Production, characterization, and potential applications of lipopeptides in food systems: A comprehensive review.

Lipopeptides are a class of lipid-peptide-conjugated compounds with differing structural features. This structural diversity is responsible for their diverse range of biological properties, including antimicrobial, antioxidant, and anti-inflammatory activities. Lipopeptides have been attracting the attention of food scientists due to their potential as food additives and preservatives. This review provides a comprehensive overview of lipopeptides, their production, structural characteristics, and functional properties. First, the classes, chemical features, structure-activity relationships, and sources of lipopeptides are summarized. Then, the gene expression and biosynthesis of lipopeptides in microbial cell factories and strategies to optimize lipopeptide production are discussed. In addition, the main methods of purification and characterization of lipopeptides have been described. Finally, some biological activities of the lipopeptides, especially those relevant to food systems along with their mechanism of action, are critically examined.

Positional Distribution of Fatty Acids in Processed Chinook Salmon Roe Lipids Determined by 13C Magnetic Resonance Spectroscopy (NMR).

Recently, there has been great interest in the lipidomic of marine lipids and their potential health benefits. Processing of seafood products can potentially modify the characteristics and composition of lipids. The present study investigated the effect of processing methods (salting and fermentation) on the positional distribution of fatty acids of Chinook salmon roe using 13C nuclear magnetic resonance spectroscopy (NMR). The NMR analysis provided information on the carbonyl atom, double bond/olefinic, glycerol backbone, aliphatic group, and chain ending methyl group regions. The obtained data showed that docosahexaenoic acid (DHA) is the main fatty acid esterified at the sn-2 position of the triacylglycerides (TAGs), while other fatty acids, such as eicosapentaenoic acid (EPA) and stearidonic acid (SDA), were randomly distributed or preferentially esterified at the sn-1 and sn-3 positions. Fermentation of salmon roe was found to enrich the level of DHA at the sn-2 position of the TAG. The processing of roe by both salt drying and fermentation did not appear to affect the proportion of EPA at the sn-2 position. This present study demonstrated that fish roe processing can enhance the proportion of DHA at the sn-2 position and potentially improve its bioavailability.

Marine Fish-Derived Lysophosphatidylcholine: Properties, Extraction, Quantification, and Brain Health Application.

Long-chain omega-3 fatty acids esterified in lysophosphatidylcholine (LPC-omega-3) are the most bioavailable omega-3 fatty acid form and are considered important for brain health. Lysophosphatidylcholine is a hydrolyzed phospholipid that is generated from the action of either phospholipase PLA1 or PLA2. There are two types of LPC; 1-LPC (where the omega-3 fatty acid at the sn-2 position is acylated) and 2-LPC (where the omega-3 fatty acid at the sn-1 position is acylated). The 2-LPC type is more highly bioavailable to the brain than the 1-LPC type. Given the biological and health aspects of LPC types, it is important to understand the structure, properties, extraction, quantification, functional role, and effect of the processing of LPC. This review examines various aspects involved in the extraction, characterization, and quantification of LPC. Further, the effects of processing methods on LPC and the potential biological roles of LPC in health and wellbeing are discussed. DHA-rich-LysoPLs, including LPC, can be enzymatically produced using lipases and phospholipases from wide microbial strains, and the highest yields were obtained by Lipozyme RM-IM®, Lipozyme TL-IM®, and Novozym 435®. Terrestrial-based phospholipids generally contain lower levels of long-chain omega-3 PUFAs, and therefore, they are considered less effective in providing the same health benefits as marine-based LPC. Processing (e.g., thermal, fermentation, and freezing) reduces the PL in fish. LPC containing omega-3 PUFA, mainly DHA (C22:6 omega-3) and eicosapentaenoic acid EPA (C20:5 omega-3) play important role in brain development and neuronal cell growth. Additionally, they have been implicated in supporting treatment programs for depression and Alzheimer's. These activities appear to be facilitated by the acute function of a major facilitator superfamily domain-containing protein 2 (Mfsd2a), expressed in BBB endothelium, as a chief transporter for LPC-DHA uptake to the brain. LPC-based delivery systems also provide the opportunity to improve the properties of some bioactive compounds during storage and absorption. Overall, LPCs have great potential for improving brain health, but their safety and potentially negative effects should also be taken into consideration.

