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.

Comparative genomics of Bifidobacteria

Chapter 2 of this thesis describes the sequence analysis of 14 bifidobacterial genomes from various species of the genus Bifidobacterium, and the determination of their open pan-genome trend. This analysis first determined the total number of genes to be considered as the reservoir of functions available to representatives of this genus. Many identified genes are still uncharacterized, but may be involved in the adaptation to the gut environment. This comparative genomic analysis also determined a pool of ortholog functions used to infer their phylogenetic relationship, thereby providing a more robust approach compared to that based solely on the16S rRNA-encoding gene. The genome sequence of an isolate from the insect hindgut Bifidobacterium asteroides PRL2011 was fully characterized in Chapter 3, surprisingly revealing a putative respiratory metabolism, which was also found to be present in other insect isolates, suggesting that respiration was an ancient feature of this genus, but also an adaptative trait to different atmosferic oxygen levels. Chapter 4 of this thesis outlines a comparative study which focused on the analysis of representatives of the Bifidobacterium breve species, revealing that the genetic variability among members of this species principally consists of genes with a role in adaptation to host environment and gut colonization. Finally, Chapter 5 describes the analysis of the genome sequence of Bifidobacterium animalis subsp. lactis BLC1, a probiotic bacterium widely used in food industries as an ingredient of functional foods, providing information that will allow future investigations of this species.

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.

Investigation of lantibiotic regulation, immunity and synergy

Due to the increasing incidence of antibiotic resistant strains, the use of novel antimicrobials, such as bacteriocins, has become an ever more likely prospect. Lacticin 3147 (of which there are two components, Ltnα and Ltnβ) and nisin belong to the subgroup of bacteriocins called the lantibiotics, which has attracted much attention in recent years. The lantibiotics are antimicrobial peptides that contain unusual amino acids resulting from a series of enzyme-mediated post translational modifications. Given that there have been relatively few examples of lantibiotic-specific resistance; these antimicrobials appear to represent valid alternatives to classical antibiotics. However, the fact that lantibiotics are naturally only produced in small amounts often hinders their commercialisation. In order to overcome this bottleneck, several approaches can be employed. For example, we can create a situation that reduces the quantity of a lantibiotic required to inhibit a target by combining it with other antimicrobials. Here, following an initial screen involving lacticin 3147 and several classical antibiotics, it was observed between lacticin 3147 and the commercial antibiotics polymyxin B/E function synergistically. This reduced the amounts of the individual antimicrobials required for kill and broadened the spectrum of inhibition of both agents. Upon combination with polymyxins, lacticin 3147, which has been associated with Gram positive targets only, actively targeted Gram negative species such as Escherichia coli and Cronobacter sp. An alternative means of addressing problems associated with lantibiotic yield is to better understand how production is regulated, and ultimately use this information to enhance peptide levels. With this in mind the regulation of lacticin 3147 production from the promoter Pbac was investigated using a green fluorescent protein (GFP) expression reporter system. This revealed that elements within both of the divergent operons of the lacticin 3147 gene cluster are involved in Pbac regulation. That is, LtnR, already established as a negative regulator of itself and the lacticin 3147 associated immunity genes, also acts as an activator of Pbac transcription. In contrast, an enhanced level of expression is observed in the absence of the lacticin 3147 structural genes, ltnA1 and ltnA2, indicating that these genes/gene products are involved in Pbac repression. In fact, through complementation of the ltnA2 gene, it was revealed that this regulation is more likely to be dependent on the presence of the gene transcript rather that the corresponding prepropeptide or modified Ltnβ. It may be that if lacticin 3147 production is successfully enhanced, the ability of the producing cell to protect itself may become an issue. To prepare for such a possibility a bioengineered derivative of the lacticin 3147 immunity protein LtnI (LtnI I81V) which provides enhanced protection was discovered through an in depth investigation involving the site and saturation mutagenesis of this protein. In addition, the creation of truncated forms of LtnI allowed the identification of important and essential regions of this immunity protein. Finally, as mentioned, self-immunity is essential to prevent self-killing. However the discovery of nisin U immunity and regulatory gene homologues (spiFEGRR’K) within the pathogenic strain S. infantarius subsp. infantarius is a cause for concern as it represents an example of immune mimicry, a form of lantibiotic-specific resistance. The ability of spiFEG to confer protection was apparent when they successfully provided protection to nisin A, F, Z, Q and U when expressed heterologously in the nisin sensitive L. lactis HP host. As a consequence of the studies presented in this thesis, it is likely that strategies will emerge that will facilitate the production of greater levels of lacticin 3147 production and lead to enhanced immunity in lactococcal backgrounds. Alternatively the need for enhanced production could be avoided through the use of antimicrobial combinations. In addition, providing awareness of the threats of the emergence of resistance through immune mimicry can allow researchers to develop strategies to prevent this phenomenon from leading to the dissemination of lantibiotic resistance.

