Antonio Guadix

Physical and oxidative stability of fish oil-in-water emulsions stabilized with fish protein hydrolysates.

The emulsifying and antioxidant properties of fish protein hydrolysates (FPH) for the physical and oxidative stabilization of 5% (by weight) fish oil-in-water emulsions were investigated. Muscle proteins from sardine (Sardina pilchardus) and small-spotted catshark (Scyliorhinus canicula) were hydrolyzed to degrees of hydrolysis (DH) of 3-4-5-6% with subtilisin. Sardine hydrolysates with low DH, 3% and 4%, presented the most effective peptides to physically stabilize emulsions with smaller droplet size. This implied more protein adsorbed at the interface to act as physical barrier against prooxidants. This fact might also be responsible for the higher oxidative stability of these emulsions, as shown by their lowest peroxide value and concentration of volatiles such as 1-penten-3-one and 1-penten-3-ol. Among the hydrolysates prepared from small-spotted catshark only the hydrolysate with DH 3% yielded a physically stable emulsion with low concentration of unsaturated aldehydes. These results show the potential of FPH as alternative protein emulsifiers for the production of oxidatively stable fish oil-in-water emulsions.

Correlation of base consumption with the degree of hydrolysis in enzymic protein hydrolysis

It is fairly easy to control the enzymic hydrolysis of proteins in alkaline conditions by measuring the base consumption required to keep the pH constant in the reactor. Unfortunately, however, base consumption is not related in any simple way to the degree of hydrolysis reached at any given moment and to establish this relationship it is essential to find out the mean pK of the alpha-amino groups released during the hydrolytic process. We have shown here that the correct mean pK value varies according to the pH of the working conditions and that the relationship between these values may depend upon the kind of protein and protease used. We have put forward a method for determining this relationship experimentally by using a given protein-protease system, consisting of an alkaline titration of the raw protein and when partially hydrolysed. We have tested the results predicted by our theoretical model by applying it to the hydrolysis of whey proteins with a bacterial protease from Bacillus licheniformis at 50 degrees C, pH 8.0. This model can easily be applied to any hydrolytic process involving the appearance of functional groups that are partially protonizable under the working conditions in question in order to follow the kinetics of the reaction via the consumption of the neutralizing agent required to keep pH constant.

Production of resistant starch by enzymatic debranching in legume flours.

Resistant starch (RS) was produced by enzymatic hydrolysis of flours from five different legumes: lentil, chickpea, faba bean, kidney bean and red kidney bean. Each legume was firstly treated thermally, then hydrolyzed with pullulanase for 24h at 50°C and pH 5 and lyophilized. At the end of each hydrolysis reaction, the RS amount ranged from 4.7% for red kidney beans to 7.5% for chickpeas. With respect to the curves of RS against hydrolysis time, a linear increase was observed initially and a plateau was generally achieved by the end of reaction. These curves were successfully modeled by a kinetic equation including three parameters: initial RS, RS at long operation time and a kinetic constant (k). Furthermore, the relative increase in hydrolysis, calculated using the kinetic parameters, was successfully correlated to the percentage of amylose.

Lipid characterization and properties of protein hydrolysates obtained from discarded Mediterranean fish species

BACKGROUND Discards are an important fraction of the by-products produced by the fishing industry. As a consequence of their low commercial acceptance, it is necessary to provide added value to these underutilized materials. In this study the lipid fraction of three discarded fish species in the western Mediterranean Sea, namely sardine (Sardina pilchardus), mackerel (Scomber colias) and horse mackerel (Trachurus trachurus), was characterized and the angiotensin I-converting enzyme (ACE)-inhibitory and antioxidative activities of their protein hydrolysates were evaluated. RESULTS Processing of these biomaterials led to oils with a high content of omega-3 polyunsaturated fatty acids (PUFAs), ranging from 220.5 g kg(-1) for horse mackerel to 306.0 g kg(-1) for sardine. Regarding the protein fraction, most of the hydrolysates presented ACE inhibition values higher than 60%, corresponding to IC50 values varying from 345 µg protein mL(-1) for mackerel to 400 µg protein mL(-1) for sardine. Moreover, most of the hydrolysates exhibited acceptable antioxidative activity, namely 35-45% inhibition of 1,1-diphenyl-2-picrylhydrazyl (DPPH). CONCLUSION This study suggests that the three discarded species evaluated are valuable raw materials for the production of bioactive ingredients such as omega-3 PUFAs and protein hydrolysates exhibiting antihypertensive and antioxidative activities.

Functional, bioactive and antigenicity properties of blue whiting protein hydrolysates: effect of enzymatic treatment and degree of hydrolysis

BACKGROUND Fish discards represent an important under-utilisation of marine resources. This study evaluated the up-grading of the protein fraction of blue whiting (Micromesistius poutassou) discards by the production of fish protein hydrolysates (FPHs) exhibiting functional, antioxidant, angiotensin-I converting enzyme (ACE)-inhibitory and antigenicity properties. RESULTS FPHs with low DH (4%) showed better emulsifying, foaming and oil binding capacities, particularly those obtained using only trypsin. FPHs with DH 4% exhibited also the stronger antioxidant activity, especially the one obtained using only subtilisin (IC50 = 1.36 mg protein mL-1 ). The presence of hydrophobic residues at the C-terminal of the FPH produced using subtilisin also led to the stronger ACE-inhibitory activity. However, FPHs with high DH (12%), which implies a higher proportion of short peptides, was required to enhance ACE-inhibition (IC50 = 172 µg protein mL-1 ). The antigenic levels of the FPH were also reduced with DH independently of the enzymatic treatment. Nevertheless, the highest degradation of fish allergens (e.g. parvalbumin) was also obtained when using only subtilisin. CONCLUSION These results suggest that added-value products for food applications can be produced from the protein fraction of discards.

