High-Throughput Metabolic Fingerprinting of Legume Silage Fermentations via Fourier Transform Infrared Spectroscopy and Chemometrics

Silage quality is typically assessed by the measurement of several individual parameters, including pH, lactic acid, acetic acid, bacterial numbers, and protein content. The objective of this study was to use a holistic metabolic fingerprinting approach, combining a high-throughput microtiter plate-based fermentation system with Fourier transform infrared (FT-IR) spectroscopy, to obtain a snapshot of the sample metabolome (typically low-molecular-weight compounds) at a given time. The aim was to study the dynamics of red clover or grass silage fermentations in response to various inoculants incorporating lactic acid bacteria (LAB). The hyperspectral multivariate datasets generated by FT-IR spectroscopy are difficult to interpret visually, so chemometrics methods were used to deconvolute the data. Two-phase principal component-discriminant function analysis allowed discrimination between herbage types and different LAB inoculants and modeling of fermentation dynamics over time. Further analysis of FT-IR spectra by the use of genetic algorithms to identify the underlying biochemical differences between treatments revealed that the amide I and amide II regions (wavenumbers of 1,550 to 1,750 cm⁻¹) of the spectra were most frequently selected (reflecting changes in proteins and free amino acids) in comparisons between control and inoculant-treated fermentations. This corresponds to the known importance of rapid fermentation for the efficient conservation of forage proteins.     

New trends and opportunities in the development and use of inoculants for silage

Abstract: Inoculants are used as silage additives to improve preservation efficiency and to enhance animal performance. In most commercially available inoculants, homofermentative lactic acid bacteria (LAB) have been used because they are fast and efficient producers of lactic acid, improving natural silage fermentation. Specific LAB inuculants may also have beneficial effects on animal performance even if there is no effect on fermentation. However, these types of inoculants are not always advantageous. They do not necessarily prevent sermentation by clostridia in moist silages, and they sometimes impair the aerobic stability of grass and small grain silages. Therefore, new criteria for silage inoculants should be established which consider the specific needs of the crop being ensiled. New approaches which are being taken to develop improved inoculants for silage include the following: (1) using LAB isolates which are more specific to the target crops; (2) inclusion of heterofermentative LAB to produce volatile fatty acids to inhibit yeasts and moulds upon aerobic exposure; (3) inclusion of organisms other than LAB in inoculants to inhibit detrimental microorganisms; (4) selection or engineering of LAB strains to inhibit specific microorganisms; and (5) cloning and expression of genes which would enable selected LAB strains to utilize polysaccharides in crops which are low in soluble carbohydrates. Many of these new strategies for formulating inoculants are being tested, but further research is needed to determine the most successful approaches.     

Influence of temperature and backslopping time on the microbiota of a type I propagated laboratory wheat sourdough fermentation

Sourdough fermentation is a cereal fermentation that is characterized by the formation of stable yeast/lactic acid bacteria (LAB) associations. It is a unique process among food fermentations in that the LAB that mostly dominate these fermentations are heterofermentative. In the present study, four wheat sourdough fermentations were carried out under different conditions of temperature and backslopping time to determine their effect on the composition of the microbiota of the final sourdoughs. A substantial effect of temperature was observed. A fermentation with 10 backsloppings (once every 24 h) at 23°C resulted in a microbiota composed of Leuconostoc citreum as the dominant species, whereas fermentations at 30 and 37°C with backslopping every 24 h resulted in ecosystems dominated by Lactobacillus fermentum. Longer backslopping times (every 48 h at 30°C) resulted in a combination of Lactobacillus fermentum and Lactobacillus plantarum. Residual maltose remained present in all fermentations, except those with longer backslopping times, and ornithine was found in almost all fermentations, indicating enhanced sourdough-typical LAB activity. The sourdough-typical species Lactobacillus sanfranciscensis was not found. Finally, a nonflour origin for this species was hypothesized.     

