Sourdough Lactic Acid Bacteria Research Articles

Proteolytic and Phytase Activity in Sourdough Lactic Acid Bacteria

ABSTRACTTwenty-six strains of lactic acid bacteria (LAB) isolated from French and Bulgarian sourdoughs were screened for their enzymatic activities, to elucidate their possible roles during the fermentation process. Phytase and phosphatase activities were measured. These activities were determined spectrophotometrically by using p-nitrophenyl phosphate and sodium phytate as substrates. SDS-PAGE and zymograme were performed to prove phytase activities and evaluate molecular weight of purified proteins. The enzyme responsible for these activities was an acid phosphatase, with a molecular mass of 70 kDa. Proteolytic activity were measured with casein and asocasein in model system and also in modified media with gluten as only one nitrogen source. Several strains have shown an interesting combination of proteolytic and phytase activities, suggesting their possible roles during dough fermentation.

Long-Term Fungal Inhibitory Activity of Water-Soluble Extracts of Phaseolus vulgaris cv. Pinto and Sourdough Lactic Acid Bacteria during Bread Storage

The antifungal activity of proteinaceous compounds from different food matrices was investigated. In initial experiments, water-soluble extracts of wheat sourdoughs, cheeses, and vegetables were screened by agar diffusion assays with Penicillium roqueforti DPPMAF1 as the indicator fungus. Water-soluble extracts of sourdough fermented with Lactobacillus brevis AM7 and Phaseolus vulgaris cv. Pinto were selected for further study. The crude water-soluble extracts of L. brevis AM7 sourdough and P. vulgaris cv. Pinto had a MIC of 40 mg of peptide/ml and 30.9 mg of protein/ml, respectively. MICs were markedly lower when chemically synthesized peptides or partially purified protein fractions were used. The water-soluble extract of P. vulgaris cv. Pinto showed inhibition toward a large number of fungal species isolated from bakeries. Phaseolin alpha-type precursor, phaseolin, and erythroagglutinating phytohemagglutinin precursor were identified in the water-soluble extract of P. vulgaris cv. Pinto by nano liquid chromatography-electrospray ionization-tandem mass spectrometry. When the antifungal activity was assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, all three proteins were inhibitory. A mixture of eight peptides was identified from the water-soluble extract of sourdough L. brevis AM7, and five of these exhibited inhibitory activity. Bread was made at the pilot plant scale by sourdough fermentation with L. brevis AM7 and addition of the water-soluble extract (27%, vol/wt; 5 mg of protein/ml) of P. vulgaris cv. Pinto. Slices of bread packed in polyethylene bags did not show contamination by fungi until at least 21 days of storage at room temperature, a level of protection comparable to that afforded by 0.3% (wt/wt) calcium propionate.

Functionality of lactic acid bacteria peptidase activities in the hydrolysis of gliadin-like fragments

To evaluate the role of the peptidase activities from sourdough lactic acid bacteria (LAB) in the degradation of alpha-gliadin fragments. Different proline-containing substrates were hydrolysed by LAB indicating pro-specific peptidase activities. Lactobacillus plantarum CRL 775 and Pediococcus pentosaceus CRL 792 displayed the highest tri- and di-peptidase activities, respectively. Lactobacillus plantarum strains hydrolysed more than 60%alpha-gliadin fragments corresponding to the 31-43 and 62-75 amino acids in the protein after 2 h. None of the LAB strains alone could hydrolyse 57-89 alpha-gliadin peptide; however, the combination of L. plantarum CRL 775 and P. pentosaceus CRL 792 led to hydrolysis (57%) of this peptide in 8 h. The capacity of LAB strains to degrade alpha-gliadin fragments was not correlated to individual peptidase activities. Several strains separately degraded the 31-43 and 62-75 alpha-gliadin fragments, while the 57-89 peptide degradation was associated with the combination of peptidase profiles from pooled LAB strains. This is the first report on the peptide hydrolase system of sourdough pediococci and its ability to reduce alpha-gliadin fragments. This study contributes to a better knowledge of sourdough LAB proteolytic system and its role in the degradation of proline-rich alpha-gliadin peptides involved in celiac disease.

