One-Step and Colorimetric Detection of Fish Freshness Indicator Hypoxanthine Based on the Peroxidase Activity of Xanthine Oxidase Grade I Ammonium Sulfate Suspension

The global food waste problem, especially aquatic product spoilage, stimulates the accurate freshness analysis of food products. However, it still remains a great challenge to realize in-field determination of fish freshness at the time of use. In the present study, a colorimetric enzyme biosensor was developed for one-step detection of hypoxanthine (Hx), which is an important intermediate of adenosine triphosphate decomposition during fish storage. We demonstrate that xanthine oxidase grade I ammonium sulfate suspension (XOD-ASS) possesses peroxidase activity. It can oxidize different peroxidase substrates, including 3,3′,5,5′-tetramethylbenzidine, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, and o-phenylenediamine in the presence of H2O2, producing visible color reactions. Further experiments indicate that XOD-ASS displayed effective peroxidase activity and could be used for H2O2 detection. Based on this, a one-step Hx detection method was established using only XOD-ASS as the catalyst. The method displays a good linear relationship in the range from 20 to 100 μM with a detection limit of 6.93 μM. Additionally, we successfully applied this method in testing Hx accumulation in sea bass fish samples of different storage times. The recovery values range from 97.44 to 102.56%. It is exciting to note that, compared with other methods, our proposed method provides a robust advantage on the economic reaction system, ease of preparation, short time consumption, and moderate reaction temperature. We believe that this method shows good application prospects for on-site fish freshness determination.

QUALITATIVE ANALYSIS OF POLYMERIC FILMS WITH A NAPHTHOQUINONE COMPLEX OF BIOLOGICALLY ACTIVE SUBSTANCES OF LITHOSPERMUM ERYTHRORHIZON

Lithospermum erythrorhizon is a promising plant, its roots synthesize biologically active substances (BAS): naphthoquinone shikonin and its esters, which have antimicrobial, wound healing, anti-inflammatory, antioxidant, antineoplastic and other types of pharmacological action. The industrial harvesting of Lithospermum erythrorhizon is difficult due to its limited area, but there is a possibility to grow its cell culture using the biotechnological method, which opens up great prospects for the further isolation of these BAS. Previously, we proposed a composition and a technological scheme for the manufacture of polymeric films containing shikonin and its esters, isolated from a culture of Lithospermum erythrorhizon cells. This study is devoted to the development of approaches to the qualitative analysis of polymeric films. Three directions of qualitative analysis are considered: evaluation of external signs, determination of technological parameters of films, confirmation of the presence of naphthoquinones. The criteria of quality and methods of analysis in each direction have been determined. The presence of shikonin and its derivatives in the polymeric films was confirmed by color reactions, thin layer chromatography and spectrophotometry.

Good-Practice Non-Radioactive Assays of Inorganic Pyrophosphatase Activities

Inorganic pyrophosphatase (PPase) is a ubiquitous enzyme that converts pyrophosphate (PPi) to phosphate and, in this way, controls numerous biosynthetic reactions that produce PPi as a byproduct. PPase activity is generally assayed by measuring the product of the hydrolysis reaction, phosphate. This reaction is reversible, allowing PPi synthesis measurements and making PPase an excellent model enzyme for the study of phosphoanhydride bond formation. Here we summarize our long-time experience in measuring PPase activity and overview three types of the assay that are found most useful for (a) low-substrate continuous monitoring of PPi hydrolysis, (b) continuous and fixed-time measurements of PPi synthesis, and (c) high-throughput procedure for screening purposes. The assays are based on the color reactions between phosphomolybdic acid and triphenylmethane dyes or use a coupled ATP sulfurylase/luciferase enzyme assay. We also provide procedures to estimate initial velocity from the product formation curve and calculate the assay medium's composition, whose components are involved in multiple equilibria.

The study on the development of the technology of the Sedum maximum juice as a biogenic stimulator for obtaining medicines

Due to the complex of biologically active substances (BAS) in the native state, juices are becoming increasingly popular in pharmacy and medicine. We were interested in the Sedum maximum plant as a powerful biogenic stimulant. Despite its wide distribution in the world and in Ukraine, plants of the Sedum L. genus are rarely used. The aim of the study is to determine the technological parameters of the process of obtaining juice from Sedum maximum for the development of technology for its production in pharmacy and industrial conditions and further use in pharmacy and medicine. Results. The optimal method of obtaining fresh juice from Sedum maximum grass using a number of technological techniques in the following sequence was substantiated: fermentation of Sedum maximum grass (keeping in a dark cool place (5 ± 3 ºC) for 5 days; pre-treatment of Sedum maximum grass raw material with boiling water; keeping the crushed raw material at a temperature of 5 ± 3 ºC during the day, purification of unclarified juice with rapid heating to 75-78 °C for 30 min followed by rapid cooling and treatment with 95 % ethanol, sedimentation of Sedum maximum grass juice at a temperature of 5 ± 3 ºC followed by stabilization of clarified juice of Sedum maximum grass with ethanol 15% and chloroethane in the amount of 0.5 %. Conclusions. The quality criteria of the obtained Sedum maximum juice were determined: appearance, pH, dry residue, identification by color reactions and quantitative determination by spectrophotometry (sum of tannins, in terms of pyrogalol). Key words: juice; Sedum maximum; technology; biogenic stimulant

