- Email: [email protected]
The use of Lactobacillus brevis PS1 to in vitro inhibit the outgrowth of Fusarium culmorum and other common Fusarium species found on barley
International Journal of Food Microbiology 141 (2010) 116–121
Contents lists available at ScienceDirect
International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o
Short Communication
The use of Lactobacillus brevis PS1 to in vitro inhibit the outgrowth of Fusarium culmorum and other common Fusarium species found on barley A. Mauch a,b, F. Dal Bello a,b, A. Coffey c, E.K. Arendt a,⁎ a b c
Department of Food Science, Food Technology and Nutrition, National University of Ireland, Cork, Ireland National Food Biotechnology Centre, National University of Ireland, Cork, Ireland Department of Biological Sciences, Cork Institute of Technology, Bishopstown, Cork, Ireland
a r t i c l e
i n f o
Article history: Received 15 December 2009 Received in revised form 15 April 2010 Accepted 1 May 2010 Keywords: Antifungal Lactobacillus Fusarium spp. Mycelia growth Macroconidia Inhibition
a b s t r a c t A total of 129 lactic acid bacteria (LAB) were screened for antifungal activity against common Fusarium spp. isolated from brewing barley. Four out of the five most inhibiting isolates were identified as Lactobacillus brevis, whereas one belonged to Weissella cibaria. L. brevis PS1, the isolate showing the largest inhibition spectrum, was selected and the influence of its freeze-dried cell-free supernatant (cfsP) on germination of macroconidia as well as mycelia growth was investigated using Fusarium culmorum as target organism. Addition of cfsP into the growth medium at concentrations ≥ 2% altered the growth morphology of F. culmorum, whereas at concentrations N 5% the outgrowth of germ tubes from macroconidia was delayed and distorted. The presence of 10% cfsP completely inhibited the outgrowth of F. culmorum macroconidia. The activity of the compounds produced by L. brevis PS1 was higher at low pH values, i.e. pH b 5. Heating and/or proteolytic treatment reduced the inhibitory activity of cfsP, indicating that L. brevis produces organic acids and proteinaceous compounds which are active against Fusarium spp. © 2010 Elsevier B.V. All rights reserved.
1. Introduction The mycotoxigenic fungi associated with the human food chain belong mainly to the three genera Aspergillus, Fusarium and Penicillium (Pitt et al., 2000). Aspergillus and Penicillium species are reported as spoilage organisms from a wide range of food and feeds, whereas Fusarium species are often found on cereal grains (Filtenborg et al., 1996; Samson et al., 2000). In grain processing, mycotoxin secretion by storage fungi like Aspergillus or Penicillium species can be prevented by the selection of appropriate storage conditions of the grains. This approach does not apply for field fungi. Therefore field fungi represent an important threat to the safety of cereal products (Noots et al., 1999). Although processing, notably heat treatment, can reduce mycotoxin concentrations significantly, it does not eliminate them completely (Bullerman and Bianchini, 2007; Ryu et al., 2002). Safety of raw materials and food products can be assured by the use of chemical preservatives. However, during the last decades the application of these compounds has been questioned. In particular, the enhanced interest in natural and free-from foods, preferentially with healthpromoting characteristics has forced the food makers to find alter-
⁎ Corresponding author. Tel.: + 353 21 4902064; fax: + 353 21 4270213. E-mail address: [email protected] (E.K. Arendt). 0168-1605/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2010.05.002
native solutions. The applications of lactic acid bacteria (LAB) as starter cultures as well as their metabolites are a matter of particular interest to perform this task. LAB have a long history of application in fermented foods because of their beneficial influence on nutritional, organoleptic, and shelf-life characteristics, and are naturally occurring in many food systems (De Vuyst and Leroy, 2007; Tamminen et al., 2004; Vaughan et al., 2001). There is an extensive knowledge about antibacterial compounds, especially bacteriocins, produced by LAB (Aso et al., 2008; De Vuyst and Leroy, 2007; Elegado et al., 2007; Ghrairi et al., 2007; Millette et al., 2008; Oguntoyinbo, 2007) whereas the number of published studies on the identification of antifungal compounds produced by LAB is rather limited. Several low molecular weight compounds, mostly organic acids, have been isolated with the ability to retard or eliminate fungal growth or spores outgrowth, either on their own or synergistically (Batish et al., 1997; Dal Bello et al., 2007; Lavermicocca et al., 2003; Lind et al., 2007; Ryan et al., 2008; Schnurer and Magnusson, 2005). Regarding the great diversity of LAB within a single species, particularly due to environmental adaptions, there is a strong justification for further studies aimed at identifying novel antifungal LAB and characterising the compounds responsible for their inhibitory activity. The ambition of this study was to find LAB isolated from different sources like cheese as well as human, mouse, pig and bovine intestinal sources exhibiting antifungal activity against a variety of important Fusarium species commonly found on barley.
A. Mauch et al. / International Journal of Food Microbiology 141 (2010) 116–121
2. Materials and methods 2.1. Fungal cultures and spore–mycelia suspension Fusarium avenaceum TMW 4.1843, F. culmorum TMW 4.2043, Fusarium graminearum TMW 4.2046, Fusarium poae TMW 4.2044 and Fusarium tricinctum TMW 4.1405 all isolates from barley were kindly provided by the culture collection of Lehrstuhl fuer Technische Mikrobiologie, TU-Muenchen Weihenstephan (TMW). Fungi were cultivated on potato-dextrose agar (PDA) plates (Fluka Chemie AG, Buchs, Switzerland) at 25 °C for 5 days and then stored at 4 °C until further use. Small pieces from PDA-plates inoculated with fusaria were transferred into 500 mL of synthetic-nutrient-poor bouillon (SNB) (Nirenberg, 1976). The suspensions were incubated at 25 °C (120 rpm) for 5–7 days to induce both microconidia and macroconidia (F. avenaceum, F. poae and F. tricinctum) or solely macroconidia (F. culmorum and F. graminearum) formation. Concentrations of 1 × 105 to 3 × 105 CFU per mL were measured by plating out serial dilutions on PDA-plates. 2.2. Bacterial cultures The LAB used were isolated from cheese as well as human, mouse, pig and bovine intestinal samples. LAB were routinely grown on MRSagar plates (Fluka Chemie AG, Buchs, Switzerland) under microaerophilic conditions for 48 h at 30 or 37 °C. Long-term storage was done in 35% glycerol at −80 °C. 2.3. Screening for antifungal LAB The screening of LAB for antifungal activity was performed by nebulising 100 μL of fungal spore–mycelia suspension (approx. 104 CFU) onto the surface of petri-dishes containing 20 mL of MRSagar modified as follows (mMRS): pH adjusted to 6.0, sodium acetate as well as potassium dihydrogenphosphate omitted. After 30 min, bacteria were inoculated as two parallel lines of 3 cm length, keeping a distance between the lines of approx. 2 cm. Plates were incubated under microaerophilic conditions at 30 as well as 37 °C for 48 h followed by an additional incubation for 48 h under aerobic conditions at 25 °C to promote fungal growth. The antifungal activity of each LAB was ascertained by measuring the size of the clear zone surrounding the bacterial streaks. Antifungal activity was scored as follows: −, clear zone size b 3 mm; +, clear zone size ≥ 3 mm; ++, clear zone size ≥ 5 mm; and +++, clear zone size ≥ 10 mm. The overall growth of the fungi was compared to that in control plates (i.e. with no LAB present) and rated as follows: a, identical growth of fungi surrounding the clear zone; b, retarded growth of fungi surrounding the clear zone; c, strongly retarded growth of fungi surrounding the clear zone. 2.4. Identification of LAB isolates Bacteria exhibiting strong antifungal activity were identified upon sequencing of the first 900 bp of the 16 S rDNA (Meroth et al., 2003). To determine the closest relatives of the partial 16 S rDNA sequences, a GeneBank DNA database search was conducted. A similarity of N98% to 16S rDNA sequences of type strains was used as the criterion for identification. 2.5. Production of freeze-dried bacterial supernatant powder Cell-free supernatant (cfs) powders of the most inhibitory strain (cfsP) and of a non-inhibitory strain belonging to the same species (cfsN) were produced to serve as base material for the experiments describing the nature of the antifungal compounds. Briefly, overnight cultures of bacteria were inoculated in 500 mL of mMRS broth to
117
