Ferulic acid, a very attractive natural antioxidant is present in beer in free form, but the main form is the bound form as feruloylated oligosaccharides. Previous research showed that feruloylated oligosaccharides more effectively inhibited lipid and Low Density Lipoprotein oxidation than free ferulic acid. The aim of the present study was to evaluate free and bound ferulic acid concentrations throughout the brewing process in experimental mashes (worts, beers during fermentation, maturation and storage), and to conduct a comparison in commercial beers. Another aim of the study was to investigate methods to increase levels of bound ferulic acid in beer due to the potential health benefits. Specifically, the influence of commercial enzyme preparations on both forms of ferulic acid contents was studied. Five commercial enzyme preparations during mashing were examined: Celluclast, Shearzyme, Viscozyme, Cereflo and Ultraflo. In all experimental beers, the concentrations of esterified ferulic acid were 4–6 fold higher than the corresponding free ferulic acid contents, depending on the enzyme preparation used. Ferulic acid contents in the ester form in experimental beers were in the range of 748.4 mg/hL to 1244.3 mg/hL, whereas the contents of free ferulic acid were in the range of 134.6 mg/hL to 275.2 mg/hL. Comparison of free and bound ferulic acid contents in experimental beers, produced using enzyme preparations and commercial beers found in a local market, showed that concentrations of bound ferulic acid in experimental beers were significantly higher than in commercial beers, whereas concentrations of free ferulic acid in experimental and commercial beers were comparable.
Abstract A proteinase produced by the human gastrointestinal isolate Lactobacillus rhamnosus strain OXY was identified and characterized. The prtR2 gene coding for proteinase activity was detected in the examined strain. The PCR primers used were constructed on the basis of the sequence of the prtR2 proteinase gene from Lactobacillus rhamnosus GG. The enzyme was purified by fast protein liquid chromatography (FPLC) using CM-Sepharose Fast Flow and Sephacryl S-300 columns. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the enzyme had a relatively low molecular mass of 60 kD. Protease activity was observed at a pH range from 6.5 to 7.5 with optimum k cat/K m values at pH 7.0 and 40°C. Maximum proteolytic activity (59 U mL−1) was achieved after 48 hr of cultivation. The activity of the enzyme was inhibited only by irreversible inhibitors specific for serine proteinases (PMSF and 3,4-dichloro-isocumarine), suggesting that the enzyme was a serine proteinase. Proteinase activity was increased by Ca2+ and Mg2+, and inhibited by Cu2+, Zn2+, Cd2+, and Fe2+. Keywords: Lactobacillus rhamnosus proteinasepurification
A simple spectrophotometric method for the determination of total iodine content in foods by modification of Sveikina procedure, based on the works of Moxon and Dixon, is reported. The modified method had about 100-fold higher sensitivity in comparison to the above mentioned methods. The method detection limit (MDL) was 0.01 ng per 100 g of food and a mean recovery test of 92%. This method was used for the analyses of total iodine content in different food products. Une methode spectrophotometrique simple pour la determination de la teneur en iode total dans les aliments par modification de la procedure de Sveikina et basee sur les travaux de Moxon et Dixon est reportee. La methode modifiee a une sensibilite 100 fois plus forte que les methodes precedentes. Sa limite de detection est de 0,01 ng/100g d'aliment et le recouvrement moyen est de 92%. Cette methode est utilisee pour la determination de l'iode total dans differents aliments.
Enterobacter sp. LU1, a wild-type bacterium originating from goat rumen, proved to be a potential succinic acid producer in previous studies. Here, the first complete genome of this strain was obtained and analyzed from a biotechnological perspective. A hybrid sequencing approach combining short (Illumina MiSeq) and long (ONT MinION) reads allowed us to obtain a single continuous chromosome 4,636,526 bp in size, with an average 55.6% GC content that lacked plasmids. A total of 4425 genes, including 4283 protein-coding genes, 25 ribosomal RNA (rRNA)-, 84 transfer RNA (tRNA)-, and 5 non-coding RNA (ncRNA)-encoding genes and 49 pseudogenes, were predicted. It has been shown that genes involved in transport and metabolism of carbohydrates and amino acids and the transcription process constitute the major group of genes, according to the Clusters of Orthologous Groups of proteins (COGs) database. The genetic ability of the LU1 strain to metabolize a wide range of industrially relevant carbon sources has been confirmed. The genome exploration indicated that Enterobacter sp. LU1 possesses all genes that encode the enzymes involved in the glycerol metabolism pathway. It has also been shown that succinate can be produced as an end product of fermentation via the reductive branch of the tricarboxylic acid cycle (TCA) and the glyoxylate pathway. The transport system involved in succinate excretion into the growth medium and the genes involved in the response to osmotic and oxidative stress have also been recognized. Furthermore, three intact prophage regions ~70.3 kb, ~20.9 kb, and ~49.8 kb in length, 45 genomic islands (GIs), and two clustered regularly interspaced short palindromic repeats (CRISPR) were recognized in the genome. Sequencing and genome analysis of Enterobacter sp. LU1 confirms many earlier results based on physiological experiments and provides insight into their genetic background. All of these findings illustrate that the LU1 strain has great potential to be an efficient platform for bio-based succinate production.
