Os compostos antimicrobianos naturais são um tema de grande interesse devido ao aumento da demanda por alimentos seguros e de alta qualidade. A utilização de lipossomas é uma alternativa interessante para proteger antimicrobianos nos alimentos, além de fornecer compostos naturais de liberação controlada. Os lipossomas revestidos com polissacarídeos apresentam melhor estabilidade, representando uma alternativa aos lipossomas convencionais. Inicialmente, os nanolipossomas que encapsulam a nisina foram preparados com fosfatidilcolina de soja (PC) e pectina ou ácido poligalacturônico. Os lipossomas desenvolvidos apresentaram alta eficiência de encapsulação, baixo índice de polidispersão e foram estáveis durante 21 dias a 7 °C e 25 °C. A atividade antimicrobiana foi observada contra cinco cepas diferentes de Listeria em placas de ágar de leite, com uma melhor eficiência contra L. innocua 6a. Em um segundo momento, as características estruturais dos lipossomas foram estudadas por dispersão de raios-X de pequeno ângulo (SAXS) e as amostras foram submetidas a ciclos de temperatura (20-60 °C). Para isso, os lipossomas foram desenvolvidos contendo pectina ou ácido poligalacturônico pelos métodos de hidratação de filme e evaporação em fase reversa, para encapsular nisina. A análise de SAXS confirmou a presença de estruturas lamelares em todas as amostras. Além disso, parte da estrutura multilamelar tornou-se cúbica, provavelmente devido à presença de nisina nos lipossomas. A adição de polissacarídeos mostrou diferenças entre as fases cúbicas formadas. Em última análise, a mistura de lisozima e nisina foi encapsulada em lipossomas contendo polissacarídeos. O diâmetro médio dos lipossomas foi de 85,6 e variou para 77,3 e 79,9 nm com a incorporação de pectina ou ácido poligalacturônico, respectivamente. O potencial zeta dos lipossomas com polissacarídeos foi de cerca de -30 mV, mostrando alta eficiência de encapsulação. A atividade antimicrobiana foi avaliada a 37 °C, mostrando que a PC-pectina reduziu a população de L. monocytogenes em 2 log UFC/mL e 5 log UFC/mL em leite integral e desnatado, respectivamente. Em refrigeração, a PC-pectina reduziu a população de L. monocytogenes para quase zero por até 25 dias em leite desnatado. Portanto, pode dizer-se que os lipossomas que contêm polissacarídeos podem ser uma tecnologia promissora para o encapsulamento da lisozima e nisina. Além disso, a existência de estrutura cúbica nos lipossomas pode proporcionar liberação controlada de antimicrobianos. / Natural antimicrobial compounds are a topic of utmost interest due to the increased demand for safe and high-quality foods. The use of liposomes is an interesting alternative to protect antimicrobials in food, also providing controlled release natural compounds. Polysaccharides coated liposomes present better stability, representing an alternative to conventional liposomes. Initially, nanoliposomes encapsulating nisin were prepared with soy phosphatidylcholine (PC) and pectin or polygalacturonic acid. The liposomes developed presented high encapsulation efficiency, low polydispersity index, and were stable for 21 days at 7°C and 25°C. The antimicrobial activity was observed against five different strains of Listeria in milk-agar plates, with a better efficiency against L. innocua 6a. In a second moment, structural characteristics of liposomes were studied by small angle X-ray scattering (SAXS) and the samples were submitted to temperature cycles (20-60°C). For this, liposomes were developed containing pectin or polygalacturonic acid by the thin-film hydration method and reverse phase evaporation method for nisin encapsulation. The analysis of SAXS confirmed the presence of lamellar structures in all the samples. In addition, part of the multilamellar structure became cubic, probably due to the presence of nisin in the liposomes. The addition of polysaccharides showed differences between the cubic phases formed. Ultimately, the mixture of lysozyme and nisin were encapsulated in liposomes containing polysaccharides. The mean diameter of the liposomes was 85.6 and varied to 77.3 and 79.9 nm with the incorporation of pectin or polygalacturonic acid, respectively. The zeta potential of liposomes with polysaccharides were around -30 mV, showing high encapsulation efficiency. The antimicrobial activity was assessed at 37 °C, showing that PC-pectin reduced the population of L. monocytogenes to 2 log CFU/mL and 5 log CFU/mL in whole and skim milk, respectively. At under refrigeration, PC-pectin reduced the population of L. monocytogenes to almost zero for up to 25 days in skim milk. Therefore, it can say that the liposomes containing polysaccharides can be a promising technology for the encapsulation of lysozyme and nisin. In addition, the existence of cubic structure in the liposomes can provide controlled release of antimicrobials.

