Whey-pectin microcapsules improve the stability of grape marc phenolics during digestion.

Grape marc (GM) is an agri-food residue from the wine industry valuable for its high content of phenolic compounds. This study aimed to develop an encapsulation system for GM extract (GME) using food-grade biopolymers resistant to gastric conditions for its potential use as a nutraceutical. For this purpose, a hydroalcoholic GME was prepared with a total phenolics content of 219.62 ± 11.50 mg gallic acid equivalents (GAE)/g dry extract and 1389.71 ± 97.33 µmol Trolox equivalents/g dry extract antioxidant capacity, assessed through ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assay. Moreover, the extract effectively neutralized reactive oxygen species in Caco-2 cells, demonstrating an intracellular antioxidant capacity comparable to Trolox. The GME was encapsulated using whey protein isolate and pectin through nano spray drying (73% yield), resulting in spherical microparticles with an average size of 1 ± 0.5 µm and a polydispersity of 0.717. The encapsulation system protected the microcapsules from simulated gastrointestinal digestion (GID), where at the end of the intestinal phase, 82% of the initial phenolics were bioaccessible compared to 54% in the free GME. Besides, the encapsulated GME displayed a higher antioxidant activity by the ferric reducing antioxidant power assay than the free extract after GID. These results show the potential of this encapsulation system for applying GME as a nutraceutical with a high antioxidant capacity and protective effect against cellular oxidation.

H4K5 Butyrylation Coexist with Acetylation during Human Spermiogenesis and Are Retained in the Mature Sperm Chromatin.

Male germ cells experience a drastic chromatin remodeling through the nucleo-histone to nucleo-protamine (NH-NP) transition necessary for proper sperm functionality. Post-translational modifications (PTMs) of H4 Lys5, such as acetylation (H4K5ac), play a crucial role in epigenetic control of nucleosome disassembly facilitating protamine incorporation into paternal DNA. It has been shown that butyrylation on the same residue (H4K5bu) participates in temporal regulation of NH-NP transition in mice, delaying the bromodomain testis specific protein (BRDT)-dependent nucleosome disassembly and potentially marking retained nucleosomes. However, no information was available so far on this modification in human sperm. Here, we report a dual behavior of H4K5bu and H4K5ac in human normal spermatogenesis, suggesting a specific role of H4K5bu during spermatid elongation, coexisting with H4K5ac although with different starting points. This pattern is stable under different testicular pathologies, suggesting a highly conserved function of these modifications. Despite a drastic decrease of both PTMs in condensed spermatids, they are retained in ejaculated sperm, with 30% of non-colocalizing nucleosome clusters, which could reflect differential paternal genome retention. Whereas no apparent effect of these PTMs was observed associated with sperm quality, their presence in mature sperm could entail a potential role in the zygote.

Natural deep eutectic solvents as a green extraction of polyphenols from spent coffee ground with enhanced bioactivities.

Conventional extraction techniques are usually based on highly pollutant and/or flammable organic solvents. Therefore, alternative environmentally friendly extraction methods are of particular interest for the recovery of bioactive compounds for their application as food ingredients and/or nutraceuticals. Natural deep eutectic solvents (NADES) are a green and nontoxic attractive alternative to hydroalcoholic extraction. NADES media primarily depends on the intermolecular interactions (hydrogen bonding) among their components to form a eutectic mixture with a much lower final melting point than its individual components. Examples of natural deep eutectic NADES solvents include aqueous solutions (25%-50% water) of choline chloride, sugars, and polyols. This study aimed to investigate the application of two NADES, namely, betaine:triethylene glycol (Bet : TEG) and choline chloride:1,2-propanediol (Chol : Prop), as sustainable green solvents for the extraction of polyphenols from spent coffee ground (SCG), a by-product of coffee processing. In particular, the extraction yield and selectivity were evaluated and compared with conventional green extractions (hot water and a hydroalcoholic solution). In addition, the effect of NADES on the antioxidant and antimicrobial activity of the extracts was investigated. The main outcomes were as follows: (i) NADES were as effective as other conventional green solvents in the extraction of polyphenols with the added advantage of operating at milder temperature conditions, without flammable solvents and with sustainable and natural compounds; (ii) the antimicrobial activity of the NADES extracts was 10 times higher than that of the ethanolic and aqueous extracts. Given the low toxicity of NADES, they could be used as formulation aid for food ingredients.

