Chelating peptides from rapeseed meal protein hydrolysates: identification and evaluation of their capacity to inhibit lipid oxidation.

An optimized proteolysis process was applied to rapeseed meal proteins (RP) and the hydrolysate was separated by membrane filtration allowing the production of highly metal-chelating peptides in the permeate. In order to identify the chemical structure of the most active obtained metal-chelating peptides, immobilized metal affinity chromatography (IMAC) was applied. The RP-IMAC peptide fraction was mainly composed of small peptides from 2 to 20 amino acids. Using the Ferrozine assay, RP-IMAC peptides showed a significant chelating efficiency higher than sodium citrate and close to that of EDTA. The peptide sequences were identified by UHPLC-MS and several possible iron binding sites were found. ß-carotene oxidation assay and lipid oxidation in bulk oils or emulsion were carried out to evaluate the potential of such peptides as efficient antioxidants to protect lipids from oxidation. While chelating peptides showed a limited efficiency in bulk oil, they performed more efficiently in emulsion.

Improving the in vitro digestibility of rapeseed albumins resistant to gastrointestinal proteolysis while preserving the functional properties using enzymatic hydrolysis.

Study of in vitro digestibility of high-quality isolate of rapeseed albumins (RA) was carried out in this work, using Size-Exclusion Chromatography. A poor in vitro digestibility of the RA isolate was highlighted (15%). The aim of this study was therefore to improve the RA in vitro digestibility by enzymatic hydrolysis while preserving its attractive functional properties. Alcalase, Flavourzyme and Prolyve were used to obtain 12 hydrolysates with various degrees of hydrolysis (DH) and compositions. All hydrolysates showed improved digestibility and those with the highest DH showed the best improvements. Techno-functional properties of these hydrolysates were also characterized. The poor emulsion capacity of initial RA was improved and results showed that extent proteolysis can be a good way to improve both digestibility and functional properties. Moreover, optimal conditions for RA proteolysis were identified to produce with Flavourzyme a partial hydrolysate (still containing 50% intact RA) that is both digestible and functional.

Optimization of Selective Hydrolysis of Cruciferins for Production of Potent Mineral Chelating Peptides and Napins Purification to Valorize Total Rapeseed Meal Proteins.

Preventing oxidation and microbial spoilage are both major concerns in food industries. In this context, this study aimed to valorize the total rapeseed meal proteins with controlled enzymatic proteolysis to generate potent mineral-chelating peptides from cruciferins while keeping intact the antimicrobial napins. Implementation of proteolysis of total rapeseed protein isolate with the Prolyve® enzyme highlighted an interesting selective hydrolysis of the cruciferins. Hence, the mechanism of this particular hydrolysis was investigated through a Design of Experiments method to obtain a model for the prediction of kinetics (cruciferin degradation and napin purity) according to the operating conditions applied. Then, multicriteria optimization was implemented to maximize the napin purity and yield while minimizing both enzymatic cost and reaction time. Antioxidant assays of the peptide fraction obtained under the optimal conditions proved the high metal-chelating activity preservation (EC50 = 247 ± 27 µg) for more than three times faster production. This fraction might counteract lipid oxidation or serve as preventing agents for micronutrient deficiencies, and the resulting purified napins may have applications in food safety against microbial contamination. These results can greatly help the development of rapeseed meal applications in food industries.

Combined Effect of Extraction and Purification Conditions on Yield, Composition and Functional and Structural Properties of Lupin Proteins.

Lupin meal presents great potential as an alternative plant-based source of proteins for human nutrition. In the present work, different conditions of extraction and purification were evaluated for production of lupin protein isolates. The results showed that the protein extraction yield was comparable at acidic and conventionally used alkaline extraction pH (37% vs. 40-45%, respectively). Proteins extracted were principally composed of globulins. The ionic strength negatively impacted the protein extractability at pH 2, whereas no significant differences were observed between extractions at 20 to 50 °C. The selected extraction conditions (pH 2 and 7) combined with purification by isoelectric precipitation or ultrafiltration process generated the isolate-grade products. Interestingly, further characterization revealed a partial denaturation of proteins extracted at pH 2 resulting in loss of protein solubility at pH 6 and 7 (10-50%), modifications in secondary structure, lower thermal stability, and formation of protein aggregates. However, foaming and emulsifying properties were generally similar for almost all lupin isolates. Further investigation might be of interest with regard to the extraction behaviours and structural and functional properties of specific lupin protein fractions.

