Encapsulation of Tenebrio molitor Hydrolysate with DPP-IV Inhibitory Activity by Electrospraying and Spray-Drying.

This study investigates the encapsulation of Tenebrio molitor hydrolysate exhibiting DPP-IV inhibitory activity by spray-drying and electrospraying techniques. First, we optimized the feed formulation and processing conditions required to obtain nano-microcapsules by electrospraying when using Arabic gum as an encapsulating agent and pullulan and Tween 20 as additives. The optimum formulation was also dried by spray-drying, where the removal of the additives was also assayed. Morphology analysis reveals that electrosprayed capsules have a smaller size (1.2 ± 0.5 µm vs. 12.4 ± 8.7 µm) and greater uniformity compared to those obtained by spray-drying. Regarding the surface nitrogen content and DPP-IV inhibitory activity, our results show no significant difference between the electrosprayed capsules and spray-dried capsules containing additives (IC50 of ~1.5 mg protein/mL). Therefore, it was concluded that adding additives during spray-drying allows for a similar encapsulation efficiency and reduced degradation during processing, as achieved by electrospraying technique but providing higher productivity. On the other hand, spray-dried capsules without additives displayed a higher surface nitrogen content percentage, which was mainly due to the absence of Tween 20 in the feed formulation. Consequently, these capsules presented a higher IC50 value (IC50 of 1.99 ± 0.03 mg protein/mL) due to the potential degradation of surface-exposed peptides.

Physical and Oxidative Stability of Emulsions Stabilized with Fractionated Potato Protein Hydrolysates Obtained from Starch Production Side Stream.

This work studies the emulsifying and antioxidant properties of potato protein hydrolysates (PPHs) fractions obtained through enzymatic hydrolysis of potato protein using trypsin followed by ultrafiltration. Unfractionated (PPH1) and fractionated (PPH2 as >10 kDa, PPH3 as 10-5 kDa, PPH4 as 5-0.8 kDa, and PPH5 as <0.8 kDa) protein hydrolysates were evaluated. Pendant drop tensiometry and dilatational rheology were applied for determining the ability of PPHs to reduce interfacial tension and affect the viscoelasticity of the interfacial films at the oil-water interface. Peptides >10 kDa showed the highest ability to decrease oil-water interfacial tension. All PPH fractions predominantly provided elastic, weak, and easily stretchable interfaces. PPH2 provided a more rigid interfacial layer than the other hydrolysates. Radical scavenging and metal chelating activities of PPHs were also tested and the highest activities were provided by the unfractionated hydrolysate and the fractions with peptides >5 kDa. Furthermore, the ability of PPHs to form physically and oxidatively stable 5% fish oil-in-water emulsions (pH 7) was investigated during 8-day storage at 20 °C. Our results generally show that the fractions with peptides >5 kDa provided the highest physicochemical stability, followed by the fraction with peptides between 5 and 0.8 kDa. Lastly, promising sensory results with mostly mild attributes were obtained even at protein concentration levels that are higher than needed to obtain functional properties. The more prominent attributes (e.g., bitterness and astringency) were within an acceptable range for PPH3 and PPH4.

Antioxidant peptides from alternative sources reduce lipid oxidation in 5% fish oil-in-water emulsions (pH 4) and fish oil-enriched mayonnaise.

Bioinformatics tools were used to predict radical scavenging and metal chelating activities of peptides derived from abundant potato, seaweed, microbial, and spinach proteins. The antioxidant activity was evaluated in 5% oil-in-water emulsions (pH4) and best-performing peptides were tested in mayonnaise and compared with EDTA. Emulsion physical stability was intact. The peptide DDDNLVLPEVYDQD showed the highest protection against oxidation in both emulsions by retarding the formation of oxidation products and depletion of tocopherols during storage, but it was less efficient than EDTA when evaluated in mayonnaise. In low-fat emulsions, formation of hydroperoxides was reduced 4-folds after 5 days compared to control. The concentration effect of the peptide was confirmed in mayonnaise at the EDTA equimolar concentration. The second-best performing peptides were NNKWVPCLEFETEHGFVYREHH in emulsion and AGDWLIGDR in mayonnaise. In general, the peptide efficacy was higher in low-fat emulsions. Results demonstrated that peptide negative net charge was important for chelating activity.

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.

Oxidation and oxidative stability in emulsions.