Whey-Derived Peptides at the Heart of the COVID-19 Pandemic.

The renin-angiotensin system (RAS) is a key regulator of blood pressure and hypertension. Angiotensin-converting enzyme 2 (ACE2) and angiotensin-converting enzyme I (ACE) are two main components of the RAS that play a major role in blood pressure homeostasis. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses ACE2 as a receptor to enter cells. Despite some controversies, numerous studies have reported a significant association between the use of ACE inhibitors and reduced risk of COVID-19. In our previous studies, we produced and identified peptide sequences present in whey hydrolysates exhibiting high ACE inhibitory activity. Therefore, the aim of this work is to obtain an improved understanding of the function of these natural peptides as RAS inhibitors and investigate their potential therapeutic role in the COVID-19 pandemic. The molecular interactions between peptides IPP, LIVTQ, IIAE, LVYPFP, and human ACE2 were assessed by employing a molecular docking approach. The results show that natural whey-derived peptides have a dual inhibitory action against both ACE and ACE2. This dual activity distinguishes these ACE inhibitory peptides from synthetic drugs, such as Captopril and Lisinopril which were not shown to inhibit ACE2 activity, and may represent a potential strategy in the treatment of COVID-19.

Whey proteins-polyphenols interactions can be exploited to reduce astringency or increase solubility and stability of bioactives in foods.

Whey proteins have very interesting properties including, high solubility in water, strong interactions with polyphenols, aggregation properties and are classified as GRAS. Here the application of whey proteins to improve food formulations is evaluated. In particular, the reduction of astringency in wine by whey proteins and peptides was assessed applying an analytical method based on determination of tannins. As shown previously for ß-lactoglobulin (ß-Lg) and ß-Lg-Caseinomacropeptides fractions, here it was shown that hydrolysates interacted with polymeric polyphenols such as tannins which, led to their complexation and precipitation effectively reducing astringency. In addition, nanoparticles of ß-Lg and sweet whey were produced by applying a method developed previously in our group based on heat induced denaturation which could consistently produce nanoparticles of 200-300 nm with high colloidal stability. Here further characterisation of ß-Lg nanoparticles showed that addition of ethanol up to 28% led to nanoparticles destabilisation mainly due to a reduction in ß-Lg overall charge. The application of centrifugal force above 863g led to the disruption of colloidal stability and ß-Lg sedimentation. However, nanoparticles were stable to freeze drying conditions. Furthermore, it was shown that sweet whey could be used instead of ß-Lg to produce nanoparticles of similar characteristics. These ß-Lg nanoparticles formed nanocomplexes with resveratrol which, resulted in improved solubility and increased antioxidant activity under pasteurisation conditions. The isomerization from trans to cis and a protective effect of nanoparticles against some chemical changes that result in reduced activity could explain these results. Thus, whey proteins show promise in their application for improved formulations of food/beverages with bioactive ingredients.

Functional characterisation and sensory evaluation of a novel synbiotic okara beverage.

This study aimed to produce four different beverages from okara (soybean by-product) previously hydrolyzed by Cynara cardunculus enzymes and fermented by probiotic bacteria or unfermented beverage. The probiotic viable cells, the isoflavones profile and organic acids were evaluated in the okara beverage. In addition, total phenolic content, antioxidant and ACE inhibitory activities were evaluated at storage time and during in vitro gastrointestinal digestion of all beverages. The probiotic was viable throughout storage in all fermented beverages. The significant bioconversion of the isoflavone glycosides into their corresponding bioactive aglycones was observed in fermented beverage. Furthermore, the beverages showed a good ACE inhibitory activity. After gastrointestinal tract, all beverages showed an increase in the antioxidant and ACE inhibitory activities. In conclusion, this study shows that the application of okara for a multifunctional beverage could be a promising strategy in the disease prevention and contribution to a zero waste approach in food industry.

