Free D- and L-Amino Acid Evolution During Sourdough Fermentation and Baking

The evolution of free D- and L-amino acids in sourdoughs started with various lactic acid bacteria (LAB) and yeasts was studied. Lactobacillus brevis subsp. lindneri CB1 and Lactobacillus plantrum DC400 had high proteolytic activity. During sourdough fermentation, Saccharomyces cerevisiae 141 and Saccharomyces exiguus M14 sequentially utilized free amino acids produced by bacterial activity. Due to increased cell yeast autolysis, more S. exiguus M14 inocula caused more free amino acids which were partially utilized by LAB without causing hydrolysis of wheat flour protein. D-alanine, D-glutamic acid and traces of other D-isomers were observed in sourdoughs fermented with L. brevis subsp. lindneri CB1 and S. cerevisiae 141. Free total D- and L-amino acid content decreased by more than 44% after baking the sourdoughs. No abiotic generation of new D-amino acid isomers was detected in the baked sourdoughs.     

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.     

Fundamental study on the effect of hydrostatic pressure treatment on the bread-making performance of oat flour

The use of sourdough in wheat and rye breads has been extensively studied; however, little is known about its potential effect when baking oat bread. Consequently, the impact of sourdough on oat bread quality was investigated. Two different sourdoughs were prepared from wholegrain oat flour without the addition of starter cultures, by continuous propagation at 28 (SD 28) or 37 °C (SD 37) until the composition of the lactic acid bacteria remained stable. The dominant LAB were identified by sequence analysis of the 16S rDNA isolated from pure cultures. LAB from SD 28 belonged to the species Leuconostoc argentinum, Pedicoccus pentosaceus and Weissella cibaria, while Lactobacillus coryniformis dominated SD 37. The isolated LAB were further used as starter cultures for the production of oat sourdoughs. Fundamental rheology revealed softening of the sourdoughs compared to non-acidified and chemically acidified controls, which could not be attributed to proteolytic activity. Incorporation of oat sourdough into an oat bread recipe resulted in significantly increased loaf-specific volume as well as improved texture, independent of addition level or sourdough type. Overall, the results of this study show that sourdoughs containing lactic acid bacteria isolated from oats have the potential to enhance oat bread quality.     

Bread quality by substituting normal and waxy hull-less barley (<Emphasis Type="Italic">Hordeum Vulgare</Emphasis> L.) flours

Substitution of regular and waxy hull-less barley flour was evaluated in pan breads prepared from the blends of barley cvs. Saessal (SSWB) and Saechalssal (SCWB) with wheat flour. Effect of barley type and barley flour level (10, 20, and 30%) was investigated on compositions, dough development, and bread qualities. Compared to 100% wheat flour, increasing barley flour increased ash from 0.36 to 0.67%, and β-glucan from 0.1 to 1.91%. Pasting viscosity exhibited higher peak viscosity, through, and breakdown in barley flour blends, showing higher viscosity in SSWB than SCWB. Optimum water absorption and mixing time were increased in barley-wheat flour blends. Substitution of 10% barley flour had no significant difference from wheat bread in bread volume and crumb firmness statistically (p<0.05). The SSWB showed better bread qualities in terms of bread volume and crumb firmness than SCWB. The β-glucan content was 0.13% in wheat bread, but ranged from 0.45 to 1.05% in barley breads.     

Identification of lactic acid bacteria isolated from oat sourdoughs and investigation into their potential for the improvement of oat bread quality

The use of sourdough in wheat and rye breads has been extensively studied; however, little is known about its potential effect when baking oat bread. Consequently, the impact of sourdough on oat bread quality was investigated. Two different sourdoughs were prepared from wholegrain oat flour without the addition of starter cultures, by continuous propagation at 28 (SD 28) or 37 °C (SD 37) until the composition of the lactic acid bacteria remained stable. The dominant LAB were identified by sequence analysis of the 16S rDNA isolated from pure cultures. LAB from SD 28 belonged to the species Leuconostoc argentinum, Pedicoccus pentosaceus and Weissella cibaria, while Lactobacillus coryniformis dominated SD 37. The isolated LAB were further used as starter cultures for the production of oat sourdoughs. Fundamental rheology revealed softening of the sourdoughs compared to non-acidified and chemically acidified controls, which could not be attributed to proteolytic activity. Incorporation of oat sourdough into an oat bread recipe resulted in significantly increased loaf-specific volume as well as improved texture, independent of addition level or sourdough type. Overall, the results of this study show that sourdoughs containing lactic acid bacteria isolated from oats have the potential to enhance oat bread quality.     

