Industrial Sourdough Research Articles

Technological and acid stress performance of yeast isolates from industrial sourdough

Yeast populations in sourdough have been poorly characterized from baking-relevant attributes as leavening activity in a stressful environment. In particular, the presence of extracellular acids represents an important challenge, which compromises the growth and fermentative metabolism of microbial starters. Here, we have analyzed the fungal diversity of six industrial sourdoughs identifying Saccharomyces cerevisiae and Kazachstania humilis as the predominant species. The volatile organic compounds (VOC) profile revealed differences between sourdough samples which can be attributed, at least in part, to their microbial composition and ingredients used in each one. In particular, K. humilis appeared to contribute to the presence octan-1-ol, 2-hepten-1-ol, 2-octen-1-ol, nonan-1-ol and 2-pentylfuran in the sourdough. A total of 45 yeast isolates were then analyzed in terms of CO2 production and tolerance to NaCl, ethanol, oxidative-, cold- and heat-stress, which allowed the selection of 5 S. cerevisiae isolates and 2 from both K. humilis and Torulaspora delbrueckii for further characterization. Overall, K. humilis isolates showed a higher total CO2 production and gassing rate than a commercial yeast strain used as a control. Hence, the isolated strains are potential candidates for the development of new sourdough starters and the basis for a further selection of variants with superior performance.

Comparison of homo- and heterofermentative lactic acid bacteria for implementation of fermented wheat bran in bread

Despite its potential health benefits, the integration of wheat bran into the food sector is difficult due to several adverse technological and sensory properties such as bitterness and grittiness.Sourdough fermentation is a promising strategy to improve the sensory quality of bran without inducing severe changes to the bran matrix. Therefore, identification of species/strains with potential for industrial sourdough fermentations is important. We compared the effects of different representatives of species of lactic acid bacteria (LAB) on wheat bran. Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus brevis, Lactobacillus sanfranciscensis and Fructobacillus fructosus produced highly individual fermentation patterns as judged from carbohydrate consumption and organic acid production. Interestingly, fructose was released during all bran fermentations and possibly influenced the fermentation profiles of obligately heterofermentative species to varying degrees. Except for the reduction of ferulic acid by L. plantarum and L. pentosus, analyses of phenolic compounds and alkylresorcinols suggested that only minor changes thereof were induced by the LAB metabolism. Sensory analysis of breads baked with fermented bran did not reveal significant differences regarding perceived bitterness and aftertaste.We conclude that in addition to more traditionally used sourdough species such as L. sanfranciscensis and L. brevis, also facultatively heterofermentative species such as L. plantarum and L. pentosus possess potential for industrial wheat bran fermentations and should be considered in further investigations.

Genomic analysis reveals Lactobacillus sanfranciscensis as stable element in traditional sourdoughs

Sourdough has played a significant role in human nutrition and culture for thousands of years and is still of eminent importance for human diet and the bakery industry. Lactobacillus sanfranciscensis is the predominant key bacterium in traditionally fermented sourdoughs.The genome of L. sanfranciscensis TMW 1.1304 isolated from an industrial sourdough fermentation was sequenced with a combined Sanger/454-pyrosequencing approach followed by gap closing by walking on fosmids. The sequencing data revealed a circular chromosomal sequence of 1,298,316 bp and two additional plasmids, pLS1 and pLS2, with sizes of 58,739 bp and 18,715 bp, which are predicted to encode 1,437, 63 and 19 orfs, respectively. The overall GC content of the chromosome is 34.71%. Several specific features appear to contribute to the ability of L. sanfranciscensis to outcompete other bacteria in the fermentation. L. sanfranciscensis contains the smallest genome within the lactobacilli and the highest density of ribosomal RNA operons per Mbp genome among all known genomes of free-living bacteria, which is important for the rapid growth characteristics of the organism. A high frequency of gene inactivation and elimination indicates a process of reductive evolution. The biosynthetic capacity for amino acids scarcely availably in cereals and exopolysaccharides reveal the molecular basis for an autochtonous sourdough organism with potential for further exploitation in functional foods. The presence of two CRISPR/cas loci versus a high number of transposable elements suggests recalcitrance to gene intrusion and high intrinsic genome plasticity.