reach an initial concentration of 104 CFU/mL. The bacteria were grown for 144 h at 37 °C (temperature at which the antifungal strain showed its highest activity). Cells were separated from the supernatant by centrifuging twice at 3000 g for 15 min at 4 °C. The cell-free supernatant was freeze dried and the powder stored at 4 °C. 2.6. D/L-lactic acid and acetate amount in cfsP and cfsN The amounts of D/L-lactic acid and acetic acid present in the cfs were determined in solutions containing 25% of cfsP or cfsN redissolved in distilled water. For this purposes D-lactic acid/L-lactic acid as well as acetic acid test kits were used according to the manufacturer instructions (R-biopharm AG, Darmstadt, Germany). 2.7. Impact of bacterial supernatant on growth of F. culmorum The effect of different concentration of cfs on the growth of F. culmorum was examined by using a microplate assay. F. culmorum was chosen as the test-fungus due to the fact that it exhibited strong sensitivity during the screening and also it develops macroconidia during spore formation, which simplifies microscopic examinations. Pure macroconidia were obtained by filtering the spore–mycelia suspension of F. culmorum through a 30 μm pore size filter paper (Filter Paper 113 wet strengthened, Whatman International Ltd, Maidstone, England). The macroconidia suspension was adjusted to 1.0 × 105 macroconidia per mL. Aliquots of 1 mL were centrifuged at 3000 g for 10 min at 4 °C and the supernatant was discarded. The conidia pellets were resuspended in 1 mL malt extract broth (MEB) (Merck KGaA, Darmstadt, Germany). The MEB, adjusted to pH 4 using equal volumes of commercial D/L-lactic acid (Sigma-Aldrich, St. Louis, USA) and variable amounts of 4 M NaOH, contained 0, 1.0, 2.5, 5.0, 7.5 or 10.0% (w/v) of cfsP or cfsN. Before adding to the pellets, the dilutions were filtered through a sterile 0.45 μm MINISART®-plus filter (Sartorius Stedim Biotech GmbH, Goettingen Germany). After resuspending the conidia pellets in MEB, wells of a sterile 96-well microplate (Sarstedt AG & Co, Nuembrecht, Germany) were filled with 200 μL and sealed with optically clear seal for QPCR (Thermo Scientific, Waltham, USA). The microplates were incubated for 48 h at 25 °C and agitated every 4 s for approximately 1 s inside a Multiskan FC microplate-reader (Thermo Scientific, Waltham, USA). The optical density at 620 nm (OD620) was automatically recorded for each well every 3 h. The changes in OD620 over time were used to generate F. culmorum growth curves at each cfs concentration. The experiment was performed in triplicate. 2.8. Macroconidia germination assay For both antifungal and control strain, 20 mL of MEB was prepared containing 0 (control), 5 or 10% (w/v) of cfsP or cfsN. The pH was adjusted to 4 as described before. Chemically acidified control (CAC) broths were prepared by adding to MEB the amount of D/L-lactic acid used to adjust the pH and the quantity of D/L-lactic acid and acetic acid present in the cfs of the antifungal strain. The pH was adjusted to 4 using 4 M NaOH. All solutions were filtered through a sterile 0.45 μm MINISART®-plus filter. Macroconidia were obtained as described before but from 20 mL spore and mycelia suspension. After centrifugation at 3000 g for 10 min at 4 °C, the macroconidia pellets were resuspended in MEB containing cfs and incubated at 25 °C on an orbital platform shaker Heidolph® Unimax 1010 (Heidolph, Schwabach, Germany) at 350 rpm. Samples of 1 mL were taken at 3 h intervals and centrifuged at 4000 g for 3 min. After discarding the supernatant, the pellet was resuspended in 100 μL glycerol:water (50:50 v/v). A volume of 5 μL was transferred onto a microscopy slide and 100 randomly chosen macroconidia were analysed with a HP 630 microscope (Zefa Laborservice GmbH, Muenchen, Germany) at 400× magnification. The number of conidia showing germ tube formation
118
A. Mauch et al. / International Journal of Food Microbiology 141 (2010) 116–121
was determined and the values were given as percentage. The tests were performed in triplicate. 2.9. Morphological observations during mycelia growth A 50% (w/w) cfsP working-solution was prepared by dissolving the powder in bi-distilled water and filter sterilised using a 0.45 μm MINISART®-plus filter. PDA-plates were prepared containing 0 (control), 0.01, 0.05, 0.1, 0.5, 1, 2 or 3% (m/v) cfsP. For each concentration, chemically acidified control (CAC) plates were prepared containing commercially available D/L-lactic acid (Sigma-Aldrich, St. Louis, USA) and glacial acetic acid (BDH Laboratory, England) complying with the amounts present in each cfsP concentration used. After cooling, approximately 100 macroconidia were inoculated as a spot in the centre of the PDA-plates. The plates were incubated for 72 h at 25 °C under aerobic conditions. The fungal growth was monitored by photoscanning the plates and measuring the growth area using an image evaluation software (image j 1.40 g, Wayne Rasband, National Institutes of Health, USA). The test was performed in triplicate. 2.10. Activity of antifungal compounds at different pH values Aliquots of 20 mL MEB containing 10% (w/v) cfsP or cfsN were prepared and the pH values adjusted to 4.0, 5.0 or 6.5 using D/L-lactic acid. The solutions were filtered through a 0.45 μm MINISART®-plus filter. The MEB solutions were inoculated with F. culmorum macroconidia and the percentage of germinated macroconidia was determined as described before. The test was performed in triplicate. 2.11. Activity of antifungal compounds after treatment with proteolytic enzymes To determine the production of proteinaceous compounds by the antifungal strain, cfsP was treated with Pronase E (Fluka Chemie AG, Buchs, Switzerland) or Proteinase K (Sigma-Aldrich, St. Louis, USA). The enzymes were prepared according to the suppliers' recommendations but SDS and TRITON X-100® were omitted from the buffer. A 25% (w/v) cfsP solution was prepared using the enzyme buffers. After adjusting the pH to 7.5 using 4 M NaOH, the samples were filtered through a 0.45 μm MINISART®-plus filter and incubated for 1 to 5 h at 37 °C. The reaction was stopped by cooling to 20 °C. The cfsP concentration was adjusted to 10% (m/v) using MEB, and the pH was adjusted to 4 using D/L-lactic acid. The solutions were inoculated with F. culmorum macroconidia and the percentage of germinated macroconidia was determined as described above. The test was performed in duplicate. 2.12. Activity of antifungal compounds after heat treatment The heat stability of the antifungal compounds produced by the antifungal strain was determined by dissolving 2 mg of cfs with bi-
distilled water to a total volume of 8 mL (20% w/v) and heating it for 1 h at 80, 90 or 100 °C. After heating, the cfs was diluted to 10% (w/v) and the macroconidia germination assay was performed as described above. 3. Results and discussion The antifungal activity of 129 LAB isolated from different sources was investigated by streaking out the bacteria in two parallel lines onto mMRS plates inoculated with Fusaria spores and mycelia. Incubation at 30 °C resulted in a higher number of LAB showing antifungal activity. At this temperature, around 58% of the isolates inhibited at least one fungus, compared to approx. 50% when the incubation temperature was 37 °C. The LAB showing no antifungal activity at any growth temperature accounted to approx. 24%. These results confirmed that LAB are generally active against fungi, in particular Fusarium spp. L. brevis was the dominant species among the most inhibitory LAB, with four out of the five most inhibitory strains belonging to this species. However, we also isolated a L. brevis strain (i.e., NS) among the non-active LAB, suggesting that antifungal activity of L. brevis is a strain dependant trait (Table 1). This observation is in agreement with the work of Gerez et al. (2009) who found two out of five L. brevis strains exhibiting inhibitory activity against F. graminearum. On account of its ability to inhibit all tested fungi at 37 °C and three out of five fungi at 30 °C, L. brevis PS1 was selected for further experiments. The non-inhibitory L. brevis NS was used as negative control. The antifungal activity of L. brevis PS1 was investigated in depth using F. culmorum as target organism. The compounds produced by L. brevis PS1 affected both mycelial growth and macroconidia germination of F. culmorum. Addition of cfsP into PDA-agar plates at concentrations lower than 2% did not affect morphology of the aerial mycelia of the fungus, and no differences were observed when compared to the growth on control plates without any additives. At concentrations ≥ 2%, a distinctive change in colour as well as a higher density of mycelia emanating from the centre of the colony fading towards the borders appeared. On CAC plates this effect was only slightly visible. The growth areas on the plates containing cfsP were significantly (P N 0.05) reduced in comparison to both CAC and control plates (Fig. 1). While determining germ tube formation in the macroconidia germination assay, it was noted that a concentration of 5% cfsP resulted in macroconidia frequently possessing more than two germ tubes although outgrowth was slightly retarded compared to the control (Fig. 3). These germ tubes mainly emerged from nonapical cells. On the contrary, the macroconidia in the cfs-free or 5% cfsN broth developed their germ tubes mainly from the apical cells. The alterations of growth morphology as well as the more frequent emergence of germ tubes from internal compartments, observed when the macroconidia were exposed to cfsP at levels of 5% indicate that F. culmorum experienced stress even under those environmental conditions. In agreement with this observation, Koch and Loffler
Table 1 Antifungal activity of the most inhibiting lactic acid bacteria against selected Fusarium species. LAB
L. brevis PS1 W. cibaria PS2 L. brevis PS3 L. brevis PS4 L. brevis PS5 L. brevis NS
F. avenaceum
F. culmorum
F. graminearum
F. poae
F. tricinctum
30 °C
37 °C
30 °C
37 °C
30 °C
37 °C
30 °C
37 °C
30 °C
37 °C
− − +, c +, b − −
++, a ++, c − − +++, c −
+, b − ++, c ++, a − −
+++, a +, c +, b − ++, a −
− ++, b − − − −
+, b +, a − ++, c − −
+, b − ++, c ++, c − −
+++, a ++, a +++, b − +++, a −
++, a − +, b ++, a ++, a −
+, a ++, a − − +, a −
The distance between the peripheral sides of the bacterial-lines and the starting growth zone was scored as follows:−, no clear zone; +, distance ≥ 3 mm; ++, distance ≥ 5 mm; +++, distance ≥ 10 mm. Growth of fungi in the surrounding zone was recorded as follows: a, identical growth of fungi surrounding the clear zone; b, retarded growth of fungi surrounding the clear zone; c, strongly retarded growth of fungi surrounding the clear zone.
