Bioprocess development to add value to canola cake used as substrate for proteolytic enzyme production

Microbial proteases are one of the largest groups of industrial enzymes. This important market has led to a need for technically and economically efficient bioprocesses for protease production that could be exploited commercially. The aim here was to develop a complete bioprocess for protease production, from microbial fermentation up to dried product formulation. Evaluation was made of the effects of operational conditions on the production of protease by a selected strain of Aspergillus oryzae, cultivated under solid-state fermentation (SSF) with canola cake as a sole carbon source in an instrumented lab-scale bioreactor. Statistical experimental design revealed that the air flow rate, inlet air relative humidity, and initial substrate moisture content had significant effects on the efficiency of protease production. The highest protease production by A. oryzae was achieved at a fixed air flow rate of 12 mL/min, with inlet air relative humidity within the range 66–94% and initial substrate moisture content between 30% and 40%. The enzymatic extract produced under the selected conditions was spray dried using different concentrations of additives (glucose, maltodextrin, and CMC). The stability of the dried enzymatic powder during shelf storage was evaluated over a period of 90 days. There was a positive effect of CMC and a negative effect of glucose on protease activity and stability, while the influence of maltodextrin was negative in enzyme recovery at time zero, but it was positive on protease stability over a longer period. The spray dried proteolytic enzymatic formulation obtained from SSF of canola cake using A. oryzae has potential for applications in the industrial sector.     

Production and Application of Novel Sterol Esterases from <Emphasis Type="Italic">Aspergillus</Emphasis> Strains by Solid State Fermentation

Among numerous mesophilic fungi screened for sterol esterase activity followed by the esterification reaction between plant β-sitosterol and lauric acid in organic solvent, six Aspergillus strains were selected as the most active producers. These fungi had not been studied previously for sterol esterase production. The fungi were cultivated under solid state fermentation (SSF) conditions. The gently dried SSF cultures as such were tested in the esterification reactions, without any special enzyme isolation and purification (downstream) processes. All the six Aspergillus SSF preparations were able to synthesize sterol esters. Sterol esterase activity of these GRAS cleared Aspergillus strains was inducible by sterol ester supplementation to the SSF medium and showed remarkably different moisture optimum during growth as compared to the production of lipase (determined by pNP-palmitate). Genome analysis revealed that sterol esterase production might be a common feature of many Aspergillus species. The synthetic usefulness of the best SSF preparations of A. oryzae NRRL 6270 and A. sojae NRRL 6271 was demonstrated by synthesis of esters of plant sterols with lauric acid resulting in 45–63% conversions (GC) and 27–38% isolated yields of steryl laurates. The isomer preference of A. oryzae NRRL 6270 towards the 10E,12Z isomer of conjugated linoleic acid (CLA) in the esterification reaction with plant sterols was also determined.     

Fungal fucoidanase production by solid-state fermentation in a rotating drum bioreactor using algal biomass as substrate

Fucoidanase enzymes able to degrade fucoidan were produced by solid-state fermentation (SSF). The fermentation assays were initially carried out in a laboratory-scale rotating drum bioreactor, and two fungal strains (Aspergillus niger PSH and Mucor sp. 3P) and three algal substrates (untreated, autohydrolyzed, and microwave processed seaweed Fucus vesiculosus) were evaluated. Additionally, fermentations were carried out under rotational (10 rpm) and static conditions in order to determine the effect of the agitation on the enzyme production. Agitated experiments showed advantages in the induction of the enzyme when compared to the static ones. The conditions that promoted the maximum fucoidanase activity (3.82 U L^?1) consisted in using Mucor sp. 3P as fungal strain, autohydrolyzed alga as substrate, and the rotational system. Such conditions were subsequently used in a 10 times larger scale rotating drum bioreactor. In this step, the effect of controlling the substrate moisture during the enzyme production by SSF was investigated. Moreover, assays combining the algal substrate with an inert support (synthetic fiber) were also carried out. Fermentation of the autohydrolyzed alga with the moisture content maintained at 80% during the fermentation with Mucor sp. 3P gave the highest enzyme activity (9.62 U L^?1).     

