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Professeur des Universités
Faculté des Sciences et Technologies - Nancy
Université de Lorraine
+33 (0)3 72 74 51 93 | Laurent.Miclo@univ-lorraine.fr
Molecules, 29 (7), 1552.
Allouche, R., Hafeez, Z., Dary-Mourot, A., Genay, M., Miclo, L.
In addition to traditional use in fermented dairy products, S. thermophilus also exhibits anti-inflammatory properties both in live and heat-inactivated form. Recent studies have highlighted that some hydrolysates from surface proteins of S. thermophilus could be responsible partially for overall anti-inflammatory activity of this bacterium. It was hypothesized that anti-inflammatory activity could also be attributed to peptides resulting from the digestion of intracellular proteins of S. thermophilus. Therefore, total intracellular proteins (TIP) from two phenotypically different strains, LMD-9 and CNRZ-21N, were recovered by sonication followed by ammonium sulphate precipitation. The molecular masses of the TIP of both strains were very close to each other as observed by SDS-PAGE. The TIP were fractionated by size exclusion fast protein liquid chromatography to obtain a 3–10 kDa intracellular protein (IP) fraction, which was then hydrolysed with pancreatic enzyme preparation, Corolase PP. The hydrolysed IP fraction from each strain exhibited anti-inflammatory activity by modulating pro-inflammatory mediators, particularly IL-1β in LPS-stimulated THP-1 macrophages. However, a decrease in IL-8 secretion was only observed with hydrolysed IP fraction from CNRZ-21N, indicating that strain could be an important parameter in obtaining active hydrolysates. Results showed that peptides from the 3–10 kDa IP fraction of S. thermophilus could therefore be considered as postbiotics with potential beneficial effects on human health. Thus, it can be used as a promising bioactive ingredient for the development of functional foods to prevent low-grade inflammation.
Pratiques en nutrition, 19 (73), pp. 36-40.
Miclo, L.
Les consommateurs qui recherchent des produits peu transformés d’origine végétale incluent de nouvelles graines dans leur alimentation. Parfois qualifiées de superaliments, elles sont parées de vertus pour la santé. Si des activités biologiques ont pu être établies dans des modèles, leur transposition à l’échelle de l’individu nécessiterait des études cliniques plus nombreuses. Certaines de ces graines contiennent des substances pouvant exercer un impact sur la biodisponibilité de nutriments ou la sécurité alimentaire.
Consumers by seeking less-processed products of plant origin, are including new seeds in their diet. Sometimes called superfoods, many health effects are attributed to them. While some biological activities was established in models, their transposition to humans would require more clinical studies. Some of these seeds contain substances that can impact the bioavailability of nutrients or food security.
Pratiques en nutrition, 19 (75), pp. 41-44.
Miclo, L.
Les messages émis par les autorités de santé concernant une consommation excessive de sucre ont été intégrés par les consommateurs qui se tournent vers des sources alternatives au sucre de canne ou de betterave, tout en recherchant une certaine naturalité du produit. Ces alternatives peuvent être des sources de fructose, glucide susceptible d’avoir un impact sur le risque de survenue de certaines pathologies. La préparation de la majorité d’entre eux repose, tout comme le sucre raffiné, sur une série de procédés technologiques.
Consumers have heeded the messages issued by health authorities regarding excessive sugar consumption, but they are nevertheless looking for cane or beet sugar substitutes with the desire to find a natural product. These substitutes can be sources of fructose from beet or cane, a carbohydrate that can have an impact on the risk of developing certain pathologies. In addition, the majority of these substitutes undergo, like refined sugar, technological processes for their preparation.
Pratiques en Nutrition, 19 (76), pp. 8-11.
Miclo, L.
Dans les pays occidentaux, la consommation d’algues est à la hausse, notamment chez les personnes qui suivent un régime végétarien. Les macroalgues sont une source de macronutriments et de micronutriments variés dont la concentration et la biodisponibilité dépendent néanmoins des espèces considérées. La forte capacité de bioaccumulation des macroalgues peut augmenter la concentration de certains nutriments et composés toxiques, ce qui implique de fixer les quantités qu’il est possible d’ingérer sans risque.
In Western countries, seaweed consumption is on the rise, especially among people following a vegetarian diet. Macroalgae are a source of numerous macronutrients and micronutrients, the concentration and bioavailability of which depend on the species considered. The high bioaccumulation capacity of macroalgae can increase the concentration of certain nutrients and toxic compounds leading to determine the quantities that can be consumed without any risk.
Nutrients, 14 (22), pp. 4777.
Allouche, R., Genay, M., Dary-Mourot, A., Hafeez, Z., Miclo, L.
Streptococcus thermophilus, a food grade bacterium, is extensively used in the manufacture of fermented products such as yogurt and cheeses. It has been shown that S. thermophilus strains exhibited varying anti-inflammatory activities in vitro. Our previous study displayed that this activity could be partially due to peptide(s) generated by trypsin hydrolysis of the surface proteins of S. thermophilus LMD-9. Surface protease PrtS could be the source of these peptides during gastrointestinal digestion. Therefore, peptide hydrolysates were obtained by shaving two phenotypically distinct strains of S. thermophilus (LMD-9 PrtS+ and CNRZ-21N PrtS−) with pepsin, a gastric protease, followed or not by trypsinolysis. The peptide hydrolysates of both strains exhibited anti-inflammatory action through the modulation of pro-inflammatory mediators in LPS-stimulated THP-1 macrophages (COX-2, Pro-IL-1β, IL-1β, and IL-8) and LPS-stimulated HT-29 cells (IL-8). Therefore, peptides released from either PrtS+ or PrtS− strains in the gastrointestinal tract during digestion of a product containing this bacterium may display anti-inflammatory effects and reduce the risk of inflammation-related chronic diseases.
Foods, 11 (8), pp. 1157.
Allouche, R., Hafeez, Z., Papier, F., Dary-Mourot, A., Genay, M., Miclo, L.
Nutrients, 14 (11), pp. 2212.
Benoit, S., Chaumontet, C., Violle, N., Boulier, A., Hafeez, Z., Cakir-Kiefer, C., Tomé, D., Schwarz, J., Miclo, L.
Pratiques en nutrition, 18 (71), pp. 40-44.
Miclo, L.
Les polyols sont des édulcorants de masse incorporés dans des produits alimentaires en substitution des sucres. Ils possèdent un pouvoir sucrant et une valeur énergétique inférieurs à ceux du saccharose mais leurs propriétés laxatives limitent leur emploi, bien que celles-ci dépendent largement du polyol considéré. Ce sont des fermentable oligo-, di-, monosaccharides and polyols, dont l’utilisation doit être surveillée chez les personnes sensibles, bien qu’ils aient des effets prébiotiques importants pour le microbiote intestinal.
Polyols are bulk sweeteners used in food products as a substitute for sugars. They have a sweetness and a caloric value lower than those of sucrose, but their laxative effects limit their use although these largely depend on the polyol in question. Being a part of Fermentable Oligo-, Di-, Monosaccharides and Polyols, their use should bear continued scrutiny in sensitive people, knowing that they have major prebiotic effects for the gut microbiota.
Microorganisms, 9 (11), pp. 2380.
Awussi, A. A., Roux, É., Humeau, C., Hafeez, Z., Maigret, B., Chang, O.K., Lecomte, X., Humbert, G., Miclo, L., Genay, M., Perrin, C., Dary-Mourot, A.
Growth of the lactic acid bacterium Streptococcus thermophilus in milk depends on its capacity to hydrolyze proteins of this medium through its surface proteolytic activity. Thus, strains exhibiting the cell envelope proteinase (CEP) PrtS are able to grow in milk at high cellular density. Due to its LPNTG motif, which is possibly the substrate of the sortase A (SrtA), PrtS is anchored to the cell wall in most S. thermophilus strains. Conversely, a soluble extracellular PrtS activity has been reported in the strain 4F44. It corresponds, in fact, to a certain proportion of PrtS that is not anchored to the cell wall but rather is released in the growth medium. The main difference between PrtS of strain 4F44 (PrtS4F44) and other PrtS concerns the absence of a 32-residue imperfect duplication in the prodomain of the CEP, postulated as being required for the maturation and correct subsequent anchoring of PrtS. In fact, both mature (without the prodomain at the N-terminal extremity) and immature (with the prodomain) forms are found in the soluble PrtS4F44 form along with an intact LPNTG at their C-terminal extremity. Investigations we present in this work show that (i) the imperfect duplication is not implied in PrtS maturation; (ii) the maturase PrtM is irrelevant in PrtS maturation which is probably automaturated; and (iii) SrtA allows for the PrtS anchoring in S. thermophilus but the SrtA of strain 4F44 (SrtA4F44) displays an altered activity.
Food & Function, 12, pp. 1415-1431.
Hafeez, Z., Benoit, S., Cakir-Kiefer, C., Dary, A., Miclo, L.
