Valérie Gagnaire

Application of chromatography and mass spectrometry to the characterization of food proteins and derived peptides.

The following review describes the development of mass spectrometry off-line and on-line coupled with liquid chromatography to the analysis of food proteins. It includes the significant results recently obtained in the field of milk, egg and cereal proteins. This paper also outlines the research carried out in the area of food protein hydrolysates, which are important components in foodstuffs due to their functional properties. Liquid chromatography and mass spectrometry have been particularly used for the characterization of food peptides and especially in dairy products.

Autolysis and related proteolysis in Swiss cheese for two Lactobacillus helveticus strains

Intracellular peptidases of Lactobacillus helveticus may play a major role in the proteolysis of Swiss cheeses, provided that they are released through bacterial lysis. Experimental Swiss cheeses were manufactured on a small scale from thermized and microfiltered milk using as starters (in addition to Streptococcus thermophilus and Propionibacterium freudenreichii) one of two Lb. helveticus strains, ITGLH1 and ITGLH77, which undergo lysis to different extents in vitro. All the cheeses were biochemically identical after pressing. The viability of Lb. helveticus ITGLH1 and ITGLH77 decreased to a similar extent (96-98%) while in the cold room, but the concomitant release of intracellular lactate dehydrogenase in cheeses made with strain ITGLH1 was 5-7-fold that in cheeses made with ITGLH77. Protein profiles and immunoblot detection of the dipeptidase PepD confirmed a greater degree of lysis of the ITGLH1 strain. Free active peptidases were detected in aqueous extracts of cheese for both strains, and proteolysis occurred principally in the warm room. Reversed-phase HPLC revealed a more extensive peptide hydrolysis for ITGLH1, which was confirmed by the greater release of free NH2 groups (+33%) and free amino acids (+75%) compared with ITGLH77. As the intracellular peptidase activities of ITGLH1 and ITGLH77 have previously been shown to be similar, our results indicated that the extent of lysis of Lb. helveticus could have a direct impact on the degree of proteolysis in Swiss cheeses.

Invited review: Proteomics of milk and bacteria used in fermented dairy products: From qualitative to quantitative advances

Proteomics is a powerful tool that can simultaneously analyze several hundred proteins in complex mixtures, either through the use of high-resolution 2-dimensional gel electrophoresis or by mono- and multi-dimensional liquid chromatography coupled with mass spectrometry. Since the last review in 2005, proteomics has mainly been applied to describe minor proteins in the bovine milk fat globule membrane and soluble proteins in human colostrum. At least 130 new minor proteins have been identified. These proteins play roles in cell signaling, host defense, and transport as suggested by sequence homology. Proteomic approaches have also been applied to milk of other species such as donkey, horse, and marsupial. Peptides produced in food matrices that can exhibit functional or bioactive properties have been identified as have the proteases leading to their release in situ. However, the most spectacular proteomic development has been in the field of bacteria used in dairy products. Proteomics has resulted in the establishment of reference maps to detect strain-to-strain variations and to elucidate the mechanisms of in vitro and in vivo adaptation to environmental conditions. Proteomic analysis of bacteria entrapped in cheese has been achieved and revealed which predominant metabolic pathways are active depending on the strain. Proteomic approaches are often evoked as time-consuming procedures that provide a list of identified proteins without efficient quantification of each one. New quantitative proteomic methods have emerged and the most promising ones and their application to dairy products and bacteria will be presented.

Phosphopeptides interacting with colloidal calcium phosphate isolated by tryptic hydrolysis of bovine casein micelles

After extended tryptic hydrolysis of large bovine casein micelles, a mineral-rich peptide fraction was recovered by ultracentrifugation. Its mineral part contained 72% of the colloidal Ca and 49% of the colloidal P1 originally present in the native micelle. Colloidal nitrogenous components were also present, amounting to 27% of the original N content. They contained most of the phosphopeptides and 82% of the micellar phosphoseryl residues. These tryptic peptides were characterized by reversed-phase HPLC on-line electrospray ion source-mass spectrometry analysis. Among the peptides produced 14 phosphopeptides were identified: alpha s2-CN(1-24), alpha s2-CN(1-21), alpha s1-CN(43-79), alpha s1-CN(35-79)7P, alpha s1-CN(35-79)8P, alpha s1-CN(37-79), alpha s1-CN(104-119), alpha s1-CN(104-124), beta-CN(1-25), beta-CN(1-28), beta-CN(1-29), beta-CN(30-97), beta-CN(33-97) and beta-CN(29-97). The proportion of the phosphopeptides interacting with colloidal calcium phosphate was correlated with their relative content of phosphoserine residues, since phosphopeptides containing more than four phosphoserine residues were consistently present within this fraction. It also appeared that other types of peptides, some of them hydrophobic in nature, were also partly or completely present within the colloidal fraction, including alpha s1-CN(91-100), alpha s1-CN(152-193), alpha s1-CN(23-34), alpha s1-CN(125-193), alpha s1-CN(125-199), beta-CN(177-209). beta-CN(184-209), beta-CN(114-169) and beta-CN(108-169). Their possible involvement in the micellar backbone is discussed.

