Current knowledge on the extraction, purification, identification and validation of bioactive peptides from seaweed

peer-reviewedSeaweed (macroalgae) is considered as a sustainable bioresource rich in high-quality nutrients such as protein. Seaweed protein can be used as an alternative to other protein sources. Furthermore, these proteins are natural reservoirs of bioactive peptides (BAPs) associated with various health benefits such as antioxidant, antihypertensive and antidiabetic activities. However, seaweed derived BAPs remain underexploited due to challenges which arise during protein extraction from algal biomass. Coupled with this, limited proteomic information exists regarding certain seaweed species. This review highlights the current state of the art of seaweed protein extraction techniques, e.g., liquid, ultrasound, microwave, pulsed electric field and high hydrostatic pressure assisted extraction. The review also focuses on the enzymatic hydrolysis of seaweed proteins and characterisation of the resultant hydrolysates/peptides using electrophoretic and chromatographic techniques. This includes reference to methods employed for separation, fractionation and purification of seaweed BAPs, as well as the methodologies used for identification, e.g., analysis by mass spectrometry. Furthermore, a bioinformatics or in silico approach to aid discovery of seaweed BAPs is discussed herein. Based on the information available to date, it is suggested that further research is required in this area for the development of seaweed BAPs for nutraceutical applications

Enzyme-assisted release of antioxidant peptides from porphyra dioica conchocelis

peer-reviewedThe conchocelis life cycle stage of P. dioica represents an unexplored source of bioactive compounds. The aim of this study was to generate and characterise, for the first time, hydrolysates of conchocelis using a specific combination of proteases (Prolyve® and Flavourzyme®). Hydrolysate molecular mass distribution and free amino acid contents were assessed, and the antioxidant activity was determined using a range of in vitro assays. The protein content and the total amino acid profiles of conchocelis were also studied. Conchocelis contained ~25% of protein (dry weight basis) and had a complete profile of essential amino acids. Direct sequential enzymatic treatment modified the profile of the generated compounds, increasing the amount of low molecular weight peptides (<1 kDa). There was a significant improvement in the antioxidant activity of the hydrolysates compared with the control (up to 2.5-fold), indicating their potential as a novel source of antioxidant ingredients

Optimisation of low temperature (8ᵒC) enzymatic hydrolysis of acid whey using design of experiments (DOE) for the generation of thermally stable whey protein hydrolysates

The impact of enzymatic hydrolysis at 8ᵒC of liquid whey protein concentrate (WPC35) derived from acid casein manufacture using Prolyve 1000®, a microbial protease preparation, under free-fall pH conditions on the physicochemical and the thermal stability properties of the resultant hydrolysates was investigated. The hydrolysis process was optimised using a design of experiments (DOE) approach. Eleven hydrolysates (H1–H11) were generated using 3 factors x 2 levels, i.e., enzyme:substrate (E:S) 0.25–1.00% (v/w), starting pH 7.5–9.0 and incubation time 10–96 h. Hydrolysate degree of hydrolysis (DH) values ranged from 0.75 (H10) to 4.74% (H6) which increased to 1.91 (H10) and 7.24% (H6) following subsequent thermal processing (mimicking the evaporation process) at 54ᵒC x 15 min and 64ᵒC x 10 min. The apparent viscosity (ηapp) of all hydrolysates was measured to assess their heat stability during heating at 85ᵒC for up to 20 min, this was shown to be E:S and pH-dependent. While the unhydrolysed samples formed a gel on heating at ~74-80ᵒC. Following DOE analysis, it was found that E:S had a significant impact on the DH, the extent of intact protein degradation (�g) and the thermal stability. The �g following processing at 64ᵒC showed a strong correlation (R2 = 0.924; p < 0.001) with ηapp, and thus this parameter may be used to predict the thermal stability of the hydrolysate samples. The optimised hydrolysis conditions for the generation of heat stable WPC35 hydrolysates during 8◦C incubation were achieved using an E:S = 0.625% at a starting pH = 8.65. Performance of protein hydrolysis reactions at low temperature has potential to enhance process sustainability.</p

Impact of pH control on peptide release and the in vitro bioactive properties of whey protein enzymatic hydrolysates

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Application of in silico approaches for the generation of milk protein-derived bioactive peptides

