The sourdough microflora Microbiological, biochemical and breadmaking characteristics of doughs fermented with freeze-dried mixed starters, freeze-dried wheat sourdough and mixed fresh-cell starters

Freeze-dried mixed starters, freeze-dried wheat sourdough and mixed fresh-cell starters made withLactobacillus sanfrancisco CBI,L. plantarum DC400 andSaccharomyces cerevisiae 141 and/orS. exiguus M14 were used for leavening wheat doughs, and their microbiological, biochemical and breadmaking characteristics were compared with those of Italian traditional doughs produced by baker's yeast. All the doughs fermented with starters had more balanced microbiological and biochemical characteristics than dough started with baker's yeast in which alcoholic fermentation end-products largely predominated. By using starters, the greatest lactic acid bacteria cell number and acetic acid production, were achieved, along with more complete profiles of volatile compounds and greater structural stability of fermented doughs. Fresh-cell starters showed higher microbial functionality and represented the only way to enrich the doughs withS. exiguus M14, some of which survived the freeze-drying process. No differences were detected between the two different types of freeze-dried starters and the subsequent use (10 times) of doughs initially produced with freezedried starters eliminated initial differences in the microbial functionality with respect to fresh-cell starters.     

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 degrees 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 2l batch fermentation bioreactor (12 h at 37 degrees 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.     

Liquid sourdough fermentation: industrial application perspectives

Sourdough fermentation is considered to play a key role to get improved flavour, texture, nutritional and shelf-life properties of bakery products. Since few years Barilla R&D has been focusing on liquid sourdough fermentation which may deserve several advantages with respect to traditional processes. The results showed that the micro-biota of sourdough markedly influences flavour and texture of bakery products. Particular attention has been paid to lactic acid bacteria and yeasts. Selected lactic acid bacteria and yeasts were tested in sourdough liquid fermentation as single strain or in association. The parameters of fermentations were optimized and standardized to set up a laboratory plant liquid fermentation. Only a few strains of lactic acid bacteria were found to be suitable for liquid fermentation alone or in association with yeasts. Fermentations were carried out at pilot plant and an industrial technology was developed. This work describes the results found for the organoleptic profile of an industrial bread started with liquid sourdough with respect to bakers' yeast bread without sourdough addition.     

Biodiversity of lactic acid bacteria and yeasts in spontaneously-fermented buckwheat and teff sourdoughs

In this study, four different laboratory scale gluten-free (GF) sourdoughs were developed from buckwheat or teff flours. The fermentations were initiated by the spontaneous biota of the flours and developed under two technological conditions (A and B). Sourdoughs were propagated by continuous back-slopping until the stability was reached. The composition of the stable biota occurring in each sourdough was assessed using both culture-dependent and -independent techniques. Overall, a broad spectrum of lactic acid bacteria (LAB) and yeasts species, belonging mainly to the genera Lactobacillus, Pediococcus, Leuconostoc, Kazachstania and Candida, were identified in the stable sourdoughs. Buckwheat and teff sourdoughs were dominated mainly by obligate or facultative heterofermentative LAB, which are commonly associated with traditional wheat or rye sourdoughs. However, the spontaneous fermentation of the GF flours resulted also in the selection of species which are not consider endemic to traditional sourdoughs, i.e. Pediococcus pentosaceus, Leuconostoc holzapfelii, Lactobacillus gallinarum, Lactobacillus vaginalis, Lactobacillus sakei, Lactobacillus graminis and Weissella cibaria. In general, the composition of the stable biota was strongly affected by the fermentation conditions, whilst Lactobacillus plantarum dominated in all buckwheat sourdoughs. Lactobacillus pontis is described for the first time as dominant species in teff sourdough. Among yeasts, Saccharomyces cerevisiae and Candida glabrata dominated teff sourdoughs, whereas the solely Kazachstania barnetti was isolated in buckwheat sourdough developed under condition A. This study allowed the identification and isolation of LAB and yeasts species which are highly competitive during fermentation of buckwheat or teff flours. Representatives of these species can be selected as starters for the production of sourdough destined to GF bread production.     

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.     

Impact of ecological factors on the stability of microbial associations in sourdough fermentation

The limits for the stability of the microbial association 1 (Lactobacillus sanfranciscensis and Candida humilis) and association 2 (Lactobacillus reuteri, Lactobacillus johnsonii and Issatchenkia orientalis) during sourdough fermentation were evaluated by investigating the effects of the ecological factors substrate, refreshment time, temperature, amount of backslopping and competing species in different combinations on their growth. Sourdoughs were fermented in 28 batches under different conditions using the associations and possible competing strains as starters. The dominating microbiota was characterized by bacteriological culture, rRNA gene sequence analysis and RAPD-PCR. Association 1 was found to be competitive in doughs with rye and wheat flour at temperatures between 20 and 30 °C, refreshment times of 12 and 24 h, amounts of backslopping dough from 5 to 20% and against all competing lactic acid bacteria and yeasts. The processing parameters for the competitiveness of the association 2 were temperatures of 35-40 °C, refreshment times of 12-24 h and the substrates rye bran, wheat and rye flour, but not in every case. Issatchenkia orientalis could only grow when enough oxygen was available. Its cell counts fell rapidly under the limit of detection when using high amounts of doughs (small ratio of surface to volume) and refreshment times of 12 h. In conclusion, the results demonstrated that the two associations were remarkably stable under most of the investigated process conditions.     

