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Edible active films based on sodium caseinate (SC) and calcium caseinate (CC) plasticized with glycerol (G) at three different concentrations and carvacrol (CRV) as active agent were prepared by solvent casting. Transparent films were obtained and their surfaces were analysed by optical microscopy and scanning electron microscopy (SEM). The influence of the addition of three different plasticizer concentrations was studied by determining tensile properties, while Fourier transformed infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA) were used to evaluate the structural and thermal behavior of such films. The addition of glycerol resulted in a reduction in the elastic modulus and tensile strength, while some increase in the elongation at break was observed. In general terms, SC films showed flexibility higher than the corresponding CC counterparts. In addition, the presence of carvacrol caused further improvements in ductile properties suggesting the presence of stronger interactions between the protein matrix and glycerol, as it was also observed in thermal degradation studies. FTIR spectra of all films showed the characteristic bands and peaks corresponding to proteins as well as to primary and secondary alcohols. In summary, the best results regarding mechanical and structural properties for caseinates-based films containing carvacrol were found for the formulations with high glycerol concentrations., Marina Patricia Arrieta thanks Fundacion MAPFRE for "Ignacio Hernando de Larramendi 2009- Medio Ambiente" fellowship (MAPFRE-IHL-01). The Spanish Ministry of Economy and Competitiveness is acknowledged by financial support (project Ref. MAT2011-28468-C02-01). Authors thank to Ferrer Alimentacion S.A., for providing caseinates and to Prof. Juan Lopez Martinez (Polytechnic University of Valencia, Spain) for his collaboration and useful discussions.

Active edible films were prepared by adding carvacrol into sodium caseinate (SC) and calcium caseinate (CC) matrices plasticized with two different glycerol concentrations (25 and 35 wt%) prepared by solvent casting. Functional characterisation of these bio-films was carried out by determination of some of their physico-chemical properties, such as colour, transparency, oxygen barrier, wettability, dye permeation properties and antibacterial effectiveness against Gram negative and Gram positive bacteria. All films exhibited good performance in terms of optical properties in the CIELab space showing high transparency. Carvacrol was able to reduce CC oxygen permeability and slightly increased the surface hydrophobicity. Dye diffusion experiments were performed to evaluate permeation properties. The diffusion of dye through films revealed that SC was more permeable than CC. The agar diffusion method was used for the evaluation of the films antimicrobial effectiveness against Escherichia cell and Staphylococcus aureus. Both SC and CC edible films with carvacrol showed inhibitory effects on both bacteria. (C) 2013 Elsevier Ltd. All rights reserved., This research was supported by the Ministry of Science and Innovation of Spain through the projects MAT2011-28468-C02-01, MAT2011-28468-C02-02 and HP2008-0080. M.P. Arrieta thanks Fundacion MAPFRE for "Ignacio Hernando de Larramendi 2009-Medio Ambiente" fellowship (MAPFRE-IHL-01). Authors thank Ferrer Alimentacion S.A., for providing the caseinates powders.

The development of phenolic-rich functional foods is often limited by the off-tastes of phenolics that might be counteracted by sequestering these compounds using a carrier, thereby preventing them to interact with bitter taste receptors and salivary proteins. A range of common animal food proteins were tested for binding of phenolics. It appeared that a proline-rich open protein structure, as in β-casein, favored binding of phenolics. Globular proteins other than bovine serum albumin showed poor potential for use as carrier. No appropriate carriers for monomeric phenolics were found. β-Casein and Na-caseinate were shown to have good bitter-masking potential for EGCG, as measured by a maximal reduction in bitter receptor activation of ~93% measured in vitro. This effective reduction in bitter receptor activation was confirmed by a sensory test. This illustrates the validity of using food proteins with good binding properties as carriers for phenolics. Different methodologies for probing the interaction between proteins and phenolics were developed: (i) ultrafiltration followed by UV quantification of unbound phenolics in the retentate, (ii) fluorescence quenching, and (iii) ultrafiltration followed by mass spectrometric quantification of unbound phenolics in the retentate. The latter method offered the opportunity to analyze preferential binding to protein of individual phenolics present in a complex mixture. With these methods, it was established that, with respect to phenolics, conformation and flexibility were important drivers of protein-phenolic interaction, besides degree of polymerization and galloylation. With respect to relatively proline-poor unstructured proteins such as α-casein and β-casein, it appeared that there should be other factors, besides proline density, explaining the interaction with phenolics.

