Evolution Of Lactic Acid Bacteria Research Articles

Reuse of almond by-products: Functionalization of traditional semolina sourdough bread with almond skin

Almond production generates large amounts of by-products rich in polyphenols. In this study, almond skin was explored as a valuable food ingredient in bread making. To this purpose, almond skin was used to produce functional products modifying a traditional sourdough bread recipe. The doughs were prepared replacing semolina with powdered almond skin (PAS) at 5 and 10 % (w/w). Sourdough inoculum was started with a mix of lactic acid bacteria (LAB) and propagated in semolina until reaching pH 3.7. The pH of PAS added breads was higher than that of control (CTR) breads before and after fermentation. Plate counts showed a similar evolution of LAB and total mesophilic microorganisms, but members of Enterobacteriaceae and coliform were detectable in PAS doughs. Illumina data clearly showed a dominance of lactobacilli in all trials, but PAS doughs displayed the presence of Bacillus. The final bread characteristics were influenced by PAS and its addition percentage; in particular, crust and crumb colour resulted darker, the alveolation decreased and, regarding sensory attributes, odour intensity increased, while bread odour diminished. In presence of PAS, bread emissions were characterized by lower percentages of alcohols and aromatic hydrocarbons and higher percentages of the other volatile compound classes, especially terpenoids like β-pinene, β-myrcene and limonene than CTR trial. After in vitro simulated digestion, the final release of phytochemicals from 10 % PAS bread was almost 100 %. Thus, PAS determined an increase of the antioxidant capacity of the breads. Phytochemicals released from digested PAS-fortified bread can provide antioxidant protection in a complex biological environment such as human intestinal-like cells. Besides the positive functional properties of PAS, this work also evidenced the hygienic issues of almond skin and, in order to avoid potential risks for the human health, highlighted the need to preserve its microbiological characteristics during storage for their reuse in bread production.

Bioinformatics and its role in the study of the evolution and probiotic potential of lactic acid bacteria.

Due to their numerous well-established applications in the food industry, there have been many studies regarding the adaptation and evolution of lactic acid bacteria (LAB) in a wide variety of hosts and environments. Progress in sequencing technology and continual decreases in its costs have led to the availability of LAB genome sequence data. Bioinformatics has been central to the extraction of valuable information from these raw genome sequence data. This paper presents the roles of bioinformatics tools and databases in understanding the adaptation and evolution of LAB, as well as the bioinformatics methods used in the initial screening of LAB for probiotic potential. Moreover, the advantages, challenges, and limitations of employing bioinformatics for these purposes are discussed.

Microbial community of natural starter of fermented dairy product

The study of natural starter cultures in different geographical areas showed a high biochemical activity of lactic acid bacteria. They have spontaneous variability being in a state of stress that determines their morphological, cultural physiological and biochemical properties. Adaptive evolution of lactic acid bacteria under the influence of adverse natural factors in its growth process affects their ability to metabolize important functional compounds. It leads to the activation of adaptive genes that provide the cell development and stability. They act as chemical agents with defined functions in the nature. Their functions affect the natural history of the organism in biochemical status. It has been established that the directed synthesis of the products depends on the producer strain, the activity of corresponding enzymes and the physic-chemical state of the nutrient medium. It has been known that the function of microorganisms is more specialized, more clearly delineated in nature than in the laboratory environments. The surface layer pigmentation of fermentable dairy products increases in density due to the transition of lactic acid bacteria cells in hypometabolism conditions. These conditions are defined by functional specialization in bacteria and the redox conditions.

Ecology of indigenous lactic acid bacteria from Rioja Alavesa red wines, focusing on biogenic amine production ability

Evolution of lactic acid bacteria (LAB) from two wineries located in Rioja Alavesa region has been analyzed during the whole winemaking process. Special attention was focused on determining their biogenic amine (BA) forming potential. From 295 isolates, 36 different genotypic profiles were identified, from which 17 belonged to Oenococccus oeni. Although LAB species evolution differed in both wineries during winemaking process, O. oeni became the predominant species in both wineries once malolactic fermentation (MLF) began. Each winery showed an exclusive ecosystem with own microbiota, as low coincident O. oeni strains were identified. Both phenotypical and molecular methods revealed the low incidence of BA producing LAB strains, as exclusively Lactobacillus hilgardii strains were positive for putrescine production. Other bacteria genus rarely found in wine environment such as Staphylococcus and Paenibacillus were also identified. Indeed, this is the first work in which those species have been reported as BA producers in wine. In addition, BA concentration was also monitored. Both wineries showed low concentrations for each BA, far from limits recommended for safety and quality principles. These results emphasize the potential application of these autochthonous strains in order to minimize BA formation during the whole winemaking process.

