Enzyme Preparation Research Articles

Characterization of Novel Multifunctional Xylanase from Rumen Metagenome and Its Effects on In Vitro Microbial Fermentation of Wheat Straw

This study investigated the characterization of a novel multifunctional enzyme, RuXyn394, derived from the metagenome of beef cattle rumen, and its impact on the in vitro microbial fermentation of wheat straw. RuXyn394, a member of the glycosyl hydrolase 11 family, displayed optimal activity under diverse pH and temperature conditions: xylanase at pH 5.5 and 50 °C, acetyl esterase at pH 6.5 and 60 °C, exoglucanase at pH 7.0 and 50 °C, and endoglucanase at pH 6.0 and 50 °C. The enzyme’s xylanase, endoglucanase, and exoglucanase activities exhibited remarkable pH stability across the range of pH 3–8 and maintained a relatively stable performance at temperatures from 20 to 50 °C, 20 to 60 °C, and 20 to 70 °C, respectively. The xylanase function, with the highest kcat/Km ratio, was identified as the predominant activity of RuXyn394. The enzyme’s various functions responded uniquely to metal ions; notably, the addition of 5 mM K+ significantly boosted the activities of xylanase, exoglucanase, and endoglucanase by 55.5%, 53.5%, and 16.4%, respectively, without affecting its acetyl esterase activity. Over the course of three time points (30 min, 60 min, 120 min), the degradation products of wheat straw xylan, including xylopentaose, xylotetraose, xylotriose, xylobiose, xylose, and total xylooligosaccharides, constituted an average of 18.4%, 33.7%, 20.6%, 22.9%, 4.3%, and 95.7% of the total products, respectively. RuXyn394 effectively hydrolyzed wheat straw, resulting in augmented volatile fatty acid production and ammonia-N levels during in vitro microbial fermentation. These findings indicate the potential of RuXyn394 as a novel and highly efficient enzyme preparation, offering promising prospects for the valorization of wheat straw, an agricultural by-product, in ruminant diets.

Antioxidants, ACE I Inhibitory Peptides, and Physicochemical Composition, with a Special Focus on Trace Elements and Pollutants, of SPRING Spawning Atlantic Herring (Clupea harengus) Milt and Hydrolysates for Functional Food Applications

Norwegian spring spawning (NVG) herring milt is a raw material with high nutritional and functional values. However, its incorporation into food presents physicochemical and sensory challenges. Its high DNA content, the presence of TMA/TMAO and possibly heavy metal and/or environmental pollutants, and its bitter taste due to amino acids or peptides requires a careful approach to food development. Hydrolysis with food-grade enzymes enable an improvement in both the functional and sensory properties of the substrate and the increased stability of the raw materials and end products. HLPC, GC-MS, and in vitro protocols were used for the characterisation of manually extracted material (sample code: HMC) and milt from a fish-filleting line from early spring/late autumn catches. Three different food-grade protein hydrolysates were prepared from these raw materials (sample codes: H1, H2, and H3) as a means to estimate their functional food development potential. Combinations of three commercial enzymatic preparations were applied, targeting specific sensory properties. Parameters related to consumer safety (e.g., the presence of heavy metals and TMA/TMAO); beneficial health effects, such as antioxidant or antihypertensive bioactivities (measured using in vitro TAC, ORAC, DPPH, and ACE I inhibitory activity assays); the presence of beneficial fatty acids and micronutrients; and the protein quality were studied. On the basis of their total amino acid compositions, freeze-dried herring milt and hydrolysates could provide high-quality protein with most of the essential amino acids and taurine. Powdered milt has a particularly high fatty acid profile of bioavailable omega-3 fatty acids (2024.06 mg/100 g docosahexaenoic acid (DHA; 22:6n-3) and 884 mg/100 g eicosapentaenoic acid (EPA; 20:5n-3)). The experimentally measured levels of arsenic (3.9 ± 1.2 mg/kg) and cadmium (0.15 ± 0.05 mg/kg) were higher than the levels of the other two heavy metals (mercury and lead). The bioactivity is concentration-dependent. Overall, this work presents complementary information for the future utilisation of C. harengus powdered milt (possibly obtained directly from a fish-filleting line) and some of its protein hydrolysates as food ingredients.

