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Bacteria are an ideal source for producing pectin lyases (PNLs) due to their amenability to laboratory cultivation and genetic manipulation, which facilitates enhanced enzyme production. Predominantly originating from various thermophilic bacteria, bacterial PNLs usually exhibit alkaline properties, although cases of acidic variants have also been documented. In particular, a thermostable alkaline pectin lyase, displaying optimal activity at 60°C, has been characterized from the thermophilic bacterium Brevibacillus borstelensis P35. Similarly, thermostable acidic PNLs have been identified in Geobacillus stearothermophilus Ah22 and Bacillus subtilis SAV-21. Thermophilic bacterial species are emerging as significant and highly efficient sources, boasting diverse enzymatic repertoires, including pectinolytic enzymes, rendering them attractive candidates for various biotechnological applications. This mini-review focuses on the characterization of pectin lyases from a thermophilic bacterium, shedding light on its biochemical properties, substrate specificity, and potential industrial applications. Enzymes exhibit outstanding biochemical properties, with optimal pH and temperature ranges conducive to industrial processes, along with notable thermostability and pH tolerance, augmenting their suitability for diverse biotechnological endeavours. Furthermore, the enzyme demonstrates specificity towards pectin, efficiently cleaving glycosidic bonds within the polysaccharide backbone. Understanding the substrate specificity of pectin lyases is crucial for its effective utilization in industrial processes, especially considering its preferences for high-methoxylated pectin while still demonstrating activity on low-methoxylated and amidated pectins, expanding its applicability. Additionally, the synergy of pectin lyases with other pectinolytic enzymes enhances the efficiency of pectin degradation, facilitating the production of valuable products such as biofuels, dietary fibers

This research deals with the characterization of a thermostable endo-1,4-beta-xylanase enzyme from Anoxybacillus vranjensis ST4, a thermophilic bacterium isolated from Vranjska Banja hot spring, Serbia. The enzyme shows a high degree of identity with the same type of enzyme from other species of the genera Anoxybacillus (97%), Geobacillus (74%) and Paenibacillus (65%). The gene for endo-1,4-beta-xylanase from the thermophilic strain ST4 was cloned into the pQE_Ek expression vector and successfully expressed and purified from the Escherichia coli M15[pREP4]. The study encompasses recombinant production, purification, and the comprehensive characterization of the enzymatic properties of endo-1,4-beta-xylanase. This is the first successful overexpression, purification and characterization of a recombinant thermostable endo-1,4-beta-xylanase enzyme from Anoxybacillus. With a monomeric structure of 38.7 kDa, the enzyme demonstrates peak activity at 70°C and pH 6.5. Notably, it exhibits remarkable stability across a wide pH range and at high temperatures, rendering it suitable for diverse industrial applications. Investigation into the enzyme’s kinetic parameters, substrate specificity, and its ability to degrade xylan into high-energy value products further enhances understanding of its biotechnological potential. These findings underscore the significance of thermophilic bacteria and their thermostable enzymes in various industrial processes.

Recently, the exploration of agricultural by-products for biotechnological applications has gained substantial interest. This study addresses the multiple potential of corn agricultural waste, focusing on its dual utility: cellulase production and enhanced polyphenols extraction. Aspergillus welwitschiae FAW1 and Aspergillus tubingensis FAT36 strains, isolated from the environment, has proven to be good producers of the cellulase enzyme complex and at the same time non-toxigenic and safe for use in food production. Due to the fact that most of the fungal enzymes are inducible, the substrates used in this solid-state fermentation were agro-waste materials rich in cellulose and hemicellulose (corn silk and corn cob). The cellulase complex was produced by growing fungal strains at 28℃for 96 h. Through enzyme assisted extraction, the release of polyphenols from corn silk was enhanced. The obtained extracts, whether we used FAW1 or FAT36 strain`s cellulase enzyme complex showed an increase in total phenolic content, ranging from 876 mg/mL to 3050 mg/mL and 2944 mg/mL, respectively, which was determined by the Folin Ciocalteu method. Increasing the amount of extracted polyphenols led to the two to three times higher antioxidant activity of the extracts, according to ABTS and FRAP methods. This enzymatic approach offers a sustainable and eco-friendly alternative to conventional extraction methods, mitigating environmental impact while enhancing product purity. These findings not only contribute to the valorization of agricultural waste, but also emphasize the potential of corn agro-waste as a versatile resource for bioprocessing applications, connecting enzyme production, natural product extraction and added value products obtaining.