A. Mauch et al. / International Journal of Food Microbiology 141 (2010) 116–121
Fig. 1. Growth areas of F. culmorum incubated for 72 h on Potato Dextrose Agar-plates containing cell-free supernatant of L. brevis PS1 (cfsP) lactic/acetic acid-mixture complying the amounts found in cfsP (CAC) or no additives.
(2009) reported distorted hyphae with different degrees of swelling when F. culmorum was grown in the presence of antifungal filtrate from cultures of Streptomyces antimycoticus. Other authors observed the more frequent germ tube emergence from internal compartments when non-lethal concentrations of known antifungal substances are present (Aleandri et al., 2008; Chitarra et al., 2005; Harris, 2005). However, growth of F. culmorum in MEB containing 10% cfsP adjusted to pH 4 as detected during determining the impact of bacterial supernatant on growth of F. culmorum as well as in the macroconidia germination assay was not only altered but completely restricted (Figs. 2 and 3). The OD620 did not change over 48 h (the examination
Fig. 2. Growth of F. culmorum in Malt Extract Broth (pH 4) containing: 10% (–♦–), 7.5% (--x--), 5% (∙∙Δ∙∙) or 0% (…○…) of cell-free supernatant of L. brevis PS1.
Fig. 3. Germination of F. culmorum macroconidia in Malt Extract Broth (pH 4) in the presence of 10% (♦) or 5% (■) of cell-free supernatant of L. brevis PS1; 10% (▲) or 5% (x) of cell-free supernatant of L. brevis NS, and MEB with no addition of supernatant (◊).
119
was continued based on a visual control for another 5 days, and during this period no turbidity was detected) and in addition no germ tube formation was observed. Reducing the concentration of cfsP in MEB to 7.5% resulted in an increase in OD620 after 45 h. When 5% cfsP was added, approximately 50% of macroconidia formed germ tubes after 6 h; and after 12 h of incubation a change in OD620 was detected in the growth assay. Concentrations of cfsP lower than 5% did not affect the growth of F. culmorum (data not shown). When 10% cfsN was added, growth of F. culmorum started after 12 h (data not shown). The restricted outgrowth of macroconidia at pH 4 and the diminishing inhibition capacity of cfsP when increasing the pH from 4.0 to 5.0 and a total loss of activity at pH 6.5 (Fig. 4) suggest that organic acids are involved in the antifungal activity. At low pH values, organic acids exist mainly in undissociated form, which makes them more penetrable to the hydrophobic cell membranes of target organisms. Similar findings were reported by De Muynck et al. (2004) when testing antifungal activity of bacterial supernatant from L. brevis LMG 6906 against a selection of common food spoilage fungi. The authors detected a complete loss of inhibition when the pH was increased from 3.5 to 5.0, 5.5 or 6.0. However, our measurement of the two optical isomers of lactic acid as well as acetic acid in solutions containing 25% of cfsP and cfsN did not show potent differences. The cfs from both our strains was found to contain similar amounts of D-lactic acid, in the region.of 10.7 mmol. The cfsP solution contained 17.2 mmol L-lactic acid and 11.2 mmol acetate compared to 11.8 mmol L-lactic acid and 7.6 mmol acetate in the cfsN solutions. Although lactic and acetic acids are known antimicrobial compounds (Kuwaki et al., 2002; Martirosyan et al., 2004), the similar amounts in cfs of the non-antifungal L. brevis NS indicate that other antifungal compounds must be specifically produced (or produced at higher levels) by the antifungal strain PS1. Synergistic effects in terms of antifungal activity of different organic acids in combination with the main metabolites lactic and acetic acids are widely reported in literature. Corsetti et al. (1998) found effects of a mixture of acetic, caproic, formic, propionic, butyric and n-valeric acids in which caproic acid played a key role in inhibiting mould growth whereas Lavermicocca et al. (2003) could observe that addition of both lactic as well as acetic acid in concentrations exceeding the amounts detected in culture filtrate of L. plantarum 21B amplified the inhibition strength of phenyllactic acid, another antifungal organic acid found in cultures of L. plantarum. Aside from the effect of organic acids, other authors refer to the participation of proteinaceous compounds in the antifungal activity of LAB (Coda et al., 2008; Magnusson and Schnurer, 2001; Roy et al., 1996). Strom et al. (2002) reported weak synergistic effects between some cyclic peptides and 3-phenyllactic acid. Thus, we investigated if proteolytic treatment could affect the antifungal activity of L brevis
Fig. 4. Germination of F. culmorum macroconidia in Malt Extract Broth containing 10% cell-free supernatant of L. brevis PS1 at pH 4.0 (♦), 5.0 (■) or 6.5 (▲) as well as in Malt Extract Broth with no addition of supernatant (x) at pH 4.0.
120
A. Mauch et al. / International Journal of Food Microbiology 141 (2010) 116–121
PS1 supernatant. Digestion of cfsP with the proteolytic enzymes Proteinase K or Pronase E reduced the inhibitory activity to different extents. Treatment with Proteinase K for 5 h resulted in the loss of inhibitory activity after 24 h. At this time point 22% of the macroconidia was germinated, and the germination increased to a maximum level of approx. 70% after 33 h. Treatment with Proteinase K for 1 h reduced the inhibitory activity to a lower extent, and germination started after 33 h of incubation. Digestion with Pronase E for 5 h affected the inhibitory activity of cfsP to the same level as when the cfsP was treated with Proteinase K for 1 h. Treatment for 1 h with Pronase E did not affect cfsP activity (data not shown). These data strongly suggest that proteinaceous compounds are involved in the antifungal activity of L brevis PS1. Antifungal proteinaceous compounds production by L brevis has been recently reported by Coda et al. (2008). These authors isolated five peptides showing antifungal activity in a partly purified extract from sourdough fermented with L. brevis AM7. Interestingly, when they treated the water soluble extract with trypsin for 5 h no loss of antifungal activity was observed. Gerez et al. (2009) also did not detect a reduction in the inhibitory activity of cfs of two L. brevis strains, treated with proteinase K for 1 h. Our study indicates that time and type of enzyme applied for the proteolytic treatment are key factors in determining the loss or not of the antifungal activity. In fact, we showed that a reduction in the activity of cfsP was achieved only when the incubation time with proteinase K was extended to 5 h. Whether a protective matrix in cfsP reduced the enzymatic activity, or if the slow degradation was due to a difficult accessibility of the reaction site, could not be determined. Since many proteinaceous compounds but also other compounds are often sensitive towards heat treatment, we tested the impact of heat on cfsP at different temperatures. Heating for 1 h at 80 °C reduced the inhibitory activity of cfsP. Under this condition, macroconidia outgrowth started after 27 h and ca. 10% of macroconidia germinated after 48 h of incubation indicating that the antifungal compounds are heat labile. However, heating at 90 or 100 °C for 1 h did not affect the inhibitory activity of cfsP, and no germ tube formation was observed after 48 h of incubation. This contradictory observation might be explained by the formation of additional antifungal compounds during heating at high temperatures. In fact, it has been reported that heat treatment of fermentation broths can result in the generation of antifungal compounds, e.g. diketopiperazines (Prasad, 1995). Taken together, our results imply that both organic acids and proteinaceous compounds are responsible for the antifungal activity of L. brevis PS1. This work further strengthens the idea that the antifungal activity of LAB is a complex phenomenon, where numerous and different compounds act synergistically against target organisms. Further studies will unravel the identity of the compounds responsible for the antifungal activity of L brevis PS1 and thus make it easier to estimate the potential of the strain to serve e.g. as a biopreservative against Fusarium growth during food processing or as a biological agent to control Fusarium head blight. Acknowledgements The authors wish to acknowledge that this project was funded by the National Development Plan 2006–2010. The authors would also like to thank Timo Hipp and Agata Pawlowska for their technical support and insight. References Aleandri, M.P., Magro, P., Chilosi, G., 2008. Influence of environmental pH modulation on efficiency of apoplastic PR proteins during Fusarium culmorum — wheat seedling interaction. Plant Pathology 57, 1017–1025. Aso, Y., Takeda, A., Sato, M., Takahashi, T., Yamamoto, T., Yoshikiyo, K., 2008. Characterization of lactic acid bacteria coexisting with a nisin Z producer in Tsuda-turnip pickles. Current Microbiology 57, 89–94.