黑曲霉Aspergillus niger P-6021浆态发酵常山胡柚皮渣产果胶霉

The peel of Citrus changshan-huyou, coupled with wheat bran, could be utilized by Aspergillus niger P-6021 in slurry-state fermentation to produce pectinase with suitable enzyme composition for application in apple juice processing. The production of pectinase is improved by additional nitrogen source substances and mineral supplements. The ratio of carbon source substances to nitrogen source substances in the medium also has significant effect on the pectinase production by A. niger P-6021 in slurry-state fermentation. In the optimized medium composition, the maximal enzyme activity could reach 42 U.L^- 1 （polymethylgalacturonase）, 6.7 U.L^- 1 （polymethygalacturatesterase）, and 4.3 U.L^-1 （polymethylgalacturonate lyase）, respectively, after 3 days at 180 r.min^- 1 and 30℃. The crude pectinase shows significant effect to improve the yield and clarification of apple juice. Keywords Aspergillus niger, slurry-state fermentation, pectinase, Citrus changshan-huyou, apple juice     

Evaluation of production and characterization of polygalacturonase by Aspergillus niger ATCC 9642

The aim of our study was to evaluate polygalacturonase (PG) production and characterization by Aspergillus niger ATCC 9642. The maximum PG activity (51.82 U/mL) was obtained using pectin, l-asparagine, and, iron sulphate concentrations of 32 g/L, 2 g/L, 0.06 g/L and 1.0 g/L, respectively; 180 rpm of stirring rate, 25 °C and an initial pH of 4.0. The kinetic study showed that the highest enzyme activity was achieved at 27 h of fermentation. The evaluation of the optimum pH and temperature permitted us to observe that highest PG activities were achieved at 37 °C and pH of 5.5. The enzymatic extract presented higher stability at 55 °C and pH of 5.0. The results showed that at low temperatures the enzyme extract kept the initial activity until 40 days of storage. The experimental design methodology permitted us to optimize the PG activity and an important aspect of this study is the characterization of the PG in terms of optimum temperature and pH using experimental design technique and also, the characterization of enzyme stability at low temperature. Such studies are very scarce in the literature and should be helpful to understand the true potentialities of pectinases in its industrial applications.     

Effect of canola meal fermentation and protein extraction method on the functional properties of resulting protein products

The present study investigated the effect of solid-state fermentation (SSF) of cold-pressed (CP) and hexane-extracted (HE) canola meals with Aspergillus niger NRRL 334 and Aspergillus oryzae NRRL 5590 on the functionalities of protein products extracted from them. After SSF, proteins were recovered using alkaline extraction-isoelectric precipitation (AE-IP) or salt extraction-dialysis (SE). SSF of the two meal types reduced the protein content of the extracts produced by AE-IP. There were varied effects to solubility, foaming, and emulsifying properties as a result of SSF under the combined influence of functionality pH, strain, meal type, and protein extraction method. The protein isolate produced from CP meal using SE had increased solubility at pH 7 (from 51.8% to 90.7%) when the meal was fermented with A. oryzae. Both strains resulted in an over twofold increase in the emulsifying activity index (at pH 7) of AE-IP products from CP meal. For both protein extraction methods, the protein products from A. niger fermented HE meal had better foaming capacity (FC) at pH 7 than the controls (non-fermented), but reduced FC at pH 3. Overall, regardless of meal fermentation, the SE products were richer in protein and had higher oil holding capacity (OHC), whereas the water holding capacity (WHC) was higher for AE-IP isolates. SSF of the meals generally improved the O/WHC of the extracted proteins. The findings suggest that canola protein functionality could be effectively modulated by SSF with different microbial strains under various processing conditions to enhance their applicability in the food industry.     

Effect of Natural and Pretreated Soybean Hulls on Enzyme Production by Trichoderma reesei

The enzyme induction utility of soybean hulls (SBH), consisting in excess of 50 wt% non-starch polysaccharides (NSP) cellulose, hemicellulose, and pectin, was studied through cultivation of the carbohydrase-producing fungus Trichoderma reesei Rut C-30. Shake flask systems of T. reesei were grown in a medium consisting of defatted soybean flour as a nitrogen source and SBH, some of which were untreated and others pretreated by liquid hot water, alkaline, and supercritical carbon dioxide, as carbon source. Cellulase, xylanase, and polygalacturonase activities were measured for the systems, and the natural hull systems were found to yield optimum enzyme production. Controlled batch fermentation experiments were carried out to compare enzyme production resulting from media with Avicel^® (FMC BioPolymer, Philadelphia, PA, USA) versus natural SBH with and without soybean flour as the nitrogen source. Soybean hull fermentations were also performed at several pH levels to observe the effects on enzyme production. Soybean hulls induced comparable levels of cellulase, and higher levels of xylanase and polygalacturonase, than Avicel^®. With SBH, cellulase and xylanase production were enhanced at higher pH levels (6.0), and polygalacturonase was enhanced at lower pH levels (4.0–4.5). Enzyme production was largely unaffected by the presence of soybean flour as the nitrogen source.     

