Angiotensin‐I converting enzyme inhibitory and antioxidant activities of peptide fractions extracted by ultrafiltration of cowpea Vigna unguiculata hydrolysates

BACKGROUND: Enzymatic proteolysis of food proteins is used to produce peptide fractions with the potential to act as physiological modulators. Fractionation of these proteins by ultrafiltration results in fractions rich in small peptides with the potential to act as functional food ingredients. The present study investigated the angiotensin‐I converting enzyme (ACE‐I) inhibitory and antioxidant activities for hydrolysates produced by hydrolyzing Vigna unguiculata protein extract as well as ultrafiltered peptide fractions from these hydrolysates. RESULTS: Alcalase^®, Flavourzyme^® and pepsin–pancreatin were used to produce extensively hydrolyzed V. unguiculata protein extract. Degree of hydrolysis (DH) differed between the enzymatic systems and ranged from 35.7% to 58.8%. Fractionation increased in vitro biological activities in the peptide fractions, with IC₅₀ (hydrolysate concentration in µg protein mL^?1 required to produce 50% ACE inhibition) value ranges of 24.3–123 (Alcalase hydrolysate, AH), 0.04–170.6 (Flavourzyme hydrolysate; FH) and 44.7–112 (pepsin–pancreatin hydrolysate, PPH) µg mL^?1, and TEAC (Trolox equivalent antioxidant coefficient) value ranges of 303.2–1457 (AH), 357.4–10 211 (FH) and 267.1–2830.4 (PPH) mmol L^?1 mg^?1 protein. CONCLUSION: The results indicate the possibility of obtaining bioactive peptides from V. unguiculata proteins by means of a controlled protein hydrolysis using Alcalase^®, Flavourzyme^® and pepsin–pancreatin. The V. unguiculata protein hydrolysates and their corresponding ultrafiltered peptide fractions might be utilized for physiologically functional foods with antihypertensive and antioxidant activities. Copyright © 2010 Society of Chemical Industry     

Production of bioactive proteins and peptides from the diatom Nitzschia laevis and comparison of their in vitro antioxidant activities with those from Spirulina platensis and Chlorella vulgaris

This research focuses on green production of bioactive proteins and hydrolysates from Nitzschia. A comparison of antioxidant activities was established between protein extracts and hydrolysates from Nitzschia and two other well‐known microalgae, chlorella and spirulina. Protein hydrolysates from these microalgae were produced using Alcalase^®, Flavourzyme^® and Trypsin. The hydrolysis process enhanced the antioxidant activities in general, especially those obtained using Alcalase^®. Nitzschia showed the highest (P < 0.05) total phenolic content/reducing capacity (2.4 ± 0.02 mg GAE/100 g) after 90 min of hydrolysis with Alcalase^®. The ABTS [2,2′‐Azino‐bis(3‐ethylbenzothiazoline‐6‐sulphonic acid)] radical scavenging activity (66.77 ± 0.00%) was highest (P < 0.05) after 120 min of hydrolysis, but DPPH (2,2‐Diphenyl‐1‐picrylhydrazyl radical) was low (29.59 ± 0.02%). A correlation between ABTS activity and total phenolic contents was the highest (P < 0.05) for protein hydrolysates from all three organisms using Alcalase^®, but superoxide anion radical scavenging activity was intermediate for Nitzschia. Therefore, Nitzschia protein hydrolysates have the potential to be used as antioxidants.     

Comparison of physicochemical properties,antioxidant and metal‐chelating activities of protein hydrolysates from Phaseolus lunatus and hard‐to‐cook Phaseolus vulgaris

Limited and extensive hydrolysates were obtained from Phaseolus lunatus (LHl and EHl) and hard‐to‐cook Phaseolus vulgaris (LHv and EHv) using the enzymes Flavourzyme^®, Alcalase^®, Pancreatin^® and a sequential Pepsin^®–Pancreatin^® system. Degrees of hydrolysis varied from 8.32% to 31.60%. SDS‐PAGE of extensive hydrolysates showed molecular weights smaller than limited hydrolysates. Differential scanning calorimeter (DSC) analysis of LHl and EHl revealed the presence of two endothermic transitions; LHv and EHv had only one. LHv presented a higher content of hydrophobic amino acids whose surface hydrophobicity was 12.17. Functional properties such as nitrogen solubility, foaming capacity and emulsifying activity index in LHv were better than LHl at different pH evaluated. However, the latter showed better foaming stabilities. Amino acids such as His, Tyr, Trp and Arg were observed in greater amounts in both extensive hydrolysates. 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) radical‐scavenging and metal‐chelating activities in EHv and EHl increased significantly compared to the source material.     

