Analysis of different European hazelnut (Corylus avellana L.) cultivars: authentication, phenotypic features, and phenolic profiles.

Hazelnuts exhibit functional properties due to their content in fatty acids and phenolic compounds that could positively affect human health. The food industry requires precise traits for morphological, chemical, and physical kernel features so that some cultivars could be more suitable for specific industrial processing. In this study, agronomical and morphological features of 29 hazelnut cultivars were evaluated and a detailed structural characterization of kernel polyphenols was performed, confirming the presence of protocatechuic acid, flavan-3-ols such as catechin, procyanidin B2, six procyanidin oligomers, flavonols, and one dihydrochalcone in all the analyzed cultivars. In addition, an innovative methodology based on the MALDI-TOF mass spectrometric analysis of peptide/protein components extracted from kernels was developed for the authentication of the most valuable cultivars. The proposed method is rapid, simple, and reliable and holds the potential to be applied in quality control processes. These results could be useful in hazelnut cultivar evaluation and choice for growers, breeders, and food industry.

The E1 beta-subunit of pyruvate dehydrogenase is surface-expressed in Lactobacillus plantarum and binds fibronectin.

Lactobacillus plantarum is among the species with a probiotic activity. Adhesion of probiotic bacteria to host tissues is an important principle for strain selection, because it represents a crucial step in the colonization process of either pathogens or commensals. Most bacterial adhesins are proteins, and a major target for them is fibronectin, an extracellular matrix glycoprotein. In this study we demonstrate that PDHB, a component of the pyruvate dehydrogenase complex, is a factor contributing to fibronectin-binding in L. plantarum LM3. By means of fibronectin overlay immunoblotting assay, we identified a L. plantarum LM3 surface protein with apparent molecular mass of 35 kDa. Mass spectrometric analysis shows that this protein is the pyruvate dehydrogenase E1 beta-subunit (PDHB). The corresponding pdhB gene is located in a 4-gene cluster encoding pyruvate dehydrogenase. In LM3-B1, carrying a null mutation in pdhB, the 35 kDa adhesin was not anymore detectable by immunoblotting assay. Nevertheless, the pdhB null mutation did not abolish pdhA, pdhC, and pdhD transcription in LM3-B1. By adhesion assays, we show that LM3-B1 cells bind to immobilized fibronectin less efficiently than wild type cells. Moreover, we show that pdhB expression is negatively regulated by the CcpA protein and is induced by bile.

Biochemical modifications of gliadins induced by microbial transglutaminase on wheat flour.

BACKGROUND: Celiac disease (CD) is an immune-mediated disorder caused by the ingestion of wheat gluten. A lifelong, gluten-free diet is required to normalize the intestinal mucosa. We previously found that transamidation by microbial transglutaminase (mTGase) suppressed the gliadin-specific immune response in intestinal T-cell lines from CD patients and in models of gluten sensitivity. METHODS: SDS-PAGE, Western blot, ELISA, tissue transglutaminase (tTGase) assay and nano-HPLC-ESI-MS/MS experiments were used to analyze prolamins isolated from treated wheat flour. RESULTS: Gliadin and glutenin yields decreased to 7.6±0.5% and 7.5±0.3%, respectively, after a two-step transamidation reaction that produced a water-soluble protein fraction (spf). SDS-PAGE, Western blot and ELISA analyses confirmed the loss of immune cross-reactivity with anti-native gliadin antibodies in residual transamidated gliadins (K-gliadins) and spf as well as the occurrence of neo-epitopes. Nano-HPLC-ESI-MS/MS experiments identified some native and transamidated forms of celiacogenic peptides including p31-49 and confirmed that mTGase had similar stereo-specificity of tTGase. Those peptides resulted to be 100% and 57% modified in spf and K-gliadins, respectively. In particular, following transamidation p31-49 lost its ability to increase tTGase activity in Caco-2 cells. Finally, bread manufactured with transamidated flour had only minor changes in baking characteristics. CONCLUSIONS: The two-step transamidation reaction modified the analyzed gliadin peptides, which are known to trigger CD, without influencing main technological properties. GENERAL SIGNIFICANCE: Our data shed further light on a detoxification strategy alternative to the gluten free diet and may have important implications for the management of CD patients.