Uncovering carbohydrate metabolism through a genotype-phenotype association study of 56 lactic acid bacteria genomes

Owing to their unique potential to ferment carbohydrates, both homo- and heterofermentative lactic acid bacteria (LAB) are widely used in the food industry. Deciphering the genetic basis that determine the LAB fermentation type, and hence carbohydrate utilization, is paramount to optimize LAB industrial processes. Deep sequencing of 24 LAB species and comparison with 32 publicly available genome sequences provided a comparative data set including five major LAB genera for further analysis. Phylogenomic reconstruction confirmed Leuconostoc and Pediococcus species as independently emerging from the Lactobacillus genus, within one of the three phylogenetic clades identified. These clades partially grouped LABs according to their fermentation types, suggesting that some metabolic capabilities were independently acquired during LAB evolution. In order to apply a genome-wide association study (GWAS) at the multigene family level, utilization of 49 carbohydrates was also profiled for these 56 LAB species. GWAS results indicated that obligately heterofermentative species lack 1-phosphofructokinase, required for d-mannose degradation in the homofermentative pathway. Heterofermentative species were found to often contain the araBAD operon, involved in l-arabinose degradation, which is important for heterofermentation. Taken together, our results provide helpful insights into the genetic determinants of LAB carbohydrate metabolism, and opens for further experimental research, aiming at validating the role of these candidate genes for industrial applications.

Combined effect of antimicrobial edible coatings with reduction of initial microbial load on the shelf‐life of fresh hake (Merluccius merluccius) medallions

SummaryTwo distinct strategies were combined to preserve fresh fish (Merluccius merluccius) under refrigeration at 4 °C for 12 days: (i) the application of an antimicrobial edible coating enriched with oregano essential oil (OEO) or carvacrol (CV) and (ii) the reduction of initial microbial load by good handling practise and the use of sodium hypochlorite (NaOCl). The action of antimicrobial coatings alone retarded the growth of Enterobacteriaceae, lactic acid bacteria (LAB) and H2S producing bacteria on fish samples. The reduction of initial microbial load by itself only affected the evolution of LAB, but not the rest of the bacterial groups. When using both techniques combined, edible antimicrobial coatings were significantly more effective with additional and significant delays in the growth of mesophilic, psychrotrophic and Pseudomonas bacteria. Thus, the use of both strategies combined resulted in a reduction of the counts of all bacterial groups after 12 days of storage which ranged from 1.5 log and 8 log, in Pseudomonas and H2S producing bacteria, respectively. Moreover, no significant differences were observed when comparing the microbiological evolution of samples treated with OEO compared to those only treated with CV.

Symposium review: Lactococcus lactis from nondairy sources: Their genetic and metabolic diversity and potential applications in cheese

The widespread dissemination of species of the lactic acid bacteria (LAB) group in different environments testifies to their extraordinary niche adaptability. Members of the LAB are present on grass and other plant material, in dairy products, on human skin, and in the gastrointestinal and reproductive tracts. The selective pressure imparted by these specific environments is a key driver in the genomic diversity observed between strains of the same species deriving from distinct habitats. Strains that are exploited in the dairy industry for the production of fermented dairy products are often referred to as "domesticated" strains. These strains, which initially may have occupied a nondairy niche, have become specialized for growth in the milk environment. In fact, comparative genome analysis of multiple LAB species and strains has revealed a central trend in LAB evolution: the loss of ancestral genes and metabolic simplification toward adaptation to nutritionally rich environments. In contrast, "environmental" strains, or those from raw milk, plants, and animals, exhibit diverse metabolic capabilities and lifestyle characteristics compared with their domesticated counterparts. Because of the limited number of established dairy strains used in fermented food production today, demand is increasing for novel strains, with concerted efforts to mine the microbiota of natural environments for strains of technological interest. Many studies have concentrated on uncovering the genomic and metabolic potential of these organisms, facilitating comparative genome analysis of strains from diverse environments and providing insight into the natural diversity of the LAB, a group of organisms that is at the core of the dairy industry. The natural biodiversity that exists in these environments may be exploited in dairy fermentations to expand flavor profiles, to produce natural "clean label" ingredients, or to develop safer products.