PHNQ from Evechinus chloroticus Sea Urchin Supplemented with Calcium Promotes Mineralization in Saos-2 Human Bone Cell Line.

Polyhydroxylated naphthoquinones (PHNQs), known as spinochromes that can be extracted from sea urchins, are bioactive compounds reported to have medicinal properties and antioxidant activity. The MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell viability assay showed that pure echinochrome A exhibited a cytotoxic effect on Saos-2 cells in a dose-dependent manner within the test concentration range (15.625-65.5 µg/mL). The PHNQ extract from New Zealand sea urchin Evechinus chloroticus did not induce any cytotoxicity within the same concentration range after 21 days of incubation. Adding calcium chloride (CaCl2) with echinochrome A increased the number of viable cells, but when CaCl2 was added with the PHNQs, cell viability decreased. The effect of PHNQs extracted on mineralized nodule formation in Saos-2 cells was investigated using xylenol orange and von Kossa staining methods. Echinochrome A decreased the mineralized nodule formation significantly (p < 0.05), while nodule formation was not affected in the PHNQ treatment group. A significant (p < 0.05) increase in mineralization was observed in the presence of PHNQs (62.5 µg/mL) supplemented with 1.5 mM CaCl2. In conclusion, the results indicate that PHNQs have the potential to improve the formation of bone mineral phase in vitro, and future research in an animal model is warranted.

Marine omega-3 (n-3) phospholipids: A comprehensive review of their properties, sources, bioavailability, and relation to brain health.

For several decades, there has been considerable interest in marine-derived long chain n-3 fatty acids (n-3 LCPUFAs) due to their outstanding health benefits. n-3 LCPUFAs can be found in nature either in triglycerides (TAGs) or in phospholipid (PL) form. From brain health point of view, PL n-3 is more bioavailable and potent compared to n-3 in TAG form, as only PL n-3 is able to cross the blood-brain barrier and can be involved in brain biochemical reactions. However, PL n-3 has been ignored in the fish oil industry and frequently removed as an impurity during degumming processes. As a result, PL products derived from marine sources are very limited compared to TAG products. Commercially, PLs are being used in pharmaceutical industries as drug carriers, in food manufacturing as emulsifiers and in cosmetic industries as skin care agents, but most of the PLs used in these applications are produced from vegetable sources that contain less (without EPA, DPA, and DHA) or sometimes no n-3 LCPUFAs. This review provides a comprehensive account of the properties, structures, and major sources of marine PLs, and provides focussed discussion of their relationship to brain health. Epidemiological, laboratory, and clinical studies on n-3 LCPUFAs enriched PLs using different model systems in relation to brain and mental health that have been published over the past few years are discussed in detail.

Modifying the Functional Properties of Egg Proteins Using Novel Processing Techniques: A Review.

Egg proteins can be used in a wide range of food products, owing to their excellent foaming, emulsifying, and gelling properties. Another important functional property is the susceptibility of egg proteins to enzymatic hydrolysis, as protein digestion is closely related to its nutritional value. These functional properties of egg proteins are likely to be changed during food processing. Conventional thermal processing can easily induce protein denaturation and aggregation and consequently reduce the functionality of egg proteins due to the presence of heat-labile proteins. Accordingly, there is interest from the food industry in seeking novel nonthermal or low-thermal techniques that sustain protein functionality. To understand how novel processing techniques, including high hydrostatic pressure, pulsed electric fields, ionizing radiation, ultraviolet light, pulsed light, ultrasound, ozone, and high pressure homogenization, affect protein functionality, this review introduces the mechanisms involved in protein structure modification and describes the structure-functionality relationships. Novel techniques differ in their mechanisms of protein structure modification and some have been shown to improve protein functionality for particular treatment conditions and product forms. Although there is considerable industrial potential for the use of novel techniques, further studies are required to make them a practical reality, as the processing of egg proteins often involves other influencing factors, such as different pH and the presence of other food additives (for example, salts, sugar, and polysaccharides).

Bioactive peptides derived from egg proteins: A review.