Human intestinal microbiota and metabolites they produce in relation to host health

The aim of this thesis was to identify selected potential probiotic characteristics of Bifidobacterium longum strains isolated from human sources, and to examine these characteristics in detail using genomic and phenotypic techniques. One strain in particular Bifidobacterium longum DPC 6315 was the main focus of the thesis and this strain was used in both the manufacture of yoghurt and an animal study. In total, 38 B. longum strains, obtained from infants and adults, were assessed in vitro for the selected probiotic traits using a combined phenotypic and molecular approach. Differentiation of the 38 strains using amplified ribosomal DNA restriction analysis (ARDRA) into subspecies indicated that of the 38 bifidobacterial strains tested, 34 were designated B. longum subsp. longum and four B. longum subsp. infantis.

Triterpenoid saponins from the cytotoxic root extract of Sideroxylon foetidissimum, an endemic Yucatecan medicinal plant

The flora of the Yucatan peninsula (Mexico) includes approximately 3000 plant species. Sideroxylon foetidissimum Jacq. subsp. gaumeri (Sapotaceae) is an endemic plant to the Yucatan peninsula; its fruit is edible and local people use the plant for medicinal purposes, although no details on its preparation or application are available [1,2]. A preliminary cytotoxic evaluation of the ethanolic root extract of S. foetidissimum revealed a potent activity against murine macrophage like cell line RAW 264.7 (IC50=39.54±4.11µg/mL). The systematic bioassay-guided fractionation of the extract resulted in the identification of the active saponin-containing fraction (IC50=33.69±6.19µg/mL). Four new triterpenoid saponins and a 1:1 mixture of two saponins were isolated from the active saponin- containing fraction. The evaluation of their cytotoxic activity revealed no activity for the tested pure saponins; however, the 1:1 mixture of saponins showed a potent activity (IC50=11.91±1.49µg/mL). The isolation of the saponins was carried out using semi-preparative HPLC. The structural assignments of the pure saponins were based on 1D (1H and 13C and DEPT-135) and 2D (COSY, HMBC, HSQC and TOCSY) NMR and mass spectrometry analyses. In this presentation, the isolation, identification and cytotoxic activity of the isolated compounds is discussed in more detail.

Contribution to the knowlegde of Galician xenoflora (NW Iberian Peninsula, Spain).

Information on 12 exotic plants of diverse interest for the Galician flora are presented. All of them were collected in Ribeira council (SW of the A Coruña province). The total includes 8 novelties at a regional level (Aeonium haworthii, Aloe mitriformis, Brugmansia × candida, Nephrolepis cordifolia, Osteospermum ecklonis, Pelargonium capitatum, Sedum mexicanum, Sparaxis tricolor), and 2 provincial novelties. In addition, information on two taxa hardly mentioned in the literature on Galician vascular flora is also included. All the cited specimens are deposited at the SANT Herbarium.

Plagiomnium T. Kop. (Plagiomniaceae, Musci) en la Península Ibérica e llles Balears

Se ha realizado la revisión de ejemplares de Plagiomnium T. Kop., en la Península Ibérica, registrados en los Herbarios de España, Portugal y Francia, así como en algunas colecciones privadas. Siete taxones de Plagiomnium están representados en España: P. affine, P. cuspidatum, P. elatum, P. ellipticum, P. medium subsp. medium, P. rostratum y P. undulatum, y en Portugal cuatro taxones: P. affine, P. medium subsp. mediurn, P. rostratum y P. undulatum. En Mallorca (Illes Balears), queda excluido P. cuspidatum y P. elatum es nuevo para estas islas. P. undulatum, P. affine y P. rostratum son las especies más comunes en los bosques y zonas montañosas de la Península; P. cuspidatum y P. elatum son frecuentes en la mitad septentrional de España, siendo muy escasos en la parte meridional; los más raros son: P. ellipticum y P. medium subsp. medium, que están restringidos a los hábitats mesofíticos, suelos oligotrofos y húmedos de Pirineos, Cordillera Cantábrica y el piso supra-oromediterráneo de los Montes de Cataluña, Sistema Ibérico septentrional (Macizo de La Demanda) y Sistema Central. Se aportan fotografías de las esporas al SEM de P. affine, P. medium subsp. medium, P. rostratum y P. undulatum así como los mapas de distribución de cada taxon.

Aproximación a la biogeografía del sector Setabense (provincia Catalano-Valenciano-Provenzal)

Se realiza una propuesta de división en subsectores del sector Setabense (Provincia biogeográfica Catalano- Valenciano-Provenzal). Las unidades reconocidas son: 1 subsector Valenciano;2 subseetor Enguerina-Cofrentino; 3 subsector Ayorano-Villenense; 4 subsector Alcoyano-Diánico. Para cada uno de ellos se aporta una breve descripción y caracterización de su flora, vegetación, paisaje vegetal, suelos, bioclimatología, usos del territorio, etc. Además se proponen dos nuevas combinaciones taxonómicas: Asperula paui Font Quer subsp. dianensis (Font Quer) de la Torre, Alcaraz & Crespo y Linaria depauperata Leresehe ex Lange subsp. hegelmaieri (Lange) de la Torre, Alcaraz & Crespo, y se valida la combinación Arctostaphyllos uva-ursi (L.) Sprengel subsp. crassifol¡a (Br.-Bl.) Rivas-Martínez ex de la Torre, Alcaraz & Crespo.