Bacteriophage ecology in commercial sauerkraut fermentations

Knowledge of bacteriophage ecology in vegetable fermentations is essential for developing phage control strategies for consistent and high quality of fermented vegetable products. The ecology of phages infecting lactic acid bacteria (LAB) in commercial sauerkraut fermentations was investigated. Brine samples were taken from four commercial sauerkraut fermentation tanks over a 60- or 100-day period in 2000 and 2001. A total of 171 phage isolates, including at least 26 distinct phages, were obtained. In addition, 28 distinct host strains were isolated and identified as LAB by restriction analysis of the intergenic transcribed spacer region and 16S rRNA sequence analysis. These host strains included Leuconostoc, Weissella, and Lactobacillus species. It was found that there were two phage-host systems in the fermentations corresponding to the population shift from heterofermentative to homofermentative LAB between 3 and 7 days after the start of the fermentations. The data suggested that phages may play an important role in the microbial ecology and succession of LAB species in vegetable fermentations. Eight phage isolates, which were independently obtained two or more times, were further characterized. They belonged to the family Myoviridae or Siphoviridae and showed distinct host ranges and DNA fingerprints. Two of the phage isolates were found to be capable of infecting two Lactobacillus species. The results from this study demonstrated for the first time the complex phage ecology present in commercial sauerkraut fermentations, providing new insights into the bioprocess of vegetable fermentations.     

An ecological study of lactic acid bacteria from Almagro eggplant fermentation brines

AIM: Identification of the predominant lactic acid bacteria (LAB) involved in spontaneous fermentations of Almagro eggplants, and evaluation of the biodiversity by molecular typing. METHODS AND RESULTS: Almagro eggplant fermentations in three factories (A, B and C) enjoying Protected Designation of Origin (PDO) status were monitored by chemical and microbiological analysis of brines. LAB isolates from brines were identified by phenotypic analysis and by species-specific PCR reactions and typed by randomly amplified polymorphic DNA (RAPD)-PCR. All isolates from factories A and C belonged to the genus Lactobacillus (Lact.), whereas isolates from factory B belonged to Lactobacillus (50%), Leuconostoc (Ln.) (25%) and Lactococcus (Lc.) (25%); 1.9% of this microbiota was considered cosmopolitan. The genera Leuconostoc and Lactococcus and the species Lact. acidophilus and Lact. paracasei had never previously been reported in Almagro eggplant fermentations. CONCLUSION: Considerable differences in the composition of the lactic acid microbiota participating in the Almagro eggplant fermentations exist. Brine NaCl concentration has a notable influence both in number and in the species participating. SIGNIFICANCE AND IMPACT OF THE STUDY: The original aspect of this work consists of an ecological study of the LAB taking part in spontaneous Almagro eggplant fermentations from different factories. Participation of Leuconostoc and Lactococcus species and of Lact. acidophilus and Lact. paracasei, which had never before been described for this pickle, and the evidence that a lactic fermentation does not always take place, were the most relevant results.     

The survival of silage inoculant lactic acid bacteria in rumen fluid

AIMS: To determine whether lactic acid bacteria (LAB) used in inoculants for silage can survive in rumen fluid (RF), and to identify those that survive best. METHODS AND RESULTS: Twelve commercial silage inoculants were added at 107 CFU ml-1 to strained RF (SRF) taken from dairy cows, with and without 5 g l-1 glucose and incubated in vitro at 39 degrees C. Changes in pH, LAB numbers and fermentation products were monitored for 72 h. In the inoculated RF with glucose, the pH decreased and numbers of LAB increased. The inoculants varied with regard to their effect on pH change and growth. In the SRF, both with and without glucose, the pH values of the inoculated samples were generally higher than those of the uninoculated controls throughout most of the incubation period. This may suggest a positive effect on the rumen environment. CONCLUSIONS: LAB used in silage inoculants can survive in RF in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first step in studying the probiotic potential of silage LAB inoculants for dairy cattle. The survival of these LAB in RF may enable them to interact with rumen microorganisms and to affect rumen functionality.     

