The Influence of Transglutaminase on Minced Muscular Fish Tissue Structure Formation After the Application of Various Protein Substrates

This research aimed to examine the effect of a microbial transglutaminase preparation (Activa® GS, Ajinomoto Co., Inc, Japan) on the structure formation of the myofibrillar protein system of a deep-sea fish species – the giant grenadier – after the addition of various protein substrates. The low content of proteins and their low water-holding capacity in this fish, subjected to various processing methods, leads to significant losses in the initial mass and decreased gelation ability in the muscle tissue system. Various concentrations of transglutaminase were used but these did not ensure the restructuring of the initial muscle tissue of the grenadier. Additional protein substrates with different molecular weights and amino acid composition were added, including gelatin, milk casein, hydrolysates of the skin and milt of the fish, and whole bivalves, which were used to create a firm structure. It was shown that the introduction of gelatin and casein at a concentration of 5% led to the formation of a firm, thermostable structure under the action of the enzyme, while hydrolyzed proteins with low molecular weight at their various concentrations enhanced the expression of water and formation of the fluid consistency. The ability of gastrointestinal tract proteases (pepsin and trypsin) to digest did not depend on the formation of protein-to-protein cross-linking in these combined products. The influence on the growth of the Tetrahymena pyriformis ciliates test culture also showed the high degree of product availability. The technology of molded products based on fermented minced muscle tissue of grenadier with added casein, both in the form of semi-finished products and in the form of ready-to-eat products, was developed. Keywords: transglutaminase, muscle tissue, structure formation, deep-sea fish

ДОКЛІНІЧНІ ВИПРОБУВАННЯ ПРЕПАРАТУ “БІОМАГН” НА ЛАБОРАТОРНИХ ТВАРИНАХ ТА З ВИКОРИСТАННЯМ КУЛЬТУРИ ІНФУЗОРІЙ TETRAHYMENA PYRIFORMIS

Вступ. Токсикологічний контроль ветеринарних препаратів запобігає можливим порушенням обміну речовин, негативному впливу на органи і тканини, виникненню побічних дій та віддалених наслідків, створює передумови для визначення оптимальних терапевтичних доз, способів і термінів застосування, шляхів та часу виведення з організму, що, у свою чергу, сприяє розробці нових високоефективних конкурентоспроможних препаратів. Мета дослідження – провести доклінічні випробування пробіотичного препарату “Біомагн”: визначити гостру токсичність, виявити його можливу шкідливість при одноразовому введенні в організм тварин і встановити токсичні й летальні дози. Методи дослідження. Токсичність і шкідливість препарату визначали в дослідах на найпростіших тест-організмах інфузоріях тетрахімен піріформіс (Tetrahymena pyriformis) та на лабораторних тваринах (білих мишах). Гостру токсичність препарату “Біомагн” вивчали на білих безпородних нелінійних мишах живою масою (22,5±0,2) г, яких годували стандартним гранульованим кормом відповідно до норм правил годівлі. Токсикологічні дослідження проводили згідно з монографією “Доклінічні дослідження ветеринарних лікарських засобів” (І. Я. Коцюмбас та ін., 2006) і СОУ 85.2-37-736:2011 “Препарати ветеринарні. Визначення гострої токсичності”. Для визначення ступеня кумуляції досліджуваного засобу використовували метод Ю. С. Кагана i В. В. Станкевича. Результати й обговорення. Під час досліджень встановлено, що імуномодулюючий пробіотичний препарат “Біомагн” на основі магнію хлориду, хітозану, суміші пробіотичних бактерій та ензимів не проявляє токсичних властивостей при взаємодії з інфузоріями впродовж 30 хв, тому що зберігається повна 100 % життєдіяльність особин культури тетрахімен, як і в контрольних пробірках. Як свідчать результати досліджень, гостра токсичність на лабораторних тваринах препарату “Біомагн” становить LD50 – 5000 мг/кг живої маси мишей. Під час клінічного спостереження за тваринами дослідних груп у всіх групах відхилень не виявлено, шкірний покрив гладкий, блискучий, відмови від корму не спостерігали, порушень роботи шлунково-кишкового тракту і центральної нервової системи в жодній із груп не відмічено, загибелі й захворювань не відзначено. Препарат “Біомагн” не має шкідливого впливу і стимулює покращення обміну речовин, що, у свою чергу, сприяє збільшенню маси тварин. Гематологічні показники периферичної крові мишей при застосуванні препарату достовірно не змінювались. Усі показники перебували в межах фізіологічної норми та залишалися такими до закінчення терміну експерименту, що вказувало на безпечність і нетоксичність досліджуваного засобу. За встановленими ознаками препарат віднесено до 4 класу щодо небезпечності. Композиція суміші пробіотичних бактерій Bacillus subtilis, Bacillus licheniformis, Enterococcus faecium, висушених продуктів ферментації мікроорганізмів Lactococcus Lactis, Bacillus subtilis, Bacillus licheniformis та магнію хлориду, хітозану, яка забезпечує нормалізацію обміну речовин тварин, впливає на підвищення загальної резистентності організму, стимулює роботу органів травлення. Висновки. За результатами досліджень, проведених з метою визначення гострої токсичності препарату “Біомагн”, можна зробити висновок, що він не токсичний (СОУ 85.2-37-736:2011). Розведення препарату “Біомагн” у різних концентраціях є нешкідливими для найпростіших Tetrahymena pyriformis.