Egg proteins have various functional and biological activities which make them potential precursor proteins for bioactive peptide production. Simulated in vitro gastrointestinal digestion and enzymatic hydrolysis using non-gastrointestinal proteases have been used as tools to produce these peptides. Bioactive peptides derived from egg proteins are reported to display various biological activities, including angiotensin I-converting enzyme (ACE) inhibitory (antihypertensive), antioxidant, antimicrobial, anti-inflammatory, antidiabetic and iron-/calcium-binding activities. More importantly, simulated in vitro gastrointestinal digestion has indicated that consumption of egg proteins has physiological benefits due to the release of such multifunctional peptides. This review encompasses studies reported to date on the bioactive peptide production from egg proteins.

Do Dairy Minerals Have a Positive Effect on Bone Health?

This review aims to examine the relationship between the consumption of dairy products, mineral absorption, and bone health, and critically evaluates the methods that have been used to investigate this relationship. As people live longer and have lives that are more active in modern societies, bone health is of concern due to the possibility for the increasing incidence of bone disorders, such as osteoporosis. It has been suggested that dairy products can play a key role in bone health due to their high levels of minerals. Whether the positive effect of dairy consumption on bone health is due solely to the concentration of minerals, the action of vitamins, proteins, and lipids present in dairy products, and complex interactions between different milk components remains to be determined. Assessment of how dairy products affect bone health is complex, with apparent contradictory conclusions being reported in the literature. To gain a better understanding of the effects that dairy products have on bone health, this review presents an evaluation of a combination of data obtained using a variety of methods. From those data, we surmise that the preferable approach to investigate the effects of milk on bone health is to obtain data from human, animal, and cell line testing. A combined approach will enable various aspects to be identified, including mechanisms and the assessment of holistic effects, which will enable the effects in the human situation to be ascertained.

Salivaricin E and abundant dextranase activity may contribute to the anti-cariogenic potential of the probiotic candidate Streptococcus salivarius JH.

Dental caries is an infectious disease that is continuing to increase in prevalence, reducing the quality of life for millions worldwide as well as causing considerable expense, with an estimated US$108 billion spent on dental care in the USA each year. Oral probiotics are now being investigated to determine whether they could play a role in the prevention and treatment of this disease. Streptococcus salivarius strain JH is a potential probiotic candidate that produces multiple proteinaceous antimicrobials (bacteriocins), the inhibitory spectrum of which includes Streptococcus mutans, one of the principal causative agents of dental caries. The genome of strain JH has previously been shown to contain the biosynthetic loci for the bacteriocins salivaricin A3, streptin and streptococcin SA-FF22. Here we show that strain JH also produces salivaricin E, a 32 aa lantibiotic with a mass of 3565.9 Da, which is responsible for the inhibition of S. mutans growth. In addition, strain JH was shown to produce dextranase, an enzyme that hydrolyses (1 → 6)-α-D-glucosidic linkages, at levels higher than any other S. salivarius tested. In vitro testing showed that partial hydrolysis of the exopolymeric substances of S. mutans, using strain JH dextranase, improved the anti-S. mutans inhibitory activity of the lytic bacteriocin, zoocin A. The multiple bacteriocin and dextranase activities of strain JH support its candidature for development as an oral probiotic.

Composition and biological activities of slaughterhouse blood from red deer, sheep, pig and cattle.

BACKGROUND: Animal blood is a large-volume by-product of the meat industry. Besides blood meal fertiliser, blood is marketed for human consumption as a supplement. Minimal comparative work on slaughterhouse animal blood fractions has been carried out. In this study, slaughterhouse deer, sheep, pig and cattle blood parameters were compared. Some blood constituents were determined. Fractionated blood was assessed for antioxidant activity (2,2-diphenyl-1-picrylhydrazyl radical scavenging, oxygen radical scavenging capacity and ferric reducing antioxidant power). Angiotensin converting enzyme (ACE) inhibitory activity and antimicrobial activity were also assessed. RESULTS: Serum iron ranged from 35.3 ± 0.6 µmol L(-1) in cattle to 16.3 ± 3.1 µmol L(-1) in deer. Cattle had the highest total plasma proteins (81.7 ± 1.5 g L(-1)). While the plasma fractions contained considerable antioxidant activity, the red blood cell fractions of all four animal species contained higher antioxidant activity (P < 0.05). Negligible levels of ACE inhibitory activity were found for all animal blood fractions. Antimicrobial activity was detected towards Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa with sheep white blood cells from which a crude neutrophil extract was obtained which demonstrated concentration-dependent inhibitory effects on the growth rates of these bacterial strains. CONCLUSION: Fractionated animal blood obtained from local slaughterhouses contains native proteins that possess antioxidant activity and antimicrobial activity.