Synthesis and partial characterization of octenylsuccinic starch from Phaseolus lunatus

Phaseolus lunatus starch was modified by esterification with octenyl succinic anhydride (OSA) and reaction effect evaluated in terms of chemical composition, gelatinization, pasting and emulsification properties. Succinylation was done using a 2³ factorial design with four replicates of the central treatment. Evaluated factors and levels were OSA concentration (1% and 3%), pH (7 and 9) and reaction time (30 and 60 min). Succinyl group percentage was the response variable. The optimum treatment was a reaction with 3% OSA at pH 7 for 30 min, which produced 0.5083% succinyl groups and 0.0083° of substitution. No significant changes were observed in proximate composition between the native and derivative starches. Apparent amylose level decreased notably from 32.4% to 23.6% due to OSA inclusion. Succinylation decreased starch gelatinization temperature (75.3–64.6 °C), decreased enthalpy (10.7–9.7 J/g), increased viscosity (700–1000 BU), increased emulsifying capacity (0.47–0.53 ml oil/ml sample), and made emulsions more stable over time. Starch modification did not, however, improve stability in heating–cooling processes.     

Composition and biological activities of the essential oil from Schisandra chinensis obtained by solvent-free microwave extraction

Applicability of solvent-free microwave extraction (SFME) for extraction of the fruits of Schisandra chinensis essential oil was examined; the composition and antioxidant activities and antibacterial activities of the essential oil were assessed in vitro. An orthogonal experiment (L₉ (3)⁴) was applied to optimize the extraction process. The optimum conditions were: extraction time, 45 min; microwave power, 800 W; diameter of powder particles, 0.25 mm; and proportion of water pretreatment, 30%. Under these conditions, the extraction yield was 1.75%. Thirty-five compounds, representing 91.12% of the oil, were identified, of which the major ones, ylangene (50.11%), β-himachalene (10.76%)，α-bergamotene (9.52%) and β-Chamigrene (5.41%), accounted for of 75.80% the oil.Antioxidant activity, IC₅₀ value of the essential oil was determined as 3.87 mg/mL by DPPH assay, and the inhibition values of the essential oil at 1.8 mg/mL was 41.88% by β-Carotene–linoleic acid bleaching assay. The essential oil was screened for antibacterial activity against both Gram positive (Staphylococcus epidermidis, Staphylococcus aureus, Bacillus subtilis) and Gram negative (Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris) bacteria. The essential oil showed antibacterial effect against all the gram (+) bacteria and gram (−) bacteria tested. These results show that S. chinensis essential oil could be considered as a natural alternative to food antioxidants and preservatives.     

Effects of fermentation conditions on the potential anti‐hypertensive peptides released from yogurt fermented by Lactobacillus helveticus and Flavourzyme®

This study investigates the effects of fermentation conditions on the production of angiotensin‐converting enzyme inhibitory (ACE‐I) peptides in yogurt by Lactobacillus helveticus 881315 (L. helveticus) in the presence or absence of Flavourzyme^®, which is derived from a mould, Aspergillus oryzae and used for protein hydrolysis in various industrial applications. Optimal conditions for peptides with the highest ACE‐I activity were 4% (v/w) inoculum size for 8 h without Flavourzyme^® supplementation, and 1% inoculum size for 12 h when combined with Flavourzyme^®. The yogurt fermented by L. helveticus resulted in IC₅₀ values (concentration of inhibitor required to inhibit 50% of ACE activity under the assayed conditions) of 1.47 ± 0.04 and 16.91 ± 0.25 mg mL^?1 with and without Flavourzyme^® respectively. Seven fractions of ACE‐I peptides from the yogurt incorporated with L. helveticus and Flavourzyme^® were separated using the preparative high‐performance liquid chromatography. Fraction (F3) showed the highest ACE‐I activity with an IC₅₀ of 35.75 ± 5.48 μg mL^?1. This study indicates that yogurt may be a valuable source of ACE‐I peptides, which may explain the outcomes observed in the experimental and clinical studies and foresee the application of fermented milk proteins into functional foods or dietary supplements.     