Effect of sourdough fermentation on stabilisation,and chemical and nutritional characteristics of wheat germ

Lactic acid bacteria strains were identified from wheat germ by 16S rRNA partial sequencing, subjected to RAPD-PCR typing and screened. Lactobacillus plantarum LB1 and Lactobacillus rossiae LB5 were used as starters to produce sourdough fermented wheat germ (SFWG). The chemical and nutritional characteristics of SFWG were compared to those of the raw wheat germ (RWG). Lipase activity in SFWG was ca. 2.6-fold lower than that found in RWG. As shown by SPME/GC/MS analysis, most of the volatile compounds derived from lipid oxidation during storage (40 days) were at markedly lower levels in SFWG compared to RWG. Fermentation of wheat germ increased of ca. 50% the concentration of free amino acids. Glu markedly decreased in SFWG, due to its conversion in GABA. The concentration of the anti-nutritional factor raffinose also decreased in SFWG. The in vitro protein digestibility, the concentration of total phenols, phytase and antioxidant activities were increased by fermentation.     

Microbiological profile of maize and rye flours, and sourdough used for the manufacture of traditional Portuguese bread

A thorough microbiological study of maize and rye flours, and sourdoughs obtained therefrom for eventual manufacture of broa – a dark sour bread typical in Northern Portugal, following artisanal practices, was carried out. Towards this purpose, samples were supplied by 14 artisanal producers, selected from 4 sub-regions, during two periods of the year. Total viable counts, as well as viable mesophilic and thermophilic microorganisms, yeasts and molds, Gram⁻ rods, endospore-forming and nonsporing Gram⁺ rods, and catalase⁺ and catalase⁻ Gram⁺ cocci were assayed for. The comprehensive experimental dataset unfolded a unique and rather complex wild microflora in flours and sourdoughs throughout the whole region, which did not discriminate among sub-regions or seasons, or flour source for that matter. However, fermentation played a major role upon the numbers of the various microbial groups: the viable counts of yeasts, lactobacilli, streptococci, lactococci, enterococci and leuconostocs increased, whereas those of molds, Enterobacteriaceae, Pseudomonadaceae, staphylococci and micrococci decreased.     

Adaptability of lactic acid bacteria and yeasts to sourdoughs prepared from cereals, pseudocereals and cassava and use of competitive strains as starters

The adaptability of lactic acid bacteria (LAB) and yeasts to sourdoughs prepared from cereals, pseudocereals and cassava was investigated using PCR-DGGE and bacteriological culture combined with rRNA gene sequence analysis. Sourdoughs were prepared either from flours of the cereals wheat, rye, oat, barley, rice, maize, and millet, or from the pseudocereals amaranth, quinoa, and buckwheat, or from cassava, using a starter consisting of various species of LAB and yeasts. Doughs were propagated until a stable microbiota was established. The dominant LAB and yeast species were Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus paralimentarius, Lactobacillus plantarum, Lactobacillus pontis, Lactobacillus spicheri, Issatchenkia orientalis and Saccharomyces cerevisiae. The proportion of the species within the microbiota varied. L. paralimentarius dominated in the pseudocereal sourdoughs, L. fermentum, L. plantarum and L. spicheri in the cassava sourdough, and L. fermentum, L. helveticus and L. pontis in the cereal sourdoughs. S. cerevisiae constituted the dominating yeast, except for quinoa sourdough, where I. orientalis also reached similar counts, and buckwheat and oat sourdoughs, where no yeasts could be detected. To assess the usefulness of competitive LAB and yeasts as starters, the fermentations were repeated using flours from rice, maize, millet and the pseudocereals, and by starting the dough fermentation with selected dominant strains. At the end of fermentation, most of starter strains belonged to the dominating microbiota. For the rice, millet and quinoa sourdoughs the species composition was similar to that of the prior fermentation, whereas in the other sourdoughs, the composition differed.     