About one in three people are affected by anxiety disorders during their lifetime. Anxiety episodes can be brief due to a stressful event, but anxiety disorders can last at least 6 months. A wide variety of therapeutic drugs is available for the treatment of anxiety disorders, but due to the associated side effects of these anxiolytics, it is interesting to find alternatives. Some food protein hydrolysates or active peptide fragments present in such hydrolysates provide a natural and promising mean for preventing certain forms of anxiety. To date, only a few numbers of hydrolysates or peptides from food proteins with anxiolytic-like activity have been characterized. Most of these hydrolysates or peptides have displayed potent anxiolytic profiles in animal or clinical studies. The results suggest that these molecules may exert their effects at different levels. This paper reviews data of the structure/activity relationship of anxiolytic peptides, their physiological effects displayed in in vitro and in vivo assays, bioavailability, and safety profiles.
Nutrients, 12 (5), 1497.
Benoit, S., Chaumontet, C., Schwarz, J., Cakir-Kiefer, C., Boulier, A., Tomé, D., Miclo, L.
α-Casozepine (α-CZP) is an anxiolytic-like bioactive decapeptide derived from bovine αs1-casein. The N-terminal peptide YLGYL was previously identified after proteolysis of the original peptide in an in vitro digestion model. Its putative anxiolytic-like properties were evaluated in a Swiss mice model using a light/dark box (LDB) after an intraperitoneal injection (0.5 mg/kg). The effect of YLGYL on c-Fos expression in brain regions linked to anxiety regulation was afterwards evaluated via immunofluorescence and compared to those of α-CZP and diazepam, a reference anxiolytic benzodiazepine. YLGYL elicited some anxiolytic-like properties in the LDB, similar to α-CZP and diazepam. The two peptides displayed some strong differences compared with diazepam in terms of c-Fos expression modulation in the prefontal cortex, the amygdala, the nucleus of the tractus solitarius, the periaqueductal grey, and the raphe magnus nucleus, implying a potentially different mode of action. Additionally, YLGYL modulated c-Fos expression in the amygdala and in one of the raphe nuclei, displaying a somewhat similar pattern of activation as α-CZP. Nevertheless, some differences were also spotted between the two peptides, making it possible to formulate the hypothesis that these peptides could act differently on anxiety regulation. Taken together, these results showed that YLGYL could contribute to the in vivo overall action of α-CZP.
Journal of Dairy Science, 102 (1), pp. 113-123.
Hafeez, Z., Cakir-Kiefer, C., Lecomte, X., Miclo, L., Dary, A.
This study addresses the hypothesis that the extracellular cell-associated X-prolyl dipeptidyl-peptidase activity initially described in Streptococcus thermophilus could be attributable to the intracellular X-prolyl dipeptidyl-peptidase PepX. For this purpose, a PepX-negative mutant of S. thermophilus LMD-9 was constructed by interrupting the pepX gene and named LMD-9-delta-pepX. When cultivated, the S. thermophilus LMD-9 wild type strain grew more rapidly than its delta-pepX mutant counterpart. Thus, the growth rate of the LMD-9-delta-pepX strain was reduced by a factor of 1.5 and 1.6 in milk and LM17 medium (M17 medium supplemented with 2% lactose), respectively. The negative effect of the PepX inactivation on the hydrolysis of β-casomorphin-7 was also observed. Indeed, when incubated with this peptide, the LMD-9-delta-pepX mutant cells were unable to hydrolyze it, whereas this peptide was completely degraded by the S. thermophilus LMD-9 wild type cells. This hydrolysis was not due to leakage of intracellular PepX, as no peptide hydrolysis was high-lighted in cell-free filtrate of wild type strain. Therefore, based on these results, it can be presumed that though lacking an export signal, the intracellular PepX might have accessed the β-casomorphin-7 externally, perhaps via its galactose-binding domain-like fold, this domain being known to help enzymes bind to several proteins and substrates. Therefore, the identification of novel distinctive features of the proteolytic system of S. thermophilus will further enhance its credibility as a starter in milk fermentation.
Journal of Functional Foods, 38 (A), pp. 464-473.
Benoit, S., Chaumontet, C., Schwarz, J., Cakir-Kiefer, C., Tomé, T., Miclo, L.
α-Casozepine, a bioactive peptide from milk casein, displays an anxiolytic-like activity in many species. Since its mode of action is still not elucidated, a study was conducted in Swiss mice to investigate c-Fos expression, a marker of neuronal activity, in different brain areas. After an intraperitoneal injection of α-casozepine (1 mg/kg), animals were placed either in a non-stressful or in an anxiety-inducing situation triggered with a light/dark box. No effect of α-casozepine on c-Fos expression was observed in the non-stressful situation. In the stressful situation, modulation of neuronal activity by α-casozepine was observed in different brain regions compared to that of vehicle. However, while diazepam, a benzodiazepine, modulated neuronal activity the same way in hippocampus, accumbens nucleus and hypothalamus, differences were observed in c-Fos expression in amygdala and prefrontal cortex compared to α-casozepine. These results strengthen the assumption that the anxiolytic mechanisms of α-casozepine differ partly of those of diazepam.
Journal of Agricultural and Food Chemistry, 63 (34), pp. 7522-7531.
Hafeez, Z., Cakir-Kiefer, C., Girardet, J.-M., Lecomte, X., Paris, C., Galia, W., Dary, A., Miclo, L.
The influence on the hydrolysis of isracidin of cell-associated extracellular aminopeptidase and X-prolyl dipeptidyl peptidase activities in addition to protease PrtS of Streptococcus thermophilus strains was investigated. S. thermophilus LMD-9 (PrtS+ phenotype) efficiently hydrolyzed the isracidin mainly through the PrtS activity, whereas strain CNRZ1066 (PrtS- phenotype) and two mutant strains LMD-9-?prtS and LMD-9-?prtS-?htrA also displayed substrate hydrolysis, but different from that of the wild type strain LMD-9. Identification by mass spectrometry of breakdown products of isracidin revealed the existence of novel cell-associated extracellular carboxypeptidase and peptidyl dipeptidase activities in all PrtS- strains, besides known cell-associated extracellular aminopeptidase and X-prolyl dipeptidyl peptidase activities. Both aminopeptidase and peptidyl dipeptidase activities were not able to cleave the isracidin at peptide bonds with proline residues. No hydrolysis of isracidin was detected in cell free filtrate for all the strains studied, indicating that no cell lysis had occurred. Taken together, these results suggested the presence of cell-associated extracellular peptidase activities in S. thermophilus strains that could be vital for the growth of PrtS- strains.
Food Chemistry, 187, pp. 305-313.
Zennia, S.S.A., Mati, A., Saulnier, F., Verdier, Y., Chiappetta, G., Mulliert, G., Miclo, L., Vinh, J., Girardet, J.-M.
Nonenzymatic deamidation of asparaginyl residues can occur spontaneously under physiological conditions principally when a glycyl residue is at the carboxyl side of Asn and leads to formation of aspartyl and isoaspartyl residues. This modification can change the biological activity of proteins or peptides and trigger an auto-immune response. The alpha-lactalbumins of members of the Camelidae family are the only of described alpha-lactalbumins that carry two AsnGly sequences. In the present study, high-resolution mass spectrometry, which enables accurate mass measurement has shown that Asn16 and Asn45 underwent a nonenzymatic deamidation, the sequence Asn45–Gly46 being deamidated spontaneously at nearneutral and basic pH and Asn16–Gly17 rather at basic pH. The 16–17 sequence was probably stabilized at near-neutral pH by hydrogen bonds according to the molecular modelisation performed with the camel protein.
International Dairy Journal, 38 (2), pp. 104-115.
Chang, O.-K., Roux, É., Awussi, A. A., Miclo, L., Jardin, J., Jameh, N., Dary, A., Humbert, G., Perrin, C.
Bioactive peptides can be produced from milk proteins in fermented products by proteases of lactic acid
bacteria. The cell envelope protease (PrtS) of Streptococcus thermophilus is anchored at the cellwall, but we
recently discovered that the 4F44 strain produces a soluble form that can be recovered in medium supernatant.
This workwas aimed at optimising the production of bioactive peptides from bovine caseins. By
growing S. thermophilus 4F44 in the newly designed YLUNi medium, a high quantity of the soluble form of
PrtS could be produced that could be directly used as the proteolytic agent on sodium caseinate. Peptide
production was monitored by reverse phase-high performance liquid chromatography and tandem mass
spectrometry; of 247 peptides identified, 143 were derived from beta-casein. Twenty-two peptides, already
reported in the literature as bioactive, include ACE-inhibitory, antioxidant, immunomodulating, or antibacterial
peptides; addition of such peptides could improve the health benefits of dairy products.
Food Research International, 63 (A), pp. 71-80.
Hafeez, Z., Cakir-Kiefer, C., Roux, É., Perrin, C., Miclo, L., Dary, A.