Angiotensin-I-converting enzyme inhibitory,antimicrobial, and antioxidant effect of bioactive peptides obtained from different varieties of common beans (Phaseolus vulgaris L.) with in vivo antihypertensive activity in spontaneously hypertensive rats

Peptides obtained from three varieties of common beans (Phaseolus vulgaris L.) named plus black (PB), azufrado higuera (AH) and pinto Saltillo (PS) presented antimicrobial, antioxidant and antihypertensive activities. Peptides were obtained from these common beans protein concentrates after treatment with Alcalase^® followed by ultrafiltration using 1-, 3- and 10-kDa molecular weight cutoff membranes. Antioxidant activity was determined by the 2,2′-azino-bis (3-etilbenzotiazolin-6-sulfonic) acid method and was expressed as Trolox equivalent antioxidant capacity (TEAC). The highest antioxidant activity (630, 550 and 517 mM TEAC/mg protein) was observed in the group of peptides with a molecular weight lower than 1 kDa (F < 1) from PB, AH and PS bean varieties, respectively. Antibacterial activity was determined as susceptibility test in which ten of twelve bacteria strains showed growth inhibition with the total hydrolysates (TH) and the peptidic fraction 3–10 kDa; subsequently, the minimum inhibitory concentration was determined with the standard microdilution assay in a 96-well plates in which the peptidic fraction F < 1 kDa presented antimicrobial activity against Shigella dysenteriae with the three beans varieties at 0.1, 0.4 and 0.3 mg/mL (for PB, AH and PS, respectively). Additionally to the antimicrobial and antioxidant activities, the TH of the PB and AH bean varieties presented high inhibition of the angiotensin-I-converting enzyme (ACE-I) (IC₅₀ = 4.34 ± 0.29 and 4.82 ± 1.59 μg/mL, respectively). And, when the peptidic fraction F3–10 kDa was tested, the AH variety showed a significant increase in the ACE-I capacity (IC₅₀ = 1.09 ± 0.04 μg/mL). Importantly, this peptidic fraction decreased the systolic blood pressure in a spontaneously hypertensive rat model after 2 h of administration by a single interperitoneally dose.     

Improvement of functional properties and antioxidant activities of cuttlefish (Sepia officinalis) muscle proteins hydrolyzed by Bacillus mojavensis A21 proteases

Functional properties and antioxidant activities of cuttlefish (Sepia officinalis) muscle protein hydrolysates, with different degrees of hydrolysis (DH from 7.3% to 18.8%), obtained by treatment with Bacillus mojavensis A21 alkaline proteases were investigated. Protein contents for all freeze-dried cuttlefish muscle protein hydrolysates (CMPHs) ranged from 80% to 86%. For the functional properties, hydrolysis by A21 proteases increased (p < 0.05) protein solubility to above 78% over a wide pH range (2.0–11.0). However, the interfacial activities (emulsion activity index, emulsion stability index, foaming capacity and foaming stability) decreased with the increase of the DH. All CMPHs exhibited significant metal chelating activity and DPPH free radical-scavenging activity, and inhibited linoleic acid peroxidation. Antioxidant properties of protein hydrolysates increased with protein hydrolysis and the highest activities were obtained at DH of 16%. The IC₅₀ values for DPPH radical-scavenging and metal chelating activities were found to be 0.52 ± 0.01 mg/ml and 0.67 ± 0.13 mg/ml. The obtained results suggested that functional properties and antioxidant activities of cuttlefish muscle protein hydrolysates were influenced by the degree of hydrolysis.The composition of amino acids of undigested and hydrolyzed proteins was determined. CMPHs have a high percentage of essential amino acids such as arginine, lysine, histidine and leucine. They have a high nutritional value and could be used as supplement to poorly balanced dietary proteins.     