Description of the microflora of sourdoughs by culture-dependent and culture-independent methods

Four types of sourdoughs (L, C, B, Q) from artisanal bakeries in Northern Italy were studied using culture-dependent and culture-independent methods. In all samples, the yeast numbers ranged from 160 to 10⁷ cfu/g, and the numbers of lactic acid bacteria (LAB) ranged from 10³ to 10⁹ cfu/g. The isolated LAB were sequenced, and a similarity was noted between two samples (C, Q), both in terms of the species that were present and in terms of the percentage of isolates. In these two samples, Lactobacillus plantarum accounted for 73% and 89% of the bacteria, and Lactobacillus brevis represented 27% and 11%. In the third sample (B), however, the dominant LAB isolate was Lb. brevis (73%), while Lb. plantarum accounted for only 27%. The fourth sourdough (L) was completely different from the others. In this sample, the most prominent isolate was Weisella cibaria (56%), followed by Lb. plantarum (36%) and Pediococcus pentosaceus (8%). In three out of four samples (L, C and Q), all of the yeasts isolated were identified as Saccharomyces cerevisiae, yet only Candida humilis (90%) and Candida milleri (10%) were isolated in the fourth sample (B). The microbial ecology of the sourdoughs was also examined with direct methods. The results obtained by culture-independent methods and DGGE analysis underline a partial correspondence between the DNA and RNA analysis. These results demonstrate the importance of using a combined analytical approach to explore the microbial communities of sourdoughs.     

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.     

Effects of two different sourdoughs on the characteristics of Pandoro, a typical Italian sweet leavened baked good

Pandoro is a traditional Italian sweet-leavened baked product, usually consumed at Christmas. It is manufactured according to specific procedures and preparation starts from a sourdough called ‘‘madre’’ (mother sponge) continuously refreshed. This sourdough is the result of a complex microbial association including yeasts and lactic acid bacteria (LAB) and its use can improve sensory quality and shelf-life of the resulting products. The aim of this work was to evaluate the effect of the use of two different sourdoughs matured at different temperature (13 and 19 °C) on some metabolites, which can affect the organoleptic characteristics of Pandoro. Different samples, taken throughout the 19 h of process, were analysed for the determination of yeast and LAB counts, pH, a_w, carbohydrates, organic acids content and volatile profile. The results showed that, at the end of fermentation process, the sourdough propagated at 19 °C reached lower pH values than that at 13 °C. This was probably due to higher LAB counts (1 log unit higher), resulting also in higher lactic acid concentration and faster sugars depletion. On the contrary, temperature of dough during maturation did not affected yeast concentrations. Different production processes strongly affected also the volatile profile, both of sourdough at the end of fermentation and of the final products.     

Lactic acid bacteria and yeasts isolated from the starter doughs for Chinese steamed buns in Thailand

Isolation of lactic acid bacteria (LAB) and yeasts from the starter dough of Chinese steamed buns from four different commercial sources in Thailand was carried out. Thirty-one lactic acid bacteria and eight yeast strains were isolated. Total counts of LAB were from 1.8 × 10⁴ to 10⁸ colonies/g sample, whilst yeasts were very low from 10 to 2.3 × 10² colonies/g sample. The pH values of all starter doughs ranged from 3.36 to 3.52 and the Total Titratable Acidity (TTA) varied from 11.1 to 17.0 ml of 0.1 N NaOH/10 g dough. All LAB isolates were identified as Lactobacillus. The phenotypic characteristics were used to cluster all the LAB isolates into two major groups (Group A and Group B), with the B group subdivided into four groups. Phylogenetic analysis, based upon partial 16S rRNA gene sequences, showed that isolates of Group A, which all contained meso-diaminopimelic acid in their cell wall and produced dl-lactic acid, were closely related to Lactobacillus plantarum, whilst the strains of Group B that produced l-lactic acid were closely related to Lactobacillus casei. For yeasts, eight isolates based on the D1/D2 domain sequences of 26S rRNA were identified as Candida tropicalis, Pichia stipitis, Candida parapsilosis, Issatchenkia orientalis and Saccharomyces cerevisiae.     

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.     

Isolation and characterization of the microbial population of different South African kefir grains

Kefir, a slightly acidic fermented milk, is produced by adding lactic acid bacteria and yeasts, in the form of grains, to milk. The bacteria and yeasts present in the kefir grains are known to vary widely. Selective growth media and morphological and biochemical characteristics were used for the isolation and identification of the microbes present in the grains from eight different sources in South Africa. The kefir grains were activated in milk for only 24 h to prevent any changes in the microbial population of the grains. The microbial numbers varied between 6.4 × 10⁴ and 8.5 × 10 ⁸  cfu/g on the media selective for the bacterial species and between 1.5 × 10 ⁵ and 3.7 × 10 ⁸   cfu/g on the media selective for the yeast species. The bacterial genera that were identified included Lactobacillus , Leuconostoc and Lactococcus and the yeast genera included Zygosaccharomyces , Candida and Saccharomyces . The distribution frequencies of the microbes in the different grains were determined and most of the grains were dominated by two microbial species. No pediococci, acetic acid bacteria or propionibacteria were detected.     

