propionibacteria for food biopreservation
2.6 Antifungal metabolites and further inhibitory mechanismsmechanisms
2.6.1 Purification and identification of antifungal metabolites
12 34 56 78 910 12 34 56 78 920 12 34 56 78 930 12 34 56 78 940 12 4344 45X
in addition to antifungal properties, e.g. texture-enhancing properties in dairy products or retrogradation-delaying (anti-staling) properties in bakery products.
A slimy growth suggested exopolysaccharide (EPS) production by protective cultures composed of Propionibacterium jensenii SM11 and Lactobacillus paracasei strains SM20, SM29, or SM63 that increased the viscosity of yoghurt samples produced with these antifungal strains and thus led to an improvement of texture (Miescher Schwenninger and Meile, 2004).
2.6 Antifungal metabolites and further inhibitory
Antifungal lactic acid bacteria and propionibacteria 43
12 34 56 78 910 12 34 56 78 920 12 34 56 78 930 12 34 56 78 940 12 4344 45X compounds are generally produced at very low levels by antifungal cultures, in
contrast to the high MIC values determined for pure compounds in antagonistic tests. The precise mechanism of antifungal activity is very complex and its elucidation difficult. Several methods were developed to purify and identify antifungal metabolites but, as of the date of this publication, the complete secret of antifungal activity has not been solved for any strain or strain combination. A prerequisite for the study of antifungal metabolites is to determine their activity in liquids, e.g. cell-free culture supernatant that will facilitate their further characterization, purification, and identification. Antifungal compounds that were identified from LAB and PAB are summarized in Table 2.1, while their molecular structures are depicted in Figure 2.5.
Low-molecular-mass antifungal compounds
A mixture of low-molecular-mass compounds including acetic, caproic, formic, propionic, butyric, and valeric acids was identified in cell-free supernatants of antifungal sourdough strain Lactobacillus sanfrancisco CB1 with gas chromatography-mass spectrometry (GC-MS) (Corsetti et al., 1998). The compounds appeared to act synergistically with caproic acid playing a key role.
Strain CB1 produced 13.75 mM (0.8 g/l) acetic acid, 0.88 mM (0.1 g/l) caproic acid, 1.43 mM (65.8 mg/l) formic acid, 0.14 mM (10.4 mg/l) propionic acid, and about 0.10 mM (8.8 mg/l) butyric acid and 0.10 mM (10.2 mg/l) valeric acid after 48 hours of growth in wheat flour hydrolysate (WHF) broth. Acetic acid was responsible for about one-half of the inhibitory activity of mixtures with pure compounds. Antifungal activity decreased markedly to about one-third when caproic acid was excluded.
Niku-Paavola et al. (1999) observed 37% growth inhibition of Fusarium avenaceum by a Lactobacillus plantarum strain. The low molecular mass fraction collected after gel chromatography of cell-free supernatant revealed only 27% inhibition. Characteristic compounds from this fraction were identified by GC-MS and included benzoic acid, methylhydantoin, mevalonolactone, and cyclo(Gly-L-Leu). Pure compounds in concentrations of 10 ppm (10 mg/l) inhibited growth of test organisms by 10–15% increasing to 20% when applied in mixtures.
Ten-fold concentrated cell-free supernatant of the sourdough strain Lactobacillus plantarum 21B grown in wheat flour hydrolysate exhibited fungicidal activity towards strains of Eurotium spp., Penicillium spp., Endomyces fibuliger, Aspergillus spp., Monilia sitophila, and Fusarium graminearum (Lavermicocca et al., 2000).
Extraction with ethyl acetate, preparative silica gel thin-layer chromatography, and GC-MS identified 3-phenyllactic and 4-hydroxyphenyllactic acids in active culture filtrates of Lacobacillus plantarum 21B. These acids are involved in phenylalanine metabolism and known antimicrobial compounds of LAB (Sato et al., 1986). 3-Phenyllactic acid has been recognized as the major component of antifungal activity in strain 21B (Lavermicocca et al., 2000) and has been shown to inhibit fungal test organisms at high concentration of about 50 g/l. Detailed microdilution tests with 23 fungal strains belonging to 14 species of bread and cereal spoilage Aspergillus, Penicillium, and Fusarium showed that less than
12 34 56 78 910 12 34 56 78 920 12 34 56 78 930 12 34 56 78 940 12 4344 45X
Fig. 2.5 Chemical structures of antifungal compounds (synonyms of the original publications are listed; additional names corresponding to IUPAC (International Union of Pure and Applied Chemistry, USA) are included in square brackets in case of differentiations).