Besides their basic nutritional role, dietary proteins contain bioactive peptides which are encrypted in their sequence and may modulate different body functions such as digestive, cardiovascular, immune and nervous systems, and therefore contribute in maintaining consumer health. Currently, milk proteins are considered to be the major source of bioactive peptides. The occurrence of these peptides has already been reported in fermented milk products such as yogurt, sour milk or kefir and some of them have been shown to confer health benefits. This review focuses on different strategies that could be employed to enhance the production of bioactive peptides from the milk proteins that will be consequently used to functionalize the fermented milk products. Three types of strategies are developed. The first exploits the proteolytic system of lactic acid bacteria (LAB) or food grade enzymes or combination of both to release the functional peptides from the milk proteins directly in the fermented milk products. The second concerns the supplementation of the fermented milk products with the bioactive peptides obtained outside of the product through the hydrolysis of the purified proteins by the same enzyme sources. Finally, the last consists in the production of the bioactive peptides, initially identified from the milk-proteins, by microorganisms using recombinant DNA technology.
Applied Microbiology and Biotechnology, 97 (22), pp. 9787-9799.
Hafeez, Z., Cakir-Kiefer, C., Girardet, J.-M., Jardin, J., Perrin, C., Dary, A., Miclo, L.
The trend to confer new functional properties to fermented dairy products by supplementation with bioactive peptides is growing in order to encounter the challenge of health-promoting foods. But these functional ingredients have not to be hydrolysed by proteases of bacteria used in the manufacture of these products. One of the two yoghurt bacteria, Streptococcus thermophilus, has long been considered as weakly proteolytic since its only cell wall-associated subtilisin-like protease, called PrtS, is not always present. Nevertheless, a recent study pointed out a possible peptidase activity in certain strains. In this present study, the stability of milk-derived bioactive peptides, e.g. the anxiolytic peptide, αs1-CN-(f91-97), in the presence of two different S. thermophilus strains with PrtS+ or PrtS− phenotype was studied. Both strains appeared to be capable of hydrolysing the αs1-CN-(f91-97) and other bioactive peptides by recurrent removal of N-terminal residues. The hydrolysis was neither due to intracellular peptidases nor to HtrA protease. Results obtained showed that the observed activity originates from the presence at the surface of both strains of an extracellular aminopeptidase activity. Moreover, a cell wall-associated X-prolyl dipeptidyl peptidase activity was also highlighted when β-casomorphin-7 was used as substrate. All of these findings suggest that, in order to use fermented milks as vector of bioactive peptides, the stability of these bioactive peptides in this kind of products implies to carefully characterize the potential action of the surface proteolytic enzymes of S. thermophilus.
Biochemistry, 52 (48), pp. 8722-8731.
Zidane, F., Zeder-Lutz, G., Altschuh, D., Girardet, J.-M., Miclo, L., Corbier, C., Cakir-Kiefer, C.
Somatic angiotensin I-converting enzyme (ACE) possesses two catalytic domains and plays a major role in the regulation of blood pressure, thus representing a therapeutic target for the treatment of hypertension. We present a comprehensive surface plasmon resonance (SPR) study of the interaction of human somatic ACE with the pharmacological inhibitors captopril and lisinopril, the bradykinin potentiating peptide BPP-11b, and the food peptidic inhibitors from bovine αs2-casein, F(174)ALPQYLK(181) and F(174)ALPQY(179). SPR binding curves recorded with the high potency inhibitors captopril, lisinopril, and BPP-11b were evaluated both by regression analysis and by kinetic distribution analysis. The results indicated that captopril and lisinopril bound ACE with two KD's differing by a factor 10-20 and >30, respectively (lowest KD = 0.1-0.3 nM for both inhibitors). This shows, for the first time in a direct binding assay with the two-domain enzyme, the existence of two binding modes of the pharmacological inhibitors, presumably with the two ACE domains. The BPP-11b-ACE binding curves were complex but showed a predominant interaction with KD in the nanomolar range. The caseinopeptides, known to inhibit ACE with an IC50 of 4.3 μM, bound to ACE with KD = 3-4 μM. Mapping of the F(174)ALPQY(179) binding site on ACE by sequential binding studies using captopril or BPP-11b indicated that it bound to (or near) the two active sites of ACE, in agreement with the stoichiometry of 2 determined from data fitting. Our results provide a detailed characterization of ACE-inhibitor binding modes and validate SPR for predicting the inhibitory potential of new compounds.
International Dairy Journal, 23 (2), pp. 91-98.
Chang, O.K., Perrin, C., Galia, W., Saulnier, F., Miclo, L., Roux, E., Driou, A., Humbert, G., Dary, A.
PrtS is the sole cell envelope protease (CEP) characterized in Streptococcus thermophilus. It is believed that it is anchored to the cell wall by sortase A (SrtA) through the LPXTG motif present at its C-terminus. Two soluble proteases corresponding to PrtS in its proenzyme and mature form were detected in the supernatant of S. thermophilus strain 4F44. In this strain, 60% of the PrtS molecules are anchored to the cell wall and 40% released in the medium. Such a release might result from a partial deficiency in the strain 4F44 of SrtA, even if its sequence slightly differs from that of S. thermophilus strain LMD-9, in which PrtS is anchored. Indeed, the presence of an intact LPXTG motif at the C-terminus of the released proteases showed that the linking process driven by SrtA did not occur and these proteases were not released by proteolysis after their anchoring.
Journal of Agricultural and Food Chemistry, 60 (2), pp 554-565.
Miclo, L., Roux, E., Genay, M., Brusseaux, E., Poirson, C., Jameh, N., Perrin, C., Dary, A.
Milk proteins contain numerous potential bioactive peptides, which may be released by digestive proteases or by the proteolytic system of lactic acid bacteria during food processing. The capacity of Streptococcus thermophilus to generate peptides, especially bioactive peptides, from bovine caseins was investigated. Strains expressing various levels of the Cell Envelope Proteinase, PrtS, were incubated either with αs1-, αs2- or β-casein. Analysis of the supernatants by LC-ESI-MS/MS showed that the β-casein was preferentially hydrolyzed first, followed by αs2-casein and then αs1-casein. Numbers and types of peptides released were strain-dependent. Hydrolysis appeared to be linked with the accessibility of different casein regions by protease. Analysis of bonds hydrolyzed in the region 1-23 of αs1-casein suggests that PrtS is at least in part responsible for the peptide production. Finally, among the generated peptides, 13 peptides from β-casein, 5 from αs2-casein and 2 from αs1-casein have been reported as bioactive, 15 of them being angiotensin-converting enzyme inhibitors.
Food Chemistry, 132 (1), pp. 391-398.
Zidane, F., Matéos, A., Cakir-Kiefer, C., Miclo, L., Rahuel-Clermont, S., Girardet, J.-M., Corbier, C.
To better understand the mechanism of metal ion transport through the gastrointestinal tract to their absorption sites, isothermal titration calorimetry (ITC) was used to investigate the binding of dicationic metals to beta-CN(1–25)4P, a beta-casein tetraphosphorylated peptide. ITC technology was found suitable for studying weak bonds between metal ions and phosphopeptides and provided a direct means of thermodynamic and stoichiometric characterisation of complex formation. Thus, one mole of beta-CN(1–25)4P binds two moles of Ca2+, Mg2+ or Zn2+ under experimental conditions close to those of the ileum (pH 8, 37°C), with rather low binding affinity constants (K = 4900–11,200 M-1). These low affinities should facilitate the release of metal ions during intestinal absorption. By contrast, Cu2+ did not bind to beta-CN(1–25)4P at pH 8, despite its reported significant affinity towards beta-casein and the 1–25 peptide at near-neutral pH.
Journal of Agricultural and Food Chemistry, 59 (9), pp. 4464-4472.
Cakir-Kiefer, C., Le Roux, Y., Balandras, F., Trabalon, M., Dary, A., Laurent, F., Gaillard, J.-L., Miclo, L.
α-Casozepine is a peptide, corresponding to the sequence 91−100 of the bovine αs1-casein, displaying anxiolytic activity in the rat. The αs1-casein tryptic hydrolysate containing this peptide decreases stress effects after oral administration in various species including man. Therefore, the stability of this peptide toward gastric and pancreatic proteases has been assessed by using pepsin, chymotrypsin/trypsin, Corolase PP, pepsin followed by chymotrypsin/trypsin or pepsin followed by Corolase PP. α-Casozepine was slowly degraded by chymotrypsin, much more sensitive to pepsin and Corolase PP but not completely destroyed after 4 h kinetics. The bonds in the region 91 to 95 of the α-casozepine were totally resistant to hydrolysis by all studied proteases. Surprisingly, a fragment, corresponding to the sequence 91−97 and found in all the hydrolysis media in significant amount, possessed an anxiolytic activity in three behavioral tests measuring this parameter. This peptide could participate in the in vivo activity of α-casozepine.
Journal of Agricultural and Food Chemistry, 59 (22), pp. 11956–11965.
Cakir-Kiefer, C., Miclo, L., Balandras, F., Dary, A., Soligot, C., Le Roux, Y.
