Penka Petrova

l(+)-Lactic acid production from starch by a novel amylolytic Lactococcus lactis subsp. lactis B84

A new Lactococcus lactis subsp. lactis B84, capable of utilizing starch as a sole carbon source and producing L(+)-lactate, was isolated from spontaneously fermented rye sourdough. Aiming at maximum lactic acid productivity, the components of the media and the cultivation conditions were varied. In MRS-starch medium (with absence of yeast and meat extracts), at 33 degrees C, agitation 200 rpm and pH 6.0 for 6 days complete starch hydrolysis occurred and 5.5 gl(-1) lactic acid were produced from 18 gl(-1) starch. The identification of strain B84 was based on genetic criteria. Amplified ribosomal DNA restriction analysis (ARDRA), PCR with species-specific primers and sequencing of the 16S rDNA proved its species affiliation. Four genes for enzymes, involved in starch degradation were detected in B84 genome: amyL, amyY, glgP and apu, coding cytoplasmic and extracellular alpha-amylases, glycogen phosphorylase and amylopullulanase, respectively. Reverse transcription PCR experiments showed that both genes, encoding alpha-amylases (amyL and amyY) were expressed into mRNAs, whereas apu and glgP were not. Amylase activity assay was performed at different pH and temperatures. The cell-bond amylase proved to be the key enzyme, involved in the starch hydrolysis with maximum activity at 45 degrees C and pH 5.4.

Structural and functional analysis of pt38, a 2.9 kb plasmid of Streptococcus thermophilus yogurt strain

The cryptic plasmid pt38 (2911 bp) of Streptococcus thermophilus ST2783, a strain isolated from Bulgarian yogurt, was subcloned and sequenced. Five ORFs (ORF1 to ORF5) were identified, although putative transcription initiation and termination signals, and Shine-Dalgarno sequence could only be localized for three of them (ORF1, ORF2, and ORF5). ORF2 would specify a 142-amino acid protein sharing a high degree of homology with plasmid-born low-molecular-weight heat stress proteins described in a variety of S. thermophilus strains. On the other hand, ORF1 would encode a 311-residue protein, which was found to be almost identical to the putative Rep proteins of previously sequenced S. thermophilus rolling circle-replicating plasmids. Intracellular single-stranded pt38 DNA was detected, showing that, in fact, the plasmid replicates via a rolling circle mechanism. A putative double-strand origin with significant homology to that of pC194, and a ssoA-type single-strand origin were also identified on the nucleotide sequence of pt38. A DNA region that can be transcribed into a small RNA (ctRNA) complementary to the leader segment of the rep (ORF1) mRNA is proposed to be involved in the control of plasmid replication. In vitro synthesis of this ctRNA was observed, and this constitutes the first report on the existence of such antisense RNAs, likely acting as regulatory elements, in S. thermophilus plasmids.

Direct conversion of chicory flour into l(+)-lactic acid by the highly effective inulinase producer Lactobacillus paracasei DSM 23505

Highly effective bio-process for lactic acid (LA) production by simultaneous saccharification and fermentation (SSF) of chicory flour was developed. The strain used, Lactobacillus paracasei DSM 23505 produced natural inulinase (EC 3.2.1.80) with molecular weight ∼130 kDa, located in the cell wall fraction. In batch fermentation with optimized medium content and fermentation conditions, a complete conversion of 136 g/L chicory flour (89.3% inulin and 10.7% mix of sucrose, fructose and glucose) into 123.7 g/L LA was achieved. These yield and conversion rate are the highest obtained by SSF for LA production from inulin. The high efficiency, the cheap fermentation broth and the simple process performance disclose the promising use of the chicory flour in industrial biotechnology for LA production.

