A Toxic Friend: Genotoxic and Mutagenic Activity of the Probiotic Strain Escherichia coli Nissle 1917.

The probiotic Escherichia coli strain Nissle 1917 (DSM 6601, Mutaflor), generally considered beneficial and safe, has been used for a century to treat various intestinal diseases. However, Nissle 1917 hosts in its genome the pks pathogenicity island that codes for the biosynthesis of the genotoxin colibactin. Colibactin is a potent DNA alkylator, suspected to play a role in colorectal cancer development. We show in this study that Nissle 1917 is functionally capable of producing colibactin and inducing interstrand cross-links in the genomic DNA of epithelial cells exposed to the probiotic. This toxicity was even exacerbated with lower doses of the probiotic, when the exposed cells started to divide again but exhibited aberrant anaphases and increased gene mutation frequency. DNA damage was confirmed in vivo in mouse models of intestinal colonization, demonstrating that Nissle 1917 produces the genotoxin in the gut lumen. Although it is possible that daily treatment of adult humans with their microbiota does not produce the same effects, administration of Nissle 1917 as a probiotic or as a chassis to deliver therapeutics might exert long-term adverse effects and thus should be considered in a risk-versus-benefit evaluation. IMPORTANCE Nissle 1917 is sold as a probiotic and considered safe even though it has been known since 2006 that it harbors the genes for colibactin synthesis. Colibactin is a potent genotoxin that is now linked to causative mutations found in human colorectal cancer. Many papers concerning the use of this strain in clinical applications ignore or elude this fact or misleadingly suggest that Nissle 1917 does not induce DNA damage. Here, we demonstrate that Nissle 1917 produces colibactin in vitro and in vivo and induces mutagenic DNA damage. This is a serious safety concern that must not be ignored in the interests of patients, the general public, health care professionals, and ethical probiotic manufacturers.

The secreted L-arabinose isomerase displays anti-hyperglycemic effects in mice.

BACKGROUND: The L-arabinose isomerase is an intracellular enzyme which converts L-arabinose into L-ribulose in living systems and D-galactose into D-tagatose in industrial processes and at industrial scales. D-tagatose is a natural ketohexose with potential uses in pharmaceutical and food industries. The D-galactose isomerization reaction is thermodynamically equilibrated, and leads to secondary subproducts at high pH. Therefore, an attractive L-arabinose isomerase should be thermoactive and acidotolerant with high catalytic efficiency. While many reports focused on the set out of a low cost process for the industrial production of D-tagatose, these procedures remain costly. When compared to intracellular enzymes, the production of extracellular ones constitutes an interesting strategy to increase the suitability of the biocatalysts. RESULTS: The L-arabinose isomerase (L-AI) from Lactobacillus sakei was expressed in Lactococcus lactis in fusion with the signal peptide of usp45 (SP(Usp45)). The L-AI protein and activity were detected only in the supernatant of the induced cultures of the recombinant L. lactis demonstrating the secretion in the medium of the intracellular L. sakei L-AI in an active form. Moreover, we showed an improvement in the enzyme secretion using either (1) L. lactis strains deficient for their two major proteases, ClpP and HtrA, or (2) an enhancer of protein secretion in L. lactis fused to the recombinant L-AI with the SP(Usp45). Th L-AI enzyme secreted by the recombinant L. lactis strains or produced intracellularly in E. coli, showed the same functional properties than the native enzyme. Furthermore, when mice are fed with the L. lactis strain secreting the L-AI and galactose, tagatose was produced in vivo and reduced the glycemia index. CONCLUSIONS: We report for the first time the secretion of the intracellular L-arabinose isomerase in the supernatant of food grade L. lactis cultures with hardly display other secreted proteins. The secreted L-AI originated from the food grade L. sakei 23 K was active and showed the same catalytic and structural properties as the intracellular enzyme. The L. lactis strains secreting the L-arabinose isomerase has the ability to produce D-tagatose in vivo and conferred an anti-hyperglycemic effect to mice.

The commensal bacterium Faecalibacterium prausnitzii is protective in DNBS-induced chronic moderate and severe colitis models.

