Antifungal properties of selected lactic acid bacteria and application in the biological control of ochratoxin A producing fungi during cocoa fermentation

Thirteen Lactic acid bacteria strains isolated from fermenting cocoa and seven reference strains were used in order to assess their antifungal properties towards three ochratoxin A (OTA) producing fungi (Aspergillus carbonarius, Aspergillus niger and Aspergillus ochraceus). Furthermore, two of the isolates strains (A19 and A21) identified as belonging to the genus of Pediococcus as well as Lactobacillus plantarum B4496, Lactobacillus brevis 207 and Lactobacillus sanfranciscensis BB12 showed interesting in vitro broad antifungal activities towards the three ochratoxin-producing fungi with inhibition percentages ranging from 15% to 66.7%. Treatment of cell-free supernatant at 100°C affected antifungal activity suggesting that the main compounds responsible for this activity were of proteic nature, and hence could be bacteriocins. Application of isolate A19 in cocoa fermentation as starter inhibited the growth of each of the OTA-producing species. At the end of fermentation in boxes inoculated with A19, A. niger was not detectable while A. carbonarius concentration was found to be 2 Log CFU/g of wet beans. The assessment of the ochratoxin produced during fermentation of cocoa inoculated with A. carbonarius indicated that the use of isolate A19 as starter could reduce their level of growth so as to have only a toxin production of 0.0012 ± 0.0005 μg/kg after 40 days of storage, while this was 2.45 ± 0.35 μg/kg of fermented and dried cocoa beans in the absence of A19. This work is a contribution for the application of biological control of OTA-producing fungi during cocoa production.     

The role of nisin in fuel ethanol production with Saccharomyces cerevisiae

Aims: To investigate the effects of nisin on lactobacilli contamination of yeast during ethanol fermentation and to determine the appropriate concentration required to control the growth of selected lactobacilli in a YP/glucose media fermentation model. Methods and Results: The lowest concentration of nisin tested (5 IU ml^?1) effectively controlled the contamination of YP/glucose media with 10⁶ CFU ml^?1 lactobacilli. Lactic acid yield decreased from 5·0 to 2·0 g l^?1 and potential ethanol yield losses owing to the growth and metabolism of Lactobacillus plantarum and Lactobacillus brevis were reduced by 11 and 7·8%, respectively. Approximately, equal concentrations of lactic acid were produced by Lact. plantarum and Lact. brevis in the presence of 5 and 2 IU ml^?1 nisin, respectively, thus demonstrating the relatively higher nisin sensitivity of Lact. brevis for the strains in this study. No differences were observed in the final ethanol concentrations produced by yeast in the absence of bacteria at any of the nisin concentrations tested. Conclusions: Metabolism of contaminating bacteria was reduced in the presence of 5 IU ml^?1 nisin, resulting in reduced lactic acid production and increased ethanol production by the yeast. Significance and Impact of the Study: Bacteriocins represent an alternative to the use of antibiotics for the control of bacterial contamination in fuel ethanol plants and may be important in preventing the emergence of antibiotic‐resistant contaminating strains.     

Flour carbohydrate catabolism and metabolite production by sourdough lactic acid bacteria

The aim of this study was to assess the mode of carbohydrate catabolism by lactic acid bacteria isolated from traditional sourdoughs, as well as to study their effect on the metabolites produced. For this purpose, single cultures of the heterofermentative lactic acid bacteria Lactobacillus sanfranciscensis, Lactobacillus brevis, Weissella cibaria, and the homofermentative Lactobacillus paralimentarius and Pediococcus pentosaceus were grown in liquid media containing glucose, fructose, maltose and sucrose, either as a single carbon source or in combination with glucose. Carbon catabolism and the production of metabolites were determined by HPLC analysis. W. cibaria could ferment all carbon sources, L. sanfranciscensis, L. paralimentarius and P. pentosaceus could not ferment sucrose, while L. brevis could only ferment maltose. The presence of glucose did not influence the utilization of fructose and maltose by L. sanfranciscensis, while it repressed the fermentation of fructose, maltose and sucrose by W. cibaria, and fructose and maltose by L. paralimentarius and P. pentosaceus. Moreover, L. sanfranciscensis and L. brevis could obtain extra ATP through the reduction of fructose to mannitol, which favored the production of acetic acid against ethanol. The utilization of fructose as an electron acceptor has a decisive effect on the prevailing of L. sanfranciscensis and L. brevis in spontaneously fermented sourdough and in the scarce appearance of the other lactic acid bacteria studied.     