Effect of inactivation of ccpA and aerobic growth in Lactobacillus plantarum: A proteomic perspective.

Lactobacillus plantarum is a facultative heterofermentative lactic acid bacterium widely used in the production of most fermented food due to its ability to thrive in several environmental niches, including the human gut. In order to cope with different growth conditions, it has developed complex molecular response mechanisms, characterized by the induction of a large set of proteins mainly regulated by HrcA and CtsR repressors as well as by global regulators such as carbon catabolite control protein A (CcpA). In this study, the role of CcpA in the regulation of growth under anaerobiosis and aerobiosis, and the adaptation to aeration in L. plantarum WCFS1 were comprehensively investigated by differential proteomics. The inactivation of ccpA, in both growth conditions, significantly changed the expression level of 76 proteins, mainly associated with carbohydrate and energy metabolism, membrane transport, nucleotide metabolism, protein biosynthesis and folding. The role of CcpA as pleiotropic regulator was particularly evident at the shift from homolactic fermentation to mixed fermentation. Proteomic results also indicated that the mutant strain was more responsive to aerobic growth condition.

Expression of the Lactobacillus plantarum malE gene is regulated by CcpA and a MalR-like protein.

Lactobacillus plantarum is commonly used in the food industry as a starter in various fermentations, especially in vegetable fermentations, in which starch is a common substrate. This polysaccharide, which is obtained from potatoes or corn and is hydrolysed mainly to maltose and glucose by acids or enzymes, is commercially used for the production of lactate by lactic acid fermentation. In this study, we describe the regulation of malE gene expression in L. plantarum. This gene, located in a 7-gene cluster, probably organized in an operon, encodes a putative maltose/maltodextrin-binding protein. We studied the expression of malE in L. plantarum LM3 (wild type) and in LM3-2 (ccpA1), which carries a null mutation in the ccpA gene, encoding the catabolite control protein A (CcpA). In the presence of glucose, expression of the MalE protein was higher in the mutant strain as compared to that in the wild-type strain. Transcription of the malE gene was induced by maltose and regulated by a CcpA-mediated carbon catabolite repression. Further, we isolated strains carrying mutations in 2 genes, lp_0172 and lp_0173, whose deduced amino acid sequences share significant identity with MalR, a regulator of the maltose operon in several gram-positive bacteria. A double mutant exhibited glucose-insensitive malE transcription, while absence of the functional Lp_0172 open reading frame had no effect on malE expression.

Proteomics for the elucidation of cold adaptation mechanisms in Listeria monocytogenes.

Listeria monocytogenes, one of the major food-related pathogens, is the aetiological agent of listeriosis, a potentially life-threatening illness. It is able to survive in hostile environments and stress conditions such as those encountered in food-processing technologies (high salt concentration, wide range of pH and temperature, low water availability) and it also thrives at temperatures ranging from -0.4 to 45 °C. In this study, expression proteomics was applied to gain insight into key cellular events that allow L. monocytogenes to survive and multiply even at refrigeration temperatures. Interestingly, we observed that the adaptation processes mainly affect biochemical pathways related to protein synthesis and folding, nutrient uptake and oxidative stress. Furthermore, proteins implicated in metabolic pathways for energy production, such as glycolysis and Pta-AckA pathway, were present to a higher level in the cells grown at 4 °C. This suggests that, on the whole, cells exhibit an enhanced demand for energy to sustain cold growth. Proteomics may represent a key tool in deciphering specific mechanisms underlying cold adaptation response and, more widely, cell machinery.

