Screening for Metal-Chelating Activity in Potato Protein Hydrolysates Using Surface Plasmon Resonance and Peptidomics.

Metal-catalyzed lipid oxidation is a major factor in food waste, as it reduces shelf life. Addressing this issue, our study investigates the potential of hydrolysates derived from potato protein, a by-product of potato starch production, as metal-chelating antioxidants. Through sequential enzymatic hydrolysis using alcalase or trypsin combined with Flavourzyme, we produced various hydrolysates, which were then fractionated using ultrafiltration. Using a combination of peptidomics and bioinformatics, we predicted the presence of metal-chelating and free radical-scavenging peptides across all hydrolysate fractions, with a trend indicating a higher content of antioxidant peptides in lower molecular weight fractions. To validate these predictions, we utilized surface plasmon resonance (SPR) and a 9-day emulsion storage experiment. While SPR demonstrated potential in identifying antioxidant activity, it faced challenges in differentiating between hydrolysate fractions due to significant standard errors. In the storage experiment, all hydrolysates showed lipid oxidation inhibition, though not as effectively as ethylenediaminetetraacetic acid (EDTA). Remarkably, one fraction (AF13) was not significantly different (p < 0.05) from EDTA in suppressing hexanal formation. These results highlight SPR and peptidomics/bioinformatics as promising yet limited methods for antioxidant screening. Importantly, this study reveals the potential of potato protein hydrolysates as antioxidants in food products, warranting further research.

Lipid oxidation in emulsions: New insights from the past two decades.

Lipid oxidation constitutes the main source of degradation of lipid-rich foods, including food emulsions. The complexity of the reactions at play combined with the increased demand from consumers for less processed and more natural foods result in additional challenges in controlling this phenomenon. This review provides an overview of the insights acquired over the past two decades on the understanding of lipid oxidation in oil-in-water (O/W) emulsions. After introducing the general structure of O/W emulsions and the classical mechanisms of lipid oxidation, the contribution of less studied oxidation products and the spatiotemporal resolution of these reactions will be discussed. We then highlight the impact of emulsion formulation on the mechanisms, taking into consideration the new trends in terms of emulsifiers as well as their own sensitivity to oxidation. Finally, novel antioxidant strategies that have emerged to meet the recent consumer's demand will be detailed. In an era defined by the pursuit of healthier, more natural, and sustainable food choices, a comprehensive understanding of lipid oxidation in emulsions is not only an academic quest, but also a crucial step towards meeting the evolving expectations of consumers and ensuring the quality and stability of lipid-rich food products.

Evaluation of primary and secondary oxidation products in fish oil-in-water emulsions: Effect of metal-complexing peptides and protein hydrolysates.

A novel approach consisting of preselection of peptides using bioinformatics tool followed by final selection using Surface Plasmon Resonance (SPR) - an efficient technique to investigate metal complexing properties of peptides/hydrolysates - was developed. Selected pea hydrolysates and synthetic metal chelating peptides potentially present in pea hydrolysates were investigated for their ability to inhibit the lipid oxidation in emulsions composed of 5 % w/w fish oil and stabilized with Tween® 20. Results indicated that addition of peptides/hydrolysates did not impact the physical stability of emulsions and led to lower level of lipid hydroperoxides. Moreover, peptide KGKSR inhibited the generation of 1-penten-3-ol and hexanal to the same level as ethylenediaminetetraacetic acid (EDTA) did and the formation of 2 ethyl-furan was lower than when EDTA was added. Peptide GRHRQKHS showed same concentration of hexanal as EDTA thus confirming efficacy of using SPR for selecting peptides/hydrolysates to use as antioxidants in emulsions.

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.

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.

Interfacial effects of gallate alkyl esters on physical and oxidative stability of high fat fish oil-in-water emulsions stabilized with sodium caseinate and OSA-modified starch.

