Avaliação biológica do hidrolisado de proteína da carne de galinha desossada mecanicamente - PDF

ORIGINAL ORIGINAL EVALUATION OF PROTEIN HYDROLYSATE 879 Biological evaluation of mechanically deboned chicken meat protein hydrolysate 1 Avaliação biológica do hidrolisado de proteína da carne de galinha

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ORIGINAL ORIGINAL EVALUATION OF PROTEIN HYDROLYSATE 879 Biological evaluation of mechanically deboned chicken meat protein hydrolysate 1 Avaliação biológica do hidrolisado de proteína da carne de galinha desossada mecanicamente Daniele Misturini ROSSI 2 Simone Hickmann FLÔRES 2 Janaína Guimarães VENZKE 3 Marco Antonio Záchia AYUB 2 A B S T R A C T Objective The objective of this study was to evaluate the biological properties of a protein hydrolysate obtained by enzymatic hydrolysis of mechanically deboned chicken meat. Methods Mechanically deboned chicken meat was hydrolysed using Alcalase 2.4 L FG and then dried in a spraydrier. Three groups (n=6) of male Wistar rats received diets containing casein, mechanically deboned chicken meat protein hydrolysate and a proteinfree diet. The rats were randomly assigned to individual cages with controlled temperature (22 C) for 12 days. Results The mechanically deboned chicken meat diet resulted in a good net protein utilization (3.74) and high true digestibility (96%)..The amino acid composition of the hydrolysate was relatively well balanced, but the concentrations of methionine and cystine were low, making them the limiting amino acids. The proximate chemical composition of the hydrolysate showed protein content to be as high as 62%. Conclusion The results obtained in this work suggest that mechanically deboned chicken meat hydrolysate can be used as a protein enhancer in food preparations such as enteral formulations, and as an edible protein enhancer in general applications. Indexing terms: Biological factors. Hydrolyzed vegetable protein. Protein. Epidemilogy, experimental. 1 Article based on the dissertation thesis by D.M. ROSSI, entitled Utilização de carne mecanicamente separada de frango para produção de um hidrolisado protéico a partir de enzimas microbianas (Use of mechanically deboned chicken meat to produce a protein hydrolysate using microbial enzymes). Universidade Federal do Rio Grande do Sul; Sponsored by Conselho Nacional de Desenvolvimento Científico e Tecnológico (Process number /20046). 2 Universidade Federal do Rio Grande do Sul, Instituto de Ciência e Tecnologia de Alimentos. Av. Bento Gonçalves, 9500, , Porto Alegre, RS, Brasil. Correspondência para/correspondence to: D.M. Rossi. 3 Universidade Federal do Rio Grande do Sul, Departamento de Medicina Interna. Porto Alegre, RS Brasil. Rev. Nutr., Campinas, 22(6):879885, nov./dez., 2009 880 D.M. ROSSI et al. R E S U M O Objetivo O objetivo do estudo foi avaliar a qualidade biológica da proteína hidrolisada obtida a partir da carne mecanicamente separada de frango. Métodos A carne mecanicamente separada de frango foi hidrolisada com a enzima Alcalase 2,4 L FG e o hidrolisado obtido foi submetido a secagem em atomizador. Foram utilizados três grupos (n=6) de ratos machos Wistar os quais receberam dietas contendo caseína, proteína hidrolisada de carne mecanicamente separada de frango ou uma dieta com proteína livre. Os animais foram distribuídos aleatoriamente em gaiolas individuais, com temperatura controlada (22 C), por um período de 12 dias. Resultados A dieta utilizando carne mecanicamente separada de frango resultou em elevada utilização líquida de proteína (3,74) e elevada digestibilidade verdadeira (96%). A composição de aminoácidos da proteína hidrolisada apresentou bons resultados, embora metionina e cistina tenham apresentado baixos valores, sendo considerados aminoácidos limitantes. A composição química mostrou altos valores de proteína no hidrolisado obtido (62%). Conclusão Os resultados obtidos neste trabalho sugerem que a proteína hidrolisada de carne mecanicamente separada de frango poderá ser utilizada como um suplemento em formulações alimentares, tais como formulações enterais, ou como fonte de complementação protéica na indústria de alimentos em geral. Termos de indexação: Fatores biológicos. Proteína hidrolisada