Batish, V.K., Roy, U., Lal, R., Grover, R., 1997. Antifungal attributes of lactic acid bacteria — a review. Critical Reviews in Biotechnology 17, 209–225. Bullerman, L.B., Bianchini, A., 2007. Stability of mycotoxins during food processing. International Journal of Food Microbiology 119, 140–146. Chitarra, G.S., Breeuwer, P., Rombouts, F.M., Abee, T., Dijksterhuis, J., 2005. Differentiation inside multicelled macroconidia of Fusarium culmorum during early germination. Fungal Genetics and Biology 42, 694–703. Coda, R., Rizzello, C.G., Nigro, F., De Angelis, M., Arnault, P., Gobbetti, M., 2008. Longterm fungal inhibitory activity of water-soluble extracts of Phaseolus vulgaris cv. Pinto and sourdough lactic acid bacteria during bread storage. Applied and Environmental Microbiology 74, 7391–7398. Corsetti, A., Gobbetti, M., Rossi, J., Damiani, P., 1998. Antimould activity of sourdough lactic acid bacteria: identification of a mixture of organic acids produced by Lactobacillus sanfrancisco CB1. Applied Microbiology and Biotechnology 50, 253–256. Dal Bello, F., Clarke, C.I., Ryan, L.A.M., Ulmer, H., Schober, T.J., Strom, K., Sjogren, J., van Sinderen, D., Schnurer, J., Arendt, E.K., 2007. Improvement of the quality and shelf life of wheat bread by fermentation with the antifungal strain Lactobacillus plantarum FST 1.7. Journal of Cereal Science 45, 309–318. De Muynck, C., Leroy, A.I.J., De Maeseneire, S., Arnaut, F., Soetaert, W., Vandamme, E.J., 2004. Potential of selected lactic acid bacteria to produce food compatible antifungal metabolites. Microbiological Research 159, 339–346. De Vuyst, L., Leroy, F., 2007. Bacteriocins from lactic acid bacteria: production, purification, and food applications. Journal of Molecular Microbiology and Biotechnology 13, 194–199. Elegado, F.B., Abuel, B.J.A., Te, J.T., Calapardo, M.R., Parungao, M.M., 2007. Antagonism against Listeria spp. and Staphyloccocus aureus by bacteriocin-producing lactic acid bacteria screened from the intestine of Philippine carabao, using polymerase chain reaction. Philippine Agricultural Scientist 90, 305–314. Filtenborg, O., Frisvad, J.C., Thrane, U., 1996. Moulds in food spoilage. International Journal of Food Microbiology 33, 85–102. Gerez, C.L., Torino, M.I., Rollán, G., Font de Valdez, G., 2009. Prevention of bread mould spoilage by using lactic acid bacteria with antifungal properties. Food Control 20, 144–148. Ghrairi, T., Frere, J., Berjeaud, J.M., Manai, M., 2007. Purification and characterisation of bacteriocins produced by Enterococcus faecium from Tunisian rigouta cheese. Food Control 19, 162–169. Harris, S.D., 2005. Morphogenesis in germinating Fusarium graminearum macroconidia. Mycologia 97, 880–887. Koch, E., Loffler, I., 2009. Partial characterization of the antimicrobial activity of Streptomyces antimycoticus FZB53. Journal of Phytopathology 157, 235–242. Kuwaki, S., Ohhira, I., Takahata, M., Murata, Y., Tada, M., 2002. Antifungal activity of the fermentation product of herbs by lactic acid bacteria against tinea. Journal of Bioscience and Bioengineering 94, 401–405. Lavermicocca, P., Valerio, F., Visconti, A., 2003. Antifungal activity of phenyllactic acid against molds isolated from bakery products. Applied and Environmental Microbiology 69, 634–640. Lind, H., Sjogren, J., Gohil, S., Kenne, L., Schnurer, J., Broberg, A., 2007. Antifungal compounds from cultures of dairy propionibacteria type strains. FEMS Microbiology Letters 271, 310–315. Magnusson, J., Schnurer, J., 2001. Lactobacillus coryniformis subsp. Coryniformis strain Si3 produces a broad-spectrum proteinaceous antifungal compound. Applied and Environmental Microbiology 67, 1–5. Martirosyan, A.O., Mndzhoyan, S.L., Charyan, L.M., Akopyan, L.G., Nikishchenko, M.N., 2004. Antimicrobial activity of lactic acid bacteria from sour milk products Narine, Karine, and Matsun. Applied Biochemistry and Microbiology 40, 178–180. Meroth, C.B., Walter, J., Hertel, C., Brandt, M.J., Hammes, W.P., 2003. Monitoring the bacterial population dynamics in sourdough fermentation processes by using PCRdenaturing gradient gel electrophoresis. Applied and Environmental Microbiology 69, 475–482. Millette, M., Cornut, G., Dupont, C., Shareck, F., Archambault, D., Lacroix, M., 2008. Capacity of human nisin- and pediocin-producing lactic acid bacteria to reduce intestinal colonization by vancomycin-resistant enterococci. Applied and Environmental Microbiology 74, 1997–2003. Nirenberg, H., 1976. Untersuchungen über die morphologische und biologische Differenzierung in der Fusarium-Sektion Liseola. Mitteilungen aus der Biologischen Bundesanstalt für Land und Forstwirtschaft 167, 111–117. Noots, I., Delcour, J.A., Michiels, C.W., 1999. From field barley to malt: detection and specification of microbial activity for quality aspects. Critical Reviews in Microbiology 25, 121–153. Oguntoyinbo, F.A., 2007. Identification and functional properties of dominant lactic acid bacteria isolated at different stages of solid state fermentation of cassava during traditional gari production. World Journal of Microbiology & Biotechnology 23, 1425–1432. Pitt, J.I., Basilico, J.C., Abarca, M.L., Lopez, C., 2000. Mycotoxins and toxigenic fungi. Medical Mycology 38, 41–46. Prasad, C., 1995. Bioactive cyclic dipeptides. Peptides 16, 151–164. Roy, U., Batish, V.K., Grover, S., Neelakantan, S., 1996. Production of antifungal substance by Lactococcus lactis subsp lactis CHD-28.3. International Journal of Food Microbiology 32, 27–34. Ryan, L.A.M., Dal Bello, F., Arendt, E.K., 2008. The use of sourdough fermented by antifungal LAB to reduce the amount of calcium propionate in bread. International Journal of Food Microbiology 125, 274–278. Ryu, D., Jackson, L.S., Bullerman, L.B., 2002. Effects of processing on zearalenone. Mycotoxins and Food Safety 504, 205–216. Samson, R.A., Hoekstra, E.S., Frisvad, J.C., Filtenborg, O., 2000. Introduction to food- and airborne fungi. Ed.(6), Centraalbureau voor Schimmelcultures.
A. Mauch et al. / International Journal of Food Microbiology 141 (2010) 116–121 Schnurer, J., Magnusson, J., 2005. Antifungal lactic acid bacteria as biopreservatives. Trends in Food Science and Technology 16, 70–78. Strom, K., Sjogren, J., Broberg, A., Schnurer, J., 2002. Lactobacillus plantarum MiLAB 393 produces the antifungal cyclic dipeptides cyclo(L-Phe-L-Pro) and cyclo(L-Phetrans-4-OH-L-Pro) and 3-phenyllactic acid. Applied and Environmental Microbiology 68, 4322–4327.
121
Tamminen, M., Joutsjoki, T., Sjoblom, M., Joutsen, M., Palva, A., Ryhanen, E.L., Joutsjoki, V., 2004. Screening of lactic acid bacteria from fermented vegetables by carbohydrate profiling and PCR-ELISA. Letters in Applied Microbiology 39, 439–444. Vaughan, A., Eijsink, V.G.H., O'Sullivan, T.F., O'Hanlon, K., van Sinderen, D., 2001. An analysis of bacteriocins produced by lactic acid bacteria isolated from malted barley. Journal of Applied Microbiology 91, 131–138.
