WALTHAM SUPPLEMENT Prediction of crude protein digestibility of animal by-product meals for dogs by the protein solubility in pepsin method* Iris M. Kawauchi1,2, Nilva K. Sakomura2, Cristiana F. F. Pontieri1, Aline Rebelato2, Thaila C. Putarov3, Euclides B. Malheiros2, Márcia de O. S. Gomes2,4, Carlos Castrillo5 and Aulus C. Carciofi2† 1Grandfood Indústria e Comércio Ltda, Dourado, SP, Brazil 2College of Agrarian and Veterinarian Sciences (FCAV), São Paulo State University (UNESP), Via de Acesso Professor Paulo Donato Castellane, Jaboticabal 14.884-900, SP, Brazil 3School of Veterinary Medicine and Animal Science (FMVZ), Sao Paulo State University (UNESP), Distrito de Rubião Junior, Botucatu 18.618-970, SP, Brazil 4Camilo Castelo Branco University (UNICASTELO), Av. Hilário da Silva Passos, Descalvado 13690-000, SP, Brazil 5Animal Production and Food Sciences Department, University of Zaragoza, Miguel Servet, 177, Zaragoza 50.013, Spain (Received 11 November 2013 – Final revision received 16 February 2014 – Accepted 26 February 2014) Journal of Nutritional Science (2014), vol. 3, e36, page 1 of 5 doi:10.1017/jns.2014.32 Abstract Animal by-product meals have large variability in crude protein (CP) content and digestibility. In vivo digestibility procedures are precise but laborious, and in vitro methods could be an alternative to evaluate and classify these ingredients. The present study reports prediction equations to estimate the CP digest- ibility of meat and bone meal (MBM) and poultry by-product meal (PM) using the protein solubility in pepsin method (PSP). Total tract CP digestibility of eight MBM and eight PM samples was determined in dogs by the substitution method. A basal diet was formulated for dog maintenance, and sixteen diets were produced by mixing 70 % of the basal diet and 30 % of each tested meal. Six dogs per diet were used to determine ingredient digestibility. In addition, PSP of the MBM and PM samples was determined using three pepsin concentrations: 0·02, 0·002 and 0·0002 %. The CP content of MBM and PM ranged from 39 to 46 % and 57 to 69 %, respectively, and their mean CP digestibility by dogs was 76 (2·4) and 85 (2·6) %, respectively. The pepsin concentration with higher Pearson correlation coefficients with the in vivo results were 0·0002 % for MBM (r 0·380; P= 0·008) and 0·02 % for PM (r 0·482; P= 0·005). The relationship between the in vivo and in vitro results was better explained by the following equations: CP digestibility of MBM= 61·7 + 0·2644 × PSP at 0·0002 % (P= 0·008; R2 0·126); and CP digestibility of PM = 54·1 + 0·3833 × PSP at 0·02 % (P= 0·005; R2 0·216). Although significant, the coefficients of determination were low, indicating that the models were weak and need to be used with caution. Key words: Dog food: In vitro methods: Meat and bone meal: Poultry by-product meal Animal by-product meals have been (and continue to be) the primary ingredient responsible for the growth and expansion of the global pet food industry because they provide most of the protein, fat and minerals of the diets(1). However, one difficulty in the use of these ingredients is that they have highly variable chemical composition and quality. Manufacturers of animal by-products use different criteria to select and determine which raw materials can be directly used for human or animal consumption, are further pro- cessed(2), or are disposed off. In addition, processing condi- tions may be markedly different between suppliers, leading to the production of meals of different quality(3). It is * This article was published as part of the WALTHAM International Nutritional Sciences Symposium Proceedings 2013. Abbreviations: CP, crude protein; MBM, meat and bone meal; PM, poultry by-product meal; PSP, protein solubility in pepsin. †Corresponding author: A. C. Carciofi, fax +55 16 3203-1226, email aulus.carciofi@gmail.com © The Author(s) 2014. The online version of this article is published within an Open Access environment subject to the conditions of the Creative Commons Attribution license . JNS JOURNAL OF NUTRITIONAL SCIENCE 1 mailto:aulus.carciofi@gmail.com recommended(4) that hair, feathers, hooves, horns, blood and other residues should be present in animal