Identification and characterization of a bacitracin resistance network in Enterococcus faecalis.

Resistance of Enterococcus faecalis against antimicrobial peptides, both of host origin and produced by other bacteria of the gut microflora, is likely to be an important factor in the bacterium's success as an intestinal commensal. The aim of this study was to identify proteins with a role in resistance against the model antimicrobial peptide bacitracin. Proteome analysis of bacitracin-treated and untreated cells showed that bacitracin stress induced the expression of cell wall-biosynthetic proteins and caused metabolic rearrangements. Among the proteins with increased production, an ATP-binding cassette (ABC) transporter with similarity to known peptide antibiotic resistance systems was identified and shown to mediate resistance against bacitracin. Expression of the transporter was dependent on a two-component regulatory system and a second ABC transporter, which were identified by genome analysis. Both resistance and the regulatory pathway could be functionally transferred to Bacillus subtilis, proving the function and sufficiency of these components for bacitracin resistance. Our data therefore show that the two ABC transporters and the two-component system form a resistance network against antimicrobial peptides in E. faecalis, where one transporter acts as the sensor that activates the TCS to induce production of the second transporter, which mediates the actual resistance.

Effect of consumption of sheep and cow milk on rat brain fatty acid and phospholipid composition.

The effect of sheep milk and cow milk on the lipid composition of rat brain was investigated in two feeding experiments of 28-days duration. Total lipids of the rat brain were extracted using ethanol-hexane, and the fatty acids and phospholipid contents analysed using gas chromatography with flame ionization detection (GC-FID) and phosphorus-31 nuclear magnetic resonance (31P NMR). Furthermore, freeze-dried pooled samples were analysed using attenuated total reflectance Fourier Transform Infrared and Fourier Transform Raman Spectroscopy and analysed with multivariate methods. A significantly (P < 0.05) higher C18:2 content was found in the cow milk group compared with sheep milk-treated groups in Study one. In Study two, a significantly (P < 0.05) lower C16:0 content was present in the sheep milk-treated group compared to the control low Ca/P group. No significant (P > 0.05) differences were observed in the spectroscopy analyses. It is concluded that sheep and cow milks fed to rats for 28-days had a low effect on the brain lipidome.

Physicochemical, biochemical and microbiological changes of jeotgal-like fermented Chinook salmon (Oncorhynchus tshawytscha) roe.

A jeotgal-like product was processed from Chinook salmon (Oncorrhynchus tshawytscha) roe. Physicochemical, biochemical, and microbiological compositions were studied during 30 days of fermentation. Fermentation decreased water activity (aw) and pH value. Total bacterial and LAB counts (log CFU/g) increased up to 12 days of processing and then no further changes occurred. Saturated fatty acids (SFA) decreased (p < 0.05), monounsaturated fatty acids (MUFA) did not change (p > 0.05), whereas fermentation time improved polyunsaturated fatty acids (PUFA) content significantly (p < 0.05). Astaxanthin, lutein and phospholipids (PC, LPC, PE, LPE and LPS) concentrations were found to increase, while cholesterol and tocopherol contents were decreased at the end of the fermentation (p < 0.05). This study indicates that the nutritional value of salmon roe can be enhanced by fermentation.

Earth Worming-An Evaluation of Earthworm (Eisenia andrei) as an Alternative Food Source.

