Dissertations / Theses on the topic 'Cleaning in place'

Industry operations require a clean plant to make safe, quality products consistently. As well as product quality, the environmental impact of processes has become increasingly important to industry and consumers. Cleaning In Place (CIP) is the ubiquitous method used to ensure plant cleanliness and hygiene. It is therefore vital the system is optimal and efficient. I.e. the correct cleaning agent is delivered to the fouled surface at the right time, temperature, flow rate and concentration. This cannot be assured without effective online measurement technologies. Fryer and Asteriadou (2009) describe how the nature of a fouling deposit can be related to the cost of cleaning. The evolution of three key deposit types has also enabled current fouling and cleaning literature to be easily classified. In the brewery there are many types of soil that need to be cleaned of which the cost of cleaning was unknown. The cost of fermenter CIP in one brewery was found to be £106 k per year. Effective fouling methods for yeast and caramel; and the relationship between flow, temperature, and caustic concentration in the removal of yeast and caramel soils seen in industry has been done. This work has helped determine effective cleaning methods for these soils from stainless steel coupons and pipes. Fermentation vessels have been found by Goode et al., (2010) to have two types of soil: A – fouling above the beer resulting from the act of fermentation, and B – fouling below the beer resulting from emptying the fermenter. The type B fouling below the beer was found to be a type 1 soil that could be removed with water. An increase in flow velocity and Reynolds number decreased cleaning time. An increase in temperature did not decrease cleaning time significantly at higher flow velocities, 0.5 m s-1. Fouling above the beer occurs when material is transported to and stick on to the wall during fermentation foaming. This happens initially and as a result the fouling has a long aging time. This yeast film represents a type 2 deposit, removed in part by water and in part by chemical. Most of the deposit could be removed by rinsing with warm water. At 50°C the greatest amount of deposit was removed in the shortest time. A visually clean surface could be achieved at all temperatures, 20, 30, 50 and 70°C, using both 2 and 0.2 wt % Advantis 210 (1 and 0.1 wt % NaOH respectively). A visually clean surface was achieved quicker at higher detergent temperatures rather than rinsing at higher flow velocity or concentration. This finding suggests most deposit can be removed with warm water and cleaned with lower detergent concentrations. Currently in the brewery 2 % NaOH is used at 70°C. Caramel represents a type 3 soil. When heated it sticks to stainless steel and requires chemical action for removal. Confectionary caramel was cooked onto pipes and coupons and the effect of flow velocity, temperature and concentration on removal determined. At high flow velocity most of the deposit could be removed from the pipe using water. There was no significant difference in the mass of caramel removed by the water however. A visually clean surface was achieved by rinsing at 80°C with 2.5% Advantis. A visually clean surface could not be achieved at lower temperatures at higher concentration, 5% Advantis, or at higher flow velocity. The measurement of online conductivity and flow rate values was invaluable during each experiment. Turbidity values did indicate the removal of yeast and caramel from pipes however offline measurements were required to confirm removal. Caramel removal could be wholly quantified by mass when cleaning pipes. The integration of the turbidity values measured during each rinse correlated well with the mass of deposit removed in most cases. Coupon cleaning was wholly quantified by area . A cost saving of £69 k can be made by optimising fermenter CIP to warm pre-rinsing followed by ambient caustic circulation. An £8 k saving can be made by optimising yeast tank CIP to pre-rinsing only and acid sanitisation. Industry must ensure effective online CIP measurements are made throughout cleaning to describe the process effectively and enable optimisation. It is crucial to have cleaning measurement information to hand because that is how we ensure our customers they are buying a quality product. Also you cannot optimise what you do not measure effectively.

Robust and efficient cleaning procedures for protein A chromatography resins used for production of monoclonal antibody based biopharmaceuticals are crucial for safe and cost efficient processes. In this master thesis the effect of different cleaning regimes with respect to ligand stability of two protein A derived media, MabSelectTM and MabSelect SuReTM, has been investigated. A 96-well format has been used for preliminary screening of different cleaning agents, contact times and temperatures. NaCl as a ligand stabilizer during cleaning-in-place (CIP) was also included as a parameter. For optimal throughput and efficiency of screening, Rectangular Experimental Design for Multi-Unit Platforms; RED-MUP, and TECAN robotic platform have been utilized. For verification of screening, selected conditions were run in column format using the parallel chromatography system ÄKTAxpressTM. In the efficiency study, where a manual preparation of CIP solutions was compared with an automated mode performed in TECAN, the total process time ended up at eight hours versus three days respectively. However, the time measured included the learning process for the TECAN platform and for further preparations the automated mode is the superior choice. The study confirmed the higher alkaline stability of MabSelect SuRe compared to MabSelect. After exposure to 0.55 M NaOH during 24h MabSelect SuRe still retained 90% of the initial capacity. In contrast MabSelect had 60% of the initial binding capacity. When CIP with 10 mM NaOH was performed at 40 °C MabSelect reduced its capacity by half while MabSelect SuRe still had a binding capacity of 80%. The 96-well screening showed that an addition of NaCl during CIP had a significant positive effect on the stability of MabSelect, but needs to be verified on column format. The correlation between results from screening in 96-well filter plate and column format was good.