Using an enzyme preparation and vitamin C to increase the meat productivity of broiler chickens

Purpose: to study the effect of vitamin C and MEK Feedbest®VGPro when introduced into the compound feed of broiler chickens with excessive background of heavy metals on the indices of meat productivity, food and biological value, ecological safety of their meat.Materials and methods. The objects of the study were meat chickens of the ROSS-508 cross. At the age of one day, 4 groups of 100 birds each were formed from them in accordance with the principle of analog groups. In the composition of the used recipes of complete compound feed in all three age periods of growing, the main grain and protein ingredients were represented by locally produced feed: corn, wheat, peas and rapeseed meal. Growing of experimental broilers for meat was completed at the age of 42 days and their control slaughter was carried out, for which 5 birds were selected from each group. The concentration of salts of heavy metals was determined in the samples of feed and meat of the experimental birds.Results. The experiment substantiated the feasibility of combined introduction of MEC Feedbest®VGPro at a rate of 100 g/t of feed and vitamin C at a rate of 500 g/t of feed into diets with excessive content of heavy metal salts. Compared to the control analogues, broilers of the 3rd experimental group showed a reliable (p<0,05) decrease in fat by 0,26 % and a simultaneous increase in dry matter, protein and the value of the protein-quality indicator in the pectoral muscle samples. Combined additions of the tested preparations contributed to a decrease in the concentration of zinc by 63,17 % (p<0,05), cadmium by 61,11 % (p<0,05) and lead by 62,34 % (p<0,05) in poultry meat samples from the 3rd experimental group, compared to the control. Moreover, in the meat samples of broilers of the 3rd experimental group, the maximum permissible concentrations of none of these elements were exceeded.

Research on the pharmacological potential of 1-alkyl derivatives of 3,5-dimethyl-4-((4-nitrobenzylidene)amino)-1,2,4-triazolium bromide

The heterocyclic system of 1,2,4-triazole and its derivatives is one of the leaders in the development of highly promising biologically active compounds. The peculiarities of the chemical structure of the derivatives of this heterocycle provide a wide range of possibilities for chemical transformations that allow to obtain really effective drugs. The involvement of several substituents in chemical transformations simultaneously, which have the properties of highly reactive centers, additionally creates favorable conditions for the formation of rational ways to create a biologically active compound. Amino-, mercapto- or hydroxogroups often play the role of such groups in chemistry. The use of these groups as substituents of 1,2,4-triazole synthon provides multifaceted opportunities for directed chemical transformation. The ability of such structural fragments to form chemical interactions and bonds with biological targets has an additional positive effect in the sense of their involvement in chemical transformations on the way to the targeted production of a biologically active substance. Thus, the combination of a heterocyclic structure with a highly reactive chemical center is endowed with theoretically sound and practically significant meaning. The aim of the work is to preliminary determine the potential for creating a biologically active substance with antifungal action based on 1-alkyl derivatives of 3,5-dimethy-l-4-((4-nitrobenzylidene)amino)-1,2,4-triazolium bromide. Materials and methods. The toxicity of the studied compounds has been predicted using the TEST program (Toxicity Estimation Software Tool), which allowed to determine the predictive level of acute toxicity, ecotoxicity and mutagenicity. The physicochemical and pharmacokinetic parameters have been predicted, and the drug-like properties and availability of the investigated substances have been assessed using the online resource SwissADME. The determination of the most favorable spatial configuration of the ligand relative to the active site of the protein and the assessment of the strength of their interaction have been realized using the computational method of molecular docking. The ligands have been prepared using MarvinSketch 6.3.0, HyperChem 8 and AutoDock Tools-1.5.6 software. The preparation of the model enzyme has been based on the use of Discovery Studio 4.0 and AutoDock Tools-1.5.6. The practical implementation of flexible molecular docking has been carried out using the software tools of the AutoDock/Vina platform. Results. In the process of step-by-step prescreening of the formed structures of a number of 1-alkyl derivatives of 3,5-dimethyl-4-((4-nitrobenzylidene)amino)-1,2,4-triazolium bromide, a number of qualitative and quantitative indicators related to the physicochemical characteristics and pharmacokinetic parameters of the studied substances have been obtained. According to the results of the first stage of research, the group of substances under consideration can be predictively considered low-toxic, but with a high risk of mutagenic properties. The next stage of the work, which involved the analysis of physicochemical parameters, pharmacokinetic parameters, general drug-like properties and bioavailability, allowed us to identify 1-alkyl derivatives of 3,5-dimethyl-4-((4-nitrobenzylidene)amino)-1,2,4-triazolium bromide as substances with a rather positive pharmacological profile. The final stage in the form of molecular docking of the structure of the studied compounds to the active site of lanosterol 14α-demethylase allowed us to determine the nature of the chemical interaction and the type of amino acid residues that may be involved in the antifungal properties of the key ligands. The analysis of the docking results allows us to determine the privileged nature of the nonyl substituent at the first Nitrogen atom of the 1,2,4-triazole synthon in the structure of the presented series of compounds for the formation of antifungal properties. Conclusions. The general prospects for the creation of a biologically active substance with antifungal properties using 1-alkyl derivatives of 3,5-dimethyl-4-((4-nitrobenzylidene)amino)-1,2,4-triazolium bromide look quite realistic. Particular attention should be paid to 3,5-dimethyl-1-nonyl-4-((4-nitrobenzylidene)amino)-1,2,4-triazolium bromide as a substance with significant potential for antifungal properties, which allows us to recommend this compound for further more constructive and extended in vitro and in vivo studies.

