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.     

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.     

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.     

Screening methods for the proteolytic breakdown of gluten by lactic acid bacteria and enzyme preparations

 Gluten is the major protein in wheat and it is largely responsible for the rheological characteristics of bread dough. The sourdough process is a traditional dough fermentation process with lactic acid bacteria. The proteolytic activity of lactic acid bacteria derived from starter cultures used in sourdough or meat fermentation was tested on gluten as a substrate. The activities of commercial enzyme preparations, which were derived from bacterial or fungal sources, were also evaluated. Proteolytic breakdown of gluten protein in an agar medium by either bacterial culture or enzymes was evident by a clear zone around wells after staining with Coomassie blue. The increase in TCA-soluble material due to gluten breakdown was measured spectrophotometrically. A third test evaluated the release of free amino acids due to exoproteolytic activity. The agar based test showed positive results for the commercial enzyme preparations and one Micrococcus strain. All sourdough strains were positive in the enzyme test whereas some starters from the meat fermentation process were not able to break down gluten under test conditions. Variations in the release of free amino acid indicated differences in the enzyme systems of the lactic acid bacteria tested. Received: 25 February 1999     

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.     

Enhancement of survival of probiotic and non-probiotic lactic acid bacteria by yeasts in fermented milk under non-refrigerated conditions

The effects of yeasts on the survival of probiotic and non-probiotic lactic acid bacteria (LAB) were studied in fermented milk under non-refrigerated conditions (30 °C) with a view to develop ambient-stable fermented milk with live LAB. Five yeasts tested (Saccharomyces bayanus, Williopsis saturnus var. saturnus, Yarrowia lipolytica, Candida kefyr and Kluyveromyces marxianus) enhanced the survival of Lactobacillus bulgaricus (but not Streptococcus thermophilus) in a mixed yoghurt culture in yoghurt by ~ 10² to 10⁵-fold. Seven yeasts examined (Candida krusei, Geotrichum candidum, Pichia subpelliculosa, Kloeckera apiculata, Pichia membranifaciens, Schizosaccharomyces pombe and Y. lipolytica) improved the survival of Lactobacillus rhamnosus in fermented milk by ~ 10³ to 10⁶-fold. W. saturnus var. saturnus enhanced the survival of Lactobacillus acidophilus, L. rhamnosus (probiotic) and Lactobacillus reuteri by up to 10⁶-fold, but the same yeast failed to improve the survival of Lactobacillus johnsonii (probiotic), S. thermophilus and L. bulgaricus in fermented milk. These results provide definitive evidence that yeasts possess stability-enhancing effects on LAB and that the specific effects of yeasts on LAB stability vary with yeasts as well as with LAB. However, the molecular mechanism of such interaction of yeasts with LAB remains to be found.     

Biodiversity of yeasts, lactic acid bacteria and acetic acid bacteria in the fermentation of "Shanxi aged vinegar", a traditional Chinese vinegar

Shanxi aged vinegar is a famous traditional Chinese vinegar made from several kinds of cereal by spontaneous solid-state fermentation techniques. In order to get a comprehensive understanding of culturable microorganism’s diversity present in its fermentation, the indigenous microorganisms including 47 yeast isolates, 28 lactic acid bacteria isolates and 58 acetic acid bacteria isolates were recovered in different fermenting time and characterized based on a combination of phenotypic and genotypic approaches including inter-delta/PCR, PCR-RFLP, ERIC/PCR analysis, as well as 16S rRNA and 26S rRNA partial gene sequencing. In the alcoholic fermentation, the dominant yeast species Saccharomyces (S.) cerevisiae (96%) exhibited low phenotypic and genotypic diversity among the isolates, while Lactobacillus (Lb.) fermentum together with Lb. plantarum, Lb. buchneri, Lb. casei, Pediococcus (P.) acidilactici, P. pentosaceus and Weissella confusa were predominated in the bacterial population at the same stage. Acetobacter (A.) pasteurianus showing great variety both in genotypic and phenotypic tests was the dominant species (76%) in the acetic acid fermentation stage, while the other acetic acid bacteria species including A. senegalensis, A. indonesiensis, A. malorum and A. orientalis, as well as Gluconobacter (G.) oxydans were detected at initial point of alcoholic and acetic acid fermentation stage respectively.     