Os compostos antimicrobianos naturais são um tema de grande interesse devido ao aumento da demanda por alimentos seguros e de alta qualidade. A utilização de lipossomas é uma alternativa interessante para proteger antimicrobianos nos alimentos, além de fornecer compostos naturais de liberação controlada. Os lipossomas revestidos com polissacarídeos apresentam melhor estabilidade, representando uma alternativa aos lipossomas convencionais. Inicialmente, os nanolipossomas que encapsulam a nisina foram preparados com fosfatidilcolina de soja (PC) e pectina ou ácido poligalacturônico. Os lipossomas desenvolvidos apresentaram alta eficiência de encapsulação, baixo índice de polidispersão e foram estáveis durante 21 dias a 7 °C e 25 °C. A atividade antimicrobiana foi observada contra cinco cepas diferentes de Listeria em placas de ágar de leite, com uma melhor eficiência contra L. innocua 6a. Em um segundo momento, as características estruturais dos lipossomas foram estudadas por dispersão de raios-X de pequeno ângulo (SAXS) e as amostras foram submetidas a ciclos de temperatura (20-60 °C). Para isso, os lipossomas foram desenvolvidos contendo pectina ou ácido poligalacturônico pelos métodos de hidratação de filme e evaporação em fase reversa, para encapsular nisina. A análise de SAXS confirmou a presença de estruturas lamelares em todas as amostras. Além disso, parte da estrutura multilamelar tornou-se cúbica, provavelmente devido à presença de nisina nos lipossomas. A adição de polissacarídeos mostrou diferenças entre as fases cúbicas formadas. Em última análise, a mistura de lisozima e nisina foi encapsulada em lipossomas contendo polissacarídeos. O diâmetro médio dos lipossomas foi de 85,6 e variou para 77,3 e 79,9 nm com a incorporação de pectina ou ácido poligalacturônico, respectivamente. O potencial zeta dos lipossomas com polissacarídeos foi de cerca de -30 mV, mostrando alta eficiência de encapsulação. A atividade antimicrobiana foi avaliada a 37 °C, mostrando que a PC-pectina reduziu a população de L. monocytogenes em 2 log UFC/mL e 5 log UFC/mL em leite integral e desnatado, respectivamente. Em refrigeração, a PC-pectina reduziu a população de L. monocytogenes para quase zero por até 25 dias em leite desnatado. Portanto, pode dizer-se que os lipossomas que contêm polissacarídeos podem ser uma tecnologia promissora para o encapsulamento da lisozima e nisina. Além disso, a existência de estrutura cúbica nos lipossomas pode proporcionar liberação controlada de antimicrobianos. / Natural antimicrobial compounds are a topic of utmost interest due to the increased demand for safe and high-quality foods. The use of liposomes is an interesting alternative to protect antimicrobials in food, also providing controlled release natural compounds. Polysaccharides coated liposomes present better stability, representing an alternative to conventional liposomes. Initially, nanoliposomes encapsulating nisin were prepared with soy phosphatidylcholine (PC) and pectin or polygalacturonic acid. The liposomes developed presented high encapsulation efficiency, low polydispersity index, and were stable for 21 days at 7°C and 25°C. The antimicrobial activity was observed against five different strains of Listeria in milk-agar plates, with a better efficiency against L. innocua 6a. In a second moment, structural characteristics of liposomes were studied by small angle X-ray scattering (SAXS) and the samples were submitted to temperature cycles (20-60°C). For this, liposomes were developed containing pectin or polygalacturonic acid by the thin-film hydration method and reverse phase evaporation method for nisin encapsulation. The analysis of SAXS confirmed the presence of lamellar structures in all the samples. In addition, part of the multilamellar structure became cubic, probably due to the presence of nisin in the liposomes. The addition of polysaccharides showed differences between the cubic phases formed. Ultimately, the mixture of lysozyme and nisin were encapsulated in liposomes containing polysaccharides. The mean diameter of the liposomes was 85.6 and varied to 77.3 and 79.9 nm with the incorporation of pectin or polygalacturonic acid, respectively. The zeta potential of liposomes with polysaccharides were around -30 mV, showing high encapsulation efficiency. The antimicrobial activity was assessed at 37 °C, showing that PC-pectin reduced the population of L. monocytogenes to 2 log CFU/mL and 5 log CFU/mL in whole and skim milk, respectively. At under refrigeration, PC-pectin reduced the population of L. monocytogenes to almost zero for up to 25 days in skim milk. Therefore, it can say that the liposomes containing polysaccharides can be a promising technology for the encapsulation of lysozyme and nisin. In addition, the existence of cubic structure in the liposomes can provide controlled release of antimicrobials.

Os compostos antimicrobianos naturais são um tema de grande interesse devido ao aumento da demanda por alimentos seguros e de alta qualidade. A utilização de lipossomas é uma alternativa interessante para proteger antimicrobianos nos alimentos, além de fornecer compostos naturais de liberação controlada. Os lipossomas revestidos com polissacarídeos apresentam melhor estabilidade, representando uma alternativa aos lipossomas convencionais. Inicialmente, os nanolipossomas que encapsulam a nisina foram preparados com fosfatidilcolina de soja (PC) e pectina ou ácido poligalacturônico. Os lipossomas desenvolvidos apresentaram alta eficiência de encapsulação, baixo índice de polidispersão e foram estáveis durante 21 dias a 7 °C e 25 °C. A atividade antimicrobiana foi observada contra cinco cepas diferentes de Listeria em placas de ágar de leite, com uma melhor eficiência contra L. innocua 6a. Em um segundo momento, as características estruturais dos lipossomas foram estudadas por dispersão de raios-X de pequeno ângulo (SAXS) e as amostras foram submetidas a ciclos de temperatura (20-60 °C). Para isso, os lipossomas foram desenvolvidos contendo pectina ou ácido poligalacturônico pelos métodos de hidratação de filme e evaporação em fase reversa, para encapsular nisina. A análise de