There are 500 Helichrysum (Asteraceae) species world wide of which 245 occur in South Africa.The South African species display great morphological diversity and are, therefore classified into 30 groups (Hilliard, 1983). Helichrysum species have been reported for their antimicrobial activities (Rios et al., 1988; Tomas-Barberan et al., 1990; Tomas-Lorente et al., 1989; Mathekga, 1998, Mathekga et al., 2000). Not much information on the bioactivity of compounds isolated from these species is available. In vitro antimicrobial screening methods provide the required preliminary observations to select among crude plant extracts those with potentially useful properties for further chemical and pharmaceutical investigations. In this study we investigated the antimicrobial activities of crude acetone extracts (shaken and homogenized) of twenty-eight Helichrysum species on ten bacteria species and six fungal species. A new phloroglucinol with significant antimicrobial properties was isolated by bioactivity guided fractionation from Helichrysum caespititium. The structure elucidation, conformation and stereochemistry of the new phloroglucinol, 2-methyl-4-[2',4',6'-trihydroxy-3'-(2-methylpropanoyl) phenyl] but-2-enyl acetate (caespitate), was established by high field NMR spectroscopic, crystallographic and MS data. The compound inhibited growth of Bacillus cereus, B. pumilus and Micrococcus kristinae at the very low concentration of 0.5 µg /ml and Staphylococcus aureus at 5.0 µg/ml. Six fungi tested were similarly inhibited at low MICs: Aspergillus flavus and A. niger (1.0 µg /ml), Cladosporium cladosporioides (5 µg/ml), C. cucumerium and C. sphaerospermum (0.5 µg /ml) and Phytophthora capsici at 1.0 µg/ml. The cytotoxicity of most currently used drugs has become a serious problem and efforts are being directed to obtaining new drugs with different structural features. One option favoured is the search for new plant derived non-toxic drugs, as was investigated in this study. Caespitate proved to be non-toxic at biologically active concentrations. Development of resistance to synthetic chemotherapeutic agents is known to occur in modern medicine; for example, resistance to some antibiotics of certain strains of microorganisms. A synergistic antibacterial bioassay demonstrated that the combination of caespitate and caespitin enhanced activity from a concentration range of 5 µg /ml to 0.5 µg /ml down to 0.1 µg /ml to 0.05 µg /ml on Gram-positive bacteria. The synergistic effect was in addition displayed against Gram-negative bacteria. The study of the morphology and ultrastructure of the epicuticular trichomes revealed that trichomes in H. caespititium originate from papillate cell outgrowths which elongate, develop and later polarise into apical, stem and basal parts and that repeated secretions of compounds probably occur from the young three-celled stage, enable us to characterise and relate our observations to their possible functional role in the production of the antimicrobial and other compounds on the leaf surface. South African Helichrysum species are a potentially good source of antimicrobial agents worthy of further investigation as efficient therapeutic compounds and in assisting the primary health care in this part of the world. / Dissertation (PhD (Plant Physiology))--University of Pretoria, 2004. / Plant Science / unrestricted

Please read the abstract in the section 00front of this document / Thesis (PhD (Food Science))--University of Pretoria, 2006. / Food Science / unrestricted

Globally, methicillin resistant Staphylococcus aureus (MRSA) has become increasingly difficult to manage in the clinic and new antibiotics are required. The structure activity relationship (SAR) study presented in this thesis forms part of an international collaborative effort to identify potent and selective inhibitors of an MRSA pyruvate kinase (PK) enzyme target. In earlier work the known marine natural product bromodeoxytopsentin (1.6), isolated from a South African marine sponge Topsentia pachastrelloides, exhibited selective and significant inhibition of MRSA PK (IC₅₀ 60 nM). Accordingly bromodeoxytopsentin provided the initial chemical scaffold around which our SAR study was developed. Following a comprehensive introduction, providing the necessary background to the research described in subsequent Chapters, this thesis has been divided into three major parts. Part one (Chapter 2) documents the synthesis of two natural imidazole containing topsentin analogues 1.40, 1.46, five new synthetic analogues 1.58—1.61, 2.104. In the process we developed a new method for the synthesis of topsentin derivatives via selenium dioxide mediated oxidation of N-Boc protected 3-acetylindoles to yield glyoxal intermediates which were subsequently cyclized and deprotected to yield the desired products. Interestingly we were able to demonstrate a delicate relationship between the relative equivalents of selenium dioxide and water used during the oxidation step, careful manipulation of which was required to prevent the uncontrolled formation of side products. Synthetic compounds 1.40, 1.46, 1.58—1.61 were found to be potent inhibitors of MRSA PK (IC₅₀ 238, 2.1, 23, 1.4, 6.3 and 3.2 nM respectively) with 1000-10000 fold