Characterization of lactic acid bacteria from musts and wines of three consecutive vintages of Ribeira Sacra

Aims: This study was designed to isolate and characterize the lactic acid microbiota of the musts and wines of a young denomination of origin area, Ribeira Sacra in north‐west Spain. Methods and Results: Over three consecutive years (2007, 2008 and 2009), we examined musts and wines from four cellars in different zones of the region. Through biochemical and genetic tests, 459 isolates of lactic acid bacteria (LAB) were identified as the following species: Lactobacillus alvei (0·7%), Lactobacillus brevis (1·7%), Lactobacillus frumenti (0·9%), Lactobacillus kunkeei (12%), Lactobacillus plantarum (6·5%), Lactobacillus pentosus (0·9%), Lactococcus lactis ssp. lactis (3%), Leuconostoc citreum (0·7%), Leuconostoc fructosum (synon. Lactobacillus fructosum) (3·7%), Leuconostoc mesenteroides ssp. mesenteroides (2·8%), Leuconostoc pseudomesenteroides (0·2%), Oenococcus oeni (59%), Pediococcus parvulus (7%) and Weisella paramesenteroides (synon. Leuconostoc paramesenteroides) (0·9%). Of these species, O. oeni was the main one responsible for malolactic fermentation (MLF) in all cellars and years with the exception of Lact. plantarum, predominant in 2007, in one cellar, and Lact. brevis, Lact. frumenti and Ped. parvulus coexisting with O. oeni in one cellar in 2009. Different strains (84) of LAB species (14) were identified by biochemical techniques (API strips, the presence of plasmids, enzyme activities and MLF performance) and molecular techniques (PCR). All assays were carried out with every one of the 459 isolates. To select candidates for use as culture starters, we assessed malolactic, β‐glucosidase and tannase activities, the presence of genes involved in biogenic amine production and plasmid content. Conclusions: A high diversity of LAB is present in the grape musts of Ribeira Sacra but few species are responsible for MLF; however, different strains of such species are involved in the process. As far as we are aware, this is the first report of Lact. frumenti thriving in wine. Significance and Impact of the Study: Information on LAB populations in must and wine is presented. A large collection of well‐characterized strains of LAB are available as starter cultures to winemakers.     

Determining the Suitability of Lactobacilli Antifungal Metabolites for Inhibiting Mould Growth

Summary In recent years, public concern about indoor mould growth has increased dramatically in the United States. In this study, lactic acid bacteria (LAB), which are known to produce antimicrobial compounds important in the biopreservation of food, were evaluated to determine if the same antimicrobial properties can be used to inhibit mould fungi that typically colonize wood. Based on biomass measurement, cell-free supernatants from Lactobacillus casei subsp. rhamnosus and Lactobacillus acidophilus grown in deMan Rogosa Sharpe (MRS) broth inhibited 95–100% growth of three mould fungi and one stain fungus associated with wood-based building materials. Lactic acid and four unknown compounds ⩽ kDa molecular weight were fractionated from the culture supernatant by thin layer chromatography and high-performance liquid chromatography. Antifungal activity, which was attributed to one or more unknown metabolites, was retained during heating and neutralization. A 1:2 dilution of L. casei supernatant inhibited 100% growth of all test fungi.     

Aggregation of Lactobacillus brevis associated with decrease in pH by glucose fermentation

ABSTRACT

Some Lactobacillus brevis strains were found to aggregate upon the addition of glucose, which resulted in glucose fermentation and pH decrease. Surface layer proteins (Slp) that represented the outermost layer of the bacteria decreased under these low pH conditions, probably because of the partial detachment of Slp from the cell surface triggered by the acidic environment. Similar observations of decreased Slp and aggregation were observed under the culture conditions, confirming that L. brevis aggregation was due to the partial Slp detachment under the acidic conditions of glucose fermentation. Such Slp detachment might affect the electrostatic nature of L. brevis cells by initiating the formation of irregular charge across the L. brevis cell surface, thereby leading to aggregation. These observations would be useful for elucidating the aggregation mechanism of lactic acid bacteria, which was considered to be involved in the probiotic effect of the bacteria.     

