Biodegradability of Food-Associated Extracellular Polysaccharides

Exopolysaccharides (EPSs) produced by lactic acid bacteria, which are common in fermented foods, are claimed to have various beneficial physiological effects on humans. Although the biodegradability of EPSs is important in relation to the bioactive properties, knowledge on this topic is limited. Therefore, the biodegradability of eight EPSs, six of which were produced by lactic acid bacteria, was compared with microorganisms from human feces or soil. EPS-degradation was determined from the decrease in polysaccharide-sugar concentration and by high-performance size exclusion chromatography (HPSEC). Xanthan, clavan, and the EPSs produced by Streptococcus thermophilus SFi 39 and SFi 12 were readily degraded, in contrast to the EPSs produced by Lactococcus lactis ssp. cremoris B40, Lactobacillus sakei 0-1, S. thermophilus SFi20, and Lactobacillus helveticus Lh59. Clearly, the susceptibility of exopolysaccharides to biological breakdown can differ greatly, implying that the physiological effects of these compounds may also vary a lot. Received: 23 August 1999 / Accepted: 5 October 1999     

Heme and menaquinone induced electron transport in lactic acid bacteria

Background  

For some lactic acid bacteria higher biomass production as a result of aerobic respiration has been reported upon supplementation with heme and menaquinone. In this report, we have studied a large number of species among lactic acid bacteria for the existence of this trait.     

Microbial interactions associated with secondary cucumber fermentation

Aims

To evaluate the interaction between selected yeasts and bacteria and associate their metabolic activity with secondary cucumber fermentation.

Methods and Results

Selected yeast and bacteria, isolated from cucumber secondary fermentations, were inoculated as single and mixed cultures in a cucumber juice model system. Our results confirmed that during storage of fermented cucumbers and in the presence of oxygen, spoilage yeasts are able to grow and utilize the lactic and acetic acids present in the medium, which results in increased brine pH and the chemical reduction in the environment. These conditions favour opportunistic bacteria that continue the degradation of lactic acid. Lactobacillus buchneri, Clostridium bifermentans and Enterobacter cloacae were able to produce acetic, butyric and propionic acids, respectively, when inoculated in the experimental medium at pH 4·6. Yeast and bacteria interactions favoured the survival of Cl. bifermentans and E. cloacae at the acidic pH typical of fermented cucumbers (3·2), but only E. cloacae was able to produce a secondary product.

Conclusions

The methodology used in this study confirmed that a complex microbiota is responsible for the changes observed during fermented cucumber secondary fermentation and that certain microbial interactions may be essential for the production of propionic and butyric acids.

Significance and Impact of the Study

Understanding the dynamics of the development of secondary cucumber fermentation aids in the identification of strategies to prevent its occurrence and economic losses for the pickling industry.     

Efficient conversion of lactic acid to butanol with pH-stat continuous lactic acid and glucose feeding method by Clostridium saccharoperbutylacetonicum

In order to achieve high butanol production by Clostridium saccharoperbutylacetonicum N1-4, the effect of lactic acid on acetone–butanol–ethanol fermentation and several fed-batch cultures in which lactic acid is fed have been investigated. When a medium containing 20 g/l glucose was supplemented with 5 g/l of closely racemic lactic acid, both the concentration and yield of butanol increased; however, supplementation with more than 10 g/l lactic acid did not increase the butanol concentration. It was found that when fed a mixture of lactic acid and glucose, the final concentration of butanol produced by a fed-batch culture was greater than that produced by a batch culture. In addition, a pH-controlled fed-batch culture resulted in not only acceleration of lactic acid consumption but also a further increase in butanol production. Finally, we obtained 15.5 g/l butanol at a production rate of 1.76 g/l/h using a fed-batch culture with a pH-stat continuous lactic acid and glucose feeding method. To confirm whether lactic acid was converted to butanol by the N1-4 strain, we performed gas chromatography–mass spectroscopy (GC-MS) analysis of butanol produced by a batch culture during fermentation in a medium containing [1,2,3-¹³C₃] lactic acid as the initial substrate. The results of the GC-MS analysis confirmed the bioconversion of lactic acid to butanol.     