Phenolic compounds profile and antioxidant activity of Dorystoechas hastata L. Boiss et Heldr

Phenolic compound profile and antioxidant activity of methanol, acetone, ethyl acetate, water and petroleum ether (b.p. 40–60 °C) extracts of Dorystoechas hastata, endemic to Turkey and being consumed as herbal tea by local inhabitants, have been investigated. HPLC–DAD analysis indicated the presence of chlorogenic, caffeic, p-coumaric, ferulic and rosmarinic acids as phenolic acids, quercetin, kaempferol and apigenin as flavonoids and carnosic acid and carnosol as diterpenoid antioxidants in the plant. Petroleum ether, methanol and water extracts of D. hastata were found to be very effective antioxidative extracts. Petroleum ether extract, having the highest amount of carnosic acid and carnosol contents (531.3 and 389.9 mg/g DW, respectively) among all solvent extracts, was found to have the highest antioxidative potential based on the individual antioxidant activity assays; DPPH, ABTS⁺, TBARS and total phenolic content, expressed as IC₅₀, TEAC, EC₅₀ and TPC values, respectively, and also on the principal component analysis. It exhibited significantly high TEAC (7.1 mM trolox) and low EC₅₀ (54.5 μg/mL) values, indicating the strong potential in in vitro radical scavenging and in inhibiting lipid oxidation. Water extract, with its extremely low IC₅₀ value of 4.9 μg/mL in DPPH radical scavenging and significant TEAC (4.8 mM trolox), EC₅₀ (64.4 μg/mL) and TPC (116.7 mg GAE/g DW) values, was found to be the second highest in antioxidative potential among all extracts. TPC value of methanol extract (147.3 mg GAE/g DW) was found to be significantly higher than the other extracts studied. The results showed that D. hastata can be used as a potential antioxidative edible source due to its different classes of phenolic compounds and strong antioxidative capacity.     

Improving antioxidant activity of black bean protein by hydrolysis with protease combinations

This study explored the use of a simplex centroid design to produce protein hydrolysates with antioxidant properties using Alcalase^® 2.4L, Flavourzyme^® 500L and Neutrase^® 0.8L. Proteases kinetic parameters and the ultrafiltration of protein hydrolysates were also investigated. The highest antioxidant activity, in the studied conditions, was reached when the mixture of Alcalase^® 2.4L and Flavourzyme^® 500L was used in the hydrolysates production. The antioxidant power of the black bean proteins, measured by the total antioxidant capacity and reducing power assay, increased after hydrolysis by 31% and 70%, respectively. The black bean proteins hydrolysates fractions (3–30 kDa) showed an antioxidant activity decrease along with a reduction in molecular weight, demonstrating that a set of varied molecular weight peptides was responsible for the antioxidant characteristics of black bean protein hydrolysates.     

Effect of aeration and dilution rate on nisin Z production during continuous fermentation with free and immobilized Lactococcus lactis UL719 in supplemented whey permeate

The influence of dilution rate (D) and aeration on soluble and cell-bound nisin Z production was investigated during continuous free (FC) and immobilized cell (IC) cultures with Lactococcus lactis subsp. lactis biovar diacetylactis UL719 in supplemented whey permeate. Maximum total bacteriocin titres during non-aerated continuous FC and IC cultures were obtained for low D, with 1490 and 1090 IU mL⁻¹ for 0.15 h⁻¹ or 0.25 and 0.5 h⁻¹, respectively. For both systems, aeration increased nisin total production with maximum titres of 2560 and 2430 IU mL⁻¹ for low D, respectively, as well as specific production. Volumetric productivity was the highest for an intermediate D of 0.4 h⁻¹ during FC cultures (460 IU mL⁻¹ h⁻¹ for both aerated and non-aerated cultures), while it increased continuously with D during IC cultures, reaching high values of 1090 and 1760 IU mL⁻¹ h⁻¹ at 2.0 h⁻¹ without and with aeration, respectively. In comparison with previous data for FC batch cultures, data from this study may indicate that during continuous fermentations at steady state, some steps in nisin biosynthesis are limiting. In these conditions, nisin production by immobilized cells is reduced.     