Functional genomics of lactic acid bacteria: from food to health.

Genome analysis using next generation sequencing technologies has revolutionized the characterization of lactic acid bacteria and complete genomes of all major groups are now available. Comparative genomics has provided new insights into the natural and laboratory evolution of lactic acid bacteria and their environmental interactions. Moreover, functional genomics approaches have been used to understand the response of lactic acid bacteria to their environment. The results have been instrumental in understanding the adaptation of lactic acid bacteria in artisanal and industrial food fermentations as well as their interactions with the human host. Collectively, this has led to a detailed analysis of genes involved in colonization, persistence, interaction and signaling towards to the human host and its health. Finally, massive parallel genome re-sequencing has provided new opportunities in applied genomics, specifically in the characterization of novel non-GMO strains that have potential to be used in the food industry. Here, we provide an overview of the state of the art of these functional genomics approaches and their impact in understanding, applying and designing lactic acid bacteria for food and health.

Evolution of lactic acid bacteria in the order Lactobacillales as depicted by analysis of glycolysis and pentose phosphate pathways

Lactic acid bacteria (LAB) represent a functional group of bacteria that are fundamental in human nutrition because of their prominent role in fermented food production and their presence as commensals in the gut. LAB co-evolution and niche-adaptation have been analyzed in several phylogenomic studies due to the availability of complete genome sequences. The aim of this study was to provide novel insights into LAB evolution through the comparative analysis of the metabolic pathways related to carbohydrate metabolism. The analysis was based on 42 LAB genome sequences of representative strains belonging to Enterococcaceae, Lactobacillaceae, Leuconostocaceae and Streptococcaceae. A reference phylogenetic tree was inferred from concatenation of 42 ribosomal proteins; then 42 genes belonging to the Embden–Meyerhof–Parnas (or glycolysis; EMPP) and pentose phosphate (PPP) pathways were analyzed in terms of their distribution and organization in the genomes. Phylogenetic analyses confirmed the paraphyly of the Lactobacillaceae family, while the distribution and organization of the EMPP and PPP genes revealed the occurrence of lineage-specific trends of gene loss/gain within the two metabolic pathways examined. In addition, the investigation of the two pathways as structures resulting from different evolutionary processes provided new information concerning the genetic bases of heterofermentative/homofermentative metabolism.

Fate of Escherichia coli O157:H7, Salmonella Enteritidis and Listeria monocytogenes during storage of fermented green table olives in brine

The survival of Escherichia coli O157:H7, Salmonella Enteritidis and Listeria monocytogenes during the storage of fermented green table olives cv. Halkidiki in brine was studied in parallel with the evolution of lactic acid bacteria (LAB), yeasts and pH. The olives were previously fermented with a starter culture (a potential probiotic strain of Lactobacillus pentosus B281 – starter process) or with the indigenous microbiota (control). After the end of fermentation, olives were placed in brine, inoculated with a cocktail of 5 strains of E. coli O157:H7, 5 strains of L. monocytogenes and 4 strains of S. Enteritidis, with a final concentration in the brine of ca. 7.0 log CFU/ml, and subsequently packaged in polyethylene pouches and stored at 20 °C. The population of E. coli O157:H7 reduced gradually and was detected in the brine until the 27th day of storage in both cases (i.e., starter and control process), and on olive fruits until the 19th and 16th days of storage in the starter and control process, respectively. S. Enteritidis population showed also a decrease and it was detected until the 21st day of storage in both brine and olive fruits in both cases. The population of L. monocytogenes declined during storage and it was detected until the 31st day of storage in both brine and olive fruits in both cases, showing a longer survival period in comparison to the other two studied pathogens. The presence of the potential probiotic starter did not affect the pathogen survival. The results demonstrated that even though the growth of the pathogenic strains was not supported, they may survive for a long period in a stressful environment of a fermented product with low pH value (4.2) and high salt concentration (6.0%). These results are a valuable contribution to risk assessment studies related to ready to eat foods in general.

