Evaluation of short-circuited electrodes in combination with dark fermentation for promoting biohydrogen production process.

Short-circuited electrodes, in combination with dark fermentation, were evaluated in a biohydrogen production process. The system is based on an innovative design of a non-compartmented electromicrobial bioreactor with a conductive tubular membrane as cathode and a graphite felt as anode. In particular, the electrode specialization occurred when the bioreactor was inoculated with manure as the whole medium and when a vacuum was applied in the tubular membrane, for allowing continuous extraction of gaseous species (H2, CH4, CO2) from the bioreactor. This specialization of the electrodes as anode and cathode was further confirmed by microbial ecology analysis of biofilms and by cyclic voltammetry measurements. In these experimental conditions, the potential of the electrochemical system (short-circuited electrodes) reached values as low as -320 mV vs. SHE, associated with a significant bioH2 production. Moreover, a higher bioH2 production occurred and a potential of the electrochemical system as low as -429 mV vs SHE was temporarily observed, when additional heat treatments of the whole manure were applied in order to remove methanogen microorganisms (i.e., hydrogen consumers). In the bioreactor, the higher production of bioH2 would be promoted by electrofermentation from the current flow observed between short-circuited anode and cathode.

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.

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.

N-acylation of L-amino acids in aqueous media: Evaluation of the catalytic performances of Streptomyces ambofaciens aminoacylases.

N-acylated amino acids are widely used as surfactants and/or actives in cosmetics and household formulations. Their industrial production is based on the use of the Schotten-Baumann chemical and unselective reaction. Faced to the growing demand for greener production processes, selective enzymatic synthesis in more environment-friendly conditions starts to be considered as a potential alternative. This study concerns the use of the aminoacylases from Streptomyces ambofaciens to selectively catalyse aminoacid acylation reaction by fatty acids in aqueous medium. The results demonstrated that, when using undecylenoic acid as acyl donor, these aminoacylases properly catalyse the acylation of 14 of the 20 proteogenic l-amino acids tested on their α amino group with a great variability depending on the nature of the amino acid (polar or not, positively/negatively charged, aromatic or not…). More precisely, the following 9 amino acids were shown to be preferentially acylated by S. ambofaciens aminoacylases as follows: lysine > arginine > leucine > methionine > phenylalanine > valine > cysteine > isoleucine > threonine. Different fatty acids were used as acyl donors and, in most cases, the fatty acid length influenced the conversion yield. The kinetic study of α-lauroy-lysine synthesis showed a positive influence of lysine concentration with Vmax and Km of 3.7 mM/h and 76 mM, respectively. Besides, the lauric acid had an inhibitory effect on the reaction with Ki of 70 mM. The addition of cobalt to the reaction medium led to a more than six-fold increase of the reaction rate. These results, achieved with the aminoacylases from S. ambofaciens represent an improved enzyme-based N-acylated amino acids production in order to provide an alternative way to the Schotten-Baumann chemical reaction currently used in the industry.

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.

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.

Diauxic growth of Clostridium acetobutylicum ATCC 824 when grown on mixtures of glucose and cellobiose.

Clostridium acetobutylicum, a promising organism for biomass transformation, has the capacity to utilize a wide variety of carbon sources. During pre-treatments of (ligno) cellulose through thermic and/or enzymatic processes, complex mixtures of oligo saccharides with beta 1,4-glycosidic bonds can be produced. In this paper, the capability of C. acetobutylicum to ferment glucose and cellobiose, alone and in mixtures was studied. Kinetic studies indicated that a diauxic growth occurs when both glucose and cellobiose are present in the medium. In mixtures, D-glucose is the preferred substrate even if cells were pre grown with cellobiose as the substrate. After the complete consumption of glucose, the growth kinetics exhibits an adaptation time, of few hours, before to be able to use cellobiose. Because of this diauxic phenomenon, the nature of the carbon source deriving from a cellulose hydrolysis pre-treatment could strongly influence the kinetic performances of a fermentation process with C. acetobutylicum.

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.

Response surface methodology applied to Supercritical Fluid Extraction (SFE) of carotenoids from Persimmon (Diospyros kaki L.).