by-products only in the minimal amounts that occur naturally. However, there are several methods of waste separation, which complicates efforts to standardise by-products. In addition, the time between ingredient production in the slaughterhouse and final render- ing may differ among processing conditions, leading to vari- able reductions in the nutrient quality after microbial degradation. All of these factors indicate the importance for pet food companies to have standard quality criteria for receiving ingre- dients. These criteria must include chemical and microbio- logical tests to help to define the nutritional value and safety of the ingredients before their receipt by companies. One major limitation, however, is that these evaluations do not pro- vide information about ingredient digestibility by dogs. In vivo methods are the standard way to determine ingredient quality, allowing determinations of digestibility and palatability, among others. However, they require a specific infrastructure, time and expensive animal care that increase costs. Moreover, there is increasing concern about animal welfare in experimen- tal settings(5). In addition, in vivo tests are time consuming, last- ing several weeks, and are not suitable for use during ingredient receipt. In vitro evaluation, on the other hand, is a practical and feasible alternative to obtain data more rapidly and relatively inexpensively. For quality control, animal by-product meals are usually evaluated by pet food companies through the protein solubility in pepsin (PSP) method. The concentration of pepsin that best represents the in vivo digestion of dogs, however, has not yet been established. The standardisation of this technique will be important to better define the protein quality of ingredients used in pet food, but the validation of in vitro methods must be based on the degree of relationship between the in vivo and in vitro results(6). Considering this need, the present study reports prediction equations to estimate the crude protein (CP) digest- ibility of meat and bone meal (MBM) and poultry by-product meal (PM) for dogs, using the PSP method. Materials and methods The study was conducted at the Laboratory of Research on Nutrition and Nutritional Diseases of Dogs and Cats, UNESP, Jaboticabal, Brazil. All of the procedures were approved by the Ethics and Animal Welfare Committee of the College of Agrarian and Veterinarian Sciences, Sao Paulo State University according to the Brazilian animal protection law (protocol no. 027570/10). Samples of eight MBM and eight PM produced in different animal by-products industries located in South-eastern and Southern Brazil were selected. All of the samples were marketed for the pet food industries and were evaluated by in vivo (digestibility trials with dogs) and in vitro (PSP) methods. Digestibility experiment Eighteen healthy adult beagle dogs with an average age of 5·7 years old (SEM 0·534) and 12·01 kg body weight (SEM 0·340) were used in digestibility trials. The digestibility trials were per- formed to assess the CP digestibility of the MBM and PM samples using the substitution method(7–9). For each protein source (MBM or PM) the dogs were distributed in a rando- mised block design with nine diets in total (a control diet plus eight diets composed of each of the protein source sam- ples in the study), two dogs per diet in each block and three blocks of eighteen animals each for a total of six replicates per diet. A control diet was formulated for dog maintenance(4) (Table 1). Sixteen diets, eight using MBM and eight using PM, were obtained by mixing 70 % of the control diet with 30 % of each animal by-product meal in the study (on as-fed basis). The final CP concentration of the diets using MBM ranged from 29·2 to 31·6 %, and the final CP concen- tration of diets using PM ranged from 33·4 to 37·9 %, both on an as fed-basis. The final diets (mixture of the control diet and the protein source) were mixed and ground in a hammer mill (Model 4, D’Andrea) fitted with a 0·8 mm screen before being extruded and kibbled under identical processing conditions in a single- screw extruder (Mab 400S, Extrucenter) in the extrusion facil- ity of the College of Agrarian and