α-Casozepine and f91–97, peptides from αs1-casein, display anxiolytic activity in rats and may have to cross the intestinal epithelium to exert this central effect. We evaluated their resistance to hydrolysis by the peptidases of Caco-2 cells and their ability to cross the cell monolayer. To mimic physiological conditions, two preparations of bile salts were used in noncytotoxic concentrations: porcine bile extract and an equimolar mixture of taurocholate, cholate, and deoxycholate. The presence and composition of bile salts appeared to modulate the peptidase activities of the Caco-2 cells involved (i) in the hydrolysis of α-casozepine, leading to much higher formation of fragments f91–99, f91–98, and f91–97, and (ii) in the hydrolysis of f91–97, leading to lower degradation of this peptide. Transport of α-casozepine across Caco-2 monolayer increased significantly, in the presence of bile extract, and of fragment f91–97, in the presence of bile salts.
International Dairy Journal, 21 (4), pp. 214-221.
Sadat, L., Cakir-Kiefer, C., N’Negue, M.-A., Gaillard, J.-L., Girardet, J.-M., Miclo, L.
The second main bovine whey protein, α-lactalbumin, was hydrolyzed by thermolysin at 70 °C, i.e., in a molten-globule conformational state susceptible to enzyme attack, for production of small peptides with potential antioxidative properties. The main thermolytic fragments were then purified by reversed-phase liquid chromatography and identified by mass spectrometry. Antioxidant activities of the protein, its thermolytic hydrolyzate, and pure peptides were evaluated using 2,2′-azinobis[3-ethylbenzothiazoline-6-sulfonate] (ABTS+) radical-scavenging activity. The whole protein and its hydrolyzate exhibited antioxidant activities comparable with that of Trolox, a vitamin E analog. Among the thermolytic fragments, five peptides, all containing at least one Tyr or Trp residue located at one of the extremities of the sequence, displayed the most efficient antioxidant activities. In particular, Ile101-Asn-Tyr-Trp104 and Leu115-Asp-Gln-Trp118 possessed remarkable radical-scavenging capacity, 5-fold and 10-fold higher, respectively, than those of gallic acid and Trolox tested under the same experimental conditions.
Rapid Communications in Mass Spectrometry, 24 (11) pp. 1533-1542.
Matéos, A., Girardet, J.-M., Mollé, D., Corbier, C., Gaillard, J.-L., Miclo, L.
Equine beta-casein is phosphorylated at variable degrees and isoforms carrying 3 to 7 phosphate groups (3P–7P) have been found in milk, but the phosphorylated amino acid residues of each isoform are not yet identified. In the present work, the different phosphorylation variants were first isolated by ionexchange chromatography and then hydrolysed by trypsin to generate caseinophosphopeptides (CPPs), each containing all the potential phosphorylation sites. The equine CPPs were prepared by metal oxide affinity chromatography, a method based on the affinity of phosphate groups towards titanium dioxide immobilized onto a micro-column. This method turned out to be an efficient tool to separate the CPPs Arg1–Lys34 and Glu4–Lys34 from non-phosphorylated peptides. Purification was achieved by reversed-phase high-performance liquid chromatography (RP-HPLC) and each CPP was hydrolyzed by endoproteinase Glu-C. Finally, the digests were analyzed by RP-HPLC/electrospray ionization mass spectrometry (RP-HPLC/ESI-MS) and identified by nano-electrospray ionization tandem mass spectrometry (nESI-MS/MS) to locate the phosphorylated sites of the beta-casein isoforms 4P–7P with accuracy. Thus, the isoform 4P was found to be phosphorylated on residues Ser9, Ser23, Ser24, and Ser25. Addition of phosphate groups on Ser18, Thr12, and Ser10 led to the formation of the isoforms 5P–7P, respectively. The results indicated that the in vivo phosphorylation of the equine beta-casein follows a sequential way and is not randomly performed.
Two-dimensional cartography of equine beta-casein variants achieved by isolation of phosphorylation isoforms and control of the deamidation phenomenon
Journal of Dairy Science, 92 (6) pp. 2389-2399.
Matéos, A., Girardet, J.-M., Mollé, D., Dary, A., Miclo, L., Gaillard, J.-L.
Because of variable degrees of phosphorylation, alternative splicing, and probable instability resulting from nonenzymatic deamidation, equine β-casein presents a complex pattern by 2-dimensional electrophoresis that needs clarification. β-Casein prepared from Haflinger mare’s milk by hydrophobic interaction chromatography was fractionated by ion-exchange chromatography according to the degree of phosphorylation. Isoforms were identified by mass spectrometry; they corresponded to the full-length protein having 3 to 7 phosphate groups and to the splicing variant involving exon 5 and containing 4 to 7 phosphate groups. Investigations of nonenzymatic deamidation showed that β-casein did not deamidate spontaneously in stored milk and during the different steps of chromatography, but deamidation could occur when 2-dimensional electrophoresis was performed, increasing the β-casein pattern complexity. This phenomenon was strongly minimized when the first dimension step was carried out at 10°C instead of at room temperature. Finally, spot attribution on 2-dimensional pattern of β-casein was achieved by mixing each phosphorylation isoform in its native state with the whole β-casein fraction.
Equine alpha-s1-casein: characterization of alternative splicing isoforms and determination of phosphorylation levels of multiple isoforms
Journal of Dairy Science, 92 (8), pp. 3604-3615.
Matéos, A., Miclo, L., Mollé, D., Dary, A., Girardet, J.-M., Gaillard, J.-L.
Alpha-s1-casein was isolated from Haflinger mare’s milk by hydrophobic interaction chromatography and displayed great micro-heterogeneity by 2-dimensional electrophoresis, probably because of a variable degree of phosphorylation and alternative splicing events. The aim of the present work was to investigate the complexity of the mare’s alpha-s1-casein. The different isoforms present in milk were submitted to a double treatment of dephosphorylation, first by using alkaline phosphatase and then acid phosphatase to achieve complete dephosphorylation. The apoforms were then analyzed by electrospray ionization mass spectrometry. The results revealed the existence of a full-length protein and 7 variants resulting from posttranscriptional modifications; that is, exon skipping involving exon 7, exon 14, or both and use of a cryptic splice site encoding a glutamine residue. The determination of the different phosphorylation degrees of the native isoforms of alpha-s1-casein was finally achieved by electrospray ionization mass spectrometry analysis after fractionation of the isoforms by ion-exchange chromatography. Thus, 36 different variants of equine alpha-s1-casein were identified with several phosphate groups ranging from 2 to 6 or 8 depending on whether exon 7 was skipped.
Milk-clotting activity of enzyme extracts from sunflower and albizia seeds and specific hydrolysis of bovine kappa-casein
International Dairy Journal, 17 (7), pp. 816-825.
Egito, A.S., Girardet, J.-M., Laguna, L.E., Poirson, C., Molle, D., Miclo, L., Humbert, G., Gaillard, J.-L.
Milk-clotting activity found in ammonium sulfate-precipitated protein extracts from Albizia lebbeck and Helianthus annuus seeds was studied. Specific clotting activity of albizia seed extract was 15 times higher than that of sunflower seed extract. Zymogram analysis revealed several proteolytic bands in albizia seed extract and one diffuse proteolytic band for sunflower seed extract. Whole bovine casein was incubated with the plant seed extracts or chymosin and some breakdown products were characterized by reversed-phase high-performance liquid chromatography and electrophoresis. Similar to chymosin, the two seed extracts exhibited proteolytic activity toward κ-casein, αs-casein and β-casein, with the highest activity observed for the albizia seed extract. Mass spectrometry analysis showed that the sunflower extract hydrolyzed κ-casein at the Phe105–Met106 bond, as does chymosin. The albizia extract also displayed activity on κ-casein, but the Lys116–Thr117 bond was its preferred target.
The primary structure of a low-Mr multiphosphorylated variant of beta-casein in equine milk
Proteomics, 7 (8), pp. 1327-1335.
Miclo, L., Girardet, J.-M., Egito, A.S., Mollé, D., Martin, P., Gaillard, J.-L.
Highly phosphorylated casein with a low molecular mass was isolated from Haflinger mare\'s milk by RP-HPLC. It accounts for 4.0% of the casein content. Its mass was determined by LC-ESI-MS before and after treatment by alkaline phosphatase. The molecular mass found for the apo-form (10 591 ± 2 Da) is in agreement with its primary structure, which was established by ESI-MS/MS from tryptic peptides. It appeared that this short protein (94 amino acid residues) is an internally truncated form of the full-length equine ![\"\"](\"http://www3.interscience.wiley.com/giflibrary/12/beta.gif\")
-casein (226 residues). This low-Mr variant of equine -casein displays a large deletion (residues 50-181), due to a cryptic splice site usage occurring within exon 7 during the course of primary transcripts processing. The phosphorylation pattern of this equine -casein variant was investigated by LC-ESI-MS and 2-DE. Seven phosphorylation forms were identified with one to seven phosphate groups with pIs ranging between 4.67 and 4.01. The major isoforms carry five and six phosphate groups.