Milk protein derived peptides have numerous well-documented bioactive properties. The conventional approach for the generation, identification and validation of bioactive peptides (BAPs) has involved (i) protein hydrolysis, (ii) bioactivity screening and (iii) validation in vivo. The low potency (in comparison to conventional drugs), susceptibility to breakdown during gastrointestinal transit and low intestinal permeability are key challenges in the development of highly bioactive food protein hydrolysates/peptides. However, the generation of potent and effective health enhancing hydrolysates/peptides can benefit from a range of in silico techniques including the application of structure bioactivity relationship modelling (e.g., quantitative structure activity relationship (QSAR) modelling), molecular docking and design of experiments (DOE) approaches to optimise BAP production and identification. Some examples of how these approaches have been employed in BAP discovery and generation will be outlined

In vitro Characterisation of the antioxidative properties of whey protein hydrolysates generated under pH- and non pH-controlled conditions

Bovine whey protein concentrate (WPC) was hydrolysed under pH-stat (ST) and non pH-controlled (free-fall, FF) conditions using Debitrase (DBT) and FlavorPro Whey (FPW). The resultant whey protein hydrolysates (WPHs) were assessed for the impact of hydrolysis conditions on the physicochemical and the in vitro antioxidant and intracellular reactive oxygen species (ROS) generation in oxidatively stressed HepG2 cells. Enzyme and hydrolysis condition dependent differences in the physicochemical properties of the hydrolysates were observed, however, the extent of hydrolysis was similar under ST and FF conditions. Significantly higher (p < 0.05) in vitro and cellular antioxidant activities were observed for the DBT compared to the FPW–WPHs. The WPHs generated under ST conditions displayed significantly higher (p < 0.05) oxygen radical absorbance capacity (ORAC) and Trolox equivalent antioxidant capacity (TEAC) values compared to the FF-WPHs. The impact of hydrolysis conditions was more pronounced in the in vitro compared to the cellular antioxidant assay. WPH peptide profiles (LC-MS/MS) were also enzyme and hydrolysis conditions dependent as illustrated in the case of .B-lactoglobulin. Therefore, variation in the profiles of the peptides released may explain the observed differences in the antioxidant activity. Targeted generation of antioxidant hydrolysates needs to consider the hydrolysis conditions and the antioxidant assessment method employed

Contribution of hydrolysis and drying conditions to whey protein hydrolysate characteristics and in vitro antioxidative properties

During the generation of functional food ingredients by enzymatic hydrolysis, parameters such as choice of enzyme, reaction pH and the drying process employed may contribute to the physicochemical and bio-functional properties of the resultant protein hydrolysate ingredients. This study characterised the properties of spray- (SD) and freeze-dried (FD) whey protein hydrolysates (WPHs) generated using Alcalase® and Prolyve® under pH-stat and free-fall pH conditions. The enzyme preparation used affected the physicochemical and antioxidative properties but had no impact on powder composition, morphology or colour. SD resulted in spherical particles with higher moisture content (~6%) compared to the FD powders (~1%), which had a glass shard-like structure. The SD-WPHs exhibited higher antioxidative properties compared to the FD-WPHs, which may be linked to a higher proportion of peptides <1 kDa in the SD-WPHs. Furthermore, the SD- and FD-WPHs had similar peptide profiles, and no evidence of Maillard reaction product formation during the SD processing was evident. The most potent in vitro antioxidative WPH was generated using Alcalase® under free-fall pH conditions, followed by SD, which had oxygen radical absorbance capacity and Trolox equivalent (TE) antioxidant capacity values of 1132 and 686 µmol TE/g, respectively. These results demonstrate that both the hydrolysis and the drying process impact the biofunctional (antioxidant) activity of WPHs

Effect of enzymatically hydrolysed brewers’ spent grain supplementation on the rheological, textural and sensory properties of muffins

Brewers’ spent grain (BSG) is the major co-product of the brewing industry having a high content of protein and fibre. Enzymatic modification improves the properties of different ingredients when they are incorporated into confectionary products. This study characterised the rheological, textural and sensory properties of unmodified BSG (BSGA) and enzymatically modified BSG (BSGB, i.e., proteinase and carbohydrase modified BSG) when substituted at different levels (5, 10 and 15%) in a muffin ingredient mix. Rheological assessment showed a lower viscosity for the BSGB compared to the BSGA batters. Baked BSGB containing muffins had lower hardness values compared to BSGA and control without added BSG. Sensory assessment showed no significant differences in liking of the different attributes tested (overall appearance, texture, smell, colour and taste) between the 5% BSGA and BSGB supplemented muffins. Incorporation of BSGB at 10% had no negative impact on the sensory attributes in comparison to BSGA 10%. Overall, low level substitution of enzymatic modified BSG beneficially reduced batter viscosity and muffin hardness and gave comparable sensory attributes to those of unmodified BSG supplemented muffins. Therefore, prior enzymatic modification can enhance the ingredient functionality of BSG in confectionary products