L’ultrafiltration (UF) du lait écrémé pour la standardisation de la teneur en protéines pour la fabrication des fromages est un procédé membranaire très répandu à l’échelle industrielle. Cependant, le colmatage des membranes par des protéines du lait écrémé provoque une baisse de la productivité et constitue un verrou de ce procédé.Ainsi, l’étape de nettoyage/désinfection bi-quotidienne est indispensable afin de restaurer les performances de la membrane et d’assurer la sécurité sanitaire ainsi que la qualité des produits. Elle est en général réalisée avec des détergents formulés alcalins et acides mais il est également possible d’utiliser des détergents enzymatiques formulés ayant la réputation d’être plus efficaces. Cependant, peu d’études fondamentales existent à ce sujet, ce que cette thèse se propose de contribuer à combler. L’objectif de cette thèse a été de développer de nouveaux détergents enzymatiques efficaces et compatibles avec la membrane en PES/PVP largement utilisée pour l’UF de lait écrémé. La cible du nettoyage est un dépôt de protéines. La démarche s’est appuyée sur une méthodologie d’aide à la formulation de détergents fondée sur la mesure des dépôts résiduels sur la membrane par ATR-FTIR: que ce soit les protéines, les enzymes ou les autres constituants des détergents étudiés. De multiples prototypes ont été formulés en collaboration avec la société Kersia. Leur évaluation a suivi 3 étapes: i) des tests rapides en réacteur fermé (14 cm²) pour sélectionner les détergents prometteurs selon leur efficacité d’élimination du colmatage protéique, leur rinçabilité et le respect de l’intégrité de la membrane à court termes, ii) la transposition des résultats prometteurs en condition de filtration (127 cm²) validant également la filtrabilité des détergents, iii) enfin, la validation de la compatibilité détergent/membrane sur le long terme grâce au vieillissement accéléré sous micro-onde (3 cm²), et au nettoyage d’une membrane spirale (6.8 m²) pendant 52 heures
Ultrafiltration (UF) of skim milk for standardization of the protein content for cheese making is a very common membrane process at industrial scale. However, fouling of the membranes with skim milk proteins causes a drop in productivity and constitutes a barrier to this process. Thus, the twice-daily cleaning/disinfection step is essential to restore the performance of the membrane and ensure health safety and product quality. It is generally carried out with alkaline and acidic formulated detergents, but it is also possible to use formulated enzymatic detergents which enjoy the reputation of being more effective. However, few fundamental studies exist on this subject, which this thesis aims to contribute to fill. The objective of this thesis was to develop new enzymatic detergents that are are effecient and compatible with the PES/PVP membrane widely used for skim milk UF. The target of the cleaning is a protein deposit. The approach was based on a methodology to aid the formulation of detergents based on the measurement of residual deposits on the membrane by ATR-FTIR: whether proteins, enzymes or other constituents of the detergents studied. Mutliple prototypes were formulated in collaboration with Kersia Company. Their evaluation followed 3 steps: i) rapid tests in a batch reactor (14 cm²) to select promising detergents according to their effectiveness in eliminating protein fouling, their cleanability and the integrity of the membrane in the short term, ii) transposition of promissing results under filtration conditions (127 cm²) also validating the filterability detergents, iii) finally, validation of detergent/membrane compatibility over the long term thanks to accelerated aging under microwave (3 cm²) and cleaning of the spiral wound membrane (6.8 cm²) for 52 hours

La régénération des solutions de lavage utilisées en Nettoyage En Place (NEP) présente un intérêt, tant économique qu’environnemental, pour les industriels laitiers. L’étape clé du NEP réside dans le lavage alcalin qui assure l’élimination des souillures organiques déposées sur les équipements. Ce lavage est généralement effectué par des lessives de soude, moins onéreuses que celles de potasse. Nos travaux ont visé à appliquer un procédé physico-chimique de régénération à des lessives de potasse en vue de rentabiliser leur intégration au NEP laitier. La régénération de solutions de potasse souillées par du lait a été examinée et comparée à celle de solutions de soude. L’efficacité du procédé à épurer les solutions de potasse en termes d’abattement de la DCO et de Ntot a été démontrée. L’analyse des solutions régénérées a par ailleurs mis en évidence l’amélioration de leurs propriétés interfaciales (tension superficielle, angle de contact). Ces propriétés, dont l’origine a pu être attribuée à l’accumulation de biotensioactifs, confèrent aux solutions de potasse régénérées un meilleur pouvoir nettoyant. Ce point a été validé lors d’essais de nettoyage de membranes organiques d’ultrafiltration colmatées par des protéines laitières. L’optimisation de la formulation de ces solutions a permis l’obtention de performances de nettoyage comparables à celles d’un détergent commercial classiquement utilisé pour ce type d’application. Enfin, les impacts économiques et environnementaux de l’intégration de lessives de potasse en substitution à celles de soude ont été évalués pour un NEP laitier industriel au travers d’une étude technico-économique et d’une Analyse de Cycle de Vie (ACV)
The regeneration of Cleaning In Place (CIP) solutions is interesting, both economically and environmentally, especially in dairy industry. The CIP’s key step lies on the alkaline washing which ensures the removal of organic contaminants deposited on the equipment. This washing is generally done by soda lyes, less expensive than potash ones. Our work aimed to apply a physicochemical regeneration process, based on adsorption-coagulation-flocculation phenomena, on potash lyes to make their integration in the dairy CIP affordable. The regeneration of potash solutions soiled with milk was examined and compared with the soda solutions regeneration. The process effectiveness to purify potash solutions in terms of COD and TN reduction has been shown. The solutions analysis has also highlighted the improvement of their interfacial properties (surface tension, contact angle). These properties, whose origin was attributed to the accumulation of biosurfactants, confer to potash regenerated solutions a best cleaning power. This point was validated during cleaning assays of organic ultrafiltration membranes fouled by milk proteins. The optimization of solutions formulation has also enabled obtaining a cleaning performance as efficient as those of a commercial detergent largely used for this type of application. Finally, economic and environmental impacts of the potash lyes integration in substitution to soda ones were evaluated for dairy industrial CIP through a techno-economic analysis and a Life Cycle Assessment (LCA)