selectivity for MRSA PK over four human orthologs. In the second part of this thesis (Chapter 3) we report the successful synthesis of a cohort of previously unknown thiazole containing bisindole topsentin analogues 1.62—1.68 via a Hantzsch thiazole synthesis. Bioassay results revealed that these compounds were only moderate inhibitors of MRSA PK (IC₅₀ 5.1—20 μM) which suggested that inhibitory activity was significantly reduced upon substitution of the central imidazole ring of topsentin type analogues with a thiazole type ring. In addition in Chapter 3 we describe unsuccessful attempts to regiospecifically synthesize oxazole and imidazole topsentin analogues through a similar Hantzsch method. As a consequence of our efforts in this regard we investigated three key reactions in depth, namely the synthesis of 2.2, 3.38, 3.40, 3.41 via α-bromination of 3-acetylindole and the synthesis of indolyl-3-carbonylnitriles 2.13, 3.45—3.47 and α-oxo-1H-indole-3-thioacetamides 3.48—3.51. The investigation of the latter led to the isolation and elucidation of two anomalous N,N-dimethyl-1H-indole-3-carboxamides 3.52 and 3.53. Finally the third part of this thesis (Chapter 4) deals with in silico assessment of the binding of both the imidazole and thiazole containing bisindole alkaloids to the MRSA PK protein which initially guided our SAR studies. In this chapter we reveal that there appears to be no correlation between in silico binding predictions and in vitro MRSA PK inhibitory bioassay data. Superficially it seems that binding energy as determined by the docking program used for these studies correlated with the size of the indole substituents and did not reflect IC₅₀ MRSA PK inhibitory data. Although this led us to computationally explore possible alternative binding sites no clear alternative has been identified.

This thesis is divided into two parts, assessing marine and synthetic compounds active firstly against Plasmodium falciparum (Chapter 3 and 4) and secondly active against methicillin resistant Staphylococcus aureus (MRSA, Chapter 5). In Chapter 3 the synthesis of nine new tricyclic podocarpanes (3.203-3.207 and 3.209-3.212) from the diterpene (+)-manool is described. Initial SAR study of synthetic podocarpanes concluded that the most active compound was a C-13 phenyl substituted podocarpane (3.204, IC₅₀ 6.6 μM). By preparing analogues with varying halogenated substituents on the phenyl ring (3.209-3.212) the antiplasmodial activity was improved (IC₅₀ 1.4 μM), while simultaneously decreasing the haemolysis previously reported for this class of compounds. Inspired by the antiplasmodial activity of Wright and Wattanapiromsakul’s tricycle marine isonitriles (2.16-2.21 and 2.24-2.27) an unsuccessfully attempt was made to convert tertiary alcohol moieties to isonitrile functionalities in compounds 3.188, 3.204-3.207 and 3.209-3.212. Over a decade ago Wright et al. proposed a putative antiplasmodial mechanism of action for marine isonitriles (2.4, 2.9, 2.15, 2.19 and 2.35) and isothiocyanate (2.34) which involved interference in haem detoxification by P. falciparum thus inhibiting the growth of the parasite. In Chapter 4 we describe how we successfully managed to scale down Egan’s β-haematin inhibition assay for the analyses of small quantities of marine natural products as potential β-haematin inhibitors. Our modified assay revealed that the most active antiplasmodial marine isonitrile 2.9 (IC₅₀ 13 nM) showed total β-haematin inhibition while 2.15 (IC₅₀ 81 nM) and 2.19 (IC₅₀ 31 nM) showed partial inhibition at three equivalents relative to haem. Using contempary molecular modelling techniques the charge on the isonitrile functionality was more accurately describe and the modified charge data sets was used to explore docking of marine isonitriles to haem using AutoDock. In Chapter 5 we describe how a lead South African marine bisindole MRSA pyruvate kinase inhibitor (5.8) was discovered in collaboration with colleagues at the University of British Columbia (UBC) and how this discovery inspired us to design a synthetic route to the dibrominated bisindole, isobromotopsentin (5.20) in an attempt to increase the bioactivity displayed by 5.8. We devised a fast and high yielding synthetic route using microwave assited organic synthesis. We first tested this synthesis using simple aryl glyoxals (5.27-5.32) as precursors to synthesize biphenylimidazoles (5.21-5.26), which later allowed us to synthesize the ascidian natural product 5.111. This method was sucessfully extended to the synthesis of deoxytopsentin (5.33) from an N-Boc protected indole methyl ketone (5.89). We subsequently were able to effectively remove the carbamate protection via thermal decomposition by heating the protected bisindole imidazole (5.90) in a microwave reactor for 5 min under argon. The synthesis of 5.20 resulted in an inseparable mixture of monoprotected and totally deprotected topsentin products, and due to time constraints we were not able to optimise this synthesis. Nonetheless our synthesis of the marine natural product 5.33 which was faster and higher yielding than previously reported routes could be extended to the synthesis of other topsentin bisindoles (5.138-5.140). Work towards this goal continues in our laboratory.