Effect of solid-state fermentation on select antinutrients and protein digestibility of cold-pressed and hexane-extracted canola meals

In this study, the effects of solid-state fermentation (SSF), including strain (Aspergillus niger NRRL 334 and A. oryzae NRRL 5590) and fermentation time (24, 48, and 72 h) on the nutritional value of cold-pressed (CP) and hexane-extracted (HE) canola meals were examined. SSF increased the protein content of both types of meals (from ~36% to ~40%) while reducing the oil content of CP meals (from ~12% to 9%). There was a significant reduction (~80%) in the phytic acid content of both types of meals after fermentation using either fungi. Overall, fermented samples showed a decrease in the total phenolic content from 2.7–3.1 to ~1.0 mg gallic acid equivalents (GAE)/g dry meal (DM) (a ~65% reduction), of which specifically the HE meal fermented with A. niger sample had the greatest decrease from 3.1 to 0.6 mg GAE/g DM (~81% reduction). Seventy-two hours of fermentation decreased the in vitro protein digestibility (IVPD) of A. oryzae fermented meals. In contrast, a shorter fermentation time (24 h) increased the IVPD for most samples as compared to the controls (from ~72%–73% to 77%–81%), with the exception of the CP meal fermented with A. niger which had similar IVPD at all fermentation times. Overall, the changes indicate that SSF using A. niger or A. oryzae can be useful to positively modify the composition of different canola meals and improve their nutritional value by significantly increasing the protein content, decreasing the levels of antinutrients, while only slightly reducing IVPD.     

Synthesis and antifungal activity of reduced graphene oxide nanosheets

Reduced graphene oxide (rGO) nanosheets were produced using a modified Hummers method. Antifungal activity of rGO nanosheets was tested against three fungi i.e., Aspergillus niger (A. niger), Aspergillus oryzae (A. oryzae) and Fusarium oxysporum (F. oxysporum). The rGO inhibits the mycelial growth of the fungi and it is believed that this is due to its sharp edges. The half maximal inhibitory concentration (IC₅₀), a measure of the effectiveness of the rGO in inhibiting the fungi, was investigated. IC₅₀ values of the rGO against F. oxysporum, A. niger, and A. oryzae are 50, 100, and 100 μg ml^?1, respectively.     

Rhizomucor miehei triglyceride lipase is processed and secreted from transformedAspergillus oryzae

The cDNA encoding the precursor of theRhizomucor miehei triglyceride lipase was inserted in anAspergillus oryzae expression vector. In this vector the expression of the lipase cDNA is under control of theAspergillus oryzae α-amylase gene promoter and theAspergillus niger glucoamylase gene terminator. The recombinant plasmid was introduced intoAspergillus oryzae, and transformed colonies were selected and screened for lipase expression. Lipase-positive transformants were grown in a small fermentor, and recombinant triglyceride lipase was purified from the culture broth. The purified enzymatically active recombinant lipase (rRML) secreted fromA. oryzae was shown to have the same characteristics with respect to mobility on reducing SDS-gels and amino acid composition as the native enzyme. N-terminal amino acid sequencing indicated that approximately 70% of the secreted rRML had the same N-terminal sequence as the nativeRhizomucor miehei enzyme, whereas 30% of the secreted rRML was one amino acid residue shorter in the N-terminal. The recombinant lipase precursor, which has a 70 amino acid propeptide, is thus processed in and secreted fromAspergillus oryzae. We have hereby demonstrated the utility of this organism as a host for the production of recombinant triglyceride lipases.     

Biochemical modification of fats by microorganisms: A preliminary survey

Over 100 different strains of bacteria, actinomycetes, fungi and yeasts were incubated at 28 C for five days in the presence of soybean oil. Some soybean oil was consumed by many microorganisms, and some was also hydrolyzed to free fatty acids.Aspergillus oryzae, two different strains ofAmylomyces rouxii andRhizopus oligosporus hydrolyzed the oil completely (95%). The fatty acids fromAspergillus flavus fermentation contained less linolenic acid than the original soybean oil. Lipase was found intra- and extracellularly when microorganisms were grown in the presence of soybean oil. Deceased.     

