Unique niche-specific adaptation of fructophilic lactic acid bacteria and proposal of three Apilactobacillus species as novel members of the group.

BACKGROUND: Fructophilic lactic acid bacteria (FLAB) found in D-fructose rich niches prefer D-fructose over D-glucose as a growth substrate. They need electron acceptors for growth on D-glucose. The organisms share carbohydrate metabolic properties. Fructobacillus spp., Apilactobacillus kunkeei, and Apilactobacillus apinorum are members of this unique group. Here we studied the fructophilic characteristics of recently described species Apilactobacillus micheneri, Apilactobacillus quenuiae, and Apilactobacillus timberlakei. RESULTS: The three species prefer D-fructose over D-glucose and only metabolize D-glucose in the presence of electron acceptors. The genomic characteristics of the three species, i.e. small genomes and thus a low number of coding DNA sequences, few genes involved in carbohydrate transport and metabolism, and partial deletion of adhE gene, are characteristic of FLAB. The three species thus are novel members of FLAB. Reduction of genes involved in carbohydrate transport and metabolism in accordance with reduction of genome size were the common characteristics of the family Lactobacillaceae, but FLAB markedly reduced the gene numbers more than other species in the family. Pan-genome analysis of genes involved in metabolism displayed a lack of specific carbohydrate metabolic pathways in FLAB, leading to a unique cluster separation. CONCLUSIONS: The present study expanded FLAB group. Fructose-rich environments have induced similar evolution in phylogenetically distant FLAB species. These are examples of convergent evolution of LAB.

How fructophilic lactic acid bacteria may reduce the FODMAPs content in wheat-derived baked goods: a proof of concept.

BACKGROUND: FODMAPs (Fermentable oligosaccharides, disaccharides, monosaccharides, and polyols) intake is associated with the onset of irritable bowel syndrome symptoms. FODMAPs in wheat-derived baked goods may be reduced via bioprocessing by endogenous enzymes and/or microbial fermentation. Because of the inherent enzyme activities, bread made by baker's yeast and sourdough may result in decreased levels of FODMAPs, whose values are, however, not enough low for people sensitive to FODMAPs. RESULTS: Our study investigated the complementary capability of targeted commercial enzymes and metabolically strictly fructophilic lactic acid bacteria (FLAB) to hydrolyze fructans and deplete fructose during wheat dough fermentation. FLAB strains displayed higher fructose consumption rate compared to conventional sourdough lactic acid bacteria. Fructose metabolism by FLAB was faster than glucose. The catabolism of mannitol with the goal of its reuse by FLAB was also investigated. Under sourdough conditions, higher fructans breakdown occurred in FLAB inoculated doughs compared to conventional sourdough bacteria. Preliminary trials allowed selecting Apilactobacillus kunkeei B23I and Fructobacillus fructosus MBIII5 as starter candidates, which were successfully applied in synergy with commercial invertase for low FODMAPs baking. CONCLUSIONS: Results of this study clearly demonstrated the potential of selected strictly FLAB to strongly reduce FODMAPs in wheat dough, especially under liquid-dough and high oxygenation conditions.

Functional characterization and in vitro screening of Fructobacillus fructosus MCC 3996 isolated from Butea monosperma flower for probiotic potential.