(a), Benzoic acid, molecular weight (MW): 122.12; (b), mevalonolactone[4-hydroxy-4-methyloxan-2-one], MW: 130.14; (c), cyclo(Gly-L-Leu) [3-(2-methylpropyl)piperazine-2,5-dione], MW: 170.21; (d), methylhydantoin [1-methylimidazolidine-2,4-dione], MW:
114.10; (e), 3-phenyllactic acid [2-hydroxy-3-phenylpropanoic acid], MW: 166.17; (f), 4-hydroxyphenyllactic acid [2-hydroxy-3-(4-hydroxyphenyl)propanoic acid], MW: 182.17;
(g), 2-pyrrolidone-5-carboxylic acid [(2S)-5-oxopyrrolidine-2-carboxylic acid], MW:
129.11; (h), cyclo(L-Phe-L-Pro) [(3R,8aS)-3-benzyl-2,3,6,7,8,8a-hexahydropyrrolo[1,2-a]
pyrazine-1,4-dione], MW: 244.29; (i), cyclo(L-Phe-trans-4-OH-L-Pro) [no IUPAC name
Antifungal lactic acid bacteria and propionibacteria 45
12 34 56 78 910 12 34 56 78 920 12 34 56 78 930 12 34 56 78 940 12 4344 45X available], MW: 260.29; (j), hydroxydecanoic acid, MW: 188.26; (k),
(R)-3-hydroxydodecanoic acid, MW: 216.32; (l), (R)-3-hydroxytetradecanoic acid, MW: 244.37;
(m), 3-hydroxy-5-cis-dodecenoic acid [no IUPAC name available], MW: 214.30; (n), propionic acid [propanoic acid], MW: 74.08; (o), acetic acid, MW: 60.05; (p), lactic acid [2-hydroxypropanoic acid], MW: 90.08; (q), succinic acid [butanedioic acid], MW: 118.09;
(r), caproic acid [hexanoic acid], MW: 116.16; (s), butyric acid [butanoic acid], MW: 88.11;
(t), valeric acid [pentanoic acid], MW: 102.13; and (u), formic acid, MW: 46.03.
7.5 g/l of 3-phenyllactic acid was required to obtain 90% (MIC90) growth inhibition for all strains (Lavermicocca et al., 2003). As with other weak acid preservatives, e.g. propionic, benzoic, and sorbic acids, antifungal activity of 3-phenyllactic acid is pH dependent and due to its rather low pKa (3.46) activity increased at lower pH.
Addition of lactic acid (15.8 g/l) increased 3-phenyllactic acid inhibitory activity about 30% (Lavermicocca et al., 2003). The active compounds identified from salami originating strain Lactobacillus plantarum VLT01 were likewise 3-phenyllactic (46.6 mg/l) and 4-hydroxyphenyllactic acids (67.6 mg/l) (Coloretti et al., 2007). Production of 3-phenyllactic acid and 4-hydroxyphenyllactic acid was also determined for 29 LAB belonging to 12 species widely used in the
12 34 56 78 910 12 34 56 78 920 12 34 56 78 930 12 34 56 78 940 12 4344
45X Table 2.1
Antifungal Lactobacillus spp. (L.) and Propionibacterium spp. (P.) and their inhibitory spectrum, antifungal compounds and MIC (minimal inhibitory concentrations) Antifungal cultureInhibitory spectrumAntifungal compound(s)Production levelMICaReference Low molecular mass compounds L. sanfrancisco CB1FusariumAcetic acid13.75 mM (825.7 mg/l)8.33 mM (0.5 g/l)Corsetti et al., 1998 graminearum 623Caproic acid0.88 mM (102.2 mg/l)4.30 mM (0.5 g/l) Formic acid1.43 mM (65.8 mg/l)19.50 mM (0.9 g/l) Propionic acid0.14 mM (10.4 mg/ml)8.10 mM (0.6 g/l) Butyric acid0.10 mM (8.8 mg/ml)9.08 mM (0.8 g/l) Valeric acid0.10 mM (10.2 mg/ml)7.83 mM (0.8 g/l) L. plantarumFusarium avenacum VTT D-80147Benzoic acidn.d.10 ppmbNiku-Paavola et al., 1999 Methylhydantoinn.d.10 ppmb Mevalonolactonen.d.10 ppmb Cyclo(Gly-L-Leu)n.d.10 ppmb L. plantarum 21BEurotium repens IBT18000 3-Phenyllactic acid 4-Hydroxyphenyllactic acid n.d. n.d.