Supercritical carbon dioxide with ethanol as co-solvent was used to extract carotenoids from persimmon fruits (Diospyros kaki L.). Based on a response surface methodology (RSM), a predicting model describing the effects of CO2 temperature, pressure, flow rate, ethanol percentage and extraction time was set up for each of the four carotenoids of interest. The best extraction yields in our experimental domain were found at 300 bars, 60°C, 25% (w/w) ethanol, 3mL/min flow rate and 30min for xanthophylls (all-trans-lutein, all-trans-zeaxanthin and all-trans-ß-cryptoxanthin). The yields were 15.46±0.56, 16.81±1.74 and 33.23±2.91µg/g of persimmon powder for all-trans-lutein, all-trans-zeaxanthin and all-trans-ß-cryptoxanthin, respectively. As a non-oxygenated carotenoid, all-trans-ß-carotene was better extracted using 100 bars, 40°C, 25% (w/w) ethanol, 1mL/min flow rate and 30min extraction time, with an extraction yield of 11.19±0.47µg/g of persimmon powder.

Size-exclusion HPLC as a sensitive and calibrationless method for complex peptide mixtures quantification.

This work describes an original methodology to quantify complex peptide mixtures by size-exclusion high-performance liquid chromatography (SE-HPLC). The methodology was first tested on simulated elutions of peptide mixtures. For this set of experiments, a good estimation of the total peptide concentration was observed (error less than 10 %). Then 30 fractions obtained by ultrafiltration of hydrolysates from two different sources were titrated by Kjeldahl or BCA analysis and analysed by SE-HPLC for an experimental validation of the methodology. Very good matchs between methods were obtained. The linear working range depends on the hydrolysate but is generally between 0.2 and 4gL(-1) (i.e. between 10 and 200µg). Moreover, the presence of organic solvents or salts in samples does not impact the accuracy of the methodology contrary to common quantification methods. Hence, the findings of this study show that total concentration of complex peptide mixture can be efficiently determinate by the proposed methodology using simple SE-HPLC analysis.

Accelerated solvent extraction of carotenoids from: Tunisian Kaki (Diospyros kaki L.), peach (Prunus persica L.) and apricot (Prunus armeniaca L.).

Extraction of carotenoids from biological matrices and quantifications remains a difficult task. Accelerated solvent extraction was used as an efficient extraction process for carotenoids extraction from three fruits cultivated in Tunisia: kaki (Diospyros kaki L.), peach (Prunus persica L.) and apricot (Prunus armeniaca L.). Based on a design of experiment (DoE) approach, and using a binary solvent consisting of methanol and tetrahydrofuran, we could identify the best extraction conditions as being 40°C, 20:80 (v:v) methanol/tetrahydrofuran and 5 min of extraction time. Surprisingly and likely due to the high extraction pressure used (103 bars), these conditions appeared to be the best ones both for extracting xanthophylls such as lutein, zeaxanthin or ß-cryptoxanthin and carotenes such as ß-carotene, which present quite different polarities. Twelve surface responses were generated for lutein, zeaxanthin, ß-cryptoxanthin and ß-carotene in kaki, peach and apricot. Further LC-MS analysis allowed comparisons in carotenoids profiles between the fruits.

Characterization and manipulation of the pathway-specific late regulator AlpW reveals Streptomyces ambofaciens as a new producer of Kinamycins.

The genome sequence of Streptomyces ambofaciens, a species known to produce the congocidine and spiramycin antibiotics, has revealed the presence of numerous gene clusters predicted to be involved in the biosynthesis of secondary metabolites. Among them, the type II polyketide synthase-encoding alp cluster was shown to be responsible for the biosynthesis of a compound with antibacterial activity. Here, by means of a deregulation approach, we gained access to workable amounts of the antibiotics for structure elucidation. These compounds, previously designated as alpomycin, were shown to be known members of kinamycin family of antibiotics. Indeed, a mutant lacking AlpW, a member of the TetR regulator family, was shown to constitutively produce kinamycins. Comparative transcriptional analyses showed that expression of alpV, the essential regulator gene required for activation of the biosynthetic genes, is strongly maintained during the stationary growth phase in the alpW mutant, a stage at which alpV transcripts and thereby transcripts of the biosynthetic genes normally drop off. Recombinant AlpW displayed DNA binding activity toward specific motifs in the promoter region of its own gene and that of alpV and alpZ. These recognition sequences are also targets for AlpZ, the γ-butyrolactone-like receptor involved in the regulation of the alp cluster. However, unlike that of AlpZ, the AlpW DNA-binding ability seemed to be insensitive to the signaling molecules controlling antibiotic biosynthesis. Together, the results presented in this study reveal S. ambofaciens to be a new producer of kinamycins and AlpW to be a key late repressor of the cellular control of kinamycin biosynthesis.