Global transcriptome analysis of Lactococcus garvieae strains in response to temperature

Lactococcus garvieae is an important fish and an opportunistic human pathogen. The genomic sequences of several L. garvieae strains have been recently published, opening the possibility of global studies on the biology of this pathogen. In this study, a whole genome DNA microarray of two strains of L. garvieae was designed and validated. This DNA microarray was used to investigate the effects of growth temperature (18°C and 37°C) on the transcriptome of two clinical strains of L. garvieae that were isolated from fish (Lg8831) and from a human case of septicemia (Lg21881). The transcriptome profiles evidenced a strain-specific response to temperature, which was more evident at 18°C. Among the most significant findings, Lg8831 was found to up-regulate at 18°C several genes encoding different cold-shock and cold-induced proteins involved in an efficient adaptive response of this strain to low-temperature conditions. Another relevant result was the description, for the first time, of respiratory metabolism in L. garvieae, whose gene expression regulation was temperature-dependent in Lg21881. This study provides new insights about how environmental factors such as temperature can affect L. garvieae gene expression. These data could improve our understanding of the regulatory networks and adaptive biology of this important pathogen.

Garvicin A, a novel class IId bacteriocin from Lactococcus garvieae that inhibits septum formation in L. garvieae strains

Lactococcus garvieae 21881, isolated in a human clinical case, produces a novel class IId bacteriocin, garvicin A (GarA), which is specifically active against other L. garvieae strains, including fish- and bovine-pathogenic isolates. Purification from active supernatants, sequence analyses, and plasmid-curing experiments identified pGL5, one of the five plasmids found in L. garvieae [M. Aguado-Urda et al., PLoS One 7(6):e40119, 2012], as the coding plasmid for the structural gene of GarA (lgnA), its putative immunity protein (lgnI), and the ABC transporter and its accessory protein (lgnC and lgnD). Interestingly, pGL5-cured strains were still resistant to GarA. Other putative bacteriocins encoded by the remaining plasmids were not detected during purification, pointing to GarA as the main inhibitor secreted by L. garvieae 21881. Mode-of-action studies revealed a potent bactericidal activity of GarA. Moreover, transmission microscopy showed that GarA seems to act by inhibiting septum formation in L. garvieae cells. This potent and species-specific inhibition by GarA holds promise for applications in the prevention or treatment of infections caused by pathogenic strains of L. garvieae in both veterinary and clinical settings.

Characterization of plasmids in a human clinical strain of Lactococcus garvieae

The present work describes the molecular characterization of five circular plasmids found in the human clinical strain Lactococcus garvieae 21881. The plasmids were designated pGL1-pGL5, with molecular sizes of 4,536 bp, 4,572 bp, 12,948 bp, 14,006 bp and 68,798 bp, respectively. Based on detailed sequence analysis, some of these plasmids appear to be mosaics composed of DNA obtained by modular exchange between different species of lactic acid bacteria. Based on sequence data and the derived presence of certain genes and proteins, the plasmid pGL2 appears to replicate via a rolling-circle mechanism, while the other four plasmids appear to belong to the group of lactococcal theta-type replicons. The plasmids pGL1, pGL2 and pGL5 encode putative proteins related with bacteriocin synthesis and bacteriocin secretion and immunity. The plasmid pGL5 harbors genes (txn, orf5 and orf25) encoding proteins that could be considered putative virulence factors. The gene txn encodes a protein with an enzymatic domain corresponding to the family actin-ADP-ribosyltransferases toxins, which are known to play a key role in pathogenesis of a variety of bacterial pathogens. The genes orf5 and orf25 encode two putative surface proteins containing the cell wall-sorting motif LPXTG, with mucin-binding and collagen-binding protein domains, respectively. These proteins could be involved in the adherence of L. garvieae to mucus from the intestine, facilitating further interaction with intestinal epithelial cells and to collagenous tissues such as the collagen-rich heart valves. To our knowledge, this is the first report on the characterization of plasmids in a human clinical strain of this pathogen.

