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.     

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.     

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.     

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.     

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.     

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.     

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.     

Characteristics of bread making doughs: influence of sourdough fermentation on the fundamental rheological properties

The proofing process is an essential step in the bread making technology in providing a link between the bubble structure created in the mixer and the final baked loaf structure. This research was carried out to test the effect of two different types of fermentation on bread making dough properties. Fundamental rheological tests were used to evaluate and compare doughs prepared by using compressed yeast and only natural yeast. Dynamic oscillation tests were performed using a controlled stress--strain rheometer; the phase angle (δ) and the value of the storage modulus (G′) were measured for all samples. Significant differences were found between the doughs which were made using commercial compressed yeast (baker's yeast (BY)) and those prepared using sourdough (SD). In particular SD was less elastic, less firm and easily extensible having higher phase angle and lower G′.     

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.     

Production of microbial exopolysaccharides in the sourdough and its effects on the rheological properties of dough

Exopolysaccharides (EPS) are exogenous microbial metabolites which are secreted mainly by bacteria and microalgae during growth. In addition to natural polysaccharides present in cereal grains flour and dough, microbial flora is usually involved in production of polysaccharide on sourdough fermentation. Total polysaccharides (microbial and flour) were extracted from sourdough and dough samples dehydrated and were added at the rate of 0%, 0.25%, 0.5%, 1%, 1.5%, 2% and 2.5% (w/w flour based) on the dough to investigate its effects on the rheological properties of the dough. Addition of polysaccharides to the dough increased the water absorption and decreased the dough softening after 20 min. Resistance to extension after 45, 90 and 135 min resting time was decreased by increasing the percentage of the added polysaccharides. Longer fermentation time for each level of polysaccharides led to greater stability. No significant differences were observed in the extensibility of dough. The overall effects of different levels of added polysaccharides resulted in a decrease in resistance to extension ratio of the samples. Energy input decreased in all cases. It seems therefore that addition of polysaccharides may be useful when bread is to be made with stronger flour and longer fermentation time is needed.     

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.     

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).     

The peptide hydrolase system of Lactobacillus reuteri.

Peptide hydrolase system of Lactobacillus reuteri CRL 1098, a lactic acid bacteria of sourdough origin, was investigated. This microorganism has a broad range of peptidases consisting of an active aminopeptidase, X-Prolyl-dipeptidylaminopeptidase, dipeptidase and tripeptidase. Aminopeptidase, iminopeptidase and endopeptidase are most likely located in the cytoplasmic fraction showing no detectable association with the cell membrane, while dipeptidase and tripeptidase are mainly associated with the latter fraction. The peptidases are metalloenzymes activated by Co²⁺ and inhibited by Cu²⁺, Hg²⁺, Cd²⁺ and by metal-complexing reagents. The aminopeptidase activity inhibited by EDTA can be restored by Mn²⁺ while that of di- and tripeptidase treated with 1,10-phenantroline can be restored by Zn²⁺ and Co²⁺, respectively.     

The peptide hydrolase system of Lactobacillus reuteri

Peptide hydrolase system of Lactobacillus reuteri CRL 1098, a lactic acid bacteria of sourdough origin, was investigated. This microorganism has a broad range of peptidases consisting of an active aminopeptidase, X-Prolyl-dipeptidylaminopeptidase, dipeptidase and tripeptidase. Aminopeptidase, iminopeptidase and endopeptidase are most likely located in the cytoplasmic fraction showing no detectable association with the cell membrane, while dipeptidase and tripeptidase are mainly associated with the latter fraction. The peptidases are metalloenzymes activated by Co²⁺ and inhibited by Cu²⁺, Hg²⁺, Cd²⁺ and by metal-complexing reagents. The aminopeptidase activity inhibited by EDTA can be restored by Mn²⁺ while that of di- and tripeptidase treated with 1,10-phenantroline can be restored by Zn²⁺ and Co²⁺, respectively.     