Spanish cheese screening and selection of lactic acid bacteria with high gamma-aminobutyric acid production

Gamma-aminobutyric acid (GABA) is a non-protein four-carbon amino acid which is considered a bioactive component known for its physiological functions, including a regulator of blood pressure, neurotransmitter, diuretic and anti-stress effects. Its use in foods might confer health benefits. Microorganisms such as yeast, fungi or bacteria can produce GABA naturally. Among them, the lactic acid bacteria are being studied for the potential development of fermented foods because their physiological activities and their designation of generally recognized as safe (GRAS). The objective of this study was to evaluate the GABA-production capacity in a whole wheat flour medium of lactic acid bacteria strains that showed a high conversion of glutamic acid to GABA in a screening conducted in 58 Spanish artisanal cheeses. Synthesis of GABA by these strains in a non-optimized whole wheat flour in water solution (1:5) was quantified by High-Performance Liquid Chromatography. The 4 strains showing the highest GABA production were genotypically and phenotypically characterized. Results indicated an interesting fermentative variability between strains. The addition of these isolated lactic acid strains in fermented food products could allow a potentially functional food for regulating hypertension.     

Ethyl carbamate production by selected yeasts and lactic acid bacteria in red wine

This study was designed to investigate the formation of ethyl carbamate during the fermentation of musts of Vitis vinifera L. cv. Tempranillo and Cabernet Sauvignon by selected yeasts in different conditions of temperature and pH. Secondary malolactic fermentation was then induced by selected lactic acid bacteria. The results indicate an increased concentration of ethyl carbamate after malolactic fermentation, irrespective of the bacteria used or the prevailing physicochemical conditions.     

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.     

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.     

Diversity and technological potential of lactic acid bacteria of wheat flours

Lactic acid bacteria (LAB) were analysed from wheat flours used in traditional bread making throughout Sicily (southern Italy). Plate counts, carried out in three different media commonly used to detect food and sourdough LAB, revealed a maximal LAB concentration of approximately 4.75 Log CFU g⁻¹. Colonies representing various morphological appearances were isolated and differentiated based on phenotypic characteristics and genetic analysis by randomly amplified polymorphic DNA (RAPD)-PCR. Fifty unique strains were identified. Analysis by 16S rRNA gene sequencing grouped the strains into 11 LAB species, which belonged to six genera: Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus and Weissella. Weissella cibaria, Lactobacillus plantarum, Leuconostoc pseudomesenteroides and Leuconostoc citreum were the most prevalent species. The strains were not geographically related. Denaturing gradient gel electrophoresis (DGGE) analysis of total DNA of flour was used to provide a more complete understanding of the LAB population; it confirmed the presence of species identified with the culture-dependent approach, but did not reveal the presence of any additional LAB species. Finally, the technological characteristics (acidifying capacity, antimicrobial production, proteolytic activity, organic acid, and volatile organic compound generation) of the 50 LAB strains were investigated. Eleven strains were selected for future in situ applications.     

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.     

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.     

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.     

臭豆腐乳酸菌多样性及耐酸乳酸菌的筛选分离

对采集的8个不同地区的臭豆腐样品中的乳酸菌多样性分析表明,臭豆腐中乳酸菌数量在6.8×104cfu/g～2.9×106cfu/g,优势乳酸菌包括乳杆菌、乳球菌和肠球菌.通过乳酸菌耐酸特性比较,表明不同来源样品中乳酸菌耐酸差异性不明显,而不同种类乳酸菌的耐酸差异性较明显,肠球菌耐酸性比例高,乳杆菌其次,乳球菌最低.经筛选获得pH 3.0、37℃培养6 h后存活率超过30%的菌株6株,对其中2株耐酸最强的杆菌和球菌进行鉴定,分别为植物乳杆菌和屎肠球菌.     

乳酸菌冷冻干燥保护剂的筛选

通过单因素比较和正交试验设计,确定适合保加利亚乳杆菌和嗜热链球菌的最佳冻干保护剂配方。通过对15种冻干保护剂单因素保护效果的比较,以及对选定的4种保护剂的正交试验结果,可以确定嗜热链球菌最佳冻干保护剂配方为脱脂乳粉12%、海藻糖1%、甘油3%、谷氨酸钠1%,保加利亚乳杆菌最佳冻干保护剂配方为脱脂乳粉8%、海藻糖1%、甘油2%、谷氨酸钠1.5%。