Synthesis of Angiotensin I-Converting Enzyme (ACE)-Inhibitory Peptides and γ-Aminobutyric Acid (GABA) during Sourdough Fermentation by Selected Lactic Acid Bacteria

This article aimed at investigating the synthesis of angiotensin I-converting enzyme (ACE)-inhibitory peptides and gamma-aminobutyric acid (GABA) during sourdough fermentation of white wheat, wholemeal wheat, and rye flours. Sourdough lactic acid bacteria, selected previously for proteinase and peptidase activities toward wheat proteins or for the capacity of synthesizing GABA, were used. The highest ACE-inhibitory activity was found by fermenting flour under semiliquid conditions (dough yield 330) and, especially, by using wholemeal wheat flour. Fourteen peptides, not previously reported as ACE-inhibitory, were identified from the water/salt-soluble extract of wholemeal wheat sourdough (IC 50 0.19-0.54 mg/mL). The major part of the identified peptides contained the well-known antihypertensive epitope VAP. The synthesis of GABA increased when the dough yield was decreased to 160. The highest synthesis of GABA (258.71 mg/kg) was found in wholemeal wheat sourdough.

Use of Selected Sourdough Strains of Lactobacillus for Removing Gluten and Enhancing the Nutritional Properties of Gluten-Free Bread

Forty-six strains of sourdough lactic acid bacteria were screened for proteolytic activity and acidification rate in gluten-free (GF) flours. The sourdough cultures consisted of Lactobacillus sanfranciscensis LS40 and LS41 and Lactobacillus plantarum CF1 and were selected and used for the manufacture of GF bread. Fermentation occurred in two steps: (i) long-time fermentation (16 h) and (ii) fast fermentation (1.5 h) using the previous fermented sourdough as inoculum (ca. 43%, wt/wt) with Saccharomyces cerevisiae (baker's yeast). GF bread started with baker's yeast alone was used as the control. Gluten was added to ingredients before fermentation to simulate contamination. Initial gluten concentration of 400 ppm was degraded to below 20 ppm only in the sourdough GF bread. Before baking, sourdough GF bread showed phytase activity ca. sixfold higher than that of GF bread started with baker's yeast alone. Atomic absorption spectrophotometric analysis revealed that the higher phytase activity resulted in an increased availability of free Ca2+, Zn2+, and Mg2+. The concentration of free amino acids also was the highest in sourdough GF bread. Sourdough GF bread had a higher specific volume and was less firm than GF bread started with baker's yeast alone. This study highlighted the use of selected sourdough cultures to eliminate risks of contamination by gluten and to enhance the nutritional properties of GF bread.

Effect of Sourdough Bacteria on the Quality and Shelf Life of Bread

Bread dough is usually fermented with yeast but in the present study sourdough lactic acid bacteria (Lactobacillus bulgaricus) alone and in combination with yeast (Saccharomyces cerevisiae) were used to determine their effect on the shelf life and sensory characteristics of bread at different intervals of storage. Lactic acid bacteria improved the sensory characteristics of bread such as volume, evenness of bake, character of crust, grain of bread, colour of bread crumb, aroma, taste and texture of bread and extended shelf life of bread by inhibiting the growth of microbes.

Cell–cell communication in sourdough lactic acid bacteria: A proteomic study in Lactobacillus sanfranciscensis CB1

The mechanisms of cell-cell communication in Lactobacillus sanfranciscensis CB1 were studied. The highest number of dead/damaged cells of L. sanfranciscensis CB1 was found in cocultures with Lactobacillus plantarum DC400 or Lactobacillus brevis CR13 when the late stationary phase of growth (18 h) was reached. 2-DE analysis was carried out. Almost the same proteins were induced in all three cocultures at the mid-exponential phase of growth (7 h). The number of induced proteins markedly increased at 18 h, especially when L. sanfranciscensis CB1 was cocultured with L. plantarum DC400 or L. brevis CR13. Nineteen overexpressed proteins were identified. These proteins had a central role in stress response mechanisms and LuxS-mediated signalling was involved in the regulation of most of them. The luxS and metF genes were partially sequenced in L. sanfranciscensis CB1. RT-PCR showed that the expression of luxS gene decreased from 7 to 12 h. It was highest in cocultures with L. plantarum DC400 and L. brevis CR13. 2(3H)dihydrofuranone-5ethyl and 2(3H)dihydrofuranone-5pentyl were identified as presumptive signalling molecules when L. sanfranciscensis CB1 was cocultured with L. brevis CR13 and, especially, L. plantarum DC400. The synthesis of other volatile compounds and peptidase activities were also influenced by the type of microbial cocultures.