Effects of sodium caseinate (SC) and its combination with OSA-modified starch (SC-OS; 1:1) alone and with n-alkyl gallates (C0-C18) on the physical and oxidative stability of high-fat fish oil-in-water emulsion were evaluated. SC emulsion contained the smallest droplets and highest viscosity due to the fast adsorption at droplet surfaces. Both emulsions had non-Newtonian and shear-thinning behavior. A lower accumulation of lipid hydroperoxides and volatile compounds was found in SC emulsion due to its better Fe2+ chelating activity. The incorporated short-chain gallates (G1 > G0 âˆ¼ G3) in SC emulsion had a strong synergistic effect against lipid oxidation compared to that of SC-OS emulsion. The better antioxidant efficiency of G1 can be related to its higher partition at the oil-water interface, while G0 and G3 had a higher partition into the aqueous phase. In contrast, G8, G12, and G16 added emulsions indicated higher lipid oxidation due to their internalization inside the oil droplets.

Gelatin from Saithe (Pollachius virens) Skin: Biochemical Characterization and Oxidative Stability in O/W Emulsions.

This study performed the extraction of gelatin from saithe (Pollachius virens) skin and compared it to commercial marine gelatin. As a first stage, we investigated the physicochemical and biochemical properties of the gelatin. SDS-PAGE analysis revealed the presence of α-chains, ß-chains, and other high-molecular-weight aggregates. DSC thermograms showed typical gelatin behavior, while the FTIR spectra were mainly situated in the amide band region (amide A, amide B, amide I, amide II, and amide III). In the second stage, we produced O/W emulsions and analyzed their physical and oxidative stability over 9 days. Oil droplets stabilized with the gelatins obtained from saithe fish skin had a size of ~500 nm and a ζ-potential ~+25 mV, which is comparable to oil droplets stabilized with commercial gelatin products. Moreover, the oxidative stability of the emulsions stabilized with gelatin from saithe fish skin showed promising results in terms of preventing the formation of some volatile compounds towards the end of the storage period compared to when using the commercial gelatins. This study indicates the potential application of fish skin gelatin in the fields of food and cosmetics, as well as suggesting that further investigations of their techno-functional properties.

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.

Physical and Oxidative Stability of Low-Fat Fish Oil-in-Water Emulsions Stabilized with Black Soldier Fly (Hermetia illucens) Larvae Protein Concentrate.

The physical and oxidative stability of fish oil-in-water (O/W) emulsions were investigated using black soldier fly larvae (BSFL) (Hermetia illucens) protein concentrate as an emulsifier. To improve the protein extraction and the techno-functionality, defatted BSFL powder was treated with ohmic heating (BSFL-OH) and a combination of ohmic heating and ultrasound (BSFL-UOH). Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) were performed in order to characterize the secondary structure and thermal stability of all protein concentrate samples. The interfacial properties were evaluated by the pendant drop technique. The lowest interfacial tension (12.95 mN/m) after 30 min was observed for BSFL-OH. Dynamic light scattering, ζ-potential and turbiscan stability index (TSI) were used to evaluate the physical stability of emulsions. BSFL-OH showed the smallest droplet size (0.68 µm) and the best emulsion stability (TSI = 8.89). The formation of primary and secondary volatile oxidation products and consumption of tocopherols were evaluated for all emulsions, revealing that OH and ultrasound treatment did not improve oxidative stability compared to the emulsion with untreated BSFL. The results revealed the promising application of BSFL proteins as emulsifiers and the ability of ohmic heating to improve the emulsifying properties of BSFL proteins.

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.

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.

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.

Modified phosphatidylcholine with different alkyl chain length and covalently attached caffeic acid affects the physical and oxidative stability of omega-3 delivery 70% oil-in-water emulsions.

