Lysogeny in Lactobacillus delbrueckii strains and characterization of two new temperate prolate-headed bacteriophages

Aims: Frequency of lysogeny in Lactobacillus delbrueckii strains (from commercial and natural starters) and preliminary characterization of temperate bacteriophages isolated from them. Methods and Results: Induction of strains (a total of 16) was made using mitomycin C (MC) (0·5 μg ml⁻¹). For 37% of the MC-treated supernatants, it was possible to detect phage particles or presence of killing activity, but only two active bacteriophages were isolated. The two temperate phages isolated were prolate-headed phages which belonged to group c of Lact. delbrueckii bacteriophages classification. Different DNA restriction patterns were obtained for each phage, while the structural protein profiles and packaging sites were identical. Distinctive one-step growth curves were exhibited by each phage. An influence of calcium ions was observed for their lysis in broth but not on the adsorption levels. Conclusions: Our study showed that lysogeny is also present in Lact. delbrueckii strains, including commercial strains. Significance and Impact of the Study: Commercial strains could be lysogenic and this fact has a great practical importance since they could contribute to the dissemination of active-phage particles in industrial environments.     

Lactic acid production by mixed cultures of Kluyveromyces marxianus, Lactobacillus delbrueckii ssp. bulgaricus and Lactobacillus helveticus

Lactic acid production using Kluyveromyces marxianus (IFO 288), Lactobacillus delbrueckii ssp. bulgaricus (ATCC 11842) and Lactobacillus helveticus (ATCC 15009) individually or as mixed culture on cheese whey in stirred or static fermentation conditions was evaluated. Lactic acid production, residual sugar and cell biomass were the main features examined. Increased lactic acid production was observed, when mixed cultures were used in comparison to individual ones. The highest lactic acid concentrations were achieved when K. marxianus yeast was combined with L. delbrueckii ssp. bulgaricus, and when all the strains were used revealing possible synergistic effects between the yeast and the two lactic acid bacteria. The same synergistic effects were further observed and verified when the mixed cultures were applied in sourdough fermentations, proving that the above microbiological system could be applied in the food fermentations where high lactic acid production is sought.     

Adsorption of temperate phages of Lactobacillus delbrueckii strains and phage resistance linked to their cell diversity

Aims: The aim of this work was to study the adsorption step of two new temperate bacteriophages (Cb1/204 and Cb1/342) of Lactobacillus delbrueckii and to isolate phage‐resistant derivatives with interesting technological properties. Methods and Results: The effect of divalent cations, pH, temperature and cell viability on adsorption step was analysed. The Ca²⁺ presence was necessary for the phage Cb1/342 but not for the phage Cb1/204. Both phages showed to be stable at pH values between 3 and 8. Their adsorption rates decreased considerably at pH 8 but remained high at acid pH values. The optimum temperatures for the adsorption step were between 30 and 40°C. For the phage Cb1/342, nonviable cells adsorbed a lower quantity of phage particles in comparison with the viable ones, a fact that could be linked to disorganization of phage receptor sites and/or to the physiological cellular state. The isolation of phage‐resistant derivatives with good technological properties from the sensitive strains and their relationship with the cell heterogeneity of the strains were also made. Conclusions: Characterization of the adsorption step for the first temperate Lact. delbrueckii phages isolated in Argentina was made, and phage‐resistant derivatives of their host strains were obtained. Significance and Impact of the Study: Some phage‐resistant derivatives isolated exhibited good technological properties with the prospective to be used at industrial level.     

Cloning and nucleotide sequence analysis of the Lactobacillus delbrueckii ssp. lactis DSM7290 cysteine aminopeptidase gene pepC

Abstract A genomic library of Lactobacillus delbrueckii ssp. lactis DSM7290 in the low copy number vector pLG339, was screened for the presence of peptidase genes. Using the chromogenic substrate gly-ala-β-naphthylamide, which is not a substrate for any of the recently cloned peptidases of DSM7290, and the multiple peptidase deficient Escherichia coli strain CM89, allowed the isolation of clones, which contained the equivalent hydrolytic activity. To identify genes encoding the conserved catalytic active site of cysteine proteases, partial nucleotide sequencing with a degenerate oligonucleotide was performed on recombinant plasmids isolated from such clones. This allowed to identify two out of nine clones to carry the Lactobacillus pepC gene. A total of 2026 nucleotides were determined, and sequence analysis revealed a gene with strong homology to the recently cloned Lb. helveticus (73.2%) and Lactococcus lactis (51.03%) pepC genes, and the derived protein showed homology with the active site of a large number of cysteine proteases. The predicted open reading frame consists of 449 codons, coding for a protein of 50 909 Da. The enzyme is functional and extremely overexpressed in E. coli .     