L’étape de nettoyage et désinfection est une étape indispensable au processus de filtration du lait. Elle permet de restaurer les performances membranaires et éliminer le colmatage ainsi que d’assurer la sécurité sanitaire et la qualité du produit. Elle nécessite du temps, de l’énergie, de l’eau et des produits chimiques. Pourtant, cette étape est encore gérée empiriquement dans les industries et demeure un verrou scientifique et technique des procédés membranaires. En industrie, l’efficacité du nettoyage est contrôlée en ligne par des mesures de flux. Cependant, ces mesures ne sont pas suffisantes pour attester d’un bon nettoyage ou même vérifier si le membrane n’est pas dégradée. En effet, lors du développement d’un détergent il faut prendre en compte son efficacité envers les colmatants résiduels (ici les protéines du lait) mais aussi son innocuité vis-à-vis de la membrane. Nos travaux se sont déroulés selon 3 axes : (i) le développement d’une méthodologie visant à prouver l’innocuité d’un détergent vis-à-vis d’une membrane (les méthodes actuelles étant trop chronophages) ainsi que la validation d’un biocide acide non oxydant (ii) la conception et la validation de deux détergents formulés alcalins (pH 12.0-12.5 et pH 11.5-12.0) et la validation de détergents commerciaux enzymatiques, l’efficacité des détergents étant déterminée par quantification des protéines résiduelles et l’innocuité validée également grâce à la technique ATR-FTIR (entre autres) (iii) puis une partie plus théorique sur le développement d’une aide à la formulation de détergents à partir de mesures d’angle de contact et de la théorie de Van Oss
The cleaning and disinfection step is an essential step in the overall milk filtration process. It restores membrane performance and eliminates fouling as well as ensuring health safety and product quality. It requires time, energy, water and chemicals. However, this stage is still managed empirically in industries and remains a scientific and technical obstacle to membrane processes. In industry, the effectiveness of cleaning is monitored online by flow measurements. However, these measures are not sufficient to attest to a good cleaning or even to check if the membrane is not degraded. Indeed, during the development of a detergent, it is necessary to take into account its effectiveness towards residual fouling (here milk proteins) but also its harmlessness towards the membrane. Our work followed 3 main lines : (i) the development of a methodology to prove the harmlessness of a detergent towards a membrane (current methods are too time-consuming) as well as the validation of a non-oxidizing acid biocide (ii) the formulation and validation of two alkaline formulated detergents (pH 12.0-12.5 and pH 11.5-12.0) and the validation of commercial enzymatic detergents, the effectiveness of the detergents being determined by quantification of the residual proteins and the harmlessness also validated thanks to the ATR-FTIR technique (iii) then a more theoretical part on the development of an assistance for detergent formulation built on contact angle measurements and the Van Oss theory

La régénération des solutions de lavage utilisées en Nettoyage En Place (NEP) présente un intérêt, tant économique qu’environnemental, pour les industriels laitiers. L’étape clé du NEP réside dans le lavage alcalin qui assure l’élimination des souillures organiques déposées sur les équipements. Ce lavage est généralement effectué par des lessives de soude, moins onéreuses que celles de potasse. Nos travaux ont visé à appliquer un procédé physico-chimique de régénération à des lessives de potasse en vue de rentabiliser leur intégration au NEP laitier. La régénération de solutions de potasse souillées par du lait a été examinée et comparée à celle de solutions de soude. L’efficacité du procédé à épurer les solutions de potasse en termes d’abattement de la DCO et de Ntot a été démontrée. L’analyse des solutions régénérées a par ailleurs mis en évidence l’amélioration de leurs propriétés interfaciales (tension superficielle, angle de contact). Ces propriétés, dont l’origine a pu être attribuée à l’accumulation de biotensioactifs, confèrent aux solutions de potasse régénérées un meilleur pouvoir nettoyant. Ce point a été validé lors d’essais de nettoyage de membranes organiques d’ultrafiltration colmatées par des protéines laitières. L’optimisation de la formulation de ces solutions a permis l’obtention de performances de nettoyage comparables à celles d’un détergent commercial classiquement utilisé pour ce type d’application. Enfin, les impacts économiques et environnementaux de l’intégration de lessives de potasse en substitution à celles de soude ont été évalués pour un NEP laitier industriel au travers d’une étude technico-économique et d’une Analyse de Cycle de Vie (ACV)
The regeneration of Cleaning In Place (CIP) solutions is interesting, both economically and environmentally, especially in dairy industry. The CIP’s key step lies on the alkaline washing which ensures the removal of organic contaminants deposited on the equipment. This washing is generally done by soda lyes, less expensive than potash ones. Our work aimed to apply a physicochemical regeneration process, based on adsorption-coagulation-flocculation phenomena, on potash lyes to make their integration in the dairy CIP affordable. The regeneration of potash solutions soiled with milk was examined and compared with the soda solutions regeneration. The process effectiveness to purify potash solutions in terms of COD and TN reduction has been shown. The solutions analysis has also highlighted the improvement of their interfacial properties (surface tension, contact angle). These properties, whose origin was attributed to the accumulation of biosurfactants, confer to potash regenerated solutions a best cleaning power. This point was validated during cleaning assays of organic ultrafiltration membranes fouled by milk proteins. The optimization of solutions formulation has also enabled obtaining a cleaning performance as efficient as those of a commercial detergent largely used for this type of application. Finally, economic and environmental impacts of the potash lyes integration in substitution to soda ones were evaluated for dairy industrial CIP through a techno-economic analysis and a Life Cycle Assessment (LCA)

