Recycling of agro waste by fungi for obtaining enzymes and prebiotics

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 waste in only one process

Amylases in the midgut of long – horned beetle (Morimus funereus) and great capricorn beetle (Cerambyx cerdo) larvae

U оvоm rаdu su ispitivаni: gајеnjе lаrvi i dоbiјаnjе rеprоduktivnо spоsоbnih јеdinki bukоvе strižibubе (Morimus funereus) u lаbоrаtоriјskim uslоvimа; аnаlizirаnjе аmilаznе аktivnоsti i izоаmilаznih prоfilа lаrvi bukоvе i vеlikе hrаstоvе strižibubе (Cerambyx cerdo) u zаvisnоsti оd hrаnjivih supstrаtа i uslоvа srеdinе; prеčišćаvаnjе i biоhеmiјskа kаrаktеrizаciја glаvnе izоfоrmе аmilаzе lаrvе bukоvе strižibubе i prеčišćаvаnjе glаvnе izоfоrmе аmilаzе lаrvi vеlikе hrаstоvе strižibubе. Оpisаnо је lаrvеnо rаzvićе bukоvе strižibubе u lаbоrаtоriјskim uslоvimа оd јаја dо rеprоduktivnо spоsоbnih јеdinki krоz dvе gеnеrаciје. Pоkаzаnо је dа sе gајеnjеm lаrvi u lаbоrаtоriјskim uslоvimа i stаlnој dоstupnоsti hrаnе skrаćuје lаrvеnо rаzvićе, i dа sе u drugој gеnеrаciјi uоčаvа vеćа uјеdnаčеnоst lаrvi pо svim pаrаmеtrimа kојimа sе оpisuје lаrvеnоrаzvićе. Nаđеnо је dа pоstоје rаzlikе u аmilаznој аktivnоsti i izоаmilаznim prоfilimа kоd lаrvi bukоvе i vеlikе hrаstоvе strižibubе u zаvisnоsti оd hrаnjivоg supstrаtа i uslоvа srеdinе gdе sе rаzviјајu, čimе је pоtvrđеnа njihоvа pоlifаgnоst i vеlikа аdаptаbilnоst. Glаvnа α-аmilаznа izоfоrmа lаrvе bukоvе strižibubе је prеčišćеnа dо hоmоgеnоsti 112 putа sа prinоsоm оd 15% i biоhеmiјski је оkаrаktеrisаnа. Оdrеđеnа јој је mоlеkulskа mаsа 33 kDа, pI vrеdnоst – 3,2, pH оptimum – 5,2 i tеmpеrаturni оptimum – 45°C. Аktivnоst α- аmilаzе lаrvе bukоvе strižibubе zаvisi оd јоnа Cа2+. Inhibirајu је inhibitоri iz pšеnicе. Pоkаzuје аktivnоst prеmа sirоvоm skrоbu. Glаvnа α-аmilаzа lаrvе vеlikе hrаstоvе strižibubе је dеlimičnо prеčišćеnа iz sirоvоg еkstrаktа, i nа izоеnzimskоm nivоu kоrišćеnjеm dvе vrstе FPLC hrоmаtоgrаfiје. Моlеkulskа mаsа јој је 34 kDа а pI mаnjа оd 3,5.This paper examined: larvae growing and getting reproductive age individuals of long – horned beetle (Morimus funereus) in laboratory conditions, analyzing the amylase activity and isoamylase profiles of M. funereus larvae and great capricorn beetle (Cerambyx cerdo) larvae depending on the nutrient substrate and environmental conditions, purification and biochemical characterization of the major amylase isoform of M. funereus larvae and purification of the major amylase isoform of C. cerdo larvae. Larval develop of M. funerus larvae in the laboratory from eggs to reproductively capable individuals in two generations were described. It is shown that growing growing larvae in laboratory conditions and constant availability of food larval development was reduced, and that in the second generation higher uniformity of larvae in all of the parameters that describe the larval development was noticed. It was found that there are differences in amylase activities and isoamylase profiles of M. funerus and C. cerdo larvae depending on the substrate and nutrient conditions in the environment where they develop, thus confirming their polyphagy and high adaptability. The main α-amylase isoform of M. funereus larvae was purified to homogeneity 112 times with a yield of 15% and was biochemically characterized. Its molecular weight is determined to be 33 kDa, pI value - 3.2, optimum pH - 5.2 and optimum temperature - 45 ° C. α-Amylase activity of M. funureus larvae is depended on concentration of Ca2 + ions. Inhibitors from wheat inhibit the M. funereus amylase. Midgut amylase of M. funereus larvae shows activity towards raw starch. The main α-amylase of C. cerdo larvae was partially purified from crude extract, and the isoenzyme pattern level using two types of FPLC chromatography. Molecular mass of midgut α-amylase of C. cerdo larvae was 34 kDa and pI was less than 3.5

