Differences in Ratio of Carbon Stable Isotopes among Barley Grain Milling Fractions with Various Concentrations of Beta-Glucans.

The grains of three barley varieties were milled and sieved to obtain respective milling fractions with a content of beta-glucans (b-G) from 1.4 to 10.7%. The enriched fraction obtained by the extraction and precipitation contained 24.7% of b-G. The differences between the ratio of stable C carbon isotopes were established. Milling fractions with coarse particles had more beta-glucans and a more negative ratio of δ13C isotope in comparison to the respective intact barley grain. However, the enriched fraction had a less negative isotope ratio. So, it is not expected that the deviation from the stable isotope ratio of grain in milling fractions is the result of the content of b-G, but it depends on other barley grain constituents. In different parts of barley grain, there are substances with different stable isotope ratios, and by milling and sieving, they are assorted to the same milling fraction with most of the b-G. The method for determining the ratio of a stable carbon isotope in diverse barley grain fractions, applied in this investigation, is potentially opening the possibility for an additional method of screening the concentration of bioactive constituents in barley grain.

Identification and Quantification of Selected Benzoxazinoids and Phenolics in Germinated Spelt (Triticum spelta).

In this study, we investigated the effects of germination on the secondary metabolite composition in spelt grains. Germination significantly increased the content of various metabolites in free and bound forms. Benzoxazinoids were the most important compounds in the free fraction of the 96 h germinated grains (MBOA content as the predominant compound was 277.61 ± 15.29 µg/g DW). The majority of phenolic acids were present in the bound fraction, with trans-ferulic acid as the main component, reaching 753.27 ± 95.87 µg/g DW. The often neglected cis-isomers of phenolic acids accounted for about 20% of the total phenolic acids. High levels of apigenin di-C-glycosides were found in spelt grains, and the schaftoside content was most affected by germination, increasing threefold. The accumulation of secondary metabolites significantly increased the antioxidant activity of germinated spelt. According to the results of this study, the content of most bioactive compounds was highest in spelt grains after 96 h of germination. These data suggest that germinated spelt could potentially be valuable for the production of functional foods.

Rapid weight loss among elite-level judo athletes: methods and nutrition in relation to competition performance.

Background: Rapid weight loss (RWL) followed by rapid weight gain (RWG) is a regular pre-competition routine in combat sports and weightlifting. With the prevalence of these sports exceeding 20% at the 2020 Tokyo Olympics, there are limited data on RWL and RWG practices and their impact on well-being and competitive success in elite-level athletes. Methods: A total of 138 elite-level female and male judokas, 7.7% of the athletes ranked as top 150 on the International Judo Federation Senior World Ranking List (WRL), completed a survey on RWL, RWG, and the consequences of these practices. Results: Our findings showed that 96% of the respondents practice RWL. The average reduced body mass percentage was 5.8 ± 2.3%. Respondents who used either of the dehydration methods - fluid restriction, sauna suit, and/or sauna/hot bath - to reduce weight were 88%, 85%, and 76%, respectively. Furthermore, 91% of the respondents reported reduced energy as a negative consequence of RWL and 21% experienced a collapse episode during the RWL period. Respondents ranked 1-20 on the WRL experienced fewer negative consequences of RWL and RWG (p = 0.002) and had more dietitian and/or medical doctor support (p = 0.040) than lower-ranked respondents. Those who started with RWL practices before the age of 16 (38%) were ranked lower on the WRL (p = 0.004) and reported more negative consequences of RWL and RWG (p = 0.014). Conclusions: This study is the first to provide insight into the RWL practices of worldwide elite-level judokas and provides valuable information for the combat sports society, especially coaches. Proper weight management and optimal timed initiation of RWL practices in a judoka's career may contribute to success at the elite level.

Accumulation and Transformation of Biogenic Amines and Gamma-Aminobutyric Acid (GABA) in Chickpea Sourdough.