Phenotypic and molecular identification and clustering of lactic acid bacteria and yeasts from wheat (species Triticum durum and Triticum aestivum) sourdoughs of Southern Italy

The microflora of 25 wheat sourdoughs from the Apulia region, Southern Italy, was characterized. The sourdoughs were mainly produced from Triticum durum wheat. The number of lactic acid bacteria and yeasts ranged from ca. log 7.5 to log 9.3 colony forming units (cfu)/g and from log 5.5 to log 8.4 cfu/g, respectively. About 38% of the 317 isolates of lactic acid bacteria were identified by conventional physiological and biochemical tests. Phenotypic identification was confirmed by 16S rDNA and 16S/23S rRNA spacer region PCR. Overall, 30% of the isolates were identified as Lactobacillus sanfranciscensis, 20% as Lb. alimentarius, 14% as Lb. brevis, 12% as Leuconostoc citreum, 7% as Lb. plantarum, 6% as Lactococcus lactis subsp. lactis, 4% as Lb. fermentum and Lb. acidophilus, 2% as Weissella confusa and 1% as Lb. delbrueckii subsp. delbrueckii. Some of these species have not been previously isolated from sourdoughs. Since bakers yeast is widely used in sourdough production, Saccharomyces cerevisiae was largely found. The phenotypical relationships within the main lactic acid bacteria identified were established by using cluster analysis. A microbial map of the 25 sourdoughs was plotted showing characteristic associations among lactic acid bacteria and differences in the lactic acid bacteria species which were mainly due to the species of wheat flour, use of bakers yeast and type of bread.     

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.     

Microbial Diversity of Type I Sourdoughs Prepared and Back‐Slopped with Wholemeal and Refined Soft (Triticum aestivum) Wheat Flours

The fermentation of type I sourdough was studied for 20 d with daily back‐slopping under laboratory and artisan bakery conditions using 1 wholemeal and 2 refined soft wheat (Triticum aestivum) flours. The sourdough bacterial and yeast diversity and dynamics were investigated by plate counting and a combination of culture‐dependent and culture‐independent PCR‐DGGE approach. The pH, total titrable acidity, and concentration of key organic acids (phytic, lactic, and acetic) were measured. Three flours differed for both chemical and rheological properties. A microbial succession was observed, with the atypical sourdough species detected at day 0 (i.e. Lactococcus lactis and Leuconostoc holzapfelii/citreum group for bacteria and Candida silvae and Wickerhamomyces anomalus for yeasts) being progressively replaced by taxa more adapted to the sourdough ecosystem (Lactobacillus brevis, Lactobacillus alimentarius/paralimentarius, Saccharomyces cerevisiae). In mature sourdoughs, a notably different species composition was observed. As sourdoughs propagated with the same flour at laboratory and artisan bakery level were compared, the influence of both the substrate and the propagation environment on microbial diversity was assumed.     

Investigation of Microbial Communities of Chinese Sourdoughs Using Culture‐Dependent and DGGE Approaches

Sourdough is typically characterized by the complex microbial communities mainly comprising of yeasts and lactic acid bacteria (LAB). The objective of this study was to explore the microbiota of Chinese traditional sourdoughs collected from different areas of China using culture‐dependent and denaturing gradient gel electrophoresis (DGGE) methods. A total of 131 yeasts, 2 molds, and 106 LAB strains were isolated and identified. Based on the culture‐dependent analysis, the populations of yeasts and LAB were at the level of 10⁵ to 10⁷ and 10⁶ to 10⁷ cfu/g, respectively. Similarly, the results of RT‐qPCR showed that the values of total yeasts and LAB populations were in the range of 10⁶ to 10⁷ and 10⁷ to 10⁸ copies/g, respectively. Using culture‐dependent method, a total of 7 yeasts, 2 molds and 7 LAB species were identified. Results showed that Saccharomyces cerevisiae and Lactobacillus plantarum were the predominant species among the yeasts and LAB microflora. Similarly, using PCR‐DGGE approach, 7 yeasts, 1 mold and 9 LAB species were detected. The yeast, S. cerevisiae, represented the predominant, while the yeast Candida tropicalis represented the subdominant species of the yeast community. Among the LAB community, Lactobacillus sanfranciscensis was the predominant species, while Lactococcus qarvieae, Enterococcus faecium, Lactobacillus delbrueckii and Enterococcus cecorum were among the less dominant species.     

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.     

Textural, Water Holding, and Sensory Properties of Low-fat Pork Bologna with Normal or Waxy Starch Hull-less Barley

All ultra-low-fat (< 1%) pork bolognas had similar cook yield and composition. Addition of 4% hull-less waxy barley flour or meal to formulations provided the greatest purge control; 4% normal starch barley, wheat flour and potato starch were intermediate; 0.25% kappa-carrageenan or 1% soy protein concentrate had little effect on water holding and texture. Expressible moisture and purge were significantly correlated to moisture content and batter viscosity. Formulations with wheat flour and waxy barley meal were scored the firmest, while bologna with potato starch required the most force to compress. For most sensory properties, barley fractions performed similarly to wheat flour; however, waxy barley provided superior water holding during storage.     

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.     