Starvation and protein deficiency increase susceptibility to infec­tions in general and affect the function of various types of cells individually. Saliva contains substances which offer protection to the oral tissues. Its composition and secretion rate may be important in preventing the development of disease, e.g. dental caries. The aim of the present study was to evaluate the effects of starvation and protein deficiency on the secretion and composition of saliva and the effects possibly induced on dental caries. The following results were found. Short-term starvation on a liquid diet reduced the secretion rate and changed the composition of saliva in healthy humans. Chewing during starvation while it did not restore secretion rate, did partially restore the composition. Total protein concentration was not greatly affected by gross mal­nutrition or protein deficiency. A more pronounced effect was observed on individual biologically active proteins. The salivary glands seem not to be prime targets for malnutrition but the rate of biosynthesis of a bacteria aggregating glycoprotein and the activity of salivary peroxidase were significantly reduced by a long-term protein deficien­cy and gross malnutrition respectively in the rat. In humans, short­term starvation on a liquid diet caused an impaired glycosylation of sialic acid to the protein and decreased lysozyme activity. Sialylation was partially restored by chewing. In the rat the establishment of a cariogenic microorganism, S. cricetus (S. mutans) strain E 49 serotype a, was facilitated by protein deficiency. Starvation in both man and the rat produced evident clinical effects. The rate of plaque formation was increased in man and in the rat there was significant increase in caries development induced by a standardi­zed cariogenic challenge. This study shows that nutrition is important for the secretion of saliva and that starvation and protein deficiency increase the cario- genicity of sucrose. / <p>S. 1-49: sammanfattning, s. 51-116: 5 uppsatser</p> / digitalisering@umu

In many vertebrate and invertebrate species mediators of innate immunity include antimicrobial peptides (AMPs) such as peptide fragments of histones and other proteins with previously ascribed different functions. Shark AMPs have not been described and this research examines the antibacterial activity of nurse shark (Ginglymostoma cirratum) peripheral blood leukocyte lysates. Screening of lysates prepared by homogenizing unstimulated peripheral blood leukocytes identified muramidase (lysozyme-like) and non-muramidase antibacterial activity. Lysates were tested for lysozyme using the lysoplate assays, and antibacterial (AB) activity was assayed for by a microdilution growth assay that was developed using Planococcus citreus as the target bacterium. Fractionation of crude lysates by ion exchange and affinity chromatography was followed by a combination of SDS-PAGE with LC/MS-MS and/or N-terminal sequence analysis of low molecular weight protein bands (kDa). This yielded several peptides with amino acid sequence similarity to lysozyme, ubiquitin, hemoglobin, human histones H2A, H2B and H4 and to antibacterial histone fragments of the catfish and the Asian toad. Not all peptide sequences corresponded to peptides potentially antibacterial. The correlation of a specific protein band in active lysate fractions was accomplished by employing the acid-urea gel overlay assays in which AB activity was seen as zones of growth inhibition on a lawn of P. citreus at a position corresponding to that of the putative AB protein band. This study is the first to describe putative AMPs in the shark and their potential role in innate immunity.

