Spray Drying of Goat Milk Protein Hydrolysates with Angiotensin Converting Enzyme Inhibitory Activity

A goat milk hydrolysate was stabilized by spray drying using different inlet air temperatures (170-230 °C) and feed flow rates (4-12 mL/min) following a central composite experimental design. In order to evaluate the effect of operational conditions on process yield, powder properties (density, moisture, hygroscopicity, and solubility) as well as angiotensin converting enzyme (ACE) inhibitory activity, experimental data were analyzed by response surface methodology. Input variables showed a significant influence on yield, density, and moisture, while hygroscopicity, solubility, and ACE inhibitory activity were not affected. The dried hydrolysate presented an average IC50 value of 273.13 μg/mL, which involved a loss of 25 % in ACE inhibitory activity with respect to the feed. The variations detected in the low molecular weight fractions of the dried hydrolysates could be responsible for the variations in ACE inhibitory activity.Consejería de Innovación, Ciencia y Empresa of Junta de Andalucía (P07-TEP-02579

Artificial neuronal networks (ANN) to model the hydrolysis of goat milk protein by subtilisin and trypsin

The enzymatic hydrolysis of milk proteins yield final products with improved properties and reduced allergenicity. The degree of hydrolysis (DH) influences both technological (e.g., solubility, water binding capacity) and biological (e.g., angiotensin-converting enzyme (ACE) inhibition, antioxidation) properties of the resulting hydrolysate. Phenomenological models are unable to reproduce the complexity of enzymatic reactions in dairy systems. However, empirical approaches offer high predictability and can be easily transposed to different substrates and enzymes. In this work, the DH of goat milk protein by subtilisin and trypsin was modelled by feedforward artificial neural networks (ANN). To this end, we produced a set of protein hydrolysates, employing various reaction temperatures and enzyme/substrate ratios, based on an experimental design. The time evolution of the DH was monitored and processed to generate the ANN models. Extensive hydrolysis is desirable because a high DH enhances some bioactivities in the final hydrolysate, such as antioxidant or antihypertensive. The optimization of both ANN models led to a maximal DH of 23·47% at 56·4 °C and enzyme–substrate ratio of 5% for subtilisin, while hydrolysis with trypsin reached a maximum of 21·3% at 35 °C and an enzyme–substrate ratio of 4%.Consejería de Economía, Innovación, Ciencia y Empleo de la Junta de Andalucía (P07-TEP-02579

Production and identification of angiotensin I-converting enzyme (ACE) inhibitory peptides from Mediterranean fish discards

The production of peptides exhibiting Angiotensin I-converting enzyme (ACE)-inhibitory activity from discarded Mediterranean fish species such as sardine, horse mackerel, axillary seabream, bogue and small-spotted catshark was studied. The evolution of the ACE-inhibitory activity with the degree of hydrolysis (DH) of protein hydrolysates was also investigated. Hydrolysates of horse mackerel and small-spotted catshark, both obtained with the simultaneous addition of subtilisin and trypsin, showed the highest antihypertensive activity (IC50 of 279 and 302μg/mL, respectively). For horse mackerel hydrolysate, fraction B (130-2350Da) exhibited the highest ACE-inhibitory activity (IC50=85μg/mL). In the case of small-spotted catshark hydrolysate, fraction D (<470Da) presented the lowest IC50 value (27μg/mL). In addition, 14 novel ACE-inhibitory peptides were identified in horse mackerel and small-spotted catshark hydrolysates. The peptide VAMPF, identified in fraction D of small-spotted catshark hydrolysate, is one of the most promising peptides according to its low IC50 value obtained by the QSAR-model (IC50=0.44μM)Spanish National Plan I + D + i (CTQ2011-23009)Andalusian Government (project P12-AGR-1993)

Evaluation of the Physical and Oxidative Stabilities of Fish Oil-in-Water-in-Olive Oil Double Emulsions (O1/W/O2) Stabilized with Whey Protein Hydrolysate

This work studied the physical and oxidative stabilities of fish oil-in-water-in-olive oil double emulsions (O1/W/O2), where whey protein hydrolysate was used as a hydrophilic emulsifier. A 20 wt.% fish oil-in-water emulsion, stabilized with whey protein hydrolysate (oil: protein ratio of 5:2 w/w) and with a zeta potential of ~�����40 mV, only slightly increased its D4,3 value during storage at 8 C for seven days (from 0.725 to 0.897 m), although it showed severe physical destabilization when stored at 25 C for seven days (D4,3 value increased from 0.706 to 9.035 m). The oxidative stability of the 20 wt.% fish oil-in-water emulsion decreased when the storage temperature increased (25 vs. 8 C) as indicated by peroxide and p-anisidine values, both in the presence or not of prooxidants (Fe2+). Confocal microscopy images confirmed the formation of 20 wt.% fish oil-in-water-in-olive oil (ratio 25:75 w/w) using Polyglycerol polyricinoleate (PGPR, 4 wt.%). Double emulsions were fairly physically stable for 7 days (both at 25 and 8 C) (Turbiscan stability index, TSI < 4). Moreover, double emulsions had low peroxide (<7 meq O2/kg oil) and p-anisidine (<7) values that did not increase during storage independently of the storage temperature (8 or 25 C) and the presence or not of prooxidants (Fe2+), which denotes oxidative stability.the I+D+i projects CTQ2017-87076-R and PID2020-114137RBI00 funded by MCIN/AEI/10.13039/50110001103

