Phospholipid nanovesicles containing a bacteriocin-like substance for control of Listeria monocytogenes

Bacillus licheniformis strain P40 produces a bacteriocin-like substance (BLS) that has potential to be used as a natural biopreservative for control of pathogenic and food microorganisms. The objective of this study was the encapsulation of BLS in phosphatidylcholine vesicles, evaluating its antimicrobial activity against Listeria monocytogenes. The size of the nanovesicles with the BLS was around 570 nm and of the nanovesicles without BLS was of approximately 484.8 nm, as determined by light scattering with a He–Ne laser (λ = 632.8 nm) as light source. The encapsulated BLS showed inhibitory activity against L. monocytogenes as observed by agar diffusion assay. Complete inhibition of L. monocytogenes growth was observed with the addition of 100 and 50 AU mL^− 1 of encapsulated and free BLS, respectively. A reduction in the number of viable cells to zero was observed after 10 min incubation with 400 AU mL^− 1 of either encapsulated or free BLS. The encapsulated BLS was stable for up to 30 days at 4 °C. These results indicate that nanovesicles containing BLS may have potential for use as food preservative.Industrial relevanceThe increased concern on minimally processed food and natural additives has been stimulated many studies on the utilization of antimicrobial peptides as biopreservatives. The incorporation of bacteriocins into nanovesicles may represents an interesting alternative for controlled release and increased stability of bacteriocins.     

Purification and characterisation of a zinc-binding peptide from oyster protein hydrolysate

A novel zinc-binding peptide produced from oyster protein hydrolysis using pepsin was purified and characterised. The hydrolysate was fractionated by immobilised metal ion affinity chromatography (IMAC-Zn²⁺). The zinc-binding peptide identified by reverse-phase high-pressure liquid chromatography (RP-HPLC) and sequenced by liquid chromatography (LC/LTQ) mass spectrometry (sequence from N to C terminal) had a molecular weight of 1882.0 Da. The zinc-binding capacity of the peptide (HLRQEEKEEVTVGSLK) was 6.56 μg mg^?1 and it was preserved at 85.98% of its original level upon in vitro simulated digestion. The UV–vis and FTIR spectra demonstrate that the amino nitrogen atoms and the oxygen atoms belonging to the carboxylate groups are the primary binding sites for Zn²⁺. The results provide a feasible approach to isolate zinc-binding peptides and contribute to clarification of binding mechanism between zinc and peptides.     

Incorporation of partially purified hen egg white lysozyme into zein films for antimicrobial food packaging

Lysozyme, partially purified from hen egg white by precipitation of non-enzyme protein with ethanol and lyophilized after dialysis, was incorporated into zein films. The recovery and specific activity of the enzyme after partial purification varied between 45% and 72% and 2173 and 3448 U/mg, whereas the activity of the lyophilized enzyme varied between 2900 and 3351 U/mg. The partially purified enzyme was very stable and lost almost no activity in lyophilized form or in zein films stored at −18 and 4 °C for up to 8 and 4 months, respectively. During partial purification and in zein film preparation, ethanol treatment caused 123–137% and 132–315% activation of the enzyme, respectively. In zein films incorporated with 187–1318 U/cm² (63–455 μg/cm²) lysozyme, the release rates at 4 °C, changed between 7 and 29 U/cm²/min, increased at high lysozyme concentrations. Zein films incorporated with partially purified lysozyme showed antimicrobial effect on Bacillus subtilis and Lactobacillus plantarum. By the addition of disodium EDTA, the films also became effective on Escherichia coli. The results of this study showed that the partially purified lysozyme may be used in antimicrobial packaging to increase food safety.     