Potential ACE-inhibitory activity and nanoLC-MS/MS sequencing of peptides derived from aflatoxin contaminated peanut meal

Our lab has developed a process for sequestering aflatoxin from contaminated peanut meal (PM) using commercial bentonite clays while protein is simultaneously extracted and hydrolyzed by a commercial protease. The objectives of this study were to sequence generated peptides and evaluate their potential ACE-inhibitory properties. Aflatoxin in the unprocessed PM was 610 μg kg⁻¹ compared to 9.7 μg kg⁻¹ on a dry weight basis in the 120 min hydrolysate. This hydrolysate displayed significant ACE-inhibitory activity with an IC₅₀ of 295.1 μg mL⁻¹. Ultrafiltration and size exclusion chromatography (SEC) improved the ACE-inhibitory properties, with the SEC fraction containing the smallest peptides having an IC₅₀ = 44.4 μg mL⁻¹. Additionally, 271 unique peptides were identified by nanoLC-MS/MS, of which 147 belonged to major seed storage proteins. This advanced characterization data will ultimately allow for more efficient production of hydrolysates with ACE-inhibitory activity or other bioactivities of interest from PM.     

Production and characterization of angiotensin converting enzyme (ACE) inhibitory peptides from apricot (Prunus armeniaca L.) kernel protein hydrolysate

Six different proteases (Flavourzyme^®, Neutrase^®, Protamex^®, Alcalase^® 2.4L, Proleather^® FG-F, and papain) were employed to hydrolyze apricot kernel protein (AKP). Alcalase^® is an inexpensive and non-specific protease that has been shown to be useful for the generation of bioactive peptides from AKP. Alcalase^® 2.4L was selected for further study on enzymatic preparation of ACE inhibitory peptide from AKP. After 60-min hydrolysis, the highest ACE inhibition was 82 ± 0.14%. Results of molecular weight distribution revealed that most of ACE inhibition activity was probably attributed to low-molecular weight peptide fraction ranging from 200 to 900 Da. Ultrafiltration on membranes with several molecular weight cutoffs (MWCFs) demonstrated that most of the ACE inhibitory activity was due to peptides with a less than 1,000 Da molecular weight: the IC₅₀ value of the 1-kDa ultrafiltrate was 0.15 ± 0.007 mg mL^?1, while it was 0.378 ± 0.015 mg mL^?1 before ultrafiltration. Additionally, further separation and purification of the ACE inhibitory peptides were carried out using gel filtration and C₁₈ RP-HPLC. The result of research can be used to optimize AKP enzymatic hydrolysis for producing ACE inhibitory peptides which could be used for food industry and nutraceuticals.     

Pyropia columbina phycocolloids as microencapsulating material improve bioaccessibility of brewers’ spent grain peptides with ACE-I inhibitory activity

The effects of wall hydrocolloids on delivery and bioaccessibility of encapsulated brewers’ spent grain ACE-I inhibitory peptides after simulated gastrointestinal digestion were assessed. Microencapsulation of peptides was carried out by spray drying using locust bean gum, P. columbina phycocolloids, or its mixtures as wall materials. Microcapsules presented round external surfaces with some concavities, negative surface charge and encapsulation efficiencies higher than 90%. The incorporation of phycocolloids to formulations increased the encapsulation efficiency, negative surface charge and resistance against digestive enzymes of microcapsules. Encapsulated peptides with P. columbina phycocolloids showed lower IC₅₀ value of ACE-I inhibition than un-encapsulated peptides (2.4 ± 0.2 vs. 7.2 ± 0.3 mg mL⁻¹ protein), but higher than that obtained for hydrolysate (1.5 ± 0.2 mg mL⁻¹ protein), indicating a 75% protection of bioactivity. Electrostatic and hydrophobic interactions between P. columbina phycocolloids and brewers’ spent grain peptides could be implicated in the protection of peptides during gastrointestinal digestion.     