The fructophilic bacterium Fructobacillus fructosus MCC 3996 described in the present investigation was isolated from the nectar of Butea monosperma flower and evaluated in vitro for the manifestation of probiotic features. The strain utilizes fructose faster than glucose and is capable to grow in the range of 1-35% fructose concentration (optimum 5% w/v) and thus denotes its fructophilic nature. In vitro assessments of the strain have examined for the endurance in acidic environment/gastric juice, the better auto-aggregation ability even in the presence of hydrolytic enzymes, co-aggregation with pathogenic bacteria, hydrophobicity properties and no haemolytic activity to elucidate its feasible probiotic use. The significant antagonistic activity against several detrimental bacteria, despite lacking the bacteriocin secretion, is an astonishing feature. Owing to the indigenous origin of the isolate, it could be used as a probiotic, starter culture, and/or the active ingredient of food formulation may contribute to improve the desirable fermentation, long-term storage and nutritional benefits of foods especially rich in fructose. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provided in vitro evidence that Fructobacillus fructosus MCC 3996 have endurance in acidic gastric juice, better co-aggregation, auto-aggregation properties, splendid antagonistic activities against several bacteria involved in food spoilage/human infections, pertinent antibiotic susceptibility profile and no haemolytic activity. Also, F. fructosus have the capability to survive in the appreciable amount of fructose, and this advocates that the strain could be used as starter culture and/or the active ingredient of fructose-rich foods. The current in vitro study provided a strong basis for further in vivo research to identify the health beneficial characteristics of F. fructosus and its potential could be effectively utilized as health-boosting ingredient in food and pharmaceutical industries.

Contribution of Leuconostocaceae to CO2-mediated bloater defect in cucumber fermentation.

Fermented cucumber bloater defect, caused by the accumulation of microbiologically produced carbon dioxide (CO2), creates significant economic losses for the pickling industry. The ability of Leuconostocaceae, indigenous to cucumber, to grow and produce CO2 during a fermentation and cause bloater defect was evaluated. Leuconostocaceae grew and produced over 40% CO2 in cucumber juice medium, used as a model for cucumber fermentation. The inoculation of Leuconostocaceae to 5 Log CFU/g in cucumber fermentations brined with 25 mM calcium chloride and 6 mM potassium sorbate resulted in no significant differences in bloater defect, colony counts from MRS and VRBG agar plates or the fermentation biochemistry; suggesting an inability of the inoculated bacterial species to prevail in the bioconversion. Acidified cucumbers were subjected to a fermentation inoculated with a Leuconostoc lactis starter culture after raising the pH to 5.9 ± 0.4. CO2 was produced in the acidified cucumber fermentations to 13.6 ± 3.5% yielding a bloater index of 21.3 ± 6.4; while 8.6 ± 0.8% CO2 and a bloater index of 5.2 ± 5.9 were observed in the non-inoculated control jars. Together the data collected demonstrate that Leuconostocaceae can produce enough CO2 to contribute to bloater defect, if not outcompeted by the leading lactic acid bacteria in a cucumber fermentation.

Dynamics and species diversity of lactic acid bacteria involved in the spontaneous fermentation of various palm tree saps during palm wine tapping in Côte d'Ivoire.

To document and speed up research on the usefulness and selection of potential health-promoting bacterial starter cultures from unexplored fermented saps of various palm species in Côte d'Ivoire, benchmark tapping processes were successfully developed and implemented at field level. Therefore, spontaneously fermented saps of three palm species (Elaeis guineensis, Raphia hookeri, Borassus aethiopum) were collected throughout tapping process and lactic acid bacteria (LAB) diversity and dynamics were studied through a multiphasic approach. Overall microbiological analysis revealed a LAB species diversity throughout tapping process. LAB isolates belonged to two main (GTG)5-PCR clusters, namely Fructobacillus durionis (40.33%) and Leuconostoc mesenteroides (45.66%), with Leuconostoc pseudomesenteroides, Lactobacillus paracasei, Lactobacillus fermentum Weissella cibaria, Enterococcus casseliflavus and Lactococcus lactis occurring occasionally. LAB diversity was higher in fermented saps from E. guineensis (8 species) than those of R. hookeri (5 species) and B. aethiopum (3 species). Dynamic study revealed that F. durionis and L. mesenteroides dominated the fermentations from the beginning until the end of tapping process in all palm wine types. But the earlier stages of the process were also populated by some species like W. cibaria, L. pseudomesenteroides and L. fermentum, which population decreased or disappeared after some days. Also, species of Enterococcus and Lactococcus genera were sporadically detected uniquely in sap from E. guineensis. This study is the first to investigate extensively the LAB diversity and dynamics throughout palm trees tapping process in Côte d'Ivoire and is relevant for future selection of health promoting bacteria.