The Use of Red Oncom Powder as Potential Production Media for Fibrinogenolytic Protease Derived from Bacillus Licheniformis RO3

The high cost of enzyme production is one of the barriers to successful application of enzyme in the industry. Selection of media is a critical factor for the enzyme production. Main factors for optimization of enzyme production include nutritional components and environmental conditions for growth and production of fibrinogenolytic protease. In this study, Bacillus licheniformis RO3 isolated from red oncom, an Indonesian fermented food was tested for its fibrinogenolytic protease production by using several media. Three types of media were analyzed, i.e. Luria-Bertani broth (LB), ½ LB + 1% skim milk (LBS), and ½ LB + 1% red oncom powder (LBO). Protease activity was tested by using spectrophotometric method with casein as a substrate and fibrinogenolytic activity was confirmed based on zymography assay using fibrinogen substrate. In LB media, B. licheniformis RO3 was able to produce protease with activity of 0.024 U/ml or 0.157 U/mg at 36 h fermentation. In LBS media, the highest protease activity was 0.022 U/ml or 0.152 U/mg at 48 h fermentation. The best result was shown by B. licheniformis RO3 grown in LBO media with the highest protease activity of 0.051 U/ml or 0.283 U/mg at 48 h. Zymographic profiles showed that crude enzyme from B. licheniformis RO3 consisted of six fibrinogenolytic bands with molecular weight of 20, 27, 32, 40, 70, and >140 kDa. These results indicate that red oncom powder can be used as a potential media for fibrinogenolytic protease production.     

Combined effects of high pressure,moderate heat and pH on the inactivation kinetics of Bacillus licheniformis spores in carrot juice

The objective of this research was to evaluate the combined effect of high pressure processing (temperature, pressure and time) and product (pH) related variables on destruction kinetics of spores of Bacillus licheniformis in carrot juice. A 3-level factorial experimental design was used with the microbial spores inoculated into carrot juice at the natural pH (6.2) and acidified pH (4.5 and 5.5), pressure (400, 500 or 600 MPa), temperature (40, 50, and 60 °C) and time (0–40 min) conditions. D values found varied from 0.6 to 14.1 min based on the temperature, pressure and pH level combinations. The corresponding temperature and pressure dependency of D values were in the range 23.3 to 31 °C and 241 to 465 MPa, respectively. The destruction pattern was also dependent on pH, with lower pH contributing to higher destruction rate. Conventional log-linear model and Weibull model were used to describe the survivor curves and for predicting processing time to achieve a 5D spore reduction. The survivor curves exhibited slightly upward concavity and therefore better described by a Weibull than the log-linear model. Treatment combinations showed significant (p ≤ 0.05) effects on D and z values of log-linear model and rate parameter (α) of Weibull model. The 5D spore count reduction times estimated using Weibull model parameters were longer than those from the log-linear model, generally demonstrating an over-treatment. Overall, the pH reduction of low acid foods showed a significant enhancement of rate of destruction of B. licheniformis spores.     

Purification and identification of antioxidative peptides from loach (Misgurnus anguillicaudatus) protein hydrolysate by consecutive chromatography and electrospray ionization-mass spectrometry

Loach protein was hydrolyzed by papain to obtain antioxidative peptides. The results showed that the loach protein hydrolysate (LPH) could scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) (IC₅₀ = 17.0 ± 0.54 mg/mL) and hydroxyl radicals (IC₅₀ = 2.64 ± 0.29 mg/mL). It could chelate cupric ion and inhibit the lipid peroxidation in a linoleic acid emulsion system. The hydrolysate was isolated and purified by ultrafiltration and consecutive chromatographic methods including ion-exchange chromatography, gel filtration chromatography and a two-step reverse high-performance liquid chromatography (RP-HPLC). The purified antioxidant peptide was identified as Pro-Ser-Tyr-Val (464.2 Da) using RP-HPLC connected on-line to an electrospray ionization (ESI) mass spectrometer. The purified peptide showed a 9.14-fold higher scavenging activity for hydroxyl radical compared with the crude LPH. Therefore, it is possible to produce natural antioxidative peptides from loach protein by enzymatic hydrolysis and purification.     

