Stanislav Kráčmar

Bioactive phenols in algae: The application of pressurized-liquid and solid-phase extraction techniques

A new extraction technique based on the off-line combination of pressurized-liquid with solid-phase extraction (PLE-SPE) is described. The method was used for the extraction of bioactive phenolic acids (protocatechuic, p-hydroxybenzoic, 2,3-dihydroxybenzoic, chlorogenic, vanillic, caffeic, p-coumaric, salicylic acid), cinnamic acid and hydroxybenzaldehydes (p-hydroxybenzaldehyde, 3,4-dihydroxybenzaldehyde, vanillin) from in vitro culture of two freshwater algae (Anabaena doliolum and Spongiochloris spongiosa) and from food products of marine macroalgae Porphyra tenera (nori) and Undaria pinnatifida (wakame). For the identification and quantification of the compounds the molecular ions [M-H](-) and specific fragments were analyzed by quadrupole mass spectrometry analyzer connected on-line with a reversed-phase HPLC system. Our analysis showed that the freshwater algae and marine algal products contained submicrogram or microgram level of above-mentioned phenols per gram of lyophilized sample. In addition, the total phenol content (Folin-Ciocalteu assay) and antioxidant activity (TEAC assay, Trolox equivalent antioxidant capacity assay) of the PLE-SPE extracts were determined and discussed.

The effect of ripening and storage conditions on the distribution of tyramine, putrescine and cadaverine in Edam-cheese.

The aim of the work was to describe the development of selected biogenic amines (histamine, tyramine, putrescine and cadaverine) in 4 layers of Dutch-type cheese (Edam-cheese) depending on 3 ripening/storage regimes during a 98-day period. Biogenic amines were analysed by means of ion-exchange chromatography. A further goal was to identify microbial sources of biogenic amines in the material analysed. Phenotype characterization and repetitive sequence-based PCR fingerprinting were used to identify the isolated bacteria. The highest content of tyramine, putrescine and cadaverine was determined in cheeses stored in a ripening cellar at a temperature of 10 degrees C during the whole observation period. Lower biogenic amines content was determined in samples which were moved into a cold storage device (5 degrees C) after 38 days of storage in a ripening cellar (10 degrees C). The lowest concentrations of biogenic amines were detected in cheeses which were moved into a cold storage device (5 degrees C) after 23 days of storage in a ripening cellar (10 degrees C). During the 98-day period, histamine was not detected in any of the regimes. Within the cheeses analysed, non-starter lactic acid bacteria Lactobacillus curvatus, Lactobacillus casei/paracasei and Lactobacillus plantarum were detected as the main producers of the biogenic amines tested. In starter bacteria Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris the decarboxylase activity tested was not detected.

Amino acid composition of algal products and its contribution to RDI

In this paper, the amino acid profiles of algal products from diverse groups (Cyanophyceae, Chlorophyta, Rhodophyta and Phaeophyta) were established. Contents of essential and non-essential amino acids varied in the range of 22.8-42.3 and 31.0-66.5 (g·16 g(-1)N), respectively. In dependence on daily algal intakes, the highest participation in recommended daily intakes (RDI; related to adult, body weight of 70 kg) of all EAAs was observed in freshwater micro-algal products, especially from Spirulina genus where contribution ranged from 12.6% (Lys, SB) to 38.8% (Thr, S). Generally, Lys was the lowest contributor to RDIs in almost all algal samples except Chlorella pyrenoidosa (C) and Palmaria palmata (D), where Ile and Leu, respectively, were established. Interestingly, the contents of sulfur AAs of both products of the Spirulina genus covered 74.5% (S) and 73.8% (SB) of their RDI. Finally, products from brown seaweeds showed the lowest contributions to the RDIs of all EAAs.

Effect of foliar application of selenium on the content of se-lected amino acids in potato tubers (Solanum tuberosum L.).

Selenium (Se) is an important element associated with the enhancement of antioxidant activity in organisms. Po- tato is very suitable for fertilisation with Se (biofortification). The experiment was performed to examine the effect of foliar application of Se as sodium selenite (200 or 400 g Se/ha) at the tuberisation stage on a spectrum of amino acids in tubers of varieties. The trends of amino acids were consistent in both years of the study. Application of Se increased the relative content of total essential (EAA) and non-essential (NEAA) amino acids relative to the con- trols (Karin: EAA 16.81-21.73% and NEAA 14.18-18.63%; Ditta: EAA 4.71-13.00% and NEAA 5.78-6.49%). The increase in the content of phenylalanine (Phe) was particularly significant (up to 48.9%) when also the contents of aspartic acid (Asp), glutamic acid (Glu), threonine (Thr), and tyrosine (Tyr) increased significantly compared with the controls. The results of changes in the content of isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), valine (Val), alanine (Ala), arginine (Arg), proline (Pro), cysteine (Cys), glycine (Gly), histidine (His), and serine (Ser) were also discussed. The highest dose of selenium is shown as a stress factor. Its toxic effects resulted in a change of amino acid contents.