Original features of cell-envelope proteinases of Lactobacillus helveticus. A review

Lactobacillus helveticus is a lactic acid bacterium very used in fermented milks and cheese. The rapid growth of L. helveticus in milk is supported by an efficient cell envelope proteinase (CEP) activity, due to subtilisin-like serine proteases. These enzymes play also crucial roles in texture and flavor formation in dairy products as well as in generating in situ bioactive peptides. In L. helveticus, several genes encoding putative CEPs were detected and characterized by a large intraspecific diversity; little is known about regulation of expression of CEP-encoding genes. Anchored at the bacterial surface, CEPs are large-sized enzymes (> 150 kDa) hydrolyzing β- and α(s1)-casein as well. Substrate cleavages occur after almost all types of amino acids residues, but mass spectrometry analysis revealed L. helveticus strains with specific profiles of substrate hydrolysis, which could explain identification of strains associated with interesting technological properties. In this review, the most recent data regarding CEP-encoding genes, CEP activities toward caseins and L. helveticus strain diversity are discussed.

prtH2, Not prtH, Is the Ubiquitous Cell Wall Proteinase Gene in Lactobacillus helveticus

ABSTRACT Lactobacillus helveticus strains possess an efficient proteolytic system that releases peptides which are essential for lactobacillus growth in various fermented dairy products and also affect textural properties or biological activities. Cell envelope proteinases (CEPs) are bacterial enzymes that hydrolyze milk proteins. In the case of L. helveticus, two CEPs with low percentages of amino acid identity have been described, i.e., PrtH and PrtH2. However, the distribution of the genes that encode CEPs still remains unclear, rendering it difficult to further control the formation of particular peptides. This study evaluated the diversity of genes that encode CEPs in a collection of strains of L. helveticus isolated from various biotopes, both in terms of the presence or absence of these genes and in terms of nucleotide sequence, and studied their transcription in dairy matrices. After defining three sets of primers for both the prtH and prtH2 genes, we studied the distribution of the genes by using PCR and Southern blotting experiments. The prtH2 gene was ubiquitous in the 29 strains of L. helveticus studied, whereas only 18 of them also exhibited the prtH gene. Sequencing of a 350-bp internal fragment of these genes revealed the existence of intraspecific diversity. Finally, expression of these two CEP-encoding genes was followed during the growth in dairy matrices of two strains, ITG LH77 and CNRZ32, which possess one and two CEP-encoding genes, respectively. Both genes were shown to be expressed by L. helveticus at each stage of growth in milk and at different stages of mini-Swiss-type cheese making and ripening.

Hydrolysis of Sequenced β-Casein Peptides Provides New Insight into Peptidase Activity from Thermophilic Lactic Acid Bacteria and Highlights Intrinsic Resistance of Phosphopeptides

ABSTRACT The peptidases of thermophilic lactic acid bacteria have a key role in the proteolysis of Swiss cheeses during warm room ripening. To compare their peptidase activities toward a dairy substrate, a tryptic/chymotryptic hydrolysate of purified β-casein was used. Thirty-four peptides from 3 to 35 amino acids, including three phosphorylated peptides, constitute the β-casein hydrolysate, as shown by tandem mass spectrometry. Cell extracts prepared fromLactobacillus helveticus ITG LH1, ITG LH77, and CNRZ 32,Lactobacillus delbrueckii subsp. lactis ITG LL14 and ITG LL51, L. delbrueckii subsp.bulgaricus CNRZ 397 and NCDO 1489, and Streptococcus thermophilus CNRZ 385, CIP 102303, and TA 060 were standardized in protein. The peptidase activities were assessed with the β-casein hydrolysate as the substrate at pH 5.5 and 24°C (conditions of warm room ripening) by (i) free amino acid release, (ii) reverse-phase chromatography, and (iii) identification of undigested peptides by mass spectrometry. Regardless of strain, L. helveticus was the most efficient in hydrolyzing β-casein peptides. Interestingly, cell extracts of S. thermophilus were not able to release a significant level of free proline from the β-casein hydrolysate, which was consistent with the identification of numerous dipeptides containing proline. With the three lactic acid bacteria tested, the phosphorylated peptides remained undigested or weakly hydrolyzed indicating their high intrinsic resistance to peptidase activities. Finally, several sets of peptides differing by a single amino acid in a C-terminal position revealed the presence of at least one carboxypeptidase in the cell extracts of these species.