Antimicrobial plastic film: Physico-chemical characterization and nisin desorption modeling

Antimicrobial active films represent an innovative concept in food packaging, developed to answer to consumer's expectation for better microbiological safety. In this study, the growth of pathogenic micro-organisms on the surface of food is proposed to be controlled by coating, on the surface of polyethylene/polyamide/polyethylene film (PE/PA/PE), a film-forming solution containing Nisaplin, a commercial form of bacteriocin produced by Lactococcus lactis subsp. lactis: nisin. The bioactivity of these multi-layer films coated with Nisaplin loaded HydroxyPropylMethylCellulose film is based on the release of this antimicrobial molecule towards a food simulant. Nisin mass transfer was studied and modeled, for different operating conditions, generally encountered in food products. pH didn't seem to interfere with nisin release kinetics, while the variation of NaCl concentration between 0.8% and 3.2% decreased the desorption coefficient (k_d) by 18% and the temperature increase from 10 °C to 28 °C resulted in an increase of k_d from 1.78 × 10^? 2 m s^? 1 to 2.10 × 10^? 2 m s^? 1. Coating of PE/PA/PE film with this antimicrobial layer induced little mechanical properties modifications without compromising industrial applications. Water barrier capacity was not altered.Industrial relevanceThis paper concerns active packaging, considered as a new approach to preserve food shelf life. Active packaging is a real gain for plastic and Food industrials. Coating was used to obtain antimicrobial packaging. The impact of coating on film characteristics is investigated.Also, antimicrobial agent desorption is determined during storage conditions.     

Peptide–peptide and protein–peptide interactions in mixtures of whey protein isolate and whey protein isolate hydrolysates

The extent of aggregation in whey protein isolate (WPI) hydrolysates induced by Bacillus licheniformis protease was quantified as a function of degree of hydrolysis (DH), temperature and ionic strength. The capacity of the hydrolysates to aggregate added intact protein was also studied. The amount of aggregated material and the size of the aggregated peptides were measured by nitrogen content and size exclusion chromatography, respectively. Aggregation increased with DH up to the practical end point of hydrolysis (DH 6.8%). The aggregates formed under the various conditions studied consisted of peptides with masses ranging from 1.4 to 7.5 kDa. The hydrolysates were also able to aggregate added WPI. The additional amount of aggregated material increased with increasing DH. Peptides involved in peptide–peptide interactions were also involved in protein–peptide interactions. It is hypothesized that hydrophobic interactions dominated peptide–peptide interactions, while protein–peptide interactions depended on the balance between hydrophobic attraction and electrostatic repulsion.     

A novel antioxidant and antimicrobial peptide from hen egg white lysozyme hydrolysates

Hen egg white lysozyme (HEWL) was hydrolyzed with papain, trypsin and a combination of the two to isolate antioxidant peptides. The prepared hydrolysates were evaluated for antioxidant activity using DPPH and ABTS radical scavenging, metal ion chelation and lipid peroxidation inhibition. The obtained hydrolysate by a combination of the two enzymes exhibited the highest antioxidant activity compared to other hydrolysates and elected for isolation of antioxidant peptides by reverse-phase high-performance liquid chromatography (RP-HPLC). A most potent fraction namely F2 fraction, identified to be NTDGSTDYGILQINSR (MW: 1753.98 ± 0.5 Da) using tandem mass spectrometry. The antimicrobial activity of the F2 peptide was tested using radial diffusion assay (RDA). Our results showed that this peptide has inhibitory effects on both Gram-negative and Gram-positive bacteria. Minimum inhibition concentration (MIC) values of the F2 peptide against Escherichia coli and Leuconostoc mesenteroides bacteria were 355.64 (±2.2) and 442.25 (±2.8) μg/ml, respectively.     

Antioxidant activity of protein hydrolysates and purified peptides from Zizyphus jujuba fruits

In recent years, there has been a considerable interest in bioactive peptides derived from food proteins which might have beneficial effects on human health. Zizyphus jujuba is a medicinal plant with well-demonstrated biological functions for which various bioactive compounds except antioxidant peptides have been reported. Therefore, the aim of this study was to determine the antioxidant activity of Z. jujuba-derived protein hydrolysates and the purified peptides. Based upon this study, it was revealed that the hydrolysates prepared from Z. jujuba fruit possessed antioxidant effects. Among the prepared hydrolysates, trypsin hydrolysate with the highest antioxidant activity was fractionated using reverse-phase high-performance liquid chromatography (RP-HPLC). The most potent antioxidant peptides, named fractions F3 and F6, identified as VGQHTR (MW: 678.36 ± 0.3 Da) and GWLK (MW: 482.27 ± 0.3 Da), respectively, using tandem mass spectrometry. This study demonstrated that the derived hydrolysates and the purified peptides from Z. jujuba proteins can prevent oxidative reactions and might be underutilised for food preservation and medicinal purposes. However, more detailed studies are required to explore their antioxidant abilities in vivo.     