Chemical composition of buckwheat plant parts and selected buckwheat products

Chemical composition plant parts (roots, stalks, leaves, blossoms) of common buckwheat ( Fagopyrum esculentum Moench) and selected products made from its seeds (peels, whole seed, wholemeal flour, broken seeds, crunchy products Natural and Cocoa, flour, and pasta) was determined. Samples were dried and ground to a fine powder. All analyses were performed according to the Commission Regulation no. 152/2009, while rutin concentration was determined by the modified HPLC method. The lowest content of moisture was found in roots (4.3%) and in peels (almost 8%) and the highest moisture (nearly 11%) was discovered in seeds. The lowest amount of crude protein (3.5%) was found in peels, the highest crude protein amount (>13%) in both flours and leaves (23%). The starch content (>50% in dry matter) differs from one sample to another. Only in peels the content of starch was about 3.5%. From all examined samples, the lowest content of fat was found in crunchy products Cocoa, 1.7%. The lowest amount of histidine was determined in all studied samples, except peels, the highest content of glutamic acid was determined in almost all samples, except peels. Whole-meal flour is very rich source of Ca and Fe. The content of these elements was 1172 mg.kg -1 and 45.9 mg.kg -1 , respectively. On the other hand, the highest content of Pb (>1 mg.kg -1 ) was found in broken seeds. The greatest concentration of rutin was determined in blossoms and leaves (83.6 and 69.9 mg.g -1 ), respectively. On the other hand, the lowest concentrations of rutin were found in buckwheat products (generally less then 1 mg.g -1 , i.e. in wholemeal flour, 702 μg.kg -1 , the lowest (almost 10 μg.kg -1 ) in pasta.

Influence of composition of feed and lactation period on mineral composition of Mare's

Effects of lactation period and feed on essential minerals composition of mares milk were studied. Average Ca, P, Na and Mg concentrations in feed DM were 0.66, 4.30, 0.13 and 2.21 g.kg-1 of DM (dry matter), respectively. In regard to milk all elements concentrations were not similar to each other due to the changes of the lactation day differences. Average Ca, P, Na and Mg concentrations (in DM) caused by breed differences and lactation days were 1.95, 1.08, 0.53 and 0.22 g.kg-1, respectively. During the milk period, a high-quality feed were maintaining the major mineral composition of mares milk in 1.5 - 2 times higher amount than milk of mare fed with pasture and a low-quality feed. The colostrum stage of mare was much shorter than other animals, the mares milk on the 2nd day of lactation showed properties of initial milk in which its composition, particularly in minerals, were two times higher when compare to that on milk periods that was started from 5th day after parturition. It seemed that some factors, except well physiological conditions, such as mares age, number of foaling, sex of foal etc. did not affect milk nutrient composition.

Effect of thermal treatment on rutin content in selected buckwheat products using calcium as an internal tracer

Reversed-phase high-performance liquid chromatography (RP-HPLC) was used for rutin (quercetin-3-rutinoside) determination in selected buckwheat products (whole meal flour, broken seeds, seed hulls, herbs and baked cereal breads). The effect of various thermal procedures on content of rutin was evaluated using calcium as an internal tracker to correct changes in mass and composition of the buckwheat products. These factors are very seldom taken into account. The results show non-significant changes in rutin levels obtained in whole meal flour and broken seed samples after thermal treatment up to 150°C. Higher temperature already caused sudden fall in the observed rutin concentrations. The evaporation of some volatile compounds and degradation products can decrease the mass of the samples and formally increase the content of rutin (35.5 ±4.7 mg per 100 g for whole meal flour and 10.2 ±0.4 mg per100 g for broken seeds at 150°C). Serious decrease of rutin contents at elevated temperatures (>150°C) can be explained by its degradation (by breaking off the C-C bond in quercetin-3-rutinoside moiety) and/or evaporation (24.3 ±1.4 mg per 100 g for whole meal flour and 3.06 ±0.3 mg per100 g for broken seeds at 180°C). In case of baked cereal breads the level of rutin changed in dependence on the ratio between buckwheat and corn flour. Longer time leaching and higher temperature implicate higher rutin content in infusions prepared from buckwheat seed hulls and herbs.