Phylogenetic group- and species-specific oligonucleotide probes for single-cell detection of lactic acid bacteria in oral biofilms

Background  

The purpose of this study was to design and evaluate fluorescent in situ hybridization (FISH) probes for the single-cell detection and enumeration of lactic acid bacteria, in particular organisms belonging to the major phylogenetic groups and species of oral lactobacilli and to Abiotrophia/Granulicatella.     

Diet shapes the ability of human intestinal microbiota to degrade phytate – in vitro studies

Aims

Investigation of intestinal bacterial groups involved in phytate degradation and the impact of diets with different phytate contents on phytase activity.

Methods and Results

Faecal samples of adults on conventional (n = 8) or vegetarian (n = 8) diets and breastfed infants (n = 6) were used as an inoculum for modified media supplemented with phytate. Populations of Gram‐positive anaerobes (GPA), lactic acid bacteria (LAB), Proteobacteria–Bacteroides (P‐B), coliforms and anaerobes were studied. The PCR‐DGGE analysis revealed a random distribution of DGGE profiles in the dendrograms of GPA, P‐B and coliforms, and a partially diet‐specific distribution in the DGGE dendrograms of LAB and anaerobes. The degradation of phytic acid (PA) was determined with HPLC method in supernatants of the cultures. Regardless of the diet, the Gram‐positive anaerobes and LAB displayed the lowest ability to degrade phytate, whereas the coliforms and P‐B cultures produced higher amounts of intermediate myo‐inositol phosphates. Bacterial populations grown in a nonselective medium were the most effective ones in phytate degradation. It was the vegetarians' microbiota that particularly degraded up to 100% phytate to myo‐inositol phosphate products lower than InsP₃.

Conclusions

A diet rich in phytate increases the potential of intestinal microbiota to degrade phytate. The co‐operation of aerobic and anaerobic bacteria is essential for the complete phytate degradation.

Significance and Impact of the Study

This study provides insights on the effect of diet on specific metabolic activity of human intestinal microbiota.     

Modeling <Emphasis Type="Italic">Lactococcus lactis</Emphasis> using a genome-scale flux model

Background  

Genome-scale flux models are useful tools to represent and analyze microbial metabolism. In this work we reconstructed the metabolic network of the lactic acid bacteria Lactococcus lactis and developed a genome-scale flux model able to simulate and analyze network capabilities and whole-cell function under aerobic and anaerobic continuous cultures. Flux balance analysis (FBA) and minimization of metabolic adjustment (MOMA) were used as modeling frameworks.     

Optimization of Exopolysaccharide Overproduction by Lactobacillus confusus in Solid State Fermentation under High Salinity Stress

It is believed that high concentrations of sodium chloride (NaCl) suppress the biosynthesis of exopolysaccharide (EPS) in lactic acid bacteria (LAB). Nevertheless, overproduction of EPSs due to high salinity stress in solid state fermentation performed on an agar surface was demonstrated in this study using a response surface methodology via a central composite design (CCD). Under optimized conditions with NaCl 4.97% and sucrose 136.5 g/L at 40.79 h of incubation, the EPS yield was 259% (86.36 g/L of EPS), higher than the maximum yield produced with the modified MRS medium containing only 120 g/L of sucrose without NaCl (33.4 g/L of EPS). Biosynthesis of EPS by Lactobacillus confusus TISTR 1498 was independent of biomass production. Our results indicated that high salinity stress can enhance EPS production in solid state fermentation.     