Optimization of sour milk fermentation for the production of ACE-inhibitory peptides and purification of a novel peptide from whey protein hydrolysate

The effect of fermentation conditions on the production of angiotensin-I converting enzyme (ACE) inhibitory peptide in sour milk fermented by Lactobacillus helveticus LB10 was investigated using response-surface methodology. Optimal conditions to produce the maximum production of ACE-inhibitory peptides were found to be 4% (v/w) inoculum, 7.5 initial pH of medium and 39.0 °C. The fermented milk resulted in 75.46% inhibition in ACE activity. The cell-envelope proteinase, assisted by X-prolyldipeptidyl aminopeptidase of Lb. helveticus LB10 produced the ACE-inhibitory peptides. A novel ACE-inhibitory peptide from whey protein hydrolysate produced by crude proteinases of Lb. helveticus LB10 was purified. The separations were performed with Sephadex^® G-75 and Sephadex G-15 gel filtration chromatography and reversed-phase, high-performance liquid chromatography. The peptide with the RLSFNP sequence was isolated from β-lactoglobulin hydrolysate and its IC₅₀ while inhibiting ACE activity was 177.39 μm.     

Formation of amyloid-like fibrils upon limited proteolysis of bovine α-lactalbumin

Bovine α-lactalbumin (α-LA) (10 g L⁻¹) was incubated with a protease from Bacillus licheniformis at pH 7.5 and 50 °C. The reaction was biphasic consisting of an initial hydrolysis of intact α-LA and formation of dimers from large hydrolysis products within 60 min followed by aggregation of dimers into aggregates of 500 kDa. The aggregates consisted primarily of fibrillar strands with a diameter of 5 nm. Formation of these strands was accompanied by a change in secondary structure towards higher β-sheet content and strong binding of thioflavin, features shared with amyloidal fibrils. The main components in these fibrils were fragments of 8.8 and 9.8 kDa shown to occur in a monomer–dimer equilibrium. These fragments were identified and a molecular mechanism involving side-by-side assembly of dimers of these fragments into fibrils is proposed.     

Identification of bioactive peptides in commercial Cheddar cheese

This study examined the presence of antimicrobial, antioxidant and antihypertensive peptides in three commercially available Australian Cheddar cheeses. Peptide extracts as well as fractionated peptide extracts were examined. Commercial cheese A peptides exhibited the greatest inhibition against Bacillus cereus and also commercial cheese A fractionated peptides greater than 10 kDa showed the highest inhibition against B. cereus. Commercial cheese A peptides also showed the highest inhibition of 2,2-diphenyl-1-picrylhydrazyl (DPPH), a free radical used to measure antioxidant activity. All cheese fractionated peptides greater than 10 kDa demonstrated higher inhibition of DPPH after fractionation. Antihypertensive peptides were determined by inhibition of the angiotensin-converting enzyme (ACE). Overall, commercial cheese A had the lowest concentration required to inhibit ACE and commercial cheese A fractionated peptides lower than 5 kDa had the lowest inhibition after fractionation. These preliminary findings suggest that peptide extracts of three commercial Australian Cheddar cheeses exhibit antimicrobial, antihypertensive and antioxidant properties.     

Purification and characterization of angiotensin I converting enzyme inhibitory peptides from jellyfish Rhopilema esculentum

Two angiotensin I converting enzyme (ACE) inhibitory peptides were isolated from jellyfish Rhopilema esculentum protein hydrolysates prepared with pepsin and papain. Consecutive purification methods, including ion-exchange chromatography, size-exclusion chromatography and reverse-phase high-performance liquid chromatography, were used for isolation of ACE inhibitory peptides. The amino acid sequence of the peptides was identified as Gln-Pro-Gly-Pro-Thr and Gly-Asp-Ile-Gly-Tyr, respectively, and the IC₅₀ value of the purified peptides for ACE inhibitory activity was 80.67 μM and 32.56 μM. The purified peptides were evaluated for the antihypertensive effect in spontaneously hypertensive rats (SHR) after oral administration. Blood pressure decreased significantly after ingestion of the peptides. The results suggest that peptides derived from jellyfish R. esculentum may be beneficial as antihypertensive compounds in functional food resources.     

