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.

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 µm), although it showed severe physical destabilization when stored at 25 °C for seven days (D4,3 value increased from 0.706 to 9.035 µm). 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.

Comparative Study on the Oxidative Stability of Encapsulated Fish Oil by Monoaxial or Coaxial Electrospraying and Spray-Drying.

The impact of the encapsulation technology on the oxidative stability of fish-oil-loaded capsules was investigated. The capsules (ca. 13 wt% oil load) were produced via monoaxial or coaxial electrospraying and spray-drying using low molecular weight carbohydrates as encapsulating agents (e.g., glucose syrup or maltodextrin). The use of spray-drying technology resulted in larger capsules with higher encapsulation efficiency (EE > 84%), whilst the use of electrospraying produced encapsulates in the sub-micron scale with poorer retention properties (EE < 72%). The coaxially electrosprayed capsules had the lowest EE values (EE = 53-59%), resulting in the lowest oxidative stability, although the lipid oxidation was significantly reduced by increasing the content of pullulan in the shell solution. The emulsion-based encapsulates (spray-dried and monoaxially electrosprayed capsules) presented high oxidative stability during storage, as confirmed by the low concentration of selected volatiles (e.g., (E,E)-2,4-heptadienal). Nonetheless, the monoaxially electrosprayed capsules were the most oxidized after production due to the emulsification process and the longer processing time.

Peptides and protein hydrolysates exhibiting anti-inflammatory activity: sources, structural features and modulation mechanisms.

Inflammation is the response of the immune system to harmful stimuli such as tissue injury, infection or toxic chemicals, which has the aim of eliminating irritants or pathogenic microorganisms and enhancing tissue repair. Uncontrolled long-lasting acute inflammation can gradually progress to chronic, causing a variety of chronic inflammatory diseases that are usually treated with anti-inflammatory drugs, but most of them are inadequate to control chronic responses and are also associated with adverse side effects. Thus, many efforts are being directed to develop alternative and more selective anti-inflammatory therapies from natural products. One main field of interest is the obtaining of bioactive peptides exhibiting anti-inflammatory activity from sustainable protein sources like edible insects or agroindustry and fishing by-products. This work highlighted the structure-activity relationship of anti-inflammatory peptides. Small peptides with molecular weight under 1 kDa and amino acid chain length between 2 to 20 residues are generally the most active because of the higher probability to be absorbed in the intestine and penetrate into cells when compared with the larger size peptides. The presence of hydrophobic (Val, Ile, Pro) and positively charged (His, Arg, Lys) amino acids is another common occurrence for anti-inflammatory peptides. Interestingly, a high percentage (77%) of these bioactive peptides can be found in alternative sustainable protein sources such as Tenebrio molitor or sunflower, apart from its original protein source. However, not all of these peptides with anti-inflammatory potential in vitro achieve good scores by the in silico bioactivity predictors studied. Therefore, it is essential to implement current bioinformatics tools, in order to complement in vitro experiments with prior prediction of potential bioactive peptides.

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., ζ-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 ζ-potential values below -35 mV and an average mean diameter D[4,3] of 0.411 ± 0.010 µm. 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.

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.

Influence of emulsifier type and encapsulating agent on the in vitro digestion of fish oil-loaded microcapsules produced by spray-drying.

The influence of the emulsifier type and the encapsulating agent on the bioaccessibility of microencapsulated fish oil was investigated. Fish oil-loaded microcapsules were produced by spray-drying using carbohydrate-based encapsulating agents (glucose syrup or maltodextrin). Whey protein concentrate hydrolysate (WPCH) or Tween 20 (TW20) were used as the emulsifiers. The microcapsules were subjected to a three-phase in vitro digestion (oral, gastric, and intestinal phase) and the changes in the physicochemical properties of the samples were monitored throughout the simulated gastrointestinal tract (oil droplet size, ζ-potential, and microstructure). The lipolysis rate and extent were evaluated at the intestinal digestion phase. Contrary to the encapsulating agent, the emulsifier used in the infeed emulsion formulation significantly influenced lipid digestion. WPCH-based interfacial layer prevented oil droplets coalescence during and after processing more efficiently than TW20, which resulted in an increased specific surface area for lipases to adsorb and thus a higher bioaccessibility of the microencapsulated oil.

Identification of dipeptidyl peptidase IV (DPP-IV) inhibitory peptides from vegetable protein sources.

Vegetable proteins are appearing as a sustainable source for human consumption. Food-derived peptides are an important field of research in terms of bioactive molecules. In this study, seven vegetable proteins were enzymatically hydrolysed following an optimised treatment (sequential hydrolysis with subtilisin-trypsin-flavourzyme) to obtain dipeptidyl peptidase IV (DPP-IV) inhibitory peptides. Hydrolysates were fractionated by size exclusion chromatography and, from the most bioactive fractions (corresponding to Glycine max, Chenopodium quinoa and Lupinus albus proteins); peptides responsible for this bioactivity were identified by mass spectrometry. Peptides with adequate molecular features and based on in silico analysis were proposed as DPP-IV inhibitors from soy (EPAAV) lupine (NPLL), and quinoa (APFTVV). These vegetable protein sources are adequate to obtain protein hydrolysates for functional food.