Whey-Derived Peptides Interactions with ACE by Molecular Docking as a Potential Predictive Tool of Natural ACE Inhibitors.

Several milk/whey derived peptides possess high in vitro angiotensin I-converting enzyme (ACE) inhibitory activity. However, in some cases, poor correlation between the in vitro ACE inhibitory activity and the in vivo antihypertensive activity has been observed. The aim of this study is to gain insight into the structure-activity relationship of peptide sequences present in whey/milk protein hydrolysates with high ACE inhibitory activity, which could lead to a better understanding and prediction of their in vivo antihypertensive activity. The potential interactions between peptides produced from whey proteins, previously reported as high ACE inhibitors such as IPP, LIVTQ, IIAE, LVYPFP, and human ACE were assessed using a molecular docking approach. The results show that peptides IIAE, LIVTQ, and LVYPFP formed strong H bonds with the amino acids Gln 259, His 331, and Thr 358 in the active site of the human ACE. Interestingly, the same residues were found to form strong hydrogen bonds with the ACE inhibitory drug Sampatrilat. Furthermore, peptides IIAE and LVYPFP interacted with the amino acid residues Gln 259 and His 331, respectively, also in common with other ACE-inhibitory drugs such as Captopril, Lisinopril and Elanapril. Additionally, IIAE interacted with the amino acid residue Asp 140 in common with Lisinopril, and LIVTQ interacted with Ala 332 in common with both Lisinopril and Elanapril. The peptides produced naturally from whey by enzymatic hydrolysis interacted with residues of the human ACE in common with potent ACE-inhibitory drugs which suggests that these natural peptides may be potent ACE inhibitors.

Antimicrobial Activity of Lipopeptide Biosurfactants Against Foodborne Pathogen and Food Spoilage Microorganisms and Their Cytotoxicity.

Lipopeptide biosurfactants produced by Bacillus sp. were assessed regarding their antimicrobial activity against foodborne pathogenic and food spoilage microorganisms. Both Gram-positive and Gram-negative bacteria were found not to be susceptible to these lipopeptides. However, mycosubtilin and mycosubtilin/surfactin mixtures were very active against the filamentous fungi Paecilomyces variotti and Byssochlamys fulva, with minimum inhibitory concentrations (MICs) of 1-16 mg/L. They were also active against Candida krusei, MIC = 16-64 mg/L. Moreover it was found that the antifungal activity of these lipopeptides was not affected by differences in isoform composition and/or purity. Furthermore their cytotoxicity tested on two different cell lines mimicking ingestion and detoxification was comparable to those of approved food preservatives such as nisin. Overall, for the first time here mycosubtilin and mycosubtilin/surfactin mixtures were found to have high antifungal activity against food relevant fungi at concentrations lower than their toxicity level hence, suggesting their application for extending the shelf-life of products susceptible to these moulds. In addition combining nisin with mycosubtilin or mycosubtiliin/surfactin mixtures proved to be an effective approach to produce antimicrobials with broader spectrum of action.

Influence of pH and ionic strength on the color parameters and antioxidant properties of an ethanolic red grape marc extract.

