Comparison of soybean hull pre-treatments to obtain cellulose and chemical derivatives: Physical chemistry characterization.

The cellulose from soybean hull, a waste without value from the argentine agriculture, was successfully obtained by using two different treatments: the traditional alkaline-bleaching pathway and from a simple pre-alkaline treatment at low temperatures. The comparison of both methods yielded similar results regarding its ability to open the lignin cellulosic structure of the hull and the total elimination of the lignin content. Fourier Transform Infrared spectroscopy (FT-IR), Thermogravimetric analysis (TGA), Scanning electron microscopy (SEM), 13C nuclear magnetic resonance (13C-RMN) and Raman spectroscopy were used to characterize the structures and the properties of cellulose. The results showed that cellulose can be easily obtained with just an alkaline pre-treatment of 5% (w/v) NaOH during 40 h at 50 °C and free of any lignin content. The attachment of different functional groups, such as -COOH and (CH3)3N+, changed the physicochemical properties of the obtained cellulose, showing mayor crystalline structure, and consequently modifying the swelling capacity and its ability to adsorb model proteins.

Control of the adsorption properties of alginate - guar gum matrix functionalized with epichlorohydrin through the addition of different flexible chain polymers as toll for the chymotrypsinogen isolation.

This work addresses the obtaining and characterization of alginate-guar gum matrix, cross-linked with epichlorohydrin in the presence of different flexible chain polymers: polyvinyl alcohol, polyvinyl pyrrolidine and Pluronic® F68. These matrixes were used for the adsorption of chymotrypsinogen and showed an increasing uptake in presence of the flexible chain polymer in the sense: none < Pluronic 68 < polyvinyl pyrrolidine < polyvinyl alcohol. The adsorption process was found to follow a first order kinetics model and was not influenced by the polymer type. It was found that Freundlich model was more suitable for our data. Polyvinyl alcohol and polyvinyl pyrrolidine addition increase the adsorption capacity of the original bed due to an increment in the rigidity of the gel caused by the formation of hydrogen bound between the polysaccharides and synthetics polymers.

Continuous method to determine the trypsin inhibitor activity in soybean flour.

The determination of trypsin inhibitor (TI) activity is of importance to evaluate the nutritional value of soybean flours. An analytical method, which involves a continuous spectrophotometric rate determination for trypsin activity against the substrate N-benzoyl-DL-arginine p-nitroanilide, is proposed as an alternative to the standard discontinuous assay. Stopping the reaction with acetic acid and a centrifugation/filtration step to decrease turbidity are not required, thus reducing costs and sample preparation time. The TI activity of different flour samples, determined by both assays, demonstrated to be statistically comparable, irrespective of the TI concentration level. The coefficients of variation of the novel method did not exceed 8% at any concentration level. The curves of progress reaction showed a non-linear behavior in samples without TI. A reduction of incubation time from 10min to 2min increased the method sensitivity and extended its linear range. A more economical, faster and simpler assay was developed.

Bioactive properties of peptides obtained from Argentinian defatted soy flour protein by Corolase PP hydrolysis.

Enzymatic hydrolysis of soybean meal protein isolate (SPI) obtained under two temperature conditions with Corolase PP was studied, assessing the impact of hydrolysis on potential antioxidant and antihypertensive activities. The protein was isolated from soybean meal under controlled conditions of time and temperature (70 °C, 1h; 90 °C, 30 min). Degree of hydrolysis assessed the progress of hydrolysis at different sampling times. For hydrolysates the antioxidant and angiotensin-converting-enzyme (ACE) inhibitory activities were measured. As observed, the DH was increasing until reaching 20% at 10h with disappearance of globular proteins and generation of low molecular weight peptides (less than 3kDa). A significant increase in antioxidant and ACE inhibitory capacities was observed. Five main peptides were identified, which may explain through their sequences the bioactive properties analyzed. Through this study was possible to obtain for the first time with Corolase PP soy hydrolysates with potential antioxidant and ACE inhibitory activities, which can be used to obtain new added value functional ingredients from soy meal.

Interaction of tannase from Aspergillus niger with polycations applied to its primary recovery.

The interaction of tannase (TAH) with chitosan, polyethyleneimine and Eudragit(®)E100 was studied. It was found that TAH selectively binds to these polycations (PC), probably due to the acid nature of the target protein. TAH could interact with these PC depending on the medium conditions. The effect of the interaction on the secondary and tertiary structure of TAH was assayed through circular dichroism and fluorescence spectroscopy. TAH was recovered from Aspergillus niger culture broth by means of precipitation and adsorption using chitosan.