Contents lists available at ScienceDirect
International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o
Short Communication
The use of Lactobacillus brevis PS1 to in vitro inhibit the outgrowth of Fusarium culmorum and other common Fusarium species found on barley A. Mauch a,b, F. Dal Bello a,b, A. Coffey c, E.K. Arendt a,⁎ a b c
Department of Food Science, Food Technology and Nutrition, National University of Ireland, Cork, Ireland National Food Biotechnology Centre, National University of Ireland, Cork, Ireland Department of Biological Sciences, Cork Institute of Technology, Bishopstown, Cork, Ireland
a r t i c l e
i n f o
Article history: Received 15 December 2009 Received in revised form 15 April 2010 Accepted 1 May 2010 Keywords: Antifungal Lactobacillus Fusarium spp. Mycelia growth Macroconidia Inhibition
a b s t r a c t A total of 129 lactic acid bacteria (LAB) were screened for antifungal activity against common Fusarium spp. isolated from brewing barley. Four out of the five most inhibiting isolates were identified as Lactobacillus brevis, whereas one belonged to Weissella cibaria. L. brevis PS1, the isolate showing the largest inhibition spectrum, was selected and the influence of its freeze-dried cell-free supernatant (cfsP) on germination of macroconidia as well as mycelia growth was investigated using Fusarium culmorum as target organism. Addition of cfsP into the growth medium at concentrations ≥ 2% altered the growth morphology of F. culmorum, whereas at concentrations N 5% the outgrowth of germ tubes from macroconidia was delayed and distorted. The presence of 10% cfsP completely inhibited the outgrowth of F. culmorum macroconidia. The activity of the compounds produced by L. brevis PS1 was higher at low pH values, i.e. pH b 5. Heating and/or proteolytic treatment reduced the inhibitory activity of cfsP, indicating that L. brevis produces organic acids and proteinaceous compounds which are active against Fusarium spp. © 2010 Elsevier B.V. All rights reserved.
1. Introduction The mycotoxigenic fungi associated with the human food chain belong mainly to the three genera Aspergillus, Fusarium and Penicillium (Pitt et al., 2000). Aspergillus and Penicillium species are reported as spoilage organisms from a wide range of food and feeds, whereas Fusarium species are often found on cereal grains (Filtenborg et al., 1996; Samson et al., 2000). In grain processing, mycotoxin secretion by storage fungi like Aspergillus or Penicillium species can be prevented by the selection of appropriate storage conditions of the grains. This approach does not apply for field fungi. Therefore field fungi represent an important threat to the safety of cereal products (Noots et al., 1999). Although processing, notably heat treatment, can reduce mycotoxin concentrations significantly, it does not eliminate them completely (Bullerman and Bianchini, 2007; Ryu et al., 2002). Safety of raw materials and food products can be assured by the use of chemical preservatives. However, during the last decades the application of these compounds has been questioned. In particular, the enhanced interest in natural and free-from foods, preferentially with healthpromoting characteristics has forced the food makers to find alter-
⁎ Corresponding author. Tel.: + 353 21 4902064; fax: + 353 21 4270213. E-mail address: [email protected] (E.K. Arendt). 0168-1605/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2010.05.002
native solutions. The applications of lactic acid bacteria (LAB) as starter cultures as well as their metabolites are a matter of particular interest to perform this task. LAB have a long history of application in fermented foods because of their beneficial influence on nutritional, organoleptic, and shelf-life characteristics, and are naturally occurring in many food systems (De Vuyst and Leroy, 2007; Tamminen et al., 2004; Vaughan et al., 2001). There is an extensive knowledge about antibacterial compounds, especially bacteriocins, produced by LAB (Aso et al., 2008; De Vuyst and Leroy, 2007; Elegado et al., 2007; Ghrairi et al., 2007; Millette et al., 2008; Oguntoyinbo, 2007) whereas the number of published studies on the identification of antifungal compounds produced by LAB is rather limited. Several low molecular weight compounds, mostly organic acids, have been isolated with the ability to retard or eliminate fungal growth or spores outgrowth, either on their own or synergistically (Batish et al., 1997; Dal Bello et al., 2007; Lavermicocca et al., 2003; Lind et al., 2007; Ryan et al., 2008; Schnurer and Magnusson, 2005). Regarding the great diversity of LAB within a single species, particularly due to environmental adaptions, there is a strong justification for further studies aimed at identifying novel antifungal LAB and characterising the compounds responsible for their inhibitory activity. The ambition of this study was to find LAB isolated from different sources like cheese as well as human, mouse, pig and bovine intestinal sources exhibiting antifungal activity against a variety of important Fusarium species commonly found on barley.
A. Mauch et al. / International Journal of Food Microbiology 141 (2010) 116–121
2. Materials and methods 2.1. Fungal cultures and spore–mycelia suspension Fusarium avenaceum TMW 4.1843, F. culmorum TMW 4.2043, Fusarium graminearum TMW 4.2046, Fusarium poae TMW 4.2044 and Fusarium tricinctum TMW 4.1405 all isolates from barley were kindly provided by the culture collection of Lehrstuhl fuer Technische Mikrobiologie, TU-Muenchen Weihenstephan (TMW). Fungi were cultivated on potato-dextrose agar (PDA) plates (Fluka Chemie AG, Buchs, Switzerland) at 25 °C for 5 days and then stored at 4 °C until further use. Small pieces from PDA-plates inoculated with fusaria were transferred into 500 mL of synthetic-nutrient-poor bouillon (SNB) (Nirenberg, 1976). The suspensions were incubated at 25 °C (120 rpm) for 5–7 days to induce both microconidia and macroconidia (F. avenaceum, F. poae and F. tricinctum) or solely macroconidia (F. culmorum and F. graminearum) formation. Concentrations of 1 × 105 to 3 × 105 CFU per mL were measured by plating out serial dilutions on PDA-plates. 2.2. Bacterial cultures The LAB used were isolated from cheese as well as human, mouse, pig and bovine intestinal samples. LAB were routinely grown on MRSagar plates (Fluka Chemie AG, Buchs, Switzerland) under microaerophilic conditions for 48 h at 30 or 37 °C. Long-term storage was done in 35% glycerol at −80 °C. 2.3. Screening for antifungal LAB The screening of LAB for antifungal activity was performed by nebulising 100 μL of fungal spore–mycelia suspension (approx. 104 CFU) onto the surface of petri-dishes containing 20 mL of MRSagar modified as follows (mMRS): pH adjusted to 6.0, sodium acetate as well as potassium dihydrogenphosphate omitted. After 30 min, bacteria were inoculated as two parallel lines of 3 cm length, keeping a distance between the lines of approx. 2 cm. Plates were incubated under microaerophilic conditions at 30 as well as 37 °C for 48 h followed by an additional incubation for 48 h under aerobic conditions at 25 °C to promote fungal growth. The antifungal activity of each LAB was ascertained by measuring the size of the clear zone surrounding the bacterial streaks. Antifungal activity was scored as follows: −, clear zone size b 3 mm; +, clear zone size ≥ 3 mm; ++, clear zone size ≥ 5 mm; and +++, clear zone size ≥ 10 mm. The overall growth of the fungi was compared to that in control plates (i.e. with no LAB present) and rated as follows: a, identical growth of fungi surrounding the clear zone; b, retarded growth of fungi surrounding the clear zone; c, strongly retarded growth of fungi surrounding the clear zone. 2.4. Identification of LAB isolates Bacteria exhibiting strong antifungal activity were identified upon sequencing of the first 900 bp of the 16 S rDNA (Meroth et al., 2003). To determine the closest relatives of the partial 16 S rDNA sequences, a GeneBank DNA database search was conducted. A similarity of N98% to 16S rDNA sequences of type strains was used as the criterion for identification. 2.5. Production of freeze-dried bacterial supernatant powder Cell-free supernatant (cfs) powders of the most inhibitory strain (cfsP) and of a non-inhibitory strain belonging to the same species (cfsN) were produced to serve as base material for the experiments describing the nature of the antifungal compounds. Briefly, overnight cultures of bacteria were inoculated in 500 mL of mMRS broth to
117
