Utjecaj miješanja na fermentaciju otpada nastalog pri proizvodnji kave s pomoću Aspergillus tamarii

Solid-state fermentation of coffee pulp with Aspergillus tamarii V12307 was carried out in laboratory scale reactors (bottle and column) to evaluate the effect of four different mixing frequencies (2.0, 2.7, 4.0 and 8.0 day^–1) on fungal growth, indirectly determined by carbon dioxide formation and the production of spores and pectin methylesterase. Coffee pulp was used as the sole source of nutrients. An increase in the fraction of bonded particles was observed in the bottle reactors after 12 h of cultivation when no mixing was applied. The use of any mixing frequency reduced the fraction of bonded particles. However, there was no significant difference in pectin methylesterase production between the mixing frequencies at the end of the fermentation. Similarly, there were no significant differences in CO2 production, oxygen uptake or sporulation, demonstrating that the mycelium was not damaged by intermittent mixing. This strategy of mixing could be used in large scale reactors in order to reduce heat and mass limitations.Fermentacija otpada nastalog pri proizvodnji kave s pomoću plijesni Aspergillus tamarii V12307 provedena je u laboratorijskim reaktorima (bocama i kolonama), te je ispitan utjecaj četiriju frekvencija miješanja (2,0; 2,7; 4,0 i 8,0 dan-1) na rast plijesni, i to indirektno, tj. mjerenjem proizvodnje ugljičnog dioksida, spora i pektin metilesteraze. Otpad nastao pri proizvodnji kave upotrijebljen je kao jedini izvor hranjiva. Povećanje udjela vezanih čestica primijećeno je nakon 12 sati uzgoja u bocama bez miješanja. Miješanje je smanjilo udjel vezanih čestica, ali nije bitno utjecalo na proizvodnju pektin metilesteraze. Također nije utjecalo na proizvodnju CO2, potrošnju kisika ili sporulaciju, što znači da povremeno miješanje supstrata nije oštetilo micelij plijesni. Takvim se miješanjem mogu smanjiti ograničenja u prijenosu topline i mase u industrijskim reaktorima

Why Solid-State Fermentation Seems to be Resistant to Catabolite Repression?

U ovom radu je dan kritički pregled fenomena otpornosti sinteze enzima na kataboličku represiju pri rastu na čvrstoj podlozi (SSF). U radu se raspravlja o praktičnoj i teorijskoj važnosti tog fenomena. Iznose se ideje koje bi ga objasnile, poput moguće prisutnosti mikroskopskih gradijenata u masi staničnih agregata, ili promjene propusnosti stanica za šećere, koje bi objasnile činjenicu da prisutnost 100 g šećera /L u čvrstoj podlozi tijekom rasta sprječava pojavu kataboličke represije, unatoč tome što u konvencionalnom submerznom uzgoju pri koncentraciji šećera većoj od 10 g/L nastaje jaka inhibicija inducibilnih enzima. Predstavljena su dva alternativna matematička modela za objašnjenje provedivosti tih hipoteza, ali i za planiranje budućih pokusa kako bi se objasnila mikroskopska fiziologija rasta na čvrstoj podlozi. Obje hipoteze objašnjavaju fenomen, ali samo ako se lokalna difuznost ili propusnost šećera tijekom rasta na čvrstoj podlozi toliko promijeni da je različita od one u submerznom uzgoju.A critical review of the phenomenon of resistance to catabolite repression of enzyme synthesis by solid-state fermentation (SSF) has been made. The practical and theoretical importance of such phenomenon is commented, together with the current ideas to explain it. Namely, the possible existence of microscopic gradients within the mass of cell aggregates, or the changes in cell permeability to sugars, which would explain the fact that no catabolite repression is observed in SSF when sugar concentration is as high as 100 g/L, despite the fact that in conventional submerged fermentation (SmF) strong inhibition of inducible enzymes is observed whenever sugar concentration is higher than 10 g/L. Two alternative mathematical models are presented in order to explore the feasibility of those hypotheses, but also to help the planning of future experiments in order to understand the microscopic physiology of SSF. A priori, both hypotheses will explain the phenomenon, but only if the local diffusivity or permeability of sugars in SSF have changed in various orders of magnitude as compared to the observed magnitudes in SmF systems