La caractérisation expérimentale et numérique du comportement rhéologique d'une mousse aqueuse s'écoulant à l'intérieur d'un tuyau horizontal avec et sans singularités (demi-expansion soudaine, et chicane) a été étudiée. Différentes conditions d'écoulement ont été étudiées en faisant varier les qualités de mousse (55-85%), et trois nombres de Reynolds (32, 65, et 97). Les mesures de la pression, de la répartition de la vitesse locale et de la contrariante et de l'épaisseur des films liquides au niveau de la paroi à l'aide respectivement de capteurs de pression, de la PIV, de la polarographie et de la conductimétrie ont montré une réorganisation de la mousse en aval du changement de géométrie, avec un film liquide plus épais au fond du conduit, des bulles de plus grande taille au sommet, ainsi qu'une plus grande fraction de vide de la mousse augmentant de la partie inférieure à la partie supérieure de la section du conduit. En outre, la mousse présenterait un caractère visco-élastique comparable à celui d'un liquide monophasique non newtonien. Des simulations de dynamique des fluides ont été entreprises pour prédire ce comportement rhéologique de la mousse, les deux modèles Herschel-Bulkley et Bingham ont été testés en tenant compte de la présence d'un film liquide sous-jacent au fond du canal. La comparaison entre les résultats expérimentaux et numériques a montré que, quelle que soit la qualité de la mousse, le modèle de Herschel-Bulkley pouvait décrire avec précision le comportement rhéologique de la mousse dans les différentes conditions d'écoulement analysées.Le deuxième objectif était d'étudier la capacité d'un écoulement de mousse humide (qualité de 50%) à nettoyer des surfaces en acier inoxydable contaminées par des micro-organismes. Pour cela, deux types de contamination ont été étudiés, des gouttelettes contenant des spores de Bacillus subtilis (soit hydrophiles Bs PY79 ou hydrophobes Bs PY79 spsA), et des biofilms produits par trois souches de bactéries (Escherichia coli SS2, Bacillus cereus 98/4, et Pseudomonas fluorescens Pf1). Différentes conditions d'écoulement ont été réalisées en faisant varier les contraintes de cisaillement pariétale (2.2 - 13.2 Pa), et la taille des bulles (0.18-0.34 mm) dans un conduit sans changement géométrique, afin d'identifier les mécanismes de libération de la contamination et ainsi mieux contrôler et optimiser le processus de nettoyage. Les résultats montrent que, par rapport au NEP conventionnel, le flux de mousse a éliminé efficacement les spores ainsi que les biofilms. De plus, la combinaison d'une contrainte de cisaillement pariétale élevée et de bulles de petite taille (<0,2 mm) s'est avérée prometteuse pour améliorer l'efficacité du nettoyage des spores. D'autre part, une nette amélioration de l'élimination des biofilms a été observée en augmentant la contrainte de cisaillement pariétale. La caractérisation de la mousse et des phénomènes d'interface a indiqué que des mécanismes tels que la fluctuation des contraintes de cisaillement locales, ou de l'épaisseur du film liquide entre les bulles et la paroi induite par le passage des bulles, l'imbibition de la mousse et le balayage de la contamination dans le film liquide, pourraient participer largement aux mécanismes d'élimination. Enfin, l'étude d'analyse du cycle de vie a démontré que le nettoyage à la mousse peut être une technique appropriée pour réduire la consommation d'eau et d'énergie (7 et 8 fois moins) présentant moins d'impacts environnementaux que les procédés CIP, avec environ 70%. Enfin, le nettoyage à la mousse peut être une méthode alternative, qui peut améliorer l'efficacité et réduire l'impact environnemental.D'autres activités menées au cours de la période de doctorat liées à la conception hygiénique sont présentées en soulignant le rôle des contaminants (spores et biofilms), du matériau (autre que l'acier inoxydable) et de la géométrie (conduits ou conception plus complexe) dans le contrôle de l'hygiène
In this research, experimental and numerical characterization of the rheological behavior of an aqueous foam flowing inside a horizontal pipe with and without singularities (presence of half-sudden expansion, and a fence) were investigated. Different conditions of foam flow were studied by varying the foam qualities (from 55% to 85%), and three Reynolds numbers (32, 65, and 97). Measurements of the pressure measurements, and at the wall the local velocity repartition and the thickness of the liquid films using respectively pressure sensors, Particle Image Velocimetry, and a conductimetry technique shown a reorganization of the foam downstream the geometry change, with a thicker liquid film at the duct bottom, larger bubble sizes at the top, as well as a larger foam void fraction increased from the bottom to the top part of the duct section. In addition, foam would present a visco-elastic character comparable to a non-Newtonian monophasic liquid. Computational Fluid Dynamics simulations were undertaken to predict this rheological behavior of the foam, the two models Herschel-Bulkley and Bingham were tested taken into account the presence of an underlying liquid film at the bottom of the channel . Comparison between experimental and numerical results showed that regardless of the foam quality, Herschel-Bulkley model could accurately describe the rheological behaviour of the aqueous foam under the different flow conditions analysed.The second target was to investigate the ability of a wet foam flow (quality of 50%) to clean stainless-steel surfaces contaminated by microorganisms. For this purpose, two different contamination patterns were studied, droplets containing Bacillus subtilis spores (either hydrophilic Bs PY79 or hydrophobic Bs PY79 spsA), and biofilms produced by three bacteria strains encountered in food industry production plants (Escherichia coli SS2, Bacillus cereus 98/4, and Pseudomonas fluorescens Pf1). Different flow conditions were performed by varying the wall shear stresses (2.2 - 13.2 Pa), and bubble sizes (0.18-0.34 mm) in a straight duct with no geometrical changes, in order to identify the mechanisms of contamination release and thus better control and optimize the foam cleaning process. Results show that compared to conventional cleaning-in-place, foam flow effectively removed Bs spores as well as Bc-98/4, Ec-SS2, and Pf1 biofilms. Moreover, the combination of high shear stress at the wall and small bubble sizes (<0.2 mm) showed promise for improving the cleaning efficiency of spores. On the other hand, a clear improvement of the biofilm removal was observed when increasing the mean wall shear stress. The characterization of the foam and the interface phenomenons (using polarography, conductimetry, and bubble size analysis methods) indicated that mechanisms such as fluctuation in local wall shear stresses, or in the liquid film thickness between the bubbles and the steel wall induced by bubble passage, foam imbibition, and sweeping of the contamination within the liquid film could participate largely to the removal mechanisms. Finally, the life cycle assessment study demonstrated that foam flow cleaning could be a suitable technique to reduce water and energy consumption (7 and 8 times less, respectively) presenting less environmental impacts than CIP processes, with about 70%. Lastly, foam flow cleaning can be an alternative method, which can improve efficiency and reduce environmental impact. Additional activities conducted during the PhD period related to hygienic design are presented highlighting the role of the contaminants (spores and biofilms), the material (other than stainless steel) and the geometry (ducts or more complex design) in hygiene monitoring

In order to produce predictable and robust systems forprotein purification and detection, well characterized, small,folded domains descending from bacterial receptors have beenused. These bacterial receptors, staphylococcal protein A (SPA)and streptococcal protein G (SPG), possess high affinity to IgGand / or HSA. They are composed of repetitive units in whicheach one binds the ligand independently. The domains foldindependently and are very stable. Since the domains also havewellknown three-dimensional structures and do not containcysteine residues, they are very suitable as frameworks forfurther protein engineering.

Streptococcal protein G (SPG) is a multidomain proteinpresent on the cell surface ofStreptococcus. X-ray crystallography has been used todetermine the binding site of the Ig-binding domain. In thisthesis the region responsible for the HSA affinity of ABD3 hasbeen determined by directed mutagenesis followed by functionaland structural analysis. The analysis shows that the HSAbindinginvolves residues mainly in the second α-helix.

Most protein-based affinity chromatography media are verysensitive towards alkaline treatment, which is the preferredmethod for regeneration and removal of contaminants from thepurification devices in industrial applications. Here, aprotein engineering strategy has been used to improve thetolerance to alkaline conditions of different domains fromprotein G, ABD3 and C2. Amino acids known to be susceptibletowards high pH were substituted for less alkali susceptibleresidues. The new, engineered variants of C2 and ABD shownhigher stability towards alkaline pH. Also, very important forthe potential use as affinity ligands, these mutated variantsretained the secondary structure and the affinity to HSA andIgG, respectively. Moreover, dimerization was performed toinvestigate whether a higher binding capacity could be obtainedby multivalency. For ABD, binding studies showed that divalentligands coupled using non-directed chemistry demonstrated anincreased molar binding capacity compared to monovalentligands. In contrast, equal molar binding capacities wereobserved for both types of ligands when using a directed ligandcoupling chemistry involving the introduction and recruitmentof a unique C-terminal cysteine residue.