BACKGROUND: The abundance of Faecalibacterium prausnitzii, an abundant and representative bacterium of Firmicutes phylum, has consistently been observed to be lower in patients with Crohn's disease than in healthy individuals. We have shown that both F. prausnitzii and its culture supernatant (SN) have anti-inflammatory and protective effects in a TNBS-induced acute colitis mouse model. Here, we tested the effects of both F. prausnitzii and its SN in moderate and severe DNBS-induced chronic colitis mouse models. METHODS: Colitis was induced by intrarectal administration of DNBS. After either 4 or 10 days of recovery (severe and moderate protocols, respectively), groups of mice were intragastrically administered either with F. prausnitzii A2-165 or with its culture SN for 7 or 10 days. Three days before being sacrificed, colitis was reactivated by administration of a lower dose of DNBS. The severity of colitis at the time of being sacrificed was assessed by weight loss and macroscopic and microscopic scores. Myeloperoxidase (MPO) activity, cytokine levels, lymphocyte populations, and changes in microbiota were studied. RESULTS: Intragastric administration of either F. prausnitzii or its SN led to a significant decrease in colitis severity in both severe and moderate chronic colitis models. The lower severity of colitis was associated with down-regulation of MPO, pro-inflammatory cytokines, and T-cell levels. CONCLUSIONS: We show, for the first time, protective effects of both F. prausnitzii and its SN during both the period of recovery from chronic colitis and colitis reactivation. These results provide further evidence that F. prausnitzii is an anti-inflammatory bacterium with therapeutic potential for patients with inflammatory bowel disease.

Faecal D/L lactate ratio is a metabolic signature of microbiota imbalance in patients with short bowel syndrome.

Our objective was to understand the functional link between the composition of faecal microbiota and the clinical characteristics of adults with short bowel syndrome (SBS). Sixteen patients suffering from type II SBS were included in the study. They displayed a total oral intake of 2661±1005 Kcal/day with superior sugar absorption (83±12%) than protein (42±13%) or fat (39±26%). These patients displayed a marked dysbiosis in faecal microbiota, with a predominance of Lactobacillus/Leuconostoc group, while Clostridium and Bacteroides were under-represented. Each patient exhibited a diverse lactic acid bacteria composition (L. delbrueckii subsp. bulgaricus, L. crispatus, L. gasseri, L. johnsonii, L. reuteri, L. mucosae), displaying specific D and L-lactate production profiles in vitro. Of 16 patients, 9/16 (56%) accumulated lactates in their faecal samples, from 2 to 110 mM of D-lactate and from 2 to 80 mM of L-lactate. The presence of lactates in faeces (56% patients) was used to define the Lactate-accumulator group (LA), while absence of faecal lactates (44% patients) defines the Non lactate-accumulator group (NLA). The LA group had a lower plasma HCO3(-) concentration (17.1±2.8 mM) than the NLA group (22.8±4.6 mM), indicating that LA and NLA groups are clinically relevant sub-types. Two patients, belonging to the LA group and who particularly accumulated faecal D-lactate, were at risk of D-encephalopathic reactions. Furthermore, all patients of the NLA group and those accumulating preferentially L isoform in the LA group had never developed D-acidosis. The D/L faecal lactate ratio seems to be the most relevant index for a higher D-encephalopathy risk, rather than D- and L-lactate faecal concentrations per se. Testing criteria that take into account HCO3(-) value, total faecal lactate and the faecal D/L lactate ratio may become useful tools for identifying SBS patients at risk for D-encephalopathy.

Identification of one novel candidate probiotic Lactobacillus plantarum strain active against influenza virus infection in mice by a large-scale screening.

In this study, we developed a large-scale screening of bacterial strains in order to identify novel candidate probiotics with immunomodulatory properties. For this, 158 strains, including a majority of lactic acid bacteria (LAB), were screened by two different cellular models: tumor necrosis factor alpha (TNF-α)-activated HT-29 cells and peripheral blood mononuclear cells (PBMCs). Different strains responsive to both models (pro- and anti-inflammatory strains) were selected, and their protective effects were tested in vivo in a murine model of influenza virus infection. Daily intragastric administrations during 10 days before and 10 days after viral challenge (100 PFU of influenza virus H1N1 strain A Puerto Rico/8/1934 [A/PR8/34]/mouse) of Lactobacillus plantarum CNRZ1997, one potentially proinflammatory probiotic strain, led to a significant improvement in mouse health by reducing weight loss, alleviating clinical symptoms, and inhibiting significantly virus proliferation in lungs. In conclusion, in this study, we have combined two cellular models to allow the screening of a large number of LAB for their immunomodulatory properties. Moreover, we identified a novel candidate probiotic strain, L. plantarum CNRZ1997, active against influenza virus infection in mice.