In general, sourdough fermentation leads to an improvement in the technological, nutritional, and sensory properties of bakery products. The use of non-conventional flours with a specific autochthonous microbiota may lead to the formation of secondary metabolites, which may even have undesirable physiological and toxicological effects. Chickpea flours from different suppliers have been used to produce sourdoughs by spontaneous and inoculated fermentations. The content of nutritionally undesirable biogenic amines (BA) and beneficial gamma-aminobutyric acid (GABA) was determined by chromatography. Fenugreek sprouts, which are a rich source of amine oxidases, were used to reduce the BA content in the sourdoughs. Spontaneous fermentation resulted in a high accumulation of cadaverine, putrescine, and tyramine for certain flours. The use of commercial starter cultures was not effective in reducing the accumulation of BA in all sourdoughs. The addition of fenugreek sprouts to the suspension of sourdough with pH raised to 6.5 resulted in a significant reduction in BA contents. Enzymatic oxidation was less efficient during kneading. Baking resulted in only a partial degradation of BA and GABA in the crust and not in the crumb. Therefore, it could be suggested to give more importance to the control of sourdough fermentation with regard to the formation of nutritionally undesirable BA and to exploit the possibilities of their degradation.

Germinated Buckwheat: Effects of Dehulling on Phenolics Profile and Antioxidant Activity of Buckwheat Seeds.

The aim was to investigate the effects of the cold dehulling of buckwheat seeds on their germination, total phenolic content (TPC), antioxidant activity (AA) and phenolics composition. Cold dehulling had no negative effects on germination rate and resulted in faster rootlet growth compared to hulled seeds. Although the dehulling of the seeds significantly decreased TPC and AA, the germination of dehulled seeds resulted in 1.8-fold and 1.9-fold higher TPC and AA compared to hulled seeds. Liquid chromatography coupled to mass spectrometry identified several phenolic compounds in free and bound forms. Rutin was the major compound in hulled seeds (98 µg/g dry weight), orientin and vitexin in 96-h germinated dehulled seeds (2205, 1869 µg/g dry weight, respectively). During germination, the increases in the major phenolic compounds were around two orders of magnitude, which were greater than the increases for TPC and AA. As well as orientin and vitexin, high levels of other phenolic compounds were detected for dehulled germinated seeds (e.g., isoorientin, rutin; 1402, 967 µg/g dry weight, respectively). These data show that dehulled germinated seeds of buckwheat have great potential for use in functional foods as a dietary source of phenolic compounds with health benefits.

Accumulation of Agmatine, Spermidine, and Spermine in Sprouts and Microgreens of Alfalfa, Fenugreek, Lentil, and Daikon Radish.

Sprouts and microgreens are a rich source of various bioactive compounds. Seeds of lentil, fenugreek, alfalfa, and daikon radish seeds were germinated and the contents of the polyamines agmatine (AGM), putrescine (PUT), cadaverine (CAD), spermidine (SPD), and spermine (SPM) in ungerminated seeds, sprouts, and microgreens were determined. In general, sprouting led to the accumulation of the total polyamine content. The highest levels of AGM (5392 mg/kg) were found in alfalfa microgreens, PUT (1079 mg/kg) and CAD (3563 mg/kg) in fenugreek sprouts, SPD (579 mg/kg) in lentil microgreens, and SPM (922 mg/kg) in fenugreek microgreens. A large increase in CAD content was observed in all three legume sprouts. Conversely, the nutritionally beneficial polyamines AGM, SPD, and SPM were accumulated in microgreens, while their contents of CAD were significantly lower. In contrast, daikon radish sprouts exhibited a nutritionally better profile of polyamines than the microgreens. Freezing and thawing of legume sprouts resulted in significant degradation of CAD, PUT, and AGM by endogenous diamine oxidases. The enzymatic potential of fenugreek sprouts can be used to degrade exogenous PUT, CAD, and tyramine at pH values above 5.

The Methodology Applied in DPPH, ABTS and Folin-Ciocalteau Assays Has a Large Influence on the Determined Antioxidant Potential.

Antioxidant potential (AOP) is not only the property of the matrix analyzed but also depends greatly on the methodology used. The chromogenic radicals 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•+), 2,2-diphenyl-1-picrylhydrazyl (DPPH•) and Folin-Ciocalteu (FC) assay were applied to estimate how the method and the composition of the assay solvent influence the AOP determined for coffee, tea, beer, apple juice and dietary supplements. Large differences between the AOP values depending on the reaction medium were observed, with the highest AOP determined mostly in the FC assay. In reactions with chromogenic radicals several fold higher values of AOP were obtained in buffer pH 7.4 than in water or methanol. The type of assay and solvent composition have similar influences on the reactivity of a particular antioxidant, either pure or as part of a complex matrix. The reaction kinetics of radicals with antioxidants in samples reveals that AOP depends strongly on incubation time, yet differently for each sample analyzed and the assay applied.

Influence of Yellow Light-Emitting Diodes at 590 nm on Storage of Apple, Tomato and Bell Pepper Fruit.