The development of phenolic-rich functional foods is often limited by the off-tastes of phenolics that might be counteracted by sequestering these compounds using a carrier, thereby preventing them to interact with bitter taste receptors and salivary proteins. A range of common animal food proteins were tested for binding of phenolics. It appeared that a proline-rich open protein structure, as in β-casein, favored binding of phenolics. Globular proteins other than bovine serum albumin showed poor potential for use as carrier. No appropriate carriers for monomeric phenolics were found. β-Casein and Na-caseinate were shown to have good bitter-masking potential for EGCG, as measured by a maximal reduction in bitter receptor activation of ~93% measured in vitro. This effective reduction in bitter receptor activation was confirmed by a sensory test. This illustrates the validity of using food proteins with good binding properties as carriers for phenolics. Different methodologies for probing the interaction between proteins and phenolics were developed: (i) ultrafiltration followed by UV quantification of unbound phenolics in the retentate, (ii) fluorescence quenching, and (iii) ultrafiltration followed by mass spectrometric quantification of unbound phenolics in the retentate. The latter method offered the opportunity to analyze preferential binding to protein of individual phenolics present in a complex mixture. With these methods, it was established that, with respect to phenolics, conformation and flexibility were important drivers of protein-phenolic interaction, besides degree of polymerization and galloylation. With respect to relatively proline-poor unstructured proteins such as α-casein and β-casein, it appeared that there should be other factors, besides proline density, explaining the interaction with phenolics.

[EN] The present work analyzes the application method and the effectiveness of edible coatings based on caseinates or chitosan in partially dehydrated pineapple for its use in dry fruit-cereal products. Commercial breakfast cereals and partially dehydrated pineapples prepared by osmotic dehydration and vacuum impregnation were used for the experiments. Four coating application techniques, working at atmospheric pressure or applying vacuum impregnation, were used for coating the pineapple samples. Coated and uncoated samples were stored, at 20 degrees C with breakfast cereals and were subsequently analyzed weekly during 6 months. Critical water activity and critical water content for the glass transition of the cereals at 20 degrees C were estimated as 0.60 and 9.1 g water/100 g of product, respectively. Results show that the application of coatings by vacuum impregnation and sample air drying after coating, considerably extends the shelf-life of the pineapple-cereal system when caseinate based coatings are used. Chitosan based coatings are less effective to limit water vapor transfer and the critical moisture content of cereals are reached during the storage period. (c) 2012 Elsevier Ltd. All rights reserved., The authors acknowledge the financial support from the Conselleria de Educacion de la Comunidad Valenciana through Project GVPRE/2008/355 and Universitat Politecnica de Valencia through Project PAID-06-08-3242 M.J. Fabra thanks the support of Campus de Excelencia Internacional from Universitat Politecnica de Valencia.

The casein and its hydrolysates have many desirable functional properties, so there is great interest in its use in food and pharmaceutical industries. The hydrolysis of casein by a new protease obtained from latex Jacaratia corumbensis was performed to obtain antimicrobial peptides. Four proteins were visualized by electrophoresis of the enzyme extract of J. corumbensis, a band of molecular weight of approximately 30 kDa with the presence of other bands of low density. The fractions as-CN, b-CN and k-CN, were subjected to hydrolysis for a period of 1 min 24 h, where aliquots were collected and analyzed by SDS-PAGE to the characterization of peptides. The hydrolysates were evaluated for antimicrobial activity by determining the Minimum Inhibitory Concentration (MIC). The hydrolysates obtained at time of 2 h showed antimicrobial activity against microorganisms Enterococcus faecalis, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Staphylococcus aureus, indicating that the peptides obtained by hydrolysis of bovine casein by latex extract J. corumbensis showed activity antimicrobial. This fraction was subjected to reverse phase chromatography for puri?cation of peptides and subsequent mass spectrometric evaluation. The peptides were sequenced and evaluated for their antimicrobial potential. Made available in DSpace on 2022-05-20T05:00:33Z (GMT). No. of bitstreams: 1 API-New-peptides.pdf: 864064 bytes, checksum: 2f8ebbdd83d26f7721233705143d6d49 (MD5) Previous issue date: 2012