Cyclic dipeptides have been well characterized for their multitude of biological activities, including antimicrobial and anticancer activities. Cyclo(His-Gly) and cyclo(His-Ala) have also recently been shown to possess significant anticancer activity against a range of cell lines, despite the limitations of these two molecules with respect to their physicochemical properties. Low Log P results in poor cell permeability which can often be problematic for drugs with intracellular mechanisms of action. It can also results in poor biodistribution, and theoretical Log P values for cyclo(His-Gly) and cyclo(His-Ala) were extremely low making them ideal candidates for inclusion into a nanoparticulate drug delivery system. The aim of this study was therefore to formulate and evaluate liposome-encapsulated cyclic dipeptides that increase the tumour-suppressive actions of the cyclic dipeptides, while showing a high degree of specificity for tumour cells. While liposomes are relatively simple to prepare, inter batch variation, low encapsulation and poor stability are often problematic in their production and this has lead to very few liposomal products on the market. This study aimed at using a comprehensive statistical methodology in optimizing liposome formulations encapsulating cyclo(His-Gly) and cyclo(His-Ala). Initial screening of potential factors was conducted using a 25-1 fractional factorial design. This design made use of two levels for each of the five factors and abbreviated the design to minimize runs. Although not much information is provided by these types of designs, the design was sufficient in identifying two critical factors that would be studies further in a more robust design. The two factors selected, based on the screening study, were cholesterol and stearylamine content. These two factors were then used in designing a response surface methodology (RSM) design making use of a central composite rotatable vii design (CCRD) at five levels (-1.5, -1, 0, 1, 1.5) for each factor in order to better understand the design space. Various factors influenced the measured responses of encapsulation efficiency, zeta potential, polydispersity index, cellular uptake and leakage, but most notable were the adverse effects of increasing stearylamine levels on encapsulations efficiency and cholesterol levels on leakage for both cyclo(His-Gly) and cyclo(His-Ala) liposomes. Optimized formulations were derived from the data and prepared. Fair correlation between the predicted and measured responses was obtained. The cytotoxic activity of the encapsulated cyclic dipeptides were assessed against HeLa and MCF-7 cells and found to have limited improvement in activity. However, modification of the polyethylene glycol (PEG) grafted to the liposome surface in order to target folate receptors showed good benefit in significantly decreasing the IC50 values recorded in all cells lines tested, particularly low folate HeLa cells with the lowest IC50 being recorded as 0.0962 mM for folate targeted cyclo(His-Ala). The results therefore indicate that hydrophilic cyclic dipeptides are ideal candidates for inclusion into targeted drug delivery systems such as liposomes. Key words: Liposomes, cyclo(His-Gly), cyclo(His-Ala), cyclic dipeptides, HeLa, MCF-7, folate receptors, factorial design, response surface methodology (RSM), central composite rotatable design (CCRD).

OBJECTIVE: To measure urinary triclosan levels and their variability across pregnancy, and to identify sources of triclosan exposure among Canadian pregnant women. METHODS: Single spot and serial urine samples, as well as consumer product use information were collected across pregnancy and post-partum from 80 healthy pregnant women in Ottawa. Analyses included descriptives, linear mixed effects and parametric trend modeling, and surrogate category analysis. RESULTS: Triclosan was detected in 87% of maternal urine samples (LOD=3.0 µg/L). Triclosan concentrations varied by time of day of urine collection (p=0.0006), season of sampling (p=0.019), and parity (p=0.038). Triclosan was included in 4% of all personal care products used by participants; 89% of these triclosan products were varying brands of toothpaste and hand soaps. CONCLUSION: This study provided the first data on temporal variability urinary triclosan levels, and on source attribution data in Canadian pregnant women. Results will assist with population-specific exposure assessment strategies.