The objective of this study is to investigate the effects of irradiation from light-emitting diodes (LEDs) on several fruits during storage. To improve storage and increase the contents of some bioactive compounds, apple, tomato and red bell pepper fruits were exposed to yellow light emitted from the diodes at 590 nm. The contents of ascorbic acid, total phenolics, total flavonoids and several pigments were investigated, along with the antioxidant potential. The colour parameters (L*, a* and b*) and firmness of the fruit were also determined. After 7 days of LED light irradiation, there was significantly higher total phenolic content and antioxidant potential in apple peel extracts. The irradiated fruit of tomato had significantly higher levels of total phenolic compounds, and the fruit of red bell pepper had significantly higher antioxidant potential. LED light had no effects on the colour parameters, although there was a tendency to accelerate colour development. Apple fruit irradiated with LED light was significantly less firm. Among twelve analysed pigments, significantly more ß-carotene was detected in LED light-irradiated apple and bell pepper fruit, more α-tocopherol and γ-tocopherol in bell pepper fruit, and more lutein in apple peel and bell pepper fruit. The applied LED light slightly accelerated the ripening of the studied fruit, and affected the synthesis of some of the secondary metabolites.

The response of aminopeptidases of Phaseolus vulgaris to drought depends on the developmental stage of the leaves.

Aminopeptidases, together with other proteases, execute and regulate the total and specifically limited protein breakdown involved in plant physiology, raising the possibility of their involvement in response to drought. We have identified, in leaves of Phaseolus vulgaris L., five aminopeptidases (E.C.3.4.11) whose levels of activity changed when three week old plants were subjected to drought. First, second and third trifoliate leaves were investigated separately. The aminopeptidases were first identified then isolated using ion exchange chromatography of leaf extracts. Three, named PvAP1, PvAP2 and PvAP4, are metallo aminopeptidases with broad substrate specificity, active against substrates conjugated to alanine and lysine. Two others, PvAP3 and PvAP5, are apparently serine aminopeptidases, the former active against substrates conjugated to phenylalanine and leucine, and the latter characterised by narrow specificity against substrates conjugated to phenylalanine. Their apparent molecular weights range from ∼37 kDa to ∼80 kDa. Levels of activity of individual aminopeptidases in both watered and drought stressed plants are shown to depend on the age of leaves. In watered plants they were generally highest in young, and very low in older, trifoliate leaves, the latter with the exception of PvAP5. Drought initiated an almost general increase of their activities, although to different extents, with the exception of PvAP4 and PvAP5 in young trifoliate leaves. Thus, in such studies it is necessary to investigate the effects of drought separately in leaves of different ages in order to elucidate the different complex and probably specific roles of aminopeptidases in the response of common bean to drought.

LC-MS analysis of phenolic compounds and antioxidant activity of buckwheat at different stages of malting.

The impact of malting on the profile of the phenolic compounds and the antioxidant properties of two buckwheat varieties was investigated. The highest relative increases in phenolic compounds were observed for isoorientin, orientin, and isovitexin, which are consequently major inducible phenolic compounds during malting. Only a minor relative increase was observed for the most abundant phenolic compound, rutin. The radical-scavenging activity of buckwheat seeds was evaluated using ABTS and DPPH assays. A considerable increase in total phenolic compounds and higher antioxidant activity were observed after 64h of germination, whereas kilning resulted in decreased total phenolic compounds and antioxidant activity. Higher antioxidant activities for extracts were found for buffered solvents than for pure methanol and water. Changes in the composition of the phenolic compounds and increased antioxidant content were confirmed by several methods, indicating that buckwheat malt can be used as a food rich in antioxidants.

Rapid Estimation of Tocopherol Content in Linseed and Sunflower Oils-Reactivity and Assay.

The reactivity of tocopherols with 2,2-diphenyl-1-picrylhydrazyl (DPPH) was studied in model systems in order to establish a method for quantifying vitamin E in plant oils. The method was optimized with respect to solvent composition of the assay medium, which has a large influence on the course of reaction of tocopherols with DPPH. The rate of reaction of α-tocopherol with DPPH is higher than that of γ-tocopherol in both protic and aprotic solvents. In ethyl acetate, routinely applied for the analysis of antioxidant potential (AOP) of plant oils, reactions of tocopherols with DPPH are slower and concentration of tocopherols in the assay has a large influence on their molar reactivity. In 2-propanol, however, two electrons are exchanged for both α- and γ-tocopherols, independent of their concentration. 2-propanol is not toxic and is fully compatible with polypropylene labware. The chromatographically determined content of tocopherols and their molar reactivity in the DPPH assay reveal that only tocopherols contribute to the AOP of sunflower oil, whereas the contribution of tocopherols to the AOP of linseed oil is 75%. The DPPH assay in 2-propanol can be applied for rapid and cheap estimation of vitamin E content in plant oils where tocopherols are major antioxidants.

