Structural and rheological characterisation of heteropolysaccharides produced by lactic acid bacteria in wheat and sorghum sourdough

Hydrocolloids improve the volume, texture, and shelf life of bread. Exopolysaccharides (EPS) produced by lactic acid bacteria (LAB) during sourdough fermentation can replace hydrocolloids. It was the aim of this study to determine whether heteropolysaccharides (HePS) synthesized intracellularly from sugar nucleotides by glycosyltransferases are produced in wheat and gluten-free sorghum sourdough at effective levels. The HePS-producing strains Lactobacillus casei FUA3185, L. casei FUA3186, and Lactobacillus buchneri FUA3154 were used; Weissella cibaria 10M producing no EPS in the absence of sucrose served as control strain. Cell suspensions of L. buchneri in MRS showed the highest viscosity at low shear rate. Glycosyltransferase genes responsible of HePS formation in LAB were expressed in sorghum and wheat sourdough. However, only HePS produced by L. buchneri influenced the rheological properties of sorghum sourdoughs but not of wheat sourdoughs. Sorghum sourdough fermented with L. buchneri exhibited a low |G^∗| compared to the control, indicating a decrease in resistance to deformation. An increase in tan δ indicated decreased elasticity.The use of LAB producing HePS expands the diversity of EPS and increases the variety of cultures for use in baking.     

Prevalence and impact of single-strain starter cultures of lactic acid bacteria on metabolite formation in sourdough

Flavour of type II sourdoughs is influenced by the ingredients, processing conditions, and starter culture composition. It is, however, not fully clear to what extent different sourdough lactic acid bacteria (LAB) contribute to flavour. Therefore, two types of flour (rye and wheat) and different LAB starter culture strains were used to prepare sourdoughs, thereby leaving the yeast microbiota uncontrolled. All LAB starter culture strains tested were shown to be prevalent and to acidify the flour/water mixture to pH values between 3.1 and 3.9 after 24 h of fermentation. Multiple aldehydes, alcohols, ketones, and carboxylic acids were produced by the sourdough-associated microbiota throughout the fermentation period. Based on the organoleptic evaluation of breads produced with these sourdoughs, five LAB strains were selected to perform prolonged wheat and rye fermentations as to their capacity to result in an acidic (Lactobacillus fermentum IMDO 130101, Lactobacillus plantarum IMDO 130201, and Lactobacillus crustorum LMG 23699), buttermilk-like (Lactobacillus amylovorus DCE 471), or fruity flavour (Lactobacillus sakei CG1). Upon prolonged fermentation, higher metabolite concentrations were produced. For instance, L. sakei CG1 produced the highest amounts of 3-methyl-1-butanol, which was further converted into 3-methylbutyl acetate. The latter compound resulted in a fruity banana flavour after 48 h of fermentation, probably due to yeast interference. Rye fermentations resulted in sourdoughs richer in volatiles than wheat, including 3-methyl-1-butanol, 2-phenylethanol, and ethyl acetate.     

Preservation of porcine blood quality by means of lactic acid bacteria

The capacity of 12 lactic acid bacteria (LAB) strains to preserve porcine blood during storage was evaluated. A general ability of LAB to prevent blood's hemolysis and to maintain the functional properties of plasma was observed. Two strains, PS99 (Enterococcus raffinosus) and TA43 (Lactobacillus reuteri), were selected for studies at 5°C according to their antibacterial activity in blood stored at 15°C. After 144h at 5°C, lower counts of coliforms, Pseudomonas spp., proteolytic and hemolytic bacteria were obtained in blood containing either PS99 or TA43 as compared to the non-inoculated blood. When inulin (2%) was added to blood, higher inhibition values were obtained and Enterococcus raffinosus (PS99) showed the best abilities for blood preservation. On the basis of these results it seems worthwhile to supplement blood with inulin and to inoculate it with an active LAB strain to avoid undesirable changes during chill storage, especially useful to prevent the effects of a cold-chain breakdown.     