Comportamiento fitosociológico de Juniperus phoenicea L. s.l. en el sur de la Península Ibérica

Se ha realizado un estudio fitosociológico de las comunidades de Juniperus phoenicea L. s.l. en el sector Rondeño. Como resultado del mismo se señala la presencia de Juniperus turbinata subsp. turbinata en territorios interiores de este sector y se describen dos nuevos sintáxones: Asparaqo horridi-Juniperetum turbinatae y Rhamno myrtifoliae-Juniperetum phoeniceae subass. rhamnetosum oleoides.

Revisión del género Saxifraga L., sect. Dactyloides Tausch en el centro y norte de la Península Ibérica

Hemos llevado a cabo la revisión del género Saxifraga L., sección Dactyloides Tausch (grex Ceratophyllae Willk., Gemmiferae Willk., y Exarato-Moschatae Engler & Itmsch.) del centro y norte de la Península Ibérica, mediante el estudio de caracteres morfológicos, palinológicos y seminológicos, de entre los que destacamos por su valor diagnóstico los siguientes: longitud y anchura máxima de las hojas basilares, longitud del peciolo real de las mismas, número total de segmentos y tipo de segmento central de las hojas basilares, anchura del segmento central de las hojas basilares, presencia-ausencia de mucrón en el ápice foliar, contorno de la lámina de las hojas basilares, forma de los segmentos laterales de la lámina de las hojas basilares, tipo de indumento de las hojas basilares, distribución de los pelos glandulares y número de células de los mismos, clasificación de las hojas en base a la presencia-ausencia de surco, longitud del tallo florífero, mitad apical o basal del tallo florífero cubierta de pelos glandulares, presencia de yemas hibemantes, forma apical de los dientes del cáliz, longitud y anchura de los pétalos, porción del pétalo que sobrepasa al sépalo, longitud y anchura de la semilla y omamentación de la cubierta seminal. Los caracteres estudiados nos han permitido reconocer un total de treinta y tres táxones de los que aportamos datos corológicos, ecológicos y fitosociológicos. Se añade una clave de identificación de todos los táxones objeto de estudio, en la que también se incluyen S. conifera Cosson, S. x martyi Luizet & Soulié, S. pubescens Pourret subsp. pubescens, S. pubescens subsp. iratiana (F.W. Schultz) Engler & Irmsch., y S. vayredana Luizet, los cuales aunque no tratados en nuestra revisión, se distribuyen por el norte de la Península Ibérica.

Datos sobre las comunidades de caméfitos espinosos en los macizos meridionales galaicos (NW España)

Se describen las comunidades de matorral dominadas por Echinospartum ibericum RivasMartínez, Sánchez-Mata & Sancho subsp. pulviniforinis (Rivas-Martínez) Rivas-Martínez, que colonizan medios rupícolas en los macizos meridionales del noroeste peninsular. Se discute su posición siníaxonómica y se propone su inclusión en la nueva asociación Festuco graniticolae-Echinospartetum pulviniformis. Se realizan también algunas consideraciones biogeográficas.

3. Murbeckiello boryi-Sperguletum pourretii Fernández Prieto 1983 corr. Izco & Ortiz 1989

Fernández-Prieto (Anales Jard. Bot. Madrid 39(2): 495. 1983) describe con inventarios de la Sierra del Teleno y de Peña Trevinca la asociación rupícula Murbeckiello herminii-Sperguletum pourretii en cuya composición florística, según el autor del sintaxon, se integra Murbeckiella boryi (Boiss.) Rothm. subsp. herminii Rivas-Martínez.

Teucrium x estevei Alcaraz, Sánchez-Gómez & Carrión hybr. nov.

Se describe un nuevo híbrido interespecifico en el género Teucrium L.: Teucrium x estevei, Alcaraz, Sánchez-Gómez & Carrión hybr. nov. (= T. libanotis Schreber x T. capitatum L. subsp. gracillimum (Rouy) Valdés-Bermejo).

Estudio cariológico en taxones del CW español pertenecientes a Quercetea ilicis

Se han determinado los números cromosómicos de 13 taxones de Quercetea ilidis del CW español; cuatro de ellos: Quercus fagineae subsp. broteroi (2n=24, n=12), Pistacia terebinthus (n=15), Rhamnus oleoides subsp. spiculosa (2n=24) y Phyllirea angustifolia (n=23), se estudian aquí por primera vez y otros cinco suponen la primera aportación cariológica sobre material español. Se discuten aspectos interesantes del estudio cariológico de los distintos taxa.