vegetal. Proteína. Estudos experimentais. I N T R O D U C T I O N Brazil is the world s third largest chicken meat producer and the leading exporter of chicken products, with a total production of 9.7 millions tons in It is reckoned that at least 20% of chicken freshcut carcasses are transformed into Mechanically Deboned Chicken Meat (MDCM), which could be used in processed meats, such as meat emulsion, paste meat and chicken nuggets 2. MDCM is a byproduct of the poultry industry increasingly used in processed meat products. It is also a source of high biological quality proteins that could be used to produce protein preparations with specific characteristics. MDCM is mostly obtained from necks and back parts of chicken and turkey carcasses 3. Protein hydrolysates constitute a promising alternative to intact proteins and elemental formulas in the development of special formulations designed to provide nutritional support for patients with different needs 4. The number of amino acid residues remaining in the peptide chains after hydrolysis is an essential characteristic in the production of protein hydrolysates with desired functional properties 5. Protein hydrolysates find applications mainly in the nutritional management of individuals who cannot digest whole/intact protein. The most prevalent application of protein hydrolysates has been in formulations for infants with food hypersensitivity, in order to improve nutritional and functional properties 6. Other possible applications of hydrolysates have been suggested and include the preparation of geriatric products, high energy supplements and weight control and therapeutic or enteric diets 7. The protein hydrolysates most commonly used in nutritional products are casein, whey protein and soy protein. Other sources of protein hydrolysates, such as fish, chickpea, shark muscle, and shrimp, were studied and can also be used in supplementation diets, high protein sports drinks, and therapeutic or enteric diets 810. Considerable attention has been given to the use of MDCM as an ingredient in the manufacture of sausages and other meat products 11, especially considering its nutritional quality 2,12,13. However, few studies have Rev. Nutr., Campinas, 22(6):879885, nov./dez., 2009 EVALUATION OF PROTEIN HYDROLYSATE 881 been published on the production and quality of MDCM protein hydrolysate. The aims of this work were to study the biological quality of MDCM protein hydrolysate and its amino acid composition in order to use it as a nutritional enhancer in food products and diets. M E T H O D S Mechanically deboned chicken meat was supplied by Avipal S.A, which is a large, local poultry processing plant. The enzyme used was Alcalase 2.4L FG, an endopeptidase from Bacillus licheniformis, provided by Novozymes Latin America, Brazil. Batches of 8L of MDCM were hydrolyzed in optimum conditions using Alcalase (50ºC, ph 8.0 and enzyme concentration of 2.0%). The hydrolysate obtained was centrifuged at 8g for 10min (Sorvall RC5B, Du Pont Instruments, USA) and concentrated to 20% of solids in a rotavap (Labortora, Heidolph 400, Germany). The hydrolysate was spraydried in a laboratory spraydryer (Model Mini Spray Dryer Buchi B 191, Sweden) with an inlet temperature of 160ºC, outlet temperature of 96ºC and feed flow rate of 160 ml.h 1, using 5% of maltodextrin as a carrier and 1% of calcium carbonate as antihumectant agent. The samples of MDCM and protein hydrolysate were collected and analyzed for moisture, ash, total fat 14 and crude protein content (N x 6. 25) by the Kjeldahl method 14. Young, 2123 days old, white male Wistar rats, weighing g, were obtained from the nursery of the Centro de Ciências Biológicas, Universidade Federal do Rio Grande do Sul, Brazil. Animals were randomly divided into three groups, each group consisting of 6 rats. Proteinfree diets were offered to the first group. The second group was fed a casein diet as control. The third group received a protein hydrolysate diet. All animals received diets formulated with casein supplementation during an acclimation period of 2 days. The diets were formulated according to Reeves et al. 15, adapted to contain 10% of protein for casein (control) and for MDCM protein hydrolysate. The diet compositions