Impact of sourdough on the texture of bread

Sourdough has been used since ancient times and its ability to improve the quality and increase the shelf-life of bread has been widely described. During sourdough fermentation, lactic acid bacteria (LAB) produce a number of metabolites which have been shown to have a positive effect on the texture and staling of bread, e.g. organic acids, exopolysaccharides (EPS) and/or enzymes. EPS produced by LAB have the potential to replace more expensive hydrocolloids used as bread improvers. Organic acids affect the protein and starch fractions of flour. Additionally, the drop in pH associated with acid production causes an increase in the proteases and amylases activity of the flour, thus leading to a reduction in staling. While improving the textural qualities of bread, sourdough fermentation also results in increased mineral bioavailability and reduced phytate content. In this review we will be discussing the effect of sourdough on wheat and rye bread as well as the potential of sourdough to improve the quality of gluten-free bread.     

The use of sourdough fermented by antifungal LAB to reduce the amount of calcium propionate in bread

Addition of sourdough is a common practice in the bakery industry to improve, among other quality parameters, the shelf life of bread. In this study, sourdough fermented by antifungal Lactobacillus plantarum strains was investigated for the ability to inhibit growth of common bread spoilage fungi. In both in vitro and sourdough wheat bread system, the antifungal sourdoughs significantly affected the outgrowth of Aspergillus niger, Fusarium culmorum, or Penicillium expansum spores, however on wheat bread outgrowth of Penicillium roqueforti spores was not affected. In an attempt to reduce the amounts of chemical additives in bread, the antifungal sourdoughs were used in combination with calcium propionate (CAP) and possible synergistic effects were evaluated. Presence of 3000 ppm CAP in the bread did not affect the outgrowth of P. roqueforti, whereas outgrowth of the other fungi was retarded. A strong synergistic effect was observed when CAP and antifungal sourdoughs were combined into the bread formulation, and outgrowth of P. roqueforti was affected. The use of reduced CAP amount (1000 ppm) showed significant inhibition only when antifungal sourdough was added. Remarkably, the increase in shelf life achieved was higher than that obtained using 3000 ppm of CAP alone. In conclusion, the results of this study clearly show that the addition of antifungal sourdough has the potential to reduce the levels of chemical additives needed in the bakery industry to ensure the microbiological safety of bread.     

Antifungal activity of sourdough fermented wheat germ used as an ingredient for bread making

This study aimed at investigating the antifungal activity of sourdough fermented (Lactobacillus plantarum LB1 and Lactobacillus rossiae LB5) wheat germ (SFWG). Preliminarily, methanol and water/salt-soluble extracts from SFWG were assayed by agar diffusion towards Penicillium roqueforti DPPMAF1. As shown by hyphal radial growth rate, the water/salt-soluble extract showed the inhibition of various fungi isolated from bakeries. The antifungal activity was attributed to a mixture of organic acids and peptides which were synthesized during fermentation. Formic (24.7 mM) acid showed the highest antifungal activity. Four peptides, having similarities with well known antifungal sequences, were identified and chemically synthesized. The minimal inhibitory concentration was 2.5–15.2 mg/ml. Slices of bread made by addition of 4% (wt/wt) of freeze dried SFWG were packed in polyethylene bags and stored at room temperature. Slices did not show contamination by fungi until at least 28 days of storage and behaved as the calcium propionate (0.3%, wt/wt).     

Taxonomic structure of the yeasts and lactic acid bacteria microbiota of pineapple (Ananas comosus L. Merr.) and use of autochthonous starters for minimally processing

Pichia guilliermondii was the only identified yeast in pineapple fruits. Lactobacillus plantarum and Lactobacillus rossiae were the main identified species of lactic acid bacteria. Typing of lactic acid bacteria differentiated isolates depending on the layers. L. plantarum 1OR12 and L. rossiae 2MR10 were selected within the lactic acid bacteria isolates based on the kinetics of growth and acidification. Five technological options, including minimal processing, were considered for pineapple: heating at 72 °C for 15 s (HP); spontaneous fermentation without (FP) or followed by heating (FHP), and fermentation by selected autochthonous L. plantarum 1OR12 and L. rossiae 2MR10 without (SP) or preceded by heating (HSP). After 30 days of storage at 4 °C, HSP and SP had a number of lactic acid bacteria 1000 to 1,000,000 times higher than the other processed pineapples. The number of yeasts was the lowest in HSP and SP. The Community Level Catabolic Profiles of processed pineapples indirectly confirmed the capacity of autochthonous starters to dominate during fermentation. HSP and SP also showed the highest antioxidant activity and firmness, the better preservation of the natural colours and were preferred for odour and overall acceptability.