β Cyclodextrin Nanocomplexes with Biologically Active Peptides from Hydrolysed Bovine Whey and Colostrum

β Cyclodextrin nanocomplexes with extensive whey and colostrum hydrolysates possessing acceptable flavor properties serve as potential sources of bioactive peptides. In this study, comparative characterization of dairy protein hydrolysates and their complexes with β cyclodextrin is presented. Antioxidant activity of studied samples was estimated by fluorometric method, the formation of clathrates with cyclic oligosaccharide was determined using thermogravimetric analysis. A significant decrease in bitterness of peptides included in cyclic oligosaccharides was established compared with samples of dairy hydrolysates. 2.1/1.3 fold increase in the antioxidant potential of β cyclodextrin clathrates with whey/colostrum hydrolysates was recorded versus unbound peptide fractions. According to toxicological tests on Tetrahymena pyriformis, the samples of whey hydrolysate and the resulting nanocomplex were referred to as non-toxic and slightly hazardous compounds, respectively. The dynamics of body weight gain and the relative weight coefficient of internal organs revealed no differences compared to the control group of Rattus norvegicus. The data on differentiation of blood cells, their death, and cytogenetic disorders demonstrated that a sample of cyclic oligosaccharides with whey peptides is non-toxic at the maximum dosages allowable for administration. β Cyclodextrin complexing with dairy peptides resulted in enhanced radical-reducing activity and improved flavor properties, making the clathrates promising and safe ingredients of special nutrition formulas.

Loss of the acetate switch in Vibrio vulnificus enhances predation defence against Tetrahymena pyriformis

