Xylanase Production by Solid-State Fermentation for the Extraction of Xylooligosaccharides from Soybean Hulls§.

Research background: The development of a novel process for the production of xylooligosaccharides (XOS) based on the 4R concept is made possible by the integration of numerous techniques, especially enzymatic modification together with the physical pretreatment of renewable materials. This study aims to integrate the use of agricultural wastes for the production of xylanase by a new strain of Penicillium sp. and value-added products, XOS. Experimental approach: For the production of xylanase, a solid-state fermentation was performed using wheat bran as substrate. To obtain the most active crude extract of xylanase, the time frame of cultivation was first adjusted. Then, the downstream process for xylanase purification was developed by combining different membrane separation units with size exclusion chromatography. Further characterisation included determination of the optimal pH and temperature, determination of the molecular mass of the purified xylanase and analysis of kinetic parameters. Subsequently, the hydrolytic ability of the partially purified xylanase in the hydrolysis of alkali-extracted hemicellulose from soybean hulls was investigated. Results and conclusions: Our results show that Penicillium rubens produced extracellular xylanase at a yield of 21 U/g during solid-state fermentation. Using two ultrafiltration membranes of 10 and 3 kDa in combination with size exclusion chromatography, a yield of 49 % and 13-fold purification of xylanase was achieved. The purified xylanase (35 kDa) cleaved linear bonds ß-(1→4) in beechwood xylan at a maximum rate of 0.64 µmol/(min·mg) and a Michaelis constant of 44 mg/mL. At pH=6 and 45 °C, the purified xylanase showed its maximum activity. The xylanase produced showed a high ability to hydrolyse the hemicellulose fraction isolated from soybean hulls, as confirmed by thin-layer chromatography. In the hydrothermally pretreated hemicellulose hydrolysate, the content of XOS with different degrees of polymerisation was detected, while in the non-pretreated hemicellulose hydrolysate, the content of xylotriose and glucose was confirmed. Novelty and scientific contribution: Future research focusing on the creation of new enzymatic pathways for use in processes to convert renewable materials into value-added products can draw on our findings.

Enzyme production by solid-state fermentation on soybean meal: A comparative study of conventional and ultrasound-assisted extraction methods.

Protease, cellulase, and α-amylase producing Bacillus subtilis strain was cultivated by solid-state fermentation technique using soybean meal as a substrate. The aim of the present study was to establish a highly efficient enzymes' extraction method as a first stage in downstream processing. The conventional extraction procedure was optimized by determining pH, stirring rate, solid/liquid ratio, and time of extraction on enzymes' recoveries from fermented soybean meal. Yields of leached enzymes were compared to the amounts of enzymes that are achieved with ultrasound-assisted extraction (UAE). UAE was established to be superior method for obtaining higher yields of proteases (up to 330 IU) and α-amylases (825 IU), under significantly shorter extraction time and gaining more concentrated product. However, the obtained model predicts that conventional process led to a product with a higher cellulolytic activity (≥7.5 IU).

Production of egg white protein hydrolysates with improved antioxidant capacity in a continuous enzymatic membrane reactor: optimization of operating parameters by statistical design.

This study focuses on the influence of operating conditions on Alcalase-catalyzed egg white protein hydrolysis performed in a continuously stirred tank reactor coupled with ultrafiltration module (10 kDa). The permeate flow rate did not significantly affect the degree of hydrolysis (DH), but a significant increase in process productivity was apparent above flow rate of 1.9 cm3 min-1. By contrast, an increase in enzyme/substrate (E/S) ratio provided an increase in DH, but a negative correlation was observed between E/S ratio and productivity. The relationship between operating conditions and antioxidant properties of the hydrolysates, measured by three methods, was studied using Box-Behnken experimental design and response surface methodology. The statistical analysis showed that each variable (impeller speed, E/S ratio, and permeate flow rate) had a significant effect on the antioxidant capacity of all tested systems. Nevertheless, obtained response functions revealed that antioxidative activity measured by DPPH, ABTS and FRAP methods were affected differently by the same operating conditions. High impeller speeds and low permeate flow rates favor ABTS while high impeller speeds and high permeate flow rates had a positive effect on the DPPH scavenging activity. On the other hand, the best results obtained with FRAP method were achieved under moderate operating conditions. The integration of the reaction and ultrafiltration membrane separation in a continuous manner appears to be a right approach to improve and intensify the enzymatic process, enabling the production of peptides with desired antioxidant activity.