Production of lactic acid from paper sludge using acid-tolerant, thermophilic Bacillus coagulan strains

Production of lactic acid from paper sludge was studied using thermophilic Bacillus coagulan strains 36D1 and P4-102B. More than 80% of lactic acid yield and more than 87% of cellulose conversion were achieved using both strains without any pH control due to the buffering effect of CaCO₃ in paper sludge. The addition of CaCO₃ as the buffering reagent in rich medium increased lactic acid yield but had little effect on cellulose conversion; when lean medium was utilized, the addition of CaCO₃ had little effect on either cellulose conversion or lactic acid yield. Lowering the fermentation temperature lowered lactic acid yield but increased cellulose conversion. Semi-continuous simultaneous saccharification and co-fermentation (SSCF) using medium containing 100 g/L cellulose equivalent paper sludge without pH control was carried out in serum bottles for up to 1000 h. When rich medium was utilized, the average lactic acid concentrations in steady state for strains 36D1 and P4-102B were 92 g/L and 91.7 g/L, respectively, and lactic acid yields were 77% and 78%. The average lactic acid concentrations produced using semi-continuous SSCF with lean medium were 77.5 g/L and 77.0 g/L for strains 36D1 and P4-102B, respectively, and lactic acid yields were 72% and 75%. The productivities at steady state were 0.96 g/L/h and 0.82 g/L/h for both strains in rich medium and lean medium, respectively. Our data support that B. coagulan strains 36D1 and P4-102B are promising for converting paper sludge to lactic acid via SSCF.     

Production of gamma-aminobutyric acid by <Emphasis Type="Italic">Lactobacillus brevis</Emphasis> NCL912 using fed-batch fermentation

Background  

Gamma-aminobutyric acid is a major inhibitory neurotransmitter in mammalian brains, and has several well-known physiological functions. Lactic acid bacteria possess special physiological activities and are generally regarded as safe. Therefore, using lactic acid bacteria as cell factories for gamma-aminobutyric acid production is a fascinating project and opens up a vast range of prospects for making use of GABA and LAB. We previously screened a high GABA-producer Lactobacillus brevis NCL912 and optimized its fermentation medium composition. The results indicated that the strain showed potential in large-scale fermentation for the production of gamma-aminobutyric acid. To increase the yielding of GABA, further study on the fermentation process is needed before the industrial application in the future. In this article we investigated the impacts of pyridoxal-5'-phosphate, pH, temperature and initial glutamate concentration on gamma-aminobutyric acid production by Lactobacillus brevis NCL912 in flask cultures. According to the data obtained in the above, a simple and effective fed-batch fermentation method was developed to highly efficiently convert glutamate to gamma-aminobutyric acid.     

Evolutionary history of the OmpR/IIIA family of signal transduction two component systems in Lactobacillaceae and Leuconostocaceae

Background  

Two component systems (TCS) are signal transduction pathways which typically consist of a sensor histidine kinase (HK) and a response regulator (RR). In this study, we have analyzed the evolution of TCS of the OmpR/IIIA family in Lactobacillaceae and Leuconostocaceae, two families belonging to the group of lactic acid bacteria (LAB). LAB colonize nutrient-rich environments such as foodstuffs, plant materials and the gastrointestinal tract of animals thus driving the study of this group of both basic and applied interest.     

Bacillus amyloliquefaciens ssp. plantarum strains as potential protective starter cultures for the production of Bikalga,an alkaline fermented food

Aims

To identify and screen dominant Bacillus spp. strains isolated from Bikalga, fermented seeds of Hibiscus sabdariffa for their antimicrobial activities in brain heart infusion (BHI) medium and in a H. sabdariffa seed‐based medium. Further, to characterize the antimicrobial substances produced.

Methods and Results

The strains were identified by gyrB gene sequencing and phenotypic tests as B. amyloliquefaciens ssp. plantarum. Their antimicrobial activity was determined by the agar spot and well assay, being inhibitory to a wide range of Gram‐positive and Gram‐negative pathogenic bacteria and fungi. Antimicrobial activity against Bacillus cereus was produced in H. sabdariffa seed‐based medium. PCR results revealed that the isolates have potential for the lipopeptides iturin, fengycin, surfactin, the polyketides difficidin, macrolactin, bacillaene and the dipeptide bacilysin production. Ultra‐high‐performance liquid chromatography‐time of flight mass spectrometry analysis of antimicrobial substance produced in BHI broth allowed identification of iturin, fengycin and surfactin.