Biodiversity in Lactobacillus helveticus strains present in natural whey starter used for Parmigiano Reggiano cheese

AIMS: Lactobacillus helveticus is the dominant microflora of the natural whey starters used for Parmigiano Reggiano cheese making. The aim of this work was to study the biodiversity of different strains of Lact. helveticus present in six cultures and to compare them with strains of the same species previously isolated from natural whey cultures used for Grana Padano and Provolone cheeses. METHODS AND RESULTS: Twenty different biotypes of Lact. helveticus strains were identified combining the results deriving from SDS-PAGE of cell surface proteins and PCR fingerprinting using M13 as a primer. The biotypes were present in varying amounts in the six natural whey starters and the biodiversity was demonstrated not only within the whey cultures, but also between the whey cultures. CONCLUSIONS: Lact. helveticus strains isolated from Parmigiano Reggiano whey cultures analysed by PCR M13, SDS-PAGE and RFLP were distinguishable from Lact. helveticus strains of different dairy origin, namely Grana Padano and Provolone natural whey starters. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of different Lact. helveticus biotypes seems to be related to the specific ecosystem of cheese making and may be considered as one of the elements contributing to the typicality of Parmigiano Reggiano cheese.     

Integration of the group c phage JCL1032 of Lactobacillus delbrueckii subsp. lactis and complex phage resistance of the host

AIMS: Sequences related to Lactobacillus delbrueckii phage JCL1032 genome integration, the maintenance of lysogeny and putative immunity genes were characterized. Phenotypic changes of the JCL1032 lysogens were investigated. METHODS AND RESULTS: Integration of JCL1032 DNA into the host genome and the location of phage and bacterial attachment sites were studied by standard molecular methods. The frequency of lysogenization was 10(-7), and stable lysogeny was an even rarer phenomenon. JCL1032 integrates its genome into two distinct host genes of unknown functions. According to EOP (efficiency of plating) and adsorption tests JCL1032 lysogens showed resistance against several virulent and temperate Lactobacillus phages at different steps of phage infection. CONCLUSIONS: Temperate JCL1032 integrates its genome into bacterial DNA with exceptionally low frequency. JCL1032 lysogens express a complex phage resistance against several Lact. delbrueckii phages. An antagonistic arms race between the temperate phage and its host is proposed. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time that the genome integration of a group c Lact. delbrueckii phage has been described. The characterized lysogens could facilitate studies on Lact. delbrueckii phage receptors and phage resistance mechanisms. The genetic information gained from this study benefits the development of integration vectors and phage resistant starters.     

Characterization of three Lactobacillus delbrueckii subsp. bulgaricus phages and the physicochemical analysis of phage adsorption

AIMS: Three indigenous Lactobacillus delbrueckii subsp. bulgaricus bacteriophages and their adsorption process were characterized. METHODS AND RESULTS: Phages belonged to Bradley's group B or the Siphoviridae family (morphotype B1). They showed low burst size and short latent periods. A remarkably high sensitivity to pH was also demonstrated. Indigenous phage genomes were linear and double-stranded DNA molecules of approx. 31-34 kbp, with distinctive restriction patterns. Only one phage genome appeared to contain cohesive ends. Calcium ions did not influence phage adsorption, but it was necessary to accelerate cell lysis and improve plaque formation. The adsorption kinetics were similar on viable and nonviable cells, and the adsorption rates were high between 0 and 50 degrees C. SDS and proteinase K treatments did not influence the phage adsorption but mutanolysin and TCA reduced it appreciably. No significant inhibitory effect on phage adsorption was observed for the saccharides tested. This study also revealed the irreversibility of phage adsorption to their hosts. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The study increases the knowledge on phages of thermophilic lactic acid bacteria.     