Veterinarian Sciences, São Paulo State University. The manufacturing process was con- trolled by adjusting the kibble density to between 390 and 455 g/l (on as-fed basis) every 20 min to ensure consistent cooking and kibble quality (size and expansion). The extruder pre-conditioning temperature was kept above 90°C. Water, steam, screw speed and ration flux were adjusted according to each diet, and the extrusion temperature varied between 107 and 119°C. During the digestibility trial, the dogs were kept in the indi- vidual stainless steel metabolic cages (100 × 100 × 100 cm3) equipped with a system to separate faeces and urine for collec- tion. The dogs were fed individually calculated amounts of food. To this end, the food metabolisable energy content was calculated from their chemical compositions (using the equation proposed by the NRC(10) that consider the crude fibre effect on energy digestibility), and the dogs were fed Table 1. Ingredient and chemical compositions (as-fed basis) of the control diet Ingredient composition % Maize 38·23 Broken rice 18·00 Soyabean meal 17·23 Maize gluten meal 9·21 Poultry fat 7·15 Poultry by-product meal 5·00 Minor ingredients* 5·18 Total 100 Chemical composition %, as-fed basis DM 89·86 Crude protein 23·78 Acid-hydrolysed fat 12·28 Gross energy (MJ/kg of diet) 18·47 *Dicalcium phosphate, calcium carbonate, potassium chloride, sodium chloride, cho- line chloride, methionine, lysine, vitamin and mineral supplement, palatability enhan- cer, mould inhibitor and antioxidant. 2 journals.cambridge.org/jns 130 kcal/kg0·75 per d(10). Water was available ad libitum. The daily food amount was divided equally between meals, placed at 08·00 and 16·00 h, and the dogs were allowed to eat for 30 min. Any remaining food was collected, and the intake was recorded. Each digestibility evaluation latest 10 d, using the first five for diet adaptation and the last 5 d for total collection of faeces(4). On the first day of collection (day 6), all of the faeces were removed from the cages and discarded before 07·30 h, and the total faecal output for each dog was collected from this point onward for the next 5 d. Faeces were collected twice per day and pooled by dog. The faecal samples were weighed and frozen (−15°C) until the laboratory analyses. Before chemical analysis, the faecal samples were thawed and dried in a forced air oven (320-SE, Fanem) at 55°C for 72 h. Dried faecal samples and diets were ground in a cutting mill (Mod MA-350) fitted with a 1 mm screen. The MBM and PM samples in the study, the seventeen diets and all of the fae- cal samples were analysed for DM and CP by standard meth- ods(11). All of the analyses were carried out in duplicate and repeated when the CV was greater than 5 %. The coefficient of total tract apparent digestibility of the CP in the control and experimental diets was calculated according to the quantitative collection of faeces method(4). The in vivo CP digestibilities of the MBM and PM samples were calculated based on the CP digestibility values of the control diet, the CP digestibility values of the experimental diets (the diets com- posed of 70 % control diet and 30 % protein source in evalu- ation), and the inclusion level of the MBM or PM, corrected for the DM-basis(7): PD of the protein source = PD control + (PD experim -- PD control)/ (The percent of total CP of experimental diets provided by the MBM or PM), where PD of the protein source is the coefficient of the total tract apparent digestibility of the CP of the MBM or PM sam- ples, PD control is the coefficient of the total tract apparent digestibility of the CP of the control diet and PD experiment is the coefficient of the total tract apparent digestibility of the CP in the diets composed of a mixture of the control diet and the protein source under study. Protein solubility by the pepsin method The PSP of the eight MBM and eight PM samples were determined at three pepsin concentrations: 0·02, 0·002 and 0·0002 %. The analytical procedure followed the method described by Association of Official Analytical Chemists(11), except for the recommended pepsin concentration. The ground MBM and PM samples were washed with petroleum diethyl ether to remove the fat from