Highly efficient production of Aspergillus niger amylase cocktail by solid-state fermentation using triticale grains as a well-balanced substrate

Triticale (x Triticosecale, Wittmack), an important industrial crop, with high grain yield, containing high amounts of starch, proteins and also major and minor mineral elements, is not yet sufficiently utilized. The simultaneous production of alpha-amylase and glucoamylase isoforms by Aspergillus niger on triticale grains, without any nutritive supplements, was developed, optimized and scaled up 10 fold for the first time. The specific combination of the examined effects led to the production of a novel glucoamylase isoform. Reduction of particle size, increase in oxygen availability and substrate height lead to an increase of 30 % in the production of amylases. Reduction of the relative humidity from 65 to 30 % increased glucoamylase production 2 fold and alpha-amylase production by 30%. The peak production of alpha-amylase (158 U g(-1)) and glucoamylase (170 U g(-1)) were obtained in Erlenmeyer flasks and in scaled-up trays. The obtained A. niger amylase cocktail was more efficient in raw starch hydrolysis from wheat flour, 29 % more efficient in glucose formation and 10 % more efficient in total reducing sugar formation, than the commercially available amylase cocktail SAN Super 240L, which is widely used in industry

Karakterizacija endopeptidaza srednjeg creva larvi Morimus funereus (Coleoptera: Cerambycidae)

Application of specific chromogenic substrates, use of class-specific inhibitors, and zymogram analysis enabled us to identify several peptidase classes in extracts of the midgut of Morimus funereus larvae. Zymogram analysis with gelatin as a peptidase substrate and phenylmethylsulfonyl fluoride as an inhibitor showed that serine peptidases were the most abundant endopeptidases in the midgut of M. funereus larvae. By zymogram analysis with gelatin as a peptidase substrate and 1,10-phenanthroline as an inhibitor, metallopeptidases were also detected. Analyses of serine peptidases with specific chromogenic substrates revealed dominance of elastase-like peptidases in extracts of the midgut of M. funereus larvae, with less pronounced chymotrypsin- and trypsin-like activities.Primenom specifičnih hromogenih supstrata, klasno-specifičnih inhibitora i zimogramske analize identifikovano je nekoliko klasa peptidaza u sirovom ekstraktu srednjeg creva larvi koleoptere Morimus funereus. Zimogramskom analizom sa želatinom kao supstratom i fenilmetilsulfonil-fluoridom kao inhibitorom utvrđeno je da su serin-peptidaze najzastupljenije peptidaze u ekstraktu srednjeg creva larvi M. funereus. Zimogramskom analizom sa želatinom kao supstratom i 1,10-fenantrolinom kaoinhibitorom takođe su detektovane metalopeptidaze. Analizom serin-peptidaza, upotrebom specifičnih hromogenih supstrata, dokazano je da su dominantni elastazi-slični enzimi u sirovom ekstraktu srednjeg creva larvi M. funereus, dok su himotripsinima- i tripsinima-slični enzimi manje zastupljeni