The biodiversity and evolution of lactic flora during ripening of the Iranian semisoft Lighvan cheese

Ninety‐five isolated strains of Lactic acid bacteria (LAB) were identified from Lighvan cheese. The LAB evolution showed the dominance of lactococci and lactobacilli in the first stage and substitution of these genera by enterococci at the end of ripening. The most predominant strains were Enterococcus faecium (22.44%), Lactococcus lactis ssp. lactis (20.4%), Lactobacillus plantarum (18.36%) and E. faecalis (14.28%), respectively. Eleven and 51 different carbohydrate fermentation profiles were observed according to API 20 STREP and API 50 CH, respectively. API 20 STREP dendogram showed identical fermentation profiles of some E. faecalis and E. faecium strains, indicating that these strains might be well adapted to the whole cheese manufacture.

Evolution of fermenting microbiota in tarhana produced under controlled technological conditions

The purpose of this study was to evaluate the evolution of lactic acid bacteria (LAB) and yeasts during the fermentation of tarhana produced with some pasteurised ingredients and carried out at 30 and 40 °C. The chemical parameters were those typical for tarhana production. Coliform bacteria were not detected during fermentation, while LAB and yeasts were in the range 107–108 colony forming units (CFU) g−1. Plate counts showed an optimal development of both fermenting microbial groups and the differences in cell concentrations were not significant (P > 0.05). LAB were isolated during fermentation and grouped on the basis of phenotypic and polymorphic characteristics. LAB isolates were identified by a combined genetic approach consisting of 16S/23S rRNA intergenic spacer region (ITS) and partial 16S rRNA gene sequencing as Pediococcus acidilactici, Lactobacillus plantarum and Lactobacillus brevis. Hence, the pasteurisation of the vegetable ingredients, excluded wheat flour, enhanced the hygienic conditions of tarhana without influencing the normal evolution of LAB. However, the fermentation at 40 °C favoured pediococci, while the production at 30 °C was mainly characterised by lactobacilli. Yeasts, identified by the restriction fragment length polymorphism (RFLP) of the 5.8S ITS rRNA gene, were mainly represented by the species Saccharomyces cerevisiae in both productions.

Caciotta della Garfagnana cheese: selection and evaluation of autochthonous mesophilic lactic acid bacteria as starter cultures

The aim of this study was to isolate, identify and select, with respect to acidification and proteolytic activities, the autochthonous mesophilic lactic acid bacteria (LAB) present in milk and Caciotta della Garfagnana, a cheese produced either with raw or thermised cow’s milk in small dairies and family plants of Garfagnana (Tuscany), to obtain LAB strains with attributes suitable to be employed as starter cultures in this type of cheese, particularly when thermised milk is used to control spoilage microflora. Samples of raw milk, curd and cheese were collected from three representative farmers of the production area and used to isolate autochthonous LAB. Phenotypic and genotypic (species-specific PCR assay) identification of isolated LAB was done. Twenty-eight strains of LAB isolated from milk, curd and cheese were screened for acidifying and proteolytic activities. LAB strains with the better attributes were used as mesophilic starter cultures in technological trials: experimental cheeses manufactured with the addition of autochthonous LAB and control cheeses were compared for LAB and pH evolution. Experimental cheeses presented a significant increase in the mesophilic lactic acid microflora up to 14 days of ripening and significantly lower pH values up to seven days of ripening. The use of wild selected mesophilic lactic acid bacteria, together with thermisation of milk, for the Caciotta della Garfagnana looks very promising and could help to both standardise the production and improve quality and traditional characteristics of this type of cheese.