SAXS confirmou a presença de estruturas lamelares em todas as amostras. Além disso, parte da estrutura multilamelar tornou-se cúbica, provavelmente devido à presença de nisina nos lipossomas. A adição de polissacarídeos mostrou diferenças entre as fases cúbicas formadas. Em última análise, a mistura de lisozima e nisina foi encapsulada em lipossomas contendo polissacarídeos. O diâmetro médio dos lipossomas foi de 85,6 e variou para 77,3 e 79,9 nm com a incorporação de pectina ou ácido poligalacturônico, respectivamente. O potencial zeta dos lipossomas com polissacarídeos foi de cerca de -30 mV, mostrando alta eficiência de encapsulação. A atividade antimicrobiana foi avaliada a 37 °C, mostrando que a PC-pectina reduziu a população de L. monocytogenes em 2 log UFC/mL e 5 log UFC/mL em leite integral e desnatado, respectivamente. Em refrigeração, a PC-pectina reduziu a população de L. monocytogenes para quase zero por até 25 dias em leite desnatado. Portanto, pode dizer-se que os lipossomas que contêm polissacarídeos podem ser uma tecnologia promissora para o encapsulamento da lisozima e nisina. Além disso, a existência de estrutura cúbica nos lipossomas pode proporcionar liberação controlada de antimicrobianos. / Natural antimicrobial compounds are a topic of utmost interest due to the increased demand for safe and high-quality foods. The use of liposomes is an interesting alternative to protect antimicrobials in food, also providing controlled release natural compounds. Polysaccharides coated liposomes present better stability, representing an alternative to conventional liposomes. Initially, nanoliposomes encapsulating nisin were prepared with soy phosphatidylcholine (PC) and pectin or polygalacturonic acid. The liposomes developed presented high encapsulation efficiency, low polydispersity index, and were stable for 21 days at 7°C and 25°C. The antimicrobial activity was observed against five different strains of Listeria in milk-agar plates, with a better efficiency against L. innocua 6a. In a second moment, structural characteristics of liposomes were studied by small angle X-ray scattering (SAXS) and the samples were submitted to temperature cycles (20-60°C). For this, liposomes were developed containing pectin or polygalacturonic acid by the thin-film hydration method and reverse phase evaporation method for nisin encapsulation. The analysis of SAXS confirmed the presence of lamellar structures in all the samples. In addition, part of the multilamellar structure became cubic, probably due to the presence of nisin in the liposomes. The addition of polysaccharides showed differences between the cubic phases formed. Ultimately, the mixture of lysozyme and nisin were encapsulated in liposomes containing polysaccharides. The mean diameter of the liposomes was 85.6 and varied to 77.3 and 79.9 nm with the incorporation of pectin or polygalacturonic acid, respectively. The zeta potential of liposomes with polysaccharides were around -30 mV, showing high encapsulation efficiency. The antimicrobial activity was assessed at 37 °C, showing that PC-pectin reduced the population of L. monocytogenes to 2 log CFU/mL and 5 log CFU/mL in whole and skim milk, respectively. At under refrigeration, PC-pectin reduced the population of L. monocytogenes to almost zero for up to 25 days in skim milk. Therefore, it can say that the liposomes containing polysaccharides can be a promising technology for the encapsulation of lysozyme and nisin. In addition, the existence of cubic structure in the liposomes can provide controlled release of antimicrobials.

Chapter I reviews current literature on gene transfer systems in lactic acid bacteria, how genetically altered microorganisms for food are presently regulated, and how nisin is used as a food preservative. Chapter II investigates previous reports which linked genes for nisin biosynthesis and sucrose utilization (Nip+Suc+) to plasmid DNA in two well characterized L· lactis subsp. lactis strains. Plasmid curing studies, conjugations, and DNA-DNA hybridizations indicated that these genes were encoded by chromosomal loci in all Nip+Suc+ strains examined. Similar results were noted in nisin-sucrose transconjugants of L. lactis subsp. cremoris and S. salivarius subsp. thermophilus in Chapters III and IV. Chapter III describes the use of conjugation to construct nisin-producing Lactococcus lactis subsp. cremoris strains. The direct-plate conjugation method was developed to facilitate transfer of Nip+Suc+ to L. lactis subsp. cremoris recipients. DNA-DNA hybridizations to transconjugant DNAs with an oligonucleotide that detected the nisin structural gene, nisA, demonstrated that this gene was transferred during conjugation. Lactococcus lactis subsp. cremoris Nip+Suc+ transconjugants retained the recipient strain phenotype with respect to bacteriophage resistance and acid production in milk. These results indicated that it would be feasible to construct nisin-producing L. lactis subsp. cremoris strains for mixed and multiple starter systems. Chapter IV investigates features of Nip+Suc+ transfer using a Lactococcus lactis subsp. lactis model system. Intergeneric transfer of nisin-sucrose genes was also achieved between lactococcal Nip+Suc+ donors and Streptococcus salivarius subsp. thermophilus recipients. Streptococcal transconjugants acquired Suc+ and nisin immunity but did not produce nisin. DNA-DNA hybridizations, however, demonstrated that nisA was present in these transconjugants. To investigate whether nisA was involved in nisin immunity, this gene was cloned and electro-transformed into Lactococcus lactis subsp. lactis LM0230. Electro-transformants did not express nisin immunity or any other trait linked to nisin production in lactococci. Results presented in Chapter V indicate that nisin may have application for control or prevention of bovine mastitis. Gram-positive pathogens which cause bovine mastitis were examined for their susceptibility to nisin. Disc diffusion assays indicated that minimum inhibitory concentrations of nisin ranged from 10 to 250 ug per ml. In addition, 50 ug of nisin per ml in milk inhibited all gram-positive pathogens tested.