Biomass is one of the main alternatives to the use of oil in plastics field. Due to their various functional properties, caseinates are considered as an interesting raw material for making biodegradable and water-soluble packaging. The transformation of sodium caseinate by the processes used for synthetic plastics industry has been demonstrated. A corotating twin-screw extruder was used to get glycerol plasticized caseinate pellets. The physicochemical properties of the obtained material have confirmed the thermal stability of this protein and demonstrated the influence of surrounding moisture on material behavior. With the Relative Humidity varying from 40 to 90% and increasing the glycerol content, an hydrous plasticizer, the mechanical properties of sodium caseinate based materials changed from those of glassy (rigid) to rubbery (soft) plastics. The pellets were then taken over to make film using a blown-film extruder. The water vapour permeability of blown film was studied and showed that sodium caseinate based films are a poor moisture barrier. The second part dealt with the sodium caseinate blend. Sodium caseinate/calcium caseinate and sodium caseinate/PBAT blends was performed. Adding calcium caseinate, started from 50%, improved the mechanical properties and delayed hydrous transfer through the film. Concerning the sodium caseinate/PBAT blends, adding PBAT increased until twice more the material's Young modulus but decreased the moisture transfer resistance because of the incompatibility between the two polymers., La biomasse représente l'une des principales alternatives à l'utilisation du pétrole dans la plasturgie. Grâce à leurs propriétés fonctionnelles, les caséinates sont une matière première prometteuse pour la fabrication de films plastiques pour des applications dans l'emballage biodégradable et hydrosoluble. La transformation du caséinate de sodium par les techniques habituellement utilisées dans la plasturgie a été démontrée. Des extrudats de caséinate plastifié au glycérol ont été obtenus au moyen d'une extrudeuse bi-vis corotative. La caractérisation physico-chimique du matériau obtenu a confirmé la thermostabilité de cette protéine et a montré la dépendance du comportement du matériau vis-à-vis de l'humidité ambiante. Pour une variation du taux d'Humidité Relative, de 40 à 90% et une augmentation de la concentration du glycérol, plastifiant hygroscopique, le matériau passe d'un état vitreux (rigide) à un état caoutchouteux (mou). Des films fins ont été ensuite réalisés, à partir des extrudats thermoplastiques, par extrusion gonflage. La perméabilité à la vapeur d'eau des films de caséinate de sodium a été étudiée et a montré que ces matériaux sont de mauvaises barrières à l'humidité. La deuxième partie a été consacré à l'étude de mélanges caséinate de sodium/caséinate de calcium d'un côté et caséinate de sodium/PBAT de l'autre. Le mélange des deux caséinates a permis d'augmenter la tenue mécanique du mélange, à partir de 50% de caséinate de calcium, et de retarder le transfert hydrique à travers le film. Dans le même sens, l'ajout du PBAT, a augmenté jusqu'à deux fois plus, le module d'Young des mélanges mais a baissé la résistance au transfert d'humidité du film à cause de l'incompatibilité des deux polymères.

Both health and sustainability are drivers for the increased interest in the creation of novel foods comprising a high protein content. The key challenge is the formation of an attractive, stable and palatable food texture, which is mainly determined by the food structure. In this research, new processing routes based on flow are explored to create innovative protein-rich structures (exceeding 10%), and in parallel, insight is gained in the relevant mechanisms of structure formation. Dense dispersions of sodium caseinate and calcium caseinate, which are derived from milk, were mixed in a conventional mixer and sheared in an in house developed shear cell device. After mixing dense (sodium or calcium) caseinate dispersions, homogenous structures were obtained of which the properties were determined by the dispersed phase added, in this case palm fat. Shearing of dense calcium caseinate dispersions in combination with solidification using the enzyme transglutaminase resulted in completely different structures; highly fibrous structures were produced, which may serve as a basis for the creation of meat analogs. In contrast, after treating dense sodium caseinate dispersions using this novel structuring process, homogenous structures were obtained. It appeared that the intrinsic properties of protein dispersions are important for the formation of fibrous structures using well-defined flow. Based on the differences in performance between the two types of caseinates, tools and parameters can be derived to optimize and control the formation of fibrous products. In conclusion, the development of equipment that is dedicated to structure food ingredients is promising for the creation of novel foods on the one hand, and for gaining scientific understanding of structure formation on the other hand.