Proteomic analysis of spontaneous mutants of Lactococcus lactis: Involvement of GAPDH and arginine deiminase pathway in H2O2 resistance.

Lactococcus lactis, one of the most commonly used dairy starters, is often subjected to oxidative stress in cheese manufacturing. A comparative proteomic analysis was performed to identify the molecular modifications responsible for the robustness of three spontaneous H(2)O(2)-resistant (SpOx) strains. In the parental strain, glyceraldehyde-3-phosphate deshydrogenase (GAPDH) activity is ensured by GapB and the second GAPDH GapA is not produced in standard growth conditions. We showed that GapA was overproduced in the highly resistant SpOx2 and SpOx3 mutants. Its overproduction in the MG1363 strain led to an increased H(2)O(2) resistance of exponential growing cells. Upon H(2)O(2) exposure, GapB was fully inactivated by oxidation in the parental strain. In SpOx mutants, it partly remained in the reduced form sustaining partially GAPDH activity. The analysis of gapA disruption in these SpOx strains indicated that additional unraveled mechanisms likely contribute to the resistance phenotype. In the SpOx1 mutant, the arginine deiminase pathway was found to be upregulated and disruption of arcA or arcB genes abolished H(2)O(2) resistance. We concluded that arginine consumption was directly responsible for the SpOx1 phenotype. Finally, these results suggest that sustaining energy supply is a major way of leading to oxidative stress resistance in L. lactis.

PpiA, a surface PPIase of the cyclophilin family in Lactococcus lactis.

BACKGROUND: Protein folding in the envelope is a crucial limiting step of protein export and secretion. In order to better understand this process in Lactococcus lactis, a lactic acid bacterium, genes encoding putative exported folding factors like Peptidyl Prolyl Isomerases (PPIases) were searched for in lactococcal genomes. RESULTS: In L. lactis, a new putative membrane PPIase of the cyclophilin subfamily, PpiA, was identified and characterized. ppiA gene was found to be constitutively expressed under normal and stress (heat shock, H(2)O(2)) conditions. Under normal conditions, PpiA protein was synthesized and released from intact cells by an exogenously added protease, showing that it was exposed at the cell surface. No obvious phenotype could be associated to a ppiA mutant strain under several laboratory conditions including stress conditions, except a very low sensitivity to H(2)O(2). Induction of a ppiA copy provided in trans had no effect i) on the thermosensitivity of an mutant strain deficient for the lactococcal surface protease HtrA and ii) on the secretion and stability on four exported proteins (a highly degraded hybrid protein and three heterologous secreted proteins) in an otherwise wild-type strain background. However, a recombinant soluble form of PpiA that had been produced and secreted in L. lactis and purified from a culture supernatant displayed both PPIase and chaperone activities. CONCLUSIONS: Although L. lactis PpiA, a protein produced and exposed at the cell surface under normal conditions, displayed a very moderate role in vivo, it was found, as a recombinant soluble form, to be endowed with folding activities in vitro.

Variations of N-acetylation level of peptidoglycan do not influence persistence of Lactococcus lactis in the gastrointestinal tract.

The food-grade Gram-positive bacterium, Lactococcus lactis, is recognized as a potential candidate to deliver proteins of medical interest by mucosal routes. The ability of carrier bacteria to persist and/or to lyse in the gastrointestinal tract needs to be considered to design optimal carrier strains to deliver proteins of interest at the mucosal level. Meyrand et al. (2007) have previously characterized in L. lactis, a peptidoglycan (PG) N-acetylglucosamine deacetylase (PgdA), which activity on PG influences bacterial sensitivity to lysozyme. Inactivation of pgdA gene in this bacterium, led to fully acetylated PG, resulting in a lysozyme-sensitive phenotype, whereas pgdA overexpression led to an increased degree of PG deacetylation, resulting in a lysozyme-resistant phenotype (Meyrand et al., 2007). In order to determine whether variations in L. lactis resistance to host lysozyme may influence its persistence in the GIT and its ability to deliver heterologous proteins in situ, we constructed L. lactis strains with different de-N-acetylation levels and producing a model antigen (the human papillomavirus type-16 E7 protein) and we compared the pharmacokinetics properties of these recombinant strains with that of a wild-type strain producing the same antigen in the GIT of mice. Our results show that there was no correlation between survival, at the ileum level, of bacteria intragastrically administered in mice and bacteria sensitivity or resistance to lysozyme. In addition, analysis of the E7-specific immune response evoked by the three strains after mucosal administration in mice suggest that neither lysozyme-sensitive nor lysozyme-resistant phenotype in L. lactis enhances significantly the potential of this bacterium as mucosal delivery live vector. In conclusion, our results suggest that either pgdA inactivation or pgdA overexpression in L. lactis leading to different levels of PG deacetylation does not confer any advantage in the persistence of this bacterium in the GIT and its ability to enhance host immune responses induced by delivered antigen in situ.