LIQUID CHROMATOGRAPHIC DETERMINATION OF POLYPHENENOLS IN CZECH BEERS DURING BREWING PROCES

High performance liquid chromatographic (HPLC/UV) method was adapted for simultaneous determination of seven polyphenols, including derivatives of benzoic (gallic and vanillic acids) and cinnamic acids (p-coumaric, ferulic and sinapic acids), flavan-3-ols (catechin) and flavonols (rutin) in worts and beers at the various stages of the brewing process. Based on the semi-quantitative HPLC analysis, total polyphenols chromatographic index (TPCI) was in the ranges of 5.18 - 19.4 mg/L and 7.37 - 20.7 mg/L for all worts and beers, respectively. The HPLC analyses showed that relatively high levels of (+)-catechin and gallic acid were in all the worts and the beers, while the values were much lower for ferulic acid, rutin, vanillic acid, sinapic acid and p-coumaric acid. Polyphenols with relatively high concentrations, that were detected in all tested worts and beers, were gallic acid (1.29 - 4.75 mg/L resp. 2.59 - 4.97 mg/L), (+)-catechin (1.66 - 7.95 mg/L resp. 4.70 - 10.0 mg/L) and ferulic acid (0.41 - 4.53 mg/L resp. 1.05 - 2.87 mg/L). On the other side, the sinapic acid (0.72 - 1.59 mg/L resp. 0.72 - 2.5 mg/L), rutin (1.17 - 2.03 mg/L resp. 1.16 - 2.85 mg/L), p-coumaric acid (ND - 4.73 mg/L resp. ND - 1.44 mg/L) and vanillic acid (ND - 1.52 mg/L resp. 0.75 - 1.81 mg/L) were detected in lowest concentrations. In both, worts and beers investigated in this study, the changes in the contents of individual polyphenols were not uniform. In the case of some polyphenols, a decrease in the content was observed after boiling the worts with hops or after the main fermentation until maturation and filtration, but with some polyphenols, the concentrations were constant until the end of the process or even increased.

Content of 4(5)-methylimidazole, caffeine and chlorogenic acid in commercial coffee brands

Content of 4(5)-methylimidazole (4-MeI), caffeine and chlorogenic acid in commercial coffee brands were determined using high-performance liquid chromatography (HPLC) with UV DAD and MS detectors. Positive ion ESI mass spectra of the 4-MeI standard yielded intense signals corresponding to [M+H] + (83.0604) and [2M+H] + ions (165.1115). Also, adducts of 4-MeI with acetonitrile from mobile were detected – [M+ACN] + ions (124.0849). The LOD of 2.5 ng mL -1 and LOQ of 8.4 ng.mL -1 were calculated according to the following formulas: LOD = 3.SD/S, and LOQ = 10.SD/S, where S is the slope of the calibration curve and SD is the standard deviation of the noise. The caffeine content was compared to the results of the standard addition, 1 st derivative and liquid-liquid extraction spectrophotometry. 4-MeI was in tens µg g -1 in the Vietnamese coffees while in units µg.g -1 in all Czech and Brazilian coffees (<2.4 µg.g -1 and <4.9 µg.g -1 , respectively). The results for caffeine were within the documented range (0.31 – 2.20%) in all coffee samples. The lower content of caffeine and chlorogenic acid was observed in Vietnamese coffees. All the methods used for determination of caffeine in the Czech and Brazilian coffees gave acceptable precision and accuracy. However, there were significant differences in the results in Vietnamese coffees. The caffeine extractability (100 °C, 3 min brewing) almost reached 100% in Czech and Brazilian coffees, while it was less than 90% in Vietnamese coffees. The Czech and Brazilian coffees tend to produce more caffeine in brews than the Vietnamese coffee because of the different composition of blends and the particle size degree.

THE EFFECT OF INDIVIDUAL PHOSPHATE EMULSIFYING SALTS AND THEIR SELECTED BINARY MIXTURES ON HARDNESS OF PROCESSED CHEESE SPREADS

The aim of this work was to observe the effects of emulsifying salts composed of trisodium citrate and sodium phosphates with different chain length (disodium phosphate (DSP), tetrasodium diphosphate (TSPP), pentasodium triphosphate (PSTP) and sodium salts of polyphosphates with 5 different mean length (n ≈ 5, 9, 13, 20, 28)) on hardness of processed cheese spreads. Hardness of processed cheese spreads with selected binary mixtures of the above mentioned salts were also studied. Measurements were performed after 2, 9 and 30 days of storage at 6 °C. Hardness of processed cheese increased with increase in chain length of individually used phosphates. Majority of applied binary mixtures of emulsifying salts had not significant influence on hardness charges in processed cheese spreads. On the other hand, a combination of phosphates salts (DSP with TSPP) was found, which had specific effect on hardness of processed cheese spreads. Textural properties of samples with trisodium citrate were similar compared to samples with DSP.