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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.

Clays are naturally occurring, environmentally friendly, chemically inert, thermostable, inexpensive resources that are easily modified into materials with tailored properties. As such, they can be used as suitable supports for enzyme immobilization and application in the food industry. Natural polysaccharides starch, xylan, pullulan, and its derivatives obtained by the action of enzymes, have numerous potentials for food industrial applications. In this work the enzyme supports were prepared from bentonite from Coal mine "Bogovina", Serbia by acid activation (AA), pillaring (P), and pillaring followed by acid activation (PAA). The characterization of the obtained materials included chemical and phase composition, surface acidity, and textural properties. After characterization, -amylase from Bacillus paralicheniformis (BliAmy), commercial xylanase from Sigma-Aldrich (Xyl), and pullulanase from B. paralicheniformis (BliPull) were immobilized on bentonite based supports by 24 h adsorption at 25 °C. The obtained biocatalysts BliAmy-AA (106 IU/g), Xyl-P (74 IU/g), and BliPull-PAA (45 IU/g) showed very good storage stability with the activity preserved after 4 weeks of testing. Products of hydrolysis were detected by TLC and indicate a promising application in the food industry.

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.

Given the growing concern about the depletion of fossil fuels, global warming, and the loss of natural resources, bioethanol made from sugar cane, molasses, and corn continues to garner interest globally and is regarded as the safest and cleanest alternative to oil. Starch is a widely available renewable carbohydrate from which bioethanol is conventionally obtained through energy demanding liquefaction and saccharification processes. A significant simplification of the process and a reduction of starch processing costs would be possible by applying raw starch hydrolysis using enzymes capable of degrading starch below the gelatinization temperature. A novel strategy for highly concentrated raw corn starch (30 % w/v) hydrolysis based on a modified simultaneous saccharification and fermentation process is optimized for the production of bioethanol. Different ratios of Bacillus paralicheniformis ATCC 9945a (BliAmy) and glucoamylase (Dextrozyme® GA), glucoamylase addition time, incubation time, and pH were investigated using a Box–Behnken experimental design to ensure high process efficiency. A two-step synergistic hydrolysis and fermentation with Saccharomyces cerevisiae at 30 °C was carried out in a single bioreactor vessel at the same pH (4.5). The obtained bioethanol concentration at 129.2 g/L, with a productivity of 2.94 g/L/h and ethanol yield (YP/S) at 0.50 g EtOH/g total sugar, equivalent to 87.8 % theoretical yield indicates the viability of the proposed innovative process.

Finding a pollen substitute for honey bees that is nutritionally adequate and affordable is a scientific and practical challenge. We attempted a new rational approach and tried to exploit honey bees’ natural cannibalistic behavior. We tested processed insect larvae as a food source that is nutritionally similar to bee brood, and which can easily be produced on a large scale. In cage experiments, monitoring bee mortality, food consumption and changes in bee body parts’ weights showed that flour obtained by grinding dried yellow mealworm larvae has the potential to become an excellent component for pollen substitution. Bees from the cage group fed Tenebrio molitor patties (TG) demonstrated overall best results in comparison to sugar patties fed bee group (CG), yeast patties fed bee group (YG) and pollen patties fed bee group (PG). They did not lose weight as rapidly as the CG, did not defecate inside cages as the YG, nor show increased mortality as the PG. At the same time, TG consumed less food (mean 13.7 g/cage) than CG (16.8 g/cage), YG (20.4 g/cage) and PG (23.9 g/cage) within the period of 28 days. Bees’ gut increase in weight was lowest in the CG, followed by TG and PG and was the highest in the YG which resulted in diarrhea after 14 days. Bees from TG did not lag behind other bees in head, thorax and abdomen weight after 28 days. We demonstrated that processed yellow mealworm larvae (T. molitor) can be used as an ingredient for honey bee feed.

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

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.