Evaluation of efficient carbon, nitrogen sources, micro and macro nutrients for dextran production by Weissella cibaria CMGDEX3 by utilizing a modified multifactorial Placket-Burman statistical design.

In the present study previously isolated Weissella cibaria CMG DEX3 capable of producing high molecular weight, water soluble dextran (Ahmed et al., 2012) is characterized for most efficient less expensive carbon, nitrogen sources, micro and macro nutrients by utilizing a multifactorial Placket-Burman statistical design for optimization of dextran production. A twelve run Plackett-Burman experimental model with slight modification was utilized to evaluate the impact of ten diverse nutrients on the production of dextran by the bacterial isolate Weissella cibaria CMG DEX3.

Fructophilic Lactic Acid Bacteria, a Unique Group of Fructose-Fermenting Microbes.

Fructophilic lactic acid bacteria (FLAB) are a recently discovered group, consisting of a few Fructobacillus and Lactobacillus species. Because of their unique characteristics, including poor growth on glucose and preference of oxygen, they are regarded as "unconventional" lactic acid bacteria (LAB). Their unusual growth characteristics are due to an incomplete gene encoding a bifunctional alcohol/acetaldehyde dehydrogenase (adhE). This results in the imbalance of NAD/NADH and the requirement of additional electron acceptors to metabolize glucose. Oxygen, fructose, and pyruvate are used as electron acceptors. FLAB have significantly fewer genes for carbohydrate metabolism than other LAB, especially due to the lack of complete phosphotransferase system (PTS) transporters. They have been isolated from fructose-rich environments, including flowers, fruits, fermented fruits, and the guts of insects that feed on plants rich in fructose, and are separated into two groups on the basis of their habitats. One group is associated with flowers, grapes, wines, and insects, and the second group is associated with ripe fruits and fruit fermentations. Species associated with insects may play a role in the health of their host and are regarded as suitable vectors for paratransgenesis in honey bees. Besides their impact on insect health, FLAB may be promising candidates for the promotion of human health. Further studies are required to explore their beneficial properties in animals and humans and their applications in the food industry.

Enhanced mannitol biosynthesis by the fruit origin strain Fructobacillus tropaeoli CRL 2034.

Mannitol is a natural low-calorie sugar alcohol produced by certain (micro)organisms applicable in foods for diabetics due to its zero glycemic index. In this work, we evaluated mannitol production and yield by the fruit origin strain Fructobacillus tropaeoli CRL 2034 using response surface methodology with central composite design (CCD) as optimization strategy. The effect of the total saccharide (glucose + fructose, 1:2) content (TSC) in the medium (75, 100, 150, 200, and 225 g/l) and stirring (S; 50, 100, 200, 300 and 350 rpm) on mannitol production and yield by this strain was evaluated by using a 22 full-factorial CCD with 4 axial points (α = 1.5) and four replications of the center point, leading to 12 random experimental runs. Fermentations were carried out at 30 °C and pH 5.0 for 24 h. Minitab-15 software was used for experimental design and data analyses. The multiple response prediction analysis established 165 g/l of TSC and 200 rpm of S as optimal culture conditions to reach 85.03 g/l [95% CI (78.68, 91.39)] of mannitol and a yield of 82.02% [95% CI (71.98, 92.06)]. Finally, a validation experiment was conducted at the predicted optimum levels. The results obtained were 81.91 g/l of mannitol with a yield of 77.47% in outstanding agreement with the expected values. The mannitol 2-dehydrogenase enzyme activity was determined with 4.6-4.9 U/mg as the highest value found. To conclude, F. tropaeoli CRL 2034 produced high amounts of high-quality mannitol from fructose, being an excellent candidate for this polyol production.

Fructophilic characteristics of Fructobacillus spp. may be due to the absence of an alcohol/acetaldehyde dehydrogenase gene (adhE).

