Evaluation of microbial adjuncts and their effect on the ripening of cheddar cheese

A bacteriocin-producing strain of Lactobacillus paracasei DPC 4715 was used as an adjunct culture in Cheddar cheese in order to control the growth of “wild” nonstarter lactic acid bacteria. No suppression of growth of the indicator strain was observed in the experimental cheese. The bacteriocin produced by Lactobacillus paracasei DPC 4715 was sensitive to chymosin and cathepsin D and it may have been cleaved by the rennet used for the cheese manufactured or by indigenous milk proteases. A series of studies were performed using various microbial adjuncts to influence cheese ripening. Microbacterium casei DPC 5281, Corynebacterium casei DPC 5293 and Corynebacterium variabile DPC 5305 were added to the cheesemilk at level of 109 cfu/ml resulting in a final concentration of 108 cfu/g in Cheddar cheese. The strains significantly increased the level of pH 4.6-soluble nitrogen, total free amino acids after 60 and 180 d of ripening and some individual free amino acids after 180 d. Yarrowia lipolytica DPC 6266, Yarrowia lipolytica DPC 6268 and Candida intermedia DPC 6271 were used to accelerate the ripening of Cheddar cheese. Strains were grown in YG broth to a final concentration of 107 cfu/ml, microfluidized, freeze-dried and added to the curd during salting at level of 2% w/w. The yeasts positively affected the primary, secondary proteolysis and lipolysis of cheeses and had aminopeptidase, dipeptidase, esterase and 5’ phosphodiestere activities that contributed to accelerate the ripening and improve the flavor of cheese. Hafia alvei was added to Cheddar cheesemilk at levels of 107 cfu/ml and 108 cfu/ml and its contribution during ripening was evaluated. The strain significantly increased the level of pH 4.6-soluble nitrogen, total free amino-acids, and some individual free amino-acids of Cheddar cheese, whereas no differences in the urea-polyacrylamide gel electrophoresis (urea-PAGE) electrophoretograms of the cheeses were detected. Hafia alvei also significantly increased the level of some biogenic amines. A low-fat Cheddar cheese was made with Bifidobacterium animalis subsp. lactis, strain BB-12® at level of 108 cfu/ml, as a probiotic adjunct culture and Hi-Maize® 260 (resistant high amylose maize starch) at level of 2% and 4% w/v, as a prebiotic fiber which also played the role of fat replacer. Bifidobacterium BB-12 decreased by 1 log cycle after 60 d of ripening and remained steady at level of ~107 cfu/g during ripening. The Young’s modulus also increased proportionally with increasing levels of Hi-maize. Hencky strain at fracture decreased over ripening and increased with increasing in fat replacer. A cheese based medium (CBM) was developed with the purpose of mimicking the cheese environment at an early ripening stage. The strains grown in CBM showed aminopeptidase activity against Gly-, Arg-, Pro- and Phe-p-nitroanalide, whereas, when grown in MRS they were active against all the substrates tested. Both Lb. danicus strains grown in MRS and in CBM had aminotransferase activity towards aromatic amino acids (Phe and Trp) and also branched-chain amino acids (Leu and Val). Esterase activity was expressed against p-nitrophenyl-acetate (C2), pnitrophenyl- butyrate (C4) and p-nitrophenyl-palmitate (C16) and was significantly higher in CBM than in MRS.

