Recent advances in the production of emulsifying peptides with the aid of proteomics and bioinformatics

Food industry aims to develop novel protein-based emulsifiers from sustainable sources (e.g. plants, seaweed/microalgae, microbial, and insects) to satisfy the clean-label demand by consumers. Enzymatic hydrolysis releases peptides with enhanced surface properties compared with the parent alternative proteins. Traditionally, a trial-and-error top-down approach, which requires extensive costs in screening analyses, has been carried out to produce emulsifying peptides. This review presents the recent advances in a novel and fundamentally orthogonal bottom-up strategy, facilitated by quantitative proteomics and bioinformatic functional prediction, to produce emulsifying peptides by targeted enzymatic hydrolysis based on in silico proteolysis. Moreover, new insights on the relation between interfacial properties of peptides and emulsifying activity, as well as impact on stability of wet and dried emulsions, are discussed.Innovation Fund Denmark (Grant nr: 7045-00021B, PROVIDE project

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

This work was part of PROVIDE (Protein valorization through informatics, hydrolysis, and separation) project, which is supported by Innovation Fund Denmark (Grant No.: 7045-00021B) . We acknowledge ISA, Centre for Storage Ring Facilities in Aarhus, Denmark, for granting the beam time (Grant No.: ISA-20-1005) . We thank Lis Berner for her help in the lab with the production and physicochemical characterization of the emulsions. We also acknowledge the companies involved and provided the original samples used as source materials: KMC Kartoffelmelcentralen amba (Brande, Denmark) , AKV Langholt amba (Langholt, Denmark) , CP Kelco (Lille Skensved, Denmark) , Unibio A/S (Odense, Denmark) , and Lihme Protein Solutions (Kgs. Lyngby, Denmark) .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.Innovation Fund Denmark 7045-00021BISA, Centre for Storage Ring Facilities in Aarhus, Denmark ISA-20-100

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.Innovation Fund Denmark (Grant No.: 7045-00021B)

Physical and oxidative stability of fish oil-in-water emulsions stabilized with emulsifier peptides derived from seaweed, methanotrophic bacteria and potato proteins

This study investigated the emulsifying and antioxidant activity of 10 peptides derived from seaweed, methanotrophic bacteria, and potatoes, which were identified as emulsifiers using quantitative proteomics and predictive bioinformatics. The factors (e.g., interfacial properties, secondary structure, net charge) behind the dualfunctionality of peptides were characterized and related to peptides’ ability to provide physical and oxidative stability in 5 % fish oil-in-water emulsions during 10 days of storage. The secondary structure of some of the peptides changed from highly disordered to more α-helical (GIIPATILEFLEGQLQEVDNNKDAR and GIIPGTILEFLEGQLQK) or β-strand (VGFACSGSAQTYLSFEGDNTGRGEEEVAI, ELQVSARVTLEIEL, KVKINETVEIKGKFHV, RSPQKKESDMMKATKFAVVLMAAGLTVGCA) structures when adsorbed at the oil-water interface in comparisonInnovation Fund Denmark (Grant no.: 7045-00021B

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

Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/antiox12081622/s1This 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 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 ffi 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.Innovation Fund Denmark, 7045-00021B (PROVIDE Project

The Use of Soy and Egg Phosphatidylcholines Modified with Caffeic Acid Enhances the Oxidative Stability of High-Fat (70%) Fish Oil-in-Water Emulsions

This study investigated the effect of the combined use of sodium caseinate (CAS), commercial phosphatidylcholine (PC), and modified PCs on the physical and oxidative stability of 70% fish oil-in-water emulsions. Caffeic acid was covalently attached to both modified PCs (PCs originated from soy and eggs) in order to increase the antioxidant activity of PCs and investigate the advantage of bringing the antioxidant activity to the close proximity of the oil-water interface. Results showed that oxidative stability was improved when part of the PC was substituted with modified soy PC or egg PC. Emulsions containing a low concentration of modified PCs (10 wt.% of total PC) resulted in a prooxidative effect on the formation of hydroperoxides compared to emulsions with free caffeic acid. On the other hand, a decrease in the formation of volatile oxidation products was observed for emulsions containing higher levels of modified PCs (60 wt.% of total PC) compared to the emulsions with free caffeic acid added at its equivalent concentration. Increased concentrations of modified PCs provided better oxidative stability in high-fat emulsions, independent of the modified PC type. Moreover, when oxidation was initiated by producing singlet oxygen near a single oil droplet using a focused laser, fluorescence imaging showed that the oxidation did not propagate from one oil droplet to another oil droplet.Danish Council for Independent Research Technology and Production Sciences for financing the project Mapping and Characterizing of Lipid Oxidation in Emulsified Systems (MAPOX)DFF–4184-0123A

Proteomic characterization of pilot scale hot-water extracts from the industrial carrageenan red seaweed Eucheuma denticulatum

Funding This work was supported by Innovation Fund Denmark (grant number 7045-00021B (PROVIDE) ) .Seaweeds have a long history as a resource for polysaccharides/hydrocolloids extraction for use in the food industry due to their functionality as stabilizing agents. In addition to the carbohydrate content, seaweeds also contains a significant amount of protein, which may find application in food and feed. Here, we present a novel combination of transcriptomics, proteomics, and bioinformatics to determine the protein composition in two pilot-scale extracts from Eucheuma denticilatum (Spinosum) obtained via hot-water extraction. Although the quality of extracted protein appeared quite poor based on SDS-PAGE analysis, extracts were characterized by qualitative and quantitative proteomics using LC-MS/MS and a de-novo transcriptome assembly for construction of a suitable protein database. A novel concept of length-normalization for relative quantification of sub-optimal protein extracts with partial, non-specific digestion is introduced and validated against conventional methods for relative quantification of proteins. Despite a limited number of protein identifications due to poor protein quality, our data suggest that the majority of quantified protein in the extracts (>75%) is constituted by merely three previously uncharacterized proteins. Putative subcellular localization for the quantified proteins was determined by bioinformatic prediction using several predictors, and by correlating with the expected copy number from the transcriptome analysis, we find that the extracts appear highly enriched in extracellular proteins. This implies that the extraction method used predominantly extracts extracellular proteins, and thus appear ineffective for cellular disruption and subsequent release of intracellular proteins. Nevertheless, the highly abundant proteins may be potential substrates for targeted hydrolysis and release of bioactive peptides. Ultimately, this study highlight the potential of quantitative proteomics for characterization of alternative protein sources intended for use in foods and evaluating protein extraction process efficiency through novel combinations with bioinformatic analysis.Innovation Fund Denmark 7045-00021