Steam explosion of Aucoumea klaineana sapwood: Membrane separation of acetylated hemicelluloses.

The fractionation of the aqueous effluent of Aucoumea klaineana Pierre (Okoumé) sapwood steam explosion was examined by a sequential-dilution type membrane diafiltration. The permeate and retentate fractions were characterized by HPLC-SEC, HSQC-NMR, FTIR, UV-visible and HPAE-PAD ion chromatography. Diafiltration with 10 kDa regenerated cellulose membrane has been shown to provide efficient fractionation without fouling. O2 and/or O3 acetylated xylans with a lower proportion of O2 and/or O3 acetylated glucomannans were isolated in the retentate (≈35% w/w and 1.08 w/w% based on initial effluent solid content and on initial dry wood respectively, including 65% w/w in the range 9-22 kDa). The molecular weights of the polysaccharides were significantly higher than those obtained by ethanolic precipitation. The permeate concentrated low molecular mass oligomers (90% w/w < 2.3 kDa, 1.88 w/w% based on initial dry wood) composed of pectic sugars, highly acetylated xylans (DS ≈ 0.9) and relatively high proportion of soluble lignin (≈40% w/w) including Lignin-Carbohydrate Complexes (LCCs).

The True Amino Acid Digestibility of 15N-Labelled Sunflower Biscuits Determined with Ileal Balance and Dual Isotope Methods in Healthy Humans.

BACKGROUND: Sunflower is a promising protein source but data on amino acid (AA) digestibility are lacking in humans. Classically, the determination of AA digestibility requires ileal digesta sampling. The dual isotope method is minimally invasive but has not been compared to the conventional approach. OBJECTIVES: This study aimed to determine the true ileal digestibility of sunflower AAs in healthy volunteers who ate biscuits containing 15nitrogen (N) protein isolate, in comparison with the dual isotope method. METHODS: Twelve healthy volunteers (men and women; 40.4 ± 10.5 years old; BMI, 23.7 ± 2.9 kg/m2) were equipped with a naso-ileal tube. For 4 hours, they consumed 9 repeated meals comprising 15N-sunflower protein biscuits together with 13carbon (C)-AAs, carried either in chocolate (SUN + Ch; n = 7) or apple puree (SUN + P; n = 5). Ileal digesta and blood were sampled throughout 8 hours after ingestion of the first meal. The 15N and 13C AA enrichments were measured in digesta to determine ileal digestibility directly and in plasma to determine lysine and threonine digestibility using the dual isotope method. Differences between methods and between vector groups were analyzed using paired and unpaired t-tests, respectively. RESULTS: The ileal digestibility of sunflower indispensable AAs (IAA) was 89% ± 5.3%, with threonine and lysine having the lowest digestibility. In the SUN + Ch meal, IAA digestibility was 3% below that of SUN + P (P < 0.05). The mean free 13C-AA ileal digestibility was 98.1% ± 0.9%. No matter which matrix was used to carry 13C-AAs, plasma 15N and 13C-AA kinetics displayed a 1-hour offset. Digestibility obtained with the dual isotope method (70.4% ± 6.0% for threonine and 75.9% ± 22.3% for lysine) was below the target values. CONCLUSIONS: The ileal digestibility of IAAs from a sunflower isolate incorporated in a biscuit was close to 90% in healthy adults. Under our experimental conditions, the dual isotope method provided lower values than the usual method. Further protocol developments are needed to validate the equivalence between both methods. This trial was registered at clinicaltrials.gov as NCT04024605.

Identification and Capture of Phenolic Compounds from a Rapeseed Meal Protein Isolate Production Process By-Product by Macroporous Resin and Valorization Their Antioxidant Properties.

In this study, phenolic compounds from an aqueous protein by-product from rapeseed meal (RSM) were identified by HPLC-DAD and HPLC-ESI-MS, including sinapine, sinapic acid, sinapoyl glucose, and 1,2-di-sinapoyl gentibiose. The main phenolic compound in this by-product was sinapine. We also performed acid hydrolysis to convert sinapine, and sinapic acid derivatives present in the permeate, to sinapic acid. The adsorption of phenolic compounds was investigated using five macroporous resins, including XAD4, XAD7, XAD16, XAD1180, and HP20. Among them, XAD16 showed the highest total phenolic contents adsorption capacities. The adsorption behavior of phenolic compounds was described by pseudo-second-order and Langmuir models. Moreover, thermodynamics tests demonstrated that the adsorption process of phenolic compounds was exothermic and spontaneous. The highest desorption ratio was obtained with 30% (v/v) and 70% (v/v) ethanol for sinapine and sinapic acid, respectively, with a desorption ratio of 63.19 ± 0.03% and 94.68 ± 0.013%. DPPH and ABTS tests revealed that the antioxidant activity of the hydrolyzed fraction was higher than the non-hydrolyzed fraction and higher than the one of vitamin C. Antioxidant tests demonstrated that these phenolic compounds could be used as natural antioxidants, which can be applied in the food industry.