Characterisation of bacteriophage-host interactions in Lactococcus lactis

Lactococcus lactis is used extensively world-wide for the production of fermented dairy products. Bacteriophages (phages) infecting L. lactis can result in slow or incomplete fermentations, or may even cause total fermentation failure. Therefore, bacteriophages disrupting L. lactis fermentation are of economic concern. This thesis employed a multifaceted approach to investigate various molecular aspects of phage-host interaction in L. lactis. The genome sequence of an Irish dairy starter strain, the prophage-cured L. lactis subsp. cremoris UC509.9, was studied. The 2,250,427 bp circular chromosome represents the smallest among its sequenced lactococcal equivalents. The genome displays clear genetic adaptation to the dairy niche in the form of extensive reductive evolution. Gene prediction identified 2066 protein-encoding genes, including 104 which showed significant homology to transposase-specifying genes. Over 9 % of the identified genes appear to be inactivated through stop codons or frame shift mutations. Many pseudogenes were found in genes that are assigned to carbohydrate and amino acid transport and metabolism orthologous groups, reflecting L. lactis UC509.9’s adaptation to the lactose and casein-rich dairy environment. Sequence analysis of the eight plasmids of L. lactis revealed extensive adaptation to the dairy environment. Key industrial phenotypes were mapped and novel lactococcal plasmid-associated genes highlighted. In addition to chromosomally-encoded bacteriophage resistance systems, six functional such systems were identified, including two abortive infection systems, AbiB and AbiD1, explaining the observed phage resistance of L. lactis UC509.9 Molecular analysis suggests that the constitutive expression of AbiB is not lethal to cells, suggesting the protein is expressed in an un/inactivated form. Analysis of 936 species phage sk1-escape mutants of AbiB revealed that all such mutants harbour mutations in orf6, which encodes the major capsid protein. Results suggest that the major capsid protein is required for activation of the AbiB system, although this requires furrther investigations. Temporal transcriptomes of L. lactis UC509.9 undergoing lytic infection with either one of two distinct bacteriophages, Tuc2009 and c2, was determined and compared to the transcriptome of uninfected UC509.9 cells. Whole genome microarrays performed at various time-points post-infection demonstrated a rather modest impact on host transcription. Alterations in the UC509.9 transcriptome during lytic infection appear phage-specific, with a relatively small number of differentially transcribed genes shared between infection with either Tuc2009 or c2. Transcriptional profiles of both bacteriophages during lytic infection was shown to generally correlate with previous studies and allowed the confirmation of previously predicted promoter sequences. Bioinformatic analysis of genomic regions encoding the presumed cell wall polysaccharide (CW PS) biosynthesis gene cluster of several strains of L. lactis was performed. Results demonstrate the presence of three dominant genetic types of this gene cluster, termed type A, B and C. These regions were used for the development of a multiplex PCR to identify CW PS genotype of various lactococcal strains. Analysis of 936 species phage receptor binding protein phylogeny (RBP) and CW PS genotype revealed an apparent correlation between RBP phylogeny and CW PS type, thereby providing a partial explanation for the observed narrow host range of 936 phages. Further analysis of the genetic locus encompassing the presumed CW PS biosynthesis operon of eight strains identified as belonging to the CW PS C (geno)type, revealed the presence of a variable region among the examined strains. The obtained comparative analysis allowed for the identification of five subgroups of the C type, named C1 to C5. We purified an acidic polysaccharide from the cell wall of L. lactis 3107 (C2 subtype) and confirmed that it is structurally different from the CW PS of the C1 subtype L. lactis MG1363. Combinations of genes from the variable region of C2 subtype were amplified from L. lactis 3107 and introduced into a mutant of the C1 subtype L. lactis NZ9000 (a direct derivative of MG1363) deficient in CW PS biosynthesis. The resulting recombinant mutant synthesized a CW PS with a composition characteristic for that of the C2 subtype L. lactis 3107 and not the wildtype C1 L. lactis NZ9000. The recombinant mutant exhibited a changed phage resistance/sensitivity profile consistent with that of L. lactis 3107, which unambiguously demonstrated that L. lactis 3107 CW PS is the host cell surface receptor of two bacteriophages belonging to the P335 species as well as phages that are member of the 936 species. The research presented in this thesis has significantly advanced our understanding of L. lactis bacteriophage-host interactions in several ways. Firstly, the examination of plasmidencoded bacteriophage resistance systems has allowed inferences to be made regarding the mode of action of AbiB, thereby providing a platform for further elucidation of the molecular trigger of this system. Secondly, the phage infection transcriptome data presented, in addition to previous work, has made L. lactis a model organism in terms of transcriptomic studies of bacteriophage-host interactions. And finally, the research described in this thesis has for the first time explicitly revealed the nature of a carbohydrate bacteriophage receptor in L. lactis, while also providing a logical explanation for the observed narrow host ranges exhibited by 936 and P335 phages. Future research in discerning the structures of other L. lactis CW PS, combined with the determination of the molecular interplay between receptor binding proteins of these phages and CW PS will allow an in depth understanding of the mechanism by which the most prevalent lactococcal phages identify and adsorb to their specific host.