Contribution of glutamate decarboxylase in Lactobacillus reuteri to acid resistance and persistence in sourdough fermentation

BackgroundAcid stress impacts the persistence of lactobacilli in industrial sourdough fermentations, and in intestinal ecosystems. However, the contribution of glutamate to acid resistance in lactobacilli has not been demonstrated experimentally, and evidence for the contribution of acid resistance to the competitiveness of lactobacilli in sourdough is lacking. It was therefore the aim of this study to investigate the ecological role of glutamate decarboxylase in L. reuteri.ResultsA gene coding for a putative glutamate decarboxylase, gadB, was identified in the genome of L. reuteri 100-23. Different from the organization of genetic loci coding for glutamate decarboxylase in other lactic acid bacteria, gadB was located adjacent to a putative glutaminase gene, gls3. An isogenic deletion mutant, L. reuteri ∆gadB, was generated by a double crossover method. L. reuteri 100-23 but not L. reuteri ∆gadB converted glutamate to γ-aminobutyrate (GABA) in phosphate butter (pH 2.5). In sourdough, both strains converted glutamine to glutamate but only L. reuteri 100-23 accumulated GABA. Glutamate addition to phosphate buffer, pH 2.5, improved survival of L. reuteri 100-23 100-fold. However, survival of L. reuteri ∆gadB remained essentially unchanged. The disruption of gadB did not affect growth of L. reuteri in mMRS or in sourdough. However, the wild type strain L. reuteri 100-23 displaced L. reuteri ∆gadB after 5 cycles of fermentation in back-slopped sourdough fermentations.ConclusionsThe conversion of glutamate to GABA by L. reuteri 100-23 contributes to acid resistance and to competitiveness in industrial sourdough fermentations. The organization of the gene cluster for glutamate conversion, and the availability of amino acids in cereals imply that glutamine rather than glutamate functions as the substrate for GABA formation. The exceptional coupling of glutamine deamidation to glutamate decarboxylation in L. reuteri likely reflects adaptation to cereal substrates.

Growth characterization of individual rye sourdough bacteria by isothermal microcalorimetry

The present work tests the feasibility of the isothermal microcalorimetry method to study the performance of individual lactic acid bacteria during solid-state fermentation in rye sourdough. Another aim was to elucidate the key factors leading to the formation of different microbial consortia in laboratory and industrial sourdough during continuous backslopping propagation. Strains of the individual LAB isolated from industrial and laboratory sourdough cycle were grown in 10 kGy irradiated rye dough in vials of an isothermal calorimeter and the power-time curves were obtained. Sugars, organic acids and free amino acids in the sourdough were measured. The OD-time curves of the LAB strains during growth in flour extract or MRS (De Man, Rogosa and Sharpe) broth were also determined. The maximum specific growth rates of Lactobacillus sakei, Lactobacillus brevis, Lactobacillus curvatus and Leuconostoc citreum strains that dominated in backslopped laboratory sourdough were higher than those of Lactobacillus helveticus, Lactobacillus panis, Lactobacillus vaginalis, Lactobacillus casei and Lactobacillus pontis strains originating from industrial sourdough. Industrial strains had higher specific growth rates below pH 4·8. It was supposed that during long-run industrial backslopping processes, the oxygen sensitive species start to dominate because of the O(2) protective effect of rye sourdough. Measurements of the power-time curves revealed that the LAB strains dominating in the industrial sourdough cycle had better acid tolerance but lower maximum growth rate and oxygen tolerance than species isolated from a laboratory sourdough cycle. Isothermal microcalorimetry combined with chemical analysis is a powerful method for characterization of sourdough fermentation process and determination of growth characteristics of individual bacteria in sourdough.

Effect of fermentation rate on endogenous leavening of Candida milleri in sour rye dough

Sourdough leavened breads rely upon an active yeast strain in the mature sourdough to provide the leavening capacity needed in the bread dough. A strain of Candida milleri yeast, previously known as a good gas producer, was grown together with a homofermentative or a heterofermentative lactobacillus in sourdough at 22, 25 or 28 °C in a one stage re-inoculation procedure. The focus was to recognize fermentation conditions of the sourdough (dough yield, DY250) favoring leavening in the final bread dough (DY200). Endogenous leavening of bread dough was enhanced by low temperature during the sourdough fermentation. The lower temperature delayed acidification of the sourdough, shortening the time that the yeast was exposed to high acidity and especially undissociated acids. This resulted in a higher leavening of the bread dough in both sourdoughs. The results suggest that it is possible to improve endogenous leavening capacity by optimizing the industrial sourdough process.