Biodiversity of lactic acid bacteria in French wheat sourdough as determined by molecular characterization using species-specific PCR

The lactic acid microflora of nine traditional wheat sourdoughs from the Midi-Pyrénées area (South western France) was previously isolated and preliminary characterized using conventional morphological and biochemical analysis. However, such phenotypic methods alone are not always reliable and have a low taxonomic resolution for identification of lactic acid bacteria species. In the present study, a total of 290 LAB isolates were identified by PCR amplification using different sets of specific primers in order to provide a thorough characterization of the lactic flora from these traditional French sourdoughs. Overall, the LAB isolates belonged to 6 genera: Lactobacillus (39%, 8 species), Pediococcus (38%, 1 species), Leuconostoc (17%, 2 species), Weissella (4%, 2 species), Lactococcus (1%, 1 species) and Enterococcus (< 1%, 1 species) and 15 different species were detected: L. plantarum, L. curvatus, L. paracasei, L. sanfranciscensis, L. pentosus, L. paraplantarum, L. sakei, L. brevis, P. pentosaceus, L. mesenteroides, L. citreum, W. cibaria, W. confusa, L. lactis and E. hirae. Facultative heterofermentative LAB represent more than 76% of the total isolates, the main species isolated herein correspond to L. plantarum and P. pentosaceus. Obligate heterofermentative lactobacilli (L. sanfranciscencis, L. brevis) represent less than 3% of the total isolates whereas Leuconostoc and Weissella species represent 21% of the total isolates and have been detected in eight of the nine samples. Detection of some LAB species was preferentially observed depending on the isolation culture medium. The number of different species within a sourdough varies from 3 to 7 and original associations of hetero- and homofermentative LAB species have been revealed. Results from this study clearly confirm the diversity encountered in the microbial community of traditional sourdough and highlight the importance of LAB cocci in the sourdough ecosystem, along with lactobacilli.     

Use of selected sourdough lactic acid bacteria to hydrolyze wheat and rye proteins responsible for cereal allergy

This work was aimed at showing the capacity of selected sourdough lactic acid bacteria to hydrolyze wheat and rye allergens. Hydrolysis was investigated after wheat sourdough fermentation and after treatment of wheat and rye sourdough breads with pepsin, trypsin and pancreatin, which mimicked the digestive process. As shown by immunoblotting with sera from allergic patients, wheat sourdough fermentation caused the disappearance of some IgE-binding proteins (albumins/globulins and gliadins mainly) with respect to the chemically acidified dough used as the control. The IgE-binding protein profile of wheat and rye sourdough breads differed from those of baker's yeast breads. The signals of the IgE-binding proteins contained in the sourdough breads disappeared after in vitro digestion with pepsin, trypsin and pancreatin. The same effect by digestive enzymes was not found for baker's yeast breads which showed persistent IgE-binding proteins. As shown by ELISA inhibition assays, the presence of IgE-binding low molecular weight proteins/peptides in sourdough breads significantly decreased with respect to baker's yeast breads. Proteolytic activity by selected sourdough lactic acid bacteria may have an importance during food processing to produce pre-digested wheat and rye dough which contains IgE-binding proteins degradable by digestive enzymes.     

Carbohydrate, peptide and lipid metabolism of lactic acid bacteria in sourdough

The metabolic pathways of lactic acid bacteria that influence bread quality are coupled to the central carbon flux by the availability of cofactors influencing the cellular and environmental redox potential. Homo- and heterofermentative metabolism differ fundamentally with respect to the requirement for regeneration of reduced cofactors, NADH or NADPH. The utilization of co-substrates such as oxygen or fructose as electron acceptors by obligate heterofermentative lactobacilli is coupled to an increased production of acetate in dough. Recently, several oxidoreductases involved in cofactor regeneration were characterized and glutathione and short-chain aldehydes derived from lipid oxidation were identified as substrates for cofactor regeneration by Lactobacillus sanfranciscensis. Based on the different metabolic requirements for cofactor regeneration, homo- and heterofermentative lactobacilli exert divergent effects on redox-reactions in sourdough that influence bread quality beyond the formation of acetate. Proteolysis, followed by peptide or amino acid metabolism by LAB is one of the key routes of flavour formation in bread flavour, and enables the strain-specific formation of antifungal metabolites. Peptide metabolism as well as the metabolism of cysteine, arginine, and phenylalanine in Lactobacillus plantarum, L. sanfranciscensis, and Lactobacillus pontis is increasingly understood and these insights provide new opportunities for the directed application of sourdough LAB for improved bread quality.     

