The sourdough microflora. Interactions between lactic acid bacteria and yeasts: metabolism of amino acids

Co-culture of Lactobacillus brevis subsp. lindneri or L. plantarum with Saccharomyces cerevisiae or S. exiguus from sourdough did not modify the yield of the yeasts but gave higher growth rates and final yields of both lactic acid bacteria (LAB) than in their respective mono-cultures. Co-cultures of L. brevis subsp. lindneri with S. cerevisiae or S. exiguus in a medium without valine or leucine, which are essential for growth of the LAB, led to growth of the LAB due to excretion of these amino acids by the yeasts.The authors are with the Institute of Dairy Microbiology, Faculty of Agriculture, University of Perugia, Via S. Costanzo, 06100 Perugia, Italy     

Presence of sourdough lactic acid bacteria in commercial total mixed ration silage as revealed by denaturing gradient gel electrophoresis analysis

Aims: To characterize the bacterial communities in commercial total mixed ration (TMR) silage, which is known to have a long bunk life after silo opening. Methods and Results: Samples were collected from four factories that produce TMR silage according to their own recipes. Three factories were sampled three times at 1‐month intervals during the summer to characterize the differences between factories; one factory was sampled 12 times, three samples each during the summer, autumn, winter and spring, to determine seasonal changes. Bacterial communities were determined by culture‐independent denaturing gradient gel electrophoresis. All silages contained lactic acid as the predominant acid, and the contents appeared stable regardless of factories and product seasons. Acetic acid and 1‐propanol contents were different between factories and indicated seasonal changes, with increases in warm seasons compared to cool seasons. Both differences and similarities existed among the bacterial communities from each factory and product season. Lactobacillus parabuchneri was found in the products from three of four factories. Various sourdough lactic acid bacteria (LAB) were identified in commercial TMR silage; Lactobacillus panis, Lactobacillus hammesii, Lactobacillus mindensis, Lactobacillus pontis, Lactobacillus frumenti and Lactobacillus farciminis were detected in many products. Moreover, changes owing to product season were distinctive, and Lact. pontis and Lact. frumenti became detectable in summer products. Conclusion: Sourdough LAB are involved in the ensiling of commercial TMR silage. Silage bacterial communities vary more by season than by factory. The LAB species Lact. parabuchneri was detected in the TMR silage but may not be essential to the product’s long bunk life after silo opening. Significance and Impact of the Study: Commercial TMR silage resembles sourdough with respect to bacterial communities and long shelf life. The roles of sourdough LAB in the ensiling process and aerobic stability are worth examining.     

Physicochemical stability of cimetidine amorphous forms estimated by isothermal microcalorimetry

The effect of humidity on the physicochemical properties of amorphous forms of cimetidine was investigated using differential scanning calorimetry, isothermal microcalorimetry, and x-ray diffraction analysis. Amorphous forms were obtained by the melting (amorphous form M [AM]) and the cotton candy (amorphous form C [AC]) methods. Thermal behaviors of AM and AC with or without seed crystals were measured using an isothermal microcalorimeter under various conditions of relative humidity (RH) and temperature, respectively. The crystallization kinetics of amorphous solids was analyzed based on 10 kinds of solid-state reaction models. AM transformed into form A at 11% RH, 50°C but transformed into a mixture of form A and monohydrate at 51% and 75% RH at 25°C. The mean crystallization times (MCTs) of the heat flow curve of AM and AC at 11% RH, 50°C were 47.82 and 32.00 hours, respectively, but at 11% RH, 25°C both were more than 4320 hours. In contrast, AC transformed into form A under all storage conditions. The MCTs of AC at 51% and 75% RH were 29.61 and 11.81 hours, respectively; whereas the MCTs of AM were 46.79 and 15.52 hours, respectively. The crystallization of amorphous solids followed the three-dimensional growth of nuclei (Avrami equation) with an induction period (IP). The IP for AM at 11% RH, 50°C was more than 2 times that for AC, but the difference in the crystal growth rate constant (CR) between AC and AM was within 10%. The IP for AM at 75% RH, 25°C was reduced to only 10% of the IP at 51% RH with increasing humidity, but the CR did not change significantly. In contrast, the IP for AC was slightly reduced at 75% RH compared with 51% RH, but the CR was about 5 times greater. At 75% RH, 25°C, the IP and CR of AM were about one-fourth the values of AC. This result suggests that the crystallization process consists of an initial stage during which the nuclei are formed and a final stage of growth.     

