Dilyana Nikolova

A novel one-pot synthesis and preliminary biological activity evaluation of cis-restricted polyhydroxy stilbenes incorporating protocatechuic acid and cinnamic acid fragments

A series of new stilbenes 4a-e, 5 were synthesized through a novel one-pot Perkin-like reaction between 6,7-dimethoxyhomophthalic anhydride and aromatic aldehydes, followed by treatment with BBr3. This synthesis is straightforward and allows polyhydroxylated cis-stilbenes gathering two well-known pharmacophoric fragments to be obtained in good yields and for short reaction times. The structure of the newly synthesized compounds was established by spectroscopic methods ((1)H NMR, (13)C NMR, IR and HRMS) and the double bond configuration was unequivocally elucidated by means of gated decoupling (13)C NMR spectra and 2D NOESY experiments. Preliminary differentiating screening of their radical scavenging, antibacterial, anti-fungal and tyrosinase inhibitory activity was further performed. The results obtained suggest that the tested compounds possess a triple biological action as potent radical scavengers, antifungal agents and tyrosinase inhibitors in micromolar concentration. The most promising bioactive compound amongst the others was 4a, acting as excellent radical scavenger against DPPH(•) radical (IC₅₀ ≤ 10 μM), antifungal agent suppressing the growth of Fusarium graminearum (89% inhibition at 0.17 μmol/mL), and tyrosinase inhibitor showing higher activity than hydroquinone at 23 μM.

Growth Parameters of Probiotic Strain Lactobacillus Plantarum, Isolated from Traditional White Cheese

ABSTRACT Different aspects, including safety, functional and technological characteristics, have to be taken into consideration in the selection of each probiotic microorganism. The aim of the present work was to determine the kinetic growth parameters of L. plantarum strain after cultivation in media with different carbon sources. The strain was isolated from traditional Bulgarian white cheese and previously characterized as putative probiotic, based on commonly accepted in vitro criteria. For further biotechnological implementation was necessary to select a suitable and economically relevant growth media. Thus, reconstituted permeate (6% w/v) and the following modification of de Man, Rogose Sharpe media (MRS): (i) MRS-glucose; (ii) MRS-lactose; (iii) MRS-galactooligosaccharide; (iv) MRS-fructooligosaccharide were used. The strain growth, lactic acid production and carbon source utilization were monitored by pH and cell number determination, and HPLC analysis at different time points of the cultivation process. The highest cell growth and carbohydrate conversation were detected in the presence of glucose and lactose. The main product of the fermentation was lactate with detectable level of acetate. The permeate and MRS-galactooligosaccharide also support good growth and lactic acid production, which indicate a great potential for industrial applications of studied L. plantarum strain into the food system.

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.

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.

Sol–gel immobilization as a suitable technique for enhancement of α-amylase activity of Aspergillus oryzae PP

Bioencapsulation of microbial cells in silica-based matrices has proved to be a good strategy to enhance the biosynthetic capabilities and viability of bioproducers. In the present study, mycelium and pellet cultures of strain Aspergillus oryzae PP were successfully immobilized in sol–gel hybrid matrices composed of tetraethylorthosilicate as an inorganic precursor, 5% (w/v) starch and 10 or 15% (w/v) polyethylene oxide, or 10% (w/v) calcium alginate as organic compounds. Biosynthetic activity of immobilized cultures was investigated by batch and fed-batch cultivation and the obtained results of 3042.04 IU cm−3 were comparable with the enzyme activity of the free cell culture. Immobilized cultures retained their viability and biosynthetic capabilities up to the 744th h during fed-batch fermentation processes. Consequently, sol–gel encapsulation in hybrid matrices could be considered as a promising technique for immobilization of Aspergillus oryzae PP in order to increase the α-amylase production.

Lactic Acid Microflora of Bulgarian Milk Products From Mountain Regions

ABSTRACT This study aimed to isolate and characterize viable lactic acid bacteria (LAB) from the most popular in Bulgaria fermented milk products. Different samples from home-made cheeses, yoghurt and katak, from ecological regions of Stara Planina, Rila and Rodopi mountains were collected. A total of 25 LAB cultures (coci and rods) were isolated and polyphasic taxonomic characterization was performed. Eight of the strains from yoghurt were phenotypically similar to Lactobacillus delbrueckii. Other seven isolates from cheese and yoghurt were identified as Lactobacillus plantarum, according to classical tests and API LAB profiles. Their species affiliation was confirmed by species-specific and multiplex PCR. The molecular- based methods allowed reclassifying two cheese isolates determined as L. paracasei. L. plantarum was the predominant species in cheese samples and referred as member of non-starter lactic acid bacteria. Two of the new isolated L. plantarum strains showed anti-E. coli activity.

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.