Isolation and characterization of Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus from plants in Bulgaria

One of the traditional ways of preparation of yogurt starter in Bulgaria is placing a branch of a particular plant species into boiled sheep's milk maintained at about 45°C, which is further incubated until a dense coagulum is obtained. To investigate the possible origin of the yogurt starter bacteria, Lactobacillus delbrueckii ssp. bulgaricus ( L. bulgaricus ) and Streptococcus thermophilus ( S. thermophilus ), the traditional way of yogurt-starter preparation was followed. Hundreds of plant samples were collected from four regions in Bulgaria and incubated in sterile skim milk. The two target bacteria at low frequencies from the plant samples collected were successfully isolated. Phenotypic and genotypic characteristics of these bacterial isolates revealed that they were identified as L. bulgaricus and S. thermophilus . Twenty isolates of L. bulgaricus and S. thermophilus , respectively, were selected from the isolated strains and further characterized with regard to their performance in yogurt production. Organoleptic and physical properties of yogurt prepared using the isolated strains from plants were not significantly different from those prepared using commercial yogurt-starter strains.
It was therefore suggested that L. bulgaricus and S. thermophilus strains widely used for commercial yogurt production could have originated from plants in Bulgaria. To our knowledge, this is the first report on the isolation and characterization of L. bulgaricus and S. thermophilus strains from plants.     

Screening and selection of exopolysaccharide-producing strains of Lactobacillus delbrueckii subsp. bulgaricus

AIMS: The selection of exopolysaccharide (EPS)-producing strains of Lactobacillus delbrueckii subsp. bulgaricus. METHODS AND RESULTS: Improved EPS-overproducing strains of L. delbrueckii subsp. bulgaricus were derived by chemical mutagenesis and selection. Initial screening of the chemically induced mutant pool relied primarily on the selection of strains with raised levels of lactic acid and reduced biomass formation. Supporting selection criteria used were ropiness and colonial mucoidy. Final screening of candidate strains undertaken in a semi-defined medium in batch culture, resulted in the selection of a mutant with a 35% improvement in specific EPS yield relative to the parent strain. CONCLUSIONS: Initial selection of mutants of L. delbrueckii subsp. bulgaricus on the basis of enhanced formation of lactate and reduced biomass formation, coupled with a ropy or mucoid phenotype, proved to be a satisfactory means of isolating strains with the potential for a higher level of specific EPS production than the parent strain. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay protocol allowed for the selection of an EPS-overproducing strain of L. delbrueckii subsp. bulgaricus. Such strains are useful for the purposes of metabolic studies related to EPS-production.     

Proteolytic activity of Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus in frozen-stored Kashkaval cheese

Proteolytic activity of Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus in Kashkaval cheeses of varying aging times, stored at −10 to −12°C for 12 months, was studied. It was established that the proteolysis of Kashkaval cheese induced by the starter culture was significantly delayed by freezing. The noncasein nitrogen (NCN/TN) and nonprotein nitrogen (NPN/TN) as a percentage of total nitrogen increased slightly during frozen storage of Kashkaval. It was found that NCN/TN and NPN/TN values increased to a larger extent in frozen-stored Kashkaval samples with shorter aging time. Enhanced proteolysis was observed during ripening of thawed Kashkaval cheese. There was greater accumulation of noncasein nitrogen in thawed Kashkaval samples compared to the control samples. The enhanced proteolysis during ripening of thawed Kashkaval cheese resulted in larger amounts of high and medium molecular weight peptides and lower amounts of low molecular weight peptides and free amino acids as compared to controls.     

Lactobacillus delbrueckii ssp. lactis and ssp. bulgaricus: a chronicle of evolution in action

Background

Lactobacillus delbrueckii ssp. lactis and ssp. bulgaricus are lactic acid producing bacteria that are largely used in dairy industries, notably in cheese-making and yogurt production. An earlier in-depth study of the first completely sequenced ssp. bulgaricus genome revealed the characteristics of a genome in an active phase of rapid evolution, in what appears to be an adaptation to the milk environment. Here we examine for the first time if the same conclusions apply to the ssp. lactis, and discuss intra- and inter-subspecies genomic diversity in the context of evolutionary adaptation.

Results

Both L. delbrueckii ssp. show the signs of reductive evolution through the elimination of superfluous genes, thereby limiting their carbohydrate metabolic capacities and amino acid biosynthesis potential. In the ssp. lactis this reductive evolution has gone less far than in the ssp. bulgaricus. Consequently, the ssp. lactis retained more extended carbohydrate metabolizing capabilities than the ssp. bulgaricus but, due to high intra-subspecies diversity, very few carbohydrate substrates, if any, allow a reliable distinction of the two ssp. We further show that one of the most important traits, lactose fermentation, of one of the economically most important dairy bacteria, L. delbruecki ssp. bulgaricus, relies on horizontally acquired rather than deep ancestral genes. In this sense this bacterium may thus be regarded as a natural GMO avant la lettre.