Evidence for Formation of Heterooligosaccharides by Lactobacillus sanfranciscensis during Growth in Wheat Sourdough

Lactobacillus sanfranciscensis is a key organism of the lactic microflora in traditional and industrial sourdough fermentations. In this paper we provide evidence for the formation of heterooligosaccharides (HeOS) by L. sanfranciscensis during growth in sourdough. To identify the HeOS based on HPAEC-PAD analysis, HeOS standards were synthesized by enzymatic reactions with L. sanfranciscensis levansucrase in a chemically defined system in the presence of raffinose, maltotriose, maltose, xylose, or arabinose as acceptor carbohydrates. The oligosaccharides known to originate from the corresponding acceptor reactions, 1(F)-beta-fructosylraffinose, 1(F)-beta-fructofuranosylmaltotriose, erlose (1(F)-beta-fructofuranosylmaltose), xylsucrose, 1(F)-beta-fructosylxylsucrose, and arabsucrose, were identified by HPAEC-PAD. Evidence for the formation of further tri-, tetra-, and pentasaccharides was provided. Wheat doughs with sucrose were fermented with L. sanfranciscesis TMW 1.392 or the isogenic, levansucrase-negative strain TMW 1.392Deltalev, and the analysis of dough extracts or invertase-treated dough extracts provided evidence for the formation of arabsucrose and erlose in sourdough in addition to 1-kestose and nystose.

The sourdough microflora: biodiversity and metabolic interactions

The production of sourdough bread can be traced back to ancient times. Sourdough is a mixture of flour and water that is fermented with lactic acid bacteria (LAB). Sourdough is an intermediate product and contains metabolically active yeast and LAB strains. The LAB that develop in the dough may originate from selected natural contaminants in the flour or from a starter culture containing one or more known species of LAB. Sourdough can be produced in bakeries or obtained from commercial suppliers. The microbial ecology of the sourdough fermentation is determined by ecological factors. Microbiological studies have revealed that more than 50 species of LAB, mostly species of the genus Lactobacillus, and more than 20 species of yeasts, especially species of the genera Saccharomyces and Candida, occur in this ecological niche. The sourdough microflora is composed of stable associations of lactobacilli and yeasts, in particular due to metabolic interactions. As shown for certain industrial sourdough processes, such microbial associations may endure for years, although the fermentation process runs under non-aseptic conditions. A reproducible and controlled composition and activity of the sourdough microflora is indispensable to achieve a constant quality of sourdough bread.

Reutericyclin: biological activity, mode of action, and potential applications.

Reutericyclin is an inhibitory compound produced by sourdough isolates of Lactobacillus reuteri that is structurally but not functionally related to naturally occurring tetramic acids. It is bacteriostatic or bactericidal to gram-positive bacteria based on its activity as a proton-ionophore, and a broad range of food-related spoilage organisms and pathogens is inhibited by reutericyclin. Gram-negative bacteria are resistant to reutericyclin because of the barrier properties of their outer membrane, and resistance of beer-spoiling lactobacilli towards hop bitter acids provides cross-protection to reutericyclin. Remarkably, reutericyclin-producing strains were shown to persist for a period of 10 years in an industrial sourdough fermentation, and reutericyclin was shown to be produced in concentrations active against competitors during growth of L. reuteri in sourdough. Based on the known properties of reutericyclin and L. reuteri, reutericyclin-producing strains may have applications in the biopreservation of foods. Furthermore, these strains were shown to colonize reconstituted lactobacilli-free mice at high levels. Therefore, they could serve as a suitable model system to evaluate a possible impact of antimicrobial compounds on the intestinal microflora of humans and animals.

Contribution of reutericyclin production to the stable persistence of Lactobacillus reuteri in an industrial sourdough fermentation

Reutericyclin is a small molecular weight antibiotic produced by the sourdough isolate Lactobacillus reuteri LTH2584. This strain was isolated from an industrial sourdough, SER, in 1988. To determine whether reutericyclin formation contributes to the stable persistence of L. reuteri in sourdough, evaluations were made on whether reutericyclin-producing strains were among L. reuteri isolates from the SER sourdough obtained in 1994 and 1998. These strains were characterised on species and strain level by physiological tests and randomly amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) patterns. Reutericyclin production in dough was evaluated by two methods, a bioassay and HPLC. Throughout 10 years of continuous propagation, reutericyclin-producing L. reuteri strains were present in SER sourdough. All isolates exhibited similar physiological properties and molecular typing revealed closely related patterns. Two isolates obtained in 1994 and 1998 were identical. Reutericyclin produced in situ by L. reuteri was active in dough against reutericyclin-sensitive L. sanfranciscensis. The reutericyclin concentration in dough fermented with L. reuteri was 5 mg kg −1. The results indicate that reutericyclin production contributed to the stable persistence of L. reuteri in sourdough. Because reutericyclin is produced in active concentrations during sourdough fermentations, it is a suitable candidate for use as natural preservative.