Distribution and genetic diversity of lactic acid bacteria from traditional fermented sausage

This study was conducted to investigate the distribution and genetic diversity of the native lactic acid bacteria (LAB) population in nem chua, a popular traditional Vietnamese uncooked fermented sausage. A total of 74 LAB isolates were identified and their molecular fingerprints were obtained using repetitive-PCR (rep-PCR) and pulse-field gel electrophoresis (PFGE). The results revealed that the majority of LAB isolates were Lactobacillus plantarum (67.6%), followed by Pediococcus pentosaceus (21.6%). A minority of LAB (9.5%) were Lactobacillus brevis and Lactobacillus farciminis (1.4%). A large genetic plasticity within the same species was also observed. Both rep-PCR and PFGE methods were found to be acceptable regarding reproducibility and reliability. However, this study demonstrated the higher discriminatory power of PFGE compared to rep-PCR, as observed by the higher number of clusters generated (17 and 12 clusters of L. plantarum respectively, seven and six clusters of P. pentosaceus respectively and four and two clusters of L. brevis respectively), and lower percentage of similarity among clusters in PFGE data analysis. These results also revealed that there was not a single LAB strain that was found to predominate in the product. To our knowledge, this is the first detailed analysis of the native LAB population in nem chua. As this traditional sausage is fermented naturally or only with back-slop, knowledge about the native LAB population in the product is very important to understand the microflora that has been active in fermenting the nem chua and to ensure the safety of this uncooked sausage.     

Comparative survival and evaluation of functional probiotic properties of spray-dried lactic acid bacteria

The effect of spray drying on the viability and retention of key probiotic properties like acid and bile tolerance and cholesterol assimilation of three probiotic lactic acid bacteria (LAB) ( Lactobacillus plantarum CFR 2191, Lactobacillus salivarius CFR 2158 and Pediococcus acidilactici CFR 2193) has been studied. More than 97% survival was exhibited by the three LAB (1% cell suspension) spray dried with maltodextrin and nonfat skimmed (NFSM) as carriers. LAB cultures spray dried with maltodextrin showed significantly greater ( P ≤  0.05) retention of functional properties than those with NFSM. The results highlight a cost-effective way of producing large quantities of selected probiotic cultures with functional properties for neutraceutical application.     

Effect of phenolic acids on glucose and organic acid metabolism by lactic acid bacteria from wine

The influence of phenolic (p-coumaric, caffeic, ferulic, gallic and protocatechuic) acids on glucose and organic acid metabolism by two strains of wine lactic acid bacteria (Oenococcus oeni VF and Lactobacillus hilgardii 5) was investigated. Cultures were grown in modified MRS medium supplemented with different phenolic acids. Cellular growth was monitored and metabolite concentrations were determined by HPLC-RI. Despite the strong inhibitory effect of most tested phenolic acids on the growth of O. oeni VF, the malolactic activity of this strain was not considerably affected by these compounds. While less affected in its growth, the capacity of L. hilgardii 5 to degrade malic acid was clearly diminished. Except for gallic acid, the addition of phenolic acids delayed the metabolism of glucose and citric acid in both strains tested. It was also found that the presence of hydroxycinnamic acids (p-coumaric, caffeic and ferulic) increased the yield of lactic and acetic acid production from glucose by O. oeni VF and not by L. hilgardii 5. The results show that important oenological characteristics of wine lactic acid bacteria, such as the malolactic activity and the production of volatile organic acids, may be differently affected by the presence of phenolic acids, depending on the bacterial species or strain.     