Immobilization of laccase on Fe3O4@SiO2 core@shell magnetic nanoparticles for methylene blue biodegradation

Here we report the preparation and characterization of novel enzyme supports based on silica-coated Fe3O4 magnetic nanoparticles. These nanomaterials were modified at their outer silica surface with isocyanate, trimethylammonium and β-cyclodextrin moieties to immobilize laccase from Trametes versicolor through covalent, electrostatic and supramolecular interactions, respectively, with protein immobilization yields ranging from 21.7% to 53.5%. The effect of the immobilization approach on the activity, optimal working conditions, stability and reusability of the resulting biocatalysts were studied. Best results were achieved for native and adamantane-modified laccase supramolecularly immobilized on β-cyclodextrin bearing supports in terms of their catalytic properties, showing 18.0 U and 14.0 U of immobilized laccase activity per gram of support. However, high thermal stability was observed for the enzyme covalently immobilized on isocyanate-modified nanoparticles, with 14.8-fold increase in the half-life time at 65ºC in comparison with native laccase. Best reusability properties were also achieved by covalent immobilization, retaining over 88% of the initial catalytic activity after 13 cycles of magnetic reuses. All enzyme derivatives were evaluated for the catalytic degradation of methylene blue as pollutant model, showing significant reduction of the dye. In special, a 68-fold increase in the removal efficacy was observed for covalently immobilized enzyme compared to the free laccase. These results suggest high potential application of these biocatalysts in wastewater treatment.

Eukaryotic expression of chitinase from dark sleeper (Odontobutis potamophila) and its effects on growth and immunity

Chitinase, an enzyme that hydrolyzes β-1,4-glycosidic bonds to degrade chitin, is essential for the digestion of chitin in fish. In this study, the chitinase OpCht from Odontobutis potamophila was expressed in Pichia pastoris, and its enzymatic properties and functional effects were evaluated. The findings revealed that OpCht exhibited optimal activity at pH 6.0 and 50 °C, with stability in the pH range of 4–8 and temperatures from 4 to 40 °C. K+, Na+, Ca2+, Mg2+, Mn2+, Hg2+, and Al3+ showed varying degrees of activation on the enzyme. At the end of the 8-week trial, the addition of OpCht significantly increased the height of intestinal villi and the thickness of the muscular layer, leading to significantly weight in the treated groups. The alleviation of intestinal inflammation also resulted in an increased survival rate (SR) of O. potamophila. High concentration treatment groups (2, 4 μg/g) showed significantly elevated digestive enzyme activities, as well as increased antioxidant enzyme activities and immune parameters. These results demonstrate that the P. pastoris expression system has successfully produced the chitinase OpCht from O. potamophila, and the addition of a certain concentration of OpCht can promote fish growth and enhance immune functions, offering a promising enzyme preparation for the aquaculture industry.