reach an initial concentration of 104 CFU/mL. The bacteria were grown for 144 h at 37 °C (temperature at which the antifungal strain showed its highest activity). Cells were separated from the supernatant by centrifuging twice at 3000 g for 15 min at 4 °C. The cell-free supernatant was freeze dried and the powder stored at 4 °C. 2.6. D/L-lactic acid and acetate amount in cfsP and cfsN The amounts of D/L-lactic acid and acetic acid present in the cfs were determined in solutions containing 25% of cfsP or cfsN redissolved in distilled water. For this purposes D-lactic acid/L-lactic acid as well as acetic acid test kits were used according to the manufacturer instructions (R-biopharm AG, Darmstadt, Germany). 2.7. Impact of bacterial supernatant on growth of F. culmorum The effect of different concentration of cfs on the growth of F. culmorum was examined by using a microplate assay. F. culmorum was chosen as the test-fungus due to the fact that it exhibited strong sensitivity during the screening and also it develops macroconidia during spore formation, which simplifies microscopic examinations. Pure macroconidia were obtained by filtering the spore–mycelia suspension of F. culmorum through a 30 μm pore size filter paper (Filter Paper 113 wet strengthened, Whatman International Ltd, Maidstone, England). The macroconidia suspension was adjusted to 1.0 × 105 macroconidia per mL. Aliquots of 1 mL were centrifuged at 3000 g for 10 min at 4 °C and the supernatant was discarded. The conidia pellets were resuspended in 1 mL malt extract broth (MEB) (Merck KGaA, Darmstadt, Germany). The MEB, adjusted to pH 4 using equal volumes of commercial D/L-lactic acid (Sigma-Aldrich, St. Louis, USA) and variable amounts of 4 M NaOH, contained 0, 1.0, 2.5, 5.0, 7.5 or 10.0% (w/v) of cfsP or cfsN. Before adding to the pellets, the dilutions were filtered through a sterile 0.45 μm MINISART®-plus filter (Sartorius Stedim Biotech GmbH, Goettingen Germany). After resuspending the conidia pellets in MEB, wells of a sterile 96-well microplate (Sarstedt AG & Co, Nuembrecht, Germany) were filled with 200 μL and sealed with optically clear seal for QPCR (Thermo Scientific, Waltham, USA). The microplates were incubated for 48 h at 25 °C and agitated every 4 s for approximately 1 s inside a Multiskan FC microplate-reader (Thermo Scientific, Waltham, USA). The optical density at 620 nm (OD620) was automatically recorded for each well every 3 h. The changes in OD620 over time were used to generate F. culmorum growth curves at each cfs concentration. The experiment was performed in triplicate. 2.8. Macroconidia germination assay For both antifungal and control strain, 20 mL of MEB was prepared containing 0 (control), 5 or 10% (w/v) of cfsP or cfsN. The pH was adjusted to 4 as described before. Chemically acidified control (CAC) broths were prepared by adding to MEB the amount of D/L-lactic acid used to adjust the pH and the quantity of D/L-lactic acid and acetic acid present in the cfs of the antifungal strain. The pH was adjusted to 4 using 4 M NaOH. All solutions were filtered through a sterile 0.45 μm MINISART®-plus filter. Macroconidia were obtained as described before but from 20 mL spore and mycelia suspension. After centrifugation at 3000 g for 10 min at 4 °C, the macroconidia pellets were resuspended in MEB containing cfs and incubated at 25 °C on an orbital platform shaker Heidolph® Unimax 1010 (Heidolph, Schwabach, Germany) at 350 rpm. Samples of 1 mL were taken at 3 h intervals and centrifuged at 4000 g for 3 min. After discarding the supernatant, the pellet was resuspended in 100 μL glycerol:water (50:50 v/v). A volume of 5 μL was transferred onto a microscopy slide and 100 randomly chosen macroconidia were analysed with a HP 630 microscope (Zefa Laborservice GmbH, Muenchen, Germany) at 400× magnification. The number of conidia showing germ tube formation
118
A. Mauch et al. / International Journal of Food Microbiology 141 (2010) 116–121
was determined and the values were given as percentage. The tests were performed in triplicate. 2.9. Morphological observations during mycelia growth A 50% (w/w) cfsP working-solution was prepared by dissolving the powder in bi-distilled water and filter sterilised using a 0.45 μm MINISART®-plus filter. PDA-plates were prepared containing 0 (control), 0.01, 0.05, 0.1, 0.5, 1, 2 or 3% (m/v) cfsP. For each concentration, chemically acidified control (CAC) plates were prepared containing commercially available D/L-lactic acid (Sigma-Aldrich, St. Louis, USA) and glacial acetic acid (BDH Laboratory, England) complying with the amounts present in each cfsP concentration used. After cooling, approximately 100 macroconidia were inoculated as a spot in the centre of the PDA-plates. The plates were incubated for 72 h at 25 °C under aerobic conditions. The fungal growth was monitored by photoscanning the plates and measuring the growth area using an image evaluation software (image j 1.40 g, Wayne Rasband, National Institutes of Health, USA). The test was performed in triplicate. 2.10. Activity of antifungal compounds at different pH values Aliquots of 20 mL MEB containing 10% (w/v) cfsP or cfsN were prepared and the pH values adjusted to 4.0, 5.0 or 6.5 using D/L-lactic acid. The solutions were filtered through a 0.45 μm MINISART®-plus filter. The MEB solutions were inoculated with F. culmorum macroconidia and the percentage of germinated macroconidia was determined as described before. The test was performed in triplicate. 2.11. Activity of antifungal compounds after treatment with proteolytic enzymes To determine the production of proteinaceous compounds by the antifungal strain, cfsP was treated with Pronase E (Fluka Chemie AG, Buchs, Switzerland) or Proteinase K (Sigma-Aldrich, St. Louis, USA). The enzymes were prepared according to the suppliers' recommendations but SDS and TRITON X-100® were omitted from the buffer. A 25% (w/v) cfsP solution was prepared using the enzyme buffers. After adjusting the pH to 7.5 using 4 M NaOH, the samples were filtered through a 0.45 μm MINISART®-plus filter and incubated for 1 to 5 h at 37 °C. The reaction was stopped by cooling to 20 °C. The cfsP concentration was adjusted to 10% (m/v) using MEB, and the pH was adjusted to 4 using D/L-lactic acid. The solutions were inoculated with F. culmorum macroconidia and the percentage of germinated macroconidia was determined as described above. The test was performed in duplicate. 2.12. Activity of antifungal compounds after heat treatment The heat stability of the antifungal compounds produced by the antifungal strain was determined by dissolving 2 mg of cfs with bi-
distilled water to a total volume of 8 mL (20% w/v) and heating it for 1 h at 80, 90 or 100 °C. After heating, the cfs was diluted to 10% (w/v) and the macroconidia germination assay was performed as described above. 3. Results and discussion The antifungal activity of 129 LAB isolated from different sources was investigated by streaking out the bacteria in two parallel lines onto mMRS plates inoculated with Fusaria spores and mycelia. Incubation at 30 °C resulted in a higher number of LAB showing antifungal activity. At this temperature, around 58% of the isolates inhibited at least one fungus, compared to approx. 50% when the incubation temperature was 37 °C. The LAB showing no antifungal activity at any growth temperature accounted to approx. 24%. These results confirmed that LAB are generally active against fungi, in particular Fusarium spp. L. brevis was the dominant species among the most inhibitory LAB, with four out of the five most inhibitory strains belonging to this species. However, we also isolated a L. brevis strain (i.e., NS) among the non-active LAB, suggesting that antifungal activity of L. brevis is a strain dependant trait (Table 1). This observation is in agreement with the work of Gerez et al. (2009) who found two out of five L. brevis strains exhibiting inhibitory activity against F. graminearum. On account of its ability to inhibit all tested fungi at 37 °C and three out of five fungi at 30 °C, L. brevis PS1 was selected for further experiments. The non-inhibitory L. brevis NS was used as negative control. The antifungal activity of L. brevis PS1 was investigated in depth using F. culmorum as target organism. The compounds produced by L. brevis PS1 affected both mycelial growth and macroconidia germination of F. culmorum. Addition of cfsP into PDA-agar plates at concentrations lower than 2% did not affect morphology of the aerial mycelia of the fungus, and no differences were observed when compared to the growth on control plates without any additives. At concentrations ≥ 2%, a distinctive change in colour as well as a higher density of mycelia emanating from the centre of the colony fading towards the borders appeared. On CAC plates this effect was only slightly visible. The growth areas on the plates containing cfsP were significantly (P N 0.05) reduced in comparison to both CAC and control plates (Fig. 1). While determining germ tube formation in the macroconidia germination assay, it was noted that a concentration of 5% cfsP resulted in macroconidia frequently possessing more than two germ tubes although outgrowth was slightly retarded compared to the control (Fig. 3). These germ tubes mainly emerged from nonapical cells. On the contrary, the macroconidia in the cfs-free or 5% cfsN broth developed their germ tubes mainly from the apical cells. The alterations of growth morphology as well as the more frequent emergence of germ tubes from internal compartments, observed when the macroconidia were exposed to cfsP at levels of 5% indicate that F. culmorum experienced stress even under those environmental conditions. In agreement with this observation, Koch and Loffler
Table 1 Antifungal activity of the most inhibiting lactic acid bacteria against selected Fusarium species. LAB
L. brevis PS1 W. cibaria PS2 L. brevis PS3 L. brevis PS4 L. brevis PS5 L. brevis NS
F. avenaceum
F. culmorum
F. graminearum
F. poae
F. tricinctum
30 °C
37 °C
30 °C
37 °C
30 °C
37 °C
30 °C
37 °C
30 °C
37 °C
− − +, c +, b − −
++, a ++, c − − +++, c −
+, b − ++, c ++, a − −
+++, a +, c +, b − ++, a −
− ++, b − − − −
+, b +, a − ++, c − −
+, b − ++, c ++, c − −
+++, a ++, a +++, b − +++, a −
++, a − +, b ++, a ++, a −
+, a ++, a − − +, a −
The distance between the peripheral sides of the bacterial-lines and the starting growth zone was scored as follows:−, no clear zone; +, distance ≥ 3 mm; ++, distance ≥ 5 mm; +++, distance ≥ 10 mm. Growth of fungi in the surrounding zone was recorded as follows: a, identical growth of fungi surrounding the clear zone; b, retarded growth of fungi surrounding the clear zone; c, strongly retarded growth of fungi surrounding the clear zone.