Enzimska ekstrakcija hidroksicinamičnih kiselina iz otpada nastalog pri proizvodnji kave

Ferulic, caffeic, p-coumaric and chlorogenic acids are classified as hydroxycinnamic acids, presenting anticarcinogenic, anti-inflammatory and antioxidant properties. In this work, enzymatic extraction has been studied in order to extract high value-added products like hydroxycinnamic acids from coffee pulp. A commercial pectinase and enzyme extract produced by Rhizomucor pusillus strain 23aIV in solid-state fermentation using olive oil or coffee pulp (CP) as an inducer of the feruloyl esterase activity were evaluated separately and mixed. The total content (covalently linked and free) of ferulic, caffeic, p-coumaric and chlorogenic acids was 5276 mg per kg of coffee pulp. Distribution was as follows (in %): chlorogenic acid 58.7, caffeic acid 37.6, ferulic acid 2.1 and p-coumaric acid 1.5. Most of the hydroxycinnamic acids were covalently bound to the cell wall (in %): p-coumaric acid 97.2, caffeic acid 94.4, chlorogenic acid 76.9 and ferulic acid 73.4. The content of covalently linked hydroxycinnamic acid was used to calculate the enzyme extraction yield. The maximum carbon dioxide rate for the solid-state fermentation using olive oil as an inducer was higher and it was reached in a short cultivation time. Nevertheless, the feruloyl esterase (FAE) activity (units per mg of protein) obtained in the fermentation using CP as an inducer was 31.8 % higher in comparison with that obtained in the fermentation using olive oil as the inducer. To our knowledge, this is the first report indicating the composition of both esterified and free ferulic, caffeic, p-coumaric and chlorogenic acids in coffee pulp. The highest yield of extraction of hydroxycinnamic acids was obtained by mixing the produced enzyme extract using coffee pulp as an inducer and a commercial pectinase. Extraction yields were as follows (in %): chlorogenic acid 54.4, ferulic acid 19.8, p-coumaric acid 7.2 and caffeic acid 2.3. An important increase in the added value of coffee pulp was mainly due to the extraction of chlorogenic acid.Ferulinska, kafeinska, p-kumarinska i klorogena kiselina pripadaju u hidroksicinamične kiseline, koje imaju antikarcinogena, protuupalna i antioksidativna svojstva. U radu je ispitana enzimska ekstrakcija hidroksicinamičnih kiselina iz otpada nastalog pri proizvodnji kave. Upotrijebljene su, zasebno i u smjesi, komercijalna pektinaza te enzimski esktrakt dobiven fermentacijom na čvrstoj podlozi od maslinovog ulja ili otpada nastalog pri proizvodnji kave (da bi se potaknula aktivnost feruloil esteraze), s pomoću Rhizomucor pusillus 23aIV. Ukupni je udio kovalentno vezanih i slobodnih hidroksicinamičnih kiselina bio 5276 mg/kg otpada. Od toga je bilo: 58,7 % klorogene; 37,6 % kafeinske; 2,1 % ferulinske i 1,5 % p-kumarinske kiseline. Većina je hidroksicinamičnih kiselina bila kovalentno vezana za staničnu stijenku, i to: 97,2 % p-kumarinske; 94,4 % kafeinske; 76,9 % klorogene i 73,4 % ferulinske kiseline. Pomoću udjela kovalentno vezanih hidroksicinamičnih kiselina izračunat je prinos ekstrahiranog enzima. Maksimalni je udio fermentacijom proizvedenog CO2 postignut na čvrstoj podlozi od maslinovog ulja, i to za kraće vrijeme nego na podlozi od otpada nastalog pri proizvodnje kave, iako je feruloil esteraza proizvedena primjenom ove druge podloge pokazala aktivnost veću za 31,8 %. Prema spoznajama autora ovo je prvo istraživanje u kojem su određeni udjeli vezanih i slobodnih hidroksicinamičnih kiselina u otpadu nastalom pri proizvodnji kave. Najveći je prinos ekstrakcije hidroksicinamičnih kiselina postignut primjenom komercijalne pektinaze i enzimskog ekstrakta. Pritom je izdvojeno 54,4 % klorogene; 19,8 % ferulinske; 7,2 % p-kumarinske i 2,3 % kafeinske kiseline, pa je zaključeno da je ovaj postupak dobar za ekstrakciju klorogene kiseline