Some coal mines suffer from problem of having huge deposits of bentonite that they regard as undesirable, since bentonite can cause problem due to its swelling property. Instead of piling stacks of bentonite and regarding it as ecological problem the bentonite can be used as enzyme support by immobilization of enzymes as the challenging new application in the field of biotechnology. The enzyme catalysts in the industrial application present lower energy costs and are more environmentally friendly in comparison to traditional chemical processes. The advantages of enzymes are even more prominent when enzymes are applied in immobilized form. In this work the enzyme supports were prepared from bentonite from Coal mine "Bogovina", Serbia where bentonite, although valuable resource, is still considered to be tailings. Bentonite was modified by common methods: acid activation (AA), pillaring (P), and pillaring followed by acid activation (PAA) and tested as enzyme support. All the obtained materials were characterized by the X-ray powder diffraction and FTIR spectroscopy. The amylase, xylanase, and pullulanase were immobilized on different bentonite based supports by 24 h adsorption at 25 °C. The experimental results revealed that under the investigated conditions AA, P, and PAA, were the most suitable for amylase (106 IU/g), xylanase (74 IU/g), and pullulanase (45 IU/g) immobilization, respectively.

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.

Finding a pollen substitute for honey bees that is nutritionally adequate and affordable is a scientific and practical challenge. We attempted a new rational approach and tried to exploit honey bees’ natural cannibalistic behavior. We tested processed insect larvae as a food source that is nutritionally similar to bee brood, and which can easily be produced on a large scale. In cage experiments, monitoring bee mortality, food consumption and changes in bee body parts’ weights showed that flour obtained by grinding dried yellow mealworm larvae has the potential to become an excellent component for pollen substitution. Bees from the cage group fed Tenebrio molitor patties (TG) demonstrated overall best results in comparison to sugar patties fed bee group (CG), yeast patties fed bee group (YG) and pollen patties fed bee group (PG). They did not lose weight as rapidly as the CG, did not defecate inside cages as the YG, nor show increased mortality as the PG. At the same time, TG consumed less food (mean 13.7 g/cage) than CG (16.8 g/cage), YG (20.4 g/cage) and PG (23.9 g/cage) within the period of 28 days. Bees’ gut increase in weight was lowest in the CG, followed by TG and PG and was the highest in the YG which resulted in diarrhea after 14 days. Bees from TG did not lag behind other bees in head, thorax and abdomen weight after 28 days. We demonstrated that processed yellow mealworm larvae (T. molitor) can be used as an ingredient for honey bee feed.

Feeding of honey bee (Apis mellifera) is a challenge for beekeepers and formulation of food supplements is improved continuously. When natural food sources are scarce or not available, supplemental foods are widely used to support and build up honey bee colonies. Influence of commercial (sugar) patty and the patties enriched with 12.5% pollen and 12.5% dried yeast on protease expression in honey bee adults is presented. This is part of a wider study aimed to compare the expression of digestive enzymes using different patties. Data collected in this study can be useful for development of higher quality of food supplements for honey bees. Honey bees were kept in an incubator for 21 days, at a temperature of 35°C and at 80% humidity. In each cage there were one hundred bees and a piece of honeycomb. Midgut samples were taken after 7, 14 and 21 days, homogenized and analyzed for protein concentration and protease activity. The highest protein concentration was detected in the midgut of pollen fed bees, and lowest in commercial patty fed group, determined by the Bradford method. Protease activity was the highest in bee’s midgut fed with pollen patties, and the lowest in bees fed commercial patties which is shown by enzyme assay and by zymograms. There were different protease isoforms present in bees fed pollen, yeast and commercial patties, but the major isoforms were the same. The observed decrease in protease activity over time in all groups is probably due to the transition to foraging tasks. Reduced protease activity in the gut of bees fed commercial patties is due to a lack of proteins or some other inducers present in food. Because pollen is honey bee natural food, we conclude that food supplements that induce similar enzyme expression as pollen (dried yeast) can be considered as appropriate food substitution, because it is superior to supplements which induce very different enzyme expression.