Varietal difference in antifungal activity of some species of Agave

Crude leaf extracts of different Agave species were eluted out with the help of 80% methanol for their antifungal activity. The activity of A. americana, A. ferox, A. montana, A. marginata, and A. scabra were evaluated against brown rot fungi Postia placenta, the causal agent of wood decay of some economically important plants using a media poising method. The percent inhibition of hyphal growth was measured after the seventh day of incubation. A. montana shows the highest percent inhibition, 69.31%, of hyphal growth. These data suggested that the different Agave species analyzed have potential as antifungal agents with a broad range of activity. In the future they may be used as biocontrol agents to cure plant diseases without harming them.     

Bacterial and fungal communities of wilted Italian ryegrass silage inoculated with and without Lactobacillus rhamnosus or Lactobacillus buchneri

Aims: To understand the effects of lactic acid bacteria (LAB) inoculation on fermentation products, aerobic stability and microbial communities of silage. Methods and Results: Wilted Italian ryegrass was stored in laboratory silos with and without inoculation of Lactobacillus rhamnosus and Lactobacillus buchneri. The silos were opened after 14, 56 and 120 days and then subjected to aerobic deterioration for 7 days. Intensive alcoholic fermentation was found in untreated silage; the sum of ethanol and 2,3‐butanediol content at day 14 was about 7 times higher than that of lactic and volatile fatty acids. Alcoholic fermentation was suppressed by L. rhamnosus and L. buchneri inoculation and lactic acid and acetic acid became the dominant fermentation products, respectively. Silages were deteriorated in untreated and L. rhamnosus‐inoculated silages, whereas no spoilage was found in L. buchneri‐inoculated silage. Enterobacteria such as Erwinia persicina, Pantoea agglomerans and Rahnella aquatilis were detected in untreated silage, whereas some of these bacteria disappeared or became faint with L. rhamnosus treatment. When silage was deteriorated, Lactobacillus brevis and Bacillus pumilus were observed in untreated and L. rhamnosus‐inoculated communities, respectively. The inoculated LAB species was detectable in addition to untreated bacterial communities. Saccharomyces cerevisiae and Pichia anomala were the main fungi in untreated and L. rhamnosus‐inoculated silages; however, P. anomala was not visibly seen in L. buchneri‐inoculated silage either at silo opening or after exposure to air. Conclusion: Inoculation with L. rhamnosus can suppress alcoholic fermentation of wilted grass silage with elimination of enterobacteria at the beginning of fermentation. Addition of L. buchneri may improve aerobic stability, with distinct inhibitory effect observed on P. anomala after silo opening. Significance and Impact of the Study: Bacterial and fungal community analyses help us to understand how inoculated LAB can function to improve the fermentation and aerobic stability of silage.     

In vitro antifungal activity of lactic acid bacteria low molecular peptides against spoilage fungi of bakery products

Bio-preservation, a promising preservation method that involves the use of “friendly” microorganisms such as lactic acid bacteria, has recently become a topic of considerable interest. In the present study, 16 lactic acid bacteria isolates were evaluated for antifungal activity against six fungi commonly associated with bread spoilage. The antifungal compounds were heat stable at 121 °C, and only four isolates, DU15, IT10, TE10, and IS10, showed partial loss of activity when supernatants were treated with proteolytic enzymes. The four isolates showed high inhibition activity at pH 3 and were identified using 16S rDNA sequencing as belonging to Leuconostoc mesenteroides DU15, Lactobacillus plantarum TE10, Lactobacillus plantarum IT10, and Lactobacillus plantarum IS10. The minimum germination inhibitions were 30 mg, 50 mg, 40 mg, and 50 mg for TE10, IT10, DU15, and IS10 respectively. The optimum conditions for the strains to produce antifungal compounds were 37 °C for 48 h for IT10, IS10, and TE10, and 30 °C for 24 h for DU15. Antifungal activity was increased threefold when supernatants were filtered using 10 KDa membranes. These findings demonstrate the potential of using lactic acid bacteria antifungal peptides as natural preservatives in bakery products to control the growth of spoilage fungi.     