UV-C inactivation of Escherichia coli at different temperatures

The influence of treatment parameters (dose and temperature), treatment medium characteristics (absorption coefficient, pH and water activity) and microbiological factors (strain, growth phase and UV damage and repair capacity) on Escherichia coli UV-C resistance has been investigated. UV-C doses to inactivate at 25 °C 99.99% of the initial population (4D) of five strains of E. coli in McIlvaine buffer of pH 7.0 with tartrazine added (absorption coefficient of 10.77 cm⁻¹) were 16.60, 14.36, 14.36, 13.22, 11.18 J/mL for strains E. coli STCC 4201, STCC 471, STCC 27325, O157:H7 and ATCC 25922, respectively. The entrance in the stationary growth phase increased the 4D value of the most resistant strain, E. coli STCC 4201, from 13.09 to 17.23 J/mL. Survivors to UV treatments showed neither oxidative damages nor injuries in cell envelopes. On the contrary, the photoreactivation by the incubation of plates for 60 min below visible light (11.15 klx) increased the dose to 18.97 J/mL. The pH and the water activity of the treatment medium did not affect the UV tolerance of E. coli STCC 4201, but the lethal effect of the treatments decreased exponentially (Log₁₀4D = − 0.0628α + 0.624) by increasing the absorption coefficient (α). A treatment of 16.94 J/mL reached 6.35, 4.35, 2.64, 1.93, 1.63, 1.20, 1.02 and 0.74 Log₁₀ cycles of inactivation with absorption coefficients of 8.56, 10.77, 12.88, 14.80, 17.12, 18.51, 20.81 and 22.28 cm⁻¹. The temperature barely changed the UV resistance up to 50.0 °C. Above this threshold, inactivation rates due to the combined process synergistically increased with the temperature. The magnitude of the synergism decreased over 57.5 °C. An UV treatment of 16.94 J/mL in media with an absorption coefficient of 22.28 cm⁻¹ reached 1.23, 1.64, 2.36, 4.01 and 6.22 Log₁₀ cycles of inactivation of E. coli STCC 4201 at 50.0, 52.5, 55.5, 57.5 and 60.0 °C, respectively.

Industrial relevance

Results obtained in this investigation show that UV light applied at mild temperatures (57.5 to 60 °C) could be an alternative to heat treatments for 5-Log₁₀ reductions of E. coli in liquid foods. Since microbial resistance to UV-C light did not depend on the pH and water activity (a_w) of the treatment media, eventual advantages of UV light for pasteurization purposes will be higher in low a_w foods. E. coli STCC 4201 could be considered as a target when UV light processing of foods.     

Autolysis-assisted production of fish protein hydrolysates with antioxidant properties from Pacific hake (Merluccius productus)

Fish protein hydrolysates (FPH) with antioxidative properties were prepared using Pacific hake fish with high endogenous proteolytic activity from Kudoa paniformis parasitic infection. Infection level of ∼10⁷K. paniformis spores/g fish mince or higher yielded FPH with high antioxidant potential by autolysis and/or Validase^® BNP or Flavourzyme^® 500L. Autolyzing fish mince containing 30 × 10⁶ spores/g for 1 h at 52 °C and pH 5.50 produced FPH (named E-1h) with Trolox equivalent antioxidant capacity (TEAC) in the 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) radical assay of 262 ± 2 μmol/g and oxygen radical absorbing capacity (ORAC) of 225 ± 17 μmol Trolox equivalents/g freeze-dried sample. E-1h FPH also exhibited a marked concentration-dependent scavenging activity on 1,1-diphenyl-2-picrylhydrazyl radical. Antioxidant activity of E-1h FPH was higher (p < 0.05) than BHA and α-tocopherol in a linoleic acid peroxidation system over prolonged storage (∼162 h). Antioxidative FPH from Pacific hake may be useful ingredients in food and nutraceutical applications.     

High nisin-Z production during repeated-cycle batch cultures in supplemented whey permeate using immobilized Lactococcus lactis UL719

Nisin-Z production was studied during repeated-cycle pH-controlled batch (RCB) cultures using Lactococcus lactis subsp. lactis biovar. diacetylactis UL719 immobilized in κ-carrageenan/locust bean gum gel beads in supplemented whey permeate. After an initial colonization of gel beads during the first two cycles, nisin-Z production in bulk medium and gel beads was very similar for 1-h and 2-h cycle RCB cultures. A very high nisin-Z production (8200 IU mL⁻¹) was measured in the broth after the 1-h cycles, with a corresponding volumetric productivity of 5730 IU mL⁻¹ h⁻¹. This productivity is much higher than maximum nisin productivities reported in literature or maximum productivities obtained previously for free-cell batch cultures (850 IU mL⁻¹ h⁻¹), and free-cell (460 IU mL⁻¹ h⁻¹) or immobilized-cell (1760 IU mL⁻¹ h⁻¹) continuous cultures, using the same strain and fermentation conditions. The stability of RCB cultures was demonstrated for 24 and 36 1-h cycles carried out over 3 and 6-day periods, respectively. Changing environmental conditions during batch cultures resulted high nisin production.     