Добијање производа додатне вредности од отпадне биомасе

Енергетска транзиција подразумева примену правилног управљања отпадним биомасама које обухвата њено смањење, поновну употребу, рециклирање, добијање енергије и/или одлагање. Наука нуди решења за ефикасну примену поновне употребе и рециклирања, где се добијају производи додатне вредности. У Србији, према проценама, у врстама отпадних биомаса доминирају жетвени остаци (10.140.268 t годишње) и остаци резидбе воћа (74.329 t годишње) а следе стајњак, отпадни материјал из прерадне индустрије и биоразградиви комунални отпад. Биопроцесовање (употреба микроба и ензима) представља најприхватљивије решење заштите животне средине, где се из отпадне биомасе добијају вредни производи (пребиотици, биоактивни пептиди, антибиотици, терпеноиди, алкалоиди, итд.) широке примене у храни, лековима и козметици. Из отпадног клипа кукуруза (кога у Србији има 1.073.780 t годишње) добијају се пребиотици, одобрени за употребу у исхрани. Искоришћени компост за гајење шампињона је погодан супстрат за микробиолошко добијање индустријски значајних ензима (амилазе, целулазе, ксиланазе). Постоје примери индустрије где се из дрвних остатака (којих има у Србији 700.000 m3 годишње) поред етанола добијају и ацеталдехид, сирћетна киселина и етилацетат. Екстракцијом из остатака индустрије прераде воћа, поврћа и винове лозе се могу добити полифеноли (антиоксиданси) и други вредни производи. Максимална искористљивост отпадне биомасе може се постићи комбиновањем метода биопроцесовања за добијање производа додатне вредности са добијањем енергије (биогаса, биоетанола).Predavanje po pozivu / Invited lectur

Enhancement of amylase production by Aspergillus sp using carbohydrates mixtures from triticale

With the purpose of finding a suitable available inducer in combination with starvation, carbohydrate mixtures from triticale were used and compared with well-known amylase inducers in fungi. Carbohydrate mixtures from triticale induced the production of an amylase cocktail (alpha-amylase and glucoamylase) in Aspergillus niger, unlike induction with well-known inducers that induce only glucoamylase, shown by zymography and TLC analysis of the carbohydrate mixtures before and after fermentation. Glucoamylase production by A. niger was the highest in the presence of the extract obtained after auto-hydrolysis of starch from triticale (95.88 U mL(-1)). Carbohydrate mixtures from triticale induced the production of alpha-amylase in A. oryzae. More alpha-amylase isoforms were detected when using a complex carbohydrate mixture, compared to induction with maltose or starch. A 48-h induction was the most efficient using a triticale extract (101.35 U mL(-1)). Carbohydrates from triticale extracts could be used as very good cheap amylase inducers. Triticale, still not fully utilized, could be taken into consideration as an inducer in amylase production by Aspergillus sp, and in such a way, it could be used as the sole substrate in fermentation

Uporedna analiza izoformi α-amilaze iz srednjeg creva larvi Cerambyx cerdo L. (Coleoptera: Cerambycidae) iz prirode i gajenih na veštačkoj podlozi

alpha-Amylase isoforms of Cerambyx cerdo larvae from the wild (ML and SL) and reared in the laboratory (ADL) were compared. Three amylase isoforms were presented in the SL and ML extracts while two isoforms were presented in the ADL according to zymogram after isoelectric focusing (IEF). All C. cerdo amylase isoforms were acidic proteins (pI lt 3.5). Seven amylase isoforms (ACC 1-7) from the midgut of C. cerdo larvae were found in the ML midgut extract, six in the SL extract, and four in the ADL extract according to native PAGE zymogram. The ADL amylase had the highest activity. All crude midgut extracts of C. cerdo larvae were fractionated on a Superose 12 HR column. The molecular mass of the ACC was estimated to be 34 kDa.Upoređene su izoforme α-amilaze larvi Cerambyx cerdo sakupljenih iz prirode (ML i SL) i gajenih na veštačkoj podlozi u laboratoriji (ADL). Zimogramskom detekcijom posle IEF-a po tri izoforme su detektovane u ML i SL ekstraktima, a u ADL dve izoforme. Sve amilazne izoforme iz C. cerdo su bile kisele (pI lt 3.5). Zimogramskom detekcijom posle nativne elektroforeze sedam izoformi je detektovano u ML ekstraktu, šest u SL ekstraktu i četiri u ADL ekstraktu. Najveća amilazna aktivnost je detektovana u ADL ekstraktu. Svi ekstrakti srednjih creva larvi C. cerdo su frakcionisani na koloni Superose 12 HR. Molekulska masa ACC-a je bila 34 kDa