Kinetic analysis of strains of lactic acid bacteria and acetic acid bacteria in cocoa pulp simulation media toward development of a starter culture for cocoa bean fermentation

The composition of cocoa pulp simulation media (PSM) was optimized with species-specific strains of lactic acid bacteria (PSM-LAB) and acetic acid bacteria (PSM-AAB). Also, laboratory fermentations were carried out in PSM to investigate growth and metabolite production of strains of Lactobacillus plantarum and Lactobacillus fermentum and of Acetobacter pasteurianus isolated from Ghanaian cocoa bean heap fermentations, in view of the development of a defined starter culture. In a first step, a selection of strains was made out of a pool of strains of these LAB and AAB species, obtained from previous studies, based on their fermentation kinetics in PSM. Also, various concentrations of citric acid in the presence of glucose and/or fructose (PSM-LAB) and of lactic acid in the presence of ethanol (PSM-AAB) were tested. These data could explain the competitiveness of particular cocoa-specific strains, namely, L. plantarum 80 (homolactic and acid tolerant), L. fermentum 222 (heterolactic, citric acid fermenting, mannitol producing, and less acid tolerant), and A. pasteurianus 386B (ethanol and lactic acid oxidizing, acetic acid overoxidizing, acid tolerant, and moderately heat tolerant), during the natural cocoa bean fermentation process. For instance, it turned out that the capacity to use citric acid, which was exhibited by L. fermentum 222, is of the utmost importance. Also, the formation of mannitol was dependent not only on the LAB strain but also on environmental conditions. A mixture of L. plantarum 80, L. fermentum 222, and A. pasteurianus 386B can now be considered a mixed-strain starter culture for better controlled and more reliable cocoa bean fermentation processes.     

Bacteriophage Ecology in Commercial Sauerkraut Fermentations

Knowledge of bacteriophage ecology in vegetable fermentations is essential for developing phage control strategies for consistent and high quality of fermented vegetable products. The ecology of phages infecting lactic acid bacteria (LAB) in commercial sauerkraut fermentations was investigated. Brine samples were taken from four commercial sauerkraut fermentation tanks over a 60- or 100-day period in 2000 and 2001. A total of 171 phage isolates, including at least 26 distinct phages, were obtained. In addition, 28 distinct host strains were isolated and identified as LAB by restriction analysis of the intergenic transcribed spacer region and 16S rRNA sequence analysis. These host strains included Leuconostoc, Weissella, and Lactobacillus species. It was found that there were two phage-host systems in the fermentations corresponding to the population shift from heterofermentative to homofermentative LAB between 3 and 7 days after the start of the fermentations. The data suggested that phages may play an important role in the microbial ecology and succession of LAB species in vegetable fermentations. Eight phage isolates, which were independently obtained two or more times, were further characterized. They belonged to the family Myoviridae or Siphoviridae and showed distinct host ranges and DNA fingerprints. Two of the phage isolates were found to be capable of infecting two Lactobacillus species. The results from this study demonstrated for the first time the complex phage ecology present in commercial sauerkraut fermentations, providing new insights into the bioprocess of vegetable fermentations.     

Oxidation of Metabolites Highlights the Microbial Interactions and Role of Acetobacter pasteurianus during Cocoa Bean Fermentation

