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

Seaweed (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 that 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 characterization 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.

Enzymatic Modification of Porphyra dioica-Derived Proteins to Improve their Antioxidant Potential.

Enzymatic hydrolysis has been employed to modify protein functional properties and discover new sources of antioxidants. In this study, the effect of different enzymatic treatments on antioxidant activity of Porphyra dioica (blades and protein isolate (PI)) was investigated. Protein nitrogen content of P. dioica blades and PI were 23 and 50% (dry weight), respectively. Blades and PI were hydrolyzed with Prolyve® and Prolyve® plus Flavourzyme®. Peptide profiles and molecular mass distribution of the hydrolysates were investigated. The hydrolysis promoted generation of peptides and low molecular mass components <1 kDa. Antioxidant activity was assessed using ferric reducing antioxidant power (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH·) scavenging, 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS·+) inhibition, and reactive oxygen species scavenging ability, i.e., oxygen radical absorbance capacity (ORAC) and hypochlorous acid (HOCl) scavenging assays. In general, enzymatic hydrolysis of P. dioica blades and PI enhanced the in vitro antioxidant activity. Direct hydrolysis of blades improved ORAC values up to 5-fold (from 610 to 3054 µmol Trolox eq./g freeze dried sample (FDS). The simultaneous release of phenolic compounds suggested a potential synergistic activity (ORAC and ABTS·+ assays). Such hydrolysates may be of value as functional food ingredients.

Volatile components and sensory characteristics of Thai traditional fermented shrimp pastes during fermentation periods.

Headspace-volatile components and sensory characteristics, including color, Maillard reaction products and free amino acid profiles, of two types of Thai traditional fermented shrimp paste, Kapi Ta Dam and Kapi Ta Deang, were investigated during the fermentation periods up to 6 months. The results showed that the colors of both products were changed with a decrease in CIELAB values over the fermentation period, except for yellowness of Kapi Ta Deang. Essential amino acids such as lysine and leucine and non-essential amino acids such as glutamic acid and alanine were found to be predominant free-amino acids in the products. After headspace volatile component extraction of the product was carried out using a SPME fiber coated with DVB/CAR/PDMS and analyzed by GC-MS, the main compounds responsible for distinct volatiles in the products were N-containing compounds, especially pyrazines which give roasted nutty odor. The results of sensory evaluation from panelists also suggest that fermentation period had an effect on sensory characteristics of the fermented shrimp pastes. Moreover, the sensory perceptions of the products would associate with their color, the Maillard reaction products, amino acid profiles and volatile compounds.

Characterization and in vitro biological activities of Thai traditional fermented shrimp pastes.

In this work, chemical and biological characteristics of two types of Thai fermented shrimp paste, Kapi Ta Dam and Kapi Ta Deang, at different fermentation periods and their raw materials were investigated. Kapi had low water activity and high proteins with high glutamic acid and lysine. Both Kapis, which had different sources, showed similar characteristics. The number of lactic acid bacteria in the products increased during the early stages of fermentation. Free α-amino acid contents in the products increased with the fermentation time. The water extracts from Kapi products showed strong antioxidative activities against ABTS(+) radical, and ACE inhibitory activity but they did not exhibit antimicrobial activity against Staphylococcus aureus, Bacillus cereus, Escherichia coli and Salmonella Typhimurium. Biological activities in Kapi could be developed by fermentation process, enzymatic hydrolysis of proteins and non-enzymatic browning reactions. Kapi could, thus, serve as a potential source of natural bioactive substances.

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.

Enzyme-Assisted Release of Antioxidant Peptides from Porphyra dioica Conchocelis.

The 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.

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 ß-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.

Effect of in vitro simulated gastrointestinal digestion on the antioxidant activity of the red seaweed Porphyra dioica.

Porphyra sp. is one of the most cultivated and commercially valuable species, recognized for its high protein content (up to 47% dry weight) and complete amino acids profile. Based on these characteristics, P. dioica produced in an integrated multitrophic aquaculture system was selected for this study. The aim was to evaluate the effect of in vitro simulated gastrointestinal digestion (SGID) on the antioxidant activity of the hydrolysates generated from dried blades and from the protein isolate (PI) extracted from them. The alkali extraction and isoelectric precipitation (pH 4.5) of P. dioica protein prior SGID allowed isolating/enriching protein, while direct SGID of blades allowed assessing the potential influence of other constituents of the sample on the bioactive properties. Overall, SGID promoted the release of smaller bioactive peptides and their in vitro antioxidant activity, which was assessed by different methods (DPPH and ABTS+ scavenging capacity, ORAC and FRAP), was improved compared to the intact samples. Blades submitted to direct SGID presented significantly higher ORAC values compared to PI (2010 ± 136 vs 542 ± 21 µmol TE/g FDS, respectively). For the remaining assays, PI presented more potent antioxidant activity, especially FRAP (131 ± 2 vs 16 ± 1 µmol TE/g FDS) and ABTS+ (1244 ± 157 vs 230 ± 15 µmol TE/g FDS). The results indicated that gastrointestinal digestion improved the antioxidant activity of P. dioica-derived hydrolysates, as they presented effective activity against different oxidative mechanisms, thus suggesting health-protecting effects.

Extraction of antioxidant and ACE inhibitory peptides from Thai traditional fermented shrimp pastes.

Antioxidant and angiotensin converting enzyme (ACE) inhibitory peptides were extracted and isolated from two different types of Thai traditional fermented shrimp pastes, Kapi Ta Dam (Kp-B6) and Kapi Ta Deang (Kp-R6). Compounds with masses less than 500Da were found to be predominantly presented in both extracts. Following fractionation with sequential anion exchange chromatography and solid phase extraction (C18 matrix), three dipeptides were identified. Ser-Val and Ile-Phe were shown to exhibit ACE inhibitory activity with IC50 values of 60.68±1.06 and 70.03±1.45µM, respectively. Trp-Pro was shown to have high 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) radical scavenging activity (EC50 17.52±0.46µM). These results indicate that Thai traditional fermented shrimp pastes are potential sources of bioactive peptides possessing ACE inhibitory and antioxidant activities.