the samples and were incubated in an incubator shaker (Innova 44 stackable shaker. Eppendorf) at 45°C and 50 rpm for 16 h with the different pepsin solutions (P7000, Sigma-Aldrich). The insolubilised residue was filtered using filter papers (Whatman ref. 10300211, pore size <2 µm, VWR International eurolab), dried, and its nitrogen content was determined. The PSP was calculated according to the procedure described by the Association of Official Analytical Chemists(11). Statistical analysis The Pearson correlation coefficients between the in vivo CP digestibility results determined in the digestibility trials with dogs and the PSP values obtained in vitro with the three pepsin concentrations were determined. The pepsin concentration that correlated better with the in vivo results was used to develop the prediction equations for the MBM and PM CP digestibility. To establish the prediction equations, simple lin- ear regressions were performed that considered the PSP results as an independent variable and the in vivo digestibility values as a dependent variable. Values of P < 0·05 were con- sidered significant. The SAS software version 9·0 (SAS Institute, Cary, NC, USA) was used for the data analysis. Results The CP concentrations of the studied MBM and PM samples ranged from 39·8 to 46·6 % and 57·8 to 69·1 %, respectively (Table 2). The in vivo digestibility coefficients of the CP of the MBM samples ranged from 72·6 to 79·4 %, and the pepsin concentration which better correlated with the in vivo results was 0·0002 % (r 0·380; P= 0·008). The prediction equation that represented this relationship was as follows: CP digestibil- ity of MBM= 61·7 + 0·2644 × PSP at 0·0002 % (P = 0·008; R2 0·126). The in vivo CP digestibility of the studied PM sam- ples ranged from 80·4 to 89·1 %, and the pepsin concentration that correlated best with the in vivo results was 0·02 % (r 0·482; P= 0·005). This relationship was expressed by the following equation: CP digestibility of PM = 54·1 + 0·3833 × PSP at 0·02 % (P = 0·005; R2 0·216). Discussion The MBM samples evaluated presented low CP contents com- pared with values described in the scientific literature(3,10,12–15). However, when compared with the values reported on Brazilian feed composition tables(16) and local studies(17), it is possible to observe that the low CP concentration of MBM is an intrinsic characteristic of the raw material used to produce this ingredient in Brazil. The MBM samples pre- sented high ash content (38·7 % on as-fed basis; data not shown), suggesting large inclusion of bones. The CP contents of the PM samples evaluated were slightly lower than the con- tents recorded by Dozier et al.(18), who evaluated ten PM clas- sified as pet food grade. Those authors collected samples of PM produced in industries located in Alabama, Delaware, Georgia, North Carolina, Tennessee and Virginia and found that the CP contents ranged from 63·0 to 69·3 % (mean of 66·1 %). In addition, in the present study, a high variation in CP concentrations of PM produced in Brazil compared with the values reported by authors in the USA was noted (SD = 4·32 v. 1·90, respectively). It has been suggested that a lower standard applies to the PM classified as pet food 3 journals.cambridge.org/jns grade in Brazil and that pet food companies in Brazil may need to pay greater attention when acquiring this ingredient. However, despite the variations in the CP contents of the PM, high CP digestibilities were observed, indicating that most of the CP in the ingredient originated from meat compo- nents and not from nitrogen-rich components of low nutri- tional value, such as feathers. Greater differences between the PSP results of the evaluated samples were observed when lower concentrations of pepsin were used for both MBM and PM. In addition, when the enzyme concentration was reduced, the protein solubilities were also reduced and numerically distanced from the in vivo data. For MBM, however, it was observed that only the pepsin percentage of 0·0002 % provided PSP values that showed sig- nificant correlation with the in vivo results, suggesting that when higher concentrations of the enzyme were used, the in vitro values were too high and did not adequately