Genome sequence of Lactococcus garvieae 8831, isolated from rainbow trout lactococcosis outbreaks in Spain

Lactococcus garvieae is the etiological agent of lactococcosis, one of the most important disease threats to the sustainability of the rainbow trout farming industry. Here, we present the draft genome sequence of Lactococcus garvieae strain 8831, isolated from diseased rainbow trout, which is composed of 2,087,276 bp with a G+C content of 38%.

Genome sequence of Lactococcus garvieae 21881, isolated in a case of human septicemia

Lactococcus garvieae is a Gram-positive bacterium considered an important opportunistic emerging human pathogen and also a well-recognized fish pathogen. Here, we present the draft genome sequence of Lactococcus garvieae strain 21881 (2,164,557 bp, with a G+C content of 37.9%), which represents the first report of a genome sequence on Lactococcus garvieae.

Utilization of lactose and presence of the phospho-β-galactosidase (lacG) gene in Lactococcus garvieae isolates from different sources

This study evaluates the utilization of lactose (Lac) and the presence of the phospho-β-galactosidase (lacG) gene as markers for distinguishing between fish (Lac-/lacG-) and dairy isolates (Lac+/lacG+) of Lactococcus garvieae, using a panel of L. garvieae isolates from different sources. None of the fish isolates produced acid from lactose (Lac-), however Lac-/lacG- isolates were observed in pigs, cows, birds and humans. Most of the dairy isolates (77.8%) were Lac+/lacG+, but some dairy isolates did not produce acid from this sugar. Data in the present study show that the ability to metabolize lactose and the presence of the lacG gene are heterogeneously scattered among L. garvieae isolates of different sources. Therefore, the use of these criteria as markers to differentiate between L. garvieae isolates of dairy and fish origin should be considered with caution.

Comparison of stool microbiota compositions, stool alpha1-antitrypsin and calprotectin concentrations, and diarrhoeal morbidity of Indonesian infants fed breast milk or probiotic/prebiotic-supplemented formula

Aim The composition of faecal microbiota of babies is known to be influenced by diet. Faecal calprotectin and α1-antitrypsin concentrations may be associated with mucosal permeability and inflammation. We aimed to assess whether there was any difference after consumption of a probiotic/prebiotic formula on faecal microbiota composition, calprotectin and α1-antitrypsin levels, and diarrhoea in comparison with breast milk-fed Indonesian infants. Methods One hundred sixty infants, 2 to 6 weeks old, were recruited to the study. They were either breastfed or formula fed (80 per group). Faecal samples were collected at recruitment and 3 months later. Bacterial groups characteristic of the human faecal microbiota were quantified in faeces by quantitative polymerase chain reaction. Calprotectin and α1-antitrypsin concentrations were measured using commercial kits. Details of diarrhoeal morbidity were documented and rated for severity. Results The compositions of the faecal microbiota of formula-fed compared with breast milk-fed children were similar except that the probiotic strain Bifidobacterium animalis subsp. lactisâ€...DR10 was more abundant after 3 months consumption of the formula. Alpha1-antitrypsin levels were higher in breastfed compared with formula-fed infants. The occurrence of diarrhoea did not differ between the groups of babies. Conclusion Feeding Indonesian babies with a probiotic/prebiotic formula did not produce marked differences in the composition of the faecal microbiota in comparison with breast milk. Detrimental effects of formula feeding on biomarkers of mucosal health were not observed.