Recent technological development has focused on addressing two major global issues: environmental protection and conservation of natural resources. High levels of dye production and use have led to significant environmental concerns due to large quantities of unmanaged colored wastewater. Textile dyes, in particular, are highly reactive and problematic when released into the environment. This study investigates the use of Aspergillus fungi biomass, a byproduct of enzyme production (xylanase, cellulase, amylase, and other hydrolytic enzymes), for removing textile dyes from wastewater. The strains Aspergillus niger, Aspergillus oryzae, Aspergillus welwitschiae, and Aspergillus tubingensis were cultivated on corn meal under optimized conditions for enzyme production. The adsorption capacity of the biomass was tested on eight textile dyes: Bezaktiv Gelb, Procion reactive yellow, Reactive yellow, Golden yellow, Procion amber, Congo red, Reactive black 5, and Remazol brilliant blue. The best results were achieved with Aspergillus oryzae biomass, particularly for Reactive black 5. Optimal adsorption conditions identified by DoE were pH 4-5 and a temperature of 25°C. Within the first hour, approximately 90% of the dye (2.16 mg/g biomass) was adsorbed, and after 24 hours, over 99% was adsorbed (2.4 mg/g biomass). The findings demonstrate that post-enzyme production biomass, typically considered waste, can effectively remove reactive dyes and other xenobiotics from wastewater. This dual benefit reduces waste and mitigates water pollution, contributing significantly to environmental protection.

Dental pulp stem cells (DPSCs) are mesenchymal stem cells that are easy to obtain from any kind of discarded teeth and have the ability to differentiate into several cell types which makes them very popular in researches regarding regenerative dentistry. In recent years, there is an emerging trend of the use of nanomaterials in medicine and dentistry and they become very attractive tool for the treatment of bone tissue defects but also as a carriers of bioactive molecules. Before clinical application, thorough preclinical studies should be conducted regarding biocompatibility and biofunctionality of synthesized nanomaterials. Therefore, the aim of this study was to examine the influence of synthesized nanomaterial, biphasic calcium phosphate coated with poly-D,L-lactide-co-glycolide (CP/PLGA), on the proliferation of DPSCs during osteogenic differentiation in cell culture in vitro. We isolated the cells from the mature healthy teeth by outgrowth of the cells from undigested pulp pieces during culturing, in standard cell culture conditions. Cells were incubated with two concentrations of CP/PLGA nanoparticles in standard cell culture media and media for osteogenic differentiation. Cell proliferation rate was assessed using MTT and Crystal violet test after one, three and seven days of cell culturing. The results showed time- and concentration-dependent differences in DPSCs proliferation in the presence of nanomaterial. Differences in cell proliferation were noticed between osteogenic and standard cell culture media as well as in results obtained by performed tests. Increased cell proliferation was observed over time in both media but this effect was dependent on nanomaterial concentration. Calcium phosphate-based nanomaterials and DPSCs, based on observed cell materials interactions, could be a promising tool in bone tissue regeneration and further studies using in vitro as well as in vivo models should be conducted to fully unravel all of its potential.