are listed in Table 1. Water and feed were offered ad libitum. The animals were individually caged and kept in a room at 22ºC and a 12h light and dark cycle. Feces were collected five times for each rat for a period of ten days. The feces were dehydrated, weighed and their nitrogen content was analyzed by the Kjeldahl method 14. Amino acid composition was determined after acid hydrolysis with 6 N HCl for 24 hours at 110ºC, following methodology described by Spackman et al. 16, using a Beckman HPLCamino Table 1. Calculated composition of the experimental diets as 10% protein of the total. Porto Alegre (RS), Brazil, Ingredients Casein Proteinfree diet g Protein hydrolysate Casein MDCM protein hydrolysate Sucrose Corn oil Cellulose Mineral mix Vitamin mix Lcystine Choline bitartrate TertButylhydroquinone Cornstarch MDCM: mechanically deboned chicken meat. Rev. Nutr., Campinas, 22(6):879885, nov./dez., 2009 882 D.M. ROSSI et al. acid analyzer. Tryptophan was analyzed according to Spies 17. The data obtained from this experiment were used to calculate True Digestibility (TD) (Eq.1) and Net Protein Retention (NPR) (Eq.2) by employing the following formulas, according to Sgarbieri 18 : TD = ((Ni NF 1 NF 2 ) /Ni) x 100 (1) Where: Ni = Nitrogen intake of animals fed the tested diet; N F1 = Nitrogen excreted in feces of animals fed the tested diet; N F2 = Nitrogen excreted in feces of animals fed the proteinfree diet. NPR = weight gain of test group + weight loss of proteinfree group / weight of test protein consumed. (2) Feed intake, protein intake and body weight were used to compute the following nutritional indices according to Sogi et al. 19 showed in equations 3 to 5: Feed efficiency (FE) = Gain in body wt (g)/feed intake (g) (3) Feed utilization (FU) = Feed intake (g)/gain in body wt (g) (4) Protein utilization (PU) = Protein intake (g)/gain in body wt (g) (5) The data were subjected to Analysis of Variance (ANOVA) in a completely randomized design to determine the significant differences among the various groups. R E S U L T S A N D D I S C U S S I O N Proximate chemical composition The chemical composition of mechanically deboned chicken meat and its spraydried hydrolysate is presented in Table 2. MDCM presented much higher fat and lower protein contents. The high amounts of fats in the MDCM might indicate a process where more skin offproducts are being used, while the unusually low ash content is indicating that less bones and cartilaginous tissues were used. The fat content in the hydrolysate was greatly reduced during the Table 2. Proximate composition of mechanically deboned chicken meat and the spraydried hydrolysate. Porto Alegre (RS), Brazil, Composition (%) Moisture Protein c Fat c Ash c MDCM a M SD a Values are means and standard deviations of quintuplicate measurements; b Values obtained using 5% of maltodextrin as a carrier for spray drying. Values are means of duplicate measurements; c Dry weight basis. MDCM: mechanically deboned chicken meat; M: mean; SD: standard deviation. exclusion of the insoluble protein fraction by centrifugal separation 20. Spraydried hydrolysate showed very high protein content (61.9%). Comparatively, Hamid et al. 21, used 2.0% Alcalase, ph 8.0 and 55 ºC to produce fish protein hydrolysate powder with 49.6% of total protein and 2.8% of fat, while Soares et al. 22, using Alcalase at 1.2%, ph 7.0 and 50ºC to hydrolyze mechanically deboned chicken meat, obtained around 40.5% of total protein after the spraydrying process. Nutritional indices Spraydried hydrolysate b M SD The protein hydrolysate showed good nutritional quality when compared with casein. Table 3 shows the nutritional indices obtained in the biological assay. Rats fed casein, with a total consumption of g of feed, gained less weight than those fed protein hydrolysate (total consumption of g of feed, although these results were not significantly different (p 0.05). Protein intake, feed efficiency, feed utilization and protein utilization were not significantly different (p 0.05) when compared with casein. Digestibility is the measure of the percentage of proteins that are hydrolyzed by digestive enzymes and absorbed as amino acids by the organism, and it determines the protein quality of the