Immunomodulatory effects of probiotic bacteria in healthy adults

Probiooteilla kantakohtaisia vaikutuksia ihmisen immuunijärjestelmään terveillä aikuisilla Probiooteilla on kantakohtaisia tulehduksen välittäjäaineita vähentäviä vaikutuksia ja probioottien yhdistelmien vaikutukset eroavat yksittäisten kantojen vaikutuksista selviää TtM Riina Kekkosen tuoreesta väitöstutkimuksesta. TtM Riina Kekkonen on selvittänyt väitöskirjassaan eri probioottikantojen vaikutuksia immuunivasteeseen valkosolumallissa sekä terveillä aikuisilla lumekontrolloiduissa kliinisissä tutkimuksissa. Aikaisemmin probioottien vaikutuksia on tutkittu lähinnä allergian ja erilaisten vatsavaivojen ehkäisyssä ja hoidossa. Probiootteja sisältäviä tuotteita käyttävät kuluttajat ovat kuitenkin useimmiten terveitä aikuisia, ja probioottien vaikutus terveiden aikuisten immuunijärjestelmään on ollut puutteellisesti selvitettyä. Valkosolumallissa probioottikantojen havaittiin poikkeavan toisistaan niiden kyvyssä aktivoida immuunivasteen välittäjäaineiden, sytokiinien, tuotantoa. Anti-inflammatorisia, eli tulehdusta lievittäviä vaikutuksia nähtiin lähinnä Bifidobacterium ja Propionibacterium sukuihin kuuluvilla kannoilla. Streptococcus ja Leuconostoc sukuihin kuuluvat kannat puolestaan aktivoivat Th1 tyyppistä, soluvälitteistä immuunivastetta. Eri probioottien kombinaatiot eivät saaneet aikaan voimakkaampaa aktivaatiota yksittäisiin kantoihin verrattuna, joka viittaa probioottien keskinäiseen kilpailuun niiden ollessa kontaktissa ihmisen solujen kanssa. Probioottikantojen valinta kliinisiin tutkimuksiin tehtiin niiden anti-inflammatoristen ominaisuuksien perusteella. Parhaita anti-inflammatorisia kantoja olivat B. lactis ssp. animalis Bb12 ja P. freudenreichii ssp. shermanii JS, joiden lisäksi tutkimuksiin valittiin myös L. rhamnosus GG (LGG) hyvin tutkittuna referenssikantana. Solutöiden tulokset eivät olleet täysin verrannollisia kliinisen työn tuloksiin, koska LGG näytti omaavan parhaat anti-inflammatoriset ominaisuudet kliinisissä tutkimuksissa vaikka solutyössä sen aikaansaamat vasteet olivat melko vaimeita. Kolmen viikon kliinisessä tutkimuksessa terveillä aikuisilla LGG alensi mm. tulehdusta kuvaavan C-reaktiivisen proteiinin ja inflammatoristen sytokiinien määrää. Pidemmässä kolmen kuukauden pituisessa kliinisessä tutkimuksessa LGG:llä ei ollut vaikutusta terveiden aikuisten infektiosairastavuuteen, mutta LGG lyhensi vatsavaivojen kestoa. Probioottien vaikutukset immuunijärjestelmään näyttävät olevan kantakohtaisia ja erityisesti Lactobacillus rhamnosus GG:llä havaittiin anti-inflammatorisia vaikutuksia. Valkosolumallia ei tulisi käyttää ainoana probioottikantojen skriinausmenetelmänä niiden immunologisia vaikutuksia selvitettäessä, koska solutöiden tulokset eivät olleet täysin verrannollisia kliinisten tutkimusten tuloksiin. Sen sijaan veren perifeeristen lymfosyyttien eristäminen ja niiden aktivoitumisen selvittäminen lyhytaikaisessa kliinisessä tutkimuksessa voisi toimia suhteellisen helppona skiinausmenetelmänä.

