Michel Piot

Rehydration of casein powders: effects of added mineral salts and salt addition methods on water transfer

Abstract Enrichment of milk with micellar casein decreases water transfer during the rehydration of milk powders. In this study, the effects of the ion environment and the ion addition method on the rehydration kinetics were found to be dependent on the changes in the micellar casein. For example, adding citrate or phosphate solution to the micellar casein suspension before drying considerably increased rehydration rates and this was related to the destruction of the micelle structure. Water transfer in the casein suspension was improved by adding NaCl during rehydration: this effect may be explained by the more hygroscopic nature of NaCl rather than by extensive modification of the micellar structure. The addition of CaCl 2 considerably affected micelle organization and led to the formation of insoluble structures during spray drying.

Dynamic analysis of the Lactococcus lactis transcriptome in cheeses made from milk concentrated by ultrafiltration reveals multiple strategies of adaptation to stresses.

ABSTRACT Lactococcus lactis is used extensively for the production of various cheeses. At every stage of cheese fabrication, L. lactis has to face several stress-generating conditions that result from its own modification of the environment as well as externally imposed conditions. We present here the first in situ global gene expression profile of L. lactis in cheeses made from milk concentrated by ultrafiltration (UF-cheeses), a key economical cheese model. The transcriptomic response of L. lactis was analyzed directly in a cheese matrix, starting from as early as 2 h and continuing for 7 days. The growth of L. lactis stopped after 24 h, but metabolic activity was maintained for 7 days. Conservation of its viability relied on an efficient proteolytic activity measured by an increasing, quantified number of free amino acids in the absence of cell lysis. Extensive downregulation of genes under CodY repression was found at day 7. L. lactis developed multiple strategies of adaptation to stressful modifications of the cheese matrix. In particular, expression of genes involved in acidic- and oxidative-stress responses was induced. L. lactis underwent unexpected carbon limitation characterized by an upregulation of genes involved in carbon starvation, principally due to the release of the CcpA control. We report for the first time that in spite of only moderately stressful conditions, lactococci phage is repressed under UF-cheese conditions.

Staphylococcus aureus virulence and metabolism are dramatically affected by Lactococcus lactis in cheese matrix

In complex environments such as cheeses, the lack of relevant information on the physiology and virulence expression of pathogenic bacteria and the impact of endogenous microbiota has hindered progress in risk assessment and control. Here, we investigated the behaviour of Staphylococcus aureus, a major foodborne pathogen, in a cheese matrix, either alone or in the presence of Lactococcus lactis, as a dominant species of cheese ecosystems. The dynamics of S. aureus was explored in situ by coupling a microbiological and, for the first time, a transcriptomic approach. Lactococcus lactis affected the carbohydrate and nitrogen metabolisms and the stress response of S. aureus by acidifying, proteolysing and decreasing the redox potential of the cheese matrix. Enterotoxin expression was positively or negatively modulated by both L. lactis and the cheese matrix itself, depending on the enterotoxin type. Among the main enterotoxins involved in staphylococcal food poisoning, sea expression was slightly favoured in the presence of L. lactis, whereas a strong repression of sec4 was observed in cheese matrix, even in the absence of L. lactis, and correlated with a reduced saeRS expression. Remarkably, the agr system was downregulated by the presence of L. lactis, in part because of the decrease in pH. This study highlights the intimate link between environment, metabolism and virulence, as illustrated by the influence of the cheese matrix context, including the presence of L. lactis, on two major virulence regulators, the agr system and saeRS.

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.

High pressure-induced gel formation of milk and whey concentrates

Abstract Gels from milk concentrates [milk and caseinate powder, ultrafiltration (UF) and microfiltration (MF)] and whey concentrates (UF) were obtained with high pressure (200 and 400 MPa, 10 and 30 min). The effects of protein concentration (66–114gkg −1 for milk and 97–127g kg −1 for whey), NaCl addition (0–8 g kg −1 ), sodium citrate addition (0–4 g kg −1 ) and pH (5.2–6.6 for milk and 7–9 for whey) were studied with a Box-Behnken design. Milk supplemented with casemate powder did not lead to gel formation by pressure. For UF or MF milk concentrates, a pH decrease towards 5.9 and a protein content increase led to firmer pressure-set gels. Gels of whey concentrates were obtained only at pH 9. A pressure increase from 200 to 400 MPa led to firmer gels, while a protein content increase did not.