Lactic acid bacteria community dynamics and metabolite production of rye sourdough fermentations share characteristics of wheat and spelt sourdough fermentations

Four spontaneous rye sourdough fermentations were performed over a period of ten days with daily back-slopping. Samples taken at all refreshment steps were used for culture-dependent and culture-independent characterization of the microbiota present. Furthermore, an extensive metabolite target analysis was performed through a combination of various chromatographic methods, including liquid chromatography coupled to mass spectrometry (LC/MS) and gas chromatography coupled to mass spectrometry (GC/MS). Spearman’s rank correlation coefficients were calculated and a principal component analysis (PCA) was performed on the data obtained in this study combined with data obtained previously for wheat and spelt sourdoughs. In general, the establishment of a stable microbial ecosystem occurred through a three-phase evolution, with mainly Lactobacillus plantarum and Lactobacillus fermentum dominating the rye sourdough ecosystems. PCA revealed that ornithine and mannitol were positively correlated with rye sourdoughs, contributing to bacterial competitiveness at the onset of sourdough production. Wheat and spelt sourdoughs showed a high degree of similarity, although certain compounds (e.g. indolelactic acid) appeared to be specific for spelt sourdoughs. The production of amino acid metabolites, mainly hydroxy acids (e.g. phenyllactic acid) and alcohols (e.g. 3-methyl-1-butanol), contributed to the equilibration of the redox balance and further enhanced the competitiveness of dominant species in stable sourdoughs.     

Fingerprint of lactic acid bacteria population in beef carpaccio is influenced by storage process and seasonal changes

We have investigated the population structure of lactic acid bacteria (LAB) for several beef carpaccio available on the market with the purpose of comparing the effect of storage process (modified-atmosphere packaging and vacuum-packaging) and of seasonal changes on this microbial population. Out of 60 samples we have characterised 214 isolates accounting for 10 LAB species and 35 isolates accounting for 11 non-LAB species. Lactobacillus sakei, Leuconostoc carnosum and Leuconostoc mesenteroides were the most prevailing LAB species with a frequency of identification within 66%, 62% and 52% of the samples respectively. These 3 species were also characterised by a phenotypic intra-species diversity of isolates based on colony morphology. We showed that the prevalence was increased 1.5 fold for L. sakei and L. mesenteroides during the summer sampling in comparison to the spring or the fall sampling suggesting an environmental origin of these two species. Seasonal variations were also observed for the prevalence of Lactobacillus fuchuensis and L. carnosum in spring (2- and 1.5-fold increase, respectively) and of Brochothrix thermosphacta in fall (6-fold increase). Finally, we demonstrated that the growth potential after the sell-by-date was favourable of 1.25 log₁₀ cfu g⁻¹ to Leuconostoc spp. in modified-atmosphere packaging and of 1.38 log₁₀ cfu g⁻¹ to Lactobacillus spp. in vacuum-packaging. In conclusion, we show that important and unsuspected traits in bacterial population dynamics can be unravelled by large sampling strategies. We discuss about the need to take this assessment into account for further studies on bacterial ecosystems of meat.     

Stable and non-competitive association of Saccharomyces cerevisiae, Candida milleri and Lactobacillus sanfranciscensis during manufacture of two traditional sourdough baked goods

The microbiota occurring in all the manufacturing phases of two Italian sourdough sweet-leavened baked goods (a typical Genoese dry biscuit, Lagaccio, and a soft stuffed North Italian typical cake, Panettone) were investigated over a period of three years. The two sourdough mother sponges were characterized by the stable presence of three dominant microbial species in potential competition for carbohydrates: Lactobacillus sanfranciscensis, Candida milleri, and Saccharomyces cerevisiae. Genotypic and phenotypic characterizations of microbial isolates pointed out that each mother sponge harbored its own strains, well distinguishable by molecular methods of analysis but not differing in their main metabolic properties from those known for the corresponding species. The microbial and biochemical evolution during the whole production protocol of both manufactures demonstrated that the three microbial species grew at almost the same growth rates, without exhausting any of the main carbon substrates (maltose, glucose and fructose). The quite similar growth dynamics under practical conditions and the constant presence of all fermentable carbohydrates were recognized as responsible for the stable non competitive association of maltose-positive and maltose-negative species in both sourdoughs. However, the two sourdoughs were characterized by quite different LAB to yeast ratio, with values significantly higher in Panettone than in Lagaccio. The cause of this difference could mainly be ascribed to the temperature of the mother sponge regeneration phase, that, in the case of Panettone manufacture, occurred under conditions of moderate refrigeration.     