Lactobacillus brevis CD2: Fermentation Strategies and Extracellular Metabolites Characterization

Functional foods and nutraceuticals frequently contain viable probiotic strains that, at certain titers, are considered to be responsible of beneficial effects on health. Recently, it was observed that secreted metabolites might play a key role in this respect, especially in immunomodulation. Exopolysaccharides produced by probiotics, for example, are used in the food, pharmaceutical, and biomedical fields, due to their unique properties. Lactobacillus brevis CD2 demonstrated the ability to inhibit oral pathogens causing mucositis and periodontal inflammation and to reduce Helycobacter pylori infections. Due to the lack of literature, for this strain, on the development of fermentation processes that can increase the titer of viable cells and associated metabolites to industrially attractive levels, different batch and fed-batch strategies were investigated in the present study. In particular, aeration was shown to improve the growth rate and the yields of lactic acid and biomass in batch cultures. The use of an exponential feeding profile in fed-batch experiments allowed to produce 9.3 ± 0.45 × 10⁹ CFU/mL in 42 h of growth, corresponding to a 20-fold increase of viable cells compared with that obtained in aerated batch processes; moreover, also increased titers of exopolysaccharides and lactic acid (260 and 150%, respectively) were observed. A purification process based on ultrafiltration, charcoal treatment, and solvent precipitation was applied to partially purify secreted metabolites and separate them into two molecular weight fractions (above and below 10 kDa). Both fractions inhibited growth of the known gut pathogen, Salmonella typhimurium, demonstrating that lactic acid plays a major role in pathogen growth inhibition, which is however further enhanced by the presence of Lact. brevis CD2 exopolysaccharides. Finally, the EPS produced from Lact. brevis CD2 was characterized by NMR for the first time up to date.

     

Immunological characterization of lignocellulose degradation

Polyclonal antibodies produced to the brown-rot fungus Poria placenta have been used with fluorescence microscopy to detect fungal hyphae colonizing wood. In addition, an enzyme-linked immunosorbent assay (ELISA) has been used to detect and quantify the extent of fungal decay in wood. ELISA values correlated with percent weight loss associated with fungal degradation and the assay was able to detect fungal colonization ten days after inoculation in solid wood. Monoclonal antibodies to extracellular metabolites of P. placenta have been produced and are being characterized. Monoclonal antibodies have also been produced to native and partially deglycosylated Mn2 peroxidase. The use of immunological probes to monitor and characterize the decay process is discussed.     

Bacterial community associated with ensilage process of wilted guinea grass

Aims: To determine the effects of wilting, storage period and bacterial inoculant on the bacterial community and ensiling fermentation of guinea grass silage. Methods and Results: Fermentation products, colony counts and denaturing gradient gel electrophoresis (DGGE) profiles were determined. There was more lactic acid than acetic acid in all silages, but the lactic acid to acetic acid ratio decreased with storage time. This shift from lactic to acetic acid was not prevented even with a combination of wilting and bacterial inoculant. The DGGE analyses suggest that facultatively heterofermentative lactic acid bacteria (Lactobacillus plantarum, Lactobacillus brevis and Lactobacillus pentosus) were involved in the shift to acetic acid fermentation. Conclusions: Lactic acid can dominate the fermentation in tropical grass silage with sufficient wilting prior to ensiling. Prolonged storage may lead to high levels of acetic acid without distinctive changes in the bacterial community. Significance and Impact of the Study: The bacterial community looks stable compared to fermentation products over the course of long storage periods in tropical grass silage. Acetic acid fermentation in tropical grass silage can be a result of the changes in bacterial metabolism rather than community structure.     

Inhibition of growth of Trichophyton tonsurans by Lactobacillus reuteri

Aim: The aims of this study were to identify antifungal lactic acid bacteria (LAB) and characterize their activity against the dermatophyte Trichophyton tonsurans. Methods and Results: A total of 165 different LAB were isolated and initially screened for anti‐Penicillium expansum activity. Five strains, which exhibited strong inhibitory activity, were then tested against the dermatophyte T. tonsurans DSM12285, where they also caused inhibition as observed by large fungal clearing on agar surface. The strongest inhibition was seen with Lactobacillus reuteri R2. When freeze‐dried cell‐free supernatant powder from this strain was incorporated in culture medium at concentrations >1%, growth of fungal colony was inhibited. Conidia germination was also inhibited under these conditions as determined by microscopy. The anti‐T. tonsurans activity of Lact. reuteri R2 was not affected neither by heat treatment nor by proteolytic treatment using pronase E and proteinase K, indicating that the responsible agent(s) were nonproteinaceous in nature. Conclusions: Lactobacillus reuteri R2 was identified as having strong inhibitory activity against the dermatophyte T. tonsurans DSMZ12285. Significance and Impact of the Study: LAB are naturally associated with many foods and are well recognized for their biopreservative properties. The use of these and/or their products may well provide alternative safe approaches for the inhibition of dermatophytic fungi.     