Conclusions

The Bacillus amyloliquefaciens ssp. plantarum exhibited broad‐spectrum antifungal and antibacterial properties. They produced several lipopeptide antibiotics and showed good potential for biological control of Bikalga.

Significance and Impact of the Study

Pathogenic bacteria often occur in spontaneous food fermentations. This is the first report to identify indigenous B. amyloliquefaciens ssp. plantarum strains as potential protective starter cultures for safeguarding Bikalga.     

Detection of the pediocin gene <Emphasis Type="Italic">pedA</Emphasis> in strains from human faeces by real-time PCR and characterization of <Emphasis Type="Italic">Pediococcus acidilactici</Emphasis>UVA1

Background  

Bacteriocin-producing lactic acid bacteria are commonly used as natural protective cultures. Among them, strains of the genus Pediococcus are particularly interesting for their ability to produce pediocin, a broad spectrum antimicrobial peptide with a strong antagonistic activity against the food-borne pathogen Listeria monocytogenes. Furthermore, there is increasing interest in isolating new bacteriocin-producing strains of human intestinal origin that could be developed for probiotic effects and inhibition of pathogenic bacteria in the gut. In this work, we typed a new strain, co-isolated from baby faeces together with a Bifidobacterium thermophilum strain, and characterized its proteinaceous compound with strong antilisterial activity.     

Primary metabolism in <Emphasis Type="Italic">Lactobacillus sakei</Emphasis> food isolates by proteomic analysis

Background  

Lactobacillus sakei is an important food-associated lactic acid bacterium commonly used as starter culture for industrial meat fermentation, and with great potential as a biopreservative in meat and fish products. Understanding the metabolic mechanisms underlying the growth performance of a strain to be used for food fermentations is important for obtaining high-quality and safe products. Proteomic analysis was used to study the primary metabolism in ten food isolates after growth on glucose and ribose, the main sugars available for L. sakei in meat and fish.     

Heterologous protein display on the cell surface of lactic acid bacteria mediated by the s-layer protein

Background

Previous studies have revealed that the C-terminal region of the S-layer protein from Lactobacillus is responsible for the cell wall anchoring, which provide an approach for targeting heterologous proteins to the cell wall of lactic acid bacteria (LAB). In this study, we developed a new surface display system in lactic acid bacteria with the C-terminal region of S-layer protein SlpB of Lactobacillus crispatus K2-4-3 isolated from chicken intestine.

Results

Multiple sequence alignment revealed that the C-terminal region (LcsB) of Lb. crispatus K2-4-3 SlpB had a high similarity with the cell wall binding domains S_A and CbsA of Lactobacillus acidophilus and Lb. crispatus. To evaluate the potential application as an anchoring protein, the green fluorescent protein (GFP) or beta-galactosidase (Gal) was fused to the N-terminus of the LcsB region, and the fused proteins were successfully produced in Escherichia coli, respectively. After mixing them with the non-genetically modified lactic acid bacteria cells, the fused GFP-LcsB and Gal-LcsB were functionally associated with the cell surface of various lactic acid bacteria tested. In addition, the binding capacity could be improved by SDS pretreatment. Moreover, both of the fused proteins could simultaneously bind to the surface of a single cell. Furthermore, when the fused DNA fragment of gfp:lcsB was inserted into the Lactococcus lactis expression vector pSec:Leiss:Nuc, the GFP could not be secreted into the medium under the control of the nisA promoter. Western blot, in-gel fluorescence assay, immunofluorescence microscopy and SDS sensitivity analysis confirmed that the GFP was successfully expressed onto the cell surface of L. lactis with the aid of the LcsB anchor.

Conclusion

The LcsB region can be used as a functional scaffold to target the heterologous proteins to the cell surfaces of lactic acid bacteria in vitro and in vivo, and has also the potential for biotechnological application.