Aside from their bioremediation roles, little is known about the food and feed value of earthworms. In this study, a comprehensive evaluation of the nutritional composition (proximate analysis and profiles of fatty acids and minerals) and techno-functional properties (foaming and emulsion stability and capacity) of earthworm (Eisenia andrei, sourced in New Zealand) powder (EAP) were investigated. Lipid nutritional indices, ω6/ω3, atherogenicity index, thrombogenicity index, hypocholesterolemic/hypercholesterolemic acid ratio, and health-promoting index of EAP lipids are also reported. The protein, fat, and carbohydrate contents of EAP were found to be 53.75%, 19.30%, and 23.26% DW, respectively. The mineral profile obtained for the EAP consisted of 11 essential minerals, 23 non-essential minerals, and 4 heavy metals. The most abundant essential minerals were potassium (8220 mg·kg-1 DW), phosphorus (8220 mg·kg-1 DW), magnesium (744.7 mg·kg-1 DW), calcium (2396.7 mg·kg-1 DW), iron (244.7 mg·kg-1 DW), and manganese (25.6 mg·kg-1 DW). Toxic metals such as vanadium (0.2 mg·kg-1 DW), lead (0.2 mg·kg-1 DW), cadmium (2.2 mg·kg-1 DW), and arsenic (2.3 mg·kg-1 DW) were found in EAP, which pose safety considerations. Lauric acid (20.3% FA), myristoleic acid (11.20% FA), and linoleic acid (7.96% FA) were the most abundant saturated, monounsaturated, and polyunsaturated fatty acids, respectively. The lipid nutritional indices, such as IT and ω-6/ω-3, of E. andrei were within limits considered to enhance human health. A protein extract derived from EAP (EAPPE), obtained by alkaline solubilisation and pH precipitation, exhibited an isoelectric pH of ~5. The total essential amino acid content and essential amino acid index of EAPPE were 373.3 mg·g-1 and 1.36 mg·g-1 protein, respectively. Techno-functional analysis of EAPPE indicated a high foaming capacity (83.3%) and emulsion stability (88.8% after 60 min). Heat coagulation of EAPPE was greater at pH 7.0 (12.6%) compared with pH 5.0 (4.83%), corroborating the pH-solubility profile and relatively high surface hydrophobicity (1061.0). These findings demonstrate the potential of EAP and EAPPE as nutrient-rich and functional ingredients suitable as alternative food and feed material. The presence of heavy metals, however, should be carefully considered.

Physicochemical Characteristics, Techno-Functionalities, and Amino Acid Profile of Prionoplus reticularis (Huhu) Larvae and Pupae Protein Extracts.

The amino acid profile, techno-functionalities (foaming stability/capacity, emulsion stability/capacity, solubility, and coagulation), and physicochemical characteristics (colour, particle size, surface hydrophobicity, Fourier-transform infrared spectroscopy, and differential scanning calorimetry) of protein extracts (PE) obtained from Prionoplus reticularis (Huhu grub) larvae (HLPE) and pupae (HPPE) were investigated. Total essential amino acid contents of 386.7 and 411.7 mg/g protein were observed in HLPE and HPPE, respectively. The essential amino acid index (EAAI) was 3.3 and 3.4 for HLPE and HPPE, respectively, demonstrating their nutritional equivalence. A unique nitrogen-to-protein conversion constant, k, and the corresponding protein content of the extracts were 6.1 and 6.4 and 72.1% and 76.5%, respectively. HLPE (37.1 J/g) had a lower enthalpy than HPPE (54.1 J/g). HPPE (1% w/v) exhibited a foaming capacity of 50.7%, which was higher than that of HLPE (41.7%) at 150 min. The foaming stability was 75.3% for HLPE and 73.1% for HPPE after 120 min. Both protein extracts (1% w/v) had emulsifying capacities that were 96.8% stable after 60 min. Therefore, protein extracts from Huhu larvae and pupae are of a good nutritional quality (based on their EAAI) and have techno-functional properties, such as foaming and emulsification, that afford them potential for certain food technology applications.

A simple method for enrichment of ß-lactoglobulin from bovine milk whey involving selective hydrolysis by two fungal protease preparations.

Two commercially available food grade fungal protease preparations (Fungal Protease 31,000 and Fungal Protease 60000) were found to hydrolyse bovine acid whey proteins but left the beta-lactoglobulin (ß-Lg) intact under the processing conditions used. Comparative analysis before and after hydrolysis of bovine acid whey, by 1D- and 2D-PAGE, RP-HPLC and intact-mass mass spectrometry showed that the ß-Lg remains intact and in high yield after hydrolysis by the fungal proteases. The ß-Lg could be separated from the whey protein peptide hydrolysate by ultrafiltration. Subjecting whey fraction to hydrolysis with the fungal protease preparations provides a procedure, under relatively mild conditions, to generate a highly enriched ß-Lg fraction. ß-Lg is recognised as a valued material in the food, pharmaceutical and cosmetic industries due to its properties such as gelling and foaming. The enriched ß-Lg preparation would also have application in areas such as nanoencapsulation.

The effect of pulsed electric fields on the extracted total lipid yield and the lipidomic profile of hoki roe.