Four cocoa-specific acetic acid bacterium (AAB) strains, namely, Acetobacter pasteurianus 386B, Acetobacter ghanensis LMG 23848^T, Acetobacter fabarum LMG 24244^T, and Acetobacter senegalensis 108B, were analyzed kinetically and metabolically during monoculture laboratory fermentations. A cocoa pulp simulation medium (CPSM) for AAB, containing ethanol, lactic acid, and mannitol, was used. All AAB strains differed in their ethanol and lactic acid oxidation kinetics, whereby only A. pasteurianus 386B performed a fast oxidation of ethanol and lactic acid into acetic acid and acetoin, respectively. Only A. pasteurianus 386B and A. ghanensis LMG 23848^T oxidized mannitol into fructose. Coculture fermentations with A. pasteurianus 386B or A. ghanensis LMG 23848^T and Lactobacillus fermentum 222 in CPSM for lactic acid bacteria (LAB) containing glucose, fructose, and citric acid revealed oxidation of lactic acid produced by the LAB strain into acetic acid and acetoin that was faster in the case of A. pasteurianus 386B. A triculture fermentation with Saccharomyces cerevisiae H5S5K23, L. fermentum 222, and A. pasteurianus 386B, using CPSM for LAB, showed oxidation of ethanol and lactic acid produced by the yeast and LAB strain, respectively, into acetic acid and acetoin. Hence, acetic acid and acetoin are the major end metabolites of cocoa bean fermentation. All data highlight that A. pasteurianus 386B displayed beneficial functional roles to be used as a starter culture, namely, a fast oxidation of ethanol and lactic acid, and that these metabolites play a key role as substrates for A. pasteurianus in its indispensable cross-feeding interactions with yeast and LAB during cocoa bean fermentation.     

Growth of yeast contaminants in an immobilized lactic acid bacteria system

Calcium alginate-immobilized lactic acid bacteria (LAB) were used to acidify milk and re-utilized for five successive fermentations. The yeast Kluyveromyces marxianus was added to the milk in order to simulate contamination of the bioreactor. Growth of the yeast was studied over the successive re-utilizations of the immobilized LAB. When the system was contaminated only in the first fermentation, the yeast population decreased in the successive re-utilizations; rinsing of the system following each fermentation was effective in further reducing yeast contamination. Yeast population increased only when contaminants were added at the beginning of each fermentation. Presence of yeast in the system did not influence acidification rate of the immobilized LAB or cell release from the alginate beads.     

Microbiological and physicochemical characterization of small-scale cocoa fermentations and screening of yeast and bacterial strains to develop a defined starter culture

Spontaneous cocoa bean fermentations performed under bench- and pilot-scale conditions were studied using an integrated microbiological approach with culture-dependent and culture-independent techniques, as well as analyses of target metabolites from both cocoa pulp and cotyledons. Both fermentation ecosystems reached equilibrium through a two-phase process, starting with the simultaneous growth of the yeasts (with Saccharomyces cerevisiae as the dominant species) and lactic acid bacteria (LAB) (Lactobacillus fermentum and Lactobacillus plantarum were the dominant species), which were gradually replaced by the acetic acid bacteria (AAB) (Acetobacter tropicalis was the dominant species). In both processes, a sequence of substrate consumption (sucrose, glucose, fructose, and citric acid) and metabolite production kinetics (ethanol, lactic acid, and acetic acid) similar to that of previous, larger-scale fermentation experiments was observed. The technological potential of yeast, LAB, and AAB isolates was evaluated using a polyphasic study that included the measurement of stress-tolerant growth and fermentation kinetic parameters in cocoa pulp media. Overall, strains L. fermentum UFLA CHBE8.12 (citric acid fermenting, lactic acid producing, and tolerant to heat, acid, lactic acid, and ethanol), S. cerevisiae UFLA CHYC7.04 (ethanol producing and tolerant to acid, heat, and ethanol), and Acetobacter tropicalis UFLA CHBE16.01 (ethanol and lactic acid oxidizing, acetic acid producing, and tolerant to acid, heat, acetic acid, and ethanol) were selected to form a cocktail starter culture that should lead to better-controlled and more-reliable cocoa bean fermentation processes.     