The staphylococcal protein A-derived domain Z is also a wellknown and thoroughly characterized fusion partner widely usedin affinity chromatography systems. This domain is consideredto be relatively tolerant towards alkaline conditions.Nevertheless, it is desirable to further improve the stabilityin order to enable an SPA-based affinity medium to withstandeven longer exposure to the harsh conditions associated withcleaning in place (CIP) procedures. For this purpose adifferent protein engineering strategy was employed. Smallchanges in stability due to the mutations would be difficult toassess. Hence, in order to enable detection of improvementsregarding the alkaline resistance of the Z domain, a by-passmutagenesis strategy was utilized, where a mutated structurallydestabilized variant, Z(F30A) was used as a surrogateframework. All eight asparagines in the domain were exchangedone-by-one. The residues were all shown to have differentimpact on the alkaline tolerance of the domain. By exchangingasparagine 23 for a threonine we were able to remarkablyincrease the stability of the Z(F30A)-domain towards alkalineconditions. Also, when grafting the N23T mutation to the Zscaffold we were able to detect an increased tolerance towardsalkaline treatment compared to the native Z molecule. In allcases, the most sensitive asparagines were found to be locatedin the loops region.

In summary, the work presented in this thesis shows theusefulness of protein engineering strategies, both to explorethe importance of different amino acids regarding stability andfunctionality and to improve the characteristics of aprotein.

Keywords:binding, affinity, human serum albumin (HSA),albumin-binding domain (ABD), affinity chromatography,deamidation, protein A, stabilization, Z-domain, capacity,protein G, cleaning-in-place (CIP), protein engineering, C2receptor.

I have been dealing with the themes of prefabricated houses and housing estates until recently. I was especially interested in their aesthetics. Order, grid and certain regularity and repeatability. At the same time, I was always interested in his social connotations, mainly because I live in these places. Gradually I became more interested in topics related to my own home. So I moved from the general themes to my own experience. But what is my home? Home is a place of utmost importance in our society. Home is made up of people, family. People have it associated with many rituals that accompany their lives often without actually being considered for them. Thanks to these rituals, we manage, among other things, the everyday influx of positive and negative influences of the surroundings and deal with them in various ways. Morning coffee, brushing your teeth, lighting a candle, wiping dust, filling a bath, scattering water on flowers, etc. Balance is the key to everything we do. And I would like to analyze, document and process these home and personal rituals in this work. The final thesis will consist of a free series of paintings with possible interventions and overlaps into video or installation, etc.

The thesis focuses on repair preparation of Ion Filters at nuclear power plants cleaning units. The thesis shortly describes cleaning units of primary and secondary circuit where Ion Filters and Ion Catchers are implemented. The thesis introduces summary of convenient method for welding of thin filtration plates from austenitic materials to collecting pipe including convenient technology for fabrication of narrow loop holes in filtration plates. The aim of thesis is to propose convenient execution of repair and manufacturing of spare parts after different kind of damage of filter internals. Part of the thesis is welded joints quality verification on the testing sample including result of provided test, its evaluation and Ion Filters repair economical evaluation as well.

Introdução: Nas últimas décadas tem-se pesquisado a saúde bucal como um importante atributo no contexto da saúde integral do indivíduo. Diante do exposto estabeleceu-se como objetivos: Avaliar a condição clínica e microbiológica da cavidade bucal do paciente com entubação orotraqueal internado em um hospital terciário, em dois momentos (até 48 horas da entubação orotraqueal e em 72 horas após a 1a coleta); caracterizar os pacientes quanto ao gênero, faixa etária, etnia, motivo de hospitalização, ocorrência de infecção nosocomial, período de hospitalização e óbito; avaliar na saliva destes pacientes (dentados e edêntulos) a presença de microrganismos epidemiologicamente importantes (Staphylococcus spp., Pseudomonas spp., Enterococos spp. e Leveduras); descrever o uso de antimicrobianos e determinar o perfil de sensibilidade das cepas isoladas aos antibióticos. Material e Método: este estudo prospectivo foi realizado em um período de quatro meses consecutivos mediante a aprovação do comitê de ética em pesquisa. Utilizou-se na coleta dos dados recursos referenciados na literatura e específicos a cada objetivo. Após a codificação apropriada de cada uma das variáveis elaborou-se o banco de dados no Microsoft Excel validado mediante dupla entrada. Utilizou-se o programa Statical Package for the Social Sciences (SPSS) versão 14.0 para análise estatística e os testes não paramétricos de McNemar e Wilcoxon, considerando um nível de significância fixo a = 0,05. Resultados: dos 68 pacientes (66,2%) masculinos, na faixa etária entre 31 a 59 anos, e 39,7% teve como principal motivo de hospitalização doenças do aparelho circulatório. Evidenciou-se que das 54,4% ocorrências das infecções hospitalares 35,2% eram respiratórias, seguidas de 27,0% vasculares. A média de hospitalização foi de 27 dias e 48,5% correspondeu à taxa de mortalidade. Quanto às condições clínicas da cavidade bucal observou-se uma degradação na saúde gengival, e no índice de higienização bucal, bem como na ocorrência de queilite, entre outros agravos. Na avaliação da microbiota, o microrganismo de maior freqüência (86,7%) foi o Staphylococcus spp., E, os antibióticos mais utilizados foram (72,3%) do grupo betalactâmico. Outro aspecto preocupante se reporta as cepas resistentes aos antibióticos cuja freqüência houve um acréscimo quando comparado os dois momentos de coleta. Conclusão: os resultados deixam transparecer alguns questionamentos sugestivos para futuras pesquisas e alertam sobre a necessidade de implementar um protocolo de higienização oral direcionado a pacientes críticos com entubação orotraqueal.
Introduction: For the last decades, oral health has been studied as an important part of an individual\'s general health. Objectives: Based on that information, this study aimed to evaluate the oral cavity clinical and microbiological status of patients with orotracheal intubation at a tertiary hospital (48 hours after orotracheal intubation and 72 hours after the first collection) as well as classify those patients according to gender, age, race, reason for being at the hospital, occurrence of nosocomial infection, period at the hospital and death. It also aimed to assess important epidemiological micro organisms (Staphylococcus spp., Pseudomonas sp., Enterococos spp and Yeasts) in (toothed and edentulous) patients\' saliva, and describe antimicrobial use and specify the characteristics of isolated strains to antibiotics. Material and Method: This study took four months and it was approved by the Research Ethics Committee. Resources from the literature, which were specific for each objective, were used when collecting data. A database was created using Microsoft Excel, validated with double input after appropriate coding of each one of the variables. Statical Package for the Social Sciences (SPSS) version 14.0 was used for statistical analysis and McNemar and Wilcoxon non-parametric tests with a = 0.05 significance. Results: out of 68 male patients (66.2%) between 31 and 59 years old, 39.7% were hospitalized mainly because of circulatory diseases. Out of 54.4% of hospital infections, 35.2% were respiratory infections and 27.0% were vascular. On average the patients were 27 days at the hospital and 48.5% of them died. It was possible to notice that gingival health and oral cleansing got worse, there was keilite and other diseases. According to the microbiota evaluation, Staphylococcus spp. was more prevalent and the antibiotics used (72.3) belonged to beta-lactamico group. The strain got more resistant to antibiotic if both collect were compared. Conclusion: Future researches with questioning were suggested and a warning about the need of a protocol of oral cleansing for critical patients with orotracheal intubation.