Both health and sustainability are drivers for the increased interest in the creation of novel foods comprising a high protein content. The key challenge is the formation of an attractive, stable and palatable food texture, which is mainly determined by the food structure. In this research, new processing routes based on flow are explored to create innovative protein-rich structures (exceeding 10%), and in parallel, insight is gained in the relevant mechanisms of structure formation. Dense dispersions of sodium caseinate and calcium caseinate, which are derived from milk, were mixed in a conventional mixer and sheared in an in house developed shear cell device. After mixing dense (sodium or calcium) caseinate dispersions, homogenous structures were obtained of which the properties were determined by the dispersed phase added, in this case palm fat. Shearing of dense calcium caseinate dispersions in combination with solidification using the enzyme transglutaminase resulted in completely different structures; highly fibrous structures were produced, which may serve as a basis for the creation of meat analogs. In contrast, after treating dense sodium caseinate dispersions using this novel structuring process, homogenous structures were obtained. It appeared that the intrinsic properties of protein dispersions are important for the formation of fibrous structures using well-defined flow. Based on the differences in performance between the two types of caseinates, tools and parameters can be derived to optimize and control the formation of fibrous products. In conclusion, the development of equipment that is dedicated to structure food ingredients is promising for the creation of novel foods on the one hand, and for gaining scientific understanding of structure formation on the other hand.

Colon cancer (cancer of the large intestine) is a worldwide problem in especially Western countries. The diet might be responsible for up to 90% of these colon cancer cases. This means that decreasing colon cancer risk should be possible by changing the diet. The research presented in this thesis concerns the question what the influence is of dietary protein sources on colon cancer risk. As described in Chapter1 , casein was compared to other dietary protein sources (mainly soy protein) for its influence on:the carcinogen scavenging capacity of proteins,early stages of colon cancer,fecal colon cancer biomarkers.Chapter2 provides information on various fields of study in the research on dietary proteins and colon cancer, including background information on the methods used. It is explained how dietary proteins can act as carcinogen scavengers by binding to carcinogens in the gastro-intestinal lumen. The carcinogen can be transported along the intestinal tract and be taken up by the body. If a carcinogen is not taken up, it will be excreted with the feces. Furthermore, the influence of the amount of protein intake in the diet on colon cancer risk is discussed. It is concluded that either no or a positive correlation between the amount of protein intake and colon cancer risk, exists. Another important research area is protein digestibility because a low protein digestibility results in a high amount of protein in the colon. Furthermore, the amino acid composition of a protein is important for two reasons. When an essential amino acid is present in a limiting amount, growth of both the individual and the tumor is inhibited. Secondly, some amino acids are needed for specific biological functions. For example, sulfur containing amino acids can stimulate the biotransformational system. The last important factor discussed in the research area of proteins on colon cancer risk is the presence of various non-protein components. As an example, saponins and isoflavones present in many soy proteins and known to be biologically active, are discussed.In Chapter 3 and 4 it is described how dietary proteins can scavenge carcinogens in vitro . In Chapter3 it is shown that carcinogen scavenging is influenced by many factors, such as heat treatment of the protein, degree of protein digestion and presence of bile acids or lipase. Protein type did not have a major influence so no effect of feeding different dietary protein sources on colon cancer risk is expected.In Chapter4 it is shown that the carcinogen scavenging capacity of proteins is also very much dependent on the type of carcinogen. Benzo[a]pyrene, a large non-reactive pro-carcinogen, interacted only with intact dietary proteins, whereas MNNG, as described in Chapter 3, strongly interacted with both intact as well as hydrolyzed proteins. Again no difference between dietary protein sources was shown, suggesting no difference in colon cancer risk.In Chapter 5 the first animal experiments are described. In Study 1, the influence of the addition of 1% methionine to the diet on colonic cell proliferation was tested. Methionine is an important methyl donor in many metabolic routes and is present in different amounts in casein and soy protein preparations. Colonic cell proliferation was used as an indication of colon cancer risk, because cell proliferation is an important phase in the process of colon cancer. The study showed no influence of methionine on colonic cell proliferation. In Study 2 rats were fed different amounts of soy non-nutrients to test the influence of these non-nutrients on several parameters in the feces. Most important fecal parameter was the fecal fat percentage, because earlier studies reported a correlation between high fecal fat and colon tumors. Study 2 showed that fecal fat excretion in rats is dependent on the amount of soy non-nutrients in the diet. However, the difference in fecal fat excretion between casein and soy protein was smaller (factor 2) than reported in literature. Therefore