Identification of novel dipeptidyl peptidase IV and α-glucosidase inhibitory peptides from Tenebrio molitor.

The exponential increase in world population is leading to a need for new sustainable protein sources that could supply the high demands without resulting in an enormous environmental impact. Bioactive peptides from food proteins are currently seen as capable of modulating physiological processes, such as diabetes. The potential of insects as a cheap source of antidiabetic peptides is a recent research topic. In this work, fractionation and identification of dipeptidyl peptidase IV (DPP-IV) and α-glucosidase inhibitory peptides from mealworm (Tenebrio molitor) was carried out. Peptides from 500 to 1600 Da showed the highest level of DPP-IV inhibition (IC50 value of 0.91 mg ml-1) and peptides below 500 Da showed the highest level of α-glucosidase inhibition (IC50 value of 2.58 mg ml-1). Numerous novel peptides were identified from the most bioactive fractions, and based on the molecular features usually described for these peptides, some of them are suggested to be the bioactive peptides responsible for the inhibition observed (e.g. APVAH for DPP-IV inhibition and CSR for α-glucosidase inhibition). Hence, these insect protein hydrolysates or their purified fractions could be used as ingredients for regulation of the glycaemic index.

Evaluation of the bioactive potential of foods fortified with fish protein hydrolysates.

Protein hydrolysates bioactivity makes them suitable for functional food ingredients. However, their chemical reactivity with different molecules, mainly reducing sugars, might lead to a modification of their structure, and thus function, after their inclusion into food matrices. The effect of food matrix and the storage time on the antihypertensive and antioxidant capacity of sardine hydrolysates was evaluated. Furthermore, bioactivity remaining after simulated gastrointestinal digested samples was also analyzed to verify its potential to be administrated orally. Sugar containing matrix improved the ACE inhibitory and DPPH radical scavenging activity of hydrolysates after thermal treatment and storage up to 80%. Maillard reaction products were responsible for the changes observed in the bioactivity of enriched food. Digested samples showed different but still adequate bioactivity depending on the matrix containing the hydrolysate. These results support the potential of fish protein hydrolysates to be used as ingredient in food formulation.

Antidiabetic Food-Derived Peptides for Functional Feeding: Production, Functionality and In Vivo Evidences.

Bioactive peptides released from the enzymatic hydrolysis of food proteins are currently a trending topic in the scientific community. Their potential as antidiabetic agents, by regulating the glycemic index, and thus to be employed in food formulation, is one of the most important functions of these peptides. In this review, we aimed to summarize the whole process that must be considered when talking about including these molecules as a bioactive ingredient. In this regard, at first, the production, purification and identification of bioactive peptides is summed up. The detailed metabolic pathways described included carbohydrate hydrolases (glucosidase and amylase) and dipeptidyl-peptidase IV inhibition, due to their importance in the food-derived peptides research field. Then, their characterization, concerning bioavailability in vitro and in situ, stability and functionality in food matrices, and ultimately, the in vivo evidence (from invertebrate animals to humans), was described. The future applicability that these molecules have due to their biological potential as functional ingredients makes them an important field of research, which could help the world population avoid suffering from several diseases, such as diabetes.

Production and identification of dipeptidyl peptidase IV (DPP-IV) inhibitory peptides from discarded Sardine pilchardus protein.

Production of bioactive peptides via enzymatic hydrolysis is a sustainable way to take advantage of proteinaceous by-products from food industry, such as fish discards. Sardine pilchardus protein was subjected to different enzymatic treatments using two endopeptidases of different selectivity and one exopeptidase in order to produce hydrolysates with antidiabetic activity. The highest dipeptidyl peptidase IV inhibitory activity was obtained by the combination of three enzymes (subtilisin, trypsin and flavourzyme) employed sequentially. This hydrolysate was subsequently purified by size exclusion chromatography to obtain fractions sorted by size (hydrodynamic volume). Peptides below 1400 Dalton had the highest activity, and these pools were analysed by mass spectrometry in order to identify the peptides responsible for that activity. Numerous peptides with adequate molecular features, it is, owning an alanine (A) as their penultimate N-terminal residue (e.g. NAPNPR, YACSVR) were identified and are proposed to be antidiabetic peptides from Sardine pilchardus muscle.

Protein derived emulsifiers with antioxidant activity for stabilization of omega-3 emulsions.