A more quantitative understanding of peptide loading and release from polyethylene oxide (PEO) brush layers will provide direction for development of new strategies for drug storage and delivery. The antimicrobial peptide nisin shows potent activity against Gram-positive bacteria including the most prevalent implant-associated pathogens, its mechanism of action minimizes the opportunity for the rise of resistant bacteria and it does not appear to be toxic to humans, suggesting good potential for its use in antibacterial coatings for selected medical devices. In this work, optical waveguide lightmode spectroscopy was used to record changes in adsorbed mass during cyclic adsorption-elution experiments with nisin, at uncoated and PEO-coated surfaces. PEO layers were prepared by radiolytic grafting of Pluronic® surfactant F108 or F68 to silanized silica surfaces, producing long- or short-chain PEO layers, respectively. Kinetic patterns were interpreted with reference to a model accounting for history-dependent adsorption, in order to evaluate rate constants for nisin adsorption and desorption, as well as the effect of pendant PEO on the lateral clustering behavior of nisin. Lateral rearrangement and clustering of adsorbed nisin was apparent on uncoated and F68-coated surfaces, but not on F108-coated surfaces. In addition, nisin showed greater resistance to elution by peptide-free buffer from uncoated and F68-coated surfaces. These results are consistent with shorter PEO chains allowing for peptide adsorption to the base substrate in the case of F68-coated surfaces, while adsorption to the F108-coated surfaces is apparently governed by the presence of a hydrophobic core within the brush layer itself. Further, these results suggest that while peptide location within the hydrophobic core provides stability against lateral rearrangement, the pendant PEO chains themselves provide no steric barrier to nisin rearrangement within the brush layer. / Graduation date: 2012

Thesis (MSc)--Stellenbosch University, 2001. / ENGLISH ABSTRACT: Lactic acid bacteria are present in many foods and beverages and are used as starter cultures in the production of a variety of fermented products. Many of these bacteria produce ribosomally synthesized antimicrobial peptides (bacteriocins), which inhibit the growth of bacteria genetically closely related to the producer cell. Since many of these target bacteria include foodbome pathogens such as Bacillus spp., Clostridium spp., Listeria spp., and Staphylococcus spp., the practical importance of these peptides as food preservatives has been well documented and, in the case of nisin and pediocin PA-I, commercially explored. The increased demand from health conscious consumers for foods with no chemical preservatives is putting renewed pressure on the producer to supply a "clean and green" product, but with the same or even an extended shelf life. Various research groups are screening lactic acid bacteria for production of novel broad-spectrum antimicrobial peptides or are exploring the possibilities of altering known bacteriocins to inhibit Gram-negative bacteria, yeasts and molds. Pediocin PD-I, produced by Pediococcus damnosus NCFB 1832, belongs to the class Ila bacteriocins, i.e. heat-stable Listeria-active peptides, containing the YGNGV -consensus sequence in the N-terminal region. Little is known about the production and mode of activity of pediocin PD-I. In this study, production of pediocin PD-I was significantly increased by optimizing the growth medium, De Man Rogosa and Sharpe (MRS) broth. Addition of bacteriological peptone (1.7%, w/v), manganous sulphate (0.014%, w/v) and Tween 80 (3%, v/v), and lowering of the pH during fermentation stimulated pediocin PD-I production and the level of organic acids produced. Maximum levels of bacteriocin activity were recorded at an initial pH of 6.7 in the latter medium. Under these conditions the specific bacteriocin activity increased by a factor of approximately six after 55 h of fermentation. The effect of pediocin PD-I, plantaricin 423, produced by Lactobacillus plantarum 423, and commercial grade nisin (Aplin and Barrett Ltd., Trowbrige, Wilts, England) was tested against planktonic cells of Oenococcus oeni and a biofilm of the cells established on stainless steel surfaces identical to those used in wineries. After 5 h of treatment with 3000 AU (arbitrary units )/ml of each bacteriocin, all planktonic cells of 0. oeni in a modified Chardonnay must medium were killed. All viable cells in the biofilm were killed after only 1 h in the presence of 3000 AU/ml of anyone of the bacteriocins. In addition, pediocin PD-I, plantaricin 423 and nisin removed the biofilms from the surfaces and reduced the biomass either completely, as in the case of pediocin PD-I, or by 58% and 50% as in the case of plantaricin 423 and nisin, respectively. These same results were recorded after 5 h of treatment with 3000 AU/ml in a modified Chardonnay must medium. To our knowledge this is the first report of controlling biofilm formation of malolactic bacteria on stainless steel surfaces with natural antimicrobial peptides. This implies that, apart from being very effective in controlling the cell numbers of free-living cells of 0. oeni, the three bacteriocins, especially pediocin PD-I, could also be used as natural sanitizers. The fact that the production and activity levels ofpediocin PD-I could be increased without genetically modifying the producer strain is an added advantage. / AFRIKAANSE OPSOMMING: Melksuurbakterieë is teenwoordig in verskeie soorte voedsel- en drankprodukte en word as suurselkulture in die produksie van 'n verskeidenheid gefermenteerde produkte gebruik. Baie van hierdie bakterieë produseer ribosomaal-vervaardigde antimikrobiese peptiede (bakteriosiene) wat die groei van ander bakterieë, geneties naverwant aan die produserende