Intragastric administration of a superoxide dismutase-producing recombinant Lactobacillus casei BL23 strain attenuates DSS colitis in mice.

Human immune cells release large amounts of reactive oxygen species (ROS) such as superoxide radical and hydrogen peroxide via respiratory burst. In inflammatory bowel diseases, a sustained and abnormal activation of the immune response results in oxidative stress of the digestive tract and in a loss of intestinal homeostasis. We previously reported that heterologous production of the Lactobacillus plantarum manganese catalase (MnKat) enhances the survival of Lb. casei BL23 when exposed to oxidative stress. Anti-inflammatory effects were observed after Lb. casei BL23 oral administrations in moderate murine dextran sodium sulfate (DSS)-induced colitis, without added effects of the MnKat production. Here, we evaluated the protective effects obtained by an improved antioxidative strategy. The Lactococcus lactis sodA gene was expressed in Lb. casei BL23 which acquired an efficient manganese superoxide dismutase (MnSOD) activity. The effects of Lb. casei MnSOD alone or in combination with Lb. casei MnKat were compared first in eukaryotic cell PMA-induced oxidative stress model and then in severe murine DSS-induced colitis. Based on ROS production assays as well as colonic histological scores, a significant reduction of both oxidative stress and inflammation was observed with Lb. casei MnSOD either alone or in combination with Lb. casei MnKat. No added effect of the presence of Lb. casei MnKat was observed. These results suggest that Lb. casei BL23 MnSOD could have anti-inflammatory effects on gut inflammation.

Analysis of Lactobacillus sakei mutants selected after adaptation to the gastrointestinal tracts of axenic mice.

We recently showed that Lactobacillus sakei, a natural meat-borne lactic acid bacterium, can colonize the gastrointestinal tracts (GIT) of axenic mice but that this colonization in the intestinal environment selects L. sakei mutants showing modified colony morphology (small and rough) and cell shape, most probably resulting from the accumulation of various mutations that confer a selective advantage for persistence in the GIT. In the present study, we analyzed such clones, issued from three different L. sakei strains, in order to determine which functions were modified in the mutants. In the elongated filamentous cells of the rough clones, transmission electron microscopy (TEM) analysis showed a septation defect and dotted and slanted black bands, suggesting the presence of a helical structure around the cells. Comparison of the cytoplasmic and cell wall/membrane proteomes of the meat isolate L. sakei 23K and of one of its rough derivatives revealed a modified expression for 38 spots. The expression of six oxidoreductases, several stress proteins, and four ABC transporters was strongly reduced in the GIT-adapted strain, while the actin-like MreB protein responsible for cell shaping was upregulated. In addition, the expression of several enzymes involved in carbohydrate metabolism was modified, which may correlate with the observation of modified growth of mutants on various carbon sources. These results suggest that the modifications leading to a better adaptation to the GIT are pleiotropic and are characterized in a rough mutant by a different stress status, a cell wall modification, and modified use of energy sources, leading to an improved fitness for the colonization of the GIT.

Inactivation of the Lactococcus lactis high-affinity phosphate transporter confers oxygen and thiol resistance and alters metal homeostasis.

Numerous strategies allowing bacteria to detect and respond to oxidative conditions depend on the cell redox state. Here we examined the ability of Lactococcus lactis to survive aerobically in the presence of the reducing agent dithiothreitol (DTT), which would be expected to modify the cell redox state and disable the oxidative stress response. DTT inhibited L. lactis growth at 37 degrees C in aerobic conditions, but not in anaerobiosis. Mutants selected as DTT resistant all mapped to the pstFEDCBA locus, encoding a high-affinity phosphate transporter. Transcription of pstFEDCBA and a downstream putative regulator of stress response, phoU, was deregulated in a pstA strain, but amounts of major oxidative stress proteins were unchanged. As metals participate in oxygen radical formation, we compared metal sensitivity of wild-type and pstA strains. The pstA mutant showed approximately 100-fold increased resistance to copper and zinc. Furthermore, copper or zinc addition exacerbated the sensitivity of a wild-type L. lactis strain to DTT. Inactivation of pstA conferred a more general resistance to oxidative stress, alleviating the oxygen- and thermo-sensitivity of a clpP mutant. This study establishes a role for the pst locus in metal homeostasis, suggesting that pst inactivation lowers intracellular reactivity of copper and zinc, which would limit bacterial sensitivity to oxygen.

Faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified by gut microbiota analysis of Crohn disease patients.

A decrease in the abundance and biodiversity of intestinal bacteria within the dominant phylum Firmicutes has been observed repeatedly in Crohn disease (CD) patients. In this study, we determined the composition of the mucosa-associated microbiota of CD patients at the time of surgical resection and 6 months later using FISH analysis. We found that a reduction of a major member of Firmicutes, Faecalibacterium prausnitzii, is associated with a higher risk of postoperative recurrence of ileal CD. A lower proportion of F. prausnitzii on resected ileal Crohn mucosa also was associated with endoscopic recurrence at 6 months. To evaluate the immunomodulatory properties of F. prausnitzii we analyzed the anti-inflammatory effects of F. prausnitzii in both in vitro (cellular models) and in vivo [2,4,6-trinitrobenzenesulphonic acid (TNBS)-induced] colitis in mice. In Caco-2 cells transfected with a reporter gene for NF-kappaB activity, F. prausnitzii had no effect on IL-1beta-induced NF-kappaB activity, whereas the supernatant abolished it. In vitro peripheral blood mononuclear cell stimulation by F. prausnitzii led to significantly lower IL-12 and IFN-gamma production levels and higher secretion of IL-10. Oral administration of either live F. prausnitzii or its supernatant markedly reduced the severity of TNBS colitis and tended to correct the dysbiosis associated with TNBS colitis, as demonstrated by real-time quantitative PCR (qPCR) analysis. F. prausnitzii exhibits anti-inflammatory effects on cellular and TNBS colitis models, partly due to secreted metabolites able to block NF-kappaB activation and IL-8 production. These results suggest that counterbalancing dysbiosis using F. prausnitzii as a probiotic is a promising strategy in CD treatment.

Anti-inflammatory effects of Lactobacillus casei BL23 producing or not a manganese-dependant catalase on DSS-induced colitis in mice.

BACKGROUND: Human immune cells generate large amounts of reactive oxygen species (ROS) throughout the respiratory burst that occurs during inflammation. In inflammatory bowel diseases, a sustained and abnormal activation of the immune system results in oxidative stress in the digestive tract and in a loss of intestinal homeostasis. We previously showed that the heterologous production of the Lactobacillus plantarum ATCC14431 manganese-dependant catalase (MnKat) in Lb. casei BL23 successfully enhances its survival when exposed to oxidative stress. In this study, we evaluated the preventive effects of this antioxidative Lb. casei strain in a murine model of dextran sodium sulfate (DSS)-induced moderate colitis. RESULTS: Either Lb. casei BL23 MnKat- or MnKat+ was administered daily to mice treated with DSS for 10 days. In contrast to control mice treated with PBS for which DSS induced bleeding diarrhea and mucosal lesions, mice treated with both Lb. casei strains presented a significant (p < 0.05) reduction of caecal and colonic inflammatory scores. CONCLUSION: No contribution of MnKat to the protective effect from epithelial damage has been observed in the tested conditions. In contrast, these results confirm the high interest of Lb. casei as an anti-inflammatory probiotic strain.

Production of a heterologous nonheme catalase by Lactobacillus casei: an efficient tool for removal of H2O2 and protection of Lactobacillus bulgaricus from oxidative stress in milk.

Lactic acid bacteria (LAB) are generally sensitive to H2O2, a compound that they can paradoxically produce themselves, as is the case for Lactobacillus bulgaricus. Lactobacillus plantarum ATCC 14431 is one of the very few LAB strains able to degrade H2O2 through the action of a nonheme, manganese-dependent catalase (hereafter called MnKat). The MnKat gene was expressed in three catalase-deficient LAB species: L. bulgaricus ATCC 11842, Lactobacillus casei BL23, and Lactococcus lactis MG1363. While the protein could be detected in all heterologous hosts, enzyme activity was observed only in L. casei. This is probably due to the differences in the Mn contents of the cells, which are reportedly similar in L. plantarum and L. casei but at least 10- and 100-fold lower in Lactococcus lactis and L. bulgaricus, respectively. The expression of the MnKat gene in L. casei conferred enhanced oxidative stress resistance, as measured by an increase in the survival rate after exposure to H2O2, and improved long-term survival in aerated cultures. In mixtures of L. casei producing MnKat and L. bulgaricus, L. casei can eliminate H2O2 from the culture medium, thereby protecting both L. casei and L. bulgaricus from its deleterious effects.