discriminate between the high and low digestibility values observed in dogs. This finding may be justified by the fact that insoluble and less digestible proteins become soluble at high concentrations of pep- sin, and consequently, the solubility results do not match with values determined in vivo and have low biological significance. It also became clear that, as was already known, the PSP is a qualitative and not a quantitative method, which may allow for discrimination between more or less soluble samples of MBM and PM. To better use the method during the in vitro evalu- ation and classification of animal by-product meals it was necessary, however, to generate an equation to translate the results for solubility in pepsin into terms of dog digestibility, as already available for other species(19,20). However, the pre- diction equations proposed in the present study, even though statistically significant, presented low determination coeffi- cients and need to be used with caution. It is possible that the small number of samples exerted some influence on this low adjustment as well as the reduced variation observed for the in vivo values, which were relatively close between samples. Furthermore, it is important to note that there are many sources of variation in studies involving in vitro methodologies, including the source of the evaluated ingredient, particular var- iations related to the different enzymes marketed, the enzyme concentration and purity, and the range of the pH during the assessment, among others. Unfortunately, no other prediction equations to estimate MBM and PM for extruded dog foods based on the PSP method are available to compare with the present findings. Therefore, the data presented are an important starting point for the use and application of the PSP method as a tool to evaluate the nutritional quality of MBM and PM, and this method deserves further study. Acknowledgements The authors acknowledge the financial support of FAPESP (No. 2010/13065–5), the scholarship of CNPq (No. 143145/2009–6) and CAPES (No. 2479/11–7) and the finan- cial and technical support of Mogiana Alimentos S.A. (Guabi) Table 2. Crude protein (CP) concentration (%, as-fed basis), in vivo CP digestibility for dogs calculated by the substitution method* and in vitro protein solubility in three pepsin concentrations of the meat and bone meal and poultry by-product meal samples under study Animal by-product meal CP concentration (%) In vivo CP digestibility(%) CP solubility in pepsin at 0·02% CP solubility in pepsin at 0·002% CP solubility in pepsin at 0·0002% Meat and bone meal A 41·0 74·0 79·4 60·7 41·6 B 39·8 76·1 87·5 74·6 53·4 C 46·1 75·1 82·2 71·3 55·1 D 44·9 77·3 85·3 69·4 54·0 E 46·6 75·4 87·5 70·0 57·6 F 43·2 78·9 89·2 77·3 62·2 G 46·5 79·4 86·4 81·8 61·4 H 41·1 72·6 93·8 79·1 52·0 Mean 43·6 76·1 86·4 73·0 54·7 SD 2·75 2·35 4·14 6·31 6·09 Pearson correlation† – – −0·006 0·218 0·380 P value‡ – – 0·969 0·137 0·008 Poultry by-product meal A 68·7 86·0 82·8 68·0 52·1 B 59·8 82·6 71·0 60·8 42·1 C 69·1 89·1 83·5 72·5 61·6 D 62·5 83·7 79·9 66·3 54·4 E 60·9 86·1 85·1 67·0 54·5 F 57·8 80·4 76·5 70·6 51·3 G 61·0 84·8 81·8 65·1 51·9 H 66·9 84·0 74·7 67·2 51·6 Mean 63·3 84·6 79·4 67·5 52·4 SD 4·32 2·63 4·64 3·47 5·02 Pearson correlation† – – 0·482 0·288 0·470 P value‡ – – 0·005 0·047 0·007 *Calculated with six dogs per diet(7). †Pearson correlation coefficient between the in vivo crude protein (CP) digestibility results determined in the digestibility trials with dogs and the pepsin solubility of CP values obtained in vitro with the three pepsin concentrations(11). ‡P value of the Pearson correlation. 