Fast electrophoretic detection method of adulteration of caprine milk by bovine milk
Arquivo Brasileiro de Medicina Veterinaria e Zootecnia, 58 (5), pp. 932-939.
Egito, A.S., Rosinha, G.M.S., Laguna, L.E., Miclo, L., Girardet, J.M., Gaillard, J.L.
Proteomics, 6 (12), pp. 3707-3717.
Girardet, J.-M., Miclo, L., Florent, S., Mollé, D., Gaillard, J.-L.
-Casein was isolated from Haflinger mare\'s milk by RP-HPLC, and displayed microheterogeneity by urea-electrophoresis and 2-DE probably due to a variable degree of phosphorylation. To investigate the degree of phosphorylation, the primary structure of equine -casein was determined by tryptic hydrolysis and MS of peptides released and by MS of the protein treated by alkaline phosphatase. The molecular mass found for the apo-form of Haflinger mare\'s -casein (25 514 ± 3 Da) was close to the theoretical mass of the reported sequence (GenBank AAG43954) modified by insertion of a region (residues 27-34) encoded by an exon sometimes out-spliced (25 511.40 Da). Hence, the -casein isolated from Haflinger mare\'s milk corresponded to a variant of 226 amino acid residues. The latter was composed by highly multi-phosphorylated isoforms with three to seven phosphate groups, and pIs, determined by 2-DE, ranging from 4.74 to 5.30. Moreover, the equine -casein was able to deamidate spontaneously, at the level of Asn in the potential deamidation motif 135Asn-Gly136. Approximately 80% of the protein was deamidated after 96 h of incubation under physiological conditions.
Proteolysis of bovine alpha-lactalbumin by thermolysin during thermal denaturation
International Dairy Journal, 16 (10), pp. 1157-1167.
N'Negue, M.-A., Miclo, L., Girardet, J.-M., Campagna, S., Molle, D., Gaillard, J.-L.
Thermolysin was used to hydrolyze bovine α-lactalbumin at 25 and 70 °C under non-reducing conditions. The breakdown products were identified by mass spectrometry. At 25 °C, the low proportion of α-lactalbumin in an unfolded state in equilibrium with the native state underwent limited hydrolysis leading to the production of peptides, that were no longer degraded (final peptides). At 70 °C, the protein was in a molten globule-like state according to circular dichroism and complete cleavage of the protein was achieved. At 70 °C, the protein was first quickly cleaved, then unfolded, leading to the release of intermediate peptides, from which final peptides were eventually produced. The amino-terminal 1–58 and carboxy-terminal 95–123 regions were readily cleaved, whereas the central region including the calcium-binding domain was more resistant. Some peptides were produced at 70 °C, but not at 25 °C. The choice of accurate experimental conditions may be of importance for the preparation of functional peptides.
International Dairy Journal, 13 (10), pp. 813-820.
Egito, A.S., Girardet, J.-M., Poirson, C., Molle, D., Humbert, G., Miclo, L., Gaillard, J.-L.
International Dairy Journal, 13 (1), pp. 15-27.
Tauzin, J., Miclo, L., Roth, S., Molle, D., Gaillard, J.-L.
Separation and characterization of mares' milk alphaS1-, beta-, kappa-caseins, gamma-casein-like, and proteose peptone component 5-like peptides
Journal of Dairy Science, 85 (4), pp. 697-706.
Egito, A.S., Miclo, L., Lopez, C., Adam, A., Girardet, J.-M., Gaillard, J.-L.
FEBS Letters, 531 (2), pp. 369-374.
Tauzin, J., Miclo, L., Gaillard, J.-L.
Lait, 81 (6), pp. 775-785.
Egito, A.S., Girardet, J.-M., Miclo, L., Gaillard, J.-L.
Susceptibility of Equine alpha- and beta-caseins to hydrolysis by chymosin
International Dairy Journal, 11 (11-12), pp. 885-893.
Egito, A.S., Girardet, J.-M., Miclo, L., Molle, D., Humbert, G., Gaillard, J.-L.
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 15 (10), pp. 1780-1782.
Miclo, L., Perrin, E., Driou, A., Papadopoulos, V., Boujrad, N., Vanderesse, R., Boudier, J.F., Desor, D., Linden, G., Gaillard, J.L.
Journal of Dairy Science, 83 (11), pp. 2410-2421.
Girardet, J.-M., Debomy, L., Courthaudon, J.-L., Miclo, L., Humbert, G., Gaillard, J.-L.
European Journal of Biochemistry, 248 (3), pp. 872-878.
Lecouvey, M., Frochot, C., Miclo, L., Orlewski, P., Driou, A., Linden, G., Gaillard, J.-L., Marraud, M., Cung, M.T., Vanderesse, R.
Letters in Peptide Science, 4 (4-6), pp. 359-364.
Lecouvey, M., Frochot, C., Miclo, L., Orlewski, P., Marraud, M., Gaillard, J.-L., Cung, M.T., Vanderesse, R.
Reproduction Nutrition Development, 36, pp. 414-415.
Perrin, E., Miclo, L., Driou, A., Gaillard, J.-L., Linden, G.
Analytical Communications, 33 (4), pp. 143-147.
Perrin, E., Miclo, L., Driou, A., Linden, G.
Amino acid composition analysis is sometimes used for the identification of peptides obtained from the hydrolysis of a protein of known sequence. Nevertheless, the interpretation of the analysis can be difficult if only one hydrochloric acid hydrolysis can be performed because of the partial destruction of some amino acids and the lack of cleavage of certain peptidic bonds. In this paper we propose combining the analysis of amino acids by two techniques (derivative UV spectrometry and retention time estimation) associated on-line with the purification step of the peptides (reversed-phase HPLC).
A set of 56 peptides from the peptic and chymotryptic hydrolysis of bovine αs1-casein (αs1-CN) were analysed in this manner. The difference between the theoretical and observed retention times was 1.9 min, for aromatic amino acids ratios the difference was 4.0%. The ambiguities coming from repeated residues in the sequence or the presence of Trp residues, destroyed by acidic hydrolysis, were solved and a fragment of the αs1-CN sequence could be attributed to each peptide.
Cow's milk proteins: a new raw material for drugs? [Les protéines du lait de vache : une nouvelle source de médicaments ?]
Cahiers de Nutrition et de Dietetique, 30 (6), pp. 359-364.
Driou, A., Jeantroux, M., Perrin, E., Miclo, L.
International Journal of Peptide and Protein Research, 46 (2), pp. 186-192.
Miclo, L., Perrin, E., Driou, A., Mellet, M., Linden, G.
Journal of Chromatography B: Biomedical Applications, 664 (1), pp. 267-276.
Perrin, E., Miclo, L., Driou, A., Linden, G.
Brevet international WO 2009/147234 publié le 10/12/2009
Balandras F., Gaillard J.-L., Laurent F., Le Roux Y., Miclo L.
Compositions pharmaceutiques et produits alimentaires comprenant des peptides dérivés de la caséine alpha-s1 du lait ayant une activitéde type benzodiazépine et notamment une activité anxiolytique.
Peptides dérivés de la caséine ayant une activité anxiolytique
Brevet international WO 2008/071755 publié le 19/06/2008
Balandras F., Miclo L., Gaillard J.-L., Le Roux Y., Laurent F.
Use of at least one alpha-s2 casein peptide with angiotensin I converting enzyme inhibiting activity for preparing medicines, food products and food complements
Brevet américain 2006/0234942 publié le 19/10/2006
Tauzin, J., Miclo, L., Lefranc, C., Boudier J.-F., Gaillard, J.-L.
Use of at least one peptide of alpha-s2 casein with inhibiting activity of ACE for the preparation of medicaments and foodstuffs
Brevet japonais JP 2005/530851 publié le 13/10/2005
Tauzin, J., Miclo, L., Lefranc, C., Boudier, J.-F., Gaillard, J.-L.
Use of at least one alpha-s2 casein peptide with angiotensin I converting enzyme inhibiting activity for preparing medicines, food products and food complements
Brevet international WO 2004/002509 publié le 08/01/2004
Tauzin J., Miclo L., Lefranc C., Boudier J-F., Gaillard J-L.
Use of at least one peptide of alpha-s2 casein with inhibiting activity of ACE for the preparation of medicaments and foodstuffs
Brevet européen EP 1374885 publié le 02/01/2004
Tauzin, J., Miclo, L., Lefranc, C., Boudier, J.-F., Gaillard, J.-L.
Use of a decapeptide with benzodiazepine-type activity for preparing medicines and food supplements
Brevet américain 5,846,939 publié le 08/12/1998
Miclo, L., Perrin, E., Driou, A., Boudier, J.-F., Iung, C., Linden, G.
