Svetla Ilieva

Production of wheat bread without preservatives using sourdough starters.

In order for the beneficial effects of sourdough application in breadmaking to take place a proper selection of lactic acid bacteria species and strains, an appropriate technology and effective control of the purity and activity of the selected cultures. Four symbiotic starters for sourdough for the production of bread were developed and probated in a production laboratory using the selected strains Lactobacillus brevis LBRZ7, L. buchneri LBRZ6, L. plantarum X2, L. paracasei RN5, L. sanfranciscensis R and L. fermentum LBRH10 and the probiotic strain Propionibacterium freudenreichii ssp. shermanii NBIMCC 327. The starter sourdoughs that include Propionibacterium freudenreichii ssp. shermanii NBIMCC 327 had greater antimicrobial activity against saprophytic microorganisms: Bacillus subtilis, B. mesentericus, Aspergillus niger, Penicillium sp. and Rhizopus sp., but none of them inhibited the growth of bakery yeasts Saccharomyces cerevisiae. It was established that in order to prevent bacterial spoilage 10% of the selected starter sourdoughs had to be added in the breadmaking process, while for prevention of mold spoilage the necessary amount of starter sourdough had to be between 15% and 20%.The application of the developed starters for the production of wheat bread guarantees longer shelf life and no adverse alterations in the features of the final bread.

IDENTIFICATION AND CHARACTERIZATION OF α-AMYLASE AND ENDOXYLANASE, PRODUCED BY ASPERGILLUS MUTANT STRAINS

ABSTRACT Filamentous fungi are widely used for the production of homologous and heterologous proteins. Recently, there has been increasing interest in Aspergillus oryzae PP and Aspergillus awamori K-1 because of its ability to produce heterologous proteins in submerged (liquid) cultures. The goal of this investigation was to determine the α-amylase and endoxylanase enzyme production ability and molecular characteristics of fungal strains Aspergillus oryzae PP and Aspergillus awamori K-1 and its mutant strains R5 and A45. The strains were cultivated in liquid culture and maximum enzyme production of parent and mutant strains was determined after 72–96 h of cultivation. Extracellulars α-amylase and endoxylanase were partially purified from the culture filtrates, using molecular sieve chromatography with Gel permeation chromatography. The molecular weight of the partial purified enzymes has been estimated to be 57 kDa for α-amylase and 31 kDa for endoxylanase on SDS-polyacrylamide gel electrophoresis. The temperature optimum of the enzyme α-amylase was 30°C, respectively for endoxylanase was 40°C and the pH optimum was 4.7 and 4.0.

Examination of the technological properties of newly isolated strains of the genus Lactobacillus and possibilities for their application in the composition of starters

The ability of four Lactobacillus strains – Lactobacillus brevis LBRZ7 (isolated from fermented cabbage), Lactobacillus plantarum LBRZ12 (isolated from fermented cabbage), Lactobacillus fermentum LBRH9 (of human origin) and Lactobacillus casei ssp. rhamnosus LBRC11 (isolated from home-made cheese) – to grow in flour/water environment and to accumulate high concentrations of viable cells was examined. Two starters for sourdough were created for lab-scale production of wheat bread: a two-strain starter and a four-strain starter. Wheat bread with improved properties – greater loaf volume, enhanced flavour and softer and brighter crumb – was obtained from the 7% four-strain starter sourdough. The addition of sourdough in the production of wheat bread affected positively the technological and organoleptic characteristics of the final bread by inhibiting the growth of wild yeasts and mold and Bacillus spores without the addition of preservatives. The inclusion of 15% of the four-strain starter sourdough in the bread-making process led to enhanced safety and longer shelf life of the baked bread.

Selected adjunct cultures remarkably increase the content of bioactive peptides in Bulgarian white brined cheese

Some lactic acid bacteria strains in milk media are capable of releasing bioactive peptides. In this study, we evaluated the angiotensin-converting enzyme (ACE)inhibitory activity of 180 lactic acid bacteria and selected several Lactobacillus helveticus, L. delbrueckii subsp. bulgaricus and L. casei strains that demonstrated strong ACE-inhibitory activity. The aim was to carry out a molecular study on the bioactive peptides released by the strains with the best ACE-inhibitory properties and by the strains demonstrating a calcium-binding effect. To the best of our knowledge, this is the first study of bioactive peptides in Bulgarian white cheese. Peptides with the strongest ACE-inhibitory activity were purified and sequenced. The strains were assessed for production of peptides with calcium-binding properties. These peptides were isolated, purified and sequenced. Two strains releasing bioactive peptides with the strongest ACE-inhibitory and calcium-binding activities were selected for development of cheese starters. The strain with the best ACE-inhibitory activity was L. helveticus A1, which releases the peptide Ala-Leu-Pro-Met as a main contributor to the ACE inhibition. The strain with the best calcium-binding activity was L. casei C3 releasing the peptide SpLSpSpSpE (fraction 15–20 of ß-casein) as a main contributor to calcium binding. After pilot production of cheeses with the developed starters, the ACE-inhibitory and calcium-binding effects were confirmed during the cheese ripening. The addition of the two selected adjunct cultures led to increased production of bioactive peptides in the cheese. In this way, it is possible to increase the functional properties of Bulgarian white brined cheese.