7.5 g/lc n.d.Lavermicocca et al., 2003 Endomyces fibuliger IDM3812 Penicillium corylophilum IBT18687 Monilia sitophila IDM/ FS5 L. plantarum MiLAB393Fusarium sprotrichioides J304
3-Phenyllactic acid L-Pro)
Cyclo(L-Phe-trans-4- OH-L-Pro)
n.d. n.d. n.d.
7.5 g/l 20 g/l n.d.
Ström et al., 2002; Broberg et al., 2007 Aspergillus fumigatus J9 Kluyveromyces marxianus J137
12 34 56 78 910 12 34 56 78 920 12 34 56 78 930 12 34 56 78 940 12 4344 45X 47
L. plantarum MiLAB14Aspergillus fumigatus J93-(R)-Hydroxydecanoic acid 3-Hydroxy-5-cis-dodecenoic
acid 3-(R)-Hydroxydodecanoic acid 3-(R)-Hydroxytetradecanoic acid
1.6 mg/l 1.0 mg/l 0.5 mg/l 0.2 mg/l
100 mg/l n.d. 25 mg/l >100 mg/l
Sjögren et al., 2003 L. rhamnosus LC705(Enterobacter cloaceae Pseudomonas fluorescens)d
2-Pyrrolidone5-carboxylic acidn.d.n.d.Yang et al., 1997 L. paracasei subsp. paracasei SM202-Pyrrolidone5-carboxylic acid7 mM (903.8 mg/l)e> 500 mM (> 64.5 g/l)fMiescher Schwenninger et al., 2008 Lactic acidn.d.e
> 500 mM (> 45.0 g/l)
f Acetic acidn.d.e
50–500 mM (3.0–30.0 g/l)
f P. jensenii SM11Candida pulcherrima 1-50/133-Phenyllactic acid1 mM (166.2 mg/l)e
50–500 mM (8.3–83.1 g/l)
fMiescher Schwenninger et al., 2008 4-Hydroxyphenyllactic acid0.2 mM (36.4 mg/l)en.d. Succinic acid29 mM (3.4 g/l)e
200–>500 mM (23.6–>59.1 g/l)
f Rhodotorula mucilaginosa FSQE63Propionic acid362 mM (26.8 g/l)e Acetic acid168 mM (10.1 g/l)e
10–200 mM (0.7–14.8 g/l)
f
50–500 mM (3.0–30.0 g/l)
f P. spp. type strainse3-Phenyllactic acid1.0–15.1 mg/ln.d.Lind et al., 2007 Proteinaceous compounds L. pentosus TV35bCandida albicans
Bacteriocin-like peptide (pentocin
TV35b)n.d.n.d.Okkers et al., 1999 L. sp. B3Penicillium spp.Possibly proteinaceoushn.d.n.d.Gourama, 1997 (Continued )
12 34 56 78 910 12 34 56 78 920 12 34 56 78 930 12 34 56 78 940 12 4344
45X aAntifungal cultureInhibitory spectrumAntifungal compound(s)Production levelMICReference L. coryniformis subsp. Aspergillus 3-kDa compoundn.d.n.d.Magnusson and coryniformis Si3fumigatus J9Schnürer, 2001 Aspergillus nidulans J10 Penicillium commune J238 Mucor hiemalis J42 Talaromyces flavus J37 Fusarium poae J24 Fusarium graminearum J114 Fusarium culmuorum J300 Fusarium sporotrichoides J319 h,iL. plantarum VLT01Aspergillus spp.Peptidic compoundsn.d.n.d.Coloretti et al., 2007 Penicillium spp.3-Phenyllactic acid46.6 mg/ln.d. Geotrichum candidum4-Hydroxyphenyllactic acid67.6 mg/ln.d. Moniliella spp.
Mucor racemosus Wallemia sebi Eurotium herbariorum a Minimal inhibitory concentration. b 10–15% inhibition by separate compounds applied at 10 ppm (10 mg/l) and maximally 20% in combinations. c MIC corresonding to 90% growth inhibition.90 d Antibacterial activity as main activity determined. e Produced with immobilized cells in a co-culture of L. paracasei subsp. paracasei SM20 with P. jensenii SM11. f MIC dependent on pH (pH 4.0, 5.0, and 6.0) and yeast. g P. jensenii DSMZ20535, P. thoenii DSMZ20276, P. acidipropionici DSMZ4900, P. freudenreichii subsp. freudenreichii DSMZ20271, P. freudenreichii subsp. shermanii DSMZ4902. h Sensitive to proteolytic enzymes (trypsin and pepsin). i Determined after autolysis of 30-day-old cultures. n.d.: not determined.