Sensitization Potency of Sunflower Seed Protein in a Mouse Model: Identification of 2S-Albumins More Allergenic Than SFA-8.

SCOPE: Food allergy to sunflower seed (SFS) protein is not frequent and only non-specific lipid transfert protein (nsLTP) Hel a 3 is officially recognized as a food allergen. Out of the eleven seed storage 2S-albumins (SESA) detected in SFS, only SFA-8 allergenicity has been investigated so far. The study aimed then to evaluate SFS protein allergenicity and particularly, to compare the sensitization potency of SESA in a mouse model. METHODS AND RESULTS: The most abundant SESA and nsLTP were isolated from SFS through a combination of chromatographic methods. Purified proteins were then used to measure specific IgG1 and IgE responses in BALB/c mice orally sensitized to different SFS protein isolates. The study, thus, confirmed the allergenicity of SFA-8 and Hel a 3 but mice were also highly sensitized to other SESA such as SESA2-1 or SESA20-2. Furthermore, competitive inhibition of IgE-binding revealed that SFA-8 IgE-reactivity was due to cross-reactivity with other SESA. 11S-globulins were weakly immunogenic and were rapidly degraded in an in vitro model of gastroduodenal digestion. In contrast, Hel a 3, SESA2-1 and SFA-8 were more resistant to proteolysis and gastroduodenal digestion did not affect their IgE-reactivity. CONCLUSIONS: SESA2-1 or SESA20-2 were more potent allergens than SFA-8 in this mouse model. Allergenicity of SESA must be now confirmed in SFS-allergic patients.

Production and antioxidant capacity of bioactive peptides from plant biomass to counteract lipid oxidation.

Preventing lipid oxidation, especially with the polyunsaturated fat-based products, is a major concern in sectors as agri-food and cosmetic. Even though the efficiency of synthetic antioxidants has been recognized, both consumers and manufacturers are looking for more innovative, healthy and quality products while rejecting synthetic additives due to their concern about safety, along with their environmental impact issues. In this context, plant biomass, which have shown to be rich in compounds, have raised interest for the isolation of novel naturally occurring antioxidants. Among their myriad of molecules, bioactive peptides, which are biologically active sequence of amino acid residues of proteins, seem to be of a great interest. Therefore, the number of identified amino acids sequences of bioactive peptides from plant biomass with potential antioxidant action is progressively increasing. Thus, this review provides a description of 129 works that have been made to produce bioactive peptides (hydrolysate, fraction and/or isolate peptide) from 55 plant biomass, along with the procedure to examine their antioxidant capacity (until 2019 included). The protein name, the process, and the method to concentrate or isolate antioxidant bioactive peptides, along with their identification and/or specificity were described. Considering the complex, dynamic and multifactorial physico-chemical mechanisms of the lipid oxidation, an appropriate in-vitro methodology should be better performed to efficiently probe the antioxidant potential of bioactive peptides. Therefore, the results were discussed, and perspective for antioxidant applications of bioactive peptides from plant biomass was argued.

A Rational Approach for the Production of Highly Soluble and Functional Sunflower Protein Hydrolysates.

Exploitation of plant proteins as an alternative to animal proteins currently presents an important challenge for food industries. In this contribution, total sunflower protein isolate from cold press meal was used as a starting material for the generation of highly soluble and functional hydrolysates that could be used in various food formulations. To do this, a rational and complete approach of controlled hydrolysis was implemented using the individual Alcalase and Prolyve enzymes. The method of stopping the hydrolysis reaction was also evaluated. The influence of operating conditions on hydrolysis kinetics and enzymatic mechanism was studied to identify the appropriate hydrolysis conditions. The gain of the solubility was then analyzed and compared to that of the initial proteins. Finally, the emulsifying and foaming properties (capacities and stabilities) of the resulting hydrolysates were also assessed. As a result, controlled enzymatic proteolysis significantly improved the sunflower protein solubility at neutral pH (twofold increase) and generated highly soluble hydrolysates. The limited proteolysis also maintained the good foam capacities and allowed an improvement in the initial foam stabilities and emulsifying capacities and stabilities of sunflower proteins. This contribution can greatly increase the value of sunflower meal and help in the development of sunflower protein products in the future.