Enzymatic synthesis of banana flavour (isoamyl acetate) by Bacillus licheniformis S-86 esterase

New carboxylesterase from organic-solvent-tolerant Bacillus licheniformis S-86 strain was characterized. The enzyme named as type II esterase showed an optimal activity in the temperature range 60–65 °C and pH 8.0. The enzyme was moderatly thermostable (half-life of 1 h at 50 °C), but remarkable stable at extremely alkaline pH, retaining 100% of its activity at pH 10.0–11.0. Furthermore, the esterase showed high stability in detergents (86% residual activity in 10% SDS), and also 0.1% ionic and non-ionic detergents are inducers of enzyme activity. PMSF, a serine protease inhibitor, did not show any effect on the activity. The immobilized type II esterase was able to synthesize isoamyl acetate from isoamyl alcohol and p-nitrophenyl acetate (acyl donor) in n-hexane. The resulting ester yield (42.8%), obtained at a low temperature (28 °C) and with a very low amount of enzyme (4.6 × 10^?5 mg ml^?1), indicates a high potential for type II esterase in isoamyl acetate synthesis for production purposes.     

Effects of drying methods and conditions on antimicrobial activity of edible chitosan films enriched with galangal extract

The aim of this work was to study the effects of drying methods and conditions (i.e., ambient drying, hot air drying at 40 °C, vacuum drying and low-pressure superheated steam drying within the temperature range of 70–90 °C at an absolute pressure of 10 kPa) as well as the concentration of galangal extract on the antimicrobial activity of edible chitosan films against Staphylococcus aureus. Galangal extract was added to the film forming solution as a natural antimicrobial agent in the concentration range of 0.3–0.9 g/100 g. Fourier transform infrared (FTIR) spectra and swelling of the films were also evaluated to investigate interaction between chitosan and the galangal extract. The antimicrobial activity of the films was evaluated by the disc diffusion and viable cell count method, while the morphology of bacteria treated with the antimicrobial films was observed via transmission electron microscopy (TEM). The antimicrobial activity, swelling and functional group interaction of the antimicrobial films were found to be affected by the drying methods and conditions as well as the concentration of the galangal extract. The electron microscopic observations revealed that cell wall and cell membrane of S. aureus treated by the antimicrobial films were significantly damaged.     

Antimicrobial and antioxidant activity of essential oil from pink pepper tree (Schinus terebinthifolius Raddi) in vitro and in cheese experimentally contaminated with Listeria monocytogenes

Natural compounds with preservative activity have gained prominence in the field of food science as an alternative to traditional additives. To be effective, biopreservatives must have antioxidant and/or antimicrobial activities, characteristics often found in the essential oils (EO). This study aimed to verify the antimicrobial and antioxidant activity of EO from pink pepper tree fruit. Antimicrobial activity was evaluated in vitro on 18 bacteria, and in situ (Minas-type fresh cheese) against Listeria monocytogenes during storage (30 days/4 °C). The EO from ripe fruit showed the greatest activity in in vitro tests (MBC of 6.8 mg/mL for L. monocytogenes) and exhibited biopreservative activity in situ, having reduced the development of L. monocytogenes by 1.3 log CFU/g in 30 days. The values of peroxides and malonaldehydes were reduced by 3 Meq O₂/Kg and 0.15 mg MDA/Kg, respectively, in 30 days. Results demonstrate that this EO has the potential to act as a preservative in food.Industrial relevanceThe pink pepper tree (Schinus terebinthifolius Raddi) is a plant with favorable features for industrial use, but little exploited by the food industry so far. In this work, the essential oil (EO) of the pink pepper tree was presented as an alternative to us of preservatives traditionally applied in food. For this, antimicrobial and antioxidant activities of the EO were evaluated and discussed, analyzing its effects initially in vitro and after in situ, in order to infer the technological potential for application this extract may have use as a food biopreservative.     