Rational Design for Enhancing Cellobiose Dehydrogenase Activity and Its Synergistic Role in Straw Degradation.

Addressing the demand for efficient biological degradation of straw, this study employed rational design coupled with structural biology and enzyme engineering techniques to enhance the catalytic activity of cellobiose dehydrogenase (PsCDH, CDH form Pycnoporus sanguineus). By predicting and modifying the active site and key amino acids of PsCDH, several CDH immobilized enzyme preparations with higher catalytic activities were successfully obtained. The excellent mutant T1 (C286Y/A461H/F464R) exhibited a 2.7-fold increase in enzyme activity compared to the wild type. Simulated calculations indicated that the enhancement of catalytic activity was primarily due to the formation of additional intermolecular interactions between CDH and the substrate, as well as the enlargement of the substrate pocket to reduce steric hindrance effects. Additionally, molecular dynamics simulation analysis revealed a potential correlation between structural stability and enzyme activity. When PsCDH was added to a multienzyme synergistic straw degradation system, the cellulose degradation rate increased by 1.84-fold. Moreover, mutant T1 further increased the degradation of lignocellulose in the mixed system. This study provides efficient enzyme sources and modification strategies for the high-efficiency biological conversion of straw and unconventional feedstock degradation, thereby possessing significant academic value and application prospects.

Direct Preparation of Alginate Oligosaccharides from Brown Algae by an Algae-Decomposing Alginate Lyase AlyP18 from the Marine Bacterium Pseudoalteromonas agarivorans A3

Alginate oligosaccharides (AOs), derived from alginate degradation, exhibit diverse biological activities and hold significant promise in various fields. The enzymatic preparation of AOs relies on alginate lyases, which offers distinct advantages. In contrast to the conventional use of sodium alginate derived from brown algae as the substrate for the enzymatic preparation of AOs, AO preparation directly from brown algae is more appealing due to its time and energy efficiency. Thus, the identification of potent alginate lyases and cost-effective brown algae substrates is crucial for optimizing AO production. Herein, we identified and characterized an alginate lyase, AlyP18, capable of efficiently decomposing algae, from a marine bacterium Pseudoalteromonas agarivorans A3 based on secretome analysis. AlyP18 is a mesothermal, endo-type and bifunctional alginate lyase with high enzymatic activity. Two brown algae substrates, Laminaria japonica roots and Macrocystis pyrifera, were used for the AO preparation by AlyP18. Upon optimization of AlyP18 hydrolysis parameters, the substrate degradation efficiency and AO production reached 53% and ~32% for L. japonica roots, respectively, and 77% and ~46.5% for M. pyrifera. The generated AOs primarily consisted of dimers to pentamers, with trimers and tetramers being dominant. This study provides an efficient alginate lyase and alternative brown algal feedstock for the bioconversion of high-value AOs from brown algae.

High keratinase and other types of hydrolase activity of the new strain of Bacillus paralicheniformis.