The performance of a whey protein hydrolysate (WPH) for producing physically and chemically stable omega-3 emulsions was compared to hydrolysates obtained from other sustainable protein sources such as soy (SPH) and blue whiting (BPH). The oxidative stability of hydrolysate-stabilized emulsions was greatly influenced by their physical stability. Emulsion stabilized with BPH suffered a constant increase in droplet size and BPH was not able to prevent omega-3 oxidation, showing high concentration of volatiles. The peroxide value of SPH emulsion increased after the first day of storage, but it had a lower concentration of volatiles. In contrast, WPH-stabilized emulsion, which did not had any change in droplet size during storage, showed the highest oxidative stability. Therefore, our results confirmed that WPH is an interesting option for physical and oxidative stabilization of omega-3 emulsions, while SPH could be used in emulsions with shorter storage time such as pre-emulsions for microencapsulation of omega-3 oils.

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.

Evaluation of Tenebrio molitor protein as a source of peptides for modulating physiological processes.

The increasing world population has led to the need to search for new protein sources, such as insects, the harvesting of which can be economical and environmentally sustainable. This study explores the biological activities (angiotensin-converting enzyme (ACE) inhibition, antioxidant capacity, and dipeptidyl peptidase IV (DPP-IV) inhibition) of Tenebrio molitor hydrolysates produced by a set of food-grade proteases, namely subtilisin, trypsin, ficin and flavourzyme, and the degree of hydrolysis (DH), ranging from 5% to 20%. Trypsin hydrolysates exhibited the highest ACE inhibitory activity at a DH of 10% (IC50 0.27 mg mL-1) in the experimental series, which was attributed to the release of short peptides containing Arg or Lys residues in the C terminus, and described as the ACE-inhibition feature. The levels of in vitro antioxidant activities were comparable to those reported for insect species. Subtilisin and trypsin hydrolysates at a DH of 10% displayed optimal DPPH scavenging and ferric reducing activities, which was attributed to the presence of 5-10-residue active peptides, as reported in the literature. Iron chelating activity was significantly favoured by increasing the DH, attaining a minimal IC50 of 0.8 mg mL-1 at a DH of 20% regardless of the enzymatic treatment. Similarly, in vitro antidiabetic activity was significantly improved by extensive hydrolysis, and, more specifically, the presence of di- and tripeptides. In this regard, the combined treatment of subtilisin-flavourzyme at a DH of 20% showed maximal DPP-IV inhibition (IC50 2.62 mg mL-1). To our knowledge, this is the first study evaluating the DPP-IV activity of Tenebrio molitor hydrolysates obtained from these commercial proteases. We conclude that Tenebrio molitor hydrolysates produced with food-grade proteases are a valuable source of active peptides that can be used as functional ingredients in food and nutraceutical preparations.

Development of Fish Oil-Loaded Microcapsules Containing Whey Protein Hydrolysate as Film-Forming Material for Fortification of Low-Fat Mayonnaise.

The influence of the carbohydrate-based wall matrix (glucose syrup, GS, and maltodextrin, MD21) and the storage temperature (4 °C or 25 °C) on the oxidative stability of microencapsulated fish oil was studied. The microcapsules (ca. 13 wt% oil load) were produced by spray-drying emulsions stabilized with whey protein hydrolysate (WPH), achieving high encapsulation efficiencies (>97%). Both encapsulating materials showed an increase in the oxidation rate with the storage temperature. The GS-based microcapsules presented the highest oxidative stability regardless of the storage temperature with a peroxide value (PV) of 3.49 ± 0.25 meq O2/kg oil and a content of 1-penten-3-ol of 48.06 ± 9.57 ng/g oil after six weeks of storage at 4 °C. Moreover, low-fat mayonnaise enriched with GS-based microcapsules loaded with fish oil and containing WPH as a film-forming material (M-GS) presented higher oxidative stability after one month of storage when compared to low-fat mayonnaise enriched with either a 5 wt% fish oil-in-water emulsion stabilized with WPH or neat fish oil. This was attributed to a higher protective effect of the carbohydrate wall once the microcapsules were incorporated into the mayonnaise matrix.

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. © 2016 Society of Chemical Industry.

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.

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.

Bile acid binding capacity of fish protein hydrolysates from discard species of the West Mediterranean Sea.

Fish protein hydrolysates (FPH), produced from the six main discard species from the West Mediterranean Sea (sardine, horse mackerel, axillary seabream, bogue, small-spotted catshark and blue whiting) were tested for their bile acid binding capacity. This capacity is directly linked to the ability to inhibit bile reabsorption in the ileum and therefore to lower cholesterol levels in the bloodstream. From each species, FPH were obtained by three different enzymatic treatments employing two serine endoproteases (subtilisin and trypsin) sequentially or in combination. The results show statistically significant differences among the fish species, attaining interesting average values of bile acid binding capacity for blue whiting (27.32% relative to cholestyramine on an equal protein basis) and horse mackerel (27.42% relative to cholestyramine on an equal protein basis). The enzymatic treatments did not significantly affect the ability of a given species to bind bile acids. These results are similar to other protein sources, such as soy protein or casein, of proven hypocholesterolemic effect. It can be concluded that fish protein hydrolysates from these discard species are suitable as ingredients in the formulation of cholesterol-lowering supplements.