Multicriteria Optimization of Phenolic Compounds Capture from a Sunflower Protein Isolate Production Process by-Product by Adsorption Column and Assessment of Their Antioxidant and Anti-Inflammatory Effects.

The aim of this study was to valorize liquid effluent from the sunflower protein isolate process by extracting phenolic compounds it contains. To do so, XAD7 resin was used. A multicriteria optimization methodology based on design of experiments showed the optimal conditions were adsorption flow rate of 15 BV/h at pH 2.7, a desorption flow rate at 120 BV/h with ethanol/water 50% (v/v). The best trade-off between purity and recovery yields resulted in the production of a fraction containing 76.05% of chlorogenic acid (CGA) whose biological properties were evaluated. DPPH and ABTS tests showed that this fraction had a higher radical scavenging capacity than vitamin C. In vitro assays have shown that this fraction, when used at a concentration corresponding to 50 or 100 µM of CGA, does not present any cytotoxicity on human THP-1 cells differentiated into macrophages. In addition, this fraction when added prior to the inflammatory stimulus (LPS) can reduce tumor necrosis factor-alpha (TNF-α) production by 22%, thereby highlighting its protective properties against future inflammation.

Sunflower Proteins at Air-Water and Oil-Water Interfaces.

The adsorption of a sunflower protein extract at two air-water and oil-water interfaces is investigated using tensiometry, dilational viscoelasticity, and ellipsometry. For both interfaces, a three step mechanism was evidenced thanks to master curve representations of the data taken at different aging times and protein concentrations. At short times, a diffusion limited adsorption of proteins at interfaces is demonstrated. First, a two-dimensional protein film is formed with a partition of the polypeptide chains in the two phases that depends strongly on the nature of the hydrophobic phase: most of the film is in the aqueous phase at the air-water interface, while it is mostly in the organic phase at the oil-water interface. Then a three-dimensional saturated monolayer of proteins is formed. At short times, adsorption mechanisms are analogous to those found with typical globular proteins, while strong divergences are observed at longer adsorption times. Following the saturation step, a thick layer expands in the aqueous phase and appears associated with the release of large objects in the bulk. The kinetic evolution of this second layer is compatible with a diffusion limited adsorption of the minor population of polymeric complexes with hydrodynamic radius RH ∼ 80 nm, evidenced in equilibrium with hexameric globulins (RH ∼ 6 nm) in solution. These complexes could result from the presence of residual polyphenols in the extract and raise the question of the role of these compounds in the interfacial properties of plant protein extracts.

Ultrafiltration Fractionation of Bovine Hemoglobin Hydrolysates: Prediction of Separation Performances for Optimal Enrichment in Antimicrobial Peptide.

Hydrolysis of bovine hemoglobin (bHb), the main constituent of bovine cruor by-product, releases a natural antimicrobial peptide (NKT) which could present a major interest for food safety. To enrich this, tangential ultrafiltration can be implemented, but ultrafiltration conditions are mainly empirically established. In this context, the application of a simulation method for predicting the NKT yield and enrichment was investigated. Ultrafiltration performances were studied for decolored bHb hydrolysates at different degrees of hydrolysis (DH; 3%, 5%, 10% and 18%) and colored hydrolysates (3% and 5% DH) with 1 and 3 kg·mol-1 regenerated cellulose membranes. The simulation method helped to identify the most promising hydrolysate (in terms of NKT enrichment, yield and productivity) as the 3% DH colored hydrolysate, and UF conditions (volumetric reduction factor of 5 and 3 with 1 and 3 kg·mol-1 membrane, respectively) for higher antimicrobial recovery. A maximal enrichment factor of about 29 and NKT purity of 70% in permeate were observed. The results showed that the antimicrobial activity was in relation with the process selectivity and NKT purity. Finally, this reliable method, applied for predicting the ultrafiltration performances to enrich peptides of interest, is part of a global approach to rationally valorize protein resources from various by-products.

Multi-objective optimization of solid/liquid extraction of total sunflower proteins from cold press meal.