Bacterial population in traditional sourdough evaluated by molecular methods

AIMS: To study the microbial communities in artisanal sourdoughs, manufactured by traditional procedure in different areas of Sicily, and to evaluate the lactic acid bacteria (LAB) population by classical and culture-independent approaches. METHODS AND RESULTS: Forty-five LAB isolates were identified both by phenotypic and molecular methods. The restriction fragment length polymorphism and 16S ribosomal DNA gene sequencing gave evidence of a variety of species with the dominance of Lactobacillus sanfranciscensis and Lactobacillus pentosus, in all sourdoughs tested. Culture-independent method, such as denaturing gradient gel electrophoresis (DGGE) of the V6-V8 regions of the 16S rDNA, was applied for microbial community fingerprint. The DGGE profiles revealed the dominance of L. sanfranciscensis species. In addition, Lactobacillus-specific primers were used to amplify the V1-V3 regions of the 16S rDNA. DGGE profiles flourished the dominance of L. sanfranciscensis and Lactobacillus fermentum in the traditional sourdoughs, and revealed that the closely related species Lactobacillus kimchii and Lactobacillus alimentarius were not discriminated. CONCLUSIONS: Lactobacillus-specific PCR-DGGE analysis is a rapid tool for rapid detection of Lactobacillus species in artisanal sourdough. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports a characterization of Lactobacillus isolates from artisanal sourdoughs and highlights the value of DGGE approach to detect uncultivable Lactobacillus species.     

Characterization of sourdough lactic acid bacteria based on genotypic and cell-wall protein analyses

AIMS: To evaluate the effectiveness of two independent methods in differentiating a large population of lactic acid bacteria (LAB) isolated from wheat flours and sourdoughs and to correlate eventual differences/similarities among strains with their geographical origin and/or process parameters. METHODS AND RESULTS: One hundred fifty strains belonging to Lactobacillus spp. and Weissella spp., plus eight type strains, one for each species, and two unidentified isolates, were characterized by randomly amplified polymorphic DNA (RAPD) and SDS-PAGE of cell-wall proteins. The RAPD analysis separated the eight type strains but did not always assign all the strains of a species to the same group, while SDS-PAGE cell-wall protein profiles were species-specific. Frequently, strains isolated from sourdoughs of the same geographical origin or produced by similar raw material/process parameters showed similar RAPD and/or cell-wall profiles. CONCLUSIONS: The combined use of the RAPD and cell-wall protein analysis represents a useful tool to classify large adventitious microbial populations and to discriminate the diversity of the strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This study represents a typing of a large collection of flour/sourdough LAB and provides evidence of the advantage of using two independent methods in the classification and traceability of microorganisms.     

Evaluation of mono or mixed cultures of lactic acid bacteria in type II sourdough system

The aim of this study was to evaluate the use of mono and mixed lactic acid bacteria (LAB) cultures to determine suitable LAB combinations for a type II sourdough system. In this context, previously isolated sourdough LAB strains with antimicrobial activity, which included Lactobacillus plantarum PFC22, Lactobacillus brevis PFC31, Pediococcus acidilactici PFC38, and Lactobacillus sanfranciscensis PFC80, were used as mono or mixed culture combinations in a fermentation system to produce type II sourdough, and subsequently in bread dough production. Compared to the monoculture fermentation of dough, the use of mixed cultures shortened the adaptation period by half. In addition, the use of mixed cultures ensured higher microbial viability, and enhanced the fruity flavor during bread dough production. It was determined that the combination of L. plantarum PFC22 + P. acidilactici PFC38 + L. sanfranciscensis PFC80 is a promising culture mixture that can be used in the production of type II sourdough systems, and that may also contribute to an increase in metabolic activity during bread production process.     