The role of probiotics in Helicobacter pylori infection

Helicobacter pylori (H. pylori) infection is a major cause of chronic gastritis and peptic ulcer disease, and it is also designated as a class-I carcinogen for stomach cancer. The role of probiotics in the treatment of gastrointestinal infections is increasingly documented as an alternative or complement to antibiotics, with the potential to decrease the use of antibiotics or reduce their adverse effects. These studies were conducted to investigate the role of probiotics in the treatment of H. pylori infection. Various aspects included: an investigation of the effects of a probiotic combination consisting of Lactobacillus rhamnosus GG, L. rhamnosus LC705, Propionibacterium freudenreichii ssp. shermanii JS and Bifidobacterium breve Bb99 or B. lactis Bb12 as a supplementation to H. pylori eradication therapy, with special reference to tolerability, effectiveness, and microbiota alterations following the treatment; discovering the role of probiotics in vivo with H. pylori infected and uninfected patients, as well as with an in vitro model of H. pylori infection. The probiotic combination therapy was able to reduce significantly the total symptom score, which takes into account both the frequency and the severity of the adverse effects, during the eradication treatment. The supplementation did not improve the success of the eradication treatment significantly, though some difference was seen in the eradication percentages (91% vs. 79%). The quantities of predominant bacterial groups were altered significantly following the triple treatment. Probiotics slightly counteracted the effects of anti-H. pylori treatment, monitored as significantly less alterations in the total numbers of aerobes and lactobacilli/enterococci group bacteria. After probiotic intervention, L. rhamnosus GG adhered to a minority of the patients upper gastrointestinal mucosa, but all of the probiotics survived well through the gastrointestinal tract transit with and without antimicrobial treatment. Probiotic intervention decreased gastrin-17 levels in H. pylori infected patients and appeared to decrease the 13C-urea breath test values. In in vitro Caco-2 cell line experiments, probiotics inhibited H. pylori adhesion to intestinal epithelial cells. Both L. rhamnosus strains, P. freudenreichii ssp. shermanii JS and the combination inhibited the H. pylori-induced acute cell leakage. Simultaneously, both L.rhamnosus strains and the combination transiently improved the epithelial barrier function. The pro-inflammatory effects prevailed when the probiotics were used in combination. According to this series of studies, probiotic combination could have some potential in reducing adverse effects induced by H. pylori eradication treatment and beneficial effects on H. pylori infected subjects.

Pathophysiological factors of irritable bowel syndrome, and the effects of probiotic supplementation

Gastrointestinal symptoms and impaired quality of life caused by irritable bowel syndrome (IBS) affect up to 20% of the adult population worldwide. The exact aetiology and pathophysiology of IBS are incompletely understood. Clinical studies suggest that supplementation with certain probiotics may be beneficial in IBS, but there is not enough evidence to make general recommendations. The aim of this thesis was to investigate microbiota- and mucosa-associated pathophysiological factors of IBS, and to evaluate the long-term effects of multispecies probiotic supplementation on symptoms, quality of life, intestinal microbiota and systemic inflammatory markers in IBS. The intestinal microbiota composition in IBS patients and healthy control subjects was analysed by quantitative polymerase chain reaction (qPCR). Significantly lower counts for the Clostridium coccoides and the Bifidobacterium catenulatum groups were found in IBS compared to controls. Quantitative differences also appeared in subgroup analysis based on the predominant bowel habit: diarrhoea patients harboured significantly lower numbers of Lactobacillus spp. than the constipation-predominant patients, while higher counts for Veillonella spp. were detected in constipation-predominant patients compared to healthy controls. Analysis of mucosal biopsies by a metabolomic approach revealed multiple differences between patients and controls. The most prominent finding was an upregulation of specific lipid species, principally lysophosphatidylcholines and ceramides, in IBS. The effects of multispecies probiotic supplementation with Lactobacillus rhamnosus GG, Lactobacillus rhamnosus Lc705, Propionibacterium freudenreichii subsp. shermanii JS, and Bifidobacterium breve Bb99 or Bifidobacterium animalis subsp. lactis Bb12 was evaluated in two, randomised, double-blind, placebo-controlled trials. Compared to placebo, the probiotic supplementation significantly reduced the total symptoms of IBS. No effects on bowel habit were seen. Health-related quality of life (HRQOL) is reduced in patients with IBS in comparison with the Finnish population on the whole. The probiotic supplementation improved one IBS-specific domain of quality of life (bowel symptoms), whereas no other effects on HRQOL were seen. The probiotics had no major effects on the predominant microbiota as measured by qPCR, but a microarray-based analysis suggested that the probiotic consumption stabilised the microbiota. No effects on serum sensitive-CRP or cytokines were detected. In conclusion, alterations in the microbiota composition and in the mucosal metabolite profile are potential pathophysiological factors of IBS. Multispecies probiotic supplementation alleviates the gastrointestinal symptoms of IBS, and improves the bowel symptoms domain of HRQOL. Probiotic supplementation in IBS is associated with a stabilisation of microbiota, but it does not influence systemic inflammatory markers.

