1,721,012 research outputs found
A mass spectrometry approach to dairy science
No full textBovine milk is a source of an array of (un)known (bioactive) compounds from a variety of molecular and chemical classes. Because of a such complex food matrix, accurate and sensitive analytical approaches are needed to identify newly formed molecules (e.g. bioactive milk non-nutrients or xenobiotics), to recognize chemical and enzymatic modifications which known milk components undergo upon processing, storage and in vitro digestion. In this perspective, tailored sample preparation followed by liquid chromatography/high resolution mass spectrometry (LC/HR-MS) represents a powerful analytical tool to solve some scientific issues in dairy sector. In this presentation, some of the current applications of MS in dairy science will be discussed with special regard to characterization of novel functional/(bio)active milk compounds [1], reliable identification of dairy markers (e.g. studying post-translational modifications [2], proteolytic phenomena [3–6], identification of bioactive non-nutrients [7–9]), traceability and confirmation of authenticity [10] of dairy products.
[1] M. Stuknytė, S. Cattaneo, F. Masotti, I. De Noni, Food Chemistry, 168, 27–33 (2015)
[2] S. Cattaneo, F. Masotti, L. Pellegrino, Journal of Agricultural and Food Chemistry, 57, 10689–10694 (2009)
[3] S. Cattaneo, M. Stuknytė, L. Pellegrino, I. De Noni, Food Chemistry, 155, 179–185 (2014)
[4] F. Masotti, I. De Noni, S. Cattaneo, M. Brasca, V. Rosi, M. Stuknyte, S. Morandi, L. Pellegrino, International Dairy Journal, 33, 90–96 (2013)
[5] F. Masotti, J. A. Hogenboom, V. Rosi, I. De Noni, L. Pellegrino, International Dairy Journal, 20, 352–359 (2010)
[6] S. Cattaneo, J. A. Hogenboom, F. Masotti, V. Rosi, L. Pellegrino, P. Resmini, Dairy Science and Technology, 88, 595–605 (2008)
[7] V. Taverniti, M. Stuknyte, M. Minuzzo, S. Arioli, I. De Noni, C. Scabiosi, Z. Martinez Cordova, I. Junttila, S. Hämäläinen, H. Turpeinen, D. Mora, M. Karp, M. Pesu, S. Guglielmetti, Applied and Environmental Microbiology, 79, 1221–1231 (2013)
[8] I. De Noni, S. Cattaneo, Food Chemistry, 119, 560–566 (2010)
[9] I. De Noni, Food Chemistry, 110, 897–903 (2008)
[10] R. Russo, V. Severino, A. Mendez, J. Lliberia, A. Parente, A. Chambery, Journal of Mass Spectrometry, 47, 1407–1414 (2012
Immunomodulatory activity of bovine casein hydrolysates produced after digestion with proteinases of lactic acid bacteria
No full textAttivitá immunomodulatoria di idrolizzati di caseina bovina ottenuti mediante digestione con proteinasi di batteri lattici. La prima parte di questa tesi di dottorato ha riguardato lo studio della attività immunomodulatoria di idrolizzati di caseina bovina (CHs), prodotti mediante digestione con proteinasi di batteri lattici. La ricerca ha evidenziato, per gli ultrafiltrati a 3 kDa dei CHs prodotti dopo digestione con proteinasi di Lactobacillus acidophilus ATCC 4356 e Lactococcus lactis subsp. lactis GR5, una significativa attività immunomodulatoria per riduzione dell’attività basale in vitro del NF-kB in cellule Caco-2. Nel caso di L. helveticus MIMLh5, l'attività immunomodulatoria è invece probabilmente attribuibile a componenti batterici presenti nel CH non ultrafiltrato.This report presents the results of the first PhD thesis topic, which dealt with the investigation of immunomodulatory activity of bovine casein hydrolysates (CHs), produced after digestion with proteinases of lactic acid bacteria. The study demonstrated that the 3 kDa-ultrafiltered CHs, produced after digestion with proteinases of Lactobacillus acidophilus ATCC 4356 and Lactococcus lactis subsp. lactis GR5, significantly decreased in vitro the basal NF-B activity in intestinal epithelial-like Caco-2 cells, demonstrating immunomodulatory activity. In the case of L. helveticus MIMLh5, the immunomodulatory activity was likely due to the bacterial cell components present in the whole CH
Lactobacillus helveticus S-layer protein specific binders from antibody libraries
No full textMany strains of Lactobacillus helveticus have been characterised as probiotics. These strains were reported to exert health benefits such as protection against infection, e.g. by modulating the immune system. L. helveticus MIMLh5, isolated from Grana Padano cheese natural whey starter, was proposed as a potential probiotic bacterium for the human pharynx with promising antagonistic and immunomodulatory properties (Guglielmetti et al., 2010). The surface layer (S-layer) protein was identified as being involved in the immunomodulatory properties of L. helveticus strains M92 (Beganović et al., 2011), MIMLh5 (Guglielmetti and Taverniti, unpublished data).