Lactic acid fermentation of food waste using integrated glucoamylase production

Commercial enzyme is usually needed for the bioconversion of organic waste or biomass. The overall cost could be reduced very significantly if enzyme production could be integrated with its application, avoiding unnecessary steps in enzyme production (such as concentration, recovery and transportation). This investigation attempted to integrate crude glucoamylase production with lactic acid fermentation of food waste. A maximum glucoamylase activity of 1850 U g^?1 was obtained with Aspergillus nigerduring solid‐state fermentation (SSF) of food waste, 14.8 times more than that obtained during submerged fermentation (SmF). The optimum pH for producing glucoamylase was 4.6, and glucoamylase retained 83.5% of peak activity at pH 3.0. Without any recovery treatment, the glucoamylase produced by SSF could be used directly for lactic acid fermentation of food waste. Lactic acid concentration reached 45.5 g L^?1 with the addition of the crude enzyme, 72% higher than the control. No side‐effects were caused by the viable A. niger in the crude enzyme. This work successfully integrated glucoamylase production with lactic acid fermentation. The enzyme produced by SSF of food waste had sufficient activity to be used directly without any treatment. The integrated process proposed in this study was very economical and may be helpful to other bioconversions. Copyright © 2008 Society of Chemical Industry     

Porosity changes during packed bed solid-state fermentation

Porosity is one of the key parameters in a typical solid-state fermentation model that represents the intra and inter-particle void spaces in bioreactor bed. In this research, the particle density and moisture content of the bed were used to determine the porosity changes of the solid substrate bed during the fermentation of Aspergillus niger in a laboratory scale packed-bed bioreactor. Biomass growth was also monitored by means of measuring CO₂ and O₂ in the bioreactors outlet gas online. As such, an experimental relationship between humidity and porosity has been developed and can be used in solid-state fermentation modeling.     

Antarctic Rahnella inusitata: A Producer of Cold-Stable β-Galactosidase Enzymes

There has been a recent increase in the exploration of cold-active β-galactosidases, as it offers new alternatives for the dairy industry, mainly in response to the current needs of lactose-intolerant consumers. Since extremophilic microbial compounds might have unique physical and chemical properties, this research aimed to study the capacity of Antarctic bacterial strains to produce cold-active β-galactosidases. A screening revealed 81 out of 304 strains with β-galactosidase activity. The strain Se8.10.12 showed the highest enzymatic activity. Morphological, biochemical, and molecular characterization based on whole-genome sequencing confirmed it as the first Rahnella inusitata isolate from the Antarctic, which retained 41–62% of its β-galactosidase activity in the cold (4 °C–15 °C). Three β-galactosidases genes were found in the R. inusitata genome, which belong to the glycoside hydrolase families GH2 (LacZ and EbgA) and GH42 (BglY). Based on molecular docking, some of these enzymes exhibited higher lactose predicted affinity than the commercial control enzyme from Aspergillus oryzae. Hence, this work reports a new Rahnella inusitata strain from the Antarctic continent as a prominent cold-active β-galactosidase producer.     

Degradation of Phytic Acid and Soy Protein in Soy Meal via Co-fermentation of Aspergillus oryzae and Aspergillus ficuum

The usage of soy meal (SM) as a protein source for young monogastric animals is limited by the presence of antinutritional factors. To address this problem, fermentation was applied to simultaneously degrade phytic acid and soy protein in SM. Aspergillusoryzae (ATCC 9362) and Aspergillusficuum (ATCC 66876), sources of protease and thermostable phytase, respectively, were co‐fermented using a two‐stage temperature protocol: 36.5 °C (0–28 h), then 50 °C (28–40 h). The two‐stage co‐fermentation approach achieved a 17 % increase of phytic acid degradation compared to A. oryzae fermentation and 72 % increase in protein degree of hydrolysis (DH) compared to A. ficuum fermentation. The two‐stage temperature fermentation produced a 27 % increase in phytic acid degradation and 90 % increase in DH compared to a single‐stage fermentation. The fermented SM with reduced levels of antinutritional factors would serve as high quality feedstuff.     