LAB-Secretome: a genome-scale comparative analysis of the predicted extracellular and surface-associated proteins of Lactic Acid Bacteria

BackgroundIn Lactic Acid Bacteria (LAB), the extracellular and surface-associated proteins can be involved in processes such as cell wall metabolism, degradation and uptake of nutrients, communication and binding to substrates or hosts. A genome-scale comparative study of these proteins (secretomes) can provide vast information towards the understanding of the molecular evolution, diversity, function and adaptation of LAB to their specific environmental niches.ResultsWe have performed an extensive prediction and comparison of the secretomes from 26 sequenced LAB genomes. A new approach to detect homolog clusters of secretome proteins (LaCOGs) was designed by integrating protein subcellular location prediction and homology clustering methods. The initial clusters were further adjusted semi-manually based on multiple sequence alignments, domain compositions, pseudogene analysis and biological function of the proteins. Ubiquitous protein families were identified, as well as species-specific, strain-specific, and niche-specific LaCOGs. Comparative analysis of protein subfamilies has shown that the distribution and functional specificity of LaCOGs could be used to explain many niche-specific phenotypes.A comprehensive and user-friendly database LAB-Secretome was constructed to store, visualize and update the extracellular proteins and LaCOGs http://www.cmbi.ru.nl/lab_secretome/. This database will be updated regularly when new bacterial genomes become available.ConclusionsThe LAB-Secretome database could be used to understand the evolution and adaptation of lactic acid bacteria to their environmental niches, to improve protein functional annotation and to serve as basis for targeted experimental studies.

Characterization of pLAC1, a cryptic plasmid isolated from Lactobacillus acidipiscis and comparative analysis with its related plasmids

The pLAC1 plasmid of Lactobacillus acidipiscis ACA-DC 1533, a strain isolated from traditional Kopanisti cheese, was characterised. Nucleotide sequence analysis revealed a circular molecule of 3478 bp with a G + C content of 37.2%. Ab initio annotation indicated four putative open reading frames ( orfs). orf1 and orf4 were found to encode a replication initiation protein (Rep) and a mobilization protein (Mob), respectively. The deduced products of orf2 and orf3 revealed no significant homology to other known proteins. However, in silico examination of the plasmid sequence supported the existence of a novel operon that includes rep, orf2 and orf3 in pLAC1 and that this operon is highly conserved also in plasmids pLB925A02, pSMA23, pLC88 and pC7. RT-PCR experiments allowed us to verify that these three genes are co-transcribed as a single polycistronic mRNA species. Furthermore, phylogenetic analysis of pLAC1 Rep and Mob proteins demonstrated that they may have derived from different plasmid origins, suggesting that pLAC1 is a product of a modular evolution process. Comparative analysis of full length nucleotide sequences of pLAC1 and related Lactobacillus plasmids showed that pLAC1 shares a very similar replication backbone with pLB925A02, pSMA23 and pLC88. In contrast, mob of pLAC1 was almost identical with the respective gene of plasmids pLAB1000, pLB4 and pPB1. These findings lead to the conclusion that pLAC1 acquired mob probably via an ancestral recombination event. Our overall work highlights the importance of characterizing plasmids deriving from non-starter ‘wild’ isolates in order to better appreciate plasmid divergence and evolution of lactic acid bacteria.

Microbiological determination of lactic acid bacteria from fermented dairy products in different storage times.

Lactic acid bacteria evolution (Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus) from two Romanian fermented dairy products (yoghurt and sana) was followed in tree stages of storage during shelf-life: in first day of storage after processing; in the middle of storage period and in the last day of storage period. An important decrease of lactic acid bacteria was observed to storage during shelf-life. This indicates a low stability of starter culture, viable germs being inhibited by other micro-organisms development (first Enterobacteriaceae then yeast and molds).

Behaviour of the constitutive biota of two types of Spanish dry-sausages ripened in a pilot-scale chamber

The behaviour of the constitutive biota in eighty four samples belonging to two different types of Spanish dry-cured sausages during the ripening process in a pilot-scale chamber was investigated. Samples were analyzed in three stages during production: fresh product, first drying stage and finished product. Lactic acid bacteria (LAB) and Coagulase-negative cocci (CNC) were identified by the API system. In general, evolution of LAB and CNC during the ripening process of Spanish dry-cured sausages increased during the first days after which numbers of these organisms remained stable. Pediococcus pentosaceus and Staphylococcus xylosus, were the dominant species. Lactobacillus plantarum, Staphylococcus saprophyticus Staphylococcus simulans and Kocuria varians were also present. The results obtained show that the ripening process in a pilot-scale chamber under controlled conditions contributes to a more homogeneous behaviour of the constitutive biota, in comparison with commercial production standards.