Design of biopolymer carriers enriched with natural emulsifiers for improved controlled release of thyme essential oil.

This work aims to characterize a novel system for thyme essential oil delivery based on the combination of natural emulsifiers (soy protein and soy lecithin) and alginate, produced using the extrusion technique. The formulations are optimized concerning alginate and soy protein concentrations (both 1 to 1.5 wt.%), and consequently lecithin amount, in order to achieve spherical beads in the range 2.0 to 2.3 mm and 1.2 to 1.4 mm, wet and dry, respectively. Fourier-transform infrared analysis was performed, proving that there are interactions between all components. Lecithin-soy protein synergistic combination improved entrapment efficiency of total polyphenols (for nearly 12%) and decreased thymol release in a simulated gastric solution for nearly 35%, in comparison with beads without lecithin. The addition of lecithin enhances the thermal properties of the polysaccharide-protein systems at 50 °C after 3 hr of heating. The mechanical stability of the biopolymer carriers is improved with lecithin addition and the elastic modulus varied from 80.06 to 123.7 kPa, depending on the formulation. Alginate/soy protein/lecithin are effective carriers for the encapsulation, protection, and controlled release of thyme essential oil. PRACTICAL APPLICATION: There is unfortunately growing human resistance to antibiotics. This work offers a novel system for effective protection and controlled release of thyme essential oil in the small intestine. The mechanical and thermal properties of the carrier were estimated as they indicate how the beads will be able to resist stress during their incorporation into food (i.e. cookies-mixing, baking). The proposed approach offers ''green advantage'' as arises from all-natural materials.

Production of enzymes by a newly isolated Bacillus sp. TMF-1 in solid state fermentation on agricultural by-products: The evaluation of substrate pretreatment methods.

Study on potential of different agro-industrial waste residues for supporting the growth of newly isolated Bacillus sp. TMF-1 strain under solid-state fermentation (SSF) was conducted aiming to produce several industrially valuable enzymes. Since the feasibility of the initial study was confirmed, further objectives included evaluation of several pretreatments of the studied agricultural by-products (soybean meal, sunflower meal, maize bran, maize pericarp, olive oil cake and wheat bran) on the microbial productivity as means of enhancing the yields of produced proteases, α-amylases, cellulases and pectinases. Among acid/alkaline treatment, ultrasound and microwave assisted methods, chemical treatments superiorly affected most of the studied substrates. Highest yields of produced proteases (50.5IUg-1) and α-amylases (50.75IUg-1) were achieved on alkaline treated corn pericarp. Alkaline treatment also promoted the secretion of cellulases on maize bran (1.19IUg-1). Highest yield of pectinases was obtained on untreated soybean meal (64.90IUg-1).

Adding value to the oil cake as a waste from oil processing industry: production of lipase and protease by Candida utilis in solid state fermentation.

Olive oil cake is a by-product from the olive oil processing industry and can be used for the lipase and protease production by Candida utilis in solid state fermentation. Different carbon and nitrogen sources were evaluated, and the results showed that the supplementation of the substrate with maltose and starch as carbon sources and yeast extract as a nitrogen source significantly increased the lipase production. The best results were obtained with maltose, whereas rather low lipase and protease activities were found with glucose and oleic acid. Response surface methodology and a five-level-three-factor central composite rotatable design were used to evaluate the effects of the initial moisture content, inoculum size and fermentation time on both lipase and protease activity levels. A lipase activity value of ≈25 U g(-1) and a protease activity value of 110 U g(-1) were obtained under the optimized fermentation conditions. An alkaline treatment of the substrate appeared to be efficient, leading to increases of 39% and 133% in the lipase and protease production, respectively. The results showed that the olive cake could be a good source for enzyme production by solid state fermentation.