Keratinases, a subclass of proteases, are used to degrade keratin thereby forming peptones and free amino acids. Bacillus paralicheniformis strain T7 was isolated from soil and exhibited high keratinase, protease, collagenase, amylase, xylanase, lipase, and phosphatase activities. Keratinases of the strain showed maximum activity at 70°C and pH 9.0 as well as high thermal stability. A mass-spectrometric analysis identified seven peptidases with molecular masses of 26.8-154.8 kDa in the secretory proteome. These peptidases are members of S8 and S41 serine peptidase families and of M14, M42, and M55 metallopeptidase families. Additionally, α-amylase (55.2 kDa), alkaline phosphatase (59.8 kDa), and esterase (26.8 kDa) were detected. The strong keratinolytic properties of the strain were confirmed by degradation of chicken and goose feathers, which got completely hydrolyzed within 4 days. Submerged fermentation by strain B. paralicheniformis T7 was carried out in a pilot bioreactor, where the highest keratinase production was noted after 19 h of cultivation. After the fermentation, in the culture fluid, the keratinase activity toward keratin azure was 63.6 ± 5.8 U/mL. The protease activity against azocasein was 715.7 ± 40.2 U/mL. The possibility of obtaining enzyme preparations in liquid and powder form was demonstrated, and their comparative characteristics are given. In the concentrate, the keratinase, protease, α-amylase, phosphatase, and esterase/lipase activities were 2,656.7 ± 170.4, 29,886.7 ± 642.9, 176.1 ± 16.3, 23.9 ± 1.8, and 510.9 ± 12.2 U/mL, respectively. In the lyophilizate, these activities were 57,733.3 ± 8,911.4, 567,066.7 ± 4,822.2, 2,823.0 ± 266.8, 364.2 ± 74.8, and 17,618.0 ± 610.3 U/g, respectively. In the preparation obtained by air flow drying at 55°C, these activities were 53,466.7 ± 757.2, 585,333.3 ± 4,277.1, 2,395.8 ± 893.7, 416.7 ± 52.4, and 15,328.1 ± 528.6 U/g, respectively. The results show high potential of B. paralicheniformis strain T7 as a producer of keratinases and other enzymes for applications in agricultural raw materials and technologies for processing of keratin-containing animal waste.

Enzymatic Preparation of Machine-Washable Antibacterial Wool through a Subtraction-Addition Technique Using Hydrolase and Transglutaminase

Enzymatic Preparation of Machine-Washable Antibacterial Wool through a Subtraction-Addition Technique Using Hydrolase and Transglutaminase

Effects of fibrolytic and amylolytic compound enzyme preparation on rumen fermentation, serum parameters and production performance in primiparous early-lactation dairy cows.

This research communication reports the effects of a compound enzyme preparation consisting of fibrolytic (cellulase 3500 CU/g, xylanase 2000 XU/g, β-glucanase 17 500 GU/g) and amylolytic (amylase 37 000 AU/g) enzymes on nutrient intake, rumen fermentation, serum parameters and production performance in primiparous early-lactation (47 ± 2 d) dairy cows. Twenty Holstein-Friesian cows in similar body condition scores were randomly divided into control (CON, n = 10) and experimental (EXP, n = 10) groups in a completely randomized single-factor design. CON was fed a basal total mixed ration diet and EXP was dietary supplemented with compound enzyme preparation at 70 g/cow/d. The experiment lasted 4 weeks, with 3 weeks for adaptation and then 1 week for measurement. Enzyme supplementation significantly increased diet non-fibrous carbohydrates (NFC) content as well as dry matter intake (DMI) and NFC intake (P < 0.05). EXP had increased ruminal butyrate and isobutyrate percentages (P < 0.01) but decreased propionate and valerate percentages (P < 0.05), as well as increased serum alkaline phosphatase activity and albumin concentration (P ≤ 0.01). Additionally, EXP had increased milk yield (0.97 kg/d), 4% fat corrected milk yield and energy corrected milk yield, as well as milk fat and protein yield (P < 0.01). In conclusion, dietary supplementation with a fibrolytic and amylolytic compound enzyme preparation increased diet NFC content, DMI and NFC intake, affected rumen fermentation by increasing butyrate proportion at the expense of propionate, and enhanced milk performance in primiparous early-lactation dairy cows.