Amylolytic Lactobacillus Strains from Bulgarian Fermented Beverage Boza

The lactic acid fermentation is a worldwide method for cereal processing. Great diversity of fermented foods and drinks is produced with the participation of amylolytic lactic acid bacteria (ALAB). In the present study the ALAB content of the Bulgarian cereal beverage “boza” was investigated. Two strains, Bom 816 and N3, were found to possess significant amylolytic activity. The strains’ identification was based on genetic criteria, namely amplified ribosomal DNA restriction analysis (ARDRA) and sequencing of the 16S rDNA. The strain Bom 816 belongs to the species Lactobacillus plantarum and N3 to Lactobacillus pentosus, being the first amylolytic representative of this species. Optimization of the media composition with starch as a sole carbon source was done. The starch hydrolysis was most efficient in medium containing 4 g/l yeast and 8 g/l meat extracts. Thus, L. plantarum Bom 816 consumed 14 g/l starch, while L. pentosus N3 consumed 17 g/l. The highest values of lactic acid reached were 9.5 g/l produced by Bom 816 and 5.5 g/l produced by N3. In the presence of yeast extract L. pentosus N3 formed 0.8 - 1 g/l succinic acid. Both strains produced mainly cell-bound enzymes with amylase activity, at a pH optimum of 5.5, ranging from 3 - 4 to 21 U/ml for L. pentosus N3 and from 0.5 to 11.5 U/ml for L. plantarum Bom 816, in dependence of the assay conditions

Antimicrobial Activity of Starch-Degrading Lactobacillus Strains Isolated from Boza

ABSTRACT The proved probiotic effects of boza consummation prompted the increasing interest in the microflora taking part in its fermentation. Boza is a rich source of probiotic lactic acid bacteria that are able to produce bacteriocins active against a number of pathogens. In the present study, three strains—Lactobacillus paracasei B41, Lactobacillus plantarum Bom 816, and Lactobacillus pentosus N3, isolated from boza and possessing significant amylolytic activity, were studied for their ability to repress the growth of common foodborne pathogens. All strains demonstrated antimicrobial activity against Escherichia coli, Klebsiella pneumoniae, Vibrio cholerae and Bacillus subtilis. The results indicated possible bacteriocin production and made the strains desirable starters with application in cereals processing. None of the tested lactobacilli inhibited the growth of Saccharomyces cerevisiae or Pichia strains, suggesting the existence of stable microbial community of yeasts and lactobacilli in boza starters.

Rapid screening of plasmid-encoded small hsp-genes in Streptococcus thermophilus

A new rapid procedure for detection of small heat shock protein genes (shsp) was developed. Using PCR-based molecular approach, single colonies of 49 Streptococcus thermophilus industrial and artisanal starters were examined. Five strains contained plasmid-encoded shsp. The nucleotide sequence analysis revealed that the genes are very conservative, as only a few nucleotide substitutions were noticed. It was shown that all new isolated plasmids belong to the pC194 family of rolling circle replicating (RCR) plasmids. We concluded that shsp genes are always inherited together with pC194-type replicative region. The viability of plasmid-bearing and plasmid-free derivatives of S. thermophilus strain ST2980 under heat shock condition was studied.

Molecular Study of the useful and the Contaminant Microflora in Fermented Dairy Products

ABSTRACT Lactic acid bacteria have been used as starter strains in the production of fermented dairy products for centuries. Most of the dairy products contain lactic acid bacteria, but also other bacteria involved as contaminant microflora. We explored the microbial content of home-made dairy products and those purchased from the market. In our study twenty-six pure cultures were isolated. The isolated strains were investigated by a set of physiological and molecular-genetic methods for their accurate species identification and genotyping. From the microorganisms, involved in fermentation and ripening of dairy products with proven health benefits to human, in studied foods predominated Lactobacillus delbrueckii, Lactobacillus helveticus and Lactobacillus plantarum. Another part of the isolated strains, representatives of the genus Kluyveromyces, Rhodotorula and Candida were contaminant microflora, as a result of poor hygiene in the manufacture and storage of the dairy products. Some of these strains were isolated from commercially available dairy products. The obtained results raise again the question about the efficacy of microbiological quality control and food safety.

