Lantibiotic Nisin and Its Detection Methods

The type A lantibiotic nisin produced by several Lactococcus lactis strains, and one Streptococcus uberis strainis a small antimicrobial peptide that inhibits the growth of a wide range of gram-positive bacteria, such as Bacillus, Clostridium, Listeria and Staphylococcus species. It is nontoxic to humans and used as a food preservative (E234) in more than 50 countries including the EU, the USA, and China. National legislations concerning maximum addition levels of nisin in different foods vary greatly. Therefore, there is a demand for non-laborious and sensitive methods to identify and quantify nisin reliably from different food matrices. The horizontal inhibition assay, based on the inhibitory effect of nisin to Micrococcus luteus is the base for most quantification methods developed so far. However, the sensitivity and accuracy of the agar diffusion method is affected by several parameters. Immunological tests have also been described. Taken into account the sensitivity of immunological methods to interfering substances within sample matrices, and possible cross-reactivities with lantibiotics structurally close to nisin, their usefulness for nisin detection from food samples remains limited. The proteins responsible for nisin biosynthesis, and producer self-immunity are encoded by genes arranged into two inducible operons, nisA/Z/QBTCIPRK and nisFEG, which also contain internal, constitutive promoters PnisI and PnisR. The transmembrane histidine kinase NisK and the response regulator NisR form a two-component signal transduction system, in which NisK autophosphorylates after exposure to extra cellular nisin, and subsequently transfers the phosphate to NisR. The phosphorylated NisR then relays the signal downstream by binding to two regulated promoters in the nisin gene cluster, i.e the nisA/Z/Qand the nisF promoters, thus activating transcription of the structural gene nisA/Z/Q and the downstream genes nisBTCIPRK from the nisA/Z/Q promoter, and the genes nisFEG from the nisF promoter. In this work two novel and highly sensitive nisin bioassays were developed. Both of these quantification methods were based on NisRK mediated, nisin induced Green Fluorescent Protein (GFP) fluorescence. The suitabilities of these assays for quantifica¬tion of nisin from food samples were evaluated in several food matrices. These bioassays had nisin sensitivities in the nanogram or picogram levels. In addition, shelf life of nisin in cooked sausages and retainment of the induction activity of nisin in intestinal chyme (intestinal content) was assessed.

Äidinmaito - terveysjuomaa ja normaalibakteereita

The chemical composition of breast milk has been studied in detail in the past decades. Hundreds of new antibacterial and antiviral components have been found. Several molecules have been found to promote the proper function of neonatal intestine. However, microbiological studies of breast milk have been, until recently, focused mainly on detecting harmful and pathogenic bacteria and viruses. Natural microbial diversity of human milk has not been widely studied before the work reported in this thesis. This is mainly because breast milk has traditionally been thought to be sterile - even if a certain amount of commensal bacteria have usually been detected in milk samples. The first part of this licentiate thesis contains a short literature review about the anatomy and physiology of breast feeding, human milk chemical and microbiological composition, mastitis, intestinal flora and bacteriocins. The second part reports on the experiments of the licentiate work, concentrating on the microbial diversity in the milk of healthy breast-feeding mothers, and the ability of these bacteria to produce antibacterial substances against pathogenic bacteria. The results indicate that human milk is a source of commensal bacteria for infant intestine. 509 random isolates from 40 breast milk samples were isolated and identified by 16S rRNA sequencing. Median bacterial count was about 600 colony forming units per milliliter. Over half of the isolates were staphylococci, and almost one third streptococci. The most common species were skin bacteria Staphylococcus epidermidis and oral bacteria Streptococcus salivarius and Streptococcus mitis. Lactic acid bacteria, identified as members of Lactobacillus-, Lactococcus- and Leuconostoc -genera, were found in five milk samples. Enterococci were found in three samples. A novel finding in this study is the capability of these commensal bacteria to inhibit the growth of pathogens. In 90 precent of the milk samples commensal bacteria inhibiting the growth of Staphylococcus aureus were found. In 40 precent of samples the colonies could block the growth completely. One fifth of the isolated Staph. epidermidis strains, half of Str. salivarius strains, and all lactic acid bacteria and enterococci could inhibit or block the growth of Staph. aureus. In further study also Listeria innocua- and Micrococcus luteus active isolates were found in 33 and 11 precent of milk samples (out of 140). Furthermore, two Lactococcus lactis isolates from the breast milk were shown to produce bacteriocin nisin, which is an antimicrobial molecule used as a food preservative. The importance of these human milk commensal bacteria in the development of newborn intestinal flora and immune system, as well as in preventing maternal breast infections, should be further explored.