Lactic acid bacteria and their antimicrobial peptides : Induction,detection,partial characterization, and potential applications

Bacteriocin-producing lactic acid bacteria and their isolated peptide bacteriocins are of value to control pathogens and spoiling microorganisms in foods and feed. Nisin is the only bacteriocin that is commonly accepted as a food preservative and has a broad spectrum of activity against Gram-positive organisms including spore forming bacteria. In this study nisin induction was studied from two perspectives, induction from inside of the cell and selection of nisin inducible strains with increased nisin induction sensitivity. The results showed that a mutation in the nisin precursor transporter NisT rendered L. lactis incapable of nisin secretion and lead to nisin accumulation inside the cells. Intracellular proteolytic activity could cleave the N-terminal leader peptide of nisin precursor, resulting in active nisin in the cells. Using a nisin sensitive GFP bioassay it could be shown, that the active intracellular nisin could function as an inducer without any detectable release from the cells. The results suggested that nisin can be inserted into the cytoplasmic membrane from inside the cell and activate NisK. This model of two-component regulation may be a general mechanism of how amphiphilic signals activate the histidine kinase sensor and would represent a novel way for a signal transduction pathway to recognize its signal. In addition, nisin induction was studied through the isolation of natural mutants of the GFPuv nisin bioassay strain L. lactis LAC275 using fl uorescence-activated cell sorting (FACS). The isolated mutant strains represent second generation of GFPuv bioassay strains which can allow the detection of nisin at lower levels. The applied aspect of this thesis was focused on the potential of bacteriocins in chicken farming. One aim was to study nisin as a potential growth promoter in chicken feed. Therefore, the lactic acid bacteria of chicken crop and the nisin sensitivity of the isolated strains were tested. It was found that in the crop Lactobacillus reuteri, L. salivarius and L. crispatus were the dominating bacteria and variation in nisin resistance level of these strains was found. This suggested that nisin may be used as growth promoter without wiping out the dominating bacterial species in the crop. As the isolated lactobacilli may serve as bacteria promoting chicken health or reducing zoonoosis and bacteriocin production is one property associated with probiotics, the isolated strains were screened for bacteriocin activity against the pathogen Campylobacter jejuni. The results showed that many of the isolated L. salivarius strains could inhibit the growth of C. jejuni. The bacteriocin of the L. salivarius LAB47 strain, with the strongest activity, was further characterized. Salivaricin 47 is heat-stable and active in pH range 3 to 8, and the molecular mass was estimated to be approximately 3.2 kDa based on tricine SDS-PAGE analysis.