Enzymatic hydrolysis of hard‐to‐cook bean (Phaseolus vulgaris L.) protein concentrates and its effects on biological and functional properties

Inadequate postharvest handling and storage under high temperature and relative humidity conditions produce the hard‐to‐cook (HTC) defect in beans. However, these can be raw material to produce hydrolysates with functional activities. Angiotensin I‐converting enzyme (ACE) inhibitory and antioxidant capacities were determined for extensively hydrolysed proteins of HTC bean produced with sequential systems Alcalase‐Flavourzyme (AF) and pepsin–pancreatin (Pep‐Pan) at 90 min ACE inhibition expressed as IC₅₀ values were 4.5 and 6.5 mg protein per mL with AF and Pep‐Pan, respectively. Antioxidant activity as Trolox equivalent antioxidant capacity (TEAC) was 8.1 mm  mg^?1 sample with AF and 6.4 mm  mg^?1 sample with Pep‐Pan. The peptides released from the protein during hydrolysis were responsible for the observed ACE inhibition and antioxidant activities. Nitrogen solubility, emulsifying capacity, emulsion stability, foaming capacity and foam stability were measured for limited hydrolysis produced with Flavourzyme and pancreatin at 15 min. The hydrolysates exhibited better functional properties than the protein concentrate.     

Comparing predicting models for heat inactivation of Listeria monocytogenes and Pseudomonas aeruginosa at different pH

Under the same experimental conditions it has been demonstrated that whereas survival curves of Listeria monocytogenes in the range of temperatures from 54 to 62 °C followed a first-order kinetic, those of Pseudomonas aeruginosa in the range of temperatures from 50 to 56 °C were not linear showing a shoulder followed by a linear region. The first order kinetic model did not describe survival curves of P. aeruginosa. A model based on the Weibull distribution (Log₁₀(N_t/N₀)=(1/−2.303)*(t/b)ⁿ)) accurately described the inactivation kinetics of both microorganisms at the three pHs of 4, 5.5, 7.4 investigated. For both microorganisms, the b value depended on the treatment temperature and the pH of the treatment medium. Whereas for L. monocytogenes the n value was independent of the treatment conditions, for P. aeruginosa the n value depended on the pH of the treatment medium.The model based on the Weibull distribution was capable of accurately predicting the treatment time to inactivate five Log₁₀ cycles of both microorganisms at the three pHs investigated.     

Separation of angiotensin I‐converting enzyme inhibitory peptides from bovine connective tissue and their stability towards temperature,pH and digestive enzymes

Bovine collagen was isolated from connective tissue, a by‐product in the meat processing industry and characterised by SDS‐PAGE. Alcalase and papain were employed to generate collagen hydrolysates with different degree of hydrolysis (DH). In vitro angiotensin I‐converting enzyme (ACE) inhibitory activities were evaluated and the two most potent hydrolysates from each enzyme were separated by two‐step purification. Both alcalase‐catalysed and papain‐catalysed hydrolysates exhibited strong ACE inhibitory capacities with IC₅₀ values of 0.17 and 0.35 mg mL^?1, respectively. Purification by ion‐exchange chromatography and gel filtration chromatography revealed higher ACE inhibitory activities in one fraction from each enzyme with IC₅₀ values of 3.95 and 7.29 μg mL^?1. These peptide fractions were characterised as 6‐12 amino acid residues by MALDI‐TOF/MS. The peptides retained their activity (>90%) after exposure to processing temperature and pH and in vitro simulated gastrointestinal digestion. The present results demonstrated that collagen peptides can be utilised for developing high value‐added ingredients, for example ACE inhibitory peptides.