Selective inhibition of IFNG-induced autophagy by Mir155- and Mir31-responsive WNT5A and SHH signaling

Autophagy is one of the major immune mechanisms engaged to clear intracellular infectious agents. However, several pathogens have evolved strategies to evade autophagy. Here, we demonstrated that Mycobacteria, Shigella, and Listeria but not Klebsiella, Staphylococcus, and Escherichia inhibit IFNG-induced autophagy in macrophages by evoking selective and robust activation of WNT and SHH pathways via MTOR. Utilization of gain- or loss-of-function analyses as well as mir155-null macrophages emphasized the role of MTOR-responsive epigenetic modifications in the induction of Mir155 and Mir31. Importantly, cellular levels of PP2A, a phosphatase, were regulated by Mir155 and Mir31 to fine-tune autophagy. Diminished expression of PP2A led to inhibition of GSK3B, thus facilitating the prolonged activation of WNT and SHH signaling pathways. Sustained WNT and SHH signaling effectuated the expression of anti-inflammatory lipoxygenases, which in tandem inhibited IFNG-induced JAK-STAT signaling and contributed to evasion of autophagy. Altogether, these results established a role for new host factors and inhibitory mechanisms employed by the pathogens to limit autophagy, which could be targeted for therapeutic interventions.

Nutraceutical and functional food bioactive peptides in beef

In recent years, the potential to positively modulate human health through dietary approaches has received considerable attention. Bioactive peptides which are released during the hydrolysis or fermentation of food proteins or following digestion may exert beneficial physiological effects in vivo. The aim of this work was to isolate, characterise and evaluate Angiotensin-І-converting enzyme (ACE-І) inhibitory, antimicrobial and antioxidant peptides from the bovine myofibrillar proteins actin and myosin. In order to generate these peptides, the myofibrillar proteins actin and myosin were hydrolysed with digestive enzymes pepsin, trypsin and α-chymotrypsin, or with the industrial thermolysin-like enzyme “Thermoase”, Amano Inc. It was found that each hydrolysate generated contained peptides which possessed ACE inhibitory, antioxidant and antimicrobial activity. The peptides responsible in part for the observed ACE inhibitory, antioxidant and antimicrobial activity of a number of hydrolysates were isolated using the method of RP-HPLC and the bioactive peptides contained within each active fraction was determined using either MALDI-TOF MS/MS or N-terminal peptide sequencing. During the course of this thesis six ACE inhibitory and five antimicrobial peptides were identified. It was determined that the reported antioxidant activity was a direct result of a number of peptides working in synergy with each other. The IC50 values of the six ACE inhibitory peptides ranged in values of 6.85 to 75.7 µM which compare favourably to values previously reported for other food derived ACE inhibitory peptides, particularly the well known milk peptides IPP and VPP, IC50 values of 5 and 9 µM respectively. All five antimicrobial peptides identified in this thesis displayed activity against Escherichia coli, Salmonella typhimurium, Staphylococcus aureus and Listeria innocua with MIC values ranging from 0.625 to10 mM. The activity of each antimicrobial peptide was strain specific. Furthermore the role and importance of charged amino acids to the activity of antimicrobial peptides was also determined. Generally the removal of charged amino acids from the sequence of antimicrobial peptides resulted in a loss of antimicrobial activity. In conclusion, this thesis revealed that a range of bioactive peptides exhibiting ACE inhibitory, antioxidant and antimicrobial activities were encrypted in bovine myofibrillar proteins that could be released using digestive and industrial enzymes. Finally enzymatic hydrolysates of muscle proteins could potentially be incorporated into functional foods; however, the potential health benefits would need to be proven in human clinical studies.