Flour carbohydrate catabolism and metabolite production by sourdough lactic acid bacteria

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

Identification of subdominant sourdough lactic acid bacteria and their evolution during laboratory-scale fermentations

Presumptive lactic acid bacterial cocci were found in six sourdoughs (out of 20) from the Abruzzo region (central Italy) and subjected to phenotypic and genotypic characterization. A total of 21 isolates, recognized as seven strains by randomly amplified polymorphic DNA (RAPD)–polymerase chain reaction (PCR) typing, were identified by a polyphasic approach, consisting of 16S rRNA gene sequencing, multiplex PCR assays and physiological features, as Enterococcus faecium and Pediococcus pentosaceus. Four strains belonging to those species and previously isolated from wheat kernels were inoculated in sterile flour to verify their capacity to grow in sourdough environment. Doughs with several dual bacterial combinations, including Lactobacillus sanfranciscensis, were propagated for 11 days and pH measurements and bacterial counts were carried out.

Glucan and Fructan Production by Sourdough Weissella cibaria and Lactobacillus plantarum

After a large screening on sourdough lactic acid bacteria, exopolysaccharide (EPS)-forming strains of Weissella cibaria, Lactobacillus plantarum, and Pediococcus pentosaceus were selected. After 6 days of incubation at 30 degrees C, the synthesis of EPS in MRS-based broth ranged from 5.54 to 7.88 mg mL-1. EPS had an apparent molecular mass of ca. 104 Da. As shown by carbohydrate consumption, the synthesis of EPS was found from sucrose only. Two types of homopolysaccharides were synthesized: glucans simultaneously with growth and fructans after 1 day of incubation. Two protein bands of ca. 180-200 kDa were in situ detected on SDS-PAGE gels incubated with sucrose. PCR products of ca. 220 bp were found for L. plantarum PL9 (100% of identity to putative priming glycosyltransferase of L. plantarum WCFS1) and W. cibaria WC4 (80% of identity to putative glycosyltransferase, epsD, of Bacillus cereus G9241) by using hybrid primers for the priming gtf genes. Degenerated primers DexreuR and DexreuV showed a unique PCR product, and the predicted amino acid sequences were identical for W. cibaria WC4 and L. plantarum PL9. The sequence had similarity with polysaccharide biosynthesis glycosyltransferases. W. cibaria WC4 or L. plantarum LP9 synthesized ca. 2.5 g kg-1 EPS during sourdough fermentation with sucrose added. Compared to the sourdough started with an EPS-negative strain, the sourdough started with W. cibaria WC4 or L. plantarum LP9 increased the viscosity, and the resulting bread had higher specific volume and lower firmness. The synthesis of EPS by selected sourdough lactic acid bacteria could be considered as a useful tool to replace the additives for improving the textural properties of baked goods.

Sourdough lactobacilli and celiac disease

Celiac disease (CD) is one of the most common food intolerance. The only effective treatment for CD is a strict adherence to a gluten-free diet throughout the patient's lifetime. Gluten-free products are not widely available and are usually more expensive than their gluten-containing counterparts. There is, therefore, an urgent need to develop safe and effective therapeutic alternatives, to develop high-quality gluten-free products and to investigate the potential of the bread making biotechnology following ancient protocols which include long-time fermentation by selected sourdough lactic acid bacteria. This review describes the most relevant results related to biotechnologies that use selected sourdough lactic acid bacteria and probiotics as starters for sourdough fermentation to investigate their potential to decrease the risk of gluten contamination in gluten-free products. As shown by studies in vitro on celiac intestinal tissue and in vivo on CD patients, the bacterial proteolytic activity is rather promising not only as currently demonstrated for eliminating traces of contaminant gluten but probably also in perspective for the manufacture of tolerated baked goods.

Biodiversity and identification of sourdough lactic acid bacteria

This review deals with recent developments on the biodiversity of sourdough lactic acid bacteria (LAB) and the recent description of new sourdough LAB species. One of the outcomes of biodiversity studies of particular sourdough ecosystems throughout Europe is the description of new taxa of LAB. During the last 3 years, several new LAB species have been isolated from traditional sourdoughs: Lactobacillus mindensis, Lactobacillus spicheri, Lactobacillus rossiae, Lactobacillus zymae, Lactobacillus acidifarinae, Lactobacillus hammesii, and Lactobacillus nantensis. Some of these species have been described on one single isolate only. Isolation of novel taxa mainly depends on the cultivation approach used, i.e. (selective) incubation media and conditions. The distribution of the taxa of LAB is highly variable from one sourdough ecosystem to another. Therefore, it is difficult to define correlations between population composition and both the type of sourdough or the geographic location. Identification of isolated strains needs a polyphasic approach, including a combination of phenotypic and genotypic methods, the latter often based on the polymerase chain reaction (PCR) and encompassing 16S rRNA sequencing and DNA–DNA hybridizations. A main obstacle in current identification approaches of LAB strains is the lack of a robust and exchangeable identification system for all LAB species. Recent studies based on complete genomes have provided the basis for establishing sets of genes useful for multi-locus sequence analysis (MLSA). Monitoring the population dynamics of sourdough ecosystems can be performed by both culture-dependent (plating and incubation) and culture-independent (e.g. PCR-Denaturing Gradient Gel Electrophoresis) methods. Although highly valuable for community fingerprinting, culture-independent methods do not always yield precise quantitative information.