The aim of present study was to investigate the influences of pH and several salts on the antioxidant activity and color of an ethanolic grape marc extract. Furthermore, the phenolic content of the extract was analyzed using HPLC and spectrophotometric methods while the total antioxidant activity was assessed by the reaction with ABTS radical. Gallic acid, procyanidins B1, B2, polydatin, catechin, epicatechin, hyperoside, ferulic, chlorogenic, and salicylic acids were among the main identified polyphenols. Different pH values had slight influence on the antioxidant activity, the highest value being determined for pH 3.7. The redness, chroma, and hue were significantly enhanced at pH 3.7 and 2.6. The chromaticity decreased at pH = 5.5 and pH = 7.4, so the extract should be used with care in products with such media. The presence of salts did not noticeably affect the antioxidant activity, except the higher concentrations of CaCl2 , which decreased the antioxidant activity but enhanced the color intensity. PRACTICAL APPLICATION: The data presented in this paper could be used for the development of a new food dye with antioxidant properties of natural origin. The optimal medium conditions (i.e., pH and ionic strength) for the use of an ethanolic red grape marc extract have been identified. The information could be used in product development and product formulation, especially when functional foodstuffs are envisaged. Consequently, this paper would be of significant interest for food chemists, food technologists, food manufacturers, and especially manufacturers of food dyes and all those using natural substances in their production process.

Protein Hydrolysate from Pterygoplichthys disjunctivus, Armoured Catfish, with High Antioxidant Activity.

Pterygoplichthys disjunctivus, locally named the armoured catfish, is a by-catch of tilapia fishing that accounts for up to 80% of total captured fish in the Adolfo Lopez Mateos dam, in Michoacán, México, affecting the economy of its surrounding communities. This invasive fish is discarded by fishermen since native people do not consume it, partly due to its appearance, yet it is rich in protein. The aim of this study was to produce hydrolysates from armoured catfish using food-grade proteases (neutrases HT and PF and alcalase PAL) and investigate the processing conditions (pH and temperature) that lead to a high degree of hydrolysis, antioxidant activity, and Angiotensin I-Converting Enzyme (ACE) Inhibitory activity. No other similar research has been reported on this underutilized fish. The antioxidant activity was measured by three different methods, ABTS, FRAP and ORAC, with relevance to food and biological systems in order to obtain a more comprehensive assessment of the activity. In addition, the main peptide sequences were identified. All enzymes produced hydrolysates with high antioxidant activity. In particular, the protease HT led to the highest antioxidant activity according to the ABTS (174.68 µmol Trolox equivalent/g fish) and FRAP (7.59 mg ascorbic acid equivalent/g fish) methods and almost the same as PAL according to the ORAC method (51.43 µmol Trolox equivalent/g fish). Moreover, maximum activity was obtained at mild pH and temperature (7.5; 50 °C). Interestingly, the ORAC values obtained here were higher than others previously reported for fish hydrolysates and similar to those reported for fruits such as blueberries, apples and oranges. The peptide sequence IEE(E) was present in several peptides in both hydrolysates; this sequence may be partly responsible for the high antioxidant activity, particularly the one based on iron-reducing power. These findings will be relevant to the valorization of other fish/fish muscle discards and could contribute to the production of food supplements and nutraceuticals.

Surfactant TWEEN20 provides stabilisation effect on anthocyanins extracted from red grape pomace.

Red grape pomace, a wine-making by-product is rich in anthocyanins and has many applications in food and pharmaceutical industry. However, anthocyanins are unstable during processing and storage. This study aimed to investigate the stability of anthocyanins obtained by hydroalcoholic extraction (with and without sorbic acid) and colloidal gas aphrons (CGA) separation; a surfactant (TWEEN20) based separation. Anthocyanins in CGA samples showed higher stability (half-life = 55 d) than in the crude extract (half-life = 43 d) and their stability increased with the concentration of TWEEN20 in the CGA fraction (6.07-8.58 mM). The anthocyanins loss in the CGA sample (with the maximum content of surfactant, 8.58 mM) was 34.90%, comparable to that in the crude ethanolic extract with sorbic acid (EE-SA) (31.53%) and lower than in the crude extract (44%). Colour stabilisation was also observed which correlated well with the stability of individual anthocyanins in the EE and CGA samples. Malvidin-3-o-glucoside was the most stable anthocyanin over time.

Protective effect of ß-lactoglobulin against heat induced loss of antioxidant activity of resveratrol.