Use of a decapeptide with a benzodiazepine activity for the preparation of medicaments and dietary supplements
Brevet européen EP 0714910 publié le 05/06/1996
Miclo, L., Perrin, E., Driou, A., Boudier, J.-F., Iung, C., Linden, G.
Brevet japonais JP 8268903 publié le 15/10/1996
Miclo, L., Perrin, E., Driou, A., Boudier J.-F., Iung, C., Linden, G.
Brevet français FR 2727315 publié le 31/05/1996
Miclo, L., Perrin, E., Driou, A., Boudier, J.-F., Iung, C., Linden, G.
Biochimie alimentaire : 6e édition de l'abrégé
Dunod ed., 260 pp.
Alais C., Linden G., Miclo L.
L'ouvrage présente une vue d'ensemble synthétique de la biochimie alimentaire. Cette sixième édition entièrement révisée décrit les principes de constitution des substances alimentaires et les caractéristiques des principaux aliments. Des notions nouvelles sont introduites çà et là sur des thèmes «à la mode» : aliments fonctionnels, oligoéléments, anti-oxydants, allergies alimentaires, additifs conformes aux nouvelles législations européennes...
Biochimie alimentaire : 5e édition de l'abrégé
Dunod ed.
Alais C., Linden G., Miclo L.
FEBS 2023 - the 47th FEBS Congress, 08-12 juillet, Tours, France
Allouche, R., Hafeez, Z., Dary, A., Genay, M., Miclo, L.
Streptococcus thermophilus is a dairy starter granted “Generally Recognized as Safe” by the FDA and “Qualified Presumption of Safety” by EFSA. A significant part of the world's population ingests this bacterium when consuming fermented products. Some strains of S. thermophilus, either in the live or heat-inactivated state, and peptides released after shaving and hydrolysis of the surface proteins of some strains of this bacterium displayed anti-inflammatory activity in vitro (Allouche et al.,2022). S. thermophilus cells could undergo lysis during their passage through the digestive tract. Consequently, its intracellular proteins could be hydrolysed by endogenous proteases leading to the release of peptides. We hypothesized that peptides generated from digestion of intracellular protein of S. thermophilus might also contribute to its overall anti-inflammatory effect. Therefore, intracellular proteins from S. thermophilus CNRZ-21N strain were recovered after sonication. After fractionation by size exclusion chromatography, the resulting 3-10 kDa protein fraction was hydrolysed by Corolase PP, a mixture of pancreatic proteases. MS-MS analysis showed that most of the identified peptides belonged to the ribosomal proteins. The hydrolysed fraction showed anti-inflammatory activity on macrophagelike THP-1 cells inflamed by LPS since their secretion of IL-8 and IL-1β cytokines and expression level of Pro-IL-1β were reduced. The results suggest that the peptides released from a fraction of intracellular proteins of S. thermophilus after digestion by Corolase PP may contribute to the anti-inflammatory activity of this bacterium and could be used as a functional ingredient to prevent lowgrade inflammation.
5th Edition of Innovations in Food Science and Human Nutrition (IFHN-2022), 20-21 septembre, Barcelone, Espagne
Allouche, R., Hafeez, Z., Dary-Mourot, A., Genay, M., Miclo, L.
23ème Colloque du Club des Bactéries Lactiques, 08-10 juin, Rennes, France
Allouche, R., Hafeez, Z., Papier, F., Dary-Mourot, A., Genay, M., Miclo, L.
Journée doctorale virtuelle franco-allemande et transfrontalière : Biotechnologies et Sciences de la Vie, 10 novembre, dématérialisée
Allouche, R., Hafeez, Z., Dary-Mourot, A., Genay, M., Miclo, L.
Séminaire de l'École Doctorale SIReNa, 13 février, Nancy, France
Allouche, R., Hafeez, Z., Dary, A., Genay, M., Miclo, L.
Streptococcus thermophilus is widely used as a starter culture in the dairy industry and has been awarded generally recognized as safe status (GRAS) by the American Food and Drug Administration. Some strains of S. thermophilus display an anti-inflammatory activity in vitro (Junjua et al., 2016). Inflammation is a part of the regular host reaction to injury or infection caused by pathogens, damaged cells, irritants and allergens. However, the mechanism of action by which this bacterium modulates inflammatory response remains unclear. It has been shown that the hydrolysis of food proteins or endogenous proteins by some digestive proteases releases peptides with various biological activities. Such peptides can also be generated by the surface proteolytic system of Lactic Acid Bacteria (Hafeez et al., 2014) as S. thermophilus, which produces bioactive peptides from bovine caseins (Miclo et al., 2012). These peptides are inactive within the sequence of the parent protein and display their activity after a hydrolysis step. Thus, the assumption that peptides generated in the gastro-intestinal tract from hydrolysis of S. thermophilus surface or intracellular proteins could display an anti?inflammatory activity and contribute to the overall anti-inflammatory effect of the bacterium can be made. Therefore, it is interesting to explore the role of such peptides in the modulation of inflammation. In a first approach, this study aims to investigate the anti-inflammatory properties of hydrolysates genrated after hydrolysis by gastrointestinal enzymes of surface proteins of S. thermophilus. The method involves the recovery of bacterial surface polypeptides by shaving with pepsin. Supernatant obtained after shaving was analysed by RP-HPLC and showed the release of peptides. The next challenge constitutes evaluation in in vitro cell model of anti-inflammatory activity of the peptides obtained and the characterisation of these peptides by mass spectrometry. This study will lead to novel insights into the modulation of host inflammatory response through probable action of peptides obtained from S. thermophilus.
22nd International Conference of Functional Food Center (FFC) - 10th International Symposium of Academic Society for Functional Foods and Bioactive Compounds (ASFFBC) at Harvard Medical School, 22-23 septembre, Boston, États-Unis
Hafeez, Z., Perrin, C., Dary, A., Chevalot, I., Kapel, R., Chatel, J.M., Miclo, L.
Background:
Inflammation, a basic host defensive response, is crucial for resistance to injury, infectious agents or other noxious stimuli. Nevertheless, excessive and persistent inflammation often leads to various chronic diseases such as cardiovascular, osteoporosis, diabetes, obesity and gastro-intestinal inflammatory diseases. According to WHO, chronic diseases are the leading cause of morbidity and mortality both in developed and developing countries, and represent 60% of all deaths in the world. This figure rises to 87% in Europe and it is expected that more people will be affected by chronic diseases over the next few decades. Increased treatment-related health care costs have made chronic diseases a real health problem to the societies and the concern to prevent or treat these illnesses through diet has increased.
Daily diet is comprised of variety of nutrients including proteins. Bioactive peptides derived from dietary proteins particularly milk may modulate different body functions both at intestinal and systemic levels and ultimately contribute in maintaining consumer health. It has been shown that casein hydrolysates or peptides present in them exhibit anti-inflammatory activity by inhibiting and/or reducing the expression of inflammatory markers and/or by modulating their activity. For example, besides the fragment 106-169 of the bovine κ-casein, anti-inflammatory activity was demonstrated for 84VPP86 and 74IPP76 peptides from the bovine β-casein which reduce in vivo the mRNA expression of inflammatory cytokines (IL-6 and IL-1β). Three ways for releasing peptides from native proteins can be: (i) in vitro enzymatic hydrolysis, (ii) during gastrointestinal digestion or (iii) during fermentation by lactic acid bacteria.
Streptococcus thermophilus, a lactic acid bacterium previously known for conferring organoleptic properties to dairy products, has also been shown to generate bioactive peptides from milk proteins through its cell envelope proteinase (PrtS), which is anchored to cell wall by the transpeptidase sortase SrtA. However, during fermentation the pH decreases due to production of lactic acid and consequently the activity of PrtS is reduced or inhibited (optimal activity at pH 7.5), resulting in lower peptide contents. Therefore, to overcome this problem, S. thermophilus LMD-9-delta-srtA mutant strain was constructed to release in growth medium PrtS, which after purification was used to hydrolyze a caseinate to produce a hydrolysate with higher peptide content, which was afterward fractionated.
Objectives:
The main goal of the study was to evaluate the immunomodulatory potential of the peptide fractions obtained after fractionation by ultrafiltration and diafiltration of a hydrolysate resulting from the hydrolysis of a caseinate by PrtS purified from the growth culture of S. thermophilus LMD-9-delta-srtA strain, using peripheral blood mononuclear cells (PBMC) from 4 human donors.