Spelt and emmer flours: characterization of the lactic acid bacteria microbiota and selection of mixed starters for bread making

Aims: This study aimed at characterizing the lactic acid bacteria microbiota and selecting mixed endogenous starters to be used for sourdough fermentation of spelt or emmer flours. Methods and Results: Identification of lactic acid bacteria was carried out by partial sequencing of the 16S rRNA, recA, 16S/23S rRNA spacer region and pheS genes. Spelt flour showed the largest biodiversity, while Lactobacillus plantarum dominated in emmer flour. Isolates were subjected to RAPD‐PCR analysis and screened based on the kinetics of growth and acidification, quotient of fermentation and liberation of free amino acids (FAA) during sourdough fermentation. After selection, mixed starters were used according to a two‐step fermentation process. Wheat flour was fermented by the same starters. Spelt and emmer sourdoughs had slightly higher pH than wheat sourdoughs but titratable acidity, concentration of FAA and phytase activity were higher. Specific volume and crumb grain of emmer and, especially, spelt breads approached those of wheat breads. Sensory analysis confirmed the suitability of spelt and emmer for bread making. Conclusions: The sourdough biotechnology was indispensable to completely exploit the potential of spelt and emmer flours. Significance and Impact of the Study: Results filled up the lack of knowledge on the lactic acid bacteria microbiota and technological performances of spelt and emmer flours.     

AFLP技术对发酵酸面团中乳酸菌多态性的研究

乳酸菌是酸面团发酵的主要细菌,它对面团的质量、营养有非常重要的作用,利用AFLP技术对分离自酸面团中的20株乳酸菌进行了研究,以期了解其中乳酸菌的多态性及其分型。研究优化并确定了AFLP技术的各种实验条件,然后利用该技术对乳酸菌的多态性进行了研究,并采用聚类分析软件ProBiosys 1.0对实验结果进行了分析。     

Molecular source tracking of predominant lactic acid bacteria in traditional Belgian sourdoughs and their production environments

Aims:  To investigate the circulation of predominant sourdough lactic acid bacteria (LAB) species in the production environment of two Belgian artisan sourdough bakeries.
Methods and Results:  Isolates were collected from sourdoughs, flour, hands of the baker and air in the bakery setting and taxonomically characterized using repetitive element sequence-based PCR fingerprinting, pheS and/or 16S rRNA gene sequencing and amplified fragment length polymorphism (AFLP) analysis. In parallel, PCR-DGGE (denaturing gradient gel electrophoresis) analysis of V3-16S rDNA amplicons was applied to visualize the predominant bacterial population in the sourdoughs and the corresponding bakery environment (flour, hands of the baker, air and bakery equipment). Both approaches revealed that sourdoughs produced at D01 and D10 were mainly dominated by Lactobacillus spicheri and L. plantarum and by L. sanfranciscensis , respectively, and that these LAB species also circulated in the corresponding bakery environment. Furthermore, AFLP fingerprinting demonstrated that sourdough and bakery environment isolates of these species were genetically indistinguishable. For more sensitive source-tracking, SYBR Green-based real-time PCR assays were developed using species-specific primers targeting the pheS gene of L. plantarum and L. sanfranciscensis, detected in air samples from D01 and D10, respectively.
Conclusions:  The results obtained in this study indicate that specific strains of LAB persist in artisan doughs over years and circulate in the bakery environment. Furthermore, the importance of air as a potential carrier of LAB in artisan bakery environments was demonstrated.
Significance and Impact of the Study:  PheS -based real-time PCR can be used to detect, quantify and/or monitor specific LAB species (e.g. starter cultures) in sourdough and bakery environment samples.     