Contribution of SO2 to antioxidant potential of white wine.

The reactivity of SO2 with the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) and in Folin Ciocalteu (FC) assays was analysed under different experimental conditions. There was significantly higher reactivity between SO2 and DPPH in buffered methanol than in methanol alone. When DPPH and FC assays were performed in a mixture of caftaric acid and SO2, there were synergistic effects that were more pronounced with the FC assay. Phenolics are an important parameter of wine quality, and their accurate characterisation in wine is essential. Analysis of white wines with DPPH and FC assays overestimates the contribution of phenolics to the antioxidant potential (AOP). SO2 contributes (from 20% to 45%) to the AOP of the white wines analysed. As SO2 reactivity depends highly on buffer composition, pH, time of incubation and other compounds, e.g. phenolics and aldehydes, different experimental protocols can produce large variations in AOPs, and therefore control of experimental conditions is extremely important.

Correlation of basic oil quality indices and electrical properties of model vegetable oil systems.

Model vegetable oil mixtures with significantly different basic oil quality indices (free fatty acid, iodine, and Totox values) were prepared by adding oleic acids, synthetic saturated triglycerides, or oxidized safflower oil ( Carthamus tinctorius ) to the oleic type of sunflower oil. Dielectric constants, dielectric loss factors, quality factors, and electrical conductivities of model lipids were determined at frequencies from 50 Hz to 2 MHz and at temperatures from 293.15 to 323.15 K. The dependence of these dielectric parameters on basic oil quality indices was investigated. Adding oleic acids to sunflower oil resulted in lower dielectric constants and conductivities and higher quality factors. Reduced iodine values resulted in increased dielectric constants and quality factors and decreased conductivities. Higher Totox values resulted in higher dielectric constants and conductivities at high frequencies and lower quality factors. Dielectric constants decreased linearly with temperature, whereas conductivities followed the Arrhenius law.

DPPH assay of vegetable oils and model antioxidants in protic and aprotic solvents.

The rate of reaction of phenolic antioxidants with DPPH depends on solvent composition. The rate constants can differ by more than two orders of magnitude for the same phenolic compound. Reactions are faster in alcohols than in ethyl acetate that is used routinely for the analysis of antioxidant potential (AOP) of nonpolar samples such as vegetable oils. Incorporation of an acid base pair into the assay solvent buffers the system against acid impurities such as free fatty acids and CO2 from the air. This is shown to increase the rate of oxidation and number of electrons of phenolic compounds exchanged with DPPH. Typically, DPPH assays are performed for predetermined time intervals at which phenolic compounds are not fully oxidized and therefore higher reaction rates result in higher values of AOP. More than twofold AOP was obtained for oleuropein, sesamol, sinapic acid, caffeic acid and protocatechuic acid in buffered alcohols than in ethyl acetate. The AOP of sesame, pumpkin seed and extra virgin olive oil is accordingly higher when determined in buffered alcohols. DPPH assays in ethyl acetate result in underestimation of AOP of unrefined vegetable oils.

Influence of solvent composition on antioxidant potential of model polyphenols and red wines determined with 2,2-diphenyl-1-picrylhydrazyl.

Solvent composition has a large influence on measured antioxidant potential (AOP) of model polyphenols and red wines with 2,2-diphenyl-1-picrylhydrazyl (DPPH). We have shown that incorporation of aqueous buffer in the assay medium results in higher reactivity of catechin and caftaric acid, which are among major polyphenolic constituents of wines. Consequently, AOPs of red wines determined after 60 min of incubation at 25 °C in buffered methanol are 1.5-1.6-fold of values determined in methanol. Even in buffered methanol, wine polyphenols are not fully oxidized after 60 min. Only about half of Trolox equivalents were determined in comparison to the Folin-Ciocalteu assay. Buffer composition, pH, time of incubation, temperature, and concentration of antioxidants and DPPH all contribute to the resulting value of the AOP being standardized or at least strictly reported because minor differences in experimental procedures can account for large variations in determined AOP with DPPH for the same samples.