A. Mauch et al. / International Journal of Food Microbiology 141 (2010) 116–121
Fig. 1. Growth areas of F. culmorum incubated for 72 h on Potato Dextrose Agar-plates containing cell-free supernatant of L. brevis PS1 (cfsP) lactic/acetic acid-mixture complying the amounts found in cfsP (CAC) or no additives.
(2009) reported distorted hyphae with different degrees of swelling when F. culmorum was grown in the presence of antifungal filtrate from cultures of Streptomyces antimycoticus. Other authors observed the more frequent germ tube emergence from internal compartments when non-lethal concentrations of known antifungal substances are present (Aleandri et al., 2008; Chitarra et al., 2005; Harris, 2005). However, growth of F. culmorum in MEB containing 10% cfsP adjusted to pH 4 as detected during determining the impact of bacterial supernatant on growth of F. culmorum as well as in the macroconidia germination assay was not only altered but completely restricted (Figs. 2 and 3). The OD620 did not change over 48 h (the examination
Fig. 2. Growth of F. culmorum in Malt Extract Broth (pH 4) containing: 10% (–♦–), 7.5% (--x--), 5% (∙∙Δ∙∙) or 0% (…○…) of cell-free supernatant of L. brevis PS1.
Fig. 3. Germination of F. culmorum macroconidia in Malt Extract Broth (pH 4) in the presence of 10% (♦) or 5% (■) of cell-free supernatant of L. brevis PS1; 10% (▲) or 5% (x) of cell-free supernatant of L. brevis NS, and MEB with no addition of supernatant (◊).
119
was continued based on a visual control for another 5 days, and during this period no turbidity was detected) and in addition no germ tube formation was observed. Reducing the concentration of cfsP in MEB to 7.5% resulted in an increase in OD620 after 45 h. When 5% cfsP was added, approximately 50% of macroconidia formed germ tubes after 6 h; and after 12 h of incubation a change in OD620 was detected in the growth assay. Concentrations of cfsP lower than 5% did not affect the growth of F. culmorum (data not shown). When 10% cfsN was added, growth of F. culmorum started after 12 h (data not shown). The restricted outgrowth of macroconidia at pH 4 and the diminishing inhibition capacity of cfsP when increasing the pH from 4.0 to 5.0 and a total loss of activity at pH 6.5 (Fig. 4) suggest that organic acids are involved in the antifungal activity. At low pH values, organic acids exist mainly in undissociated form, which makes them more penetrable to the hydrophobic cell membranes of target organisms. Similar findings were reported by De Muynck et al. (2004) when testing antifungal activity of bacterial supernatant from L. brevis LMG 6906 against a selection of common food spoilage fungi. The authors detected a complete loss of inhibition when the pH was increased from 3.5 to 5.0, 5.5 or 6.0. However, our measurement of the two optical isomers of lactic acid as well as acetic acid in solutions containing 25% of cfsP and cfsN did not show potent differences. The cfs from both our strains was found to contain similar amounts of D-lactic acid, in the region.of 10.7 mmol. The cfsP solution contained 17.2 mmol L-lactic acid and 11.2 mmol acetate compared to 11.8 mmol L-lactic acid and 7.6 mmol acetate in the cfsN solutions. Although lactic and acetic acids are known antimicrobial compounds (Kuwaki et al., 2002; Martirosyan et al., 2004), the similar amounts in cfs of the non-antifungal L. brevis NS indicate that other antifungal compounds must be specifically produced (or produced at higher levels) by the antifungal strain PS1. Synergistic effects in terms of antifungal activity of different organic acids in combination with the main metabolites lactic and acetic acids are widely reported in literature. Corsetti et al. (1998) found effects of a mixture of acetic, caproic, formic, propionic, butyric and n-valeric acids in which caproic acid played a key role in inhibiting mould growth whereas Lavermicocca et al. (2003) could observe that addition of both lactic as well as acetic acid in concentrations exceeding the amounts detected in culture filtrate of L. plantarum 21B amplified the inhibition strength of phenyllactic acid, another antifungal organic acid found in cultures of L. plantarum. Aside from the effect of organic acids, other authors refer to the participation of proteinaceous compounds in the antifungal activity of LAB (Coda et al., 2008; Magnusson and Schnurer, 2001; Roy et al., 1996). Strom et al. (2002) reported weak synergistic effects between some cyclic peptides and 3-phenyllactic acid. Thus, we investigated if proteolytic treatment could affect the antifungal activity of L brevis
Fig. 4. Germination of F. culmorum macroconidia in Malt Extract Broth containing 10% cell-free supernatant of L. brevis PS1 at pH 4.0 (♦), 5.0 (■) or 6.5 (▲) as well as in Malt Extract Broth with no addition of supernatant (x) at pH 4.0.
120
A. Mauch et al. / International Journal of Food Microbiology 141 (2010) 116–121
PS1 supernatant. Digestion of cfsP with the proteolytic enzymes Proteinase K or Pronase E reduced the inhibitory activity to different extents. Treatment with Proteinase K for 5 h resulted in the loss of inhibitory activity after 24 h. At this time point 22% of the macroconidia was germinated, and the germination increased to a maximum level of approx. 70% after 33 h. Treatment with Proteinase K for 1 h reduced the inhibitory activity to a lower extent, and germination started after 33 h of incubation. Digestion with Pronase E for 5 h affected the inhibitory activity of cfsP to the same level as when the cfsP was treated with Proteinase K for 1 h. Treatment for 1 h with Pronase E did not affect cfsP activity (data not shown). These data strongly suggest that proteinaceous compounds are involved in the antifungal activity of L brevis PS1. Antifungal proteinaceous compounds production by L brevis has been recently reported by Coda et al. (2008). These authors isolated five peptides showing antifungal activity in a partly purified extract from sourdough fermented with L. brevis AM7. Interestingly, when they treated the water soluble extract with trypsin for 5 h no loss of antifungal activity was observed. Gerez et al. (2009) also did not detect a reduction in the inhibitory activity of cfs of two L. brevis strains, treated with proteinase K for 1 h. Our study indicates that time and type of enzyme applied for the proteolytic treatment are key factors in determining the loss or not of the antifungal activity. In fact, we showed that a reduction in the activity of cfsP was achieved only when the incubation time with proteinase K was extended to 5 h. Whether a protective matrix in cfsP reduced the enzymatic activity, or if the slow degradation was due to a difficult accessibility of the reaction site, could not be determined. Since many proteinaceous compounds but also other compounds are often sensitive towards heat treatment, we tested the impact of heat on cfsP at different temperatures. Heating for 1 h at 80 °C reduced the inhibitory activity of cfsP. Under this condition, macroconidia outgrowth started after 27 h and ca. 10% of macroconidia germinated after 48 h of incubation indicating that the antifungal compounds are heat labile. However, heating at 90 or 100 °C for 1 h did not affect the inhibitory activity of cfsP, and no germ tube formation was observed after 48 h of incubation. This contradictory observation might be explained by the formation of additional antifungal compounds during heating at high temperatures. In fact, it has been reported that heat treatment of fermentation broths can result in the generation of antifungal compounds, e.g. diketopiperazines (Prasad, 1995). Taken together, our results imply that both organic acids and proteinaceous compounds are responsible for the antifungal activity of L. brevis PS1. This work further strengthens the idea that the antifungal activity of LAB is a complex phenomenon, where numerous and different compounds act synergistically against target organisms. Further studies will unravel the identity of the compounds responsible for the antifungal activity of L brevis PS1 and thus make it easier to estimate the potential of the strain to serve e.g. as a biopreservative against Fusarium growth during food processing or as a biological agent to control Fusarium head blight. Acknowledgements The authors wish to acknowledge that this project was funded by the National Development Plan 2006–2010. The authors would also like to thank Timo Hipp and Agata Pawlowska for their technical support and insight. References Aleandri, M.P., Magro, P., Chilosi, G., 2008. Influence of environmental pH modulation on efficiency of apoplastic PR proteins during Fusarium culmorum — wheat seedling interaction. Plant Pathology 57, 1017–1025. Aso, Y., Takeda, A., Sato, M., Takahashi, T., Yamamoto, T., Yoshikiyo, K., 2008. Characterization of lactic acid bacteria coexisting with a nisin Z producer in Tsuda-turnip pickles. Current Microbiology 57, 89–94.