Optimisation of the hydrolysis of goat milk protein for the production of ACE-inhibitory peptides

Goat milk protein was hydrolysed with subtilisin and trypsin. As input variables, temperature was assayed in the interval 45-70 °C for subtilisin and 30-55 °C for trypsin, while the enzyme-substrate ratio varied from 1 to 5%. The effect of the input variables on the degree of hydrolysis and ACE-inhibitory activity (output variables) was modelled by second order polynomials, which were able to fit the experimental data with deviations below 10%. The individual maximum values of the degree of hydrolysis and the ACE-inhibitory activity were found at conflicting conditions of temperature and enzyme-substrate ratio. Since such maximum values could not be reached simultaneously, a bi-objective optimisation procedure was undertaken, producing a set of non-inferior solutions that weighted both objectives.

Development of an up-grading process to produce MLM structured lipids from sardine discards

The aim of the work was to produce MLM structured lipids with caprylic acid (M) as medium chain fatty acid located at the external bonds of the glycerol backbone and concentrated polyunsaturated fatty acids (L) from sardine discards (Sardine pilchardus) in the central bond of the glycerol. To that end, the following steps were conducted: (i) fish oil extraction, (ii) Omega-3 free fatty acids (FFA) concentration (low temperature winterization), (iii) two-steps enzymatic esterification and (iv) triacylglycerols (TAG) purification (liquid column chromatography). The resultant purified triacylglycerols accomplished with the oxidative state (peroxide and anisidine value, PV and AV) required for refined oils. As enzymatic treatment, Omega-3 concentrate FFA (Omega-3>600mg Omega-3 per g oil) were esterified with dicaprylic glycerol employing Novozyme 435. This process presented high regioselectivity, with ∼80mol% of concentrated fatty acids esterified at the sn-2 position.

Artificial neural networks to model the production of blood protein hydrolysates for plant fertilisation

BACKGROUND Amino acid-based fertilisers increase the bioavailability of nitrogen in plants and help withstand stress conditions. Additionally, porcine blood protein hydrolysates are able to supply iron, which is involved in chlorophyll synthesis and improves the availability of nutrients in soil. A high degree of hydrolysis is desirable when producing a protein hydrolysate intended for fertilisation, since it assures a high supply of free amino acids. Given the complexity of enzyme reactions, empirical approaches such as artificial neural networks (ANNs) are preferred for modelisation. RESULTS Porcine blood meal was hydrolysed for 3 h with subtilisin. The time evolution of the degree of hydrolysis was successfully modelled by means of a feedforward ANN comprising 10 neurons in the hidden layer and trained by the Levenberg-Marquardt algorithm. The ANN model described adequately the influence of pH, temperature, enzyme concentration and reaction time upon the degree of hydrolysis, and was used to estimate the optimal operation conditions (pH 6.67, 56.9 °C, enzyme to substrate ratio of 10 g kg(-1) and 3 h of reaction) leading to the maximum degree of hydrolysis (35.12%). CONCLUSIONS ANN modelling was a useful tool to model enzymatic reactions and was successfully employed to optimise the degree of hydrolysis.

A lumped model of the lipase catalyzed hydrolysis of sardine oil to maximize polyunsaturated fatty acids content in acylglycerols

The aim of this work was to produce diacylglycerols (DAG) and monoacylglycerols (MAG) with a high content of polyunsaturated fatty acids (PUFA). Rhizomucor miehei lipase mediated-hydrolysis of sardine oil was conducted at several water activities. The system was mechanistically modeled to predict the time evolution of the concentration of triacylglycerols, DAG, MAG and free fatty acids (FFA) and the concentration of saturated, mono- and polyunsaturated fatty acids. The release of the first fatty acid from the triacylglycerol was independent on the unsaturation degree. Contrary, the hydrolysis of the second one was highly affected by the degree of unsaturation, PUFA being the fatty acids that showed the highest resistance to hydrolysis. MAG percentage was maximum (7mol%) at lower water activities, while DAG content was favored at higher water activities (35mol%), achieving a 2-fold concentration of DHA.

1H, 13C NMR, X‐ray and conformational studies of new 1‐alkyl‐3‐benzoyl‐pyrazole and 1‐alkyl‐3‐benzoyl‐pyrazoline derivatives

The 1H and 13C NMR resonances of 22 1‐alkyl‐pyrazole and 25 1‐alkyl‐pyrazoline derivatives were assigned completely using the concerted application of one‐ and two‐dimensional experiments (DEPT, gs‐HMQC and gs‐HMBC). Nuclear Overhauser enhancement (NOE) effects, conformational analysis and X‐ray crystallography confirm the preferred conformation of those compounds. Copyright