Agro waste materials are agricultural residues (steam, stalks, husks...) and food industrial residues (potato, orange, apple and etc peels and soyabean, coconut etc cakes). 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 sorrces of nutrients emerge, such as carbohydrates, proteins and minerals. Proper waste biomass management is one of priority in EU and in world. Filamentous fungi are most potent microorganisms for bioprocessing of waste materials in purpose to obtain value added products such as enzymes, biofuel, bioactive compounds, prebiotics, chemicals, antibiotics... Genus Aspergillus and Trichoderma are well studied and recognized as potent enzyme producers. Enzyme produced by filamentous fungi such as cellulase, amylase, xylanase, glucosidase, are widely used in industry. Environmentfriendly and cost effect solution for their obtaining is utilization of agro waste material as solid substrate for fungal growth. Prebiotics are oligosaccharides with 2-6 units defined as "a substrate that is selectively utilized by host microorganisms conferring a health benefit". Epidemiologic studies have significantly recognized prebiotics as an essential constituent of a healthy diet. Prebiotics, e.g. FOS are prepared by effective fungal enzymes. Usage of biomass residues as start material for prebiotics production is preferable from the standpoint of ecology and as cheap production process. There are number of agro waste materials that could be used for this propose. Corn cob can be considered as the main source for XOS production. Fungi are saprophytic eukaryotic organisms, habitat lignocellulosic material in nature and they are capable to induce enzymes depending on growth substrate, which actually enables their usage for in situ prebiotics production, which is new and trending research in this field. This approach unites eu4tme and prebiotics obtaining from agro was

Fungal inulinases have wide application in industrial biotechnology, and it is presumed that their expression is regulated at the transcriptional level via promoter. It is also known that different sugars have an inducing effect on gene expression in fungal genome, including inulinases. Aim of this work was to determine which of the sugars used in growth medium, as the only carbon source, induce the extracellular exoinulinase gene inuE expression in Aspergillus welwitschiae FAW1. Inulin, rafinose, sucrose, glucose and fructose were used as carbon sources, and expression of inuE was monitored during 72 h of cultivation (tested after 24, 36, 48 and 72 h). Both, presence of mRNA in the mycelia and extracellular enzyme activity in the growth media were monitored. Interestingly, obtained results showed that inuE was induced by fructose, sucrose and rafinose and not by inulin. In all cases, the highest mRNA was detected after 24 h of cultivation, while extracellular exoinulinase activity increased from 24 h with a peak in 72 h. Further experiments are necessary for a comprehensive understanding of the regulation mechanisms of AweinuE promoter for its more purposeful application in biotechnology.

Research background. Inulinases are used for fructooligosaccharide production and they are of interest for both scientific community and industry. Black aspergilli represent a diverse group of species that has use for enzyme production, in particular some species are known as potent inulinase producers. Finding new potential producers from the environment is as important as improving the production with known strains. Safe use of enzymes produced by aspergilli in food industry is placed ahead of their benefit for inulinase production. Experimental approach. Here we show a specific approach to finding/screening of newly isolated fungal inulinase producers that combines a newly developed screening method and an equally important assessment of the toxigenic potential of the fungus. In this study 39 black aspergilli collected from different substrates in Serbia were identified and assessed for inulinase production. Results and conclusions. The most common species were Aspergillus tubingensis (51.2 %), followed by A. niger (23.1 %), A. welwitschiae (23.1 %) and A. uvarum (2.6 %). The isolates for inulinase production were selected using a cheap and easy, fast and non-hazardous alternative inulinase screening test developed in this work. Enzymatic activity of selected inulinase-producing strains was confirmed spectrophotometrically. Since some A. niger and A. welwitschiae strains are able to produce mycotoxins ochratoxin A (OTA) and fumonisins (FB), the toxigenic potential of selected inulinase producers was assessed analytically and genetically. Fungal enzyme producer can be considered safe for use in food industry only after comparing the results of both approaches for investigating toxic potential, the direct presence of mycotoxins in the enzyme preparation (analytically) and the presence of mycotoxin gene clusters (genetically). In some strains the absence of OTA and FB production capability was molecularly confirmed by the absence of complete or critical parts of bio