Dance of the waves

An Impressionistic piece. Dance of the Waves uses and expansive structure (A, A2, B, A2, Solos, D, C2, D). The piece is an atmospheric soundscape which evokes imagery of the ocean.

Adsorption of 2-mercaptobenzimidazole on a Au(1 1 1) electrode

The description of the monolayer formed at Au(1 1 1) by 2-mercaptobenzimidazole (MBI) under potential control has been based on electrochemical data (charge measurements) and spectroscopic information from the subtractively normalized interfacial Fourier transform infrared spectroscopy method (SNIFTIRS). From the quantitative analysis of the SNIFTIR spectra, a surface coverage Γ/Γmax was extracted for each sample potential. The evolution of the coverage with potential was in full agreement with the charge density curve. The shift of the pzc in the presence of MBI indicates that the adsorbed molecules have a nonzero component of the permanent dipole moment in the direction perpendicular to the electrode surface. Thanks to the high quality of the spectra, it was possible to determine the orientation of MBI molecules at the surface in the monolayer and submonolayer range. The angle between the C2-axis of the molecule and the direction normal to the surface is close to 64 ± 4° and its small change (<15°) with potential indicates that the orientation of the molecules is chiefly controlled by the chemical interaction between the sulphur atom and the gold surface. © 2005 Elsevier Ltd. All rights reserved.

Electrochemical and FTIR characterization of the self-assembled monolayer of 2-mercaptobenzimidazole on Au(1 1 1)

The behaviour of a self-assembled monolayer of 2-mercaptobenzimidazole (MBI) at the Au(111) electrode has been examined using cyclic voltammetry and in situ FTIR spectroscopy. The charge associated with the reductive desorption is pH independent while the oxidative partial redeposition charge increases when the pH is lowered. This is due to differences between the nature and the solubility of the MBI desorption product. In alkaline and neutral media MBI desorbs as the thiolate. In contrast, in acidic solutions the thiol is the desorbed product. Subtractively normalized interfacial reflection Fourier transform absorption spectroscopy (SNIFTIRS) has been applied to investigate the MBI monolayer in contact with aqueous solutions of different pH. The SNIFTIRS data are in agreement with the electrochemical results. Moreover, quantitative analysis of the IR data provided evidence that adsorbed MBI molecules assume a tilted orientation with an angle of 60±5° between the C2 axis of the molecule and the direction normal to the gold surface. © 2003 Elsevier B.V. All rights reserved.

Purification, crystallization and preliminary X-ray diffraction studies of a complex between G protein-coupled receptor kinase 2 and Gbeta1gamma2.

G protein-coupled receptor kinase 2 (GRK2) phosphorylates activated G protein-coupled receptors (GPCRs), which ultimately leads to their desensitization and/or downregulation. The enzyme is recruited to the plasma membrane via the interaction of its carboxyl-terminal pleckstrin-homology (PH) domain with the beta and gamma subunits of heterotrimeric G proteins (Gbetagamma). An improved purification scheme for GRK2 has been developed, conditions under which GRK2 forms a complex with Gbeta(1)gamma(2) have been determined and the complex has been crystallized in CHAPS detergent micelles. Crystals of the GRK2-Gbetagamma complex belong to space group C2 and have unit-cell parameters a = 187.0, b = 72.1, c = 122.0 A, beta = 115.2 degrees. A complete data set has been collected to 3.2 A resolution with Cu Kalpha radiation.

An approach to the study of G-protein-coupled receptor kinases: an in vitro-purified membrane assay reveals differential receptor specificity and regulation by G beta gamma subunits.