Avaliação da iluminação UV-C como sistema de desinfeção eco-inovador em laranja minimamente processada

Dissertação de mest., Engenharia Biológica, Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2011

Propriedades biotecnológicas de microrganismos isolados de processos fermentativos

Dissertação de mestrado, Tecnologia dos Alimentos, Instituto Superior de Engenharia, Universidade do Algarve, 2014

Formulação de um biofilme para controlo da listéria em queijos

Dissertação de Mestrado, Tecnologia e Segurança Alimentar, 12 Fevereiro de 2016, Universidade dos Açores.

Diversity and effectiveness of tropical mangrove soil microflora on the degradation of polythene carry bags

The diversity and load of heterotrophic bacteria and fungi associated with the mangrove soil from Suva, Fiji Islands, was determined by using the plate count method. The ability of the bacterial isolates to produce various hydrolytic enzymes such as amylase, gelatinase and lipase were determined using the plate assay. The heterotrophic bacterial load was considerably higher than the fungal load. There was a predominance of the gram positive genus, Bacillus. Other genera encountered included Staphylococcus, Micrococcus, Listeria and Vibrio. Their effectiveness on the degradation of commercial polythene carry bags made of high density polyethylene (HDPE) and low density polyethylene (LDPE) was studied over a period of eight weeks in the laboratory. Biodegradation was measured in terms of mean weight loss, which was nearly 5 % after a period of eight weeks. There was a significant increase in the bacterial load of the soil attached to class 2 (HDPE) polythene. After eight weeks of submergence in mangrove soil, soil attached to class 1 and class 3 polythene mostly had Bacillus (Staphylococcus predominated in class 2 polythene). While most of the isolates were capable of producing hydrolytic enzymes such as amylase and gelatinase, lipolytic activity was low. Class 2 HDPE suffered the greatest biodegradation.

SPATIOTEMPORAL VARIATION AND ANTIBACTERIAL ACTIVITY OF ACTINOMYCETES ISOLATED FROM HIGH ALTITUDE GRASSLAND SOILS OF TROPICAL MONTANE FOREST – KERALA, INDIA

TThe invention of novel antibiotics and other bioactive microbial metabolites continues to be an important aim in new drug discovery programmes. Actinomycetes have the potential to synthesize lots of diverse biologically vigorous secondary metabolites and in the last decades actinomycetes became the most productive source for antibiotics. Therefore in the present study we analyze the antibacterial activity of the actinomycetes isolated from grassland soil samples of Tropical Montane forest. A total of 33 actinomycete strains isolated were characterized and screened for antibacterial activities using well diffusion method against six specific pathogenic organisms. Identification of the isolates revealed that the majority of them were belonging to Streptomycetes followed by Nocardia, Micromonospora, Pseudonocardia, Streptosporangium, Nocardiopsis and Saccharomonospora. Among the 33 isolates, Gr1 strain showed antagonistic activity against all checked pathogens. Nine strains showed antibacaterial activity against Listeria, Vibrio cholera, Bacillus cereus, Staphylococcus aureus and Salmonella typhi and only 2 strains (Gr1and Gr25) showed antagonism to E. coli. The overall percentage of activity of actinomycetes isolates against each pathogenic bacterium was also calculated. While 63.63% of the actinomycetes were antagoinistic against Listeria, Vibrio cholerae, and Bacillus cereus, 60.6% of them were antagonistic to Staphylococcus aureus. Very few isolates (6.06%) showed antibacterial activity against E. coli. In general most of the actinomycetes isolates were antagonistic to grampositive bacteria such as Listeria, Bacillus and Staphylococcus than Gram-negative bacteria Vibrio cholerae, E. coli and Salmonella

Estudio de las comunidades microbianas de embutidos fermentados ligeramente acidificados mediante técnicas moleculares. Estandarización, seguridad y mejora tecnológica.

