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

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

Valorisation of blood protein from livestock to produce haem iron-fortified hydrolysates with antioxidant activity

This work was supported by the project PY20_00021 from the Andalusian Regional Government, Spain. The authors would like to thank APC Europe (Barcelona, Spain) for kindly providing the porcine blood meal powder employed for the experiments.Blood is the main by-product from slaughterhouses bearing high levels of suspended solids (18% w/w) and BOD5 (250 000–375 000 mg O2/L), which makes difficult its handling and disposal. This study proposes the valorisation of blood protein to produce enzymatic hydrolysates rich in haemic iron and antioxidant peptides. Haemic iron presents higher bioavailability compared to its inorganic form, but its incorporation into foodstuffs is restrained by its high tendency to oxidation. Six commercial proteases of animal, plant and bacterial origin were employed to produce the enzymatic hydrolysates. Subtilisin and trypsin treatments were able to recover 70% of haem iron as soluble peptides, in contrast with plant enzymes where the proteolysis was reduced. Moreover, some enzymatic treatments led to hydrolysates with high levels of some in vitro antioxidant activities such as radical scavenging (Protamex, IC50 3.52 mg/mL) or metal chelating activity (trypsin, IC50 0.27 mg/mL). We conclude the enzymatic valorisation of blood protein increases haemic iron bioavailability and produces antioxidant peptides. Both properties are of interest for their use as iron fortification supplements.Andalusian Regional Government, Spain PY20_0002

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

This work was supported by the Spanish Ministry of Economy, Industry and Competitiveness and by FEDER (project CTQ2017-87076-R). The authors would like to thank MealFoodEurope (Salamanca, Spain) for kindly providing Tenebrio molitor meal.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.Spanish Ministry of Economy, Industry and CompetitivenessEuropean Commission CTQ2017-87076-

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

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

Bi-objective optimization of tuna protein hydrolysis to produce aquaculture feed ingredients

Fish meal is commonly employed as protein source in aquaculture diets. The enrichment of this ingredient with fish protein hydrolysates (FPH) and free amino acids has proved to improve larval development and feed assimilation. In this work, we produced tuna head hydrolysates using a sequential enzymatic treatment employing Alcalase and Flavourzyme. Statistical modelization coupled with bi-objective optimization were employed to optimize the operating parameters (i.e. pH, temperature and duration of the Flavourzyme treatment) for producing a FPH with a desired molecular weight profile. More specifically, this work focused on the content of small peptides between 700–2500 Da (F 2500 ) and that of free amino acids (F 250 ), supported by their benefits as aquaculture feed ingredients. The optimal reaction conditions for maximimizing the release of free amino acids F 250 (i.e, pH 7.2, 43–49 °C, Flavourzyme treatment above 160 min) were detrimental for the content of F 2500 . A bi-objective optimization approach was then proposed, able to find a set of intermediary solutions (Pareto Front) presenting maximal F 2500 for a range of free amino acids level between 2–30%. This allows the selection of the operating parameters for producing a FPH with a desired weight profile, based on the specific needs of the farmed species

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

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

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

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

Encapsulation of Bioactive Peptides by Spray-Drying and Electrospraying

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

Effect of digestive enzymes on the bioactive properties of goat milk protein hydrolysates

The aim of this research was to study the influence of the gastrointestinal digestion on the bioactivity of goat milk protein hydrolysates prepared with subtilisin, trypsin and a combination of these two enzymes. All hydrolysates had excellent angiotensin converting enzyme (ACE) inhibitory activity, antioxidant activity and bile acid-binding capacity. Peptide profiles and bioactivities were mainly altered during the intestinal digestion, whereas the effect of the gastric digestion was negligible. The influence of the intestinal digestion varied depending on the hydrolysate and the bioactivity studied. In the case of ACE inhibitory activity, it exclusively decreased when peptides were produced with trypsin. In contrast, antioxidant activity and bile acid-binding capacity improved after the gastrointestinal digestion, regardless the enzymatic treatment conducted. Hydrolysis employing mixtures of subtilisin and trypsin is considered a good approach to produce peptides that maintain, or even enhance, their bioactivity after digestion.Consejería de Innovación, Ciencia y Empresa of Junta de Andalucía (project P12-AGR-1993)Consejería de Innovación, Ciencia y Empresa of Junta de Andalucía (postdoctoral position of F. Javier Espejo-Carpio - project TEP-02579

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

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