Screening and selection of lactic acid bacteria strains suitable for ensiling grass

AIM: Lactic acid bacteria (LAB) strains shown to have broad-spectrum antimicrobial activity were screened for potential as grass silage inoculants. The strains capable of rapidly lowering the pH of the grass matrix and with low proteolytic activity were assessed in laboratory-scale silos in a grass matrix containing natural microbial flora. METHODS AND RESULTS: Screening of nine candidate strains was performed first in a grass extract medium. The four most promising strains were selected on the basis of growth rate in the medium, capacity to reduce pH and ability to limit the formation of ammonia-N. The efficiency of the selected strains was further assessed in a laboratory-scale ensiling experiment. Untreated (no additive) and formic acid served as controls. All tested inoculants improved silage quality compared with untreated. With one exception (Pediococcus parvulus E315) the fermentation losses in the inoculated silages were even lower than in the acid-treated control silage. Pure lactic acid fermentation was obtained in the timothy-meadow fescue silage with all inoculants. The results obtained in the ensiling experiments were consistent with those of the screening procedure, which appeared to predict correctly the potential of LAB as silage inoculants. The strains with a low ammonia production rate in the grass extract medium behaved similarly in the silage. Especially in this respect the strain Lactobacillus plantarum E76 was superior to the other candidates. CONCLUSIONS: The screening method using grass extract proved to be useful in strain selection. SIGNIFICANCE AND IMPACT OF THE STUDY: The rapid screening method developed for the LAB strains provides a useful tool for more systematic product development of commercial inoculant preparations. Time consuming and laborious ensiling experiments can be limited only to the most promising strains.     

Growth and survival of lactic acid bacteria in lucerne silage

A rifampicin-resistant variant of two strains of Lactobacillus plantarum, one strain of Pediococcus acidilactici, and one strain of Enterococcus faecium were used for the experimental production of lucerne silage. Laboratory silage without inoculants served as a control. Counts of total anaerobes, total lactic acid bacteria (LAB), lactobacilli, pediococci, and enterococci were determined on days 14, 21, 30, 49, and 60 of lucerne fermentation. LAB dominated in silage microflora, reaching a percentage between 59 and 95 % of total anaerobes. Lactobacilli were found as a predominant group of LAB during the whole study. Lactobacilli reached numbers 8.74 log CFU/g in treated silage and 8.89 log CFU/g in the control at the first observation. Their counts decreased to 4.23 and 4.92 log CFU/g in treated silage and the control, respectively, on day 63 of fermentation. Similar decreases were observed in all bacterial groups. The treated silage samples possessed lower pH (4.2 vs. 4.5 in control samples) and contained more lactic acid compared to control silage. The identity of re-isolated rifampicin-resistant bacteria with those inoculated to the lucerne was evaluated by fingerprinting techniques. The fingerprint profiles of re-isolated bacteria corresponded to the profiles of strains used for the treatment. It could be concluded that supplemented LAB dominated in laboratory silage and overgrew naturally occurring LAB.     

Ecology of lactic acid bacteria in Italian fermented sausages: isolation, identification and molecular characterization

In this study, the ecology of the lactic acid bacteria (LAB) of three naturally fermented sausages produced in the Friuli-Venezia-Giulia region, in the North East of Italy, was investigated. A total of 465 strains isolated from three fermentations were identified by molecular methods and 12 different species of LAB were detected. Lactobacillus curvatus and Lactobacillus sakei were the most numerous (67 and 353 strains isolated, respectively) and they were subjected to RAPD-PCR. Clusters containing strains isolated from different plants were observed, underlining a coherent population distribution in three different fermentations. However, we also observed clusters formed by strains isolated from a specific fermentation, only. This could be explained considering the different technologies and recipes used for the production in three plants. Ingredient composition, fermentation and maturation parameters could play an important role in the selection of specific populations adapted in a specific environment.     

