Biorefinery cascade processing for creating added value on tomato industrial by-products from Tunisia

Background

In today’s consumer perception of industrial processes and food production, aspects like food quality, human health, environmental safety, and energy security have become the keywords. Therefore, much effort has been extended toward adding value to biowastes of agri-food industries through biorefinery processing approaches. This study focused, for the first time, on the valorization of tomato by-products of a Tunisian industry for the recovery of value-added compounds using biorefinery cascade processing.

Results

The process integrated supercritical CO₂ extraction of carotenoids within the oil fractions from tomato seeds (TS) and tomato peels (TP), followed by a batch isolation of protein from the residues. The remaining lignocellulosic matter from both fractions was then submitted to a liquid hot water (LHW) hydrolysis. Supercritical CO₂ experiments extracted 5.79% oleoresin, 410.53 mg lycopene/kg, and 31.38 mg β-carotene/kg from TP and 26.29% oil, 27.84 mg lycopene/kg, and 5.25 mg β-carotene/kg from TS, on dry weights. Protein extraction yields, nearing 30% of the initial protein contents equal to 13.28% in TP and 39.26% in TS, revealed that TP and TS are a rich source of essential amino acids. LHW treatment run at 120–200 °C, 50 bar for 30 min showed that a temperature of 160 °C was the most convenient for cellulose and hemicellulose hydrolysis from TP and TS, while keeping the degradation products low.

Conclusions

Results indicated that tomato by-products are not only a green source of lycopene-rich oleoresin and tomato seed oil (TSO) and of protein with good nutritional quality but also a source of lignocellulosic matter with potential for bioethanol production. This study would provide an important reference for the concept and the feasibility of the cascade fractionation of valuable compounds from tomato industrial by-products.

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Graphical abstract Schema of biorefinery cascade processing of tomato industrial by-products toward isolation of valuable fractions.

     

Moroccan mitochondrial genetic background suggests prehistoric human migrations across the Gibraltar Strait

Migrations into Africa from the Levant have greatly determined the mitochondrial genetic landscape of North Africa. After analyzing samples from North Morocco to Spain, we show that three fourths of the Moroccan individuals belong to Western Eurasian haplogroups and the frequencies of these are much more similar to those of the Iberian Peninsula than to those of the Middle East. This is particularly true for the mitochondrial haplogroups H1, H3 and V, which experienced a late-glacial expansion from this region, that repopulated much of Central and Northern Europe. Iberian Peninsula was also a source for prehistoric migrations to North Africa.     

Kinetic Study of Neuropeptide Y (NPY) Proteolysis in Blood and Identification of NPY3�C35: A NEW PEPTIDE GENERATED BY PLASMA KALLIKREIN*