The impact of pH (6-9) and NaCl concentration (0-0.5 mol.L-1) on sunflower protein extraction was studied through design of experiments. The considered criteria were protein extraction yield (total proteins, helianthinin and albumins), chlorogenic acids covalently bound to proteins, and free chlorogenic acid concentration in the aqueous extract. Statistical analysis showed that the obtained by design of experiments the polynomial models of each extraction criteria were reliable for predicting the responses. They were employed in an original multi-objective optimization methodology. The optimal conditions revealed to be pH 7.3/0.3 mol.L-1 NaCl yielded 46.83% and 59.16% of total protein and albumin extraction yield, 1.730 and 1.998 mg.g-1 of chlorogenic acids covalently bound to helianthinin and albumins in aqueous extract, respectively. The sunflower protein isolate obtained after extraction in this condition had good solubility (40-80% at pH 5-8), functional properties (foaming and emulsifying) and a satisfying color.

15N and ²H Intrinsic Labeling Demonstrate That Real Digestibility in Rats of Proteins and Amino Acids from Sunflower Protein Isolate Is Almost as High as That of Goat Whey.

BACKGROUND: In the context of developing plant protein sources for humans, sunflower is a good candidate in its form as an oilseed coproduct. OBJECTIVES: We aimed to compare the real digestibility in rats of a sunflower isolate to that of goat whey protein. We also studied the efficiency of 15N and 2H intrinsic labeling in this assessment. METHODS: Sunflower seeds and goat milk were labeled with 15N and 2H. Male Wistar rats (10 wk old) were fed a meal containing 12% of either sunflower isolate (n = 8) or whey (n = 8). Six hours after meal ingestion, protein and amino acid digestibility were assessed by measuring nitrogen, hydrogen, and amino acids in the digesta, as well as isotope enrichments in the bulk and individual amino acids. The differences between groups and isotopes were respectively tested with an unpaired and a paired t test. RESULTS: Protein isolate purity was 87% for whey and 94% for sunflower. 2H and 15N enrichments were, respectively, 0.12 atom % (AP) and 1.06 AP in sunflower isolate and 0.18 AP and 0.95 AP in whey. Fecal 15N protein digestibility was 97.2 ± 0.2% for whey and 95.1 ± 0.5% for sunflower isolate. The use of 2H resulted in a lower digestibility estimate than 15N for whey (96.9 ± 0.2%, P < 0.05) and sunflower (94.2 ± 0.5%, P < 0.01). For both isotopes, protein digestibility was about 2% higher for whey than for sunflower isolate. Mean 15N amino acid caecal digestibility was 97.5 ± 0.2% for whey and 96.3 ± 0.2% for sunflower isolate. The values obtained with 15N and 2H resulted in significant differences ranging from -0.1% to 3.5%. The DIAAS was >1.0 for whey and 0.84 for sunflower (lysine). CONCLUSIONS: The protein and amino acid digestibility of sunflower isolate was high but its DIAAS reflected a moderate lysine imbalance. Despite slight differences with 15N, deuterium produced comparable results, making it suitable for in vivo digestion studies.

A new size-exclusion chromatography method for fast rapeseed albumin and globulin quantification.

The method described in the article aims at the quantification of both main storage proteins, globulins and albumins, in aqueous extract from rapeseed, as an alternative to the current reference methods, Kjeldahl and SDS-PAGE electrophoresis. The new method lies on the analytical separation of extracted compounds by Size-Exclusion High Performance Liquid Chromatography (SE-HPLC) (Biosep-SEC-s2000, 5 µm). The elution of rapeseed extracts with water/acetonitrile/trifluoroacetic acid (45/55/0.1% v/v) during 30 min yields two distinct peaks for the main proteins of rapeseed. Based on the protein extinction coefficients, a calibrationless methodology was developed for their quantification on the basis of the UV signal. The SE-HPLC method was successfully compared to references: Kjeldahl and SDS-PAGE densitometry for the determination of the proportion of each protein. Then, it was successfully applied on two other oleoproteagineous plants, linseed and sunflower.

A new SE-HPLC method for simultaneous quantification of proteins and main phenolic compounds from sunflower meal aqueous extracts.