no major effect on colon cancer risk is expected. The influence of soy non-nutrients on processes in the colon was shown in Study 3. In this in vitro study, soy non-nutrients strongly reduced bile acid induced cell damage, suggesting a small protective effect of dietary soy protein.In Chapter 6 two animal studies on the influence of casein and soy protein on colonic cell proliferation are described. The studies showed no difference in colonic cell proliferation between these two protein sources. However different results for the casein and soy protein diets were obtained for several parameters that were measured in the feces of the animals. These fecal parameters were measured because they show how important steps in the process of colon cancer development are affected. A high fecal fat percentage correlates with a high number of tumors in the colon. Fecal alkaline phosphatase (ALP) activity represents damage to cells of the colonic epithelium because epithelial cells exhibited a high ALP activity. Bile acids and free fatty acids can damage the colonic epithelium because of their lytic potential. The cytolytic activity measures this damaging capacity, because it measures the lytic potential of the colonic contents towards cells in an in vitro test. The pH of the fecal contents was measured because a low pH represents a healthy colonic environment. Fecalb-glucuronidase andb-glucosidase activities were measured because they represent the capacity of the colonic contents to liberate toxicants in the colonic lumen that were earlier detoxified by the liver.Results on cytolytic activity confirmed the lack of difference between casein and soy on colonic cell proliferation because there was no difference between diets. Fecal fat excretion was doubled after feeding soy protein compared to casein. After feeding soy protein, fecal water bile acid concentrations and ALP activity were decreased. However,b-glucuronidase andb-glucosidase were increased 10-100 fold. Overall, no consistent conclusion on fecal parameters was possible.In Chapter 7 the influence of casein, soy protein isolate and soy flour on colonic aberrant crypt foci (ACF) is tested. ACFs are considered to be early stages of colonic tumors. Overall no consistent difference of casein and soy protein on aberrant crypt foci was detected. Againb-glucuronidase,b-glucosidase and ALP activities were measured in feces.b-Glucuronidase andb-glucosidase activities were significantly increased after feeding soy protein suggesting an increased risk after soy feeding.In Chapter 8 the influence of casein, soy protein isolate, soy protein flour and red meat is tested on the occurrence of intestinal polyps in the Apc Minmodel. In this model, mice with a defect Apc gene spontaneously develop polyps in both small and large intestine. The four diets showed no difference in the occurrence of polyps. Again several differences were observed in the fecal parameters.b-Glucuronidase andb-glucosidase were strongly increased after soy protein compared to the other diets. The pH of the colonic contents was decreased after soy flour feeding indicating fermentation of fibers present in soy flour preparations has a major influence. Furthermore it was shown that heme in the form of meat protein is not nearly as lytic as heme added to the diet in the pure form. No differences between the diets were found for free fatty acids. The concentrations of bile acids in fecal water were decreased for especially the soy protein diets. Overall, fecal parameters showed marked differences, but again no consistent protective or risk inducing effects.In Chapter 9 all studies performed are compared and discussed with an emphasis on the animal studies. Overall, fecal fat excretion was consistently doubled after feeding soy protein.b-Glucuronidase andb-glucosidase, able to release toxicants, were consistently increased after feeding soy protein. Bile acids in fecal water, able to damage colonic epithelium, were consistently decreased after soy protein feeding. Other fecal parameters either showed no difference between diets (pH, cytolytic activity) or variable results (ALP, free fatty acids). It was shown that fecal magnesium excretion has no predictive value for colonic cell proliferation. Fecalb-glucuronidase,b-glucosidase, ALP, bile acids and free fatty acids showed large differences in absolute values between animal experiments. Possibly the effect caused by some protein sources on fecal parameters, is also partly dependent on interaction with other dietary constituents such as fat and fiber or non-protein components. Because no differences on cell proliferation, aberrant crypt foci and intestinal polyps were found, a difference in colon cancer risk after consumption of casein or soy protein is unlikely. Correlation studies revealed that none of the fecal parameters tested consistently predicted the outcome of the colonic parameters tested, stressing the need for further research in this area. Because of the variable composition and results obtained with soy, it was concluded that one should be very cautious concerning the interpretation of studies in which soy protein preparations are used.The main conclusion from the studies performed is that many significant differences occurred between casein and other dietary protein sources such as soy. Differences specifially occurred on parameters related to the carcinogen scavenging capacity of proteins and on fecal parameters such as fecal bile acid, ALP andb-glucuronidase excretion. However, no consistent in vivo protective effect of casein occurred on colon cancer, based on markers as colonic cell proliferation, aberrant crypt foci formation and polyp formation. Therefore results do not support an advice on consuming either more or less casein or soy protein containing products.