     

The impact of ColRS two-component system and TtgABC efflux pump on phenol tolerance of <Emphasis Type="Italic">Pseudomonas putida</Emphasis> becomes evident only in growing bacteria

Background  

We have recently found that Pseudomonas putida deficient in ColRS two-component system is sensitive to phenol and displays a serious defect on solid glucose medium where subpopulation of bacteria lyses. The latter phenotype is significantly enhanced by the presence of phenol in growth medium. Here, we focused on identification of factors affecting phenol tolerance of the colR-deficient P. putida.     

Optimisation of batch culture conditions for cyclodextrin glucanotransferase production from Bacillus circulans DF 9R

Background  

The extracellular enzyme cyclodextrin glucanotransferase (CGTase) synthesizes cyclic malto-oligosaccharides called cyclodextrins (CDs) from starch and related α-1,4-glucans. CGTases are produced by a variety of bacteria, mainly Bacillus species, by submerged culture in complex medium. CGTases differ in the amount and types of CDs produced. In addition, CGTase production is highly dependent on the strain, medium composition and culture conditions. Therefore we undertook this study with a newly isolated strain of Bacillus circulans.     

Growth,acid production and bacteriocin production by probiotic candidates under simulated colonic conditions

Aims

The aim of this study is to evaluate the capacity of three bacteriocin producers, namely Lactococcus lactis subsp. lactis biovar diacetylactis UL719 (nisin Z producer), L. lactis ATCC 11454 (nisin A producer) and Pediococcus acidilactici UL5 (pediocin PA‐1 producer), and to grow and produce their active bacteriocins in Macfarlane broth, which mimics the nutrient composition encountered in the human large intestine.

Methods and Results

The three bacteriocin‐producing strains were grown in Macfarlane broth and in De Man–Rogosa–Sharpe (MRS) broth. For each strain, the bacterial count, pH drop and production of organic acids and bacteriocins were measured for different period of time. The ability of the probiotic candidates to inhibit Listeria ivanovii HPB 28 in co‐culture in Macfarlane broth was also examined. Lactococcus lactis subsp. lactis biovar diacetylactis UL719, L. lactis ATCC 11454 and Ped. acidilactici UL5 were able to grow and produce their bacteriocins in MRS broth and in Macfarlane broth. Each of the three candidates inhibited L. ivanovii HPB 28, and this inhibition activity was correlated with bacteriocin production. The role of bacteriocin production in the inhibition of L. ivanovii in Macfarlane broth was confirmed for Ped. acidilactici UL5 using a pediocin nonproducer mutant.

Conclusions

The data provide some evidence that these bacteria can produce bacteriocins in a complex medium with carbon source similar to those found in the colon.

Significance and Impact of the Study

This study demonstrates the capacity of lactic acid bacteria to produce their bacteriocins in a medium simulating the nutrient composition of the large intestine.     

Colanic acid is an exopolysaccharide common to many enterobacteria isolated from paper-machine slimes

In this study, polysaccharide-producing bacteria were isolated from slimes collected from two Finnish and one Spanish paper mill and the exopolysaccharides (EPSs) produced by 18 isolates were characterised. Most of the isolates, selected on the bases of slimy colony morphology, were members of the family Enterobacteriaceae most frequently belonging to the genera Enterobacter and Klebsiella including Raoultella. All of the EPSs analysed showed the presence of charged groups in the form of uronic acid or pyruvate revealing the polyanionic nature of these polysaccharides. Further results of the carbohydrate analysis showed that the EPS produced by nine of the enterobacteria was colanic acid.     

Characterization of proton production and consumption associated with microbial metabolism

Background  

Production or consumption of protons in growth medium during microbial metabolism plays an important role in determining the pH of the environment. Such pH changes resulting from microbial metabolism may influence the geochemical speciation of many elements in subsurface environments. Protons produced or consumed during microbial growth were measured by determining the amount of acid or base added in a 5 L batch bioreactor equipped with pH control for different species including Escherichia coli, Geobacter sulfurreducens, and Geobacter metallireducens.