Resveratrol exhibits many health benefits; however, low water-solubility and instability under processing conditions such as heating can be some of the main challenges for its processing and formulation. Here the complexation of ß-lactoglobulin (ß-Lg) with resveratrol was investigated to improve its solubility and stability. The solubility of resveratrol in water was determined as 7 mg/100 ml. Resveratrol-ß-Lg nanoparticles (181.8 nm) were produced at pH 6 and 75 °C for 45 min. Heating resveratrol solutions at 75 °C for 45 min resulted in isomerization of resveratrol and reduced antioxidant activity. However, resveratrol-ß-Lg nanocomplexes which had undergone the same heat treatment exhibited improved antioxidant activity. Heating under pasteurisation conditions led to similar results and both native ß-Lg and nanoparticles exhibited a protective effect against heat-induced chemical changes in resveratrol, resulting in enhanced antioxidant activity. Fluorescence measurements revealed strong interactions of resveratrol with both native protein and nanoparticles.

Bioaccessible peptides released by in vitro gastrointestinal digestion of fermented goat milks.

In this study, ultrafiltered goat milks fermented with the classical starter bacteria Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarus subsp. thermophilus or with the classical starter plus the Lactobacillus plantarum C4 probiotic strain were analyzed using ultra-high performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS) and/or high performance liquid chromatography-ion trap (HPLC-IT-MS/MS). Partial overlapping of the identified sequences with regard to fermentation culture was observed. Evaluation of the cleavage specificity suggested a lower proteolytic activity of the probiotic strain. Some of the potentially identified peptides had been previously reported as angiotensin-converting enzyme (ACE) inhibitory, antioxidant, and antibacterial and might account for the in vitro activity previously reported for these fermented milks. Simulated digestion of the products was conducted in the presence of a dialysis membrane to retrieve the bioaccessible peptide fraction. Some sequences with reported physiological activity resisted digestion but were found in the non-dialyzable fraction. However, new forms released by digestion, such as the antioxidant αs1-casein 144YFYPQL149, the antihypertensive αs2-casein 90YQKFPQY96, and the antibacterial αs2-casein 165LKKISQ170, were found in the dialyzable fraction of both fermented milks. Moreover, in the fermented milk including the probiotic strain, the k-casein dipeptidyl peptidase IV inhibitor (DPP-IV) 51INNQFLPYPY60 as well as additional ACE inhibitory or antioxidant sequences could be identified. With the aim of anticipating further biological outcomes, quantitative structure activity relationship (QSAR) analysis was applied to the bioaccessible fragments and led to potential ACE inhibitory sequences being proposed. Graphical abstract Ultrafiltered goat milks were fermented with the classical starter bacteria (St) and with St plus the L. plantarum C4 probiotic strain. Samples were analyzed using HPLC-IT-MS/MS and UPLC-Q-TOF-MS/MS. After simulated digestion and dialysis, some of the active sequences remained and new peptides with reported beneficial activities were released.

Antioxidant, ACE-inhibitory and antimicrobial activity of fermented goat milk: activity and physicochemical property relationship of the peptide components.

Increasing evidence on goat milk and the health benefits of its derived products beyond its nutritional value show its potential as a functional food. In this study, goat milk fractions were tested for their total antioxidant capacity using different methods (ORAC, ABTS, DPPH and FRAP), as well as their angiotensin-I-converting-enzyme inhibitory and antimicrobial (against Escherichia coli and Micrococcus luteus) activities. Different whey fractions (whey, cation exchange membrane permeate P and retentate R) of two fermented skimmed goat milks (ultrafiltered goat milk fermented with the classical starter bacteria or with the classical starter plus the Lactobacillus plantarum C4 probiotic strain) were assessed. Additionally, P fractions were divided into two sub-fractions after being passed through a 3 kDa cut-off membrane: (a) the permeate with peptides of MW <3 kDa (P < 3); and (b) the retentate with peptides and proteins of MW >3 kDa (P > 3). No differences in biological activities were observed between the two fermented milks. However, the biological peptides present in the P < 3 fraction showed the highest total antioxidant capacity (for the ORAC assay) and angiotensin-I-converting-enzyme inhibitory activity. Those present in the R fraction showed the highest total antioxidant capacity against ABTS˙+ and DPPH˙ radicals. Some antimicrobial activity against E. coli was observed for the fermented milk containing the probiotic, which could be due to some peptides being released by the probiotic strain. In conclusion, small and non-basic bioactive peptides could be responsible for most of the angiotensin-I-converting-enzyme inhibitory and antioxidant activities. These findings reinforce the potential benefits of the consumption of fermented goat milk in the prevention of cardiovascular diseases associated with oxidative stress and hypertension.