A novel mucosal vaccine based on live Lactococci expressing E7 antigen and IL-12 induces systemic and mucosal immune responses and protects mice against human papillomavirus type 16-induced tumors.

Current strategies to prevent or treat human papillomavirus type 16 (HPV-16) infection are promising, but remain costly. More economical but efficient vaccines are thus needed. In this study, we evaluated the protective effects of mucosally coadministered live Lactococcus lactis strains expressing cell wall-anchored E7 Ag and a secreted form of IL-12 to treat HPV-16-induced tumors in a murine model. When challenged with lethal levels of tumor cell line TC-1 expressing E7, immunized mice showed full prevention of TC-1-induced tumors, even after a second challenge, suggesting that this prophylactic immunization can provide long-lasting immunity. Therapeutic immunization with L. lactis recombinant strains, i.e., 7 days after TC-1 injection, induced regression of palpable tumors in treated mice. The antitumor effects of vaccination occurred through a CTL response, which is CD4+ and CD8+ dependent. Furthermore, immunized mice developed an E7-specific mucosal immune response. These preclinical results suggest the feasibility of the low-cost mucosal vaccination and/or immunotherapy strategies against HPV-related cervical cancer in humans.

Lactococcus lactis SpOx spontaneous mutants: a family of oxidative-stress-resistant dairy strains.

Numerous industrial bacteria generate hydrogen peroxide (H(2)O(2)), which may inhibit the growth of other bacteria in mixed ecosystems. We isolated spontaneous oxidative-stress-resistant (SpOx) Lactococcus lactis mutants by using a natural selection method with milk-adapted strains on dairy culture medium containing H(2)O(2). Three SpOx mutants displayed greater H(2)O(2) resistance. One of them, SpOx3, demonstrated better behavior in different oxidative-stress situations: (i) higher long-term survival upon aeration in LM17 and milk and (ii) the ability to grow with H(2)O(2)-producing Lactobacillus delbrueckii subsp. delbrueckii strains. Furthermore, the transit kinetics of the SpOx3 mutant in the digestive tract of a human flora-associated mouse model was not affected.

Oxidative stress in Lactococcus lactis.

Lactococcus lactis, the most extensively characterized lactic acid bacterium, is a mesophilic- and microaerophilic-fermenting microorganism widely used for the production of fermented food products. During industrial processes, L. lactis is often exposed to multiple environmental stresses (low and high temperature, low pH, high osmotic pressure, nutrient starvation and oxidation) that can cause loss or reduction of bacterial viability, reproducibility, as well as organoleptic and/or fermentative qualities. Among these stress factors, oxidation can be considered one of the most deleterious to the cell, causing cellular damage at both molecular and metabolic levels. During the last two decades, considerable efforts have been made to improve our knowledge of oxidative stress in L. lactis. Many genes involved with both oxidative stress resistance and control mechanisms have been identified; functionally they seem to overlap. The finding of new genes, and a better understanding of the molecular mechanisms of stress resistance in L. lactis and other lactic acid bacterium, will lead to the construction and isolation of stress-resistant strains. Such strains could be exploited for both traditional and probiotic uses.

Optimization of headspace solid-phase Microextraction (SPME) for the odor analysis of surface-ripened cheese.

Fifty volatile compounds of surface smear-ripened cheese were detected and identified using headspace solid-phase microextraction (HS-SPME) and vacuum distillation coupled to gas chromatography-mass spectrometry. Changes in the headspace of aroma compounds were monitored over the whole packaging period (47 days) using the HS-SPME method. Initially, the concentration of methanethiol increased before reaching a plateau. This evolution could be linked to the growth of Brevibacterium linens. During the shelf life of cheese, levels of acetic acid and 3-methylbutanoic acid remained constant, whereas butane-2,3-dione, 3-hydroxybutan-2-one, and hydroxypropan-2-one levels gradually declined and acetone and 3-methylbutanol levels dropped sharply to a plateau. Changes in odor could be related to changes of the rind, which behaved as a barrier, strongly influencing the distribution of volatile compounds in the headspace. Using a gas chromatography-olfactometry technique without separation, it was shown that the SPME extract was representative of the cheese odor.