Los embutidos fermentados ligeramente acidificados son un grupo de productos tradicionales mediterráneos, caracterizados por un pH superior a 5,3. Para un control eficiente de la seguridad microbiológica de los embutidos se necesitan técnicas rápidas para la identificación y recuento de los microorganismos patógenos a estudiar. En el presente trabajo, se desarrolló una técnica para la enumeración de L. monocytogenes que combinó el método del número más probable y la identificación mediante PCR específica. Para la detección de Salmonella spp. y L. monocytogenes se desarrolló un sistema de PCR-multiplex que permitió la identificación de ambos patógenos de forma simultánea en una sola reacción. El estudio de la calidad microbiológica de los embutidos fermentados ligeramente acidificados se completó con la caracterización de las comunidades microbianas más importantes en estos productos. Se identificaron a nivel de especie los aislados de bacterias del ácido láctico (BAL), de enterococos y de cocos gram-positivos catalasa-positivos (CGC+). Posteriormente se realizó una tipificación molecular de los mismos mediante RAPD y análisis del perfil plasmídico y se estudiaron las principales características de interés higiénico-sanitario y tecnológico de las cepas. Mediante PCR se identificó Lactobacillus sakei como la especie predominante (74%), seguida por Lactobacillus curvatus (21,2%). La actividad aminoácido-descarboxilasa se asoció a la especie L. curvatus (el 66% de los aislados presentaron esta actividad). La identificación de los enterococos se realizó mediante PCR-multiplex y por secuenciación del gen sodA. Enterococcus faecium fue la especie de enterococos predominante (51,9%) seguida por Enterococcus faecalis (14,2%). Todas las cepas de E. faecalis presentaron genes asociados a factores de virulencia. E. faecalis presentó mayor resistencia a antibióticos que el resto de las especies de enterococos estudiadas. Tan sólo una cepa de E. faecium presentó el genotipo vanA (que confiere resistencia de alto nivel a la vancomicina). La identificación de los aislados de CGC+ (mediante PCR específica y amplificación de la región intergénica 16S-23S ARNr) demostró que Staphylococcus xylosus es la especie predominante en los embutidos fermentados ligeramente acidificados (80,8%). La amina biógena más común en los CGC+ fue la feniletilamina, producida por un 10,8% de aislados. Un pequeño porcentaje de aislados fueron mecA+ (4,6%), presentando además resistencia a múltiples antibióticos. El potencial enterotoxigénico de las cepas de CGC+ fue muy reducido (3,3% de los aislados), detectándose únicamente el gen entC. El estudio pormenorizado de las comunidades bacterianas de interés permitió la selección de 2 cepas de L. sakei y 2 cepas de S. xylosus con características tecnológicas e higiénico-sanitarias óptimas. Para evaluar su efectividad como cultivos iniciadores se elaboraron dos tipos de embutidos ligeramente ácidos, chorizo y fuet, inoculados con microorganismos patógenos (Salmonella spp., L. monocytogenes y S. aureus). El uso de cultivos iniciadores permitió el control de L. monocytogenes, Enterobacteriaceae y Enterococcus así como del contenido en aminas biógenas. Los recuentos de Salmonella spp. disminuyeron de forma significante durante la maduración de los embutidos, independientemente del uso de cultivos iniciadores. El uso del tratamiento de alta presión (400 MPa) en los embutidos madurados consiguió la ausencia de Salmonella spp. en los lotes tratados.

Mejora de la seguridad alimentaria en productos cárnicos listos para el consumo mediante la aplicación combinada de tecnologías de conservación emergentes

La demanda creixent de productes mínimament processats i llestos per al consum planteja un important repte per a la seguretat alimentaria i ha conduit al desenvolupament de tractaments suaus que permetin inhibir el creixement microbià conservant la qualitat dels aliments. Els treballs recollits a la present tesi van plantejar diverses estratègies consistents en la combinació d'obstacles al creixement microbià per a millorar la seguretat de productes carnis llestos per al consum. Amb l'objectiu de millorar la seguretat dels embotits poc àcids, es va valorar l'aplicació del tractament per alta pressió hidrostàtica (APH) i l'addició de cultius iniciadors en embotits poc àcids. Per altra banda, per a reduir el risc de L. monocytogenes durant la conservació del pernil cuit llescat, es va avaluar l'efecte combinat de l'addició d'antimicrobians naturals (lactatat-diacetat i enterocines), afegits directament o a través de l'envasament antimicrobià, i el tractament per alta pressió hidrostàtica.