Antagonistic activities of lactic acid bacteria in food and feed fermentations

Abstract Many factors contribute to a successful natural fermentation of carbohydrate-rich food and feed products. Metabolic activities of lactic acid bacteria (LAB) play a leading role. Their ability to rapidly produce copious amounts of acidic end products with a concomitant pH reduction is the major factor in these fermentations. Although their specific effects are difficult to quantitate, other LAB metabolic products such as hydrogen peroxide and diacetyl can also contribute to the overall antibiosis and preservative potential of these products. The contribution of bacteriocins is also difficult to evaluate. It is suggested that they may play a role in selecting the microflora which initiates the fermentation. Bacteriocins are believed to be important in the ability of LAB to compete in non-fermentative ecosystems such as the gastro-intestinal tract. During the past few decades interest has arisen in the use of the varied antagonistic activities of LAB to extent the shelf-life of protein-rich products such as meats and fish. Recent findings indicate that the newly discovered Lactobacillus reuteri reuterin system may be used for this purpose.     

Optimization of fed-batch production of the model recombinant protein GFP in Lactococcus lactis

Optimization of recombinant protein production using lactic acid bacteria (LAB) remains an important obstacle on the road to realizing LAB as oral vaccine delivery vehicles. Despite this, there have been few published investigations to explore the higher limits of LAB recombinant protein expression in fed-batch fermentations. In this study, results from response surface experiments suggested an optimal set of conditions for expression of green fluorescent protein (GFP), a model recombinant protein, in bench-scale, fed-batch Lactococcus lactis IL1403 fermentations. The 48 4-L fed-batch fermentations in this set of experiments, along with preliminary studies, investigated the effects of pH, temperature, hemin concentration, concentration of the nisin inducer per cell, and time of induction. Cell densities in this data set ranged from 2.9 to 7.4 g/L and maximum GFP expression per cell ranged from 0.1 to 4.4 relative fluorescence units (RFU)/g. The optimal 4-L, fed-batch fermentation process found here yields growth and protein expression values that dramatically improve upon results from traditional test tube and flask processes. Relative to the traditional process, the experimental optimum conditions yield 4.9 times the cell density, 1.6 times the protein per cell mass, and 8 times the total protein concentration. Unexpectedly, experiments also revealed that the compound hemin, known previously to improve growth and survival of Lactococcus lactis (L. lactis), negatively impacted recombinant protein production when added in concentrations from 5 to 20 microg/mL with this strain. The improvement in protein expression over traditional processes demonstrated here is an important step toward commercial development of LAB for oral delivery of recombinant vaccines and therapeutic proteins.     

Antagonistic activities of lactic acid bacteria in food and feed fermentations

Many factors contribute to a successful natural fermentation of carbohydrate-rich food and feed products. Metabolic activities of lactic acid bacteria (LAB) play a leading role. Their ability to rapidly produce copious amounts of acidic end products with a concomitant pH reduction is the major factor in these fermentations. Although their specific effects are difficult to quantitate, other LAB metabolic products such as hydrogen peroxide and diacetyl can also contribute to the overall antibiosis and preservative potential of these products. The contribution of bacteriocins is also difficult to evaluate. It is suggested that they may play a role in selecting the microflora which initiates the fermentation. Bacteriocins are believed to be important in the ability of LAB to compete in non-fermentative ecosystems such as the gastrointestinal tract. During the past few decades interest has arisen in the use of the varied antagonistic activities of LAB to extend the shelf-life of protein-rich products such as meats and fish. Recent findings indicate that the newly discovered Lactobacillus reuteri reuterin system may be used for this purpose.     

Next-generation sequencing reveals significant bacterial diversity of botrytized wine