There is little information on how neuropeptide Y (NPY) proteolysis by peptidases occurs in serum, in part because reliable techniques are lacking to distinguish different NPY immunoreactive forms and also because the factors affecting the expression of these enzymes have been poorly studied. In the present study, LC-MS/MS was used to identify and quantify NPY fragments resulting from peptidolytic cleavage of NPY_1–36 upon incubation with human serum. Kinetic studies indicated that NPY_1–36 is rapidly cleaved in serum into 3 main fragments with the following order of efficacy: NPY_3–36 ≫ NPY_3–35 > NPY_2–36. Trace amounts of additional NPY forms were identified by accurate mass spectrometry. Specific inhibitors of dipeptidyl peptidase IV, kallikrein, and aminopeptidase P prevented the production of NPY_3–36, NPY_3–35, and NPY_2–36, respectively. Plasma kallikrein at physiological concentrations converted NPY_3–36 into NPY_3–35. Receptor binding assays revealed that NPY_3–35 is unable to bind to NPY Y1, Y2, and Y5 receptors; thus NPY_3–35 may represent the major metabolic clearance product of the Y2/Y5 agonist, NPY_3–36.Neuropeptide Y (NPY)² is a 36-amino acid peptide involved in the central and peripheral control of blood pressure (1–4) and in feeding behavior and obesity (5–9). NPY stimulates at least 6 types of receptors, called Y1, Y2, Y3, Y4, Y5, and y6 (10–12). The Y1 receptor has high affinity for full-length NPY, while Y2 and Y5 receptors bind and are stimulated by full-length and N-terminally truncated NPY. The physiological effects associated to the Y1 and Y2 receptors are the best known; exposure to a Y1 agonist causes an increase in blood pressure and potentiates postsynaptically the action of other vasoactive substances (1, 4, 13), whereas Y2 receptors are mainly located presynaptically, and upon stimulation mediate the inhibition of neurotransmitter release (14, 15). NPY is a prototype of peptide whose function can be altered by proteases. Among peptidases displaying a high affinity for NPY, the primary role appears to be played by dipeptidyl peptidase IV (DPPIV, EC 3.4.14.5), a serine-type protease, also known as CD26, that releases an N-terminal dipeptide, Xaa-Xab- -Xac, preferentially when Xab is a proline or an alanine residue (16). By cleaving the Tyr-Pro dipeptide off the NPY N-terminal extremity, DPPIV generates NPY_3–36, a truncated form that loses its affinity for the Y1 receptor and becomes a Y2/Y5 receptor agonist (17, 18).NPY can also be degraded by aminopeptidase P (AmP, EC 3.4.11.9), a metalloprotease that hydrolyzes the peptide bond between the first and the second amino acid residue at the N terminus of proteins, if the second amino acid is a proline (19). AmP removes the N-terminal tyrosine from NPY to generate NPY_2–36, a selective Y2 agonist (18, 20). There is little information on how NPY cleavage by these enzymes occurs in serum, in part because reliable techniques are lacking to distinguish different NPY immunoreactive (NPYir) forms and also because the factors affecting the expression of these enzymes have been poorly studied. Recently, Frerker et al. (21) reported by MALDI-TOF mass spectrometry that NPY_1–36 is exclusively degraded by DPPIV into NPY_3–36 in EDTA-plasma but they did not provide kinetics of NPY cleavage efficiency of DPPIV. Beck-Sickinger and co-workers (22) studied with the same technique the metabolic stability of fluorescent N-terminally labeled NPY analogues incubated in human plasma and found that the 36th, 35th, and 33rd residues of NPY analogues may also be removed by unknown carboxypeptidases.We have set up a method using liquid chromatography coupled with tandem mass spectrometry (LC-MSⁿ) to selectively quantify NPY and its C-terminal fragments NPY_2–36 and NPY_3–36 digested by human serum. The assays used the internal standard methodology with stable isotopes NPY_1–36 (IDA) (23, 24) or porcine NPY_1–36 as internal standard.The goal of this work was: 1) to determine to which extent NPY_1–36 is degraded by proteases present in human serum and whether an inhibition of DPPIV and AmP by vildagliptin and apstatin (two specific protease inhibitors), respectively, may affect the metabolism of NPY in serum; 2) to assign kinetic values to the proteases involved in the cleavage process toward NPY; and 3) to characterize new NPY-truncated forms and to check for their possible binding capacities on NPY receptors.     

Characterisation of 5-HT3C, 5-HT3D and 5-HT3E receptor subunits: evolution, distribution and function