Analysis of morphological features and preliminary assessment of arbutin content in food varieties of pears

In this article, the authors conducted a macro-diagnostic study of pear fruits of three varieties. A qualitative analysis of raw materials was carried out using color reactions, thin-layer chromatography in pear fruits most widely cultivated in the territory of the Russian Federation. Arbutin was identified in the raw material, which allows us to consider this type of raw material as a promising alternative to lingonberry and bearberry leaves used in official medicine and actualizes further research aimed at developing modern methods of standardization with subsequent inclusion in the developed regulatory documentation.

Influence of gibberellin on cultural and growth characteristics of vegetative mycelium of fungi strains of the genus Pleurotus during cultivation on agarized nutrient media

Very slow introduction into the culture of new for Ukraine species of edible fungi is associated primarily with more difficult conditions for the cultivation of these fungi, slow growth of mycelium, difficulties in obtaining a pure culture, sterilization of the substrate and others. Therefore, it is important to study the possibility of using growth stimulants used in crop production in industrial mushroom growing in order to intensify the process of obtaining the mycelium and fruiting bodies of mushrooms. The aim of this experiment was to study the nature of the influence of gibberellin on the cultural and growth characteristics of the vegetative mycelium of strains of the genus Pleurotus, which are classified as medium- and slow-growing on agar nutrient media. The objects of the study were industrial strains of Pleurotus pulmonarius (Fr.) Quél. (strain IBK-230) (medium-growing), Pleurotus eryngii (DC.) Quél. (strains IBK-2011 and IBK-1972) (slow-growing) obtained from the Collection of cap mushrooms of the Institute of Botany named after N.G. Kholodny NAS of Ukraine. The following nutrient media were used in the study: corn agar (CA), crushed sunflower husk agar (SHA), glucose-ammonium (GAM) and glucose-asparagine (GAS) agars. Gibberellin was added to the nutrient media in the following concentrations: 1, 10, 50, 100 mg / dm3. The control media did not contain growth stimulant.Preparation and sterilization of nutrient media, tests for individual enzymes were performed according to conventional methods. 9-day crops grown on corn agar on Petri dishes were used as inoculum. Surface cultivation of the mycelium of the studied strains of mushrooms was performed in a thermostat at a temperature of 26 ± 1 °C.During the cultivation process, the radius of colonies on Petri dishes was measured daily, morphological description was performed, the duration of the lag-phase of mycelial growth on media with different concentrations of gibberellin, the average rate of radial mycelial growth (mm / day) and qualitative color reactions to phenoloxidases (laccases, tyrosinases, peroxidases) was determined. Data processing was performed by methods of mathematical statistics.According to the results of the study of cultural and morphological characteristics of colonies of P. pulmonarius, P. eryngii, it was determined that the applied concentrations of gibberellin do not have a significant effect on the morphological parameters of fungal colonies on agar nutrient media.Taking into account the data of the average daily growth of mycelium of the studied Pleurotus strains on agar nutrient media and the average duration of the lag-phase of growth of fungal colonies, the following can be stated. For P. pulmonarius, a significant positive effect of gibberellin on the lag-phase of growth was observed on the media CA, SHA, GAM – for a stimulant concentration of 1 mg / dm3 (9, 10 and 18%, respectively), CA and SHA – for a concentration of 10 mg / dm3 (9 and 10%, respectively), and on the SHA – for a concentration of 50 mg / dm3 – 20% compared with the control. Gibberellin had the best effect on the duration of the lag-phase of growth of the mycelium of P. eryngii IBK-2011, where the reduction of the lag-phase of growth ranged from 8 to 18% for all concentrations of growth regulator compared to the control.The average rate of radial growth of the mycelium of P. pulmonarius IBK-230 significantly increased compared with the control under the influence of all studied concentrations of gibberellin on the media CA (from 16.8 to 18.8%) and GAM (from 26.3 to 52.6%). The best indicator of linear growth rate was recorded on CA and GAM media with a concentration of gibberellin of 50 mg / dm3. The obtained data from the average radial growth rate of mycelium of slow-growing strains of P. eryngii (IBK-2011, IBK-1972) showed that these strains respond better to the growth stimulator gibberellin: on all nutrient media there was an increase of the growth rate for almost all concentrations of gibberellin from 3.1 to 60%. The most effective concentrations of gibberellin were 10 and 50 mg / dm3 (on the nutrient media of CA and GAM) for all strains of the studied fungi, on GAS – 1, 10 and 50 mg / dm3.The enzymatic activity of the studied strains differed depending on the type of mushrooms, nutrient medium and different concentrations of gibberellin. Thus, for P. pulmonarius no significant difference was found in the manifestation of color reactions to phenoloxidases, except for the enzyme tyrosinase, where the color appeared after 30 minutes on SHA (the concentration of gibberellin – 10 and 50 mg / dm3) and GAM (the concentration of 1 and 10 mg / dm3), on others the corresponding color appeared within 3 hours. The manifestation of color reactions to phenoloxidases for P. eryngii (IBK-2011, IBK-1972) for 30 minutes was observed on CA medium (concentration of gibberellin – 10, 50 and 100 mg / dm3), on SHA – 10 and 50 mg / dm3, on GAM – 1 and 10 mg / dm3, on GAS – 1, 10, 50 and 100 mg / dm3. Thus, slow-growing strains are more responsive to synthetic activity on this growth stimulant.As a result of the experiment it was found that the growth stimulant gibberellin affects the culture and growth characteristics of the vegetative mycelium of industrial strains of the genus Pleurotus, belonging to the medium- and slow-growing – P. pulmonarius (strain ІВК-230), P. eryngii (strains ІВК-2011, ІВК-1972): the lag-phase of mycelial growth decreases (on average from 8 to 18%), the average radial growth rate of mycelium increases from 16.8 to 57.9% for P. pulmonarius, and from 10.5 to 60% for strains P. eryngii, increases enzymatic activity. This growth stimulant can be used in biotechnology of edible fungi to obtain uterine mycelium of slow-growing species, increase the synthesis of biologically active substances by macromycetes, as well as increase the yield of fruiting bodies of mushrooms, which are poorly represented in the consumer market of Ukraine.