Corn is widely used in the diet all over the world which generate corn cob as one of the most common agrowaste material. Corn cob mainly consists of lignocellulose with a high quantity of xylan; therefore, it can be considered the main source for xylooligosaccharides (XOS) production. XOS are prebiotic, functional food ingredients, which stand out on the market of oligosaccharide prebiotics because of their properties (antitumor effect, immunomodulatory effect, antioxidant power, and high pH and thermal stability). The European Commission has approved XOS produced from corn cob as safe for human consumption. For XOS production highly efficient xylanase obtained by Aspergillus tubingensis FAT35 under SSF on corn cob medium was used. Three methods of XOS production were evaluated: by hydrolysis of xylan alkali isolated from corn cob, by direct hydrolysis of xylan in corn cob and simultaneously production during fungal growth on corn cob (in situ). XOS were separated by ethanol precipitation from corn cob hydrolysate and from fermentation extract. In this way, the enzyme is concentrated in the precipitate, and XOS (remaining in the supernatant) are partially purified (separated from unhydrolyzed xylan and long chain oligomers). All prepared XOS were characterized by TLC and HPLC and antioxidant potential. Thanks to the high antioxidant potential (proven by DPPH, ABTS, FRAP, and ORAC tests) obtained XOS are good candidates for a functional food additive. Although the highest yield of XOS was obtained by the standard method (enzymatic hydrolysis of isolated xylan), additional methods represent the direct use of agro-waste material in an environmentally and economically suitable way. Special attention attracts in situ production because after isolation of XOS by ethanol precipitation the enzyme remains functional active and available for further use in next process.

The main goal of this study was to examine the efficiency of a newly isolated fungus from quince, Aspergillus tubingensis FAT43, to produce the pectinolytic complex using agricultural and industrial waste as the substrate for solid state fermentation. Sugar beet pulp was the most effective substrate inducer of pectinolytic complex synthesis out of all the waste residues examined. For endo-pectinolytic and total pectinolytic activity, respectively, statistical optimization using Placked-Burman Design and Optimal (Custom) Design increased production by 2.22 and 2.15-fold, respectively. Liquification, clarification, and an increase in the amount of reducing sugar in fruit juices (apple, banana, apricot, orange, and quince) processed with pectinolytic complex were identified. Enzymatic pre-treatment considerably increases yield (14%–22%) and clarification (90%). After enzymatic treatment, the best liquefaction was observed in orange juice, whereas the best clarification was obtained in apricot juice. Additionally, the pectinolytic treatment of apricot juice resulted in the highest increase in reducing sugar concentration (11%) compared to all other enzymatically treated juices. Optimizing the production of a highly active pectinolytic complex and its efficient utilization in the processing of fruit juices, including the generation of an increasing amount of waste, are the significant outcomes of this research.

Regenerative medicine (RM) exploits the innate potential of the human body to effectively repair and regenerate damaged tissues and organs with the help of various biomaterials. Tissue engineering (TE) makes it possible to replace damaged tissues and organs with new ones. Research in the field of biomaterials has significantly improved the area of RM and TE. Biomaterials are used as orthopedic, dental, cardiovascular implants, medical devices, in the fields of reconstructive and regenerative medicine, among others. Important preconditions for the biomaterial to be used for implantation are its biocompatibility and biofunctionality. Biomaterials should enable adhesion, migration, proliferation and differentiation of cells. The biological properties of biomaterials are a reflection of their physicochemical properties, such as internal architecture, surface characteristics and charge. Biomaterials used in tissue regeneration should mimic the natural structure of the extracellular matrix and represent a physiological microenvironment for normal cellular functions. These biomaterials should also have adequate biodegradability properties to facilitate the formation and growth of new tissue. Biomaterials for use in RM can be of natural or synthetic origin, polymers by structural properties, ceramic and composite type, and based on bioreactivity they can be bioinert or bioactive. In RM and TE, polymers of different classes, natural and synthetic, are used, which can be made as intelligent materials. The structure of hydrogels in the form of a porous network represents a good matrix for cell activity. Ceramic biomaterials based on hydroxyapatite (HAp) are selected for use in RM and TE, especially solid tissues. Properties, such as composition, particle size, material shape, porosity, surface charge, topography, etc., are relevant for the proper use of HAp materials. The properties of HAp allow modification of its structure, surface, particle size design at the micro and nano