Les plaies chroniques représentent un problème de santé publique dont la prévalence augmente avec l'âge, l'état de santé du patient et la sédentarité. En effet, 1,69% des personnes âgées de plus de 65 ans est atteint d'ulcères de la jambe dans les pays occidentaux. De plus, l'apparition de plaies chroniques augmente chez les patients atteints de diabète avec une probabilité de développer un ulcère du pied de 2,2%/an pour les patients diabétiques atteints de neuropathie (diabète de type I et II confondus). Le risque d'infection des plaies chroniques augmentant avec le temps de guérison de la plaie, il est donc nécessaire d'apporter des soins réguliers afin de prévenir ou lutter contre l'infection de ces plaies jusqu'à leur complète guérison. En cas d'infection, la colonisation critique ou infection localisée peut conduire, en cas d'absence de traitement adapté, à la nécessité d'une intervention chirurgicale afin de prévenir l'infection systémique voire la mort du patient. Cependant, la plaie chronique représente un site de colonisation favorable pour les agents pathogènes qui s'y développent rapidement. La prolifération des bactéries peut alors aboutir à la formation d'un biofilm protecteur diminuant l'efficacité des défenses de l'organisme et des agents antimicrobiens. L'objectif de ce travail de thèse est donc de concevoir un dispositif médical sous forme de pansement capable de libérer de façon prolongée un principe actif antibactérien à large spectre d'action. Le dispositif textile utilisé dans ce projet est un textile non-tissé à base de polyéthylène téréphtalate (PET) fonctionnalisé par le chitosane (CHT) par la méthode "Pad-Dry-Cure". La fonctionnalisation du CHT est réalisée par l'utilisation de deux agents réticulants, la génipine (Gpn) ou l'acide citrique (CTR), qui permet de créer un réseau de chitosane réticulé à la surface des fibres textiles. Le coating apporte ainsi des fonctions ioniques à la surface du PET; positives lorsque l’agent réticulant est la Gpn, positives ou négatives en fonction du ratio de CTR introduit dans la solution quand l’agent réticulant est le CTR.Les charges ioniques du support PET fonctionnalisé par le CHT réticulé par la Gpn (PET-Gpn/CHT) ont été utilisées pour la construction d'un système multicouche (MC) "Layer-by-Layer" auto-assemblé. Les polyélectrolytes (PE) utilisés pour la construction de ce MC par "Dip-Coating" sont le chitosan en tant que PE cationique et un polymère de cyclodextrine (CD) en tant que PE anionique, élaboré et étudié par le laboratoire lors de travaux précédents. La capacité de formation de complexe d'inclusion des CD, apportée par le polymère de CD incorporé au revêtement multicouche, a ensuite été utilisée afin de charger un antiseptique à large spectre d'action, la chlorhexidine (Chx). La construction du système MC a d'abord été optimisée puis une évaluation physico-chimique, biologique et microbiologique a été réalisée sur les textiles PET-Gpn/CHT recouvert d'un système MC chargé par la Chx. Une étude a aussi été menée sur le PET fonctionnalisé uniquement par le CHT réticulé par le CTR (PET-CTR/CHT). Ce système anionique ou cationique en fonction de la quantité d’agent réticulant introduite, a été chargé respectivement par l'argent (Ag+) ou l’'iode (I3-). Une évaluation physico-chimique et microbiologique a été réalisée sur ces supports textiles pour évaluer leur efficacité antibactérienne. Finalement un système multicouche a été construit sur le système PET-CTR anionique chargé d’argent afin de limiter sa libération hors du système MC. Ceci a été ensuite vérifié à travers une évaluation chimique complétée par des tests microbiologiques. / The chronic wounds represent a public health problem health which the prevalence increases with age, patient health and sedentary lifestyle. Indeed, in the occidental countries, 1.69% of the elderly person aged over 65 years is suffering from leg ulcers . Moreover, the chronic wounds appearance increases for the diabetic patients who suffering neuropathy (diabetic type I and II together) with 2.2%/years of probability to developp a leg ulcer. The infection of the chronic wounds increases with wound healing delay, thus, cares need to be repeated to prevent or fight wound infection until their complete healing. In the case of infected wound, the critical colonization or localized infection can result without adapted treatment to surgery operation in order to prevent systemic infection or death. Nevertheless, the chronic wound represents a favourable site for the rapid developpment of the pathogen agents. The bacterial proliferation can lead to the biofilm formation which increases the bacterial protection against the body\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\'s natural defences and antimicrobial agents. So, the aim of this work is elaborate a medical device as wound dressing for extended broad spectrum antibacterial drug release. The textile device used in this project is a