GENETIC DIVERSITY OF BACTERIOPHAGES HIGHLY SPECIFIC FOR STREPTOCOCCUS THERMOPHILUS STRAIN LBB.A

ABSTRACT Eight virulent bacteriophages φA1, φA2, φA5, φA6, φDmA, φSfA, φPtA and φA7, that showed high specificity for Streptococcus thermophilus strain LBB.A, the streptococcal component of an industrial yogurt starter, were isolated. The comparison of the HindIII and EcoRV restriction patterns of the phage DNA revealed limited relatedness between the isolates, clustering together only φA5 with φA6 and indicating similarity between φSfA, φPtA and φA7. All eight bacteriophages were classified as costype phages. An internal region within a conserved 2.2 kbp EcoRI DNA fragment typical for the majority of S. thermophilus bacteriophages was present in six of the phage isolates and was absent in φDmA and φA1. PCR amplification of the VR2 region of the antireceptor gene yielded an 800 bp product for seven of the phages and a 700 bp product for φA2. The genome size of the eight phage isolates was in the range of 33.4–37.3 kbp. Of a total of 62 other S. thermophilus cultures φA1, φDmA and φPtA were able to infect only two strains with reduced effectiveness of plaquing. Despite the broad genetic diversity of phages of S. thermophilus strain LBB.A, a two-step selection procedure, involving successive challenge of the bacterial culture with φA1 and φA7, was sufficient to obtain mutants resistant to all eight phases.

Microbial diversity and health-promoting properties of the traditional Bulgarian yogurt

ABSTRACT Bulgarian yogurt is usually associated with good health and longevity. This study aimed to analyse the current microbial content of homemade Bulgarian yogurt. Identification by 16S rDNA sequencing revealed that out of 76 isolated strains, 53% belonged to Lactobacillus delbrueckii subsp. bulgaricus, 14% to other lactobacilli, and 32% to lactic acid cocci (Streptococcus thermophilus, Pediococcus acidilactici, Enterococcus faecium and Leuconostoc). All isolates inhibited the growth of pathogenic bacteria (10% of them by the putative action of bacteriocins); 18 isolates were able to produce extracellular exopolysaccharides (EPS), whereas 24% of them demonstrated extremely high proteolytic activity. Remarkably, 10 Lb. bulgaricus strains produced prebiotic galactooligosaccharides (GOS). High-resolution accurate mass spectrometry (HRAMS) analysis revealed production of tri- and tetrasaccharides containing atypical β(1→4) glycosidic bonds, reported for the first time for Lb. bulgaricus. Based on the beneficial features, along with good technological behaviour, we recommend several isolates as highly promising for Bulgarian yogurt starters.

Prebiotic–Probiotic Relationship: The Genetic Fundamentals of Polysaccharides Conversion by Bifidobacterium and Lactobacillus Genera

Abstract Current trends in nutrition science aim to improve foods by the addition of prebiotic carbohydrates. This approach provides better appearance, taste, and texture to the product, along with beneficial effects on the consumers’ heath. In this chapter, the food industry’s most frequently used probiotic strains and prebiotic carbohydrates are described. The applications of inulin, fructo-olygosaccharides (FOS), galactooligosaccharides (GOS), isomalto-oligosaccharides (IMO), resistant starch (RS), xylo-oligosaccharides (XOS), and soybeanoligosaccharides (SOS) are revealed in terms of their prebiotic effects. Besides, the potential routes and the mechanisms of prebiotics’ microbial hydrolysis are discussed. The genetic fundamentals of prebiotic–probiotic interrelations were clarified by comparison of the latest bioinformatics data, concerning genes and enzymes involved in carbohydrates hydrolysis by lactobacilli and bifidobacteria. The presence and expression of genes encoding glycoside-hydrolase members of different families are reported. Different approaches for prebiotics industrial manufacturing were summarized. This chapter offers deep insights into the prebiotic–probiotic partnership, giving extensive information about the genetic prerequisites allowing in vivo probiotics multiplication.