The aim of this research was to develop a method for simultaneous quantification of proteins and main polyphenolic compounds extracted from oleaginous meal by aqueous media. Size exclusion chromatography with a Biosep column (exclusion range from 1 to 300 kDa) and acetonitrile/water/formic acid (10:89.9:0.1 v/v) eluent at 0.6 mL min-1 yielded the most efficient separation of sunflower proteins and chlorogenic acid monoisomers (3-caffeoylquinic acid, 5-caffeoylquinic acid, and 4-caffeoylquinic acid). After a study of the stability of the extract components, the incorporation of a stabilization buffer (0.5 mol L-1 tris(hydroxymethyl)aminomethane-hydrochloric acid/1.0 mol L-1 sodium chloride at pH 7) was proposed to avoid polyphenol-protein interactions and/or isomeric transformation. The use of 214 nm as the wavelength for protein quantification was also included to minimize the effect of interference from polyphenol-protein interactions on the quantification. Under the used experimental conditions, the protein and chlorogenic acid monoisomer signals remained stable during 300 min at 20 °C (95-125% of the starting value). The developed method was validated and parameters such as specificity, sensitivity, precision, and accuracy were determined. The results from size exclusion chromatography correlated well with the results of protein determination by the reference Kjeldahl method. The proposed method was successfully applied for rapeseed extract analysis making simultaneous quantification of proteins and major rapeseed polyphenols (sinapine and sinapic acid) possible. Graphical abstract.

Simultaneous quantification of the degree of hydrolysis, protein conversion rate and mean molar weight of peptides released in the course of enzymatic proteolysis.

This paper describes an original analytical methodology for a simultaneous measurement of the protein conversion rate, the mean molar weight of peptide and the degree of hydrolysis in the course of proteolysis by Size-Exclusion High-Performance Liquid Chromatography. Peak area of dead volume eluents reflects the non-converted protein. The protein conversion rate is thus determined by comparing the area at a given time to the initial area. The peptide signal allows determining the peptide molar weight distribution and degree of hydrolysis of hydrolysates. As a first step, the approach was tested on the hydrolysis of bovine serum albumin, lysozyme and rapeseed albumin by Alcalase 2.4L. Values of degree of hydrolysis were also determined by TNBS and pH-stat methods. Most of the hydrolysate obtained showed relative differences < 20% with the reference methods. The method was also adapted to fit the TNBS assay. 39 experimental validation tests were analyzed by size-exclusion chromatography, TNBS and pH stat methods. 90% of the validation data show non-significant differences between the degree of hydrolysis predicted and the degree of hydrolysis measured by TNBS method. Hence, the proposed methodology can be efficient to study the process of enzymatic proteolysis while minimizing time and quantity of sample assay required.

An aminoacylase activity from Streptomyces ambofaciens catalyzes the acylation of lysine on α-position and peptides on N-terminal position.

The presence of aminoacylase activities was investigated in a crude extract of Streptomyces ambofaciens ATCC23877. First activities catalyzing the hydrolysis of N-α or ε-acetyl-L-lysine were identified. Furthermore, the acylation of lysine and different peptides was studied and compared with results obtained with lipase B of Candida antarctica (CALB). Different regioselectivities were demonstrated for the two classes of enzymes. CALB was able to catalyze acylation only on the ε-position whereas the crude extract from S. ambofaciens possessed the rare ability to catalyze the N-acylation on the α-position of the lysine or of the amino-acid in N-terminal position of peptides. Two genes, SAM23877_1485 and SAM23877_1734, were identified in the genome of Streptomyces ambofaciens ATCC23877 whose products show similarities with the previously identified aminoacylases from Streptomyces mobaraensis. The proteins encoded by these two genes were responsible for the major aminoacylase hydrolytic activities. Furthermore, we show that the hydrolysis of N-α-acetyl-L-lysine could be attributed to the product of SAM23877_1734 gene.

Recombinant pediocin in Lactococcus lactis: increased production by propeptide fusion and improved potency by co-production with PedC.

We describe the impact of two propeptides and PedC on the production yield and the potency of recombinant pediocins produced in Lactococcus lactis. On the one hand, the sequences encoding the propeptides SD or LEISSTCDA were inserted between the sequence encoding the signal peptide of Usp45 and the structural gene of the mature pediocin PA-1. On the other hand, the putative thiol-disulfide oxidoreductase PedC was coexpressed with pediocin. The concentration of recombinant pediocins produced in supernatants was determined by enzyme-linked immunosorbent assay. The potency of recombinant pediocins was investigated by measuring the minimal inhibitory concentration by agar well diffusion assay. The results show that propeptides SD or LEISSTCDA lead to an improved secretion of recombinant pediocins with apparently no effect on the antibacterial potency and that PedC increases the potency of recombinant pediocin. To our knowledge, this study reveals for the first time that pediocin tolerates fusions at the N-terminal end. Furthermore, it reveals that only expressing the pediocin structural gene in a heterologous host is not sufficient to get an optimal potency and requires the accessory protein PedC. In addition, it can be speculated that PedC catalyses the correct formation of disulfide bonds in pediocin.