In the industrial sector, it is possible to observe unexpected behaviours of processes, which were not anticipated in the design stage. Such is the case of a secondary cooling process existing within a tobacco manufacturing plant, which was initially designed to cool and recover clean process water with chilled water for posterior recirculation into the main cooling process. It has been observed that under the design flowrates the plate and frame heat exchanger where the process water is cooled is rapidly clogged by suspended solids coming from the main cooling process, resulting in both high maintenance costs and frequent product ion downtime due to necessary intervention for mechanical cleaning. The aim of this thesis project is to evaluate and design process improvements providing sustainable solutions, reducing the operative costs existing nowadays and avoiding any damage on the plate and frame heat exchanger. Any process design solution asks for a capital investment, which was also considered in the overall analysis of the alternatives within this study. Within the evaluation of the possible modifications, solid/liquid separation was mostly studied, together with the selection and design of an appropriate heat exchanger for the given process. After a preliminary screening, a reduced number of alternatives were analysed in detail to assess their possible application. Few alternatives to solving the problem have been studied, in spite of having a wide variety of options.

Currently, many developing countries are experiencing rising prevalences of caries associated with changes in lifestyle and living conditions. Objectives: To describe the hygiene, eating habits, and oral health status of Nepalese children. Materials and Methods: A stratifiedsample of 231 children 5–7, 11–13, and 15–16 years of age (53% boys, 47% girls) who attended public high schools in the rural area of the Lalitpur District, Nepal was selected. The study was a field study combining a clinical examination (plaque, gingivitis, calculus, and caries) and a questionnaire. The questions concerned sanitary conditions, health support, personal hygiene, tooth cleaning, and eating habits. Results: During the school day, half of the children ate nothing at all. General personal hygiene was associated with tooth-cleaning frequency.Four out of five children in the entire sample cleaned their teeth once/day or more, using their own toothbrush. The use of fluoride toothpaste was rare.More frequent tooth cleaning and lower plaque indices were seen among girls and older children. More plaque was found on the occlusal surfaces of erupting permanent molars than on fully occluded permanent molars. Most children had a low prevalence of manifest caries in the primary and the permanent dentitions. However, every fifth 5–7-yr-old had manifest caries in three or more primary teeth. The occlusal surfaces of molars accounted for almost all registered caries in both dentitions. Conclusion: Although the prevalence of manifest caries was low, the low level of preventive activities may cause an increase in the prevalence of caries, as in other developing countries. The presumed risk scenario needs to be met by comprehensive and systematic health promotion and preventive measures.
Sammanfattning: I många utvecklingsländer sker förändringar av livsstil och levnadsförhållanden med samtidig ökad förekomst av karies. Mål: Att beskriva hygien, matvanor och munhälsa hos nepalesiska barn. Material och Metod: Ett stratifierat urval av 231 barn, som i åldrarna 5-7, 11-13 och 15-16 år (53% pojkar, 47% flickor), elever i statliga grundskolor på landsbygden, Lalitpur distriktet Nepal, användes. Studien utformades som en fältstudie med klinisk undersökning (plack, gingivit, tandsten och karies) kombinerad med en enkätstudie. Frågorna rörde sanitära förhållanden, hälsostöd från hemmet, personlig hygien, tandrengörings- och matvanor. Resultat: Under skoldagen åt hälften av barnen ingenting alls. Allmän personlig hygien var associerad med tandrengörings frekvens. Av alla barn, som användande sin egen tandborste, borstade fyra av fem, en gång om dagen eller mer. Äldre barn och flickor rengjorde tänderna oftare och hade ett lägre plackindex. Mer plack fanns på erupterande molarers occlusalytor jämfört med molarer i full ocklusion. De flesta barnen hade en låg frekvens manifest karies i primära och permanenta bettet. Dock hade en femtedel av 5-7 åringarna tre eller fler manifesta kariesangrepp i primära bettet. Ocklusal karies på molarerna utgjorde nästan all registrerad karies i båda dentitionerna. Slutsats: Låg frekvens av manifest karies, men en låg grad av förebyggande aktiviteter, kan medföra en ökad kariesfrekvens liknande den i andra utvecklingsländer. Den förmodande risken måste bemötas med behovsinriktade och systematiska hälsobefrämjande och preventiva åtgärder.