Recombinant production of Paenibacillus wynnii β-galactosidase with Komagataella phaffii

BackgroundThe β-galactosidase from Paenibacillus wynnii (β-gal-Pw) is a promising candidate for lactose hydrolysis in milk and dairy products, as it has a higher affinity for the substrate lactose (low KM value) compared to industrially used β-galactosidases and is not inhibited by the hydrolysis-generated product D-galactose. However, β-gal-Pw must firstly be produced cost-effectively for any potential industrial application. Accordingly, the yeast Komagataella phaffii was chosen to investigate its feasibility to recombinantly produce β-gal-Pw since it is approved for the regulated production of food enzymes. The aim of this study was to find the most suitable way to produce the β-gal-Pw in K. phaffii either extracellularly or intracellularly.ResultsFirstly, 11 different signal peptides were tested for extracellular production of β-gal-Pw by K. phaffii under the control of the constitutive GAP promoter. None of the signal peptides resulted in a secretion of β-gal-Pw, indicating problems within the secretory pathway of this enzyme. Therefore, intracellular β-gal-Pw production was investigated using the GAP or methanol-inducible AOX1 promoter. A four-fold higher volumetric β-galactosidase activity of 7537 ± 66 µkatoNPGal/Lculture was achieved by the K. phaffii clone 27 using the AOX1 promoter in fed-batch bioreactor cultivations, compared to the clone 5 using the GAP promoter. However, a two-fold higher specific productivity of 3.14 ± 0.05 µkatoNPGal/gDCW/h was achieved when using the GAP promoter for β-gal-Pw production compared to the AOX1 promoter. After partial purification, a β-gal-Pw enzyme preparation with a total β-galactosidase activity of 3082 ± 98 µkatoNPGal was obtained from 1 L of recombinant K. phaffii culture (using AOX1 promoter).ConclusionThis study showed that the β-gal-Pw was produced intracellularly by K. phaffii, but the secretion was not achieved with the signal peptides chosen. Nevertheless, a straightforward approach to improve the intracellular β-gal-Pw production with K. phaffii by using either the GAP or AOX1 promoter in bioreactor cultivations was demonstrated, offering insights into alternative production methods for this enzyme.

Improving the functional and technological properties of minced pork using a proteolytic enzyme

The use of pork obtained from intensively growing hybrid animals as the main raw material in meat processing, in particular sausage production, is accompanied by undesirable variability of consumer characteristics of sausage products and a decrease in the economic indicators of enterprise performance. The main technological disadvantages of processed pork are reduced water-binding capacity and significant loss of meat fluid, which in practice is usually compensated by the increased use of food additives and non-meat ingredients. The aim of the study was to assess the possibility of targeted improvement of the technological properties of minced pork using a proteolytic enzyme of animal origin. Model samples of minced meat were developed and cured for 24 hours at a temperature of 4 °C. The following control samples were used: minced meat with the addition of 2% edible salt and minced meat with the addition of 2% edible salt and 0.2% sodium bicarbonate. Moreover, 0.0001% chymotrypsin was added to the test samples containing similar curing ingredients. During electrophoretic study, in samples with the enzyme, an increase in low-molecular fractions (20 kDa, 15 kDa and lower) was observed, which indicated the manifestation of proteolytic activity of chymotrypsin in model systems. Enzymatic treatment led to an increase in pH and water-binding capacity. Cooking loss was reduced by 3 to 6 times, compared to Control 1. After cooking, histological studies of model systems showed that the test samples subjected to enzymatic treatment were characterized by a denser arrangement of structural elements, less pronounced cellular components of muscle tissue and the presence of glutin formed as a result of protein breakdown, filling the microcapillaries. Thus, the use of an enzyme preparation provides an opportunity for targeted improvement of the technological properties of pork obtained from intensively growing hybrid animals.

Novel Antarctic Endo-Polygalacturonase for Pectin Extraction and Vegetal Tissue Maceration at Mild Temperatures.