This study investigated the effects of modified phosphatidylcholine (PC) with different alkyl chain lengths (PC_C14 and PC_C16) and covalently attached caffeic acid on the physical and oxidative stability of 70% fish oil-in-water emulsions. High fat emulsions were produced using different amounts of modified PCs in combination with sodium caseinate and soy-PC. Results showed that the physical stability of the emulsions was improved with increasing concentrations of modified PCs, due to their high surface activity. Emulsion stabilized with PC_C14 led to smaller droplets and higher viscosity, whereas PC_C16 had higher protein surface load, which may result in a thicker interfacial layer. Modified PCs enhanced the oxidative stability of the emulsions due to the attachment of caffeic acid to the glycerol backbone of PC, which brings the antioxidant in the vicinity of oil-water interface. PC_C16 led to less formation of primary and secondary oxidation products compared to PC_14 at their equivalent concentrations.

Physical and oxidative stability of high fat fish oil-in-water emulsions stabilized with sodium caseinate and phosphatidylcholine as emulsifiers.

The physical and oxidative stability of high-fat omega-3 delivery systems such as fish oil-in-water emulsions stabilized with combinations of sodium caseinate (CAS) and phosphatidylcholine (PC) was optimized. The influence of fish oil content (50, 60 and 70%, w/w), amount of total emulsifier CAS + PC (1.4, 2.1 and 2.8%, w/w) and ratio between CAS and PC (0.4, 1.2 and 2) on physical and oxidative parameters was investigated. Creaming and droplet size significantly decreased when the amount of fish oil, total emulsifier and ratio of CAS to PC were increased. Viscosity decreased significantly with decreasing fish oil content, whereas the ratio of CAS to PC did not have a significant influence. Decreasing the ratio of CAS to PC led to emulsions with a significantly lower concentration of 1-penten-3-ol, while no significant effect was found for other volatiles such as (E)-2-pentenal, (E)-2-hexenal and (E,E)-2,4-heptadienal.

Effects of Modified DATEMs with Different Alkyl Chain Lengths on Improving Oxidative and Physical Stability of 70% Fish Oil-in-Water Emulsions.

The objective of this study was to produce oxidatively and physically stable 70% fish oil-in-water emulsions by combined use of sodium caseinate (CAS), commercial diacetyl tartaric acid esters of mono- and diglycerides (DATEM), and modified DATEM. First, the optimal formula was determined using DATEM and CAS. Subsequently, modified DATEMs (DATEM C12 and DATEM C14) were designed for investigating both the effects of different alkyl chain lengths and caffeic acid conjugation to the emulsifier on physical and oxidative stability of the emulsions. Emulsions produced with modified DATEMs showed better oxidative stability compared with emulsion using commercial DATEM plus an equivalent amount of free caffeic acid, confirming the advantage of having antioxidant covalently attached to the emulsifier. Results indicated that DATEM_C14 replaced more CAS compared with DATEM_C12 from the interface in 70% fish oil-in-water emulsion. Emulsions produced with DATEM_C14 had significantly decreased amounts of primary and secondary oxidation products compared with emulsions using DATEM_C12.

Combination of sodium caseinate and succinylated alginate improved stability of high fat fish oil-in-water emulsions.

Sodium caseinate (CAS) and commercial sodium alginate (CA), long chain modified alginate (LCMA) or short chain modified alginate (SCMA) were used in combination for emulsifying and stabilizing high fat (50-70%) fish oil-in-water emulsions. Physical (creaming, droplet size, viscosity and protein determination) and oxidative (primary and secondary oxidation products) stabilities of the emulsions were studied during 12 days of storage. Creaming stability was higher for emulsions produced with alginates and CAS compared to emulsions prepared with only CAS. Combined use of CAS + LCMA performed better in terms of physical stability compared to emulsions produced with only CAS. However, the oxidative stability of this emulsion was inferior probably due to the presence of an unsaturated carbon chain in LCMA structure. CAS + SCMA emulsions not only showed better physical stability such as smaller droplet size, lower creaming and higher viscosity, but also had an improved oxidative stability than emulsions produced with only CAS.