Kinetics and energetics of ligand binding determined by microcalorimetry: insights into active site mobility in a psychrophilic alpha-amylase

A new microcalorimetric method for recording the kinetic parameters k(cat), K(m) and K(i) of alpha-amylases using polysaccharides and oligosaccharides as substrates is described. This method is based on the heat released by glycosidic bond hydrolysis. The method has been developed to study the active site properties of the cold-active alpha-amylase produced by an Antarctic psychrophilic bacterium in comparison with its closest structural homolog from pig pancreas. It is shown that the psychrophilic alpha-amylase is more active on large macromolecular substrates and that the higher rate constants k(cat) are gained at the expense of a lower affinity for the substrate. The active site is able to accommodate larger inhibitory complexes, resulting in a mixed-type inhibition of starch hydrolysis by maltose. A method for recording the binding enthalpies by isothermal titration calorimetry in a low-affinity system has been developed, allowing analysis of the energetics of weak ligand binding using the allosteric activator chloride. It is shown that the low affinity of the psychrophilic alpha-amylase for chloride is entropically driven. The high enthalpic and entropic contributions of activator binding suggest large structural fluctuations between the free and the bound states of the cold-active enzyme. The kinetic and thermodynamic data for the psychrophilic alpha-amylase indicate that the strictly conserved side-chains involved in substrate binding and catalysis possess an improved mobility, responsible for activity in the cold, and resulting from the disappearance of stabilizing interactions far from the active site.     

一株能降解胆固醇的乳酸菌的选育

利用选择性培养基从成年人的粪便中分离出1株能降解胆固醇的乳酸菌,该菌能以胆固醇作为生长的唯一能源。在10％牛奶的发酵试验中,该菌能使牛奶发酵并凝固。在液体发酵中,胆固醇的降解率为34．6％。经初步鉴定,该菌为双歧杆菌（Bifidoacterium sp.)。     

Lactic acid bacteria isolated from rye sourdoughs produce bacteriocin-like inhibitory substances active against Bacillus subtilis and fungi

Aim: To screen five strains of lactic acid bacteria (LAB) isolated from rye sourdoughs for the potential production of antimicrobial substances. Methods and Results: Lactobacillus sakei KTU05‐06, Pediococcus acidilactici KTU05‐7, Pediococcus pentosaceus KTU05‐8, KTU05‐9 and KTU05‐10 isolated from rye sourdoughs were investigated for the production of bacteriocin‐like inhibitory substances (BLIS). The supernatants of analysed LAB inhibited growth of up to 15 out of 25 indicator bacteria strains as well as up to 25 out of 56 LAB strains isolated from rye sourdoughs. Moreover, these five LAB were active against ropes‐producing Bacillus subtilis and the main bread mould spoilage causing fungi –Aspergillus, Fusarium, Mucor and Penicillium. Lactobacillus sakei KTU05‐6 demonstrated the best antibacterial properties and is resistant towards heat treatment even at 100°C for 60 min. Conclusions: The use of LAB‐producing antibacterial substances may be a good choice as a co‐starter culture to ensure the stability of sourdoughs and to avoid the bacterial and fungi spoilage of the end product. Significance and Impact of the Study: The antimicrobial compounds designated as sakacin KTU05‐6, pediocin KTU05‐8 KTU05‐9, KTU05‐10 and AcKTU05‐67 were not identical to any other known BLIS, and this finding leads up to the assumption that they might be the novel.     

Exocellular polysaccharides produced by lactic acid bacteria

Abstract The production of homopolysaccharides (dextrans, mutans) and heteropolysaccharides by lactic acid bacteria, their chemical composition, their structure and their synthesis are outlined. Mutans streptococci, which include Streptococcus mutans and S. sobrinus produce soluble and insoluble α-glucans. The latter may contain as much as 90%α-1–3 linkages and possess a marked ability to promote adherence to the smooth tooth surface causing dental plaque. Dextrans produced by Leuconostoc mesenteroides are high molecular weight α-glucans having 1–6, 1–4 and 1–3 linkages, varying from slightly to highly branched; 1–6 linkages are predominant. Emphasis is put on exopolysaccharide producing thermophilic and mesophilic lactic acid bacteria, which are important in the dairy industry. The produced polymers play a key role in the rheological behaviour and the texture of fermented milks. One of the main problems in this field is the transitory nature of the thickening trait. This instability is not yet completely understood. Controversial results exist on the sugar composition of the slime produced, but galactose and glucose have always been identified with galactose predominating in most cases.     