Antibodies constitute a powerful tool to study protein function, protein localisation and protein-protein interactions, as well as for diagnostic and therapeutic purposes. Single chain variable fragment antibodies (scFvs) have considerable potential for the immunological detection of small molecules, as well as proteins, in localisation of bacterial surface structures (Bradbury et al., 2011). In this study, a mix of two large human synthetic phage displayed libraries, protein-directed (Brockmann et al., 2011) and hapten-directed, was used to select scFvs against L. helveticus MIMLh5 S-layer protein. After three rounds of panning, four monoclonal scFvs capable of binding with the L. helveticus MIMLh5 S-layer protein and one capable of binding also with the S-layer protein of L. helveticus ATCC 15009T, which is different only in five amino acids, were obtained. All five identified novel anti-S-layer scFvs were expressed in Escherichia coli XL1-Blue as alkaline phosphatase fusions (Krebber et al., 1997). ScFv-PolyH4 was used to develop an assay, based on Western blot analysis, for detection of the S-layer protein in Grana Padano cheese ripened for 2-5 months. These results showed promising applications of the scFv-PolyH4 for detecting immunologically important S-layer protein of L. helveticus in this cheese and in other food matrices and underlined the high value of the utilised phage display libraries for obtaining the extremely specific scFvs.
References:
Beganović, J., Frece, J., Kos, B., Leboš Pavunc, A., Habjanič, K., Šušković, J. (2011) Functionality of the S-layer protein from the probiotic strain Lactobacillus helveticus M92. Antonie Van Leeuwenhoek. 100: 43-53.
Bradbury, A. R. M., Sidhu, S., Dübel, S., McCafferty, J. (2011) Beyond natural antibodies: the power of in vitro display technologies. Nature Biotechnol. 29:245-254.
Brockmann, E. C., Akter, S., Savukoski, T., Huovinen, T., Lehmusvuori, A., Leivo, J., Saavalainen, O., Azhayev, A., Lövgren, T., Hellman, J., Lamminmäki, U. (2011) Synthetic single-framework antibody library integrated with rapid affinity maturation by VL shuffling. Protein Eng Des Sel. 24: 691-700.
Guglielmetti, S., Taverniti, V., Minuzzo, M., Arioli, S., Zanoni, I., Stuknyte, M., Granucci, F., Karp, M., Mora, D. (2010) A dairy bacterium displays in vitro probiotic properties for the pharyngeal mucosa by antagonizing group A streptococci and modulating the immune response. Infect Immun. 78: 4734-4743.
Krebber, A., Bornhauser, S., Burmester, J., Honegger, A., Willuda, J., Bosshard, H. R., Plückthun, A. (1997) Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. J Immunol Methods. 201: 35-55
A mass spectrometry approach to dairy science
No full textBovine milk is a source of an array of (un)known (bioactive) compounds from a variety of molecular and chemical classes. Because of a such complex food matrix, accurate and sensitive analytical approaches are needed to identify newly formed molecules (e.g. bioactive milk non-nutrients or xenobiotics), and to recognize chemical and enzymatic modifications which known milk components undergo upon processing and in vitro digestion. In this perspective, tailored sample preparation followed by LC/HR-MS represents a powerful analytical tool to solve some scientific issues in dairy sector. In this presentation, some of the current applications of MS in dairy science will be discussed with special regard to characterization of novel functional/(bio)active milk compounds, reliable identification of dairy markers (e.g. studying post-translational modifications, proteolytic phenomena, identification of bioactive non-nutrients), traceability and confirmation of authenticity of dairy products
Identification of beta-casomorphins 3 to 7 in cheeses and in their in vitro gastrointestinal digestates