Conclusions

The dairy lactic acid producing bacteria L. delbrueckii ssp. lactis and ssp. bulgaricus appear to represent different points on the same evolutionary track of adaptation to the milk environment through the loss of superfluous functions and the acquisition of functions that allow an optimized utilization of milk resources, where the ssp. bulgaricus has progressed further away from the common ancestor.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-407) contains supplementary material, which is available to authorized users.     

Cloning of the D-lactate dehydrogenase gene from Lactobacillus delbrueckii subsp. bulgaricus by complementation in Escherichia coli

A strain of Escherichia coli (FMJ144) deficient for pyruvate formate lyase and lactate dehydrogenase (LDH) was complemented with a genomic DNA library from Lactobacillus delbrueckii subsp. bulgaricus. One positive clone showed LDH activity and production of D(−)lactate was demonstrated. The nucleotide sequence of the D-LDH gene (ldhA) revealed the spontaneous insertion of an E. coli insertion sequence IS2 upstream of the gene coding region. The open reading frame encoded a 333-amino acid protein, showing no similarity with known L-LDH sequences but closely related to L. casei D-hydroxyisocaproate dehydrogenase (D-HicDH).     

Technological properties of Lactobacillus fermentum involved in the processing of dolo and pito, West African sorghum beers, for the selection of starter cultures

Aim: Technological properties of Lactobacillus fermentum isolates involved in spontaneous fermentation of dolo and pito wort were examined to select starter cultures. Methods and Results: 264 isolates were screened for antimicrobial activity, acidifying activity, exopolysaccharides (EPSs) and amylase production. An antimicrobial activity was detected for 33·3%, 31·8%, 22·7% and 15·9% of the isolates towards Staphylococcus aureus enterotoxin A producer, Staphylococcus aureus enterotoxin A and B producer, Escherichia coli and Listeria innocua, respectively. A similarity was found between the isolates which were clustered in four groups according to their rates of acidification of sorghum malt broth. The faster acidifying group of isolates (43·48%) had a rate of acidification evaluated as ΔpH of 1·14 ± 0·15 pH unit after 6 h of fermentation, followed by a second group of isolates (38·08%) with a similar rate of acidification after 9 h of fermentation. From the isolates endowed with an antimicrobial activity, 5·76% belonged to the faster acidifying group and 40·38% belonged to the second group. 88·7% of the isolates had the ability for producing EPSs but not amylase. Conclusion: Lactobacillus fermentum ferments dolo and pito wort by lowering the pH and providing organic acids, EPSs and antimicrobial compounds. Significance and Impact of the Study: Strains with a rapid rate of acidification, an antimicrobial activity and producing EPSs are suggested to have potential for starter cultures.     

Bacillus amyloliquefaciens ssp. plantarum strains as potential protective starter cultures for the production of Bikalga,an alkaline fermented food

Aims

To identify and screen dominant Bacillus spp. strains isolated from Bikalga, fermented seeds of Hibiscus sabdariffa for their antimicrobial activities in brain heart infusion (BHI) medium and in a H. sabdariffa seed‐based medium. Further, to characterize the antimicrobial substances produced.

Methods and Results

The strains were identified by gyrB gene sequencing and phenotypic tests as B. amyloliquefaciens ssp. plantarum. Their antimicrobial activity was determined by the agar spot and well assay, being inhibitory to a wide range of Gram‐positive and Gram‐negative pathogenic bacteria and fungi. Antimicrobial activity against Bacillus cereus was produced in H. sabdariffa seed‐based medium. PCR results revealed that the isolates have potential for the lipopeptides iturin, fengycin, surfactin, the polyketides difficidin, macrolactin, bacillaene and the dipeptide bacilysin production. Ultra‐high‐performance liquid chromatography‐time of flight mass spectrometry analysis of antimicrobial substance produced in BHI broth allowed identification of iturin, fengycin and surfactin.

Conclusions

The Bacillus amyloliquefaciens ssp. plantarum exhibited broad‐spectrum antifungal and antibacterial properties. They produced several lipopeptide antibiotics and showed good potential for biological control of Bikalga.

Significance and Impact of the Study

Pathogenic bacteria often occur in spontaneous food fermentations. This is the first report to identify indigenous B. amyloliquefaciens ssp. plantarum strains as potential protective starter cultures for safeguarding Bikalga.     