Employment of autochthonous microflora in Pecorino Sardo cheese manufacturing and evolution of physicochemical parameters during ripening

The isolation and identification of lactic acid bacteria (LAB) from raw ewes' milk and traditional Pecorino Sardo cheese made from this milk without the addition of starter culture was carried out to define the autochthonous lactic microflora present in milk and the evolution of LAB during cheese ripening. Isolation of 275 strains belonging to different Lactococcus, Lactobacillus, Streptococcus and Enterococcus species was achieved. Coccal-shaped LAB were found to predominate during cheese fermentation, while lactobacilli were preponderate during the latter phase of ripening. The technological selection of a total of 174 LAB strains belonging to the species Lactococcus lactis, Streptococcus thermophilus, Lactobacillus helveticus and Lb. casei allowed an experimental starter to be prepared, in which a potentially probiotic species, Lb. casei was used. The suitability of the autochthonous starter culture was tested in cheese-making trials, using thermised ewes' milk, by comparing experimental Pecorino Sardo cheese with a control cheese produced at industrial scale using a whey starter culture from previous batches of manufacture. In particular, microbiological and physicochemical parameters were determined over 210 days of cheese ripening. Although sensory evaluation did not show any significant difference between experimental and control Pecorino Sardo cheeses, the use of the selected autochthonous starter allowed the production of experimental cheese with a significantly higher level of free amino acids, in particular essential amino acids, in comparison with the Pecorino Sardo control cheeses.

Lactic acid bacteria evolution during winemaking: Use of rpoB gene as a target for PCR-DGGE analysis

Evolution of the microbial population during winemaking is crucial. Winemakers are more and more attentive to microbial aspects during fermentation. During aging, microbial stabilization is preponderant to avoid development of spoilage yeast and bacteria. Therefore, it is necessary to improve methods to study the evolution of micro-organisms and for early detection of undesirable strain. The aim of this study was to develop a culture-independent method for identifying lactic acid bacteria (LAB) and to monitoring predominant species. The benefits of PCR-DGGE for the analysis of microbial changes during winemaking were clearly demonstrated. Targeting rpoB gene allowed a reliable discrimination of each species. The primers were able to avoid the interspecies heterogeneity problem caused by the use of the 16S rRNA gene. This method was applied to study the influence of different oenological practices on LAB population and their evolution during winemaking.

Article

Capnophiles are bacteria stimulated by bicarbonate and CO2, the two major forms of inorganic carbon (IC) in physiological neutral liquids. Capnophiles are often pathogenic heterotrophs found in IC-rich ecological niches such as human cavities. Like capnophiles, the growth of lactic acid bacteria (LAB) such as Lactobacillus plantarum and Enterococcus faecalis is stimulated by IC. CO2 or HC are substrates in carbamoyl phosphate (CP) synthesis and other carboxylation reactions in amino acid and nucleotide biosynthesis. When media were supplemented with nucleotides and all the amino acids, potassium bicarbonate still stimulated L. plantarum growth. This suggests that IC may be involved in other aspects of L. plantarum physiology besides its implication as a substrate in carboxylation reactions. Carbonic anhydrase (CA) catalyses the hydration of CO2 into bicarbonate. Since inorganic carbon stimulated L. plantarum growth, we searched for CA encoding genes in LAB genomes. CA can be classified into three classes according to their protein relatedness: α, β and γ . A class α CA was found in the L. plantarum, Leuconostoc mesenteroides, Streptococcus thermophilus, Oenococcus oeni, Enterococcus faecalis and Enterococcus faecium. These enterococci harboured a second CA encoding gene belonging to the γ class. No CA encoding gene was found in the Lactococcus lactis genome. These observations are discussed with regard to LAB evolution and ecological niches, which are often rich in IC. Lactic acid bacteria / Lactobacillus plantarum / carbon dioxide / bicarbonate / carbonic anhydrase