No full textThe occurrence of the bovine beta-casein-derived casomorphins YPF (f60-62, BCM3), YPFP (f60- 63, BCM4), YPFPG (f60-64, BCM5), YPFPGP (f60-65, BCM6) and YPFPGPI (f60-66, BCM7) was investigated in different cheeses by Ultra Performance Liquid Chromatography/High-Resolution Mass Spectrometry (UPLC/HR-MS). A total BCMs content was revealed in the range 0 e 11.74 mg/kg. The amount of BCM7 varied from 0 to 7.63 mg/kg. Gorgonzola cheese presented the highest BCM7 quantity, and it was the only sample to contain all the searched BCMs. Cheddar, Gorgonzola, Maasdam and Grana Padano were assessed for BCMs content following a recently proposed standardized in vitro static gastrointestinal digestion method (Minekus et al., 2014). After the gastric step, BCM7 was found only in Cheddar and Gorgonzola (0.25 mg/kg and 2.87 mg/kg, respectively), along with BCM4. Only the latter peptide was detected in Grana Padano, whereas none of the studied BCMs were identified in the digested Maasdam. At the end of intestinal phase, all samples contained BCM7 (0.12-1.26 mg/kg); the BCM4 was released
only in digested Gorgonzola. Based on these results, in vitro digestion of a serving size of the studied cheeses does not potentially release a BCM7 amount compatible with an in vivo opioid activity
BIOACTIVES IN MILK-DERIVED PRODUCTS: BACTERIAL PRODUCTION OF IMMUNOMODULATORY CASEIN HYDROLYSATES AND TOOLS FOR IDENTIFICATION OF IMMUNOGENIC BACTERIAL PROTEIN
Full text linkDuring hydrolysis of bovine milk caseins, cell envelope-associated proteinases (CEPs) of lactic acid bacteria (LAB) may be able to produce hydrolysates that possess in vitro immunomodulatory activity. We studied the proteolytic activity against bovine milk caseins exerted by specific strains of LAB selected on the basis of a preliminary genomic screening for the presence of CEPs. The tested strains demonstrated diverse hydrolytic ability against different casein fractions, alphaS1- and beta-casein in particular. The 3 kDa-ultrafiltered casein hydrolysates (CHs), produced after digestion with proteinases of Lactobacillus acidophilus ATCC 4356 and Lactococcus lactis subsp. lactis GR5 demonstrated immunomodulatory activity in vitro by significantly decreasing the basal NF-kB activity in recombinant Caco-2 cell layers. Both the strains digested beta-casein mostly. However, in the case of Lactobacillus helveticus MIMLh5, the investigation proved that the immunomodulatory activity of obtained CH was comparable to the one exerted by bacteria themselves (in the absence of caseinate) and therefore, was not due to the presence of casein-derived peptides. Indeed, the surface layer (S-layer) protein of L. helveticus MIMLh5 was identified as a molecule responsible for the immunomodulation. Overall, these results emphasize the importance of this bacterial cell-derived protein while evaluating the immunological activity of CHs, and it underlines the necessity to remove such molecule in order to properly assess the bioactivity of CHs deriving from the proteolytic activity of LAB.
The second part of the research was focussed on the selection of S-layer-specific single-chain variable fragment antibodies (scFvs), a new powerful tool for the study of the S-layer of L. helveticus MIMLh5, the immunologically active protein, and for its identification in milk-derived products containing L. helveticus as a starter or non starter LAB. In this study, a mix of two human synthetic phage displayed libraries (protein-directed and hapten-directed) was used to select scFvs against L. helveticus MIMLh5 S-layer protein. After three rounds of panning, four monoclonal scFv binders capable of binding to the L. helveticus MIMLh5 S-layer protein and one capable of binding not only to the mentioned protein, but also to the S-layer protein of L. helveticus ATCC 15009, which is different only in five amino acids, were obtained. All five identified novel anti-S-layer scFvs were expressed in Escherichia coli XL1-Blue, and their basic characterisation was performed. Anti-S-layer-scFv PolyH4 was used to develop an assay, based on Western blot analysis, for detection of the S-layer protein in Grana Padano samples. These results showed promising applications of the method for the detection of the S-layer protein in food matrices
Development of bioprocesses for the production of food protein hydrolysates containing bioactive peptides
No full textSviluppo di bioprocessi per l’ottenimento di idrolizzati di proteine alimentari contenenti peptidi ad attività biologica.