Astringency reduction in red wine by whey proteins.

Whey is a by-product of cheese manufacturing and therefore investigating new applications of whey proteins will contribute towards the valorisation of whey and hence waste reduction. This study shows for the first time a detailed comparison of the effectiveness of gelatin and ß-lactoglobulin (ß-LG) as fining agents. Gelatin was more reactive than whey proteins to tannic acid as shown by both the astringency method (with ovalbumin as a precipitant) and the tannins determination method (with methylcellulose as a precipitant). The two proteins showed similar selectivity for polyphenols but ß-LG did not remove as much catechin. The fining agent was removed completely or to a trace level after centrifugation followed by filtration which minimises its potential allergenicity. In addition, improved understanding of protein-tannin interactions was obtained by fluorescence, size measurement and isothermal titration calorimetry (ITC). Overall this study demonstrates that whey proteins have the potential of reducing astringency in red wine and can find a place in enology.

Novel probiotic-fermented milk with angiotensin I-converting enzyme inhibitory peptides produced by Bifidobacterium bifidum MF 20/5.

In previous research, we have demonstrated that Bifidobacterium bifidum MF 20/5 fermented milk possessed stronger angiotensin converting enzyme (ACE) inhibitory activity than other lactic acid bacteria, including Lactobacillus helveticus DSM 13137, which produces the hypotensive casokinins Ile-Pro-Pro (IPP) and Val-Pro-Pro (VPP). The aim of this study is to investigate the ACE-inhibitory peptides released in B. bifidum MF 20/5 fermented milk. The novel ACE-inhibitory peptide LVYPFP (IC50 = 132 µM) is reported here for the first time. Additionally, other bioactive peptides such as the ACE-inhibitor LPLP (IC50 = 703 µM), and the antioxidant VLPVPQK were identified. Moreover, the peptide and amino acid profiles, the ACE-inhibitory activity (ACEi), pH, and degree of hydrolysis of the fermented milk were determined and compared with those obtained in milk fermented by L. helveticus DSM 13137. The sequences of the major bioactive peptides present in fermented milk of B. bifidum and L. helveticus were identified and quantified. B. bifidum released a larger amount of peptides than L. helveticus but no IPP or VPP were detected in B. bifidum fermented milk. Also the lactotripeptide concentrations and ACEi were higher in L. helveticus fermented milk when the pH was maintained at 4.6. This may represent a technical advantage for B. bifidum that reduces the pH at a slow enough rate to facilitate the peptide generation without the need for pH control. Thus these findings show the potential for the use of this probiotic strain to produce fermented milk with a wider range of health benefits including reduction of blood pressure.

Antioxidant, antibacterial and ACE-inhibitory activity of four monofloral honeys in relation to their chemical composition.

Different monofloral honeys from Castilla-La Mancha (Spain) have been studied in order to determine their main functional and biological properties. Thyme honey and chestnut honey possess the highest antioxidant capacity, which is due to their high vitamin C (in thyme honey) and total polyphenolic content (in chestnut honey). On the other hand, chestnut honey showed high antimicrobial activity against Staphylococcus aureus and Escherichia coli, whilst others had no activity against S. aureus and showed very small activity against E. coli. Moreover it was found that the antimicrobial activity measured in chestnut honey was partly due to its lysozyme content. In addition the angiotensin I-converting enzyme (ACE) inhibitory activity was measured, and the ACE inhibition is one mechanism by which antihypertensive activity is exerted in vivo. All the types of honey showed some activity but chestnut honey had the highest ACE inhibitory activity.