Table 2.1Continued
Antifungal lactic acid bacteria and propionibacteria 49
12 34 56 78 910 12 34 56 78 920 12 34 56 78 930 12 34 56 78 940 12 4344 45X production of fermented foods (Valerio et al., 2004). According to analysis of
variance, strains were divided into three groups comprising 15 strains that produced both metabolites (0.16–0.46 mM corresponding to 26.6–76.4 mg/l 3-phenyllactic acid and 0.07–0.29 mM corresponding to 12.8–52.8 mg/l 4-hydroxyphenyllactic acid), five strains accumulating only 3-phenyllactic acid (0.17–0.57 mM corresponding to 28.3–94.7 mg/l), and nine non-producer strains (≤0.10 mM corresponding to ≤16.6 mg/l 3-phenyllactic acid and ≤0.02 mM corresponding to ≤3.6 mg/l 4-hydroxyphenyllactic acid). 3-Phenyllactic acid production was increased in Lactobacillus plantarum ITM21B (identical to Lactobacillus plantarum 21B) by increasing the concentration of phenylalanine in culture and using low amounts of tyrosine. A direct correlation between phenylalanine and 3-phenyllactic acid, tyrosine and 4-hydroxyphenyllactic acid was suggested for Lactobacillus plantarum ITM21B, as it was described in the conversion of amino acids to cheese flavor compounds by Lactococcus lactis subsp. cremoris (Yvon et al., 1998).
Ström et al. (2002) identified 3-phenyllactic acid as the key antifungal compound of Lactobacillus plantarum MiLAB 393 isolated from grass silage.
3-Phenyllactic acid was also found in grass silage inoculated with strain MiLAB 393 (Broberg et al., 2007). Fractionation of cell-free supernatant of Lactobacillus plantarum MiLAB 393 on a C18 column followed by further separation on a preparative high-performance liquid chromatography (HPLC) C18 and a porous graphitic carbon column, as well as structure determination by nuclear magnetic resonance spectroscopy (NMR), mass spectrometry (MS), and gas chromatography (GC) revealed the presence of antifungal L-Pro) and cyclo(L-Phe-trans-4-OH-L-Pro) in addition to 3-phenyllactic acid. Minimal inhibitory concentrations (MIC) against Aspergillus fumigatus and Penicillium roqueforti were 20 g/l and 7.5 g/l for cyclo(L-Phe-L-Pro) and 3-phenyllactic acid, respectively, and weak synergistic effects were proposed. Synergistic effects of cyclo(L-Leu-L-Pro) and cyclo(L-Phe-L-Pro) were similarly described as inhibitors of pathogenic microorganisms including Candida albicans as well as anti-mutagenic effects in Salmonella strains (Rhee, 2004).
We identified a pool of low-molecular-mass compounds including 3-phenyllactic acid, 4-hydroxyphenyllactic acid, 2-pyrrolidone5-carboxylic acid, succinic acid as well as propionic, acetic, and lactic acids in cell-free culture supernatants of the protective co-culture Lactobacillus paracasei subsp. paracasei SM20 and Propionibacterium jensenii SM11 (Miescher Schwenninger et al., 2008).
Purification was achieved with a microplate bioassay controlled procedure with solid-phase extraction (C18) followed by either gel filtration chromatography or semipreparative reverse-phase high-performance liquid chromatography (RP-HPLC) and identification by LC-MS. A fermentation process with separate cell immobilization of the two strains was developed to produce high antagonistic activity expression, as observed on semisolid or solid matrices. Only low concentrations of 2-pyrrolidone-5-carboxylic acid (7 mM corresponding to 0.9 g/l), 3-phenyllactic acid (1 mM corresponding to 0.2 g/l), and 4-hydroxyphenyllactic acid (0.2 mM corresponding to 36.4 mg/l) were produced during fermentation
12 34 56 78 910 12 34 56 78 920 12 34 56 78 930 12 34 56 78 940 12 4344 45X
which were in contrast to relatively high MIC values of 50 (e.g. corresponding to 8.3 g/l for 3-phenyllactic acid) to more than 500 mM (83.1 g/l) determined with increasing pH from 4.0–6.0 for strains of Candida pulcherrima and Rhodotorula mucilaginosa (Table 2.1). Succinic acid was present at higher concentrations (29 mM corresponding to 3.4 g/l) but with comparable high MICs of 200 (23.6 g/l) to more than 500 mM (59.1 g/l) for pH 4.0–6.0. We therefore assumed synergistic effects between several low-molecular-mass compounds that were heat resistant (121 °C for 15 min) and resistant to protein degrading enzymes.