Phenotypic characterization of lactic acid bacteria from sourdoughs for Altamura bread produced in Apulia (Southern Italy)

In order to study the composition of the lactic acid bacteria (LAB) community of sourdoughs used for the manufacture of Altamura bread, a traditional durum wheat bread produced in Apulia (Southern Italy), 111 strains of LAB were isolated and characterized. The phenotypic characterization of the isolates, carried out using a set of 29 tests, allowed the identification of 15 clusters at the 80% similarity level by hierarchical cluster analysis. Of the isolates, 88% were identified as facultatively heterofermentative LAB (Lactobacillus plantarum, Lb. paracasei, Lb. casei) and 12% as heterofermentative LAB (Lb. brevis, Leuconostoc mesenteroides). SDS-PAGE profiles of whole cell proteins of 68 strains confirmed the identification. Both the diversity and structure of the lactic microflora for sourdoughs for Altamura bread varied among samples.     

不同发酵基质的酸面团对酵母面团体系面包烘焙及老化特性的影响

应用分离自我国传统酸面团的区域特色乳酸菌--旧金山乳杆菌分别发酵小麦粉和小麦麸皮基质制成（小麦/麦麸）酸面团,研究了两种不同发酵基质的酸面团及其添加量对酵母面团体系面包烘焙及老化特性的影响。结果表明：与小麦粉制作的空白组面包相比,小麦酸面团可以明显改善面包的比容和感官品质;添加未发酵麦麸制作的非酸面团麦麸面包品质低于空白组,但引入麦麸酸面团（10%、20%、30%）后面包比容和感官评定得分均高于相对应的非酸面团麦麸面包。小麦酸面团和麦麸酸面团以及小麦麸皮均可以改善面包的老化特性,在相同贮藏期内,酸面团面包和麦麸面包的硬度增加量、水分迁移量和老化焓值都低于空白组,并且添加麦麸酸面团的面包其硬度和老化焓值都低于相对应的非酸面团麦麸面包。     

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.     

CELLULASES OF PLANT AND MICROBIAL ORIGIN IN GERMINATING BARLEY

Cellulase activity in crude extracts of germinating barley is at least five-fold higher in hulled than in hull-less cultivars. Surface sterilization of the grain prior to germination reduces cellulase to levels that are similar in all cultivars. In hulled barley most of the cellulase detected during germination is probably of microbial origin, but there is circumstantial evidence to suggest that significant activity can also be attributed to cellulases of plant origin. The micro-organisms associated with germinating grain reside largely in the hull and the major fungal species present on the hulled variety Clipper were Aureobasidium pullulans, Rhizopus stolonifer and Penicillium spp. Surface sterilization of the grain with silver nitrate, followed by steeping in a solution containing antibiotics, completely eliminated microbial growth on hull-less cultivars. After similar treatment of a hulled barley two yeasts could be detected. The results emphasize the importance of eliminating microbial growth prior to studies of polysaccharide hydrolases which develop during the germination of barley and which are assumed to be of plant origin.     

Effect of flour blending on functional, baking and organoleptic characteristics of bread

Soybean (full‐fat and defatted) and barley flours were incorporated into wheat flour at 5, 10, 15 and 20% substitution levels. The gluten content, sedimentation value and water absorption capacity of the flour blends and the mixing time of the dough decreased with increase in the level of soybean and barley flour separately and in combinations. Protein and glutelin contents increased significantly on blending of soyflour (full‐fat and defatted) to bread wheat flour. The breads prepared from the blends also varied in their loaf weight, loaf volume and sensory characteristics. The bread volume decreased with increasing amount of non‐wheat flour substitution. The crumb colour changed from creamish white to dull brown and a gradual hardening of crumb texture was observed as the addition of soybean (full‐fat and defatted) and barley flours increased. At the higher levels, the acceptability declined because of the compact texture of the crumb and the strong flavour of the product. The addition of 10% of soyflour (full‐fat and defatted) or 15% of barley flour, full‐fat soy + barley or defatted soy + barley flour to bread flour produced acceptable bread.     

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.     

Effects of barley flour and barley protein isolate on chemical, functional, nutritional and biological properties of Pita bread

This study was carried out to investigate the effects of fortification of wheat flour with barley flour (BF) and barley protein isolate (BPI) at three levels; 5, 10 and 15% levels on the chemical composition, nutritional evaluation and biological properties of pita bread. Proteins fractions such as globulin, prolamin, glutelin-1 and glutelin-2 as well as protein isolates were extracted from barley flour and evaluated for protein yield, chemical composition and nutritional quality. Highest yield and essential amino acids contents were obtained in barley protein isolate. SDS-PAGE gels electrophoresis indicated that fortified wheat flour with BPI and BF consists of proteins coming from wheat flour and barley proteins. The contents of essential limiting amino acids in bread were increased from 1.38 to 3.10 g/100 g for lysine and from 0.86 to 1.73 g/100 g for methionine as the ratio of fortification with BF and BPI increased from 0 to 15%. The highest content of total phenolics, antioxidant activity, and inhibitory activity for both angiotensin converting enzyme (ACE) and α-amylase were found in fortified bread with BPI at 15%. Results indicated that bread made from fortification of wheat flour with BF and BPI at 15% showed superior chemical, physico-chemical, nutritional and biological properties.