Batish, V.K., Roy, U., Lal, R., Grover, R., 1997. Antifungal attributes of lactic acid bacteria — a review. Critical Reviews in Biotechnology 17, 209–225. Bullerman, L.B., Bianchini, A., 2007. Stability of mycotoxins during food processing. International Journal of Food Microbiology 119, 140–146. Chitarra, G.S., Breeuwer, P., Rombouts, F.M., Abee, T., Dijksterhuis, J., 2005. Differentiation inside multicelled macroconidia of Fusarium culmorum during early germination. Fungal Genetics and Biology 42, 694–703. Coda, R., Rizzello, C.G., Nigro, F., De Angelis, M., Arnault, P., Gobbetti, M., 2008. Longterm fungal inhibitory activity of water-soluble extracts of Phaseolus vulgaris cv. Pinto and sourdough lactic acid bacteria during bread storage. Applied and Environmental Microbiology 74, 7391–7398. Corsetti, A., Gobbetti, M., Rossi, J., Damiani, P., 1998. Antimould activity of sourdough lactic acid bacteria: identification of a mixture of organic acids produced by Lactobacillus sanfrancisco CB1. Applied Microbiology and Biotechnology 50, 253–256. Dal Bello, F., Clarke, C.I., Ryan, L.A.M., Ulmer, H., Schober, T.J., Strom, K., Sjogren, J., van Sinderen, D., Schnurer, J., Arendt, E.K., 2007. Improvement of the quality and shelf life of wheat bread by fermentation with the antifungal strain Lactobacillus plantarum FST 1.7. Journal of Cereal Science 45, 309–318. De Muynck, C., Leroy, A.I.J., De Maeseneire, S., Arnaut, F., Soetaert, W., Vandamme, E.J., 2004. Potential of selected lactic acid bacteria to produce food compatible antifungal metabolites. Microbiological Research 159, 339–346. De Vuyst, L., Leroy, F., 2007. Bacteriocins from lactic acid bacteria: production, purification, and food applications. Journal of Molecular Microbiology and Biotechnology 13, 194–199. Elegado, F.B., Abuel, B.J.A., Te, J.T., Calapardo, M.R., Parungao, M.M., 2007. Antagonism against Listeria spp. and Staphyloccocus aureus by bacteriocin-producing lactic acid bacteria screened from the intestine of Philippine carabao, using polymerase chain reaction. Philippine Agricultural Scientist 90, 305–314. Filtenborg, O., Frisvad, J.C., Thrane, U., 1996. Moulds in food spoilage. International Journal of Food Microbiology 33, 85–102. Gerez, C.L., Torino, M.I., Rollán, G., Font de Valdez, G., 2009. Prevention of bread mould spoilage by using lactic acid bacteria with antifungal properties. Food Control 20, 144–148. Ghrairi, T., Frere, J., Berjeaud, J.M., Manai, M., 2007. Purification and characterisation of bacteriocins produced by Enterococcus faecium from Tunisian rigouta cheese. Food Control 19, 162–169. Harris, S.D., 2005. Morphogenesis in germinating Fusarium graminearum macroconidia. Mycologia 97, 880–887. Koch, E., Loffler, I., 2009. Partial characterization of the antimicrobial activity of Streptomyces antimycoticus FZB53. Journal of Phytopathology 157, 235–242. Kuwaki, S., Ohhira, I., Takahata, M., Murata, Y., Tada, M., 2002. Antifungal activity of the fermentation product of herbs by lactic acid bacteria against tinea. Journal of Bioscience and Bioengineering 94, 401–405. Lavermicocca, P., Valerio, F., Visconti, A., 2003. Antifungal activity of phenyllactic acid against molds isolated from bakery products. Applied and Environmental Microbiology 69, 634–640. Lind, H., Sjogren, J., Gohil, S., Kenne, L., Schnurer, J., Broberg, A., 2007. Antifungal compounds from cultures of dairy propionibacteria type strains. FEMS Microbiology Letters 271, 310–315. Magnusson, J., Schnurer, J., 2001. Lactobacillus coryniformis subsp. Coryniformis strain Si3 produces a broad-spectrum proteinaceous antifungal compound. Applied and Environmental Microbiology 67, 1–5. Martirosyan, A.O., Mndzhoyan, S.L., Charyan, L.M., Akopyan, L.G., Nikishchenko, M.N., 2004. Antimicrobial activity of lactic acid bacteria from sour milk products Narine, Karine, and Matsun. Applied Biochemistry and Microbiology 40, 178–180. Meroth, C.B., Walter, J., Hertel, C., Brandt, M.J., Hammes, W.P., 2003. Monitoring the bacterial population dynamics in sourdough fermentation processes by using PCRdenaturing gradient gel electrophoresis. Applied and Environmental Microbiology 69, 475–482. Millette, M., Cornut, G., Dupont, C., Shareck, F., Archambault, D., Lacroix, M., 2008. Capacity of human nisin- and pediocin-producing lactic acid bacteria to reduce intestinal colonization by vancomycin-resistant enterococci. Applied and Environmental Microbiology 74, 1997–2003. Nirenberg, H., 1976. Untersuchungen über die morphologische und biologische Differenzierung in der Fusarium-Sektion Liseola. Mitteilungen aus der Biologischen Bundesanstalt für Land und Forstwirtschaft 167, 111–117. Noots, I., Delcour, J.A., Michiels, C.W., 1999. From field barley to malt: detection and specification of microbial activity for quality aspects. Critical Reviews in Microbiology 25, 121–153. Oguntoyinbo, F.A., 2007. Identification and functional properties of dominant lactic acid bacteria isolated at different stages of solid state fermentation of cassava during traditional gari production. World Journal of Microbiology & Biotechnology 23, 1425–1432. Pitt, J.I., Basilico, J.C., Abarca, M.L., Lopez, C., 2000. Mycotoxins and toxigenic fungi. Medical Mycology 38, 41–46. Prasad, C., 1995. Bioactive cyclic dipeptides. Peptides 16, 151–164. Roy, U., Batish, V.K., Grover, S., Neelakantan, S., 1996. Production of antifungal substance by Lactococcus lactis subsp lactis CHD-28.3. International Journal of Food Microbiology 32, 27–34. Ryan, L.A.M., Dal Bello, F., Arendt, E.K., 2008. The use of sourdough fermented by antifungal LAB to reduce the amount of calcium propionate in bread. International Journal of Food Microbiology 125, 274–278. Ryu, D., Jackson, L.S., Bullerman, L.B., 2002. Effects of processing on zearalenone. Mycotoxins and Food Safety 504, 205–216. Samson, R.A., Hoekstra, E.S., Frisvad, J.C., Filtenborg, O., 2000. Introduction to food- and airborne fungi. Ed.(6), Centraalbureau voor Schimmelcultures.
A. Mauch et al. / International Journal of Food Microbiology 141 (2010) 116–121 Schnurer, J., Magnusson, J., 2005. Antifungal lactic acid bacteria as biopreservatives. Trends in Food Science and Technology 16, 70–78. Strom, K., Sjogren, J., Broberg, A., Schnurer, J., 2002. Lactobacillus plantarum MiLAB 393 produces the antifungal cyclic dipeptides cyclo(L-Phe-L-Pro) and cyclo(L-Phetrans-4-OH-L-Pro) and 3-phenyllactic acid. Applied and Environmental Microbiology 68, 4322–4327.
121
Tamminen, M., Joutsjoki, T., Sjoblom, M., Joutsen, M., Palva, A., Ryhanen, E.L., Joutsjoki, V., 2004. Screening of lactic acid bacteria from fermented vegetables by carbohydrate profiling and PCR-ELISA. Letters in Applied Microbiology 39, 439–444. Vaughan, A., Eijsink, V.G.H., O'Sullivan, T.F., O'Hanlon, K., van Sinderen, D., 2001. An analysis of bacteriocins produced by lactic acid bacteria isolated from malted barley. Journal of Applied Microbiology 91, 131–138.