Characterization of bacteriocin-like inhibitory substances (BLIS) from sourdough lactic acid bacteria and evaluation of their in vitro and in situ activity

AIMS: To identify and characterize bacteriocion-producing lactic acid bacteria (LAB) in sourdoughs and to compare in vitro and in situ bacteriocin activity of sourdough- and nonsourdough LAB. METHODS AND RESULTS: Production of antimicrobial compounds by 437 Lactobacillus strains isolated from 70 sourdoughs was investigated. Five strains (Lactobacillus pentosus 2MF8 and 8CF, Lb. plantarum 4DE and 3DM and Lactobacillus spp. CS1) were found to produce distinct bacteriocin-like inhibitory substances (BLIS). BLIS-producing Lactococcus lactis isolated from raw barley showed a wider inhibitory spectrum than sourdough LAB, but they did not inhibit all strains of the key sourdough bacterium Lb. sanfranciscensis. Antimicrobial production by Lb. pentosus 2MF8 and Lc. lactis M30 was also demonstrated in situ. CONCLUSIONS: BLIS production by sourdough LAB appears to occur at a low frequency, showing limited inhibitory spectrum when compared with BLIS-producing Lc. lactis. Nevertheless, they are active BLIS producers under sourdough and bread-making conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The activity of BLIS has been demonstrated in situ. It may influence the complex sourdough microflora and support the implantation and stability of selected insensitive bacteria, such as Lb. sanfranciscensis, useful to confer good characteristics to the dough.     

Development and potential of starter lactobacilli resulting from exploration of the sourdough ecosystem

Lactic acid bacteria are widely used as starter organisms in food fermentations. The development of such cultures as organisms fulfilling all metabolic, technical and handling requirements is the result of a multidisciplinary approach, i.e. to analyse, follow and direct the microbial ecology in food fermentations by molecular biology tools, gene cloning, biochemical and physiological analyses, pilot trials and modelling of behaviour and metabolism. The possibilities and restrictions of such an approach is given for cereal fermentations, namely sourdoughs. In this environment highly adapted lactobacilli are predominant, sharing the environment with yeasts present in traditional preparations. The competitiveness of these lactobacilli and their contribution to flavour, machineability and prebiosis of doughs and bread relies on their maltose and amino acid metabolism, use of electron acceptors and EPS formation. Their reactions on environmental stresses can be used to embed these recalcitrant organisms into starter culture preparations. Beyond the cereal environment the described strategy can be generally applied to understand ecosystems in food fermentations and finally control them. This revised version was published online in August 2006 with corrections to the Cover Date.     

Influence of baking enzymes on antimicrobial activity of five bacteriocin‐like inhibitory substances produced by lactic acid bacteria isolated from Lithuanian sourdoughs

Aim: To evaluate the effect of four different baking enzymes on the inhibitory activity of five bacteriocin‐like inhibitory substances (BLIS) produced by lactic acid bacteria (LAB) isolated from Lithuanian sourdoughs. Methods and Results: The overlay assay and the Bioscreen methods revealed that the five BLIS exhibited an inhibitory effect against spore germination and vegetative outgrowth of Bacillus subtilis, the predominant species causing ropiness in bread. The possibility that the observed antibacterial activity of BLIS might be lost after treatment with enzymes used for baking purposes was also examined. Conclusions: The enzymes tested; hemicellulase, lipase, amyloglucosidase and amylase had little or no effect on the majority of the antimicrobial activities associated with the five BLIS studied. Significance and Impact of the Study: This study suggests a potential application in the sourdough baking industry for these antimicrobial producing LAB strains in the control of B. subtilis spore germination and vegetative outgrowth.     

Glutamine deamidation by cereal-associated lactic acid bacteria

AIMS: It was the aim of our work to investigate glutamine deamidation by lactic acid bacteria isolated from cereal fermentations and to elucidate the ecological and technological relevance in baking of the conversion of glutamine to glutamate. METHODS AND RESULTS: Lactobacillus sanfranciscensis and Lact. reuteri were found to display glutaminase activity. The addition of glutamine to modified Man, Rogosa and Sharp medium increased the cell yields of Lact. sanfranciscensis, as well as the production of lactic and acetic acid. The final pH; however, was increased in the glutamine-containing medium. The addition of 47 mmol kg(-1) glutamate to chemically acidified doughs significantly changed the bread flavour. In sourdoughs with enhanced proteolytic activity, strain-dependent production of 27-120 mmol glutamate per kilogram sourdough was observed. CONCLUSIONS: Lactobacillus sanfranciscensis and Lact. reuteri converted glutamine into glutamate; this conversion improves the acid tolerance of lactobacilli and significantly influences wheat bread flavour. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper illustrates the complex interaction of sourdough-lactobacilli with their environment: the flour provides substrates for metabolic activities that enable the lactobacilli to reach higher cell counts, and the produced metabolite may be one of the reasons why the flavour of fermented breads is different to the flavour of chemically acidified breads.     