Effect of Mixing on the Solid-State Fermentation of Coffee Pulp with Aspergillus tamarii

Solid-state fermentation of coffee pulp with Aspergillus tamarii V12307 was carried out in laboratory scale reactors (bottle and column) to evaluate the effect of four different mixing frequencies (2.0, 2.7, 4.0 and 8.0 day^–1) on fungal growth, indirectly determined by carbon dioxide formation and the production of spores and pectin methylesterase. Coffee pulp was used as the sole source of nutrients. An increase in the fraction of bonded particles was observed in the bottle reactors after 12 h of cultivation when no mixing was applied. The use of any mixing frequency reduced the fraction of bonded particles. However, there was no significant difference in pectin methylesterase production between the mixing frequencies at the end of the fermentation. Similarly, there were no significant differences in CO2 production, oxygen uptake or sporulation, demonstrating that the mycelium was not damaged by intermittent mixing. This strategy of mixing could be used in large scale reactors in order to reduce heat and mass limitations

Jasmonic acid biosynthesis by fungi: derivatives, first evidence on biochemical pathways and culture conditions for production

Jasmonic acid (JA) and its derivatives called jasmonates (JAs) are lipid-derived signalling molecules that are produced by plants and certain fungi. Beside this function, JAs have a great variety of applications in flavours and fragrances production. In addition, they may have a high potential in agriculture. JAs protect plants against infections. Although there is much information on the biosynthesis and function of JA concerning plants, knowledge on these aspects is still scarce for fungi. Taking into account the practical importance of JAs, the objective of this review is to summarize knowledge on the occurrence of JAs from fungal culture media, their biosynthetic pathways and the culture conditions for optimal JA production as an alternative source for the production of these valuable metabolites

Use of water hyacinth as a substrate for the production of filamentous fungal hydrolytic enzymes in solid-state fermentation

The objective of the present work was to evaluate the water hyacinth (WH) as a substrate for the production of hydrolytic enzymes (cellulases and hemicellulases) of 100 strains of filamentous fungi under conditions of solid growth. Five fungal strains, identified as Trichoderma harzianum, Trichoderma atroviride, Penicillium griseofulvum, Penicillium commune and Aspergillus versicolor, were selected and studied for their ability to grow on water hyacinth as a substrate and carbon source only, evaluating hydrolytic enzymatic activities (α-l-arabinofuranosidase, cellulase, xylanase and β-d-xylopyranosidase) and extracellular protein per g of water hyacinth dry matter (gdm). The five strains selected were able to produce the four enzymes studied; however, T. harzianum strain PBCA produces the highest xylanase (149.3 ± 14.3 IU/gdm at 108 h), cellulase (16.4 ± 0.6 IU/gdm at 84 h) and β-d-xylopyranosidase (127.7 ± 14.8 IU/gdm at 48 h). In contrast, the fungus with the highest α-l-arabinofuranosidase activity was A. versicolor, with 129.8 ± 13.3 IU/gdm after 108 h. In conclusion, T. harzianum showed the best production of the hydrolytic enzymes studied, using as a matrix and carbon source, water hyacinth. In addition, catalytic activities of arabinofuranosidase and xylopyranosidase were reported for the first time in T. versicolor and T. harzianum.FONCICYT-C002-2008-1 ALA/127249 projec