Fructophilic strains of Leuconostoc spp. have recently been reclassified to a new genus, i.e., Fructobacillus. Members of the genus are differentiated from Leuconostoc spp. by their preference for fructose on growth, requirement of an electron acceptor for glucose metabolism, and the inability to produce ethanol from the fermentation of glucose. In the present study, enzyme activities and genes involved in ethanol production were studied, since this is the key pathway for NAD(+)/NADH cycling in heterofermentative lactic acid bacteria. Fructobacillus spp. has a weak alcohol dehydrogenase activity and has no acetaldehyde dehydrogenase activity, whereas both enzymes are active in Leuconostoc mesenteroides. The bifunctional alcohol/acetaldehyde dehydrogenase gene, adhE, was described in Leuconostoc spp., but not in Fructobacillus spp. These results suggested that, due to the deficiency of the adhE gene, the normal pathway for ethanol production is absent in Fructobacillus spp. This leads to a shortage of NAD(+), and the requirement for an electron acceptor in glucose metabolism. Fructophilic characteristics, as observed for Fructobacillus spp., are thus due to the absence of the adhE gene, and a phenotype that most likely evolved as a result of regressive evolution.

Genomic diversity in Fructobacillus spp. isolated from fructose-rich niches.

The Fructobacillus genus is a group of obligately fructophilic lactic acid bacteria (FLAB) that requires the use of fructose or another electron acceptor for their growth. In this work, we performed a comparative genomic analysis within the genus Fructobacillus by using 24 available genomes to evaluate genomic and metabolic differences among these organisms. In the genome of these strains, which varies between 1.15- and 1.75-Mbp, nineteen intact prophage regions, and seven complete CRISPR-Cas type II systems were found. Phylogenetic analyses located the studied genomes in two different clades. A pangenome analysis and a functional classification of their genes revealed that genomes of the first clade presented fewer genes involved in the synthesis of amino acids and other nitrogen compounds. Moreover, the presence of genes strictly related to the use of fructose and electron acceptors was variable within the genus, although these variations were not always related to the phylogeny.

Antimicrobial Properties, Functional Characterisation and Application of Fructobacillus fructosus and Lactiplantibacillus plantarum Isolated from Artisanal Honey.

Honey is a valuable reservoir of lactic acid bacteria (LAB) and, particularly, of fructophilic LAB (FLAB), a relatively novel subgroup of LAB whose functional potential for human and food application has yet to be explored. In this study, FLAB and LAB strains have been isolated from honeys of different floral origins and selected for their broad antimicrobial activity against typical foodborne pathogenic bacteria and spoilage filamentous fungi. The best candidates, two strains belonging to the species Lactiplantibacillus plantarum and Fructobacillus fructosus, were submitted to partial characterisation of their cell free supernatants (CFS) in order to identify the secreted metabolites with antimicrobial activity. Besides, these strains were examined to assess some major functional features, including in vitro tolerance to the oro-gastrointestinal conditions, potential cytotoxicity against HT-29 cells, adhesion to human enterocyte-like cells and capability to stimulate macrophages. Moreover, when the tested strains were applied on table grapes artificially contaminated with pathogenic bacteria or filamentous fungi, they showed a good ability to antagonise the growth of undesired microbes, as well as to survive on the fruit surface at a concentration that is recommended to develop a probiotic effect. In conclusion, both LAB and FLAB honey-isolated strains characterised in this work exhibit functional properties that validate their potential use as biocontrol agents and for the design of novel functional foods. We reported antimicrobial activity, cytotoxic evaluation, probiotic properties and direct food application of a F. fructosus strain, improving the knowledge of this species, in particular, and on FLAB, more generally.