Simultaneous presence of PrtH and PrtH2 proteinases in Lactobacillus helveticus strains improves breakdown of the pure αs1-casein

ABSTRACT Lactobacillus helveticus can possess one or two cell envelope proteinases (CEPs), called PrtH2 and PrtH. The aim of this work was to explore the diversity of 15 strains of L. helveticus, isolated from various origins, in terms of their proteolytic activities and specificities on pure caseins or on milk casein micelles. CEP activity differed 14-fold when the strains were assayed on a synthetic substrate, but no significant differences were detected between strains possessing one or two CEPs. No correlation was observed between the proteolytic activities of the strains and their rates of acidification in milk. The kinetics of hydrolysis of purified αs1- and β-casein by L. helveticus whole cells was monitored using Tris-Tricine sodium dodecyl sulfate (SDS) electrophoresis, and for four strains, the peptides released were identified using mass spectrometry. While rapid hydrolysis of pure β-casein was observed for all strains, the hydrolysis kinetics of αs1-casein was the only criterion capable of distinguishing between the strains based on the number of CEPs. Fifty-four to 74 peptides were identified for each strain. When only PrtH2 was present, 22 to 30% of the peptides originated from αs1-casein. The percentage increased to 41 to 49% for strains in which both CEPs were expressed. The peptide size ranged from 6 to 33 amino acids, revealing a broad range of cleavage specificities, involving all classes of amino acids (Leu, Val, Ala, Ile, Glu, Gln, Lys, Arg, Met, and Pro). Regions resistant to proteolysis were identified in both caseins. When strains were grown in milk, a drastic reduction in the number of peptides was observed, reflecting changes in accessibility and/or peptide assimilation during growth.

Free active peptidases are detected in Emmental juice extracted before ripening in the warm room

In Swiss-type cheese such as Emmental, proteolysis is one of the major phenomena occurring during ripening. Among the proteolytic agents involved in cheese ripening, the free enzymes originally present in milk and those arising from bacterial autolysis can act directly on the casein network. In order to understand the contribution of the bacterial enzymes and especially those arising from the thermophilic starters, the juice of an Emmental cheese entering the warm room was extracted by pressure, then sterilized by filtration and incubated at 24°C for 20 d under anaerobiosis. At different times, the peptides and free amino acids were determined in the sterile cheese juice. In parallel, in order to gather information about the nature of the enzymes present, the sterile juice was also incubated with β-naphthylamide derivatives as substrates. We have demonstrated a continuous increase in free NH 2 groups and in free amino acids throughout the 20 d incubation time. The main peptidase activity was due to aminopeptidase(s) and X-prolyldipeptidyl aminopeptidase(s) whose activities were recovered after non-denaturing polyacrylamide gel electrophoresis. Most of the enzymes found in the juice would have their origin in thermophilic starters. As they are generally intracellularly located, their release could be explained by the autolysis of these starters. Finally, the main free amino acids released in the juice (Pro, Glu, Ala, Val, Leu and Lys) corresponded to those previously found in Emmental cheese, suggesting that the enzymes detected in this study participate significantly in peptide degradation during ripening.

Quantitative proteomic analysis of bacterial enzymes released in cheese during ripening.

Due to increasingly available bacterial genomes in databases, proteomic tools have recently been used to screen proteins expressed by micro-organisms in food in order to better understand their metabolism in situ. While the main objective is the systematic identification of proteins, the next step will be to bridge the gap between identification and quantification of these proteins. For that purpose, a new mass spectrometry-based approach was applied, using isobaric tagging reagent for quantitative proteomic analysis (iTRAQ), which are amine specific and yield labelled peptides identical in mass. Experimental Swiss-type cheeses were manufactured from microfiltered milk using Streptococcus thermophilus ITG ST20 and Lactobacillus helveticus ITG LH1 as lactic acid starters. At three ripening times (7, 20 and 69 days), cheese aqueous phases were extracted and enriched in bacterial proteins by fractionation. Each sample, standardised in protein amount prior to proteomic analyses, was: i) analysed by 2D-electrophoresis for qualitative analysis and ii) submitted to trypsinolysis, and labelled with specific iTRAQ tag, one per ripening time. The three labelled samples were mixed together and analysed by nano-LC coupled on-line with ESI-QTOF mass spectrometer. Thirty proteins, both from bacterial or bovine origin, were identified and efficiently quantified. The free bacterial proteins detected were enzymes from the central carbon metabolism as well as stress proteins. Depending on the protein considered, the quantity of these proteins in the cheese aqueous extract increased from 2.5 to 20 fold in concentration from day 7 to day 69 of ripening.