Most of the functional oligosaccharides (OS) consist of monomers, present in varying degrees of polymerization (DP) ranging between 3 and 10 units. DP of inulin-type fructooligosaccharides has a great impact on fermentability and their utilization by probiotic bacteria such is Bifidobacteria, thus they have a great impact on their health-promoting effect. Technological properties of fructooligosaccharides (FOS) can improve the physicochemical and sensory characteristics of food products, leading to their increased application in the food industry. It has been found that microbial endoinulinase plays an important role in production of inulin-type fructooligosaccharides. Aspergillus welwitschiae FAW1 strain has proven to be non-toxigenic with the absence of biosynthetic gene clusters for mycotoxins (ochratoxins and fumonisins) and therefore safe for use in food production [5]. Growing on the natural substrate, triticale (Triticosecale sp) FAW1 strain produced inulinase complex from which endoinulinase (InuA) was purified by chromatographic techniques. FOS was prepared by time-controlled hydrolysis of inulin. Monitoring kinetics and determining the amount of obtained FOS by TLC and HPLC methods led to a conclusion that FOS production by hydrolysis of inulin is kinetic dependent reaction. Depending on the reaction time, FOS with different compositions are obtained.The largest amount of produced FOS (DP 2-6) has been in 15-20 minutes of the reaction, where the resulting mixture contains small amount of mono- and disaccharides. The obtained FOS were characterized on antioxidant capacity. Produced FOS showed significant antioxidant potential according to ORAC method which classifies them as potent candidate as additives in functional food. Endoinulinase (InuA) form A. welvitscihae FAW1 considered as key enzyme in FOS preparation. The composition and lenght of the produced FOS can be varied by controlling the reaction time, depending on the needs of of the market and their

Pectinases are a type of enzymes frequently used in the food industry to clarify, liquefy, and stabilize fruit juices. The main challenge in fruit juice production is the cloudiness of the juice, which is largely caused by the presence of pectic polysaccharides. Endo-pectinases are enzymes that hydrolyze the glycosidic bonds in pectic polymers. Commercial pectinolytic enzymes are typically produced by fungi, with Aspergillus spp. being the most commonly used. The aim of this research was the production and characterization of a novel endo-pectinase from the Aspergillus tubingensis strain for use in liquefying and clarifying different types of fruit juice. To accomplish this, solid-state fermentation was conducted on agricultural waste, such as sugar beet pulp and wheat bran, to produce pectinolytic enzymes. The resulting crude extract was concentrated via ultrafiltration and used to isolate the endo-pectinase via ammonium sulfate and ethanol precipitation methods. Ion-exchange chromatography technique on DEAE Sephadex A-25 matrix was used for further purification of the endo-pectinase. The purified enzyme was characterized by the determination of total pectinolytic activity, specific pectinolytic activity, and SDS-PAA gel electrophoresis. The activity of the endo-pectinase was confirmed by a diffusion test and zymography with Ruthenium Red visualization. The resulting enzyme was used to liquefy apricot, banana, apple, quince, strawberry, and orange pulp, with juice yields ranging from 71% to 83%, depending on the fruit used. The juices treated with endo-pectinase showed much higher clarification compared to untreated juices. Additionally, the treated juices demonstrated more pronounced antioxidant properties, as determined through the DPPH assay.

Xylan makes a significant part of cereals and fruits, which are used in the food industry. Therefore, enzymes that hydrolyze xylan (xylanases) have found application in the modification of cereal-based food, improving the digestibility of animal feed, and improving the texture of bakery products. In the juice industry, the main problems are turbidity, viscosity, and sedimentation during standing, which are caused by polysaccharides present in fruit (pectins, cellulose, and hemicellulose (xylan)). Pineapple, apple, orange, and tomato have a high content of hemicellulose, so xylanases are suitable for improving the properties of these juices. The Aspergillus tubingensis FAT 35 strain (considered safe for use in the food industry) growing on SSF medium composed of corn cob produced a high level of xylanase enzyme (4.03 U∕mL) and not that high pectinase (1.02 U∕mL) and cellulase (1.43 U∕mL) activities at pH 3 which is pH of freshly prepared apple, pineapple and organge juice.. The fermentation extract was used for clarification of pineapple, apple, and orange juice and for increasing the filtration rate and yield of these juices. Results indicate that A. tubigensis xylanase could be used for clarification and improvement of properties of juices of fruits that contain hemicellulose in high proportion.