Effects of essential oil of Satureja bachtiarica, Satureja khuzestanica and Mentha longifolia on the growth of Actococcus garvieae in rainbow trout fillet

The present research was done with the aim of studing the effects of some medicinal plant essential oils against Lactococcus garvieae and on some spoilage indexes in rainbow trout fillets in 4°C during 18 days. Essential oils of Mentha longifola (20, 40 and 80 μg g-1), Satureja bachtiarica (0.125, 0.25 and 0.50 μg g-1) and Satrureja khuzestanica (0.06, 0.12, 0.25 and 0.50 μg g-1) were used for this study. According to the results, the highest and the lowest microbial load were observed in control group and the groups contained 80 and 40 μg g-1 of Mentha longifola, respectively (P≤0.05). The spoilage indexes were increased after 18 days showing a statistical difference for protein, fat, TVN, FFA and TBA (P≤0.05). The most and the least changes were observed in the the control group and the fillets containing 80 μg g-1 of Mentha longifola, respectively (P≤0.05). The results indicated that, in comparison with control group, using herbal essential oils leads to reduction of Lactococcus garvieae population and spoilage process of the fillets through decreasing in TVN, FFA and TBA. Meanwhile, Mentha longifola having 80 μg g-1 was found to be more effective

Nisin高产菌株选育及应用的研究

本论文以牛奶为原料分离出42株球菌，经鉴定其中15株为乳链球菌（Streptococcus lactis）。对15株乳链球菌进行Nisin效价测定后，确定SN-21为诱变的出发菌株（598.1IU/ml,NO.95培养基），经过硫酸二乙酯和紫外线的多次诱变后，选育出一株Nisin高产菌株（1514IU/ml,CM培养基），命名为S. L. 21 (Streptococcus lactis 21)。通过对S. L. 21发酵条件的选优，其Nisin效价达到1862IU/ml。采用紫外吸收光谱法确定S. L. 21发酵产物中的抑菌物质为Nisin。在Nisin高产菌株选育的同时，开创了Nisin应用于蕨菜罐头的加工贮藏研究。通过在蕨菜罐头中添加0.1g/kg的Nisin，降低了蕨菜罐头的杀菌温度（100 ℃）和杀菌时间（15min），保证了蕨菜的品质，提高了蕨菜罐头的贮藏安全性，这为低酸易软烂野生蔬菜食品的加工贮藏提供了一条重要途径。100 ℃，15min杀菌条件下，在加工的蕨罐头中，未添加Nisin的处理和添加苯甲酸钾（1.0g/kg）的处理，均出现胀罐，爆罐现象。经分析确定蕨菜罐头胀罐原因是由污染的细菌产气引起的。通过分离污染菌优势类群得到8株芽孢杆菌，经系统的细菌学鉴定，4~#菌株为巨大芽孢杆菌（Bacillus megaterium），8~#菌株为短小芽孢杆菌（Bacillus pumilus），其余为枯草芽孢杆菌（Bacillus subtilis）。经进一步产气试验证明5~#菌株产气快，产气量大，是引起蕨菜罐头胀罐的污染优势种，经研究发现蕨菜罐头污染菌优势种是一种枯草芽孢杆菌的新变种，命名为枯草芽孢杆菌嗜热耐盐新变种（Bacillus sutilis n. var. sp.），这株新变种具有耐热（90 ℃），耐盐（10%NaCl），产生的特点。对蕨菜罐头污染菌优势种（5~#）进行防腐剂抑菌试验发现其对Nisin敏感（MIC 500ppm），而对苯甲酸钠（MIC 1.5%）和山梨酸钾（MIC 3%）不敏感，抑菌率试验进一步证明，在允许应用范围内，只有Nisin对蕨菜罐头防腐有良好的效果，而苯甲酸钠和山梨酸钾抑菌效果均不理想。在Nisin对污染菌优势种（5~#）的溶菌作用试验中，通过电镜可以观察到，Nisin的作用首先是破坏细胞壁，细胞膜，造成细胞内物质外流，严重溶菌结果，导致污染细菌死亡。以上关于Nisin应用于蕨菜罐头防腐，蕨菜罐头污染菌优势类群的分析，污染菌优势种的研究的试验结果均为国内外首次报导。总之，通过在蕨菜罐头中添加Nisin来抑制污染菌优势种的生长敏殖，进而达到降低杀菌强度的目的，对于保证蕨菜固有的品质及今后蕨菜等野生资源的开发具有重要的意义。

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.