Questo progetto di tesi di dottorato intende mettere a punto bioprocessi basati sullo sfruttamento dell'attività delle proteinasi di parete (Cell Envelope-associated Proteinases, CEP) di batteri food-grade, LAB in particolare, per la preparazione di idrolizzati di proteine alimentari (FPHs) contenenti peptidi ad attività biologica. L'obiettivo ultimo è la definizione di bioprocessi completamente food-grade, trasferibili a livello industriale e finalizzati alla preparazione di FPHs utilizzabili come ingredienti in formulazioni alimentari.The PhD thesis research project is aimed at development of bioprocesses for the production of food protein hydrolysates (FPHs) containing peptides with specific biological activities by exploiting the activity of Cell Envelope-associated Proteinases (CEP) of food-grade bacteria, in particular of lactic acid bacteria (LAB). The final object is the definition of completely food-grade bioprocesses, transferable to the industrial level and addressed to preparation of FPHs, which could be used as ingredients in functional foods
Occurrence and fate of ACE-inhibitor peptides in cheeses and in their digestates following in vitro static gastrointestinal digestion
No full textThe occurrence of the casein-derived angiotensin converting enzyme-inhibitor (ACE-I) peptides VPP, IPP, RYLGY, RYLG, AYFYPEL, AYFYPE, LHLPLP and HLPLP were investigated in 12 different cheese samples by Ultra Performance Liquid Chromatography/High-Resolution Mass Spectrometry. The total amount of ACE-I peptides was in the range 0.87–331 mg kg 1 . VPP and IPP largely prevailed in almost all cheeses.
Following in vitro static gastrointestinal digestion of Cheddar, Gorgonzola, Maasdam and Grana Padano cheeses, type and amount of ACE-I peptides changed, and only VPP, IPP, HLPLP and LHLPLP were detected in the intestinal digestates. The results evidenced that the degree of proteolysis itself cannot be regarded as a promoting or hindering factor for ACE-I peptide release during cheese digestion. Moreover, the data indicated that the ACE-I potential of cheeses cannot be inferred based on the type and amount of ACE-I peptides present in undigested samples
Purification and partial characterization of a novel bacteriocin produced by a thermophilic endospore-forming strain Geobacillus stearothermophilus 32A
Full text linkThe murB gene encodes UDP-N-acetylenolpyruvylglucosamine reductase and functions in bacterial peptidoglycan biosynthesis. A plasmid carrying the murB gene restored the temperature-sensitive growth of six Staphylococcus aureus mutants, in which peptidoglycan biosynthesis stopped at a restrictive temperature. Specific activity of UDP-N-acetylenolpyruvylglucosamine reductase in extracts from the mutants was lower than that from wild-type cells. Nucleotide sequence determination revealed that each mutant had a single amino acid substitution in the murB gene and five of six mutations were located within domain 3, where the proposed substrate binding site is located. These results suggest that the murB gene is essential for growth of S. aureus and that domain 3 is important for the MurB activity
Development of bioprocesses for the production of food protein hydrolysates containing bioactive peptides
No full textSviluppo di bioprocessi per l’ottenimento di idrolizzati di proteine alimentari contenenti peptidi ad attività biologica.
Sono descritte le prime due attività previste nel progetto di tesi di dottorato. Inizialmente, è stata effettuata la selezione di alcuni ceppi di batteri lattici valutando la presenza e il tipo di proteinasi di parete delle cellule batteriche (CEPs) e testando l'attività proteolitica dei ceppi proteasi-positivi sulle caseine del latte. Il profilo peptidico degli idrolizzati di caseina (CHs) è stato studiato mediante cromatografia liquida ad alta prestazione (HPLC). In seguito, l’attività immunomodulante degli stessi CHs è stata valutata in vitro usando un sistema reporter basato sull’attività dell’enzima luciferasi. Questo sistema è costituito dal plasmide pNiFty2-Luc, inducibile dal fattore nucleare kappa B (NF-κB), introdotto in cellule umane di adenocarcinoma del colon Caco-2.The first two activities of the PhD thesis project are described. Firstly, we performed the selection of lactic acid bacteria by screening bacterial cell envelope-associated proteinases (CEPs) at the genetic level as well as by testing the proteolytic activity on milk caseins. Moreover, fractionation of casein hydrolysates (CHs) by means of high-performance liquid chromatography (HPLC) was performed. Secondly, we developed a protocol for the in vitro biological analysis of CHs, addressed to determine their immunomodulatory activity by using a luciferase reporter gene system. This system is based on a nuclear factor kappa B (NF-κB)-inducible reporter plasmid, pNiFty2-Luc, in human colonic adenocarcinoma cells Caco-2
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