Monitoring the growth of Lactobacillus species during a rye flour fermentation

The natural microbial community conducting an industrial sourdough fermentation was investigated by molecular biological methods using the following strategy: strains were isolated and subjected to RAPD (randomly-amplified polymorphic DNA) PCR. After computer-supported pattern analysis and clustering of the strains the 16S rDNA of members of each distinct cluster were partially (530 bp) or completely (1570 bp) sequenced and identified by comparative sequence analysis. The predominant strains of this fermentation could be allotted to the species Lactobacillus amylovorus, Lactobacillus pontis and a species, which phylogenetically takes an intermediate position between L. pontis and L. panis. Sporadically, strains were identified as L. reuteri. In a second step the effect of external factors was investigated under the controlled conditions of a lab-scale process. Fermentations were carried out at 34°C, 40°C and 46°C. The development of the flora was consistent in independent fermentations as proved by RAPD typing of randomly-picked colonies. The microbial community in these fermentations was identical to those found in an industrial scale. The qualitative composition of the flora was not affected by the temperature. L. amylovorus was the dominant species. With increasing fermentation time, a shift toward the predominance of heterofermentative lactobacilli was observed. This finding was underlined by metabolic studies and stoichiometric calculations of the metabolic pathways. With increasing temperature the percentage of homofermentative organisms was reduced. Furthermore, the growth rate and the metabolic activity increased, followed by an immediate decrease of the growth rate at 46°C and lower terminal values of lactate, acetate and ethanol, respectively.

Lactobacillus frumenti sp. nov., a new lactic acid bacterium isolated from rye-bran fermentations with a long fermentation period.

Within the framework of the characterization of the microflora of an industrial sourdough fermentation, strains of Lactobacillus amylovorus, Lactobacillus pontis and two other strains were isolated which could not be associated with a valid species. These latter strains were Gram-positive, catalase-negative, non-spore-forming, non-motile rods that could be clearly differentiated from known species by 16S rDNA sequence analysis. For further characterization, the morphological, physiological (sugar fermentation, formation of DL-lactate, hydrolysis of arginine, growth temperature, CO2 production) and chemotaxonomic (G+C content, cell wall composition, SDS-PAGE of whole-cell proteins) properties were determined. Fitting of the complete 16S rDNA sequence into alignments of such sequences, together with the subsequent phylogenetic calculations, allowed the reconstruction of a phylogenetic tree. These data showed that the two strains were phylogenetically related but formed an independent cluster distinct from their closest neighbours, L. pontis, Lactobacillus panis, Lactobacillus oris, Lactobacillus vaginalis and Lactobacillus reuteri. The results of DNA-DNA hybridization experiments indicated that the two isolates represent a new Lactobacillus species, for which the name Lactobacillus frumenti is proposed; the type strain of this species is DSM 13145T (= LMG 19473T).

Multiplex PCR for the detection of Lactobacillus pontis and two related species in a sourdough fermentation.

A specific multiplex PCR assay based on the amplification of parts of the 16S rRNA molecule was designed. Primers derived from variable regions of the 16S rRNA provided a means of easily differentiating the species Lactobacillus pontis and Lactobacillus panis. They could be clearly discriminated from the phylogenetically related species Lactobacillus vaginalis, Lactobacillus oris, and Lactobacillus reuteri and from other lactobacilli commonly known to be present in sourdough. Other strains isolated together with L. pontis from an industrial sourdough fermentation could be clearly separated from these species by comparative sequence analysis and construction of a specific PCR primer. For a fast identification a DNA isolation protocol based on the ultrasonic lysis of cells from single colonies was developed. To demonstrate the potential of such techniques for tracking these organisms in a laboratory-scale fermentation, we combined the specific PCR assay with direct DNA extraction from the organisms in the sourdough without previous cultivation.

A Polyphasic Study on the Taxonomic Position of Industrial Sour Dough Yeasts

The sour dough bread making process is extensively used to produce wholesome palatable rye bread. The process is traditionally done using a back-slopping procedure. Traditional sour doughs in Finland comprise of lactic acid bacteria and yeasts. The yeasts present in these doughs have been enriched in the doughs due to their metabolic activities, e.g. acid tolerance. We characterized the yeasts in five major sour bread bakeries in Finland. We found that most of the commercial sour doughs contained yeasts which were similar to Candida milleri on the basis of 18S rDNA and EF-3 PCR-RFLP patterns and metabolic activities. Some of the bakery yeasts exhibited extensive karyotype polymorphism. The minimum growth temperature was 8 degrees C for C. milleri and also for most of sour dough yeasts.