The effect of pulsed electric fields (PEF) at different field strengths (0.62, 1.25, 1.875 kV/cm) and frequencies (25, 50, 100 Hz) on total lipid extraction from hoki roe was investigated, along with the lipidomic profile (total lipid, phospholipid, fatty acid, phospholipid composition, and positional distribution of EPA and DHA). High PEF input (112 kJ/kg, 1.875 kV/cm and 100 Hz) yielded the highest total lipid (16.2% wet weight (WW)), and phospholipid (46 µmol/g WW) contents, without affecting n-3 fatty acid content (32%), and generated the highest LDPG, LPE, LPS and LPC contents (1.1, 0.41, 6.13 and 2.15 µmol/g WW, respectively). However, this PEF treatment resulted in sn-2 phospholipid EPA and DHA to be relocated to the sn-1,3 positions. Despite the good yield of n-3 fatty acids and PL, high PEF intensity treatment was found to result in negative structural changes in hoki roe lipids.

Use of fungal and bacterial protease preparations to enhance extraction of lipid from fish roe: Effect on lipidomic profile of extracted oil.

The present study investigated the hydrolysis of protein in hoki roe homogenate using a HT (bacterial), a FP-II (fungal) protease preparations and Alcalase (bacterial) to enhance lipid yield extraction. The degree of hydrolysis was determined at various pH, temperature and time using casein and hoki roe. Total lipid extraction and lipidomic analysis was carried out following proteolysis of hoki roe homogenate. The degree of hydrolysis and SDS-PAGE revealed that the hydrolytic capability of Alcalase was better than HT and FPII. The total extracted lipid yield was better following hydrolysis with Alcalase (19.29 %), compared to HT (18.29 %) and FPII (18.33 %). However, the total phospholipid (PL) and n-3 fatty acid yields were better from HT hydrolysed hoki roe homogenate (PL = 30.7 µmol/g; n-3 = 10.5 %), compared to Alcalase (PL = 22 µmol/g; n-3 = 5.95 %). Overall, this study indicates that HT protease preparation hydrolysis of fish roe homogenate can both enhance lipid extraction and retain lipid quality.

Effect of Pulsed Electric Fields on the Lipidomic Profile of Lipid Extracted from Hoki Fish Male Gonad.

Processing of hoki, a commercially important fish species, generates substantial quantities of co-products, including male gonad, which contains valuable lipids, such as phospholipids, that could be recovered and utilised. Hoki fish male gonads (HMG) were subjected to pulsed electric fields (PEF) treatment at varying field strengths (0.625, 1.25, and 1.875 kV/cm) and frequencies (25, 50, and 100 Hz), at a fixed pulse width of 20 µs. The total lipid was extracted using an ethanol-hexane-based (ETHEX) extraction method, and the phospholipid and fatty acid compositions were determined using 31P NMR and GC-FID, respectively. The total lipid yield was increased from 4.1% to 6.7% by a relatively mild PEF pre-treatment at a field strength of 1.25 kV/cm and frequency of 50 Hz. A higher amount of EPA (8.2%), DPA (2.7%), and DHA (35.7%) were obtained by that treatment, compared to both un-heated (EPA: 8%; DPA: 2.5%; DHA: 35.2%) and heat-treated controls (EPA: 7.9%; DPA: 2.5%; DHA: 34%). No significant changes to the content of the major phospholipids were observed. PEF pre-treatment under mild conditions has potential for improving the total lipid yield extracted from fish male gonad.

Analysis of peptides in a sheep beta lactoglobulin hydrolysate as a model to evaluate the effect of peptide amino acid sequence on bioactivity.

Fungal protease FPII was found to hydrolyse sheep ß-lactoglobulin (ß-Lg), and the hydrolysate exhibited substantial antioxidant and ACE inhibition bioactivities. From analysis of the peptide sequences in the hydrolysate in relation to bioactivity, synthetic peptides corresponding to four regions of sequence in ß-Lg (LAFNPTQLEGQCHV, DTDYKKYLLF, LDAQSAPLRVY and VEELKPTPE) were analysed for bioactivity. Additional synthetic peptides were designed to examine the bioactivity of different parts of the above four sequences, and the effect of amino acid substitutions on bioactivity. The results show that parts of the peptide sequences contribute differently to bioactivity and substitution of amino acids has a substantial effect on antioxidant and ACE inhibition activities.