organisme, inhibeer. Omdat baie van hierdie bakterieë voedselpatogene soos Bacillus spp., Clostridium spp., Listeria spp. en Staphylococcus spp. insluit, is die praktiese belang van hierdie peptiede reeds deeglik ondersoek en word, soos in die geval van nisien en pediosien PA-I, kommersieel gebruik. Die toenemende behoefte van die verbruiker na voedselprodukte met geen chemiese preserveermiddels plaas nuwe druk op die vervaardiger om veilige voedselprodukte te produseer, maar met dieselfde of selfs langer rakleeftyd. Verskeie navorsingsgroepe bestudeer melksuurbakterieë vir die produksie van unieke antimikrobiese peptiede met 'n wye spektrum van inhibisie en ondersoek ook die moontlikhede om hierdie bakteriosiene geneties te manipuleer ten einde Gram-negatiewe bakterieë, giste en swamme te inhibeer. Pediosien PD-l, geproduseer deur Pediococcus damnosus NCFB 1832, word as 'n klass na bakteriosien geklassifiseer. Hierdie groep sluit in die hitte-stabiele Listeria-aktiewe peptiede, met 'n YGNGV-konsensus volgorde in die N-terminale deel van die peptied. Min is egter bekend oor die meganisme van werking van hierdie bakteriosiene. In hierdie studie is die produksie van pediosien PD-l betekenisvol verhoog met die optimalisering van die vloeibare groeimedium De Man Rogosa en Sharpe (MRS). Die toevoeging van bakteriologiese peptone (1.7%, miv), mangaan sulfaat (0.014%, miv) en Tween 80 (3.0%, v/v) en 'n afname in die pH gedurende groei het pediosien PD-l-poduksie gestimuleer en sodoende ook die vlak van organiese sure wat geproduseer is. Maksimum vlakke van bakteriosien-aktiwiteit is in hierdie medium met 'n aanvangs-pH van 6.7 waargeneem. Onder hierdie omstandighede, en na 55 uur van fermentasie, het die spesifieke aktiwiteit van die bakteriosien met 'n faktor van ongeveer ses verhoog. Die effek van pediosien PD-l, plantarisien 423, geproduseer deur Lactobacillus plantarum 423, en 'n kommersiële graad nisien (Aplin and Barrett Ltd., Trowbride, Wilts, Engeland) is teen die planktoniese selle van Oenococcus oeni en 'n biofilm van hierdie selle, gevestig op 'n vlekvrye staaloppervlak identies aan wat in wynkelders gebruik word, getoets. Na 5 ure van behandeling met 3000 AB (arbitrêre eenhede)/ml van elke bakteriosien, is al die planktoniese selle van O. oeni in 'n gemodifiseerde Chardonnay mos-medium vernietig. Alle lewensvatbare selle in die biofilm is ook na slegs 1 uur in die teenwoordigheid van 3000 AE/ml van enige een van hierdie bakteriosiene vernietig. Verdermeer het pediosien PD-I, plantarisien 423 en nisien ook die biofilm op die vlekvrye staal-oppervlak verwyder. In die geval van pediosien PD-I is 'n totale afname van die biomassa-oppervlak waargeneem, terwyl plantarisien 423 en nisien 58% en 50% van die totale biomassa verwyder het. Hierdie resultate is na 5 ure van behandeling (3000 AE/ml) in 'n gemodifiseerde Chardonnay mos-medium waargeneem. Sover ons kennis strek is hierdie die eerste verslag rakende die gebruik van natuurlike antimikrobiese peptiede om biofilm-vorming deur appel-melksuurbakterieë op vlekvrye staal oppervlaktes te beheer. Dit impliseer dat bakteriosiene, spesifiek pediosien PD-I, benewens die beheer van planktoniese selle van appel-melksuurbakterieë, ook as natuurlike oppervlak-reinigers gebruik kan word. Die feit dat die produksie en aktiwiteitsvlakke van pediosien PD-I verhoog kon word sonder om die organisme geneties te modifiseer is 'n verdere voordeel.

Thesis (PhD)-- Stellenbosch University, 2013. / ENGLISH ABSTRACT: Bacterial resistance against antibiotic treatments is a global concern and resistance to almost every known antibiotic has already been reported. There is thus a significant need for the development of novel antimicrobial drugs. In addition to probiotic traits, certain bacteria have the ability to produce antimicrobial peptides, referred to as bacteriocins. Lantibiotics, a group of small ribosomally synthesized bacteriocins, recently gained interest for their application in the medical field. Lantibiotics have a very specific structure, including lanthionine rings, that stabilise the peptides. Due to their small size and specific action, these peptides reach specific sites of infection without affecting the composition of the host’s natural microbiota. As with any therapeutic agent, antimicrobial peptides are also prone to in vivo degradation, binding, clearance via immune action and development of bacterial resistance. Nisin F, a class Ia lantibiotic produced by Lactococcus lactis subsp. lactis F10, has already shown activity against the well-known pathogens Stapylococcus aureus, Listeria monocytogenes and various antibiotic resistant strains. The aim of this study was to assess the antimicrobial activity of nisin F against systemic S. aureus infections in mice and possible immune responses elicited by the peptide. A single administration of nisin F to the peritoneal cavity protected mice from S. aureus infection for at least 15 min. After continuous administration, the peptide showed no significant antimicrobial activity against S. aureus. The peptide did, however, convey some degree of protection to infected mice by stimulating a pro-inflammatory action through lymphocyte protection. When administered to uninfected mice, nisin F had an immune boosting effect via interleukin (IL)-6 and IL-10 without being detrimental to the host. The ex vivo effects of nisin F was compared to nisin A, a natural nisin variant, and Nisaplin®, a commercially purified form of nisin A. None of the three peptides inhibited the functional capacity of leukocytes in terms of 1L-1β en IL-6 production, not even in the presence of an external stimulus (lipopolysaccharides from Escherichia coli). Cytotoxicity was detected in response to high dosages of nisin F. Serum inhibited the antimicrobial effect of nisin F and nisin A, but Nisaplin® remained unaffected. Nisin F was