Isolation of lactic acid bacteria with inhibitory activity against pathogens and spoilage organisms associated with fresh meat

The use of lactic acid bacteria (LAB) as protective cultures in vacuum-packed chill-stored meat has potential application for assuring and improving food quality, safety and market access. In a study to identify candidate strains suitable for evaluation in a meat model, agar-based methods were employed to screen 181 chilled meat and meat process-related LAB for strains inhibitory to pathogens and spoilage organisms of importance to the meat industry. Six meat-derived strains, including Lactobacillus sakei and Lactococcus lactis, were found to be inhibitory to one or more of the target strains Listeria monocytogenes, Brochothrix thermosphacta, Campylobacter jejuni and Clostridium estertheticum. The inhibitory agents appeared to be either cell-associated or molecules released extracellularly with bacteriocin-like properties. Variations detected in the antimicrobial activity of LAB associated with changes to test parameters such as substrate composition underlined the importance of further in situ evaluation of the inhibitory strains in stored meat trials.     

Comparison of the effect of exopolysaccharide-producing lactic acid bacteria from sourdough on dough characteristics and steamed bread quality

The use of exopolysaccharide (EPS)-producing lactic acid bacteria (LAB) is promising in sourdough fermentation. However, the knowledge of the effects of various species of LAB on steamed bread making remains limited. In this study, the effects of two LAB with high EPS-producing capacity, namely Weissella cibaria L32 and Lactobacillus brevis L17 on dough fermentation and steamed bread quality were estimated. The addition of these two LAB strains significantly increased the titratable acidity and protease activity during the dough fermentation, especially L. brevis L17. Although the in situ EPS synthesised by LAB could improve the steamed bread quality, excessive acidification of L. brevis L17 would still increase the protease activity and thus destroy its gluten network structure. As a result, the steamed bread fermented with L. brevis L17 had the lowest specific volume and hardest texture in comparison with the steamed bread fermented with W. cibaria L32 and with added EPS produced by W. cibaria L32 and L. brevis L17. These results indicated that different EPS-producing LAB exhibited distinctive dough fermentation characteristics, and the in situ EPS-producing W. cibaria L32 could improve steamed bread quality, which confirmed its potential application in steamed bread making.     

Spanish cheese screening and selection of lactic acid bacteria with high gamma-aminobutyric acid production

Gamma-aminobutyric acid (GABA) is a non-protein four-carbon amino acid which is considered a bioactive component known for its physiological functions, including a regulator of blood pressure, neurotransmitter, diuretic and anti-stress effects. Its use in foods might confer health benefits. Microorganisms such as yeast, fungi or bacteria can produce GABA naturally. Among them, the lactic acid bacteria are being studied for the potential development of fermented foods because their physiological activities and their designation of generally recognized as safe (GRAS). The objective of this study was to evaluate the GABA-production capacity in a whole wheat flour medium of lactic acid bacteria strains that showed a high conversion of glutamic acid to GABA in a screening conducted in 58 Spanish artisanal cheeses. Synthesis of GABA by these strains in a non-optimized whole wheat flour in water solution (1:5) was quantified by High-Performance Liquid Chromatography. The 4 strains showing the highest GABA production were genotypically and phenotypically characterized. Results indicated an interesting fermentative variability between strains. The addition of these isolated lactic acid strains in fermented food products could allow a potentially functional food for regulating hypertension.     