Emulsions are implemented in the fabrication of a wide array of foods and therefore are of great importance in food science. However, the application of emulsions in food production is restricted by two main obstacles, that is, physical and oxidative stability. The former has been comprehensively reviewed somewhere else, but our literature review indicated that there is a prominent ground for reviewing the latter across all kinds of emulsions. Therefore, the present study was formulated in order to review oxidation and oxidative stability in emulsions. In doing so, different measures to render oxidative stability to emulsions are reviewed after introducing lipid oxidation reactions and methods to measure lipid oxidation. These strategies are scrutinized in four main categories, namely storage conditions, emulsifiers, optimization of production methods, and antioxidants. Afterward, oxidation in all types of emulsions, including conventional ones (oil-in-water and water-in-oil) and uncommon emulsions in food production (oil-in-oil), is reviewed. Furthermore, the oxidation and oxidative stability of multiple emulsions, nanoemulsions, and Pickering emulsions are taken into account. Finally, oxidative processes across different parent and food emulsions were explained taking a comparative approach.

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.

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.

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.

Antioxidant peptides derived from potato, seaweed, microbial and spinach proteins: Oxidative stability of 5% fish oil-in-water emulsions.

In this study, we used a combination of quantitative proteomics and bioinformatic prediction for identifying novel antioxidant peptides. Thirty-five peptides from potato, seaweed, microbial, and spinach proteins were investigated. Based on high DPPH radical scavenging activity (IC50 ≤ 16 mg/mL), metal chelation activity, isoelectric point, and high relative abundance in the parent protein sources, 11 peptides were selected. Lipid oxidation retardation was evaluated in 5% fish oil-in-water emulsions stabilized with Tween 20, where emulsion physical stability was unaffected by peptide addition. The secondary structure of selected peptides was similar in the aqueous solution and emulsions, as confirmed by synchrotron radiation circular dichroism spectroscopy. The emulsions containing the selected peptides had lower levels of hydroperoxides and volatile compounds during storage compared to the control (without peptide). This study contributes to elucidating the effect of antioxidant peptides in emulsions and demonstrates the ability of quantitative proteomics and bioinformatics prediction to identify peptides with strong antioxidant properties.

Emulsifier peptides derived from seaweed, methanotrophic bacteria, and potato proteins identified by quantitative proteomics and bioinformatics.

Global focus on sustainability has accelerated research into alternative non-animal sources of food protein and functional food ingredients. Amphiphilic peptides represent a class of promising biomolecules to replace chemical emulsifiers in food emulsions. In contrast to traditional trial-and-error enzymatic hydrolysis, this study utilizes a bottom-up approach combining quantitative proteomics, bioinformatics prediction, and functional validation to identify novel emulsifier peptides from seaweed, methanotrophic bacteria, and potatoes. In vitro functional validation reveal that all protein sources contained embedded novel emulsifier peptides comparable to or better than sodium caseinate (CAS). Thus, peptides efficiently reduced oil-water interfacial tension and generated physically stable emulsions with higher net zeta potential and smaller droplet sizes than CAS. In silico structure modelling provided further insight on peptide structure and the link to emulsifying potential. This study clearly demonstrates the potential and broad applicability of the bottom-up approach for identification of abundant and potent emulsifier peptides.

Enrichment of mayonnaise with a high fat fish oil-in-water emulsion stabilized with modified DATEM C14 enhances oxidative stability.

Enrichment of mayonnaise using delivery emulsions (DEs) containing 70% fish oil versus neat fish oil was investigated. DEs were produced with combined use of sodium caseinate, diacetyl tartaric acid esters of mono- and diglycerides (DATEM), and/or modified DATEMs with different length (C12 or C14) and covalently attached caffeic acid. Physical and oxidative stability of the mayonnaises were analyzed based on parameters including droplet size, viscosity, peroxide value, volatile compounds, and sensory properties. DEs addition to mayonnaise resulted in larger droplets and lower viscosity compared to neat fish oil. However, zeta potential was higher in mayonnaises with DEs containing DATEMs. Mayonnaise containing DATEM C14 had higher protein surface load leading to a thicker interfacial layer, lower formation of hexanal, (E)-2-hexenal, and (E)-2-heptenal as well as lower rancid odour intensity compared to mayonnaise containing DATEM and free caffeic acid, and thus benefitted from the location of the antioxidant at the interface.

AnOxPePred: using deep learning for the prediction of antioxidative properties of peptides.