The 5‐HT₃ receptor is a member of the ‘Cys‐loop’ family of ligand‐gated ion channels that mediate fast excitatory and inhibitory transmission in the nervous system. Current evidence points towards native 5‐HT₃ receptors originating from homomeric assemblies of 5‐HT_3A or heteromeric assembly of 5‐HT_3A and 5‐HT_3B. Novel genes encoding 5‐HT_3C, 5‐HT_3D, and 5‐HT_3E have recently been described but the functional importance of these proteins is unknown. In the present study, in silico analysis (confirmed by partial cloning) indicated that 5‐HT_3C, 5‐HT_3D, and 5‐HT_3E are not human–specific as previously reported: they are conserved in multiple mammalian species but are absent in rodents. Expression profiles of the novel human genes indicated high levels in the gastrointestinal tract but also in the brain, Dorsal Root Ganglion (DRG) and other tissues. Following the demonstration that these subunits are expressed at the cell membrane, the functional properties of the recombinant human subunits were investigated using patch clamp electrophysiology. 5‐HT_3C, 5‐HT_3D, and 5‐HT_3E were all non‐functional when expressed alone. Co‐transfection studies to determine potential novel heteromeric receptor interactions with 5‐HT_3A demonstrated that the expression or function of the receptor was modified by 5‐HT_3C and 5‐HT_3E, but not 5‐HT_3D. The lack of distinct effects on current rectification, kinetics or pharmacology of 5‐HT_3A receptors does not however provide unequivocal evidence to support a direct contribution of 5‐HT_3C or 5‐HT_3E to the lining of the ion channel pore of novel heteromeric receptors. The functional and pharmacological contributions of these novel subunits to human biology and diseases such as irritable bowel syndrome for which 5‐HT₃ receptor antagonists have major clinical usage, therefore remains to be fully determined.     

The δ-Opioid Receptor in SK-N-BE Human Neuroblastoma Cell Line Undergoes Heterologous Desensitization

Abstract: The human neuroblastoma cell line SK-N-BE expresses δ-opioid receptors negatively coupled to adenylyl cyclase. Prolonged treatment (2 h) of the cells with 100 n M etorphine leads to an almost complete desensitization (8.2 ± 5.9 vs. 45.8 ± 8.7% for the control). Other receptors negatively coupled to adenylyl cyclase, namely, D2-dopaminergic, α₂-adrenergic, and m2/m4-muscarinic, were identified by screening of these cells, and it was shown that prolonged treatment (2 h) with 1 µ M 2-bromo-α-ergocryptine or 1 µ M arterenol resulted in a marked desensitization of D2-dopaminergic and α₂-adrenergic receptors, respectively. Cross-desensitization experiments revealed that pretreatment with etorphine desensitized with the same efficiency the δ-opioid receptor and the D2-dopaminergic receptor, and pretreatment with 2-bromo-α-ergocryptine also desensitized both receptors. In contrast, pretreatment with etorphine desensitized only partly the α₂-adrenergic receptor response, whereas pretreatment with 1 µ M arterenol partly desensitized the δ-opioid receptor response. It is concluded that the δ-opioid receptor-mediated inhibitory response of adenylyl cyclase undergoes heterologous desensitization, and it is suggested that δ-opioid and D2-dopaminergic receptors are coupled to adenylyl cyclase via a G_i2 protein, whereas α₂-adrenergic receptor could be coupled to the enzyme via two G proteins, G_i2 and another member of the G_i/G_o family.     

DEVELOPMENTAL INSTABILITY IN WILD CHROMOSOMAL HYBRIDS OF THE HOUSE MOUSE

In wild populations of the house mouse from Tunisia, fluctuating asymmetry and character size of tooth traits were compared between chromosomal races (2n = 40, all acrocentric standard karyotype, and 2n = 22, with nine fixed Robertsonian fusions) and their natural hybrids. Developmental stability was impaired in hybrids compared to both parental groups. Because genetic divergence measured by allozyme markers was low, genomic incompatibilities were not expected between the chromosomal races. This suggests that differentiation of gene systems specifically involved in development may have occurred between the chromosomal races. Support for the latter was found in the study of character size which showed that the 2n = 22 mice had smaller teeth than either the hybrid or the standard mice. The study of Tunisian chromosomal races thus shows that chromosomal evolution may lead to important changes in coadapted gene systems without involving extensive genic differentiation.     

Old and new questions about cholinesterases

Cholinesterases have been intensively studied for a long time, but still offer many fascinating and fundamental questions regarding their evolution, activity, biosynthesis, folding, post-translational modifications, association with structural proteins (ColQ, PRiMA and maybe others), export or degradation. They constitute an excellent model to study these processes, particularly because of the sensitivity and specificity of enzymic assays. In addition, a number of provocative ideas concerning their proposed non-conventional, or non-catalytic functions deserve to be further documented.