Propionic acid production by cofermentation of Lactobacillus buchneri and Lactobacillus diolivorans in sourdough

Cooperative metabolism of lactobacilli in silage fermentation converts lactate to propionate. This study aimed to determine whether propionate production by Lactobacillus buchneri and Lactobacillus diolivorans can be applied for bread preservation. Propionate formation was observed in cofermentation with L. buchneri and L. diolivorans in modified MRS broth as well as sourdough with low, medium and high ash contents. 48 mM of propionate was formed in sourdough with medium ash content, but only 9 and 28 mM propionate were formed in sourdoughs prepared from white wheat flour or whole wheat flour, respectively. Acetate levels were comparable in all three sourdoughs and ranged from 160 to 175 mM. Sourdough fermented with L. buchneri and L. diolivorans was used in breadmaking and its effect on fungal spoilage was compared to traditional sourdough or propionate addition to straight doughs. Bread slices were inoculated with Aspergillus clavatus, Cladosporium spp., Mortierella spp. or Penicillium roquefortii. The use of 20% experimental sourdough inhibited growth of three of the four moulds for more than 12 days. The use of 10% experimental sourdough deferred growth of two moulds by one day. Bread from traditional sourdough with added acetate had less effect in inhibiting mould growth.     

Identification of lactic acid bacteria involved in the spoilage of pasteurized “foie gras” products

The spoiling microflora of a re-packaged French “foie gras” product was studied. A total of 54 isolates, originating from two different factories, were identified using phenotypical and molecular methods (partial 16S rDNA sequencing). Weissella viridescens was the main species detected in the products from factory 1 (64% of the isolates). These products had a low lactic acid concentration and were considered as non-spoiled. The microflora of factory 2 was dominated mainly by the genus Lactobacillus (95% of the isolates), and the high lactic acid concentration of these products was linked with a strong spoilage. Among the 30 Lactobacillus strains, three species were predominant: Lactobacillus sakei (nine isolates), Lactobacillus coryniformis (eight isolates) and Lactobacillus paraplantarum (five isolates). Challenge tests were performed to confirm the involvement of the Lactobacillus strains in the spoilage of the product. Sterile “foie gras” samples were inoculated with 14 LAB strains from the collection. The most acidifying strains belonged to the species L. sakei, Lactobacillus plantarum and L. paraplantarum. This confirmed the role of the strains from the Lactobacillus genus as the main spoilers of “foie gras” products and will be useful to design new quality protocols and extend the shelf-life of these products.     

Modelling the survival of starter lactic acid bacteria and Bifidobacterium bifidum in single and simultaneous cultures

The inactivation kinetics at 4 degrees C of Bifidobacterium bifidum, Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus, cultured alone or consociated in a laboratory medium (modified MRS broth), were modelled through the Weibull model and a second-order polynomial equation. The initial cell number of S. thermophilus and L. delbrueckii subsp. bulgaricus was approximately 6-7log (CFU/ml); the viability loss after 30 days of storage was 2.87 and 1.99log (CFU/l) for L. delbruckii subsp. bulgaricus and S. thermophilus, cultured alone, respectively; whereas the consociation of lactobacilli and streptococci with bifidobacteria reduced viability loss during storage (0.28 and 0.54log CFU/l for lactobacilli and streptococci, respectively). Finally, the consociation of lactobacilli and streptococci with B. bifidum improved their oxygen uptake.     

Characterization of glucan-producing Leuconostoc strains isolated from sourdough

Sourdough was previously demonstrated to be a fruitful biotope for isolation of lactic acid bacteria producing exopolysaccharides and more accurately diverse glycan polymers which have interesting applications as texturing agents or prebiotics. Characterization of polymers by ¹H and ¹³C NMR spectroscopy analysis demonstrated that these strains could synthesize glucans of high structural variety and containing different amounts of α-(1 → 2), α-(1 → 3) and α-(1 → 6) linkages. In this study, fifteen glucan-producing Leuconostoc mesenteroides and L. citreum strains from sourdoughs were characterized according to carbohydrate fermentation, rep-PCR fingerprinting using (GTG)₅ primers and glycansucrase activity (soluble or cell-associated). Enzyme characterization using SDS-PAGE and in situ polymer production after incubation with sucrose correlated with synthesis of classical or α-(1 → 2) branched dextrans, alternan and levan. In addition, the presence of genes coding for alternansucrase was detected by PCR and partially characterized by sequence analysis. We thus provide new information on the biodiversity of glucan production by sourdough Leuconostoc strains.