Physicochemical characterization of iron-supplemented skim milk

Abstract The physicochemical characterization and study of technological properties of skim milk samples supplemented with FeCl 2 or FeCl 3 were carried out. The iron concentrations ranged from 0 to 1.5 mM. For both salts used, iron was mainly bound to the micellar phase with a probable change of casein structure. In parallel, the mineral distribution and the casein micelle hydration changed differently as a function of the initial oxidation state of iron (Fe 2+ or Fe 3+ ). After acidification (between 6.7 and 4.0) or after heat treatments (95 °C—15 and 30 min), no release of iron was found. Rennet coagulation parameters (clotting time, aggregation time and curd firmness) of the different iron-supplemented skim milk samples were more modified when fortifications were performed with FeCl 2 than with FeCl 3 . The results were discussed in relation with proteins and minerals modifications.

Mur-LH, the Broad-Spectrum Endolysin of Lactobacillus helveticus Temperate Bacteriophage φ-0303

ABSTRACT φ-0303 is a temperate bacteriophage isolated from Lactobacillus helveticus CNRZ 303 strain after mitomycin C induction. In this work, the gene coding for a lytic protein of this bacteriophage was cloned using a library of φ-0303 in Escherichia coli DH5α. The lytic activity was detected by its expression, using whole cells of the sensitive strain L. helveticus CNRZ 892 as the substrate. The lysin gene was within a 4.1-kb DNA fragment of φ-0303 containing six open reading frames (ORFs) and two truncated ORFs. No sequence homology with holin genes was found within the cloned fragment. An integrase-encoding gene was also present in the fragment, but it was transcribed in a direction opposite that of the lysin gene. The lysin-encoding lys gene was verified by PCR amplification from the total phage DNA and subcloned. The lys gene is a 1,122-bp sequence encoding a protein of 373 amino acids (Mur-LH), whose product had a deduced molecular mass of 40,207 Da. Comparisons with sequences in sequence databases showed homology with numerous endolysins of other bacteriophages. Mur-LH was expressed in E. coli BL21, and by renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis with L. helveticus CNRZ 892 as the substrate, the recombinant protein showed an apparent molecular mass of 40 kDa. The N-terminal sequence of the protein confirmed the start codon. Hydrolysis of cell walls of L. helveticus CNRZ 303 by the endolysin and biochemical analysis of the residues produced demonstrated that Mur-LH has N-acetylmuramidase activity. Last, the endolysin exhibited a broad spectrum of lytic activity, as it was active on different species, mainly thermophilic lactobacilli but also lactococci, pediococci, Bacillus subtilis, Brevibacterium linens, and Enterococcus faecium.

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.

DEHYDRATION BY DESORPTION AND BY SPRAY DRYING OF DAIRY PROTEINS: INFLUENCE OF THE MINERAL ENVIRONMENT

ABSTRACT A drying method by desorption in a water activity meter was used to simulate the conditions of spray drying and to determine the water transfer inside dairy concentrates towards the surface and from the surface to the drying air. The concentrates were also spray dried and solubility index of powders were determined during reconstitution. Whey protein concentrates (WPC) and native phosphocaseinate suspensions (NPCS) were used to study the effect of NaCl (420 mM), CaCl2 (222 raM), sodium phosphate (173 mM) and sodium citrate (238 mM) on the water transfers. The decrease in water transfer during drying was explained by the high hygroscopicity of added mineral salts to WPC. NaCl addition to NPCS decreased the water transfer during drying, but increased the solubility index. Citrate and phosphate addition to NPCS increased the water transfer during drying and reconstitution. CaCl2 increased the water transfer during drying but the solubility index was always low. Results are discussed as a function of...

Comparison of electrospray and matrix-assisted laser desorption ionization on the same hybrid quadrupole time-of-flight tandem mass spectrometer: application to bidimensional liquid chromatography of proteins from bovine milk fraction.

Recently, two ionization sources, electrospray (ESI) and matrix-assisted laser desorption (MALDI) have been used in parallel to exploit their complementary nature and to increase proteome coverage. In this study, a method using bidimensional (2D) nanoLC coupled online with ESI quadrupole time-of-flight (Q-TOF) with the simultaneous collection of fractions for analyses by LC-MALDI Q-TOF-MS/MS was developed. A total of 39 bovine proteins were identified to a high degree of confidence. To help in differentiating peptide detection following ESI and MALDI with the same mass spectrometer, we compared physico-chemical characteristics of the peptides (molecular mass, charge and size) by principal component analysis (PCA) and analysis of variance on the results of PCA. More hydrophobic peptides with a wider mass coverage were identified when ESI was used, whereas more basic and smaller peptides were identified when MALDI was used. However, the generally accepted differentiation between ESI and MALDI according to the presence of basic amino acids residues Lys and Arg and the ratio Lys/Arg was not shown as significant in this study. Moreover, we pointed out the importance of the type of mass spectrometer used in complement to both ionization sources for achieving a global increase of proteome coverage.