The aim of the present work was to partially purify and characterize an Antarctic polygalacturonase and to determine the enzyme's potential in pectin extraction and vegetal maceration at 20°C. Polygalacturonase was purified by chromatography to obtain an enzymatic preparation of specific activity 30.3 U.mg-1. Optimal conditions for the polygalacturonase activity were 45°C and pH 5.0-6.0, and the activation energy for the reaction was 41.8kJ.mol-1. Of the enzyme activity, 100% was retained after 3h at 40°C. The enzyme was remarkably stable for an hour over a wide range of pH (2.0-12.0). Polygalacturonase activity was slightly reduced in the presence of Ca+2, Fe+3, K+, Mn+2, and Zn+2, whereas Hg+2 reduced the activity by 60%, suggesting a thiol-dependent catalysis. The apparent molecular weight of the enzyme was 33kDa. The kinetic constants evaluated against polygalacturonic acid were 0.17mg.ml-1 (Km), 480s-1 (Kcat), and 7.9µmol.mg-1.min-1 (Vmax). The enzyme was active against different pectic substrates. Thin-layer chromatography revealed an endo-mechanism of action. Polygalacturonase digested lime pomace to aid the extraction of high-methoxylated pectin at 20°C and increased the vegetal maceration of Capsicum annuum by 24% over the control values.

Influence of physicochemical factors on the specific activity of proteases used in cheesemaking biotechnology

The formation of a clot under the influence of proteolytic enzymes is an essential stage in cheesemaking technology. Depending on their composition, milk-clotting enzyme preparations have a different effect on the quality characteristics of the clot, whey separation, and yield of the finished product. At present, the Russian market offers a wide range of domestic and imported milk-clotting enzyme preparations. In this connection, the choice of a milk coagulant by cheese makers should be carried out in accordance with the cheese group produced by the enterprise. The milk-clotting activity of enzyme preparations depends not only on the active acidity of the mixture, but also on the temperature of the solution used to obtain them. It is the solution temperature that has a determining effect on the duration of the coagulation process and, therefore, the quality of the formed clot. Research into the specific activity of milk-clotting enzyme preparations, obtained in a working solution with different active acidity (pH from 4.0 to 8.0), showed their milk-clotting activity to decrease significantly upon pH shifting to the alkaline region. Application of solutions with a pH of 4.0 led to an increase in the milk-clotting activity of bovine pepsin, VNIIMS SG-50 rennet-bovine preparation, and rennet enzyme by 94, 72, and 36%, respectively, relative to the control. Bovine pepsin demonstrated the highest sensitivity to fluctuations in active acidity. A study into the effect of substrate temperature in the range from 30 to 40 °C established rennet to be the most sensitive preparation to temperature changes. It is concluded that a properly selected enzyme composition is an effective means for affecting the quality of cheese products.

The use of protein hydrolysate from fish waste as part of microbiological culture media

The need to involve in the processing of significant amount of secondary protein-containing raw materials formed during fish cutting, on the one hand, and on the other hand, the shortage of a source of the protein component of microbiological nutrient media determines the relevance of the work. The goal was to study the possibility of using fish protein hydrolyzate obtained by enzymatic hydrolysis from secondary fish raw materials in microbiological nutrient media. Microbiological and physicochemical methods were used to conduct research. When optimizing the developed algorithm for the fermentation process, a mathematical model of the experiment was used with a minimum number of experiments. An algorithm has been developed for obtaining fish protein hydrolyzate from meat and bone waste from cutting pelagic fish (cod). The hydrolysis parameters were optimized: the concentration of the enzyme preparation is 1.33 % of the total mass of waste, the duration of the fermentation process – 3 hours. The characteristics of the resulting fish hydrolyzate are determined: the mass fraction of total nitrogen – 13 %, amine – 3.6 %, water – 4.6 %, sodium chloride – 2.7 %, fat – 0.3 %. An experimental fish hydrolyzate has been studied as a source of protein nitrogen in accordance with the formulation of a microbiological medium used for counting when growing colonies of microorganisms. The effectiveness of the prepared nutrient media was assessed by comparing the performance coefficients of the experimental and control nutrient media, as well as by identifying test microorganisms grown on the experimental medium. It was established that the growth pattern of test cultures both on the studied nutrient medium and on the control medium was almost the same. Test microorganisms retained their biochemical, morphological and cultural characteristics. The research results showed the possibility of using fish protein hydrolyzate obtained by enzymatic hydrolysis from secondary fish raw materials in microbiological nutrient media.