ISBN 91-7997-151-2

One of most major concerns from both cost-effective and technical point of view in membrane process industry is membrane cleaning. The aim of the project was to investigate the variations in membrane surface properties and separation efficiency of reverse osmosis membrane. Compativtive analysis have to be performed on four RO membrane before and after exposing the virgin membrane into chemical cleaning to identify and analysis the impact of the chemical cleaning on the performance of RO membrane. Commerical chemical cleaning used in this project were caustic and acidic cleaning agent. The project’s aim is the investigation of simulation software’s precision for the four membranes performance projection at different conditions of the feed water. The assessment of the membranes performance was done in the Innovation Cluster at pilot plant that was industrial in size. The main commercial elements used were the thin-film composite membranes with a spiral-wound of 8-inch polyamide. Ultrafiltration (UF) and seawater RO membrane pretreatment process was done for the red sea sourced feed water. A pressure vessel dimensioned at 8-inch was operated in conjunction with an individual element at 8 -20 m3/hr feed flow rate, with an 8 to 12 % recovery and an average 35,000-42,000 mg/L of total dissolved solids (TDS) composition for the feed water. To achieve the project’s aim in assessing the membranes, three phase experimental stages were completed. The membranes performance was assessed in terms of their water flux, salt rejection, boron rejection, bicarbonate rejection and permeate quality. In addition, the membrane surfaces were characterized after exposing the fresh membranes with a chemical cleaning reagent. The experimental results showed an increase in both permeate flow and salt passage for all studied elements. The changes in the membranes performance were systematically explained based on the changes in the charge density and chemical structure of the membranes surface. The experimental results showed that both the physical and chemical surface properties of the membranes do not significantly alter under standard industrial conditions. These results shed some light on the effects of chemical cleaning in a pilot-scale RO plant and improve our understanding to provide a potential research direction for cleaning methods of membranes.

No que diz respeito à desinfeção de equipamentos industriais, embora seja um assunto já abordado por alguns autores, na prática não existem muito dados sobre situações reais (o melhor processo, o melhor desinfetante, o tempo de contacto ideal, as condições ótimas, etc.) em particular para indústrias de fabrico de produtos de limpeza, como a Mistolin Company. Por este motivo esta dissertação teve como objetivo possibilitar uma melhor adequação dos métodos de desinfeção, de modo a permitir uma redução da probabilidade de contaminação do produto final. Para tal foram identificadas as informações atualmente disponíveis no que se refere à higienização de equipamentos industriais, tais como procedimentos, contaminantes e avaliação de grau de contaminação e legislação aplicável. Após acompanhamento e monitorização do procedimento de produção da fábrica em estudo e dos seus ciclos de higienização, foram realizadas análises microbiológicas. Por fim foi realizado um diagnóstico e foram propostas soluções. Para a higienização de equipamentos como depósitos de armazenamento e algumas linhas de enchimento foram implementadas alterações no procedimento de desinfeção. Sugeriu-se também o desenvolvimento de estudos adicionais para a adequação de parâmetros como a concentração da solução desinfetante.
Regarding the hygienization of industrial equipments, although it is a subject already addressed by some authors, there is not much data on actual situations, in particular for cleaning products industries, such as the Mistolin Company. For this reason, this dissertation aimed to enable a better adequacy of disinfection methods, in order to reduce the likelihood of contamination of the final product. To this end, the information currently available regarding the hygiene of industrial equipment, such as procedures, contaminants and evaluation of the degree of contamination and applicable legislation was reviewed. Microbiological analyses were carried out after monitoring the production procedure of the factory under study and its hygiene cycles. Finally, a diagnosis was made and solutions were proposed. For the sanitization of equipment such as storage deposits and some filling lines, alterations in the disinfection procedure were implemented. It was also suggested the realization of additional studies for the adequacy of parameters such as the concentration of the disinfectant solution.
Mestrado em Biotecnologia

Dissertação de mestrado integrado em Engenharia e Gestão Industrial
A filosofia Lean demonstra que uma empresa se pode destacar ser um verdadeiro competidor neste crescimento da economia mundial. A filosofia Lean tem como primeiro princípio o que é o valor do produto ou serviço na visão do cliente, a empresa tem de assegurar que o cliente vai receber o que pretende no tempo previsto. Então, esta filosofia tem como objetivo providenciar produtos e/ou serviços com a melhor qualidade possível no tempo pretendido pelo cliente. O presente projeto de dissertação tem como objetivo providenciar contentores lavados e esterilizados, utilizados no embalamento do produto final, sempre que necessário, conseguindo evitar atrasos nas linhas de produção ou reprocessamento do produto. Com base neste objetivo, foi efetuado um estudo na unidade fabril no interior da BetoFruta S.A., para a identificação e implementação de melhorias na secção de (Cleaning-in-Place) CIP/ (Sterilization-in-Place) SIP. Inicialmente, tive formação sobre todo o processo de produção e todos os produtos na unidade fabril. Identificaram-se os Mudas (desperdícios) existentes e foram propostas melhorias. Pela necessidade de contentores lavados e esterilizados, que ocorre na secção de CIP/SIP, foram implementadas as propostas de melhoria, ficando os restantes setores para um futuro próximo. Utilizou-se a filosofia Lean para resolver os problemas encontrados, usando ferramentas como os 5S, TPM, padronização do trabalho, poka-yoke, trabalho em células para fluxo do processo e organização do trabalho em equipa. Os objetivos foram cumpridos, conseguindo o CIP/SIP mais organizado e com todos os elementos devidamente identificados, o incentivo do trabalho em equipa e de operações de manutenção de simples execução, a uniformização do trabalho e, consequentemente, um maior número de contentores lavados e esterilizados. Atingindo mais 60 contentores por dia sendo 300 contentores por semana. Aumentando a produção em 33,33%.
The Lean philosophy shows that a company can stand out and be a real competitor in the growing world economy. Lean philosophy has as first principle what the value of the product or service is in the client’s point of view. The company assures that the client will have what he wants in the foreseen time. So, this philosophy’s main purpose is to provide products and/or services with the best possible quality in the time provided by the client. This dissertation project has as its main objective to provide washed and sterilized containers, which are used to pack the finished product, when they are needed so as to avoid delays in the production lines or the reprocessing of the product. With this objective in mind, a study was conducted in a factory situated in the interior of Portugal called “BetoFruta S.A.”, to identify and implement improvements in the (Cleaning-In-Place) CIP/ (Sterilization-In-Place)SIP sections. Initially, I had training to understand all the production processes and all the company’s products. The wastes were identified and improvement proposals were presented. Because of the necessity of washed and sterilized containers that occurs in the CIP/SIP section. The improvement proposals were implemented, the others were left to be applied in a close future. Lean philosophy and Work Study were used to solve the identified problems, using tools like the 5’S, work standardization, poka-yoke, work on cells to find process flow and organization of team work. The objectives were accomplished, resulting in a more organized section with all the tools and elements properly identified. Improvement of team work and execution of simple maintenence tasks were encouraged, as well as the standardization of processes, which in the end resulted on a greater number of washed and sterilized containers in the same time period as before. Acomplishing 60 more sterilized containers per day, what means 300 more per week. Improving production in 33.33%.