Dietary antioxidants are an important preservative in food and have been suggested to help in disease prevention. With consumer demands for less synthetic and safer additives in food products, the food industry is searching for antioxidants that can be marketed as natural. Peptides derived from natural proteins show promise, as they are generally regarded as safe and potentially contain other beneficial bioactivities. Antioxidative peptides are usually obtained by testing various peptides derived from hydrolysis of proteins by a selection of proteases. This slow and cumbersome trial-and-error approach to identify antioxidative peptides has increased interest in developing computational approaches for prediction of antioxidant activity and thereby reduce laboratory work. A few antioxidant predictors exist, however, no tool predicting the antioxidative properties of peptides is, to the best of our knowledge, currently available as a web-server. We here present the AnOxPePred tool and web-server ( http://services.bioinformatics.dtu.dk/service.php?AnOxPePred-1.0 ) that uses deep learning to predict the antioxidant properties of peptides. Our model was trained on a curated dataset consisting of experimentally-tested antioxidant and non-antioxidant peptides. For a variety of metrics our method displays a prediction performance better than a k-NN sequence identity-based approach. Furthermore, the developed tool will be a good benchmark for future predictors of antioxidant peptides.

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.

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.

Small-Angle Neutron Scattering Study of High Fat Fish Oil-In-Water Emulsion Stabilized with Sodium Caseinate and Phosphatidylcholine.

We report on small-angle neutron scattering (SANS) investigations of separate phase domains in high fat (70%) oil-in-water emulsions emulsified with the combination of sodium caseinate (CAS) and phosphatidylcholine (PC). The emulsion as a whole was studied by contrast variation to identify scattering components dominated by individual emulsifiers. The emulsion was subsequently separated into the aqueous phase and the oil-rich droplet phase, which were characterized separately. Emulsions produced with 1.05% (w/w) CAS and PC fraction which varies between 1.75% (w/w) and 0.35% (w/w) provided droplets between 10 and 19 µm in surface weighted mean in 70% fish oil-in-water emulsions. At least two-third of the overall CAS is associated with the interface, while the rest remains with the aqueous phase. Six percent of PC formed a monolayer in the interface, while the rest of the PC remains in the droplet phase in the form of multilayers. When the separated components were resuspended, the resuspended emulsion showed similar characteristics compared to the original emulsion in terms of droplet size distribution and neutron scattering. Instead, CAS in the aqueous phase separated from the emulsion shows aggregation not present in the corresponding CAS-in-D2O system.

Identification of emulsifier potato peptides by bioinformatics: application to omega-3 delivery emulsions and release from potato industry side streams.

In this work, we developed a novel approach combining bioinformatics, testing of functionality and bottom-up proteomics to obtain peptide emulsifiers from potato side-streams. This is a significant advancement in the process to obtain emulsifier peptides and it is applicable to any type of protein. Our results indicated that structure at the interface is the major determining factor of the emulsifying activity of peptide emulsifiers. Fish oil-in-water emulsions with high physical stability were stabilized with peptides to be predicted to have facial amphiphilicity: (i) peptides with predominantly α-helix conformation at the interface and having 18-29 amino acids, and (ii) peptides with predominantly ß-strand conformation at the interface and having 13-15 amino acids. In addition, high physically stable emulsions were obtained with peptides that were predicted to have axial hydrophobic/hydrophilic regions. Peptides containing the sequence FCLKVGV showed high in vitro antioxidant activity and led to emulsions with high oxidative stability. Peptide-level proteomics data and sequence analysis revealed the feasibility to obtain the potent emulsifier peptides found in this study (e.g. γ-1) by trypsin-based hydrolysis of different side streams in the potato industry.

Interfacial structure of 70% fish oil-in-water emulsions stabilized with combinations of sodium caseinate and phosphatidylcholine.

We report on the structural evaluation of high fat fish oil-in-water emulsions emulsified with sodium caseinate (CAS) and phosphatidylcholine (PC). The microemulsions contained 70% (w/w) fish oil with 1.05-1.4% (w/w) CAS and 0.4-1.75% (w/w) PC and were studied by the combination of light scattering together with small-angle X-ray and neutron scattering (SAXS/SANS). Aqueous CAS forms aggregates having a denser core of about 100 kDa and less dense shell about 400 kDa with the hard sphere diameter of 20.4 nm. PC appears as multilayers whose coherence length spans from 40 to 100 nm. PC monolayer separates oil and water phases. Moreover, 80% CAS particles are loosely bound to the interface but are not forming continuous coverage. The distance between aggregated CAS particles in microemulsion is increased compared to CAS aggregates in pure CAS-in-water system. PC multilayers become larger in the presence of oil-water interface compared to the pure PC mixtures. Bilayers become larger with increasing PC concentration. This study forms a structural base for the combination of CAS and PC emulsifiers forming a well-defined thin and dense PC layer together with thick but less dense CAS layer, which is assumed to explain its better oxidative stability compared to single emulsifiers.