Application of enzyme preparations in the treatment of sawdust spent mushrooms substrate by Protaetia brevitarsis larvae

Application of enzyme preparations in the treatment of sawdust spent mushrooms substrate by Protaetia brevitarsis larvae

Mouse Wound Models and Preparation of Single-Cell Suspensions.

The management of acute full-thickness skin injuries presents a considerable challenge in clinical practice. The complexity of wound healing, involving diverse cell populations and the intricate wound microenvironment, complicates the assessment of treatment effects and their interactions using traditional experimental approaches. Single-cell transcriptome sequencing technology has revolutionized the understanding of cellular functions and mechanisms during wound healing. However, the variability in methodologies for creating mouse wound models introduces confounding factors that can impact experimental results. Furthermore, the process of dissociating mouse skin tissues presents significant technical hurdles, highlighting the critical need for high-quality single-cell suspensions for accurate transcriptome sequencing. Therefore, this study aims to optimize the construction of mouse wound models to accurately assess changes in wound closure while eliminating variables that could influence the subsequent sequencing result. Additionally, the preparation of single-cell suspensions was performed using both enzyme preparation protocols and test kit protocols to optimize cost and effectiveness.

Aroma enhancement of dealcoholized wines using enzyme treatment and glycosidic aroma precursors

This research has studied the treatment of dealcoholized wines with glycosidic enzymatic preparation and glycosidic aroma precursors extract isolated from aromatic grapes in order to enhance the aroma profile of non-alcoholic white wines. The effect of the addition of the precursors extract and the enzymatic treatment was studied after 15 and 30 d of storage, using dealcoholized wine as control. The analysis of free and bound volatile compounds was carried out by gas chromatography-mass spectrometry (GC-MS) after isolation using solid-phase extraction (SPE). The application of multivariate statistical analysis confirmed differences in the free and bound volatile profile of the studied samples when applicated the different treatments after 15 d and 30 d of storage. The combination of both treatments increased in a significant way the aromatic profile of Verdejo dealcoholized wine, showing high concentrations of terpenic and bencenic compounds due to the addition of precursors extract from an aromatic grape variety. This brings to light that this technique could be considered as a possible way of enhancing the aroma profile of dealcoholized wines in La Mancha region, as well as a way of using by-products from the wine industry, thus contributing to the concept of circular economy.

Enzymatic-Assisted Recovery of Antioxidants from Chicory and Fennel by-Products

AbstractThe work was aimed to evaluate the antioxidant extraction efficiency of enzymatic-assisted extractions, alone or combined with sonication, applied to chicory and fennel by-products. Three enzymatic preparations were tested: enzyme mix 1, made of pectinlyase, polygalacturonase, pectinesterase, and arabinase; enzyme mix 2, composed of pectinlyase, cellulase, and acid proteas; xylanase. They were applied alone or coupled with a successive sonication performed according to the following conditions: 100 g/L; 45 °C; 40 min; 72 W. The results were compared with those of a solid-liquid extraction performed with a 70% ethanolic solution whose conditions were previously optimized to maximize the extraction of antioxidants from the specific matrix (chicory: 60–180 min, antioxidant content 6.15 ± 0.20 mg/g dm; fennel: 60–90 min, antioxidant content 7.60 ± 0.70 mg/g dm). Concerning chicory by-products, the three enzymatic treatments coupled with ultrasounds allowed the obtainment of extracts having approximately the same amounts of antioxidants than the conventional extraction (6.10–6.22 mg/g dm). Regarding fennel, all the enzymatic-assisted extractions (coupled or not with ultrasounds) gave lower antioxidant concentrations than the hydroalcoholic extraction. Enzymatic extraction and sonication significantly influenced type and quantity of phenolics in the extracts, highlighted that compounds other than phenolics contributed to their antioxidant properties.