Self-assembly of three bacterially-derived bioactive lipopeptides.

The self-assembly in aqueous solution of three lipopeptides obtained from Bacillus subtilis has been investigated. The lipopeptides surfactin, plipastatin and mycosubtilin contain distinct cyclic peptide headgroups as well as differences in alkyl chain length, branching and chain length distribution. Cryogenic transmission electron microscopy and X-ray scattering reveal that surfactin and plipastatin aggregate into 2 nm-radius spherical micelles, whereas in complete contrast mycosubtilin self-assembles into extended nanotapes based on bilayer ordering of the lipopeptides. Circular dichroism and FTIR spectroscopy indicate the presence of turn structures in the cyclic peptide headgroup. The unexpected distinct mode of self-assembly of mycosubtilin compared to the other two lipopeptides is ascribed to differences in the surfactant packing parameter. This in turn is due to specific features of the conformation of the peptide headgroup and alkyl chain branching.

Production of angiotensin-I-converting enzyme inhibitory peptides from ß-lactoglobulin- and casein-derived peptides: an integrative approach.

Angiotensin I-converting enzyme (ACE) inhibition is one of the mechanisms by which reduction in blood pressure is exerted. Whey proteins are a rich source of ACE inhibitory peptides and have shown a blood pressure reduction effect i.e. antihypertensive activity. The aim of this work was to develop a simplified process using a combination of adsorption and microfiltration steps for the production of hydrolysates from whey with high ACE inhibitory activity and potency; the latter was measured as the IC50, which is the peptide concentration required to reduce ACE activity by half. This process integrates the selective separation of ß-lactoglobulin- and casein-derived peptides (CDP) from rennet whey and their hydrolysis, which results in partially pure, less complex hydrolysates with high bioactive potency. Hydrolysis was carried out with protease N "Amano" in a thermostatically controlled membrane reactor operated in a batch mode. By applying the integrative approach it was possible to produce from the same feedstock two different hydrolysates that exhibited high ACE inhibition. One hydrolysate was mainly composed of casein-derived peptides with IC50=285 µg/mL. In this hydrolysate we identified the well-known potent ACE-inhibitor and antihypertensive tripeptide Ile-Pro-Pro (IPP) and another novel octapeptide Gln-Asp-Lys-Thr-Glu-Ile-Pro-Thr (QDKTEIPT). The second hydrolysate was mainly composed of ß-lactoglobulin derived peptides with IC50=28 µg/mL. This hydrolysate contained a tetrapeptide (Ile-Ile-Ala-Glu) IIAE as one of the two major peptides. A further advantage to this process is that enzyme activity was substantially increased as enzyme product inhibition was reduced.

Chemical characterisation and determination of sensory attributes of hydrolysates produced by enzymatic hydrolysis of whey proteins following a novel integrative process.

The overall aim of this work was to characterise the major angiotensin-converting enzyme (ACE) inhibitory peptides produced by enzymatic hydrolysis of whey proteins, through the application of a novel integrative process. This process consisted of the combination of adsorption and microfiltration within a stirred cell unit for the selective immobilisation of ß-lactoglobulin and casein-derived peptides (CDP) from whey. The adsorbed proteins were hydrolysed in situ, which resulted in the separation of peptide products from the substrate and fractionation of peptides. Two different hydrolysates were produced: (i) from CDP (IC(50)=287 µg/mL) and (ii) from ß-lactoglobulin (IC(50)=128 µg/mL). The well-known antihypertensive peptide IPP and several novel peptides that have structural similarities with reported ACE inhibitory peptides were identified and characterised in both hydrolysates. Furthermore, the hydrolysates were assessed for bitterness. No significant difference was found between the bitterness of the control (milk with no hydrolysate) and hydrolysate samples at different concentrations (at, below and above the IC(50)).