Defined multi-species semi-liquid ready-to-use sourdough starter

This paper aimed at setting up a protocol for the manufacture of a defined multi-species semi-liquid ready-to-use sourdough starter consisting of lactic acid bacteria (LAB) selected on the basis of their capacity of rapid acidification and adaptation to several technological factors. Preliminarily, 56 strains of sourdough LAB were screened based on the acidification kinetics during sourdough fermentation. The influence of temperature (25–37 °C), NaCl (2%, w/w, of wheat flour), initial cell number (10 7–10 9 cfu g −1), dough yield (150–180), substrate for cell cultivation (mMRS or SDB media) and phase of cell growth (exponential or stationary phases) on the kinetics of acidification was also determined. Lactobacillus casei DPPMA27, Weissella confusa DPPMA20 and Lactobacillus fructivorans DPPMA8 were selected and used to produce a defined multi-species semi-liquid sourdough starter. Fermentation was carried out in a 2 l batch fermentation bioreactor (12 h at 37 °C), under controlled pH, by cultivating cells in a medium made of water (60%), flour and other nutrients (40%). The semi-liquid sourdough starter was stored for 30 days under different conditions and used for dough fermentation (dough yield 160). The microbial composition, acidifying activity and fermentation end-products were kept constant during 21 days of storage. The findings of this study may be useful to increase the use of sourdough in bakery industries.

Fermentation by selected sourdough lactic acid bacteria to decrease coeliac intolerance to rye flour

A pool of selected lactic acid bacteria was used to ferment suspensions of rye flour. Two-dimensional electrophoresis showed that 109 of the 129 ethanol-soluble rye polypeptides were hydrolysed almost totally by lactic acid bacteria. Matrix-assisted laser desorption ionization—time of flight mass spectrometry and reversed-phase high performance liquid chromatography analysis confirmed the hydrolysis of prolamins. After 48 h fermentation, no prolamin polypeptides were recognized by R5-Western analysis. HPLC analysis of glutelin polymers showed a very low bacterial proteolysis but a pH dependent hydrolysis probably due to activation of rye enzymes. Prolamins were extracted from rye flour and used to produce a peptic–tryptic (PT)-digest for in vitro tests with K 562 (S) sub-clone and Caco-2/TC7 cells of human origin. The PT-digest was also treated with lactic acid bacteria before assay. The Minimal Agglutinating Capacity increased ca. 8-times when K 562 (S) sub-clone cells were exposed to rye PT-digest treated with lactic acid bacteria. Hydrolysis of rye PT-digest by lactic acid bacteria decreased the toxicity of PT-digest itself towards Caco-2/TC7 cells as estimated by cell viability, caspase-3 activity and release of nitric oxide. Rye prolamins and glutelins were extracted from doughs and subjected to PT digestion. Compared to PT-digests from chemically acidified dough, coeliac jejunal biopsies exposed to the PT-digest from the dough fermented by lactic acid bacteria did not show an increase of the infiltration of CD3 + intraepithelial lymphocytes. The same was found for epithelial cell Fas expression. Long-time fermentation of dough by selected lactic acid bacteria could be considered as a potential tool to decrease the risk of rye contamination of gluten-free products for coeliac patients.

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.

In situ activity of a bacteriocin-producing Lactococcus lactis strain. Influence on the interactions between lactic acid bacteria during sourdough fermentation

To biochemically characterize the bacteriocin produced by Lactococcus lactis ssp. lactis M30 and demonstrate its effect on lactic acid bacteria (LAB) during sourdough propagation. A two-peptide bacteriocin produced by L. lactis ssp. lactis M30 was purified by ion exchange, hydrophobic interaction and reversed phase chromatography. Mass spectrometry of the two peptides and sequence analysis of the ltnA2 gene showed that the bacteriocin was almost identical to lacticin 3147. During a 20-day period of sourdough propagation the stability of L. lactis M30 was demonstrated, with concomitant inhibition of the indicator strain Lactobacillus plantarum 20, as well as the non-interference with the growth of the starter strain Lact. sanfranciscensis CB1. In situ active bacteriocins influence the microbial consortium of sourdough LAB and can "support" the dominance of insensitive strains during sourdough fermentation. The in situ bacteriocinogenic activity of selected lactococci enables the persistence of insensitive Lact. sanfranciscensis strains, useful to confer good characteristics to the dough, at a higher cell concentration with respect to other LAB of the same ecosystem.