Colon cancer (cancer of the large intestine) is a worldwide problem in especially Western countries. The diet might be responsible for up to 90% of these colon cancer cases. This means that decreasing colon cancer risk should be possible by changing the diet. The research presented in this thesis concerns the question what the influence is of dietary protein sources on colon cancer risk. As described in Chapter1 , casein was compared to other dietary protein sources (mainly soy protein) for its influence on:the carcinogen scavenging capacity of proteins,early stages of colon cancer,fecal colon cancer biomarkers.Chapter2 provides information on various fields of study in the research on dietary proteins and colon cancer, including background information on the methods used. It is explained how dietary proteins can act as carcinogen scavengers by binding to carcinogens in the gastro-intestinal lumen. The carcinogen can be transported along the intestinal tract and be taken up by the body. If a carcinogen is not taken up, it will be excreted with the feces. Furthermore, the influence of the amount of protein intake in the diet on colon cancer risk is discussed. It is concluded that either no or a positive correlation between the amount of protein intake and colon cancer risk, exists. Another important research area is protein digestibility because a low protein digestibility results in a high amount of protein in the colon. Furthermore, the amino acid composition of a protein is important for two reasons. When an essential amino acid is present in a limiting amount, growth of both the individual and the tumor is inhibited. Secondly, some amino acids are needed for specific biological functions. For example, sulfur containing amino acids can stimulate the biotransformational system. The last important factor discussed in the research area of proteins on colon cancer risk is the presence of various non-protein components. As an example, saponins and isoflavones present in many soy proteins and known to be biologically active, are discussed.In Chapter 3 and 4 it is described how dietary proteins can scavenge carcinogens in vitro . In Chapter3 it is shown that carcinogen scavenging is influenced by many factors, such as heat treatment of the protein, degree of protein digestion and presence of bile acids or lipase. Protein type did not have a major influence so no effect of feeding different dietary protein sources on colon cancer risk is expected.In Chapter4 it is shown that the carcinogen scavenging capacity of proteins is also very much dependent on the type of carcinogen. Benzo[a]pyrene, a large non-reactive pro-carcinogen, interacted only with intact dietary proteins, whereas MNNG, as described in Chapter 3, strongly interacted with both intact as well as hydrolyzed proteins. Again no difference between dietary protein sources was shown, suggesting no difference in colon cancer risk.In Chapter 5 the first animal experiments are described. In Study 1, the influence of the addition of 1% methionine to the diet on colonic cell proliferation was tested. Methionine is an important methyl donor in many metabolic routes and is present in different amounts in casein and soy protein preparations. Colonic cell proliferation was used as an indication of colon cancer risk, because cell proliferation is an important phase in the process of colon cancer. The study showed no influence of methionine on colonic cell proliferation. In Study 2 rats were fed different amounts of soy non-nutrients to test the influence of these non-nutrients on several parameters in the feces. Most important fecal parameter was the fecal fat percentage, because earlier studies reported a correlation between high fecal fat and colon tumors. Study 2 showed that fecal fat excretion in rats is dependent on the amount of soy non-nutrients in the diet. However, the difference in fecal fat excretion between casein and soy protein was smaller (factor 2) than reported in literature. Therefore no major effect on colon cancer risk is expected. The influence of soy non-nutrients on processes in the colon was shown in Study 3. In this in vitro study, soy non-nutrients strongly reduced bile acid induced cell damage, suggesting a small protective effect of dietary soy protein.In Chapter 6 two animal studies on the influence of casein and soy protein on colonic cell proliferation are described. The studies showed no difference in colonic cell proliferation between these two protein sources. However different results for the casein and soy protein diets were obtained for several parameters that were measured in the feces of the animals. These fecal parameters were measured because they show how important steps in the process of colon cancer development are affected. A high fecal fat percentage correlates with a high number of tumors in the colon. Fecal alkaline phosphatase (ALP) activity represents damage to cells of the colonic epithelium because epithelial cells exhibited a high ALP activity. Bile acids and