Bacterial population in traditional sourdough evaluated by molecular methods

AIMS: To study the microbial communities in artisanal sourdoughs, manufactured by traditional procedure in different areas of Sicily, and to evaluate the lactic acid bacteria (LAB) population by classical and culture-independent approaches. METHODS AND RESULTS: Forty-five LAB isolates were identified both by phenotypic and molecular methods. The restriction fragment length polymorphism and 16S ribosomal DNA gene sequencing gave evidence of a variety of species with the dominance of Lactobacillus sanfranciscensis and Lactobacillus pentosus, in all sourdoughs tested. Culture-independent method, such as denaturing gradient gel electrophoresis (DGGE) of the V6-V8 regions of the 16S rDNA, was applied for microbial community fingerprint. The DGGE profiles revealed the dominance of L. sanfranciscensis species. In addition, Lactobacillus-specific primers were used to amplify the V1-V3 regions of the 16S rDNA. DGGE profiles flourished the dominance of L. sanfranciscensis and Lactobacillus fermentum in the traditional sourdoughs, and revealed that the closely related species Lactobacillus kimchii and Lactobacillus alimentarius were not discriminated. CONCLUSIONS: Lactobacillus-specific PCR-DGGE analysis is a rapid tool for rapid detection of Lactobacillus species in artisanal sourdough. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports a characterization of Lactobacillus isolates from artisanal sourdoughs and highlights the value of DGGE approach to detect uncultivable Lactobacillus species.     

Evaluation of mono or mixed cultures of lactic acid bacteria in type II sourdough system

The aim of this study was to evaluate the use of mono and mixed lactic acid bacteria (LAB) cultures to determine suitable LAB combinations for a type II sourdough system. In this context, previously isolated sourdough LAB strains with antimicrobial activity, which included Lactobacillus plantarum PFC22, Lactobacillus brevis PFC31, Pediococcus acidilactici PFC38, and Lactobacillus sanfranciscensis PFC80, were used as mono or mixed culture combinations in a fermentation system to produce type II sourdough, and subsequently in bread dough production. Compared to the monoculture fermentation of dough, the use of mixed cultures shortened the adaptation period by half. In addition, the use of mixed cultures ensured higher microbial viability, and enhanced the fruity flavor during bread dough production. It was determined that the combination of L. plantarum PFC22 + P. acidilactici PFC38 + L. sanfranciscensis PFC80 is a promising culture mixture that can be used in the production of type II sourdough systems, and that may also contribute to an increase in metabolic activity during bread production process.     

Technological and molecular diversity of Lactobacillus plantarum strains isolated from naturally fermented sourdoughs

Thirty Lactobacillus (L.) plantarum strains, isolated from sourdough, were identified by biochemical tests as well as 16S rDNA sequencing and differentiated on the basis of technological properties, such as amylase, protease, phytase and antirope activities. These properties were shown to be widely differing among the strains, indicating a significant technological diversity. Genetic differentiation was achieved by restriction endonuclease analysis-pulsed field gel electrophoresis (REA-PFGE) that allowed the L. plantarum strains to be divided into 10 different genomic groups. Moreover, 32 different starters were employed in dough making experiments; each starter consisted of a single strain of L. plantarum associated with a maltose positive or a maltose negative yeast. The technological properties of the doughs were greatly influenced by the type of strain included in the starter. The time of leavening and the acidification activities detected in the dough were enhanced by the presence of L. plantarum strains. The bacterial and yeast contents and fermentation properties were statistically treated by principal component analysis (PCA), which allowed the discrimination of different typologies of dough. The study of the peculiar characteristics of different strains of L. plantarum is fundamental for a better understanding of their potential in affecting the nutritional value, quality and stability of the baked goods. L. plantarum strains are able to differentially influence the dough quality when employed as starters.     

Genotypic and phenotypic diversity of Lactobacillus rossiae strains isolated from sourdough

AIM: To characterize the genetic and phenotypic diversity of 33 strains of Lactobacillus rossiae. METHODS AND RESULTS: Genotypic identification was carried out by partial 16S rRNA gene sequence analysis. Genetic diversity was evaluated by RAPD-PCR analysis. Phenotypic diversity was evaluated through fermentative profile by Biolog system, proteinase and peptidase activities using synthetic substrates, and acidification capacity and amino acid profile during sourdough fermentation. The genetic analyses excluded clonal relatedness among the strains used. A large phenotypic diversity was found. It mainly concerned the capacity to use carbon sources available in sourdough during fermentation, the quotient of fermentation and the peptidase activities, especially towards proline containing synthetic substrates. The free amino acid profiles differed either for the total concentration or for the type of amino acids. With a few exceptions, proteinase activity towards wheat albumins and globulins was weak. CONCLUSIONS: Overall, no relationships between genetic and physiological analyses were found, and the strains examined showed a marked genetic and phenotypic heterogeneity. L. rossiae strains had interesting properties for application in sourdough fermentation. Although some strains combined several technological traits, the association of more strains seemed to be a requisite to get optimal sourdough characteristics. SIGNIFICANCE AND IMPACT OF THE STUDY: It represents the first characterization of the diversity within the L. rossiae species. Besides, it may represent an example of computerized analysis of genotypic and phenotypic information to select strains for improving sourdough characteristics.