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. RESULTS: The genomes of 19 strains belonging to 16 different species have been analyzed. The number of TCS encoded by the strains considered in this study varied between 4 in Lactobacillus helveticus and 17 in Lactobacillus casei. The OmpR/IIIA family was the most prevalent in Lactobacillaceae accounting for 71% of the TCS present in this group. The phylogenetic analysis shows that no new TCS of this family has recently evolved in these Lactobacillaceae by either lineage-specific gene expansion or domain shuffling. Furthermore, no clear evidence of non-orthologous replacements of either RR or HK partners has been obtained, thus indicating that coevolution of cognate RR and HKs has been prevalent in Lactobacillaceae. CONCLUSIONS: The results obtained suggest that vertical inheritance of TCS present in the last common ancestor and lineage-specific gene losses appear as the main evolutionary forces involved in their evolution in Lactobacillaceae, although some HGT events cannot be ruled out. This would agree with the genomic analyses of Lactobacillales which show that gene losses have been a major trend in the evolution of this group.

Fructobacillus tropaeoli sp. nov., a fructophilic lactic acid bacterium isolated from a flower.

A fructophilic lactic acid bacterium, designated strain F214-1(T), was isolated from a flower of Tropaeolum majus in South Africa. Based on phylogenetic analysis of 16S rRNA gene sequences, the strain formed a subcluster with Fructobacillus ficulneus and Fructobacillus pseudoficulneus and, based on recA gene sequences, the strain formed a subcluster with F. ficulneus. DNA-DNA hybridization studies showed that strain F214-1(T) was phylogenetically distinct from its closest relatives. Acid was produced from the fermentation of d-glucose, d-fructose and d-mannitol only. d-Fructose was the preferred sole carbon and energy source and was fermented more rapidly than d-glucose. Growth of the strain on d-glucose under anaerobic conditions was very weak but external electron acceptors such as oxygen and pyruvate enhanced growth on d-glucose. Lactic acid and acetic acid were produced from d-glucose in equimolar amounts. Ethanol was produced at very low levels, despite the strain's obligately heterofermentative metabolism. Based on these data, strain F214-1(T) represents a novel species of fructophilic bacteria in the genus Fructobacillus, for which the name Fructobacillus tropaeoli sp. nov. is proposed. The type strain is F214-1(T) ( = JCM 16675(T)  = DSM 23246(T)).

Identification of potential causative agents of the CO2-mediated bloater defect in low salt cucumber fermentation.

Development of bloater defect in cucumber fermentations is the result of carbon dioxide (CO2) production by the indigenous microbiota. The amounts of CO2 needed to cause bloater defect in cucumber fermentations brined with low salt and potential microbial contributors of the gas were identified. The carbonation of acidified cucumbers showed that 28.68 ± 6.04 mM (12%) or higher dissolved CO2 induces bloater defect. The microbiome and biochemistry of cucumber fermentations (n = 9) brined with 25 mM calcium chloride (CaCl2) and 345 mM sodium chloride (NaCl) or 1.06 M NaCl were monitored on day 0, 2, 3, 5, 8, 15 and 21 using culture dependent and independent microbiological techniques and High-Performance Liquid Chromatography. Changes in pH, CO2 concentrations and the incidence of bloater defect were also followed. The enumeration of Enterobacteriaceae on Violet Red Bile Glucose agar plates detected a cell density of 5.2 ± 0.7 log CFU/g on day 2, which declined to undetectable levels by day 8. A metagenomic analysis identified Leuconostocaceae in all fermentations at 10 to 62%. The presence of both bacterial families in fermentations brined with CaCl2 and NaCl coincided with a bloater index of 24.0 ± 10.3 to 58.8 ± 23.9. The prevalence of Lactobacillaceae in a cucumber fermentation brined with NaCl with a bloater index of 41.7 on day 5 suggests a contribution to bloater defect. This study identifies the utilization of sugars and malic acid by the cucumber indigenous Lactobacillaceae, Leuconostocaceae and Enterobacteriaceae as potential contributors to CO2 production during cucumber fermentation and the consequent bloater defect.

Selenium bio-enrichment of Mediterranean fruit juices through lactic acid fermentation.