There is an urgent need to increase the daily intake of insoluble dietary fiber, and at the same time to find new sources and new production technologies. We hypothesized that fungal enzymes directly involved in lignocellulosic material hydrolysis (Aspergillus and Trichoderma enzyme cocktails) will change the fiber structure particularly efficiently after the action of laccase (Trametes versicolor enzyme cocktail). Enzymes production on an inducing substrate (same as starting material for obtainment of insoluble dietary fibers) and their usage resulted in obtainment of novel insoluble dietary fibers with better characteristics, 24% higher swelling, 43% higher WRC and 57% higher ORC compared to insoluble dietary fibers from triticale (already proven to be a good food additive). Changes in structure were analyzed by FTIR and microscopic analysis. Antioxidative performance of the obtained products, new insoluble and released soluble dietary fibers, was analyzed in detail. Newly obtained soluble dietary fibers demonstrated up to 20 times higher antioxidant activity compared to untreated fibers (ABTS and DPPH tests). These results suggest their good performance as a future food additive. At the same time, they prove the hypothesis that the use of enzyme cocktails rich in laccase is a good choice for biological pretreatment in this process.

Duckweed (L. minor) is a cosmopolitan aquatic plant of simplified morphology and rapid vegetative reproduction. In this study, an H. paralvei bacterial strain and its influence on the antioxidative response of the duckweeds to phenol, a recalcitrant environmental pollutant, were investigated. Sterile duckweed cultures were inoculated with H. paralvei in vitro and cultivated in the presence or absence of phenol (500 mg L−1), in order to investigate bacterial effects on plant oxidative stress during 5 days. Total soluble proteins, guaiacol peroxidase expression, concentration of hydrogen peroxide and malondialdehyde as well as the total ascorbic acid of the plants were monitored. Moreover, bacterial production of indole-3-acetic acid (IAA) was measured in order to investigate H. paralvei’s influence on plant growth. In general, the addition of phenol elevated all biochemical parameters in L. minor except AsA and total soluble proteins. Phenol as well as bacteria influenced the expression of guaiacol peroxidase. Different isoforms were associated with phenol compared to isoforms expressed in phenol-free medium. Considering that duckweeds showed increased antioxidative parameters in the presence of phenol, it can be assumed that the measured parameters might be involved in the plant’s defense system. H. paralvei is an IAA producer and its presence in the rhizosphere of duckweeds decreased the oxidative stress of the plants, which can be taken as evidence that this bacterial strain acts protectively on the plants during phenol exposure.

FTIR spectra of IDFT treatment with fungal enzymatic complex.

Fortification of fermented dairy products with insoluble dietary fiber is an interesting way to increase consumers' fiber intake. The objective of this study was to evaluate the sensory characteristics and consumer acceptance of low-fat unsweetened yoghurt, fortified at levels of 15 and 30 g/kg, with insoluble triticale, wheat or oat fibers. The addition of insoluble triticale fiber resulted in yellowish-brown color, grainy flavor, and pronounced sandiness/grittiness of the fortified yoghurts. The products were classified into the 'very good' quality category, despite the lower quality scores given to the 30 g/kg fiber fortified yoghurts, caused primarily by a gritty/sandy texture and some bitterness. Three distinct consumer subgroups were revealed by the clustering analysis, one of which showed a preference for the triticale-yoghurts. Insoluble dietary fiber from triticale showed promising potential to be used as a fortifying ingredient in the production of fiber-enriched fermented dairy products such as yoghurt.