Lactic acid bacteria in the quality improvement and depreciation of wine

The winemaking process includes two main steps: lactic acid bacteria are responsible for the malolactic fermentation which follows the alcoholic fermentation by yeasts. Both types of microorganisms are present on grapes and on cellar equipment. Yeasts are better adapted to growth in grape must than lactic acid bacteria, so the alcoholic fermentation starts quickly. In must, up to ten lactic acid bacteria species can be identified. They belong to the Lactobacillus, Pediococcus, Leuconostoc and Oenococcus genera. Throughout alcoholic fermentation, a natural selection occurs and finally the dominant species is O. oeni, due to interactions between yeasts and bacteria and between bacteria themselves. After bacterial growth, when the population is over 10⁶CFU/ml, malolactic transformation is the obvious change in wine composition. However, many other substrates can be metabolized. Some like remaining sugars and citric acid are always assimilated by lactic acid bacteri a, thus providing them with energy and carbon. Other substrates such as some amino acids may be used following pathways restricted to strains carrying the adequate enzymes. Some strains can also produce exopolysaccharides. All these transformations greatly influence the sensory and hygienic quality of wine. Malic acid transformation is encouraged because it induces deacidification. Diacetyl produced from citric acid is also helpful to some extent. Sensory analyses show that many other reactions change the aromas and make malolactic fermentation beneficial, but they are as yet unknown. On the contrary, an excess of acetic acid, the synthesis of glucane, biogenic amines and precursors of ethylcarbamate are undesirable. Fortunately, lactic acid bacteria normally multiply in dry wines; moreover some of these activities are not widespread. Moreover, the most striking trait of wine lactic acid bacteria is their capacity to adapt to a hostile environment. The mechanisms for this are not yet c ompletely elucidated . Molecular biology has provided some explanations for the behaviour and the metabolism of bacteria in wine. New tools are now available to detect the presence of desirable and undesirable strains. Even if much remains unknown, winemakers and oenologists can nowadays better control the process. By acting upon the diverse microflora and grape musts, they are more able to produce healthy and pleasant wines.     

Growth and exopolysaccharide (EPS) production by Oenococcus oeni I4 and structural characterization of their EPSs

AIMS: To study the influence of medium constituents on growth, and exopolysaccharide (EPS) production by a strain of Oenococcus oeni. The structure of one of the EPSs has also been characterized. METHODS AND RESULTS: EPS concentration was estimated by the phenol/sulfuric acid method. After purification and fractionation of crude EPSs, the sugar composition was determined by GLC-MS of the TMS methyl glycosides. The major polysaccharide is 2-substituted-(1-3)-beta-D-glucan. This structure was determined by methylation analysis and conventional (1)H- and (13)C-nuclear magnetic resonance spectroscopy. In addition, O. oeni synthesized two heteropolysaccharides, although a lesser proportion, constituted by galactose and glucose, and one of them also showed rhamnose. The sugar source has a clear influence on growth and EPS synthesis, and EPS production was not enhanced by adding ethanol or increasing the nitrogen source. EPS biosynthesis starts in the exponential growth phase, and continued during the stationary growth phase. CONCLUSIONS: Higher EPS yields were obtained on cultures grown on glucose + fructose. O. oeni produces a beta-glucan, as the predominant EPS, and it is also able to produce two heteropolysaccharides. Significance and Impact of the Study: This work provides a better understanding of EPS synthesis by O. oeni and shows the first EPS structure described for this species.