Generation of aroma compounds in sourdough: effects of stress exposure and lactobacilli-yeasts interactions

The effects of the interaction between Saccharomyces cerevisiae LBS and Lactobacillus sanfranciscensis LSCE1 and of their responses to acid, oxidative or osmotic stress on alcohol and aroma production were assessed. The exposure of S. cerevisiae LBS and L. sanfranciscensis LSCE1 cells to oxidative, acid or osmotic sub-lethal stress gave rise to a common or specific responses. Gamma-decalactone, 2(5H)-furanones and aldehydes were overproduced by LAB following oxidative stress. The acid stress induced both in yeasts and LAB, as well as in their co-cultures, a relevant accumulation of isovaleric and acetic acids and higher alcohols. A cross-exposure of yeasts and LAB to their preconditioned media, generated in S. cerevisiae a release of esters including esters of long-chain unsaturated fatty acids coming from membrane phospholipids. These esters were excreted also by yeasts following a pressure stress.     

Effects of pectinolytic yeast on the microbial composition and spoilage of olive fermentations

This study resulted in the identification of pectinolytic yeasts in directly brined Sicilian-style green olive fermentations and examination of the influence of those yeasts on the microbial composition and quality of fermented olives. Firstly, defective olives processed in Northern California from 2007 to 2008 and characterized by high levels of mesocarp tissue degradation were found to contain distinct yeast and bacterial populations according to DNA sequence-based analyses. Strains of (pectinolytic) Saccharomyces cerevisiae, Pichia manshurica, Pichia kudriavzevii, and Candida boidinii isolated from directly brined olives were then inoculated into laboratory-scale olive fermentations to quantify the effects of individual yeast strains on the olives. The pH, titratable acidity, and numbers of lactic acid bacteria (LAB) and yeasts varied between the fermentations and fermentations inoculated with P. kudriavzevii and C. boidinii promoted the development of LAB populations. Olive tissue structural integrity declined significantly within 30, 74, and 192 days after the inoculation of pectinolytic S. cerevisiae, P. manshurica and C. boidinii, respectively. In comparison, tissue integrity of olives in control fermentations remained intact although pectinolytic yeasts were present. Notably, pectinolytic yeasts were not found in fermentations inoculated with (non-pectinolytic) P. kudriavzevii and olives exposed to a 1:1 ratio of P. kudriavzevii and P. manshurica exhibited no significant tissue defects. This study showed that pectinolytic yeast are important components of directly brined green olive fermentations and damage caused by pectinolytic yeasts might be prevented by other microbial colonists of the olives.     

The effects of wheat sourdough on glutenin patterns, dough rheology and bread properties

Sourdough was prepared with cellular suspension containing 10⁹ cfu of each strain mL⁻¹ and incubated at 28 °C for 24 h and at 37 °C for 4 h. Two different sourdough levels (20 and 40%) were used in bread dough preparation. The bread doughs were proofed at 30 °C and 85% relative humidity for 60/120/180 min. When glutenin changes that occurred in samples 17, 18, 19, and 20 (40% SD 28) are compared with those that appeared in controls, it is obvious that, the relative intensities of some of the protein bands slightly decreased and a few fainter protein bands appeared (which did not exist in controls). A few fainter protein bands corresponding to the MM ≈ 25 kDa (high-mobility region) and the MM ≈ 66 kDa (low-mobility region) were appeared in the same samples. In the samples prepared with 20% sourdoughs incubated at 28 or 37 °C, the bands were still evident after 180 min of proof. This can be explained that glutenin fractions were not hydrolysed in these applications due to the delay in pH drop. The use of 40% sourdough incubated at 28 °C for 24 h resulted in sticky doughs and breads with lower volume, harder texture, unsatisfactory crumb grain and unpleasant flavour than the rest of the samples. The use of sourdoughs incubated at 37 °C for 4 h caused positive effect on loaf volumes, specific loaf volumes and crumb structure.