applied against systemic infection for the first time and the immunological effect of the peptide was investigated. Nisin F partially protected mice against S. aureus infections through immunomodulatory effects. This study provided valuable knowledge on the in vivo application of nisin F. With further optimization of nisin F preparation and application systems, the peptide might be more effective against in vivo infections. / AFRIKAANSE OPSOMMING: Bakteriële weerstand teen antibiotika wek wêreldwyd kommer en weerstand teen amper elke bekende antibiotikum is reeds aangemeld. Daar is dus 'n groot behoefte vir die ontwikkeling van nuwe antimikrobiese middels. Bykomend tot probiotiese eienskappe, het sekere bakterieë die vermoë om antimikrobiese peptiede, bekend as bakteriosiene, te produseer. ‘n Groep klein ribosomaal-gesintetiseerde bakteriosiene, lantibitiotika, is onlangs vir mediese toepassing oorweeg. Lantibiotika beskik oor 'n baie spesifieke struktuur, insluitend lantionien ringstrukture, wat die peptied stabiliseer. Weens hul klein grootte en spesifieke aksie is hierdie peptiede daartoe in staat om spesifieke areas van infeksie te bereik sonder om die gasheer se natuurlike mikrobepopulasie te beïnvloed. Soos met enige terapeutiese middel, is bakteriosiene ook geneig tot in vivo afbreking, binding, klaring via die immuunsisteem en ontwikkeling van bakteriële weerstand. Nisien F, 'n klas Ia lantibiotikum, deur Lactococcus lactis subsp. lactis F10 geproduseer, het reeds aktiwiteit teen die bekende patogene Stapylococcus aureus, Listeria monocytogenes en verskeie antibiotika-weerstandige stamme getoon. Die doel van hierdie studie was om die antimikrobiese aktiwiteit van nisien F teen sistemiese S. aureus infeksies in muise te bepaal, asook die moontlike immuunreaksies wat die peptied mag veroorsaak. 'n Enkele toediening van nisien F het muise vir ten minste 15 min teen S. aureus beskerm. Na deurlopende administrasie het die peptied geen beduidende antimikrobiese aktiwiteit teen S. aureus getoon nie. Die peptied het egter 'n mate van beskerming aan geinfekteerde muise verleen deur ‘n pro-inflammatoriese aksie te inisieer deur limfosiet beskerming. Met toediening aan gesonde diere, het nisien F 'n immuunversterkende effek teweeg gebring via interleukin (IL)-6 en IL-10 vlakke, sonder nadelige uitwerking op die gasheer. Die ex vivo effek van nisien F is ook vergelyk met nisien A, 'n natuurlike variant van nisien, asook Nisaplin®, 'n kommersieël-gesuiwerde vorm van nisien A. Nie een van die drie peptide het leukosiete se funksionele kapasiteit in terme van 1L-1β en IL-6 produksie inhibeer nie, selfs nie in die teenwoordigheid van ‘n eksterne stimulus (lipopolisakkariede van Escherichia coli) nie. Seltoksisiteit is na blootstelling aan hoë dosisse van nisien F waargeneem. Serum het die antimikrobiese effek van beide nisien F en nisien A geïnhibeer, terwyl die werking van Nisaplin® nie beïnvloed is nie. Nisien F is vir die eerste keer teen sistemiese infeksies ingespan en die immunologiese impak van die peptied is ondersoek. Nisien F het gedeeltelike beskerming aan muise met S. aureus infeksies verleen deur die immuunsisteem te versterk. Die resultate het ‘n waardevolle bydrae gelewer tot die in vivo toediening van nisien F. Met verdere optimisering van nisien F voorbereiding en toedieningsisteme, mag die peptied moontlik meer effektief teen in vivo infeksies aangewend word. / The National Research Foundation (NRF) of South Africa for financial support and funding of the research

Os produtos cárneos prontos para consumo estão entre os principais alimentos associados a surtos de listeriose, uma doença causada por Listeria monocytogenes. Este estudo objetivou avaliar a ação antimicrobiana do lactato de sódio e de duas formulações comerciais à base de nisina (produtos A e B) em mortadela, e do lactato de sódio e fumaça líquida em salsicha, experimentalmente contaminadas com um pool de seis cepas de L. monocytogenes, durante o armazenamento à vácuo à 8 ºC. Os aditivos com melhor atividade anti-listeria também foram avaliados quanto ao controle de bactérias láticas (BAL) e bolores e leveduras em produto cárneo, durante o armazenamento à 8 ºC. O produto A é uma mistura de nisina encapsulada, nisina livre e extrato de alecrim, enquanto o produto B é uma mistura de nisina livre e extrato de alecrim. L. monocytogenes foi capaz de se multiplicar em todas as formulações de mortadela ao longo do período de armazenamento. Entretanto, o produto A exerceu efeito antimicrobiano contra este patógeno. Após 10 dias de armazenamento, as contagens de L. monocytogenes nas mortadelas formuladas com o produto A foram 3 log UFC/g inferiores às contagens observadas nas demais formulações avaliadas (produto B, lactato de sódio e controle), além disso, o produto A foi capaz de reduzir a velocidade de multiplicação deste microrganismo. Em relação às salsichas tratadas com fumaça líquida, as contagens de L. monocytogenes, no oitavo dia de armazenamento foram 3 log UFC/g inferiores às contagens de L. monocytogenes nas salsichas formuladas com lactato de sódio e controle. Ao final do período de armazenamento (28 dias) observou-se diferença de 2 log UFC/g nas contagens de L. monocytogenes entre as salsichas formuladas com lactato de sódio + fumaça líquida e as demais formulações. A fumaça líquida foi capaz de inibir a multiplicação de bolores e leveduras por 21 dias de armazenamento, observando-se diferença de 2 log UFC/g entre as contagens observadas nas salsichas imersas em fumaça líquida e não imersas. As contagens de BAL permaneceram baixas ao longo do período de armazenamento (< 2 log UFC/g). Também não foi observada alteração nos valores de pH e atividade de água dos produtos cárneos, o que permite concluir que as diferenças observadas nas contagens de L. monocytogenes e bolores e leveduras são devidas à ação dos