Potential use of different agroindustrial by-products as supports for fungal ellagitannase production under solid-state fermentation

Ellagitannase is a novel enzyme responsible for biodegradation of ellagitannins and ellagic acid production. Ellagic acid is a bioactive compound with great potential in food, pharmaceutical and cosmetic industries. This work describes the ellagitannase enzyme production from partial purified ellagitannins as inducers by Aspergillus niger GH1 grown on solid-state fermentation. Solid-state fermentation was carried out on four different lignocellulosic materials (sugarcane bagasse, corn cobs, coconut husks and candelilla stalks) as matrix support and production of ellagitannase enzyme was evaluated. All lignocellulosic materials were characterized in terms of water absorption index and critical humidity point. The best lignocellulosic materials for ellagitannase production were sugarcane bagasse and corn cobs (1400 U L⁻¹ and 1200 U L⁻¹, respectively). The lowest values were obtained with candelilla stalks (500 UL-1). The highest specific productivity was obtained with corn cobs (2.5 U mg⁻¹ h⁻¹) which enable increase ellagitannase productivity up to 140 times. Corn cobs have great potential as support matrix for production of fungal ellagitannase in SSF.     

Synthesis and Antimicrobial Activity of p-tetranitrocalix[4]arene Derivative

The present study demonstrates the synthesis and antimicrobial activity of the p-tetranitrocalix[4]arene (3). The microbial activity was determined against a variety of microorganisms, i.e., Gram-positive and Gram-negative bacterial strains such as Staphylococcus aureus ATCC 10231, Streptococcus viridans ATCC 12392, Escherichia coli ATCC 8739, as well as some fungal species including Aspergillus niger ATCC 16404, Aspergillus flavus ATCC 90906, and Candida albicans ATCC 32333. Kirby-Bauer well agar diffusion method was employed for the determination of antimicrobial activity. All the microorganisms were applied to a selective agar medium (Mueller Hinton Agar) for growth. It was observed that compound 3 is considerably effective against selected microorganisms. The MIC values were also evaluated. Thus, from the results it could be deduced that compound 3 may be a valuable addition to the therapeutic index.     

Protease production by Aspergillus oryzae in solid‐state fermentation using agroindustrial substrates

BACKGROUND: An inexpensive and readily available agroindustrial substrate such as rice bran can be used to produce cheap commercial enzymes by solid‐state fermentation. This work investigates the production of food‐grade proteases by solid‐state fermentation using readily available Thai rice bran. RESULTS: A local strain of Aspergillus oryzae (Ozykat‐1) was used to produce proteases. Rice bran used alone proved to have poor substrate morphology (insufficient porosity) for satisfactory solid‐state fermentation. A certain amount of wheat bran was necessary to improve the morphology of the substrate. The following variables affected protease production: substrate composition, initial moisture content and initial pH. A high protease activity (∼1200 U g⁻¹ dry solids) was obtained on a substrate that had a wheat bran to rice bran ratio of 0.33 by dry weight, a moisture content of 50%, initial pH of 7.5, and incubation temperature of 30 °C. CONCLUSION: Nutritionally, rice bran used alone was as good a substrate as mixed bran for producing protease, but rice bran had poor morphological characteristics for consistent fermentation. A substrate that had a wheat bran to rice bran ratio of 0.33 by dry weight was best for producing protease. Copyright © 2008 Society of Chemical Industry     

Cellulolytic properties of Chaetomium crispatum

The cellulolytic properties of a Chaetomium crispatum strain were investigated. The cellulolytic enzyme complex i.e.: exo-1,4-β-glucosidase (EC 3.2.1.74); endo-1,4-β-glucanase (EC 3.2.1.4.) and β-glucosidase or cellobiase (EC 3.2.1.21) displayed optimal activity at pH 5.0 and 25°C. Although carboxymethyl-celluloses are the usual pseudo-substrates for this enzyme complex, those with a high degree of substitution gave rise to poor growth and low cellulase activity. Insoluble crude cellulosics such as newsprint, recycled paper, rice and flax straw were substantially solubilised at 28°C within 3–5 days of fermentation. A study of the cellulase-complex formation during the growth cycle revealed that β-glucosidase was produced mainly intracellularly in the early exponential phase, while the overall exo-1,4-β-glucosidase and endo-1,4-β-glucanase formation gradually increased during the total fermentation cycle. The mycelial protein of Chaetomium crispatum grown on crude cellulosics displayed a favourable amino acid pattern, indicating its potential value as a source of single cell protein (SCP).