4 journals.cambridge.org/jns to the Laboratory of Research in Nutrition and Nutritional Diseases of Dogs and Cats ‘Professor Dr Flávio Prada’. None of the authors has any conflicts of interest. I. M. K., N. K. S. and A. C. C. conceived the present study. C. F. F. P., A. R., T. C. P. and M. O. S. G. conducted diet preparation, feeding trials and chemical analysis. I. M. K. and C. C. conducted the in vitro solubility in pepsin analysis. E. B. M. did all statistical analysis and help in study design. I. M. K. and A. C. C. drafted the manuscript. All of the authors contributed to the critical revision of the manuscript. This paper was published as part of the WALTHAM International Nutritional Sciences Symposium Proceedings 2013, publication of which was supported by an unrestricted educational grant from Mars Incorporated. The papers included in these proceedings were invited by the Guest Editor and have undergone the standard journal formal review process. They may be cited. References 1. Corbin JE (1992) Inedible meat, poultry, and fish by-products in pet foods. In Inedible Meat By-Products, Advances in Meat Research, vol. 8, pp. 329–347 [AM Pearson and TR Dutson, editors]. London: Elsevier Applied Science. 2. Murray SM, Patil AR, Fahey GC Jr, et al. (1997) Raw and rendered animal by-products as ingredients in dog diets. J Anim Sci 75, 2497–2505. 3. Adedokun SA & Adeola O (2005) Apparent metabolizable energy value of meat and bone meal for white pekin ducks. Poult Sci 84, 1539–1946. 4. Association of American Feed Control Officials (2009) Dog and Cat Nutrient Profiles. Official Publication of the Association of American Feed Control Officials Incorporated. Oxford: AAFCO Incorporated. 5. Cramer KR, Greenwood MW, Moritz JS, et al. (2007) Protein qual- ity of various raw and rendered by-product meals commonly incor- porated into companion animal diets. J Anim Sci 85, 3285–3293. 6. Boisen S & Eggum BO (1991) Critical evaluation of in vitro meth- ods for estimating digestibility in simple-stomach animals. Nutri Res Rev 4, 141–162. 7. Matterson LD, Potter LM, Stutuz NW, et al. (1965) The Metabolizable Energy of Feed Ingredients for Chickens, pp. 3–11. Storrs: The University of Connecticut, Agricultural Experiment Station. 8. Sá-Fortes CML, Carciofi AC, Sakomura NK, et al. (2010) Digestibility and metabolizable energy of some carbohydrate sources for dogs. Anim Feed Sci Technol 156, 121–125. 9. Kawauchi IM, Sakomura NK, Vasconcellos RS, et al. (2011) Digestibility and metabolizable energy of maize gluten feed for dogs measured by two different techniques. Anim Feed Sci Technol 169, 96–103. 10. National Research Council (2006) Nutrient Requirements of Dogs and Cats. Washington: National Academy Press. 11. Association of Official Analytical Chemists International (1995). Official Methods of Analysis, 16th ed. Arlington: AOAC International. 12. National Research Council (1994) Nutrient Requirements of Poultry, 9th ed. Washington: National Academy Press. 13. Parsons CM, Castanon F & Han Y (1997) Protein and amino acid quality of meat and bone meal. Poult Sci 76, 361–368. 14. Sauvant D, Perez JM & Tran G (editors) (2004) Tablas de composición y de valor nutritivo de las materias primas destinadas a los animales de interés ganadero, 3rd ed. Madrid: Mundi-Prensa. 15. Hendriks WH, Butts CA, Thomas DV, et al. (2002) Nutritional quality and variation of meat and bone meal. Asian-Aust. J. Anim. Sci. 15, 1507–1516. 16. Rostagno HS, Albino LFT, Donzele JL, et al. (2005) Tabelas Brasileiras para Aves e Suínos. Composição de Alimentos e Exigências Nutricionais, 2nd ed. Viçosa: UFV. 17. De-Oliveira LD, Picinato MAC, Kawauchi IM, et al. (2012) Digestibility for dogs and cats of meat and bone meal processed at two different temperature and pressure levels. J Anim Physiol Anim Nutri 96, 1136–1146. 18. Dozier WA, Dale NM, Dove CR (2003) Nutrient composition of feed-grade and pet-food-grade poultry by-product meal. J Appl Poult Res 12, 526–530. 19. Bellaver C, Zanotto DL, Guidoni AL, et al. (2000) In vitro solubility of meat and bone meal protein with different pepsin concentra- tions. Cienc Rural 30, 489–492. 20. Rochell SJ, Kuhlers DL & Dozier WA (2013) Relationship between in vitro assays and standardized ileal amino acid digestibility of animal protein meals in broilers. Poult Sci 92, 158–170. 