antimicrobianos adicionados aos produtos cárneos. A partir destes resultados é possível concluir que o produto A e a fumaça líquida são uma barreira adicional para o controle de L. monocytogenes em mortadela e salsichas, respectivamente, durante armazenamento a 8ºC. / Ready-to-eat meat products are among the foods most frequently associated to outbreaks of listeriosis, a disease caused by Listeria monocytogenes. The objective of the present study was to evaluate the antimicrobial activity of sodium lactate and two nisin-based commercial products (A and B) in bologna, and sodium lactate and liquid smoke in frankfurters, experimentally contaminated with a pool of six strains of L. monocytogenes, during storage at 8 ºC under vacuum. The antimicrobial with better activity against L. monocytogenes was also evaluated against yeasts and molds and lactic acid bacteria. Product A is a mixture of encapsulated nisin, free nisin and rosemary extract, and product B is a mixture of free nisin and rosemary extract. L. monocytogenes was able to grow in all bologna formulations during storage. However, product A was capable of reducing the growth rate of the microorganism. Besides, L. monocytogenes populations in bologna formulated with product A were 3 log CFU/g lower than the population observed for control samples, after 10 days of storage at 8 ºC. Regarding frankfurter samples, the use of liquid smoke alone or in combination with sodium lactate resulted in a 3 log CFU/g difference between L. monocytogenes counts found in control and treated samples, after 8 days of storage at 8 ºC. However, after 28 days only the samples treated with liquid smoke combined with sodium lactate presented lower counts than controls (2 log CFU/g). Liquid smoke was also able to inhibit the growth of yeasts and molds in frankfurters, for 21 days of storage at 8 ºC. Low counts of lactic acid bacteria (< 2 log CFU/g) were detected in frankfurters during the storage. There was no variation on pH and water activity values in the meat products during storage period; therefore, the differences found in counts of L. monocytogenes in the meat products can be attributed to the presence of the antimicrobials added in the formulations. Hence, product A and liquid smoke appear to be an additional barrier to control L. monocytogenes in bologna and frankfurters, respectively, during storage at 8 ºC.

Nisina, um antimicrobiano produzido por cepas de Lactococcus lactis subsp. Lactis, pode ser utilizada como preservante em alimentos e, possivelmente, como agente antimicrobiano. Para aumentar sua produção por Lactococcus lactis ATCC 11454 em meio MRS (pH = 6,5) realizamos ensaios em agitador rotativo (100 rpm/ 30 °C/ 36h) a partir de 3 grupos de ensaios, designados por desenho fatorial de dois níveis (2 (4-1) no grupo 01) e 2(3) nos grupos 2 e 3. As concentrações de sacarose (2,2 a 15 g/L), asparagina (7,5 a 75 g/L), fosfato de potássio (6 a 18 g/L) e Tween 80 (1,0 a 6,6 g/L) foram adicionadas ao meio MRS (pH = 6,5). A produção de nisina foi acompanhada a partir do método de difusão em ágar a partir de utilização de Lactobacillus sake ATCC 9924 como microorganismo sensível. Os melhores níveis de nisina atingidos foram de 1,6 x 104 AU/mL (grupo 2) a 1,8 x 104 AU/mL (grupo 1) e obtidos para os níveis máximos de sacarose e asparagina. Dependendo das proporções entre as concentrações dos outros nutrientes, quando a concentração final de sacarose variou entre 0,33 g/L a 3,64 g/L, o pH oscilou entre 5,10 to 6,01, o acido lático produzido variou de 1,19 g/L a 4,44 g/L e a concentração celular variou de 0,99 a 4,184 mg/L. produção de nisina mostrou-se independente das velocidades de crescimento, as quais variaram de 0,0070 h-1 a 0,0401 h-1. A analise de regressão mostrou se adequar a um modelo polinomial quadrático: Log AU/mL = 1,98905 - 0,213558 x2 + 1,80028 x3 - 1,40776 x4 + 0,23436 x1 x2 + 0,35936 x1 x3 +1,47217 x3 x4 (equação principal). Onde os índices representam as concentrações de sacarose; asparagina; fosfato e tween 80 respectivamente. A análise de variância foi realizada para os parâmetros fixados para demonstrar a adequação do modelo proposto. (x são os índices dos nutrientes adicionados). / Nisin, an antimicrobial substance tha is produced by Lactococcus lactis, can be utilized as a preservative and therapeutic agent. To improve its production by Lactococcus lactis ATCC 11454 in MRS broth (pH =6.5), in rotatory shaker (100 rpm/ 30 °C/ 36h), three groups of assays, set up by a fractional factorial design of two-levels (2 (4-1) in group 01), and 2(3) in groups 2 and 3 were carried out. The minimum and maximum concentrations of sucrose (5 to 12.5 g/L), asparagine (7.5 to 75 g/L), potassium phosphate (6 to 18 g/L) and Tween 80 (1.0 to 6.6 g/L) were added to the MRS broth (pH = 6.5). The nisin production was accompanied through the agar diffusion assay using Lactobacillus sake ATCC 9924 as a sensitive microorganism. The best nisin activities ranged from 1.67 x 104 AU/mL (group 2) to 1.84 x 104 AU/mL (group 1) and were obtained for maximum levels of sucrose and asparagine, dependending of the proportions of the other nutrients concentrations, when the final sucrose content varied from 0.33 g/L to 3.64 g/L, the pH value oscillated beTween 5.10 to 6.01, the produced lactic acid was ranged from 1.19 g/L to 4.44 g/L and the dry-cell content was about 0.99 to 4,184 mg (DCW)/L. Nisin production was shown to be independent of growth rates, which ranged from 0.0070 h-1 to 0.0401 h-1. The regression analysis attained a quadratic polynomial model: Log AU/mL = 1.98905 - 0.213558 x2 + 1.80028 x3 - 1.40776 x4 + 0.23436 x1 x2 + 0.35936 x1 x3 +1.47217 x3 x4 (main equation). The analysis of variance performed to the fitted parameters of the independent variables was performed for checking model fitting results adequacy. (x are the index for the nutrients added).