Influence of nutrient substrates on the expression of cellulases in Cerambyx cerdo L. (Coleoptera: Cerambycidae) larvae

The expression and distribution of digestive cellulases along the midgut of Cerambyx cerdo larvae were analyzed for the first time and are presented in this article. Four groups of larvae were examined: larvae developed in the wild; larvae taken from the wild and successively reared on an artificial diet based on polenta; and larvae hatched in the laboratory and reared on two different artificial diets. Seven endocellulase and seven β-D-glucosidase isoforms were detected in all midgut extracts of C. cerdo with a zymogram after native PAGE. We observed that C. cerdo larvae are capable of producing cellulase isoforms with different PAGE mobilities depending on the nutrient substrate. From our findings it can be assumed that, depending on the distribution of endocellulase and β-D-glucosidase, cellulose molecules are first fragmented in the anterior and middle midgut by endo-β-1,4-glucanase; subsequently, the obtained fragments are broken down by β-D-glucosidase mostly in middle midgut

Comparison of α-amylase isoforms from the midgut of Cerambyx cerdo L. (Coleoptera: Cerambycidae) larvae developed in the wild and on an artificial diet

α-Amylase isoforms of Cerambyx cerdo larvae from the wild (ML and SL) and reared in the laboratory (ADL) were compared. Three amylase isoforms were presented in the SL and ML extracts while two isoforms were presented in the ADL according to zymogram after isoelectric focusing (IEF). All C. cerdo amylase isoforms were acidic proteins (pI < 3.5). Seven amylase isoforms (ACC 1-7) from the midgut of C. cerdo larvae were found in the ML midgut extract, six in the SL extract, and four in the ADL extract according to native PAGE zymogram. The ADL amylase had the highest activity. All crude midgut extracts of C. cerdo larvae were fractionated on a Superose 12 HR column. The molecular mass of the ACC was estimated to be 34 kDa.Upoređene su izoforme α-amilaze larvi Cerambyx cerdo sakupljenih iz prirode (ML i SL) i gajenih na veštačkoj podlozi u laboratoriji (ADL). Zimogramskom detekcijom posle IEF-a po tri izoforme su detektovane u ML i SL ekstraktima, a u ADL dve izoforme. Sve amilazne izoforme iz C. cerdo su bile kisele (pI < 3.5). Zimogramskom detekcijom posle nativne elektroforeze sedam izoformi je detektovano u ML ekstraktu, šest u SL ekstraktu i četiri u ADL ekstraktu. Najveća amilazna aktivnost je detektovana u ADL ekstraktu. Svi ekstrakti srednjih creva larvi C. cerdo su frakcionisani na koloni Superose 12 HR. Molekulska masa ACC-a je bila 34 kDa.Projekat ministarstva br. 142026

Selection of Non-Mycotoxigenic Inulinase Producers in the Group of Black Aspergilli for Use in Food Processing

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 biosynthetic gene clusters, respectively. The two best inulinase producers and mycotoxin non-producers (without mycotoxin production capability as additional safety) were selected as potential candidates for further development of enzyme production. Novelty and scientific contribution. The presented innovative approach for the selection of potential fungal enzyme producer shows that only non-toxigenic fungi could be considered as useful in food industry. Although this study was done on local isolates, the approach is applicable globally