3-Phenyllactic acid, 4-hydroxyphenyllactic acid, and succinic acid production were associated with Propionibacterium jensenii SM11 and 2-pyrrolidone-5-carboxylic acid with Lactobacillus paracasei SM20 (Miescher Schwenninger et al., 2008).
Lind et al. (2007) likewise observed the production of 3-phenyllactic acid in five type strains of dairy propionibacteria, at extremely low concentrations ranging from 1.0 mg/l (Propionibacterium freudenreichii subsp. shermanii) to 15.1 mg/l (Propionibacterium thoenii).
2-Pyrrolidone-5-carboxylic acid is a widespread pyroglutamic acid and can be synthesized from glutamic acid by a heating process (Airaudo et al., 1987;
Mijin et al., 1989). LAB are known producers of 2-pyrrolidone-5-carboxylic acid which has antibacterial activity against Enterobacter cloacae, Pseudomonas fluorescens, Pseudomonas pudida, and Bacillus subtilis (Huttunen et al., 1995;
Yang et al., 1997). Purification of 2-pyrrolidone-5-carboxylic acid from cell-free supernatants was achieved by ethanol precipitation, gel filtration, anion exchange, RP-HPLC, NMR, and MS. Yang et al. (1997) observed complete inhibition of the bacterial indicator strains in a concentration range of 2-pyrrolidone-5-carboxylic acid from 6–23 mM (corresponding to 0.8–3.0 g/l) for pH 5.0 and 5.5. Although most of the preceding studies have suggested that antifungal activity is mainly based on 3-phenyllactic acid in combination with organic acid, e.g.
lactic and acetic acids, Yang and Clausen (2005) isolated high antifungal strains of Lactobacillus casei and Lactobacillus acidophilus that did not produce 3-phenyllactic acid but instead, at least four unknown heat resistant antifungal metabolites were recognized.
Using the isolation procedure described by Ström et al. (2002), Sjögren et al.
(2003) identified extremely low amounts of four hydroxylated fatty acids with antifungal activity after 78 h of growth of Lactobacillus plantarum MiLAB 14, i.e. 3-(R)-hydroxydecanoic acid (1.6 mg/l), 3-hydroxy-5-cis-dodecenoic acid (1.0 mg/l), 3-(R)-hydroxydodecanoic acid (0.5 mg/l), and 3-(R)-hydroxytetradecanoic acid (0.2 mg/l). MICs for total growth inhibition of yeasts and moulds were 10–100 mg/l for the racemic forms.
None of the above low-molecular-mass compounds was found to be solely responsible for high antifungal activity of its producer. These substances may however be important for more target-oriented screenings and also for the development and optimization of fermentation processes aimed to produce highly active protective cultures for food applications. Further research in this field is thus of the highest importance.
Antifungal lactic acid bacteria and propionibacteria 51
12 34 56 78 910 12 34 56 78 920 12 34 56 78 930 12 34 56 78 940 12 4344 45X Proteinaceous antifungal compounds
In addition to these characteristic low molecular mass antifungal metabolites, often observed in LAB and PAB, a few studies have described the production of proteinaceous antifungal compounds. The Propionibacterium bacteriocin propionicin PLG-1 was isolated by freezing and centrifugation of soft-agar (0.4%, w/v) cultures since its activity was never determined in cell-free supernatants (Lyon and Glatz, 1991). Propionicin PLG-1 showed broad antagonistic activities including yeast, moulds, Gram-positive, and Gram-negative bacteria. Lactobacillus pentosus TV35b was shown to produce a 3.9-kDa bacteriocin-like peptide with fungistatic effects against Candida albicans (Okkers et al., 1999). Lactobacillus coryniformis subsp.
coryniformis was observed to produce heat stable (121 °C for 15 min) proteinaceous compound(s) of about 3 kDa, which were inactivated after treatment with proteolytic enzymes (Magnusson and Schnürer, 2001). Gourama (1997) similarly described antifungal compounds that were sensitive to proteolytic enzymes such as trypsin and pepsin suggesting proteinaceous molecules. Lactobacillus plantarum strain VLT32 isolated from salami did not show any antifungal activity after 48 h of growth at 30 °C, but an inhibitory activity was determined in 30-day cultures which suggested the release of peptidic compounds after autolysis (Coloretti et al., 2007).