BI-OBJECTIVE OPTIMISATION OF THE ENZYMATIC HYDROLYSIS OF PORCINE BLOOD PROTEIN

Protein from porcine blood meal was hydrolysed with Alcalase to obtain a final revalorised product suitable, for example, to take part in the composition of an organic fertiliser. Three experimental factors of the reaction (pH, temperature and enzyme-substrate ratio) were optimised by means of a statistically designed experiment and response surface methodology. The goal of the optimisation problem was to maximise both the degree of hydrolysis and solubilisation of the substrate, obtaining a maximum degree of hydrolysis (28.89%) with pH 6.24, 54.2 °C and enzyme-substrate ratio of 10%. Regarding the content of suspended solids, its minimum value (30.29% related to the initial weight of blood meal) was attained at pH 7.5, 59.8 °C and enzyme-substrate ratio of 10%. The controversial effects of pH and temperature on the substrate solubilisation and the final degree of hydrolysis, suggested employing a multiobjective optimisation technique. A Pareto Front was generated in order to find a set of intermediate solutions which satisfied both objectives in an adequate degree.Ministerio de Ciencia e Innovación ( CTQ2008-02978

Optimization of the Emulsifying Properties of Food Protein Hydrolysates for the Production of Fish Oil-in-Water Emulsions

The incorporation of lipid ingredients into food matrices presents a main drawback—their susceptibility to oxidation—which is associated with the loss of nutritional properties and the generation of undesirable flavors and odors. Oil-in-water emulsions are able to stabilize and protect lipid compounds from oxidation. Driven by consumers’ demand, the search for natural emulsifiers, such as proteins, is gaining much interest in food industries. This paper evaluates the in vitro emulsifying properties of protein hydrolysates from animal (whey protein concentrate) and vegetal origin (a soy protein isolate). By means of statistical modelling and bi-objective optimization, the experimental variables, namely, the protein source, enzyme (i.e., subtilisin, trypsin), degree of hydrolysis (2–14%) and emulsion pH (2–8), were optimized to obtain their maximal in vitro emulsifying properties. This procedure concluded that the emulsion prepared from the soy protein hydrolysate (degree of hydrolysis (DH) 6.5%, trypsin) at pH 8 presented an optimal combination of emulsifying properties (i.e., the emulsifying activity index and emulsifying stability index). For validation purposes, a fish oil-in-water emulsion was prepared under optimal conditions, evaluating its physical and oxidative stability for ten days of storage. This study confirmed that the use of soy protein hydrolysate as an emulsifier stabilized the droplet size distribution and retarded lipid oxidation within the storage period, compared to the use of a non-hydrolyzed soy protein isolate.Spanish Government CTQ2017-87076-

Structure of whey protein hydrolysate used as emulsifier in wet and dried oil delivery systems: Effect of pH and drying processing

The secondary structure of whey protein concentrate hydrolysate (WPCH), used as an emulsifier in oil delivery systems, was investigated using Synchrotron Radiation Circular Dichroism (SRCD). The effect of pH on the conformation of peptides in solution and adsorbed at the oil/water interface, as well as the thermal stability of the systems was studied. Furthermore, oil-loaded microcapsules were produced by spray-drying or electrospraying to investigate the influence of encapsulating agents (glucose syrup, maltodextrin) and drying technique on the secondary structure of WPCH at the oil/water interface. Enzymatic hydrolysis resulted in peptides with a highly unordered structure (~60% turns and unordered regions) in solution. However, WPCH adsorption onto the oil/water interface increased the α-helical content resulting in an improved thermal stability. The encapsulating agents and spray-drying process did not modify the conformation of WPCH at the oil/water interface. Nonetheless, electrospraying affected the SRCD spectra obtained for WPCH adsorbed at the oil/water interface.I + D + i project CTQ2017-87076-R MCIN/AEI/10.13039/501100011033 PRE2018-084861 730872European Commissio

pH influences the interfacial properties of blue whiting (M. poutassou) and whey protein hydrolysates determining the physical stability of fish oil-in-water emulsions