Phosphorylation of GTP-binding-regulatory (G)-protein-coupled receptors by specific G-protein-coupled receptor kinases (GRKs) is a major mechanism responsible for agonist-mediated desensitization of signal transduction processes. However, to date, studies of the specificity of these enzymes have been hampered by the difficulty of preparing the purified and reconstituted receptor preparations required as substrates. Here we describe an approach that obviates this problem by utilizing highly purified membrane preparations from Sf9 and 293 cells overexpressing G-protein-coupled receptors. We use this technique to demonstrate specificity of several GRKs with respect to both receptor substrates and the enhancing effects of G-protein beta gamma subunits on phosphorylation. Enriched membrane preparations of the beta 2- and alpha 2-C2-adrenergic receptors (ARs, where alpha 2-C2-AR refers to the AR whose gene is located on human chromosome 2) prepared by sucrose density gradient centrifugation from Sf9 or 293 cells contain the receptor at 100-300 pmol/mg of protein and serve as efficient substrates for agonist-dependent phosphorylation by beta-AR kinase 1 (GRK2), beta-AR kinase 2 (GRK3), or GRK5. Stoichiometries of agonist-mediated phosphorylation of the receptors by GRK2 (beta-AR kinase 1), in the absence and presence of G beta gamma, are 1 and 3 mol/mol, respectively. The rate of phosphorylation of the membrane receptors is 3 times faster than that of purified and reconstituted receptors. While phosphorylation of the beta 2-AR by GRK2, -3, and -5 is similar, the activity of GRK2 and -3 is enhanced by G beta gamma whereas that of GRK5 is not. In contrast, whereas GRK2 and -3 efficiently phosphorylate alpha 2-C2-AR, GRK5 is quite weak. The availability of a simple direct phosphorylation assay applicable to any cloned G-protein-coupled receptor should greatly facilitate elucidation of the mechanisms of regulation of these receptors by the expanding family of GRKs.

Search for maximal flavor violating scalars in same-charge lepton pairs in pp̄ collisions at s=1.96TeV

Models of maximal flavor violation (MxFV) in elementary particle physics may contain at least one new scalar SU(2) doublet field ΦFV=(η0,η+) that couples the first and third generation quarks (q1, q3) via a Lagrangian term LFV=ξ13ΦFVq1q3. These models have a distinctive signature of same-charge top-quark pairs and evade flavor-changing limits from meson mixing measurements. Data corresponding to 2fb-1 collected by the Collider Dectector at Fermilab II detector in pp̄ collisions at s=1.96TeV are analyzed for evidence of the MxFV signature. For a neutral scalar η0 with mη0=200GeV/c2 and coupling ξ13=1, ∼11 signal events are expected over a background of 2.1±1.8 events. Three events are observed in the data, consistent with background expectations, and limits are set on the coupling ξ13 for mη0=180-300GeV/c2. © 2009 The American Physical Society.

¿Es el agua un espejismo? algunas reflexiones desde la perspectiva de la educación matemática crítica

Teniendo en cuenta que la educación tradicional es vista como un modelo pedagógico que entre otras: i) se enfoca en desarrollar en los estudiantes conocimientos algorítmicos, ii) hace un énfasis en la ejercitación de procedimientos, iii) no tiene en cuenta el desarrollo social del individuo dentro de una comunidad y tampoco se enfoca en el proceso que tiene un estudiante al desarrollar una actividad con determinado objeto matemático; hoy en día se propende por buscar perspectivas que le permitan a los estudiantes encontrarle sentido a las actividades que el profesor lleva al aula. A la luz de lo anterior, en Colombia han surgido diversas tendencias que han buscado la renovación pedagógica, didáctica y conceptual en la educación escolar, enmarcadas –la mayoría de estas propuestas– dentro de la idea de que los estudiantes se relacionen directamente con el conocimiento, mientras que el profesor toma una postura de orientador del proceso de aprendizaje del estudiante. Teniendo en cuenta lo anterior, muchos profesores han buscado cambiar sus prácticas tradicionales de enseñanza, un ejemplo de ello lo encontramos en el colectivo de profesores de la Institución Educativa Distrital Colegio Paulo Freire de la localidad de Usme (Bogotá, Colombia); donde los profesores –en concordancia con las ideas del pedagogo Paulo Freire– comparten, como parte de su proyecto educativo, el hecho de ver a la enseñanza como un proceso que debe generar en los estudiantes una comprensión crítica de la realidad social, política y económica en la que él está inmerso.