Materials and Methods:
S. thermophilus LMD-9-delta-srtA strain was cultured for 8 h in yeast-lactose (YL) medium to obtain PrtS-rich supernatant. Followed by batch chromatography using diethylaminoethyl (DEAE) cellulose DE23 (Pharmacia, Uppsala, Sweden) resin and discontinuous gradient of NaCl, PrtS-rich fraction was concentrated by ultrafiltration using Amicon® Ultrafiltration system (cutoff threshold 50 kDa, Milipore, Jaffery, USA). The concentrated PrtS-rich fraction was then used to hydrolyze an industrial caseinate comprising of 87% proteins (92% casein and 8% whey proteins). This initial hydrolyzate (F1) was further fractionated by ultrafiltration using a 3 kDa cut-off membrane to isolate non-hydrolysed proteins in the form of retentate (F2) and peptide fraction as permeate (F3). The F3 fraction was then concentrated by ultrafiltration and diafiltration using a 1 kDa cutoff membrane to obtain retentate (F4) and permeate (F5). The peptide fractions were co-incubated in vitro with PBMCs (Clinisciences, Nanterre, France) and secretion of the IL-10 anti-inflammatory cytokine and of the IL-12 pro-inflammatory cytokine was measured in the cellular medium. The IL-10/IL-12 ratio makes it possible to evaluate the immunomodulatory potential of the peptide fractions.
Results:
Using DEAE cellulose DE23 batch chromatography with NaCl gradient, the PrtS was predominantly recovered in the fraction eluted by 0.4 M NaCl since about 90% of the initial activity was found in this fraction. Hydrolysis of caseinate by PrtS was not total since size-exclusion chromatography showed that a significant proportion of proteins remained unhydrolyzed under the conditions used. The protein concentration of the hydrolyzate was about 5 g/L with about 1.6 g/L of peptides. F4 fraction containing peptides whose molecular mass was between 1000 and 3000 Da was free of any salts contrary to F3 and F5 fractions. Anti-inflammatory activity of the five peptide fractions was evaluated by quantifying IL-10 and IL-12 production in vitro after co-incubating individually with PBMCs obtained from four healthy donors who did not use anti-inflammatory drugs for a significant period of time. All the fractions tested at concentrations of 0.2 and 1.0 mg of protein matter / mL led to a secretion of IL-10 by PBMCs except F3 and F5 where the secretion of IL-10 was observed only for concentration of 0.2 mg/mL. The absence of secretion with these fractions tested at 1.0 mg/mL was probably due to an excess of salt content. However, F4 fraction induced higher level of IL-10 production (between 70 and 400 pg/mL according to the PBMC donor) followed by F2 fraction regardless of concentration used. No IL-10 was detected when PBS alone was applied. Similarly, none of the peptide fraction favored the release of the pro-inflammatory cytokine IL-12. Results obtained with the cells of all the donors gave the same conclusions.
Conclusion:
PBMC treated with F4 fraction, among all peptide fractions, secreted higher levels of IL-10 in vitro and, therefore, this fraction displayed potential anti-inflammatory activity, as it did not trigger any secretion of IL-12. This peptide fraction is rich in peptides with molecular mass ranging between 1 and 3 kDa. Hence, milk proteins represent a promising source of peptides with potential anti-inflammatory activity and these peptides could be released directly by S. thermophilus or by its protease used as a biotechnological tool. It could be interesting to identify the peptide sequences of the F4 fraction in order to characterize it with the aim of using it in the development of functional fermented milk products.
15th workshop on bioactive peptides, 23-25 juin, Naples, Italie
Benoit, S., Chaumontet, C., Cakir-Kiefer, C., Schwarz, J., Tomé, D., Miclo, L.
Experimental Biology, 02-06 avril, San Diego, CA, États-Unis
Benoit, S., Chaumontet, C., Schwarz, J., Cakir-Kiefer, C., Tomé, D., Miclo, L.
Journées Francophones de Nutrition, 09-11 décembre, Marseille, France
Benoit, S., Chaumontet, C., Cakir-Kiefer, C., Schwarz, J., Tomé, D., Miclo, L.
Actes du séminaire de l'École Doctorale RP2E, 15 janvier, Nancy, France
Benoit, S., Chaumontet, C., Cakir-Kiefer, C., Tomé, D., Miclo, L.
7th International Whey Conference, 07-09 septembre, Rotterdam, Pays-Bas
Si Ahmed Zennia, S., Mati, A., Miclo, L., Girardet, J.-M.
Printemps de la Cardiologie, 24-25 avril, Strasbourg, France
Zeder-Lutz, G., Zidane, F., Legrani, K., Dary, A., Miclo, L., Altschuh, D., Cakir-Kiefer, C.
Angiotensin I-converting enzyme (ACE), which is a key enzyme of the renin-angiotensin system, is one of target for antihypertensive molecules. Indeed, ACE is well known for its involvement in hypertension which is the main risk factor involved in the development of cardiovascular and kidney diseases and is a major cause of morbidity and mortality. Currently, many pharmacological ACE inhibitors (captopril, lisinopril…) are used for hypertension treatment, but their administration over a long period is associated with some undesirable side effects. Furthermore, there are many publications dealing with antihypertensive peptides from food proteins. ACE-inhibitory peptides, generated by hydrolysis of food proteins, may be a natural alternative to prevent hypertension appearance. However, among the bioactive peptides published in the literature as ACE inhibitors, a very small number really displays an antihypertensive activity in vivo in animals. Moreover, their mechanism of action at the molecular level is still misunderstood.
The objective of our work was to characterize the molecular interactions between ACE and some peptides described as ACE-inhibitors, which have or not a true antihypertensive activity in vivo.
For this purpose, a methodology already developed in our teams was employed (Zidane et al., 2013). It is based on the use, for the first time, of Biacore® technology (SPR). This real time technology provides some important molecular information such as the rate constants of association and dissociation, the stoichiometry and the site of the interaction on ACE. In our study, the direct interaction between ACE and inhibitors (without substrate or ligand) showed dissociation constants (KD) of the same order of magnitude as IC50. Moreover, the formed ACE-inhibitor complexes are unstable. Given these results, it is difficult to attribute significant antihypertensive effect demonstrated in vivo for these peptides (for example IPP, VPP) to the only ACE inhibition.
Journées Scientifiques A2F-OTELo, 08 février, Nancy, France
Miclo, L.
8th NIZO dairy conference, 11-13 Septembre, Papendal, Pays-Bas.
Roux, É., Miclo, L., Chang, O.-K., Humbert, G., Dary, A., Perrin, C.
EuroFoodChem XVII, 07-10 mai, Istanbul, Turquie
Zidane, F., Zeder-Lutz, G., Altschuh, D., Dary, A., Miclo, L., Cakir-Kiefer, C.
18e Colloque du Club des Bactéries Lactiques, 22-24 mai, Clermont-Ferrand, France
Chang, O.-K., Perrin, C., Roux, É., Miclo, L., Humbert, G., Dary, A.
Colloque Adebiotech-SFGP "Peptides issus des procédés d'hydrolyse - Filières Industrielles", 2-3 octobre, Romainville, France
El Hatmi, H., Cakir-Kiefer, C., Miclo, L., Dary, A., Girardet, J.-M.
16th World Congress of Food Science and Technology (IUFoST) & XVII Latin American Seminar of Food Science and Technology (ALACCTA), 5-9 août 2012, Foz do Iguaçu, Brésil
Hafeez, Z., Cakir-Kiefer, C., Girardet, J.-M., Dary, A., Miclo, L.
Traditional foods can be functionalized by bioactive peptides either by natural fermentation or ripening and/or by their addition in the form of food ingredients. Milk proteins had been shown as a main source of bioactive peptides. Alpha-casozepine, alpha-s1-CN(f91-100), is a bioactive peptide released by tryptic hydrolysis of bovine alpha-s1-casein that exhibits in vivo anxiolytic activity in three different behavioral tests in rats. The hydrolysate containing this peptide proved effective in clinical studies. In vitro hydrolysis of alpha-casozepine with proteolytic enzymes such as pepsin and chymotrypsin released N-terminal shorter fragment alpha-s1-CN-(f91-97) named heptapeptide that also possesses the same activity. Currently, it has been shown that various Streptococcus thermophilus strains were able to hydrolyze alpha-s1-, alpha-s2- and beta-casein with different efficiency and to release many bioactive peptides but neither alpha-casozepine nor heptapeptide was liberated by the 30 strains of our collection tested. Therefore, to develop a functional food containing such peptides, they could be added directly in dairy products (e.g. fermented milks) and should be resistant to degradation by the proteolytic system of S. thermophilus. The experiments revealed that many bioactive peptides including the heptapeptide were degraded by different S. thermophilus strains even when the strains were lacking the cell surface-associated protease PrtS. Indeed, cell surface-associated aminopeptidase and X-prolyl-dipeptidyl aminopeptidase activities were detected. Therefore, to overcome this problem, we are developing a strategy in which we clone a multicopy of anxiolytic heptapeptide in a plasmid and introduce it into bacteria to produce this peptide as a multimer directly in food product.
18e Colloque du Club des Bactéries Lactiques, 22-24 mai, Clermont-Ferrand, France
Hafeez, Z., Cakir-Kiefer, C., Girardet, J.-M., Dary, A., Miclo, L.