Proteomic investigation of the aggregation phenomenon in Lactobacillus crispatus.

Aggregation process affects the ability of Lactobacillus crispatus, a probiotic, to survive into the gastro-intestinal environment and to adhere to the intestinal mucosa. To elucidate mechanisms underlying this process, a comparative proteomic study was carried out on a wild type strain M247 and its spontaneous isogenic mutant Mu5, which had lost the aggregative phenotype. Results highlighted an overall lower amount of enzymes involved in carbohydrate transport and metabolism in strain M247 compared to strain Mu5, suggesting a reduction in the general growth rate, probably caused by nutrient limitation in cell aggregates, coherently with the phenotypic traits of the strains. Moreover, the up-regulation of a putative elongation factor Tu in the wild type M247 strain could suggest a role of this particular protein in the adhesion mechanism of L. crispatus.

Transamidation of wheat flour inhibits the response to gliadin of intestinal T cells in celiac disease.

BACKGROUND & AIMS: Celiac disease is characterized by activation of HLA-DQ2/DQ8-restricted intestinal gluten-specific CD4(+) T cells. In particular, gluten becomes a better T-cell antigen following deamidation catalyzed by tissue transglutaminase. To date, the only available therapy is represented by adherence to a gluten-free diet. Here, we examined a new enzyme strategy to preventively abolish gluten activity. METHODS: Enzyme modifications of the immunodominant alpha-gliadin peptide p56-68 were analyzed by mass spectrometry, and peptide binding to HLA-DQ2 was simulated by modeling studies. Wheat flour was treated with microbial transglutaminase and lysine methyl ester; gliadin was subsequently extracted, digested, and deamidated. Gliadin-specific intestinal T-cell lines (iTCLs) were generated from biopsy specimens from 12 adult patients with celiac disease and challenged in vitro with different antigen preparations. RESULTS: Tissue transglutaminase-mediated transamidation with lysine or lysine methyl ester of p56-68 or gliadin in alkaline conditions inhibited the interferon gamma expression in iTCLs; also, binding to DQ2 was reduced but not abolished, as suggested by in silico analysis. Lysine methyl ester was particularly effective in abrogating the activity of gliadin. Notably, a block in the response was observed when iTCLs were challenged with gliadin extracted from flour pretreated with microbial transglutaminase and lysine methyl ester. CONCLUSIONS: Transamidation of wheat flour with a food-grade enzyme and an appropriate amine donor can be used to block the T cell-mediated gliadin activity. Considering the crucial role of adaptive immunity in celiac disease, our findings highlight the potential of the proposed treatment to prevent cereal toxicity.

CcpA affects expression of the groESL and dnaK operons in Lactobacillus plantarum.

BACKGROUND: Lactic acid bacteria (LAB) are widely used in food industry and their growth performance is important for the quality of the fermented product. During industrial processes changes in temperature may represent an environmental stress to be overcome by starters and non-starters LAB. Studies on adaptation to heat shock have shown the involvement of the chaperon system-proteins in various gram-positive bacteria. The corresponding operons, namely the dnaK and groESL operons, are controlled by a negative mechanism involving the HrcA repressor protein binding to the cis acting element CIRCE. RESULTS: We studied adaptation to heat shock in the lactic acid bacterium Lactobacillus plantarum. The LM3-2 strain, carrying a null mutation in the ccpA gene, encoding the catabolite control protein A (CcpA), showed a lower percent of survival to high temperature with respect to the LM3 wild type strain. Among proteins differentially expressed in the two strains, the GroES chaperon was more abundant in the wild type strain compared to the mutant strain under standard growth conditions. Transcriptional studies showed that class I heat shock operons were differentially expressed upon heat shock in both strains. Indeed, the dnaK and groESL operons were induced about two times more in the LM3 strain compared to the LM3-2 strain. Analysis of the regulatory region of the two operons showed the presence of cre sequences, putative binding sites for the CcpA protein. CONCLUSION: The L. plantarum dnaK and groESL operons are characterized by the presence of the cis acting sequence CIRCE in the promoter region, suggesting a negative regulation by the HrcA/CIRCE system, which is a common type of control among the class I heat shock operons of gram-positive bacteria. We found an additional system of regulation, based on a positive control exerted by the CcpA protein, which would interact with cre sequences present in the regulatory region of the dnaK and groESL operons. The absence of the CcpA protein results in a lower induction of the chaperon coding operons, with a consequent lower percent of survival of the LM3-2 mutant strain population with respect to the wild type when challenged with a heat insult.