Biochemical characterisation of an aminopeptidase with highest preference for lysine from Japanese flounder skeletal muscle

An aminopeptidase was purified from Japanese flounder skeletal muscle to homogeneity by ammonium sulphate fractionation and three chromatographies. The enzyme was approximately 100 kDa with isoelectric point of 5.7 as estimated by two-dimensional polyacrylamide gel electrophoresis. Its optimum temperature and pH were 45 °C and 7.5, respectively. According to peptide mass fingerprinting study, the enzyme revealed high identity to a puromycin-sensitive aminopeptidase. It had a broad specificity toward aminopeptidase substrates and preferred to hydrolyse Lys-MCA with k_cat/K_m of 8.1 × 10⁶ M^?1 s^?1, and the activation energy (E_a) of 72.5 kJ M^?1. Metal-chelating agents effectively inhibited the enzyme activity, and Zn²⁺, Mn²⁺ and Co²⁺ significantly restored the apoenzymatic activity dialysed by EDTA, whilst inhibitors to other proteinases did not show much effect. Furthermore, bestatin strongly inhibited its activity. These results indicate that the purified enzyme is a metalloaminopeptidase which would possibly contribute to free amino acids increase in fish muscle.     

Removal of meat biofilms from surfaces by ultrasounds combined with enzymes and/or a chelating agent

A curved ultrasonic transducer was devised to standardise biofilm removal for hygiene testing in internal or curved food contact surfaces. Meat biofilms made with Escherichia coli and Staphylococcus aureus on stainless steel sheets were studied. Ultrasounds (10 s at 40 kHz) alone failed to completely remove biofilms: 49 ± 5% and 39 ± 5% recovery rates were obtained for E. coli and S. aureus biofilms, respectively. A combined treatment, which involved the application of ultrasounds to EDTA and/or in enzymes solutions, allowed to remove up to 75 ± 4% and 100 ± 15% of E. coli and S. aureus biofilms, respectively. This application was in agreement with an industrial control i.e. a combined treatment: ultrasound generation in enzymes preparation restricted to an active chamber area with a fast and good reproducible recovery.Industrial relevanceThe biofilm phenomenon has been under intensive research for several years in food industry. A curved ultrasonic transducer was devised to standardise biofilm removal for hygiene testing in internal or curved food contact surfaces. This apparatus uses the mechanical effects of ultrasonic cavitation produced at 40 KHz (10 s) for the non-destructive detection of biofilms in food processing equipment. We report the utilisation of a combined treatment, which involved the application of ultrasounds to EDTA and/or in enzymes solutions on meat biofilms made with E. coli and S. aureus on stainless steel sheets. This application was in agreement with an industrial control i.e. a combined treatment: ultrasound generation in EDTA and/or enzymes preparation restricted to an active chamber area with a fast and good reproducible recovery.     

Phenolic composition,antioxidant and antimicrobial activities of free and bound phenolic extracts of Moringa oleifera seed flour

Phenolic compounds, antioxidant and antibacterial activities of defatted Moringa oleifera seed flour (DMF) were investigated. The total phenolic and flavonoid contents were measured using colorimetric methods. Free phenolic acid and flavonoid profiles were analyzed by high performance liquid chromatography, while antioxidant capacities were evaluated using scavenging assays of 1,1-diphenyl-2-picrylhydrazyl radical (DPPH), ferric reducing antioxidant power and total antioxidant capacity. The results showed that extractability of phenolic compounds was significantly higher (p < 0.05) in bound phenolic extract (4173.00 ± 32.22 mg gallic acid equivalents (GAE)/100 g) than in free phenolic extract (780.00 ± 14.2 mg GAE/100 g) and it showed higher antioxidant and antimicrobial activities. The IC₅₀ value for DPPH radical scavenging activity was 0.9 ± 0.05 and 14.9 ± 0.07 mg/mL for bound phenolic and free phenolic extracts, respectively. Bound phenolic extract was more effective (minimum inhibitory concentration (MIC), 0.06–0.157%) than free phenolic extract (MIC, 0.117–0.191%) against tested bacteria. Ten phenolic compounds (gallic acid, p-coumaric acid, ferulic acid, caffeic acid, protocatechuic acid, cinnamic acid, catechin, epicatechin, vanillin and quercetin) were identified and quantified in both extracts. These natural plant phenolics from Moringa seeds could be a good source of antioxidants and antibacterials for food and pharmaceutical industries.