Os ramnolipídeos (RL) são biossurfatantes que apresentam características de grande interesse para a indústria de alimentos, tais como alta biodegradabilidade, baixa toxicidade e propriedades emulsionantes. Este trabalho teve como objetivo avaliar a atividade antimicrobiana do ramnolipídeo comercial (RL-Com - Jeneil Co.) e do ramnolipídeo produzido e purificado no Laboratório de Biotecnologia Microbiana (RL-LBM) sobre culturas de Listeria monocytogenes, através da determinação da concentração inibitória mínima (CIM) utilizando o método de microdiluição em caldo. Os valores de CIM dos RLs variaram entre 78,1 &mu;g mL-1 e 2500 &mu;g mL-1 sendo que a CIM predominante foi de 2500 &mu;g mL-1. Dentre as 32 L. monocytogenes testadas 68,7% foram sensíveis ao RL-LBM e 90,6% sensíveis ao RL-Com. O efeito do ramnolipídeo sobre L. monocytogenes mostrou-se predominantemente bacteriostático. O RL aumentou a permeabilidade celular de L. monocytogenes, contudo este efeito não foi relacionado à sua atividade antimicrobiana. A interação entre os antimicrobianos RL-Com e nisina foi avaliada sobre dois isolados de L. monocytogenes com sensibilidades diferentes ao RL, o L17 menos sensível, com CIM de 2500 &mu;g mL-1, e o L12 mais sensível, com CIM de 156,2 &mu;g mL-1. O índice CIF obtido para os isolados foi de 0,078 e 0,18 , para L17 e L12 respectivamente, caracterizando o efeito sinérgico da combinação entre RL e nisina. As curvas de sobrevivência dos isolados L12 e L17 mostraram que a interação entre a nisina e o RL foi bactericida em concentrações menores que as obtidas individualmente para cada antimicrobiano. Para a L12 a combinação de 78,1 &mu;g mL-1 de RL e 160 UI mL-1 de nisina reduziu completamente a população em 30 min de tratamento, e para a L17 a combinação de 156,2 &mu;g mL-1 de RL e 320 UI mL-1 de nisina foi bactericida após 2 h. Os resultados obtidos neste trabalho demonstraram que o ramnolipídeo possui potencial como agente de controle de L. monocytogenes assim como efeito sinérgico quando combinado à nisina. / The rhamnolipids (RL) are biodegradable biosurfactants which have low toxicity and surface activity properties that can be useful for food processing industries. The objective of this study was to evaluate the susceptibility of Listeria monocytogenes with two rhamnolipids products: the rhamnolipid from P. aeruginosa LBI that was produced and purified in the Microbial Biotechnology Laboratory (RLMBL) and a commercial product (RL-Com - Jeneil Co.). Susceptibility tests were performed by the minimal inhibitory concentration (MIC) using the micro-broth dilution technique. The MIC values varied from 78.1 &mu;g mL-1 to 2500 &mu;g mL-1 and the 2500 &mu;g mL-1 concentration was the predominant value . Among the 32 tested cultures, 68.7% were susceptible to RL-MBL and 90.6% to RL-Com. Results showed that the rhamnolipid activity was primarily bacteriostatic. The RL increases the membrane permeability of L. monocytogenes, however this effect was not directly related to its antimicrobial activity. The combined effect of nisin and RL-Com was evaluated against two wild-type isolates of L. monocytogenes, L12 more sensitive (MIC 156.2 &mu;g mL-1) and L17 less sensitive (2500 &mu;g mL-1). The FIC index for the isolates were 0.18 and 0.078 for L12 and L17 respectively, indicating a particular synergistic effect. The survival curve of isolates L12 and L17 showed that the combination between nisin and RL was bactericidal at lower concentration than for the individual antimicrobials. For L12 isolate 78.1 &mu;g mL-1 of RL and 160 UI mL-1 of nisin eliminated the population after 30 min of incubation. The combination of 156.2 &mu;g mL-1 of RL and 320 UI mL-1 of nisin reduced completely L17 population after 2 h of incubation. This work demonstrated the potential antimicrobial activity of rhamnolipids against L. monocytogenes, as well as the synergistic effect of this biosurfactant with nisin.