Partition in aqueous two-phase system: its application in downstream processing of tannase from Aspergillus niger.

Tannase from Aspergillus niger was partitioned in aqueous two-phase systems composed by polyethyleneglycol of molar mass 400, 600 and 1000 and potassium phosphate. Tannase was found to be partitioned toward the salt-rich phase in all systems, with partition coefficients lower than 0.5. Partition coefficients values and low entropic and enthalpic changes associated with tannase partition suggest that the entropic effect may be the driving force of the concentration of the enzyme in the bottom phase due to the high molar mass of the enzyme. The process was significantly influenced by the top phase/bottom phase volume ratio. When the fungal culture broth was partitioned in these systems, a good performance was found, since the enzyme recovery in the bottom phase of the system composed by polyethyleneglycol 1000 was around 96% with a 7.0-fold increase in purity.

Role of polymer-protein interaction on partitioning pattern of bovine pancreatic trypsinogen and alpha-chymotrypsinogen in polyethyleneglycol/sodium tartrate aqueous two-phase systems.

The partitioning pattern of bovine trypsinogen (TRPz) and alpha-chymotrypsinogen (ChTRPz) was investigated in a low impact aqueous two-phase system formed by polyethyleneglycol (PEG) and sodium tartrate (NaTart) pH 5.00. ChTRPz exhibited higher partition coefficients than TRPz did in all the assayed systems. The decrease in PEG molecular weight and the increase in tie line length were observed to displace the partitioning equilibrium of both proteins to the top phase, while phase volume ratios in the range 0.5-1.5 showed not to affect protein partitioning behaviour. Systems formed by PEG of molecular weight 600 with composition corresponding to a high tie line length (PEG 12.93%, w/w and NaTart 21.20%, w/w) are able to recover most of both zymogens in the polymer-enriched phase. A crucial role of PEG-protein interaction in the partitioning mechanism was evidenced by isothermal calorimetric titrations. The major content of highly exposed tryptophan rests, present in ChTRPz molecule, could be considered to be determinant of its higher partition coefficient due to a selective charge transfer interaction with PEG molecule. A satisfactory correlation between partition coefficient and protein surface hydrophobicity was observed in systems formed with PEGs of molecular weight above 4000, this finding being relevant in the design of an extraction process employing aqueous two-phase systems.

Extraction of trypsin from bovine pancreas by applying polyethyleneglycol/sodium citrate aqueous two-phase systems.

The goal of this work was to determine the optimal conditions for separating trypsin (TRP) from alpha-chymotrypsin (ChTRP) and to apply them for trypsin purification from bovine pancreas by liquid-liquid extraction with polyethyleneglycol/sodium citrate (PEG/NaCit) aqueous two-phase systems. Partitioning behaviours of TRP and ChTRP are demonstrated to be very sensitive to variables such as PEG molecular weight, pH and tie line length. Aqueous two-phase systems (ATPSs) formed by PEG of MW 3350 and NaCit pH 5.20 showed the best separation capability. The addition of NaCl up to a final concentration of 7% (w/w) and the decrease of top/bottom volume ratio to 0.1 led to the recovery of 60% of pancreatic TRP in a concentrated form in the top phase with a 3-fold purification. Biomass presence up to 25% (w/w) of the total system mass did not affect significantly yield and purification parameters.

Partition of whey milk proteins in aqueous two-phase systems of polyethylene glycol-phosphate as a starting point to isolate proteins expressed in transgenic milk.

Partitioning behaviour of the bovine whey proteins (bovine serum albumin, alpha lactoalbumin and beta lactoglobulin) and alpha-1 antitrypsin in aqueous two-phase systems prepared with polyethyleneglycol (molecular masses: 1000; 1500 and 3350)-potassium phosphate was analysed. Bovine serum albumin and alpha lactoalbumin concentrated in the polyethyleneglycol rich phase with a partition coefficient of 10.0 and 27.0, respectively, while beta lactoglubulin and alpha-1 antitrypsin showed affinity for the phosphate-rich phase with a partition coefficient of 0.07 and 0.01, respectively. An increase of medium pH induced an increase of the partition coefficient of these proteins while the increase in polyethyleneglycol molecular mass induced the opposite behaviour. The system polyethyleneglycol 1500-pH 6.3 showed the best capacity for recovering the alpha-1 antitrypsin with a yield of 80% and a purification factor between 1.5 and 1.8 from an artificial mixture of the milk whey proteins and alpha-1 antitrypsin. The method appears to be suitable as a starting point to isolate proteins expressed in transgenic milk.