This work was carried out to elaborate selenium (Se) bio-enriched fermented Mediterranean fruit juices. To this purpose, pomegranate and table red grape juices were added with sodium selenite (Na2SeO3) and fermented by Levilactobacillus brevis CRL 2051 and Fructobacillus tropaeoli CRL 2034 individually or combined. To better evaluate the effect of selenite addition and starter strain inoculums on the total bacterial community of the fruit juices, fermentation trials were performed with raw and pasteurized fruit juices. No statistical significant differences were observed for total mesophilic microorganisms (TMM) and rod-shaped lactic acid bacteria (LAB) levels among raw and pasteurized juices inoculated with the starter strains, while significant differences between those juices with and without selenite were registered. LAB cocci, Pseudomonadaceae and yeasts were detected only for the raw juice preparations. The dominance of L. brevis CRL 2051 and F. tropaeoli CRL 2034 was confirmed by randomly amplified polymorphic DNA (RAPD)-PCR analysis. After fermentation, pH dropped for all inoculated trials and control raw juices. The soluble solid content (SSC) levels of the raw juices were higher than the corresponding pasteurized trials. The thermal treatment affected consistently yellowness of grape juice trials and redness of pomegranate juices. No microbial Se accumulation was registered for pomegranate juices, while F. tropaeoli CRL 2034 accumulated the highest amount of Se (65.5 µg/L) in the grape juice. For this reason, only trials carried out with raw grape juices were investigated by metagenomics analysis by Illumina MiSeq technology. Non-inoculated grape juices were massively fermented by acetic acid bacteria while Fructobacillus and Lactobacillus (previous genus name of Levilactobacillus) represented the highest operational taxonomy units (OTUs) relative abundance % of the trials inoculated with the starter strains as confirmed by this technique.

Exopolysaccharide synthesized by Lactobacillus reuteri decreases the ability of enterotoxigenic Escherichia coli to bind to porcine erythrocytes.

This study investigated the therapeutic potential of bacterial polysaccharides by employing a model system based on enteroxigenic Escherichia coli (ETEC)-induced hemagglutination of erythrocytes. Exopolysaccharides produced by strains of Lactobacillus reuteri inhibited ETEC-induced hemagglutination of porcine erythrocytes. No effect was observed for dextran produced from Weissella cibaria and commercially available oligo- and polysaccharides.

Fluorescence microscopy to monitor wine malolactic fermentation.

Malolactic fermentation (MLF) is a natural and biological deacidification of wines and a required step for making premium red wines. MLF is carried out by lactic acid bacteria (LAB) that are present in the fermenting wines. Currently, real-time control of MLF is an issue of great interest as the classical plate count technique for assessing bacterial populations requires long incubation times that are not compatible with a tight control of MLF. The aim of this study was to apply fluorescence microscopy and the bacteria staining kit Live/Dead BacLight™ to quantify viable LAB populations in red wines undergoing MLF. This method proved to be a fast and reliable culture-independent method to monitor wine MLF. Moreover, comparison of bacterial population data obtained by fluorescence microscopy and classical plate counts of LAB populations allowed discriminating a population of fully active and culturable cells, from total viable cells that include cells in an intermediate unculturable state.

Survival of selenium-enriched lactic acid bacteria in a fermented drink under storage and simulated gastro-intestinal digestion.