While wine fermentation has long been known to involve complex microbial communities, the composition and role of bacteria other than a select set of lactic acid bacteria (LAB) has often been assumed either negligible or detrimental. This study served as a pilot study for using barcoded amplicon next-generation sequencing to profile bacterial community structure in wines and grape musts, comparing the taxonomic depth achieved by sequencing two different domains of prokaryotic 16S rDNA (V4 and V5). This study was designed to serve two goals: 1) to empirically determine the most taxonomically informative 16S rDNA target region for barcoded amplicon sequencing of wine, comparing V4 and V5 domains of bacterial 16S rDNA to terminal restriction fragment length polymorphism (TRFLP) of LAB communities; and 2) to explore the bacterial communities of wine fermentation to better understand the biodiversity of wine at a depth previously unattainable using other techniques. Analysis of amplicons from the V4 and V5 provided similar views of the bacterial communities of botrytized wine fermentations, revealing a broad diversity of low-abundance taxa not traditionally associated with wine, as well as atypical LAB communities initially detected by TRFLP. The V4 domain was determined as the more suitable read for wine ecology studies, as it provided greater taxonomic depth for profiling LAB communities. In addition, targeted enrichment was used to isolate two species of Alphaproteobacteria from a finished fermentation. Significant differences in diversity between inoculated and uninoculated samples suggest that Saccharomyces inoculation exerts selective pressure on bacterial diversity in these fermentations, most notably suppressing abundance of acetic acid bacteria. These results determine the bacterial diversity of botrytized wines to be far higher than previously realized, providing further insight into the fermentation dynamics of these wines, and demonstrate the utility of next-generation sequencing for wine ecology studies.     

Antibacterial activity of lactic acid bacteria included in inoculants for silage and in silages treated with these inoculants

AIMS: To determine antibacterial activity in lactic acid bacteria (LAB) silage inoculants and in wheat and corn silages which were treated with these inoculants. METHODS AND RESULTS: Wheat and two corn silages were prepared in 0.25 l sealed glass jars. Inoculant treatments were prepared for each type of silage with each of 10 LAB silage inoculants at inoculation rate of 10(6) CFU g(-1). Untreated silages served as controls. Antibacterial activity was determined in the inoculants and in their respective silages with Micrococcus luteus and Pseudomonas aeruginosa. Antibacterial activity was detected in nine of the 10 inoculants whereas such activity in the silages varied. Control silages did not have antibacterial activity. CONCLUSIONS: Many LAB silage inoculants have antibacterial activity and in some cases this activity is imparted on inoculated silages. SIGNIFICANCE AND IMPACT OF THE STUDY: This study was conducted as part of a broader research objective, which is to find out how LAB silage inoculants enhance ruminant performance. The results of this study indicate that LAB silage inoculants produce antibacterial activity, and therefore, have a potential to inhibit detrimental micro-organisms in the silage or in the rumen.     

The biodiversity of lactic acid bacteria in Greek traditional wheat sourdoughs is reflected in both composition and metabolite formation

Lactic acid bacteria (LAB) were isolated from Greek traditional wheat sourdoughs manufactured without the addition of baker's yeast. Application of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total cell protein, randomly amplified polymorphic DNA-PCR, DNA-DNA hybridization, and 16S ribosomal DNA sequence analysis, in combination with physiological traits such as fructose fermentation and mannitol production, allowed us to classify the isolated bacteria into the species Lactobacillus sanfranciscensis, Lactobacillus brevis, Lactobacillus paralimentarius, and Weissella cibaria. This consortium seems to be unique for the Greek traditional wheat sourdoughs studied. Strains of the species W. cibaria have not been isolated from sourdoughs previously. No Lactobacillus pontis or Lactobacillus panis strains were found. An L. brevis-like isolate (ACA-DC 3411 t1) could not be identified properly and might be a new sourdough LAB species. In addition, fermentation capabilities associated with the LAB detected have been studied. During laboratory fermentations, all heterofermentative sourdough LAB strains produced lactic acid, acetic acid, and ethanol. Mannitol was produced from fructose that served as an additional electron acceptor. In addition to glucose, almost all of the LAB isolates fermented maltose, while fructose as the sole carbohydrate source was fermented by all sourdough LAB tested except L. sanfranciscensis. Two of the L. paralimentarius isolates tested did not ferment maltose; all strains were homofermentative. In the presence of both maltose and fructose in the medium, induction of hexokinase activity occurred in all sourdough LAB species mentioned above, explaining why no glucose accumulation was found extracellularly. No maltose phosphorylase activity was found either. These data produced a variable fermentation coefficient and a unique sourdough metabolite composition.