Since each region of the world has different agricultural sectors, specific residues are generating and at the same time a wide range of alternative novel sources of nutrients emerge, such as carbohydrates, proteins and minerals. Usage of biomass residues as start material for prebiotics production is preferable from the standpoint of ecology and as cheap production process. Corn cob can be considered as the main source for xylooligosaccharides (XOS) production. XOS are prebiotic, functional food ingredients, with biological benefits such as immunomodulatory and anti-inflammatory properties, anticancer and antioxidant activity. The European Commission has approved XOS produced from corn cob as a safe for human consumption. The corn cob mainly consists of lignocellulose. Chemical treatment is used to extract the hemicelullose from corn cob which is then hydrolyzed to XOS by fungal xylanases. Xylanase produced by fungi genera Aspergillus and Trichoderma are considered key enzymes for XOS production. Fermentation enzyme extract of strain Aspergillus tubingensis FAT35, obtained after SSF on corn cob as substrate, was used for hydrolysis of corn cob xylan to XOS. A. tubigensis FAT35 produced high level of xylanase (350 U/mL). Obtained XOS (2-10 units) were characterized by TLC and by antioxidant activity (ORAC, DPPH, FRAP, and ABTS). Significant antioxidant potential was shown by all used antioxidant tests. The obtained XOS are suitable to be a functional food additive and are obtained from agro-waste material by environmentally and economically suitable way.

When textile dyes are released into the water, they can have a range of negative effects on the environment, for example, azo dyes can break down into carcinogenic compounds when exposed to sunlight. Remazol Brilliant Blue R (RBBR) and Reactive Black 5 (RB5) are reactive dyes. Unfortunately, the discharge of untreated effluents from these industries into nature can result in the contamination of water, which can have harmful effects on the environment. Saccharomyces cerevisiae is widely used in different fields of biotechnology. In this work, the cell walls of used yeast were reused for the biosorption of textile dyes, and the possibility of their utilization in wastewater treatment was investigated. The response surface methodology approach was applied to determine the optimal levels of three input variables - pH, temperature, and time, and also to identify the relationship between the response functions and process variables. Five levels of three variables were used, pH range 2–13, temperature range 15–40°C and time 0.15–30 h. The decolorization processes were optimized using the Central Composite Design. A significant impact of pH and temperature was found, while satisfactory results in decolorization (≥ 90%) were shown in a short time (up to 1 h). The maximum adsorption capacity (MAC) of the used Saccharomyces cerevisiae cell wall for removing RBBR and RB5 from wastewater was tested according to results obtained by CCD considering the economy of the process (pH 4, 25°C and 1 hour). Experimentally obtained MAC was 400 mg/g and 200 mg/g for RBBR and RB5, respectively.

Fish processing generates a large amount of waste products; nearly 75 % of the total fish weight consists of scales, skins, heads, viscera, and bones. These waste by-products are often dumped in landfills or the ocean, contributing to environmental pollution. Solid fish waste has a high collagen content and can be utilized for the extraction of different products such as gelatine, which can be used in different food and pharmaceutical industries. Fish gelatine contains all biogenic amino acids and can be used as a dietary supplement, thus increasing the utilization of fish waste and reducing environmental pollution. Recently, a strong worldwide focus has been directed towards the discovery of new microbial proteases for industrial applications. For that purpose, the Bacillus sp. strains from the laboratory bank were screened for the ability to secrete alkaline proteases. Bacterial strains that produced a larger clear zone on the gelatine agar plate were selected for further study. Selected bacterial strains were cultivated under submerged conditions for the purpose of producing proteases. The supernatant from each isolate was used as a crude enzyme extract for testing gelatine proteolytic activity and hydrolysis products. Bacillus sp. strains 12B, 16A, 17B and 24B showed a high degree of hydrolysis of the gelatine waste and the ability to form proteins of small molecular masses after one hour of hydrolysis. Of these, sp. 12B was the most potent, having twice the proteolytic activity determined by the TNBS method.