This work was funded by the project CTQ2017-87076-R from the Spanish Ministry of Science and Innovation. Julia Maldonado-Valderrama and Teresa del Castillo-Santaella acknowledge financialsupport from project RTI2018-101309-B-C21. The authors are also very grateful to F. Javier Espejo-Carpio and Marta Padial-Dominguez for providing the whey and blue whiting protein hydrolysates. Funding for open access charge: Universidad de Granada/CBUA.This work investigates the influence of the interfacial properties of whey protein (WPH) and blue whiting protein (BPH) hydrolysates on the physical stability of fish oil-in-water emulsions stabilized with these hydrolysates at pH 2 or 8. Measurements of interfacial tension and dilatational rheology confirmed that pH is a key factor affecting these interfacial properties of WPH and BPH. WPH, when tested at 1 and 10 mg/mL, showed a higher interfacial activity at pH 8 when compared to pH 2 or to BPH at pH 8 or 2, despite having a lower protein content. Moreover, when tested at 0.1 and 1 mg/mL, the dilatational modulus of WPH was significantly higher at pH 8 than at pH 2. These findings correlate with the formation of smaller oil droplets and a more resistant interfacial peptide layer for WPH at pH 8, hence explaining the improved physical stability of the 5% fish oil-in water emulsion stabilized with WPH at pH 8. BPH did not show significant differences in interfacial activity with pH but exhibited significantly higher dilatational elasticity and viscosity at pH 2 compared to pH 8 (when measured at 0.1 mg/mL and 0.01 or 0.1 Hz). This correlates with the formation of stable 5% fish oil-in-water emulsions with BPH at pH 2 but not at pH 8.Spanish Government CTQ2017-87076-

Evaluation of Plant Protein Hydrolysates as Natural Antioxidants in Fish Oil-In-Water Emulsions

In this work, we evaluated the physical and oxidative stabilities of 5% w/w fish oil-in-water emulsions stabilized with 1%wt Tween20 and containing 2 mg/mL of protein hydrolysates from olive seed (OSM-H), sunflower (SFSM-H), rapeseed (RSM-H) and lupin (LUM-H) meals. To this end, the plant-based substrates were hydrolyzed at a 20% degree of hydrolysis (DH) employing a mixture 1:1 of subtilisin: trypsin. The hydrolysates were characterized in terms of molecular weight profile and in vitro antioxidant activities (i.e., DPPH scavenging and ferrous ion chelation). After incorporation of the plant protein hydrolysates as water-soluble antioxidants in the emulsions, a 14-day storage study was conducted to evaluate both the physical (i.e., zeta-potential, droplet size and emulsion stability index) and oxidative (e.g., peroxide and anisidine value) stabilities. The highest in vitro DPPH scavenging and iron (II)-chelating activities were exhibited by SFSM-H (IC50 = 0.05 +/- 0.01 mg/mL) and RSM-H (IC50 = 0.41 +/- 0.06 mg/mL). All the emulsions were physically stable within the storage period, with zeta-potential values below -35 mV and an average mean diameter D[4,3] of 0.411 +/- 0.010 mu m. Although LUM-H did not prevent lipid oxidation in emulsions, OSM-H and SFSM-H exhibited a remarkable ability to retard the formation of primary and secondary lipid oxidation products during storage when compared with the control emulsion without antioxidants. Overall, our findings show that plant-based enzymatic hydrolysates are an interesting alternative to be employed as natural antioxidants to retard lipid oxidation in food emulsions.Regional Ministry of Economic Transformation, Industry, Knowledge, and Universities of Andalusia (Spain) PY20_00021Colombian Ministry of Science, Technology and Innovatio

Encapsulation of Bioactive Peptides by Spray-Drying and Electrospraying

Bioactive peptides derived from enzymatic hydrolysis are gaining attention for the production of supplements, pharmaceutical compounds, and functional foods. However, their inclusion in oral delivery systems is constrained by their high susceptibility to degradation during human gastrointestinal digestion. Encapsulating techniques can be used to stabilize functional ingredients, helping to maintain their activity after processing, storage, and digestion, thus improving their bioaccessibility. Monoaxial spray-drying and electrospraying are common and economical techniques used for the encapsulation of nutrients and bioactive compounds in both the pharmaceutical and food industries. Although less studied, the coaxial configuration of both techniques could potentially improve the stabilization of protein-based bioactives via the formation of shell–core structures. This article reviews the application of these techniques, both monoaxial and coaxial configurations, for the encapsulation of bioactive peptides and protein hydrolysates, focusing on the factors affecting the properties of the encapsulates, such as the formulation of the feed solution, selection of carrier and solvent, as well as the processing conditions used. Furthermore, this review covers the release, retention of bioactivity, and stability of peptide-loaded encapsulates after processing and digestion.I+D+i project PID2020-114137RBI00 funded by MCIN/AEI/10.13039/501100011033