Quantitative Assessment of the Dominant Genome in Fusant Cultures

ABSTRACT Selection of fusant cultures, producers of bioactive compounds is of significant importance for the biotechnology industry. Protoplast fusion between prototrophic strains of Aspergillus oryzae PP, producer of alfa-amylase and Apergillus awamori K-1, producer of xylanase was obtained. The interspecific nature of the fusants was determined by morphology and screening for combined enzymatic activity. The aim of this investigation was to quantitate the dominant parental genome in the fusants by applying whole genome typing strategy based on the Amplified Fragment Length Polymorphisms (AFLP) technique. In addition we investigated the genome decomposition of the fusant cultures from first to fourth generation. Our results demonstrate that the appearances of the fusants varied but they basically resembled Aspergillus awamori. On the bases of more than 80 polymorphic markers, the genomic analysis confirmed that Aspergillus awamori is the dominant genome (>85%) in the fusants. The stability of the fusants was examined by successive subcultures. The genomic decomposition of the fusants between first and forth generation is as little as 3%. From our studies we could conclude that AFLP is an efficient and discriminatory method for evaluation of the dominant genome in fusant cultures and the extent of genomic decomposition in the generations.

Production of β-Xylanase from Aspergillus Awamori K-1

ABSTRACT The production and hydrolytic potential of xylanase and β-xylosidase from Aspergillus awamori, strain K-1 were studied. The fermentation medium was optimised for maximum extracellular xylanase and β-xylosidase production on shake-flask and laboratory fermentor scale. Of the carbon sources studied, oat xylan and a mixture of ball-milled corn cobs and wheat bran (mixture ratio 2:1) produced the highest titres of xylanase and β-xylosidase. The temperature optima for maximal production of xylanase and β-xylosidase were 30 °C and 25 °C, respectively, and the pH optimum for both enzymes was 3.5–4.0. Under the optimised culture medium and conditions a considerable xylanase activity level of 100 IU/ml was measured in the culture filtrates. Crude xylanase demonstrated a certain potential to liberate reducing sugars from α-cellulose. Xylose and xylobiose were established as the main products of the enzymatic hydrolysis of larch wood xylan.

Characterization of Enzyme Endoxylanase Produced by Mutants Strains of Aspergillus Awamori K-1

ABSTRACT Xylanolytic enzymes of microbial origin have received great attention due to their biotechnological utility and potential applications in a range of industrial processes but the observed effects vary depending on xylanase specificities. The goal of this investigation was to determine the endoxylanase enzyme production ability and molecular characteristics of fungal strains Aspergillus awamori K-1 and its mutant strains A59, A50, A45 and A60. The strains were cultivated in liquid culture with 1% wheat bran and 2% ground corn-cobs as inducer. Maximum enzyme production of parent strain Aspergillus awamori K-1 of 42,35 IU/ml and of mutant strains A59 - 96,91 IU/ml, A50 - 71,67 IU/ml, A45 - 74,04 IU/ml and A60 - 62,59 IU/ml was determined after 72–96 h of cultivation. Extracellular endoxylanase was partially purified from the culture filtrates of five strains, using molecular sieve chromatography with two type of Sephadex—G 50 and G100. The purification conditions were optimized and the main endoxylanase activity was determined in second fraction in case of Sephadex G100. The partial purified enzyme fractions showed the same band for all strains on SDS polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular weight of 32 kDa.

Molecular Taxonomic Characterisation of Probiotic Strain Lactobacillus Sp. 50P1

ABSTRACT In recent years interest in the probiotic lactobacilli has been stimulated by the use of these bacteria in products that are claimed to confer health benefits on the consumer. The probiotic effects are usually strain-specific, meaning that a correct identification is important to link the strain to the specific health effect. Taxonomical characterization of probiotic strains only in phenotypic and phisyological characteristics is often with low level of discrimination, probably due to their co-evolution in the same ecological niches. Thus, the nucleotide base techniques provide an accurate basis for phylogenetic analysis and identification. With this aim a probiotic strain Lactobacillus spp. 50P1 was studied. Trough the physiological characterization with API 50 CH the strain 50P1, was determinate as Lactobacillus helveticus, with low probability- 77,5%. Three PCR-based methods, species-specific PCR, of 16S rRNA gene sequencing, and restriction enzyme of 16S rDNA ARDRA analysis were used for reliable taxonomic characterization of probiotic strain Lactobacillus sp. 50P1. The sequence obtained from the strain was compared to those of Lactobacillus species held in GenBank and the belonging of the strain 50P1 to the species Lactobacillus helveticus was confirmed.