Molecular taxonomy and genetics of sourdough lactic acid bacteria

The exploitation of lactic acid bacteria with their long history in sourdough fermentations forms an emerging field for baking applications and design of added-value food. The deliberate use of functional traits within these bacteria is supported by knowledge on their phylogenetic and environmental status, the characterization of their genome structure and flexibility, gene regulation and metabolic potential. Molecular taxonomy and genetics are major contributors to this development.

Lactobacillus rossii sp. nov., isolated from wheat sourdough

Screening of sourdough lactic acid bacteria for bacteriocin production resulted in the isolation of a Gram-positive, catalase-negative, non-spore-forming, non-motile rod bacterium (strain CS1T) that could not be associated with any previously described species. Comparative 16S rRNA gene sequence analysis recognized strain CS1T as a distinct member of the genus Lactobacillus. By a species-specific PCR strategy, five additional strains previously isolated from sourdoughs were found to belong to the same species as strain CS1T, as confirmed by 16S rRNA gene sequence analysis. The closest related species were Lactobacillus durianis, Lactobacillus malefermentans and Lactobacillus suebicus, with which strain CS1T shared 93 % sequence similarity. For a further characterization of strain CS1T, physiological (growth temperature, CO2 production, hydrolysis of arginine, isomeric type of lactate, sugar fermentation) and chemotaxonomic (G+C content and peptidoglycan structure) properties were determined. Phenotypic characterization showed that strain CS1T was a member of the obligately heterofermentative group of the genus Lactobacillus. The DNA G+C content was 44.6 mol%. The peptidoglycan was of the A3alpha (L-Lys-L-Ser-L-Ala2) type. Physiological, biochemical and genotypic data, as well as results of DNA-DNA hybridization of genomic DNA with one of the closest phylogenetic relatives, L. durianis (34.3 %), indicated that strain CS1T represents a novel species of the genus Lactobacillus for which the name Lactobacillus rossii sp. nov. is proposed. The type strain of this species is CS1T (=ATCC BAA-822T=DSM 15814T).

Biochemistry and physiology of sourdough lactic acid bacteria

Lactic acid bacteria (LAB) are the dominant microorganisms in sourdoughs, and the rheology, flavour and nutritional properties of sourdough-based baked products greatly rely on the activity of LAB. The newer developments on the biochemistry and physiology of this group of bacteria are considered here, with particular emphasis on carbohydrate and nitrogen metabolism, responses to environmental stresses, production of anti-microbial compounds and nutritional implications.

Partial purification and characterization of an X-prolyl dipeptidyl aminopeptidase from Lactobacillus sanfranciscensis CB1

An X-prolyl dipeptidyl aminopeptidase (PepX) from Lactobacillus sanfranciscensis CB1, a key sourdough lactic acid bacterium, was partially purified by five chromatographic steps. As estimated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration, the enzyme appeared to be a 49.2–56 kDa monomer. Optimal activity occurred at pH 6.0 and 30 °C, with K m of 0.6 mM and V max of 99.6 nmol mg −1 min −1. The D value, calculated at 45 °C, was ∼5.46 s. The enzyme hydrolyzed (almost exclusively) substrates with a X-Pro N-terminal sequence. It did not possess prolidase, aminopeptidase or endopeptidase activities. No hydrolysis of the Pro-rich 33-mer epitope (a potent inducer of gut-derived human T-cell lines in celiac patients) was found when it was treated with PepX alone. When the general aminopeptidase type N was combined with PepX, the hydrolysis of 33-mer peptide (0.2 mM) was complete after 24 h of incubation at 30 °C. Leucine and glutamine residues were liberated from the Pro-rich 33-mer peptide by aminopeptidase type N, thus favouring the subsequent PepX activity. PepX was inactivated by p-chloromercuribenzoate, 3,4-dichloroisocoumarin and phenanthroline which exerted a competitive mode of inhibition. Among divalent cations, only Zn 2+, Hg 2+ and Mn 2+ markedly decreased the enzyme activity. Quadratic response surface methodology was used to study the individual and interactive effects of temperature, pH and NaCl on the PepX activity. The enzyme maintained considerable activity under the environmental conditions which characterize the sourdough fermentation.