RESEARCH ON THE DEVELOPMENT OF ME DICINE BASED ON NATURAL COMPOUNDS

The relevance of the search for effective hypoglycemic drugs is undeniable, due to the growth of patients with diabetes mellitus, accompanied by various diabetic complications. The purpose of the studies was to develop the composition, methods of analysis, quality standards with hypoglycemic and detoxifying effects. Developed drug includes total preparation of inulin and pectin and taurine. Qualitative analysis was carried out on the identification of pectoinline and taurine. To detect pectoinulin, the total preparation of inulin and pectin, color reactions were used (with a solution of α-naphthol alcohol 20% and with a solution of carbazole alcohol 0.5%), as well as a precipitation reaction with a solution of lead (II) acetate of the main 10%. To detect taurine, a color reaction with ninhydrin was used. The chosen procedures make it possible to establish the authenticity of taurine in the composition of the proposed drug in the joint presence of ingredients. The quantitative determination of inulin was carried out by the Bertran titrimetric method in the Maken and Shoorl modifications. Quantitative determination of pectin is proposed to be carried out by calcium-pectate method. Spectrophotometric method is proposed for determination of taurine in composition. The proposed methods of identification and quantitative determination of the ingredients of the composition are valid according to the main criteria. The studies made it possible to propose drug quality standards that are included in the draft regulatory documentation for the drug. As a result of all the conducted studies, the composition was developed and the optimal methods of qualitative and quantitative analysis of the ingredients of the proposed drug were selected, which make it possible to determine them in the joint presence, which are included in the draft pharmacopoeia article of the enterprise.

Colorimetric Quantification Methods for Peracetic Acid together with Hydrogen Peroxide for Water Disinfection Process Control

Peracetic acid (PAA) water solutions is applied for disinfection of industry systems, food products and non-potable water. Commercially available peracetic acid is always supplied mixed with hydrogen peroxide (H2O2). H2O2 degrade slower than the peracetic acid which creates a need to quantify both peroxides separately to gauge the disinfection power of the solution and the residuals. Two combinations of colorimetric reactions are presented that allows simultaneous quantification at the mg·L−1 level used in disinfection liquids and water disinfection. The first dichromic reaction use titanium oxide oxalate (TiO-Ox) which only react with H2O2 followed by addition of N,N-diethyl-p-phenylenediamine with iodide (DPD/I−) and the concentrations are read by simultaneously measuring the absorbance at 400 and 515 nm. Limit of quantification (LOQ) and maximal concentration determined was 4.6 µg·L−1 and 2.5 mg·L−1 for PAA and 9.1 µg·L−1 and 5 mg·L−1 for H2O2. The two color reactions didn’t interfere with each other when the reagent addition was consecutive. Another combination of colorimetric reaction also used where TiO-Ox was used to first measure H2O2 at 400 nm, before addition of 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS)) and reading the absorbance at 405 nm. ABTS changes the absorbance at 405 nm necessitating the two measurements be done separately. LOQ and maximal concentration determined using ABTS colorimetric assay was 42.5 µg·L−1 and 30 mg·L−1 for PAA and for titanium oxide oxalate colorimetric assay was 12.7 µg·L−1 and 75 mg·L−1 for H2O2. Both methods tested satisfactory in typical water samples (Tap, sea, lake, and biological treated sewage) spiked with peracetic acid and H2O2, separately.