Produção, caracterização, purificação parcial e aplicação de um peptídeo antimicrobiano produzido por Bacillus licheniformis P40

Uma bactéria identificada como Bacillus licheniformis P40 isolada de intestino de peixe (Leporinus sp.) da bacia amazônica foi estudada quanto à sua capacidade de produzir antimicrobianos. O sobrenadante da cultura obtido em caldo de cérebro e coração (BHI) foi caracterizado, sendo ativo contra importantes bactérias patogênicas e deteriorantes como L. monocytogenes, B. cereus, E. carotovora e isolados clínicos de Streptococcus. Este foi parcialmente purificado através de precipitação com sulfato de amônio e cromatografia de gel filtração e de troca iônica. Foram assim isoladas duas substâncias com atividade antimicrobiana, sendo uma delas de natureza protéica. Esta foi estável a altas temperaturas (100o C), numa ampla faixa de pH e mostrou propriedades de biosurfactante. O sobrenadante parcialmente purificado foi utilizado para o combate a um importante fitopatógeno: Erwinia carotovora. Uma dose de 6400 UA/mL foi bactericida para uma concentração de 107 UFC/mL em 20 minutos in vitro. A substância foi capaz de evitar a formação da podridão mole em batatas (in vivo). Foi estudada a produção da atividade antimicrobiana em resíduos e sub-produtos da indústria de alimentos, sendo escolhido o soro de queijo para otimizar a produção através de um experimento fatorial 23 variando as condições de temperatura, pH e concentração de soro de queijo em pó. As melhores condições foram para temperaturas entre 26 e 37o C e pH entre 6,5 e 7,5 para uma concentração de soro de 7%, sendo que aumentos na concentração levaram a aumentos na produção.

Estudo de bacteriocinas produzidas por espécies de Enterococcus

As enterocinas são compostos de natureza protéica que apresentam atividade antimicrobiana contra bactérias patogênicas e deteriorantes de alimentos. Por esta razão, nos últimos anos, o interesse por estas substâncias tem aumentado devido ao seu uso potencial como biopreservante de alimentos. Realizando uma triagem com 352 cepas de Enterococcus spp para detectar atividade antimicrobiana, foram encontradas 18 cepas (5%) produtoras de enterocinas, pertencentes às espécies E. faecalis, E. faecium e E. mundtii todas provenientes de amostras de fezes de humanos. Destas cepas foram produzidos sobrenadantes livres de células e realizados testes para caracterização das enterocinas produzidas. As enterocinas produzidas pelas 18 cepas apresentaram o mesmo espectro de atividade antimicrobiana, inibindo 4 espécies do gênero Listeria, Lactobacillus plantarum e Salmonella Enteritidis. Todas foram parcialmente estáveis ao aquecimento (121ºC por 10 minutos), a variações de pH de 2 a 10 e a estocagem por 60 dias a 4ºC e a -20ºC. A sensibilidade a proteases confirmou a natureza protéica destas substâncias antimicrobianas. Com cinco sobrenadantes livres de células foram realizadas curvas de produção de enterocinas e todas iniciaram a produção na fase logarítmica de crescimento, indicando cinética de metabólito primário. Com estes sobrenadantes foi determinado o peso molecular aproximado de 3 KDa, utilizando a técnica de SDS-PAGE. As cepas com atividade antimicrobiana no sobrenadante livre de células não apresentaram os genes para produção das enterocinas A e B, mas 14 cepas de E. faecium apresentaram o gene para enterocina A e 9 para enterocina B, sendo que apenas uma cepa apresentou ambos os genes. Nenhuma das 18 cepas produtoras de enterocinas apresentou atividade hemolítica e presença de adesinas, todas apresentaram cápsula. Os resultados obtidos neste trabalho indicam que estas enterocinas demonstram um potencial aplicabilidade em novos estudos relacionados aos biopreservantes.

Microbiological counting in lamb carcasses from an abattoir in São Paulo, Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Estudo microbiológico em carcaças bovinas e influência da refrigeração sobre a microbiota contaminante

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)