Micronized Copper Wood Preservatives: Efficacy of Ion,Nano, and Bulk Copper against the Brown Rot Fungus Rhodonia placenta

Recently introduced micronized copper (MC) formulations, consisting of a nanosized fraction of basic copper (Cu) carbonate (CuCO₃·Cu(OH)₂) nanoparticles (NPs), were introduced to the market for wood protection. Cu NPs may presumably be more effective against wood-destroying fungi than bulk or ionic Cu compounds. In particular, Cu- tolerant wood-destroying fungi may not recognize NPs, which may penetrate into fungal cell walls and membranes and exert their impact. The objective of this study was to assess if MC wood preservative formulations have a superior efficacy against Cu-tolerant wood-destroying fungi due to nano effects than conventional Cu biocides. After screening a range of wood-destroying fungi for their resistance to Cu, we investigated fungal growth of the Cu-tolerant fungus Rhodonia placenta in solid and liquid media and on wood treated with MC azole (MCA). In liquid cultures we evaluated the fungal response to ion, nano and bulk Cu distinguishing the ionic and particle effects by means of the Cu²⁺ chelator ammonium tetrathiomolybdate (TTM) and measuring fungal biomass, oxalic acid production and laccase activity of R. placenta. Our results do not support the presence of particular nano effects of MCA against R. placenta that would account for an increased antifungal efficacy, but provide evidence that attribute the main effectiveness of MCA to azoles.     

Biodeterioration of brazilwood Caesalpinia echinata Lam. (Leguminosae—Caesalpinioideae) by rot fungi and termites

The heartwood of Caesalpinia echinata Lam. (Leguminosae) (commonly called brazilwood) is used for violin bow manufacture due to the unique vibrational and physical properties found in the wood. In the present work, the effects of Pycnoporus sanguineus (white-rot fungus), Gloeophyllum trabeum (brown-rot fungus), Chaetomium globosum (soft-rot fungus), and Cryptotermes brevis (dry-wood termite) on weight losses and chemical composition of extractives and cell-wall polysaccharides of C. echinata wood were investigated under laboratory conditions and compared to those obtained for Anadenanthera macrocarpa, Eucalyptus grandis, and Pinus elliottii. The heartwood of C. echinata was found to be as resistant as A. macrocarpa to the decay fungi tested and to the attack of the dry-wood termite. Pinitol and galactopinitol A were the main sugar alcohols found in the extractives of wood of C. echinata, their presence, however, did not appear related to the resistance to fungal decay. Although only incipient stages of decay were found, the modifications in cell-wall polysaccharide composition of heartwood of C. echinata by rot fungi were related to decrease in polymers other than xylans. The high resistance of C. echinata to xylophages is probably due to the presence of toxic extractives in the wood.     

An FT-IR study of the changes in chemical composition of bamboo degraded by brown-rot fungi

The objective of this study was to use FT-IR analysis to investigate the chemical composition of aged and un-aged bamboo specimens, with and without node sections, decayed by brown-rot fungi. Specimens were exposed to two brown-rot fungi, Coniophora puteana and Poria placenta, for 8 weeks after which decay was assessed by weight loss and FT-IR spectra analysis. Depending on the bamboo section examined, the aging process reduced decay resistance of specimens. Weight loss (measured as a percentage) decreased from the top to the bottom portion of bamboo culms. The presence of nodes in the specimens increased weight loss caused by P. placenta attack, and caused only a slight increase in weight loss from C. puteana attack. Significant chemical changes in bamboo were observed after fungal degradation, as revealed by FT-IR analyses. Consistent with the degradation mechanism of brown-rot fungi, lignin was essentially un-degraded or modified. Both brown-rot fungi caused a sharp decrease in the carbonyl absorption area. Surprisingly, cellulose peaks of degraded specimens were nearly similar to the peaks of control specimens. Aging treatments and biodegradation affected the crystalline structure of bamboo specimens. Poria placenta degraded wood components faster and changed the crystallinity more than C. puteana did, in accordance with the weight losses due to decay.     