Vibrio vulnificus is an opportunistic human pathogen and autochthonous inhabitant of coastal marine environments, where the bacterium is under constant predation by heterotrophic protists or protozoans. As a result of this selection pressure, genetic variants with anti-predation mechanisms are selected for and persist in the environment. Such natural variants may also be pathogenic to animal or human hosts, making it important to understand these defence mechanisms. To identify anti-predator strategies, thirteen V. vulnificus strains of different genotypes isolated from diverse environments were exposed to predation by the ciliated protozoan, Tetrahymena pyriformis , and only strain ENV1 was resistant to predation. Further investigation of the cell-free supernatant showed that ENV1 acidifies the environment by the excretion of organic acids, which is toxic to T. pyriformis . As this predation resistance was dependent on the availability of iron, transcriptomes of V. vulnificus in iron-replete and iron-deplete conditions were compared. This analysis revealed that ENV1 ferments pyruvate and the resultant acetyl-CoA leads to acetate synthesis under aerobic conditions, a hallmark of overflow metabolism. The anaerobic respiration global regulator, arcA , was upregulated when iron was available. An Δ arcA deletion mutant of ENV1 accumulated less acetate and importantly, was sensitive to grazing by T. pyriformis . Based on the transcriptome response and quantification of metabolites, we conclude that ENV1 has adapted to overflow metabolism and has lost a control switch that shifts metabolism from acetate excretion to acetate assimilation, enabling it to excrete acetate continuously. We show that overflow metabolism and the acetate switch contribute to prey-predator interactions. Importance Bacteria in the environment, including Vibrio spp., interact with protozoan predators. To defend against predation, bacteria evolve anti-predator mechanisms ranging from changing morphology, biofilm formation and secretion of toxins or virulence factors. Some of these adaptations may result in strains that are pathogenic to humans. Therefore, it is important to study predator defence strategies of environmental bacteria. V. vulnificus thrives in coastal waters and infects humans. Very little is know about the defence mechanisms V. vulnificus expresses against predation. Here we show that a V. vulnificus strain (ENV1) has rewired the central carbon metabolism enabling the production of excess organic acid that is toxic to the protozoan predator, T. pyriformis . This is a previously unknown mechanism of predation defence that protects against protozoan predators.

Accumulation of Tetrahymena pyriformis on Interfaces

The behavior of ciliates has been studied for many years through environmental biology and the ethology of microorganisms, and recent hydrodynamic studies of microswimmers have greatly advanced our understanding of the behavioral dynamics at the single-cell level. However, the association between single-cell dynamics captured by microscopic observation and pattern dynamics obtained by macroscopic observation is not always obvious. Hence, to bridge the gap between the two, there is a need for experimental results on swarming dynamics at the mesoscopic scale. In this study, we investigated the spatial population dynamics of the ciliate, Tetrahymena pyriformis, based on quantitative data analysis. We combined the image processing of 3D micrographs and machine learning to obtain the positional data of individual cells of T. pyriformis and examined their statistical properties based on spatio-temporal data. According to the 3D spatial distribution of cells and their temporal evolution, cells accumulated both on the solid wall at the bottom surface and underneath the air–liquid interface at the top. Furthermore, we quantitatively clarified the difference in accumulation levels between the bulk and the interface by creating a simple behavioral model that incorporated quantitative accumulation coefficients in its solution. The accumulation coefficients can be compared under different conditions and between different species.

Alleviate Effect of Pomegranate Peel Extract in Ameliorating Fluoride-Induced Cytotoxicity, Oxidative Stress in Tetrahymena pyriformis Model

Fluoride is a major oligo element found in nature, at excessive amounts can cause enormous harm in mammalian cells. Fruits peel, considered most often as a waste of juice processing, could play an important role in attenuating metal cytotoxicity. The present study evaluated the effect of pomegranate peel (Punica granatum. L) methanolic extract (PPE) on the Fluoride-induced toxicity and redox status in the protozoa Tetrahymena pyriformis. Polyphenols of peel extract were extracted using methanol and characterized by spectrophotometric methods, total phenolic content (TPC), total flavonoids content (TFC), and in vitro, antioxidant properties were assessed using the Folin-Ciocalteu method and DPPH, ABTS, and FRAP. Pomegranate peel is a rich source of phenolic compounds TP (223.21 ± 15 mg GAE/g dw), TF (52.12 ± 1.36 mg Qu/g dw) and showed high antioxidant properties DPPH (EC50 0.043 ± 0.06 mg/ml), ABTS (EC50 0.06 ± 0.01 mg/ml) and FRAP (1.47 ± 0.01 mg AA equivalents/g dw). Cells were incubated with fluoride alone and in combination with PPE. NaF (0.8 mM) significantly decreased the cell viability, induced oxidative stress by decreasing antioxidants enzyme activities, and increased intracellular fluoride content. Treatment with NaF in combination with PPE decreases CAT, SOD, and GPx activities and alleviates GSH content. These findings suggest that pomegranate peel biomolecules may have a protective effect against fluoride induced-toxicity.