Identification of immunodominant epitopes of alpha-gliadin in HLA-DQ8 transgenic mice following oral immunization.

Celiac disease, triggered by wheat gliadin and related prolamins from barley and rye, is characterized by a strong association with HLA-DQ2 and HLA-DQ8 genes. Gliadin is a mixture of many proteins that makes difficult the identification of major immunodominant epitopes. To address this issue, we expressed in Escherichia coli a recombinant alpha-gliadin (r-alpha-gliadin) showing the most conserved sequence among the fraction of alpha-gliadins. HLA-DQ8 mice, on a gluten-free diet, were intragastrically immunized with a chymotryptic digest of r-alpha-gliadin along with cholera toxin as adjuvant. Spleen and mesenteric lymph node T cell responses were analyzed for in vitro proliferative assay using a panel of synthetic peptides encompassing the entire sequence of r-alpha-gliadin. Two immunodominant epitopes corresponding to peptide p13 (aa 120-139) and p23 (aa 220-239) were identified. The response was restricted to DQ and mediated by CD4+ T cells. In vitro tissue transglutaminase deamidation of both peptides did not increase the response; furthermore, tissue transglutaminase catalyzed extensive deamidation in vitro along the entire r-alpha-gliadin molecule, but failed to elicit new immunogenic determinants. Surprisingly, the analysis of the cytokine profile showed that both deamidated and native peptides induced preferentially IFN-gamma secretion, despite the use of cholera toxin, a mucosal adjuvant that normally induces a Th2 response to bystander Ags. Taken together, these data suggest that, in this model of gluten hypersensitivity, deamidation is not a prerequisite for the initiation of gluten responses.

Intranasal administration of a recombinant alpha-gliadin down-regulates the immune response to wheat gliadin in DQ8 transgenic mice.

The mucosal lesion in coeliac disease (CD) is an immune-mediated injury triggered by gliadin and associated with HLA-DQ2 and HLA-DQ8. In view of this, an approach that re-induces tolerance to this antigen should be considered as possible alternative to a strict gluten-free diet in treating CD. However, T-cell activation to multiple antigens, as a consequence of the chemical complexity shown by the antigen gliadin, could hamper the efforts to identify single component(s) useful for tolerance induction. To address this issue, a recombinant alpha-gliadin was tested in tolerance experiments in HLA-DQ8 transgenic mice. As tissue transglutaminase (tTG) treatment of gliadin, previously reported to enhance its stimulatory activity in CD, did not increase its immunogenicity when parenterally administered in these mice, untreated gliadin was used as immunogen. A decrease in systemic T cell responses to the recombinant alpha-gliadin was found after nasal administration of antigen, reflected by lymphocytes proliferation assay. Interestingly, while the immunisation protocol induced transcription of both Th1 (IFN-gamma) and Th2 (IL-4 and IL-10) cytokines, the tolerisation protocol down-regulated significantly only the IFN-gamma mRNA expression. More important, the recombinant alpha-gliadin induced a similar down-regulatory effect in mice immunised with a commercial preparation of wheat gliadin, that is a mixture of many different gliadin components. As the Th1 phenotype and the HLA-DQ8 molecule play a role in the pathogenesis of CD, our data underlined the potential usefulness of this recombinant protein for the immunomodulation of this disease.