Zur Bildung von Butanol-2 durch Lactobacillen und Hefen

Zusammenfassung Ein asporogener Stamm vonSaccharomyces cerevisiae und sechs Stämme vonLactobacillus brevis bildeten in einem vollsynthetischen Medium Butanol-2 aus Butandiol-2,3. Hierbei wurde meso-Butandiol-2,3 verbraucht. Die verschiedenen Lactobacillen bilden unterschiedliche Mengen an Butanol-2 (bis 810 mg/l).

---

On the formation of butanol-2 by lactic acid bacteria and yeast

Butanol-2 was formed from butanediol-2,3 in a synthetic medium by an asporogenous strain ofSaccharomyces cerevisiae and 6 strains ofLactobacillus brevis. The mesoform of butanediol-2,3 is degraded to butanol-2. The lactic acid bacteria form different amounts of butanol-2 (up to 810 mg/l).

---

Abkürzungen r.A. reiner Alkohol     

Conversion of rice straw to bio-based chemicals: an integrated process using Lactobacillus brevis

Commercialization of lignocellulosic biomass as a feedstock for bio-based chemical production is problematic due to the high processing costs of pretreatment and saccharifying enzymes combined with low product yields. Such low product yield can be attributed, in large part, to the incomplete utilization of the various carbohydrate sugars found in the lignocellulosic biomass. In this study, we demonstrate that Lactobacillus brevis is able to simultaneously metabolize all fermentable carbohydrates in acid pre-processed rice straw hydrolysate, thereby allowing complete utilization of all released sugars. Inhibitors present in rice straw hydrolysate did not affect lactic acid production. Moreover, the activity of exogenously added cellulases was not reduced in the presence of growing cultures of L. brevis. These factors enabled the use of L. brevis in a process termed simultaneous saccharification and mixed sugar fermentation (SSMSF). In SSMSF with L. brevis, sugars present in rice straw hydrolysate were completely utilized while the cellulase maintained its maximum activity due to the lack of feedback inhibition from glucose and/or cellobiose. By comparison to a sequential hydrolysis and fermentation process, SSMSF reduced operation time and the amount of cellulase enzyme necessary to produce the same amount of lactic acid.     

Optimisation of physical factors on the production of active metabolites by Bacillus subtilis 355 against wood surface contaminant fungi

A Surface Response Model was used to study the effect of pH, temperature and agitation on growth, sporulation and production of antifungal metabolites by Bacillus subtilis CCMI 355.Strong agitation, temperature between 27 and 34 °C and pH 6 favoured cell growth. Alkaline pH, strong agitation and temperature between 28 and 34 °C favoured spore formation. No relationship was found between sporulation and the production of antifungal metabolites. According to the model, pH 8, 37 °C and the absence of agitation were the optimal conditions for the production of broad-spectrum antifungal metabolites against Botrytis cinerea, Penicillium expansum, Trichoderma sp, Trichoderma harzianum, Trichoderma koningii and Trichoderma virgatum.In situ assays using green wood impregnated with Bacillus subtilis CCMI 355 inoculated in Yeast Extract Glucose Broth medium in the conditions above, displayed an efficient protection against wood surface contaminant fungi.     

Control of spoilage fungi by lactic acid bacteria

The evaluation of the potentiality of lactic acid bacteria (LAB) strains isolated from different origins to inhibit mould growth and to identify and characterize the antifungal metabolites were the aims of this study. From a total of ninety-one LAB strains tested, ten were selected due to their high inhibitory effect (>80%). The antifungal activity of the majority of the selected LAB strains was lost after the neutralization treatment determining the acidic nature of the antifungal metabolites. Lactic, acetic and phenyllactic (PLA) acids were identified as being responsible for antifungal effect in the 10 cell-free supernatants (CFS) evaluated. Amongst the strains evaluated, only Lactobacillus fermentum CRL 251 produced fungus inhibitory peptide/s, smaller than 10 kDa, thermostable, active in the pH range of 4–7 and sensitive to trypsin. This is the first report on antifungal peptide/s produced by a L. fermentum strain.