free fatty acids can damage the colonic epithelium because of their lytic potential. The cytolytic activity measures this damaging capacity, because it measures the lytic potential of the colonic contents towards cells in an in vitro test. The pH of the fecal contents was measured because a low pH represents a healthy colonic environment. Fecalb-glucuronidase andb-glucosidase activities were measured because they represent the capacity of the colonic contents to liberate toxicants in the colonic lumen that were earlier detoxified by the liver.Results on cytolytic activity confirmed the lack of difference between casein and soy on colonic cell proliferation because there was no difference between diets. Fecal fat excretion was doubled after feeding soy protein compared to casein. After feeding soy protein, fecal water bile acid concentrations and ALP activity were decreased. However,b-glucuronidase andb-glucosidase were increased 10-100 fold. Overall, no consistent conclusion on fecal parameters was possible.In Chapter 7 the influence of casein, soy protein isolate and soy flour on colonic aberrant crypt foci (ACF) is tested. ACFs are considered to be early stages of colonic tumors. Overall no consistent difference of casein and soy protein on aberrant crypt foci was detected. Againb-glucuronidase,b-glucosidase and ALP activities were measured in feces.b-Glucuronidase andb-glucosidase activities were significantly increased after feeding soy protein suggesting an increased risk after soy feeding.In Chapter 8 the influence of casein, soy protein isolate, soy protein flour and red meat is tested on the occurrence of intestinal polyps in the Apc Minmodel. In this model, mice with a defect Apc gene spontaneously develop polyps in both small and large intestine. The four diets showed no difference in the occurrence of polyps. Again several differences were observed in the fecal parameters.b-Glucuronidase andb-glucosidase were strongly increased after soy protein compared to the other diets. The pH of the colonic contents was decreased after soy flour feeding indicating fermentation of fibers present in soy flour preparations has a major influence. Furthermore it was shown that heme in the form of meat protein is not nearly as lytic as heme added to the diet in the pure form. No differences between the diets were found for free fatty acids. The concentrations of bile acids in fecal water were decreased for especially the soy protein diets. Overall, fecal parameters showed marked differences, but again no consistent protective or risk inducing effects.In Chapter 9 all studies performed are compared and discussed with an emphasis on the animal studies. Overall, fecal fat excretion was consistently doubled after feeding soy protein.b-Glucuronidase andb-glucosidase, able to release toxicants, were consistently increased after feeding soy protein. Bile acids in fecal water, able to damage colonic epithelium, were consistently decreased after soy protein feeding. Other fecal parameters either showed no difference between diets (pH, cytolytic activity) or variable results (ALP, free fatty acids). It was shown that fecal magnesium excretion has no predictive value for colonic cell proliferation. Fecalb-glucuronidase,b-glucosidase, ALP, bile acids and free fatty acids showed large differences in absolute values between animal experiments. Possibly the effect caused by some protein sources on fecal parameters, is also partly dependent on interaction with other dietary constituents such as fat and fiber or non-protein components. Because no differences on cell proliferation, aberrant crypt foci and intestinal polyps were found, a difference in colon cancer risk after consumption of casein or soy protein is unlikely. Correlation studies revealed that none of the fecal parameters tested consistently predicted the outcome of the colonic parameters tested, stressing the need for further research in this area. Because of the variable composition and results obtained with soy, it was concluded that one should be very cautious concerning the interpretation of studies in which soy protein preparations are used.The main conclusion from the studies performed is that many significant differences occurred between casein and other dietary protein sources such as soy. Differences specifially occurred on parameters related to the carcinogen scavenging capacity of proteins and on fecal parameters such as fecal bile acid, ALP andb-glucuronidase excretion. However, no consistent in vivo protective effect of casein occurred on colon cancer, based on markers as colonic cell proliferation, aberrant crypt foci formation and polyp formation. Therefore results do not support an advice on consuming either more or less casein or soy protein containing products.

Dit onderzoek is uitgevoerd om na te gaan of de impedantiemeting gebruikt kan worden als snelle screeningsmethode voor het mesofiel aeroob kiemgetal van caseines en caseinaten. De experimenten zijn uitgevoerd met een Malthus Growth Analyser.