Stability and transformation of products formed from dimeric dehydroascorbic acid at low pH.

Dehydroascorbic acid dimer (DDHA) is the commercially available form of oxidised vitaminC. When DDHA enters an aqueous environment, it will interact with water, but the details of this process are not well defined. Hydration of DDHA was analyzed as a function of concentration, pH and temperature. Above pH 5 only hydrated dehydroascorbic acid monomer (DHA) and its degradation product 2,3-diketogulonic acid (DKG) were observed. At pH lower than4 and in MilliQ water, relatively stable hydrated dimeric species were additionally detected. They are intermediates formed from DDHA and transformed into DKG, either directly or through DHA. A lower temperature of dissolution favours the formation of hydrated dimeric products, while the concentration of DDHA has no effect on their yield. All products were separated chromatographically and analyzed by three detectors: diode array, refractive index and electrospray ionisation mass spectrometry. The stability and transformations of products were studied in the mixture and in the isolated fractions. Two initially formed monohydrated products are transformed to a dihydrated product that is degraded to DHA and DKG. Once formed, the dihydrated product and one of the monohydrated products are sufficiently stable to be identified at neutral pH. All three hydrated products are interconvertible and can be also formed after dissolution of dried DHA.

A quantitative technique for determining proteases and their substrate specificities and pH optima in crude enzyme extracts.

A zymography technique based on native polyacrylamide gel electrophoresis (PAGE) has been devised, which enables the substrate specificities, content and pH profiles of proteolytic enzymes to be determined in an unfractionated tissue extract. Enzymes were visualized by exogenous application of small molecule substrates that fluoresce when hydrolyzed. The linearity of response, treatment of background fluorescence, and effects of diffusion of substrate and enzyme were taken into account. Based on these studies, successive application of different substrates on the same gel has enabled the presence and specificity of individual enzymes to be determined. Differences in the concentrations and profiles of enzymes, resulting from environmental factors or ontogeny of the organism, can be assessed from crude extracts on a single gel. The technique was applied to aminopeptidases and peptidases in crude Phaseolus vulgaris leaf extracts. One enzyme active against Ala-AMC (7-amino-4-methylcoumarin), one enzyme active against Z-Arg-AMC, several enzymes active against Leu-AMC, and (for the first time in plants) several enzymes active against Phe-AMC were identified. The technique is very sensitive, and microgram quantities of total protein led to picomoles of liberated AMC, with a linear response over a 32-fold range of concentration. The experimental procedure, including electrophoresis, is rapid, taking approximately 1 h.

Reduction of dehydroascorbic acid at low pH.

Ascorbic acid and dehydroascorbic acid are unstable in aqueous solution in the presence of copper and iron ions, causing problems in the routine analysis of vitamin C. Their stability can be improved by lowering the pH below 2, preferably with metaphosphoric acid. Dehydroascorbic acid, an oxidised form of vitamin C, gives a relatively low response on the majority of chromatographic detectors, and is therefore routinely determined as the increase of ascorbic acid formed after reduction. The reduction step is routinely performed at a pH that is suboptimal for the stability of both forms. In this paper, the reduction of dehydroascorbic acid with tris-[2-carboxyethyl] phosphine (TCEP) at pH below 2 is evaluated. Dehydroascorbic acid is fully reduced with TCEP in metaphosphoric acid in less than 20 min, and yields of ascorbic acid are the same as at higher pH. TCEP and ascorbic acid formed by reduction, are more stable in metaphosphoric acid than in acetate or citrate buffers at pH 5, in the presence of redox active copper ions. The simple experimental procedure and low probability of artefacts are major benefits of this method, over those currently applied in a routine assay of vitamin C, performed on large number of samples.

Optimization of growth for the hyperthermophilic archaeon Aeropyrum pernix on a small-batch scale.

Growth of Aeropyrum pernix, the first reported aerobic neutrophilic hyperthermophilic archaeon, was investigated under different cultivation parameters. Different sources of seawater, pH, and the cultivation methods were tested with the aim to improve the biomass production. A 1-L glass flask fitted with a condenser and air diffuser was used as a bioreactor. The optimum conditions for maximizing A. pernix biomass were obtained when Na2S2O3.5H2O (1 g/L) with added marine broth 2216 at pH 7.0 (20 mmol HEPES buffer/L) was used as a growing medium in a 1-L flask. The biomass production was 0.45 g dry cell mass/L in 40 h under the optimum conditions, which is more than the 0.42 g dry cell mass/L in 60 h previously obtained.