Selenium (Se), which is present as SeCys in seleno-proteins, is involved in cancer prevention, thyroid functioning, and pathogen inhibition. Se is incorporated in the diet through Se-containing foods. Some lactic acid bacteria (LAB) can biotransform selenite (toxic) into Se-nanoparticles (SeNPs) and Se-amino acids. To exert their beneficial properties in the host, bacteria should survive the harsh conditions of the gastrointestinal tract and during food storage. We evaluated whether selenization of LAB influenced bacterial growth and survival during gastrointestinal digestion and after storage when present in a fermented fruit juice-milk (FJM) beverage. Lactobacillus brevis CRL 2051 and Fructobacillus tropaeoli CRL 2034 were grown in MRS with and without selenite, and used to inoculate the FJM matrix. Selenization had no effect on LAB growth (9.54-9.9 log CFU/mL) in the FJM drink. The presence of SeNPs was confirmed for both selenized strains in the FJM beverage; however, the highest Se concentration (100 µg/L) was detected for the fermented beverage with selenized L. brevis. Under storage conditions 1.1 log CFU/ml decrease in cell count of selenized cells of L. brevis was observed, while no effect on cell viability was detected for non-selenized L. brevis or both selenized and control cells of F. tropaeoli. Resistance of L. brevis during digestion of the fermented FJM beverage was not affected by selenization. Contrarily, an increase (1 log CFU/mL) in the resistance of F. tropaeoli was observed when cells were selenized. After digestion, Se was detected in the soluble fraction of the beverage fermented by both strains, being higher for L. brevis (23.6 µg/L). Although selenization did not exert a drastic effect on strains´ survival during storage and digestion, microbial selenization previous to food fermentation could be an interesting tool for Se enrichment avoiding thus the addition of toxic Se salts.

Improvement of taste and shelf life of yeasted low-salt bread containing functional sourdoughs using Lactobacillus amylovorus DSM 19280 and Weisella cibaria MG1.

The challenge remains for the baking industry to reduce salt levels in yeasted bread as directed by governments, retailers and consumers around the world. The two main problems associated with the reduction of salt are a lack of salty taste and the reduction in shelf-life. Both of these issues are addressed in the presented work. A range of breads containing different levels of salt (0.0%, 0.3% and 1.2% of NaCl) in combination with various levels of sourdough (0%, 6%, 12%, 18%, 24%) was produced. The different doughs were analysed for their rheological behaviour. The bread quality characteristics such as loaf volume, crumb structure, staling rate and microbial shelf life were also determined. The sourdoughs were analysed for their different metabolites: organic acids, sugars, exopolysaccharides (EPS), and antifungal compounds. A trained sensory panel was used to perform descriptive analysis of the bread samples. The object of this paper is to use functional sourdoughs, containing Lactobacillus amylovorus DSM 19280 and Weisella cibaria MG1 to compensate for the quality problems that occur when salt is reduced in yeasted bread. The application of functional sourdoughs containing exopolysaccharides and/or antifungal substances in salt reduced breads significantly improved the quality. The application of functional sourdoughs allows the reduction of salt to a level of 0.3%.

Introduction of bifunctional alcohol/acetaldehyde dehydrogenase gene (adhE) in Fructobacillus fructosus settled its fructophilic characteristics.

Fructophilic lactic acid bacteria (FLAB) are unique in the sense that they prefer D-fructose over D-glucose as main carbon source. If D-glucose is metabolised, electron acceptors are required and significant levels of acetate are produced. These bacteria are found in environments rich in D-fructose, such as flowers, fruits and the gastrointestinal tract of insects feeding on fructose-rich diets. Fructobacillus spp. are representatives of this unique group, and their fructophilic characteristics are well conserved. In this study, the bifunctional alcohol/acetaldehyde dehydrogenase gene (adhE) from Leuconostoc mesenteroides NRIC 1541T was cloned into a plasmid and transferred to Fructobacillus fructosus NRIC 1058T. Differences in biochemical characteristics between the parental strain (NRIC 1058T) and the transformants were compared. Strain 1-11, transformed with the adhE gene, did not show any fructophilic characteristics, and the strain grew well on D-glucose without external electron acceptors. Accumulation of acetic acid, which was originally seen in the parental strain, was replaced with ethanol in the transformed strain. Furthermore, in silico analyses revealed that strain NRIC 1058T lacked the sugar transporters/permeases and enzymes required for conversion of metabolic intermediates. This may be the reason for poor carbohydrate metabolic properties recorded for FLAB.