A Indumape – Industrialização de Fruta, S.A. é uma empresa sediada em Pombal. A Indumape é considerada a maior transformadora de fruta portuguesa, sendo especializada na produção de sumos concentrados de maçã, pera e baga de sabugueiro, bem como dos seus respetivos aromas. Tendo em vista o sucesso e crescimento empresarial, a Indumape pretende inserir-se em novos mercados, nomeadamente no mercado dos purés de fruta e legumes e, ainda, do baby food. Desta forma, o trabalho realizado no âmbito da presente Dissertação de Mestrado teve como principal objetivo o desenvolvimento e a respetiva validação de novos produtos alimentares, produzidos na linha de produção de purés de fruta armazenados em bidões. Antes de iniciar a produção dos purés, o processo de limpeza Cleaning-in-Place da linha foi validado e monitorizado durante todas as suas execuções. Ao longo do estágio na Indumape, foram acompanhados todos os ensaios produtivos realizados na linha de produção de purés: seis ensaios de puré de maçã, cinco de puré de pera, um de puré de marmelo, um de puré de kiwi e um ensaio de puré de morango. A qualidade e segurança alimentar de cada produto foram avaliadas um dia após a respetiva produção e ao longo do tempo de armazenamento à temperatura ambiente. Os parâmetros físico-químicos analisados foram: ºBrix, pH, acidez titulável, concentração de ácido ascórbico, cor (expressa como L*, a*, b* e ΔE) e viscosidade; e os microrganismos pesquisados incluíram mesófilos aeróbios, bolores, leveduras, coliformes, Escherichia coli e Alicyclobacillus. Através das análises sensoriais, os provadores avaliaram a cor, a textura, o aroma e o sabor de cada puré produzido. As diversas propriedades de cada produto foram diretamente influenciadas pela matéria-prima e condições do processo produtivo, pelo que, sempre que necessário, foram realizados ajustes nas produções seguintes. De um modo geral, a cor dos purés foi o parâmetro mais afetado ao longo do tempo de armazenamento. O processo produtivo implementado para cada tipo de puré, demonstrou ser eficiente na inativação dos microrganismos pesquisados, com a exceção única do primeiro ensaio de puré de pera. Além disso, a maioria dos ensaios produtivos obteve classificação “aceitável” ou superior nas quatro propriedades organoléticas avaliadas pelo painel de provadores. Todas as análises realizadas permitiram verificar a conformidade dos produtos ao nível físico-químico, microbiológico e sensorial, permitindo assim a validação dos purés de maçã, pera, marmelo, kiwi e morango produzidos na Indumape.
Indumape – Industrialização de Fruta, S.A. is a company located in Pombal. Indumape is considered the largest Portuguese fruit processor. It is specialized in the production of concentrated apple, pear, and elderberry juices and their respective aromas. To business success and growth, Indumape intends to enter new markets, namely in the market of fruit and vegetable purees and, also, baby food. Thus, this Thesis of Master had as its main objective the development and respective validation of new food products, obtained in the production line of fruit purees stored in drums. Before starting the production of purees, the line's Cleaning-in-Place process was validated and monitored during all its runs. During the internship at Indumape, all the productive tests carried out on the puree production line were followed up: six tests of apple puree, five of pear puree, one of quince puree, one of kiwi puree, and one puree test of strawberry. The food quality and safety of each product were evaluated one day after the puree production and, over the storage time at room temperature. The physicochemical parameters analyzed were: ºBrix, pH, titratable acidity, ascorbic acid concentration, color (expressed as L*, a*, b* and ΔE), and viscosity; and the microorganisms searched included aerobic mesophiles, molds, yeasts, coliforms, Escherichia coli, and Alicyclobacillus. Through sensory analysis, the tasters evaluated the color, texture, aroma, and flavor of each puree produced. The various properties of each product were directly influenced by the raw material and conditions of the production process so that, whenever necessary, adjustments were made to subsequent productions. In general, the color of purees was the most affected parameter over the storage time. The production process implemented for each type of puree proved to be efficient in inactivating the searched microorganisms, with the sole exception of the first pear puree test. In addition, most of the production tests obtained an «Acceptable» or higher rating in the four organoleptic properties evaluated by the panel of tasters. All the analyses carried out made it possible to verify the conformity of the products at the physical-chemical, microbiological and sensory properties, thus allowing the validation of apple, pear, quince, kiwi, and strawberry purees produced at Indumape.
Mestrado em Biotecnologia

碩士
國立屏東科技大學
食品科學系
92
Abstract Effect of washing (cleaning water, cleaning agent, cleaning method and blanching) on the napiergrass of the total plate count, chlorophyll and DPPH scavenging ability were studied. The fresh leaves and juice were further investigated for their pH, soluble solid, A420, total phenolics and DPPH scavenging ability in storage. Results showed that the total plate count is highest when napiergrass was sampled at 8 AM. The best way to decrease the total plate count was to clean the samples with the running water and blanched for 8 seconds, which reduced total plate count to 2.74 Log CFU/g. Both chlorophyll concentration and DPPH scavenging ability were reduced after washing, and they were positively correlated with coefficient value of 0.92. Storing the fresh grass, the shelf life of the samples blanched for 6 seconds was 4 days under 4 ℃, while stored under 25℃ could last only 1 days. Un-blanched sample can store 7 days under 4℃, and 3 days under 25℃. Its green color and chlorophyll content decreased during the storage, especially for the samples without blanching and stored under 25℃. The residual chlorophyll content was only 223.4 mg/L left (24.5% loss) after 8 days storage. The DPPH scavenging ability maintained well when stored under 4℃ (62.4% after 12 days storage). The pH graduate raised and the total phenolics decreased during the storage. obviously during the storage. The changes of storied napiergrass juice were in the same trend as in fresh grass. The chlorophyll decreased dramatically, especially in the blanched sample as compared with the sample without blanching (38.5 mg/L and 229.7 mg/L respectively). The changes of total phenolics, DPPH scavenging ability was similar to chlorophyll. The pH value decreased obviously during the 25℃ storage. When napiergrass juice was stored under 25℃, the total plate count would exceed 5 log/CFU/g after 12 hours.