Enzymatic Extraction of Hydroxycinnamic Acids from Coffee Pulp

Ferulic, caffeic, p-coumaric and chlorogenic acids are classified as hydroxycinnamic acids, presenting anticarcinogenic, anti-inflammatory and antioxidant properties. In this work, enzymatic extraction has been studied in order to extract high value-added products like hydroxycinnamic acids from coffee pulp. A commercial pectinase and enzyme extract produced by Rhizomucor pusillus strain 23aIV in solid-state fermentation using olive oil or coffee pulp (CP) as an inducer of the feruloyl esterase activity were evaluated separately and mixed. The total content (covalently linked and free) of ferulic, caffeic, p-coumaric and chlorogenic acids was 5276 mg per kg of coffee pulp. Distribution was as follows (in %): chlorogenic acid 58.7, caffeic acid 37.6, ferulic acid 2.1 and p-coumaric acid 1.5. Most of the hydroxycinnamic acids were covalently bound to the cell wall (in %): p-coumaric acid 97.2, caffeic acid 94.4, chlorogenic acid 76.9 and ferulic acid 73.4. The content of covalently linked hydroxycinnamic acid was used to calculate the enzyme extraction yield. The maximum carbon dioxide rate for the solid-state fermentation using olive oil as an inducer was higher and it was reached in a short cultivation time. Nevertheless, the feruloyl esterase (FAE) activity (units per mg of protein) obtained in the fermentation using CP as an inducer was 31.8 % higher in comparison with that obtained in the fermentation using olive oil as the inducer. To our knowledge, this is the first report indicating the composition of both esterified and free ferulic, caffeic, p-coumaric and chlorogenic acids in coffee pulp. The highest yield of extraction of hydroxycinnamic acids was obtained by mixing the produced enzyme extract using coffee pulp as an inducer and a commercial pectinase. Extraction yields were as follows (in %): chlorogenic acid 54.4, ferulic acid 19.8, p-coumaric acid 7.2 and caffeic acid 2.3. An important increase in the added value of coffee pulp was mainly due to the extraction of chlorogenic acid

Relationship between lipopeptide biosurfactant and primary metabolite production by Bacillus strains in solid-state and submerged fermentation.

peer reviewedThe relationship between lipopeptide and primary metabolite production by Bacillus spp. in solid-state fermentation (SSF) and submerged fermentation (SmF) was evaluated. Four wild-type strains and one mutant strain (unable to develop biofilm) were assessed in SSF and SmF, using a defined medium and polyurethane foam as inert support for SSF. Strain ATCC 21,332 in SSF presented the highest lipopeptide production. The wild-type strains revealed higher lipopeptide and biomass production and lower synthesis of primary metabolites in SSF than in SmF. However, the mutant strain showed a slightly higher production of primary metabolites in SSF than in SmF. Carbon balance analysis showed that the carbon flux was mainly directed to lipopeptides in SSF, whereas in SmF, it was directed to the production of primary metabolites and the carbon flux to lipopeptides is inversely related to primary metabolites in both types of cultures

Carrier-free immobilization of lipase from candida rugosa with polyethyleneimines by carboxyl-activated cross-linking

Carrier-free immobilization of Candida rugosa lipase (CRL) and polymers containing primary amino groups were cross-linked using carbodiimide. To accomplish this, the free carboxyl groups of the enzyme were activated with carbodiimide-succinimide in organic medium, and then the activated proteins were cross-linked with different polyethylenimines (PEIs). The effect of the cross-linker chain length, the amount of added bovine serum albumin (BSA), and carbodiimide concentration on the catalytic properties of resulting cross-linked enzyme aggregates (CLEAs) was investigated. The CLEAs’ size, shape, specific activity, activity recovery, thermostability and enantioselectivity significantly varied according to the preparation procedure. The highest thermostable CRL-CLEA preparation was obtained with 1.3 kDa polyethyleneimine as cross-linker, 10 mg of BSA and 28 mM of carbodiimide. This preparation is 1.3-fold more active and thermostable than CLEAs prepared by the traditional method of amino cross-linking with glutaraldehyde, and retains 60% of residual activity after 22 h at 50 °C. Additionally, the CRL-CLEA preparation showed an enantioselectivity of 91% enantiomeric excess (ee). This immobilization procedure provides an alternative strategy for CLEA production, particularly for enzymes where the traditional method of cross-linking via lysine residues leads to enzyme inactivation.The Mexican Council for Science and Technology (CONACyT) supported this work (Project No. 154004). S.V.-L. is grateful for the scholarship received from CONACyT. We would like to thank the Spanish Government for the Juan de la Cierva fellowship and Basque country government for the Ikerbasque fellowship that funded F.L.-G.Peer Reviewe