El infinito: concepciones de los estudiantes que transitan del colegio a la universidad

En el presente trabajo nos interesa principalmente determinar qué concepciones sobre el infinito han desarrollado estudiantes de último año de secundaria y estudiantes universitarios de primer año. Aunque este concepto no aparece como un contenido específico del currículo de matemáticas, sobre él se desarrollan diferentes concepciones en escenarios no escolares que de una u otra manera afectan la construcción de conceptos matemáticos relacionados con él. Además, nos interesa confrontar las ideas que surgen cuando se habla de infinito en lo grande e infinito en lo pequeño, ya que aunque se trata de la construcción de un mismo concepto sus concepciones emergen de manera diferente en la mente de los individuos (Núñez, 1997). Lo que se puede justificar considerando que es más fácil comprender el infinito en lo grande como un proceso que continua sin parar y que no tiene fin, que el infinito en lo pequeño, en donde a pesar de conservarse el hecho de un proceso sin fin, aparece una nueva situación que sugiere que dicho proceso tiene un límite.

Problemas de ecuaciones de primer grado con una incógnita

La utilización de recursos visuales y manipulativos potencializan nuestra capacidad para resolver problemas mediante ecuaciones de primer grado con una incógnita.

Método gráfico para resolver sistemas de ecuaciones lineales 2x2

El presente documento corresponde al trabajo final de la concentración en Educación Matemática de la Maestría en Educación de la Universidad de los Andes. El trabajo fue elaborado por cuatro profesores licenciados en matemáticas que ejercen en instituciones educativas públicas y privadas en la ciudad de Bogotá y en el departamento de Cundinamarca. Este informe describe el diseño fundamentado y justificado, la implementación y el balance estratégico de la unidad didáctica titulada “Método gráfico para resolver sistemas de ecuaciones lineales 2x2”. El diseño de la unidad didáctica surgió de la selección de un tema matemático que a su vez hace parte de los contenidos incluidos en el currículo oficial para los grados octavo y noveno de educación básica como lo establece el documento de Estándares Básicos de Competencias (Ministerio de Educación Nacional [MEN], 2006a). El diseño se fundamenta a partir del procedimiento de análisis didáctico que constituyó el contenido central de la maestría. Dicho procedimiento permitió concretar elementos previos a la aplicación y la descripción junto con el balance estratégico de la implementación de la unidad didáctica.

Gasta sólo lo que puedes pagar: una experiencia de optimización con alumnos de secundaria

La experiencia que se presenta pretende valorar la intuición optimizadora en estudiantes de secundaria obligatoria. El problema que se aborda es, dado un conjunto de cantidades, elegir entre ellas las que sumen una cantidad exacta o lo más cercana a ella. El resultado de la experiencia de aula en un contexto específico ha permitido identificar la poca preparación de los estudiantes para este tipo de tarea. La principal conclusión es que los estudiantes están preparados para sumar cantidades, pero les resulta muy difícil elegir los sumandos que sumen una determinada cantidad, desconocen estrategias y son incapaces de inventar heurísticos que les lleve a conseguir el objetivo. La reflexión, consecuencia de la experiencia realizada, es que a los problemas de optimización se les dedica poca atención en la enseñanza obligatoria a pesar de ser de gran utilidad en la vida cotidiana.

Análisis de problemas en el libro de texto del estudiante de matemática en 11º año de enseñanza media en relación al eje datos y azar en el período escolar 2007 y 2011

El desarrollo de las habilidades para un conocimiento estadístico necesario es posible desarrollarlo y fortalecerlo por medio de variados recursos didácticos dispuestos para la enseñanza y aprendizaje. Dentro de los recursos disponibles es el texto de matemática el más utilizado por profesores y estudiantes. El texto debe entregar herramientas que permita a los estudiantes desarrollar una alfabetización matemática, realizando una focalización más explícita en los conocimientos, comprensión y habilidades requeridas para funcionar efectivamente en la vida diaria (PISA Chile, 2009).

Recommendations on the nature of the passenger response time distribution to be used in the MSC 1033 assembly time analysis based on data derived from sea trials

The passenger response time distributions adopted by the International Maritime Organisation (IMO)in their assessment of the assembly time for passanger ships involves two key assumptions. The first is that the response time distribution assumes the form of a uniform random distribution and the second concerns the actual response times. These two assumptions are core to the validity of the IMO analysis but are not based on real data, being the recommendations of an IMO committee. In this paper, response time data collected from assembly trials conducted at sea on a real passanger vessel using actual passangers are presented and discussed. Unlike the IMO specified response time distributions, the data collected from these trials displays a log-normal distribution, similar to that found in land based environments. Based on this data, response time distributions for use in the IMO assesmbly for the day and night scenarios are suggested