Enterobacter sp. LU1 could efficiently convert glycerol to succinic acid under anaerobic conditions after the addition of lactose. In this study, media constituents affecting both Enterobacter sp. LU1 biomass and succinic acid production were investigated employing response surface methodology (RSM) with central composite design. Statistical methods led to the development of an efficient and inexpensive microbiological media based on crude glycerol, whey permeate as carbon sources and urea as a nitrogen source. The optimized production of bacterial biomass in aerobic conditions was predicted and the interactive effects between crude glycerol, urea and magnesium sulfate were investigated. As a result, a model for predicting the concentration of bacterial biocatalyst biomass was developed with crude glycerol as a sole carbon source. In addition, it was observed that the interactive effect between crude glycerol and urea was statistically significant. Response surface methodology was also employed to develop the model for predicting the concentration of succinic acid produced. Validity of the model was confirmed during verification experiments wherein actual results differed from predicted values by 0.77%. The applied statistical methods proved the feasibility for anaerobic succinic acid production on crude glycerol without expensive yeast extract addition. In conclusion, the RSM method can provide valuable information for succinic acid scale-up fermentation using Enterobacter sp. LU1.
The newly-isolated strain Enterobacter sp. LU1, which has previously been shown to be an effective producer of succinic acid on glycerol with the addition of lactose, was used for further intensive works aimed at improving the production parameters of the said process. The introduction of an initial stage of gentle culture aeration allowed almost 47 g/L of succinic acid to be obtained after 168 h of incubation, which is almost two times faster than the time previously taken to obtain this amount. Furthermore, the replacement of glycerol with crude glycerin and the replacement of lactose with whey permeate allowed the final concentration of succinic acid to be increased to 54 g/L. Considering the high content of yeast extract (YE) in the culture medium, tests were also performed with a reduced YE content via its partial substitution with urea. Although this substitution led to a deterioration of the kinetic parameters of the production process, using the fed-batch strategy, it allowed a succinic acid concentration of 69 g/L to be obtained in the culture medium, the highest concentration ever achieved using this process. Furthermore, the use of microaerophilic conditions meant that the addition of lactose to the medium was not required, with 37 g/L of succinic acid being produced on crude glycerol alone.
Continuous cultivation of Trichoderma reesei M-7 mutant was performed at different temperatures (26, 30 and 34°C) with 1% lactose or glucose alone or a total of 1% mixtures of both sugars at different ratio. The secretion of individual enzymes was affected by the ratio of cellulase inducer (lactose) to the repressor (glucose). The ratio of enzyme secretions was additionally modified by the temperature of cultivations. An insignificant increase of nonspecific activity of cellulases (FPU) and significant increase of aryl-β-glucosidase activity were observed at 26°C with lactose/glucose ratio of 3:1 and 1:1. However, when cultivated both at 30 or 32°C, the cellulolytic activities (FPU) increased significantly in the presence of 1% glucose alone. The activity of xylanases increased significantly (about 8-fold) with lactose/glucose ratio of 1:1 at 30°C. The increased synthesis of lytic enzymes (proteases, β-1,3-glucanases) correlated with increased glucose concentration in the feeding medium and this effect potentiated at 34°C of cultivation. Cette etude examine les facteurs affectant la composition des secretions d'un complexe enzymatique extracellulaire par un mutant Trichoderma reesei M-7. La secretion des enzymes individuelles est influencee par le taux de l'inducteur de la cellulase (lactose) sur le represseur (glucose). Le ratio des secretions enzymatiques est modifie en plus par la temperature des cultures.
This study analyzes the application of degenerative primers for the screening of bile salt hydrolase-encoding genes (bsh) in various intestinal bifidobacteria. In the first stage, the design and evaluation of the universal PCR primers for amplifying the partial coding sequence of bile salt hydrolase in bifidobacteria were performed. The amplified bsh gene fragments were sequenced and the obtained sequences were compared to the bsh genes present in GenBank. The determined results showed the utility of the designed PCR primers for the amplification of partial gene encoding bile salt hydrolase in different intestinal bifidobacteria. Moreover, sequence analysis revealed that bile salt hydrolase-encoding genes may be used as valuable molecular markers for phylogenetic studies and identification of even closely related members of the genus Bifidobacterium.
The aim of the presented work was to evaluate changes in free ferulic and coumaric acid concentrations during malting. In general, malting is performed at 14°C and in steeping water of pH 7.4. The influence of an elevated temperature of the malting process (22°C, Activated Germination Malting technique) as well as a decreased pH value of steeping water (pH 5.2) on free ferulic acid and coumaric acid contents in kilned malts was estimated. The application of Activated Germination Malting caused a limited increase in the concentrations of ferulic acid and coumaric acid in malt Rudzik, whereas a higher temperature during malting of Krona barley resulted in approx. 2-fold higher concentration of both free phenolic acids in kilned malt. The use of steeping water of pH 5.2 instead of 7.4 resulted in a significant increase in the contents of free ferulic and coumaric acids in malt of both tested varieties, which could lead to an increase in the contents of these phenolic acids in beer.
The effect of temperature in the rangę of 26-38°C on the production of cellulases, xylanases and lytic enzymes by four mutant strains of <i>Trichoderma reesei</i> was analysed. On the basis of these investigations three thermosensitive strains (M-7. RUT C 30 and VTT-D-78085) which showed reduced excretion of the above mentioned enzymes as well as protein and a thermoresistant mutant (VTT-D-79I24) which grew within a temperature range of 26-34°C were characterized. Higher temperature caused an increase in the level of xylanolytic enzymes produced by the four mutants. In addition. it effected the complex composition of cellulolytic enzymes secreted by VTT-D-79l 24 (i.c. increased and reduced excertion of (β-glucosidase and β-1,4-endoglucanase respectively).