In the last few decades, increased consumers awareness about the health benefits of food products has encouraged the development of various health promoting functional foods. These foods not only contribute to the nutritional requirements but also prevent certain diseases, in order to improve the physical and mental well-being of consumers. In some cases, a bioactive peptide of food origin constitutes the active component of these products. Milk proteins are known to be a major source of bioactive peptides that may impart extra-nutritional benefits. These peptides are encrypted in the sequence of caseins and are released only after protein proteolysis in vitro or by gastric or pancreatic enzymes during the digestion of proteins in vivo or by the enzymes of lactic acid bacteria during manufacture of fermented dairy products. S. thermophilus, that possesses its own proteolytic system, is one of the most widely used lactic acid bacteria in the manufacture of fermented dairy products (yoghurt, cheese). The proteolytic system of S. thermophilus is comprised of cell surface proteinase, peptide transport system and a pool of intracellular peptidases. Thus, the question arises whether bioactive peptides added in the fermented dairy products could remain intact or not in presence of this bacterium. To answer this question, two S. thermophilus strains differing in their cell surface proteolytic activity were evaluated: strain LMD9 which possesses cell envelope proteinase activity (PrtS+) and strain CNRZ1066 without this activity (PrtS-). We studied the resistance of some milk protein derived peptides with different biological activities to S. thermophilus cell wall proteolytic system. We then incubated the peptides, namely anxiolytic [YLGYLEQ - alpha-s1-CN-(f91-97)], antihypertensive [TTMPLW - alpha-s1-CN-(f194-199), FALPQYLK - alpha-s2-CN-(f174-181)] and opioid peptide (YPFPGPI - beta-casomorphin-7, RYLGYLE - alpha-s1-CN-(f90-96)] with the non proliferating cells of LMD-9 and CNRZ1066 strains in phosphate / acetate buffer (12.5 mM, pH 6.5). Surprisingly, two different cell wall-anchored peptidase activities were highlighted. The peptides YLGYLEQ, TTMPLW, RYLGYLE, and FALPQYLK were hydrolyzed by both the strains in a similar way. Liquid chromatography coupled to mass spectrometry analysis revealed that breakdown fragments were generated after successive cleavages of amino acids from the N-terminal of these peptides which provides an evidence of an aminopeptidase activity. However, in case of beta-casomorphin-7, an X-prolyl dipeptidyl aminopeptidase activity was observed which is involved in removal of N-terminal dipeptides containing N-terminal prolyl residue. All the peptides remained stable upon incubation in cell free filtrate of these strains which showed that no intracellular peptidases were released and thus involved in the proteolysis process. Therefore, these peptidase activities may help in optimal bacterial growth which consequently increases the milk fermentation rate but at the same time may also cause degradation of bioactive peptides present or added in the fermented milk products. Hence, screening a strain of S. thermophilus having reduced cell wall proteolytic activity would be required for the production of functional fermented dairy products.
Colloque Adebiotech-SFGP "Peptides issus des procédés d'hydrolyse - Filières Industrielles", 2-3 octobre, Romainville, France
Le Roux, Y., Miclo, L.
Colloque Adebiotech-SFGP "Peptides issus des procédés d'hydrolyse - Filières Industrielles", 2-3 octobre, Romainville, France
Zidane, F., Zeder-Lutz, G., Altschuh, D., Girardet, J.-M., Miclo, L., Corbier, C., Cakir-Kiefer, C.
Actes du séminaire de l'École Doctorale RP2E, 19 janvier, Nancy, France
Hafeez, Z., Miclo, L., Girardet, J.-M., Dary, A., Cakir-Kiefer, C.
Actes du séminaire de l'École Doctorale RP2E, 19 janvier, Nancy, France
Hafeez, Z., Miclo, L., Girardet, J.-M., Dary, A., Cakir-Kiefer, C.
17e Congrès du Groupe Français des Peptides et des Protéines (GFPP), 30 janvier - 4 février, Aussois, France
Zidane, F., Cakir-Kiefer, C., Girardet, J.-M., Miclo, L., Corbier, C.
Journée d'Animation Scientifique INRA
Miclo, L.
Actes du Séminaire de l'École Doctorale RP2E, 20 janvier, Nancy, France
Zidane, F., Cakir-Kiefer, C., Girardet, J.-M., Miclo, L., Corbier C.
Bénéfices nutrition santé des micro-organismes d'intérêt laitier. Travaux de la Mission Scientifique Syndifrais, 28 mai, Paris, France
Miclo, L.
17e Colloque du Club des Bactéries Lactiques (CBL), 27-29 octobre, Nancy, France
Dary, A., Miclo, L., Cakir-Kiefer, C., Roux, E., Humbert, G., Driou, A., Perrin, C.
17e Colloque du Club des Bactéries Lactiques (CBL), 27-29 octobre, Nancy, France
Hafeez, Z., Cakir-Kiefer, C., Perrin, C., Dary, A., Miclo, L.
17e Colloque du Club des Bactéries Lactiques (CBL), 27-29 octobre, Nancy, France
Roux, E., Miclo, L., Brusseaux, E., Poirson, C., Perrin, C., Dary, A.
16e Colloque du Club des Bactéries Lactiques, 27-29 mai, Toulouse, France
Chang O.K., Perrin C., Saulnier F., Driou A., Galia W., Humbert G., Miclo L., Dary A.
Titre : Existence d'une forme extracellulaire de PrtS chez Streptococcus
thermophilus.
Auteur(s) : O. K. Chang, C. Perrin, F. Saulnier, A. Driou, W. Galia, G.
Humbert,
L. Miclo et A. Dary
16e Colloque du Club des Bactéries Lactiques, 27-29 mai 2009, Toulouse, France.
Actes du séminaire de l'école doctorale RP2E, 15 janvier, Nancy, France
Chang, O.K., Perrin, C., Driou, A., Galia, W., Humbert, G., Miclo, L., Dary, A.
Preparation of partially dephosphorylated bovine beta-casein, a model for the human homologous protein
First international symposium on minerals and dairy products, 1-3 Octobre, Saint-Malo, France.
Matéos, A., Girardet, J.-M., Bouzobra, F., Miclo, L., Gaillard, J.-L.
Characterization of isoforms of equine alpha-s1 casein: post-transcriptional variants and phosphorylation post-translational variants
First international symposium on minerals and dairy products, 1-3 Octobre, Saint-Malo, France.
Matéos, A., Miclo, L., Mollé, D., Girardet, J.-M., Gaillard, J.-L.
Phosphorylation variants of equine beta-casein: purification of caseinophosphopeptides and determination of the phosphorylation sites
First international symposium on minerals and dairy products, 1-3 Octobre, Saint-Malo, France.
Matéos, A., Miclo, L., Mollé, D., Girardet, J.-M., Gaillard, J.-L.
Caractérisation des isoformes de la caséine alpha-s1 équine : variants post-transcriptionnels et post-traductionnels
Actes du séminaire de l'école doctorale RP2E, 17 janvier, Nancy, France.
Matéos, A., Girardet, J.-M., Mollé, D., Miclo, L., Gaillard, J.-L.
Les peptides du lait : un potentiel fonctionnel ?
Congrès Fonctionnalités des aliments. Congrès d’ouverture au 10ème colloque Européen de nutrition de la FENS, 9 Juillet, Nancy, France.
Miclo L.,
Analyse protéomique des variants post-transcriptionnels et post-traductionnels de la caséine alpha-s1 équine
34e Forum des Jeunes Chercheurs, 15-17 Octobre, Iles des Embiez, France.
Matéos, A., Miclo, L., Mollé, D., Girardet, J.-M., Gaillard, J.-L.
Effets d’un hydrolysat trypsique de caséine alpha-s1 bovine sur la réponse physiologique comportementale à un stress aigu chez le rat Wistar
Réunion de la Société de Circulation et Métabolisme du Cerveau, 26 Janvier, Paris, France
Violle, N., Miclo, L., Desor, D., Gaillard, J.L., Schroeder, H.
Effets d'un hydrolysat trypsique de caséine alpha-s1 bovine sur la réponse physiologique et comportementale au stress chez le rat Wistar
Séminaire de l’Ecole Doctorale RP2E, 11 Janvier, Nancy, France
Violle, N., Miclo, L., Desor, D., Gaillard, J.L., Schroeder, H.
Rapport de contrat avec le Centre Interprofessionnel de l’Économie Laitière (CNIEL), 55 p.
Miclo, L., Brusseaux, E., Poirson, C., Perrin, C., Dary, A.
Miclo, L.
1990 - 2017
Travail collaboratif avec la société Ingredia sur l'alpha-casozépine, peptide anxiolytique dérivé de la caséine alpha-s1 bovine, et les peptides qui en sont issus. Participation à la mise au point du Lactium.
1990 - 2017
Collaborative work with the company Ingredia on alpha-casozepine, an anxiolytic peptide derived from bovine alpha-s1 casein, and the peptides derived from it. Participation in the development of Lactium.
GenBank: GU196267.1 (12-11-2009).
Martin, P.D., Miclo, L., Rebours, E., Matéos, A., Miranda, G.
Université de Lorraine, soutenue le 3 juillet 2013
Miclo, L.