5 journals.cambridge.org/jns Prediction of crude protein digestibility of animal by-product meals for dogs by the protein solubility in pepsin method* Materials and methods Digestibility experiment Protein solubility by the pepsin method Statistical analysis Results Discussion Acknowledgements References << /ASCII85EncodePages false /AllowTransparency false /AutoPositionEPSFiles false /AutoRotatePages /None /Binding /Left /CalGrayProfile (Dot Gain 20%) /CalRGBProfile (sRGB IEC61966-2.1) /CalCMYKProfile (U.S. Web Coated \050SWOP\051 v2) /sRGBProfile (sRGB IEC61966-2.1) /CannotEmbedFontPolicy /Error /CompatibilityLevel 1.3 /CompressObjects /Tags /CompressPages true /ConvertImagesToIndexed true /PassThroughJPEGImages true /CreateJobTicket false /DefaultRenderingIntent /Default /DetectBlends true /DetectCurves 0.1000 /ColorConversionStrategy /LeaveColorUnchanged /DoThumbnails false /EmbedAllFonts true /EmbedOpenType false /ParseICCProfilesInComments true /EmbedJobOptions true /DSCReportingLevel 0 /EmitDSCWarnings false /EndPage -1 /ImageMemory 1048576 /LockDistillerParams true /MaxSubsetPct 100 /Optimize false /OPM 1 /ParseDSCComments true /ParseDSCCommentsForDocInfo true /PreserveCopyPage false /PreserveDICMYKValues true /PreserveEPSInfo false /PreserveFlatness true /PreserveHalftoneInfo false /PreserveOPIComments false /PreserveOverprintSettings true /StartPage 1 /SubsetFonts true /TransferFunctionInfo /Remove /UCRandBGInfo /Remove /UsePrologue false /ColorSettingsFile () /AlwaysEmbed [ true ] /NeverEmbed [ true ] /AntiAliasColorImages false /CropColorImages true /ColorImageMinResolution 150 /ColorImageMinResolutionPolicy /OK /DownsampleColorImages true /ColorImageDownsampleType /Bicubic /ColorImageResolution 300 /ColorImageDepth -1 /ColorImageMinDownsampleDepth 1 /ColorImageDownsampleThreshold 1.50000 /EncodeColorImages true /ColorImageFilter /DCTEncode /AutoFilterColorImages true /ColorImageAutoFilterStrategy /JPEG /ColorACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /ColorImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000ColorACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000ColorImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasGrayImages false /CropGrayImages true /GrayImageMinResolution 150 /GrayImageMinResolutionPolicy /OK /DownsampleGrayImages true /GrayImageDownsampleType /Bicubic /GrayImageResolution 300 /GrayImageDepth -1 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /GrayImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000GrayACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000GrayImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasMonoImages false /CropMonoImages true /MonoImageMinResolution 400 /MonoImageMinResolutionPolicy /OK /DownsampleMonoImages true /MonoImageDownsampleType /Bicubic /MonoImageResolution 1200 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict << /K -1 >> /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile (None) /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped /False /CreateJDFFile false /Description << /CHS /CHT /DAN /DEU /ESP /FRA /ITA /JPN /KOR /NLD (Gebruik deze instellingen om Adobe PDF-documenten te maken die zijn geoptimaliseerd voor prepress-afdrukken van hoge kwaliteit. De gemaakte PDF-documenten kunnen worden geopend met Acrobat en Adobe Reader 5.0 en hoger.) /NOR /PTB /SUO /SVE /ENU (Use these settings to create Adobe PDF documents best suited for high-quality prepress printing. Created PDF documents can be opened with Acrobat and Adobe Reader 5.0 and later.) >> /Namespace [ (Adobe) (Common) (1.0) ] /OtherNamespaces [ << /AsReaderSpreads false /CropImagesToFrames true /ErrorControl /WarnAndContinue /FlattenerIgnoreSpreadOverrides false /IncludeGuidesGrids false /IncludeNonPrinting false /IncludeSlug false /Namespace [ (Adobe) (InDesign) (4.0) ] /OmitPlacedBitmaps false /OmitPlacedEPS false /OmitPlacedPDF false /SimulateOverprint /Legacy >> << /AddBleedMarks false /AddColorBars false /AddCropMarks false /AddPageInfo false /AddRegMarks false /ConvertColors /ConvertToCMYK /DestinationProfileName () /DestinationProfileSelector /DocumentCMYK /Downsample16BitImages true /FlattenerPreset << /PresetSelector /MediumResolution >> /FormElements false /GenerateStructure false /IncludeBookmarks false /IncludeHyperlinks false /IncludeInteractive false /IncludeLayers false /IncludeProfiles false /MultimediaHandling /UseObjectSettings /Namespace [ (Adobe) (CreativeSuite) (2.0) ] /PDFXOutputIntentProfileSelector /DocumentCMYK /PreserveEditing true /UntaggedCMYKHandling /LeaveUntagged /UntaggedRGBHandling /UseDocumentProfile /UseDocumentBleed false >> ] >> setdistillerparams << /HWResolution [2400 2400] /PageSize [612.000 792.000] >> setpagedevice