Technological and molecular diversity of Lactobacillus plantarum strains isolated from naturally fermented sourdoughs

Thirty Lactobacillus (L.) plantarum strains, isolated from sourdough, were identified by biochemical tests as well as 16S rDNA sequencing and differentiated on the basis of technological properties, such as amylase, protease, phytase and antirope activities. These properties were shown to be widely differing among the strains, indicating a significant technological diversity. Genetic differentiation was achieved by restriction endonuclease analysis-pulsed field gel electrophoresis (REA-PFGE) that allowed the L. plantarum strains to be divided into 10 different genomic groups. Moreover, 32 different starters were employed in dough making experiments; each starter consisted of a single strain of L. plantarum associated with a maltose positive or a maltose negative yeast. The technological properties of the doughs were greatly influenced by the type of strain included in the starter. The time of leavening and the acidification activities detected in the dough were enhanced by the presence of L. plantarum strains. The bacterial and yeast contents and fermentation properties were statistically treated by principal component analysis (PCA), which allowed the discrimination of different typologies of dough. The study of the peculiar characteristics of different strains of L. plantarum is fundamental for a better understanding of their potential in affecting the nutritional value, quality and stability of the baked goods. L. plantarum strains are able to differentially influence the dough quality when employed as starters.     

Isolation, taxonomic identification and hydrogen peroxide production by Lactobacillus delbrueckii subsp. lactis T31, isolated from Mongolian yoghurt: inhibitory activity on food-borne pathogens

AIMS: The aim of this work was to isolate lactic acid bacteria (LAB) strains from Mongolian tarag (a traditionally homemade yoghurt) displaying antimicrobial activities against food-borne pathogens, identify inhibitory substances and study the kinetics of their production. METHODS AND RESULTS: Inhibitory substance-producing bacterial strains were isolated from tarag. From 300 bacterial clones, 31 were able to inhibit the growth of the indicator strain Lactobacillus bulgaricus 340. One of the most active strains was identified as Lactobacillus delbrueckii subsp. lactis strain T31 by using cluster analysis of amplified fragment length polymorphism (AFLP) DNA fingerprints. The antimicrobial substance was inactivated by catalase, demonstrating the production of hydrogen peroxide (H(2)O(2)). Production of H(2)O(2) was studied under aerated and nonaerated culture conditions. The amount of H(2)O(2) in the culture supernatant increased during bacterial growth and reached a maximum (5.12 mmol l(-1)) at the early stationary phase under aerated conditions (agitated cultures). H(2)O(2) was not detected in the culture performed without agitation. In mixed cultures performed in milk with either Lact. delbrueckii subsp. lactis T31 in the presence of Escherichia coli, or Lact. delbrueckii subsp. lactis T31 in the presence of Listeria innocua under aerated and nonaerated conditions, a significant decrease in pathogen count was observed in aerated cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: The significant decrease in Listeria viability observed in aerated mixed cultures of Lact. delbrueckii subsp. lactis T31 is mainly because of H(2)O(2) production. Lactobacillus delbrueckii subsp. lactis T31 could be used as a protective culture in food industries or as a probiotic to prevent intestinal and urogenital infections.     

Hydrolysis of whey proteins by Lactobacillus acidophilus, Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus grown in a chemically defined medium

AIMS: To evaluate the ability of themophilic lactic acid bacteria (LAB) to hydrolyse the whey proteins beta-lactoglobulin (BLG) and alpha-lactalbumin (ALA) in a chemically defined medium (CDM). METHODS AND RESULTS: The ability of three LAB strains to hydrolyse BLG and ALA was studied in a CDM supplemented with these proteins or whey protein concentrate (WPC). Protein hydrolysis was determined by Tricine/SDS-PAGE and RP-HPLC. Maximum BLG (21%) and ALA (26%) degradation by LAB was observed using WPC. Under starving conditions, BLG degradation was greater for Lactobacillus delbrueckii ssp. bulgaricus CRL 454 than for Lactobacillus acidophilus CRL 636 and Streptococcus thermophilus CRL 804. All three strains showed different peptide profiles and were not able to hydrolyse ALA under starvation. CONCLUSIONS: The assayed LAB strains were able to degrade BLG during growth in a CDM and under starving conditions. The different peptide profiles obtained indicate distinct protease specificities. SIGNIFICANCE AND IMPACT OF THE STUDY: These strains could be used as adjunct cultures to increase BLG digestibility in whey-based or whey-containing foods. To our knowledge, this is the first report on the ability of a Lact. acidophilus strain to degrade BLG.