contamination of sourdough baked products has interesting potential applications.
ACKNOWLEDGMENTS
This work was supported by the European Project FAIR-CT98-4075 “Natural antifungal systems for prevention of mould spoilage in bakery products.”
The valuable technical assistance of S. L. Lonigro is gratefully acknowledged.
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