non-woven textile based on polyethylene terephtalate (PET) functionalized with Pad-Dry-Cure method with chitosan (CHT). In the present work, two crosslinking agents are evaluated, genipin (Gpn) and citric acid (CTR), which allows obtaining a CHT network coated at the surface of the textile fibers. Thus, there are ionic functions at the surface of the PET; positive charges when the crosslink agent is Gpn, positive or negative charges in function of the amount of the CTR used in the solution.The ionic charges from the PET functionalized by CHT crosslinked by Gpn (PET-Gpn/CHT), are used to build a multilayer system layer-by layer self-assembly. The polyelectrolytes (PE) used to build this multilayer system by dip-coating are: the CHT, as cationic PE, and cyclodextrin (CD) polymer, as anionic PE (it was developed and studied by the team in previously work). The ability of the CD to form inclusion complex, from the CD polymer included in the multilayer coating, is used in the case of PET-Gpn/CHT to load one antiseptic with broad spectrum, the chlorhexidine (Chx). This Chx loaded multilayer system is optimized and evaluated physic-chemically, biologically and microbiologically. The study is also realized on the functionalized PET-CTR/CHT. This system, anionic or cationic, in function of the quantity of the crosslinking agent used, is loaded respectively by silver (Ag+) or iodide (I3-). A physico-chemical and microbiological evaluation are realized to evaluate their antibacterial efficiency. Finally, the multilayer system is built-up on the anionic PET-CTR/CHT loaded by silver to reduce its release out of the multilayer system. Chemical completed by microbiology tests are realized to verify the influence on the antibacterial activity and the kinetic of the release of the multilayer system

Les aliments et les emballages sont interdépendants. Plusieurs réactions chimiques et physiques existent entre les aliments, l’emballage et environnement, lesquelles peuvent changer la composition, la qualité et les propriétés physiques des aliments, voire de l’emballage. L’emballage actif ou antimicrobien permet la distribution d’un aliment dans le monde entier sans perte de qualité, pendant une longue période de transport. Les antimicrobiens naturels comme la nisine, le lysozyme ou l’acide lactique contrôlent la contamination microbienne d’un aliment, améliorent son stockage, éliminent les pathogènes indésirables et retardent la prolifération microbienne. L’amélioration d’un emballage en papier, contenant de la nisine, le lysozyme, l’acide lactique, ainsi que l’étude de la synergie d’action entre antimicrobiens constituent les objectifs de cette thèse.La synergie entre nisine, lysozyme et l’acide lactique a etait déterminée avec Listeria monocyotgenes CIP 82.110 mais n’a pas observée pour Staphylococcus aureus CIP 4.83 par analyses statistiques. Le papier « activé » avec un mélange de nisine, lysozyme et acide lactique permet d’assurer une action antimicrobienne vis à vis de Listeria monocytogenes CIP 82.110 et de Staphylococcus aureus CIP 4.93. La diffusion de la nisine du papier vers un aliment simulé a était démontrée pendant 48h à la vitesse de 0.03 mg/h. En 5 jours, seulement 30% de la nisine a migré vers l’agarose. Des analyses complémentaires de diffusion vont permettre de mieux comprendre l’efficacité d’un emballage antimicrobien contenant un mélange de nisine, de lysozyme et d’acide lactique / Food and packaging are closely related. Many chemical and physical reactions exist between a food, its packages and the environment, which alter the composition, quality and physical properties of the food and/or the package. Thanks to active or antimicrobial packaging, food products can be distributed over a wide geographical area over a long period of time without unacceptable quality loss. Natural antimicrobials such as nisin, lysozyme or lactic acid improve shelf-life, eliminate undesirable pathogens and delay microbial spoilage. The present PhD thesis was focused on the improvement of paper wrapping materials by adding nisin, lactic acid and lysozyme in combination and on the determination of the synergism, which could occur between these antimicrobial agents. Synergy between nisin, lysozyme and lactic acid, which was proved against Listeria monocytogenes CIP 82.110 and was not confirmed against Staphylococcus aureus CIP 4.83 by statistical analysis. The paper, containing a mixture of nisin, lysozyme and lactic acid could ensure a antimicrobial activity against Listeria monocytogenes CIP 82.110 or Staphylococcus aureus CIP 4.93. Nisin diffusion from packaging to simulated food was demonstrated. It was proved that nisin in cellulose was only able to diffuse for 48 h at a speed of 0.03 mg/h. Only 30% of nisin was diffused from paper to gel matrix after 5 days. Extra diffusion analyses on combination of nisin, lysozyme and lactic acid should be performed to confirm the antimicrobial effectiveness of this packaging