diet. The compared digestibility of Rev. Nutr., Campinas, 22(6):879885, nov./dez., 2009 EVALUATION OF PROTEIN HYDROLYSATE 883 Table 3. Nutritional indices * of casein and protein hydrolysate diets. Porto Alegre (RS), Brazil, Parameter * M Casein SD M Protein hydrolysate SD Feed intake (g) Protein intake(g) b b Initial weight (g) Weight gain (g) Feed efficiency b b b Feed utilization Protein utilization NPR b b b TD (%) a b * Values are means and standard deviations of 6 rats per group over an experimental period of 10 days; a,b Different superscripts are significantly different (p 0.05) according to the Student s t test. M: mean; SD: standard deviation; NPR: net protein retention; TD: true digestibility. protein hydrolysate (96.43%) and casein (97.74%) were proximately the same, indicating that protein hydrolysate is exceptionally good as a source of amino acids, rendering highly digestible protein meals. The Net Protein Ratio method estimates the protein used for growth and maintenance. The NPR value of protein hydrolysate (3.74) was greater than that of casein (3.21), although not significantly different (p 0.05). Our results showed that MDCM protein hydrolysate, as prepared in this work, was a much better source of protein when compared with other preparations listed in the literature. Rebeca et al. 23, who used eviscerated mullet to produce fish protein hydrolysate, found NPR values of Negrão et al. 2, using mechanically deboned chicken flour, reported NPR values of 3.19 and true digestibility of 92.90%, which was lower than that of MDCM protein hydrolysate, suggesting that hydrolysis can contribute to better digestibility because its absorption by the organism might be more effective. Amino acid composition The nutritional value of dietary proteins is determined by the pattern and quantity of essential amino acids present. The presence of one or more of the essential amino acids in adequate amounts increases the nutritive value of the protein 24. The quality of MDCM protein hydrolysate as source of amino acids can be assessed by comparing the essential amino acids with the World Health Organization s (WHO) 25 recommended standard, and is shown in Table 4. The amino acid profile of the mechanically deboned chicken meat protein hydrolysate contained higher amounts of all essential amino acids, except for methionine and cystine when compared with the WHO s reference for adults. Considering the demands for children (25yearolds), the results indicated that MDCM protein hydrolysate contained adequate amounts of lysine, treonine, isoleucine and histidine 25. The low concentrations of methionine and cystine are reported not only in animal proteins, which are generally considered reference proteins, but also in vegetable sources, normally lacking sulfur amino acids 18. This low content of methionine and cystine can be increased by adding other food ingredients containing high or moderate amounts of sulfur amino acids or by supplementing Lmethionine to the formulation, especially when used for infants. However, a good protein needs more than just containing amino acids in adequate proportions to meet all the requirements of an organism. The amino acids also need to have high bioavailability, i.e., they can be absorbed in a metabolic active form, in order to achieve specific functions in the body. Despite the relatively low amounts of methionine and cystine in MDCM protein hydrolysate, animals in our study had good Rev. Nutr., Campinas, 22(6):879885, nov./dez., 2009 884 D.M. ROSSI et al. Table 4. Amino acid composition of mechanically deboned chicken meat (MDCM) protein hydrolysate. Porto Alegre (RS), Brazil, Amino acid Protein hydrolysate (mg/g protein) FAO (25 years) FAO (adults) Essential Histidine Isoleucine Leucine Lysine Metionine+Cystine* Phenylalanine+Tyrosine Treonine Tryptophan Valine Non essential Aspartic acid Glutamic acid Serine Proline Glycine Alanine Arginine * limiting amino acid. MDCM: mechanically deboned chicken meat; FAO: Food and Agriculture Organization. weight gain and the NPR and the true digestibility of MDCM protein hydrolysate values were excellent, suggesting that the amino acids were promptly available for digestion, rendering this source as adequate. A C K N O W L E D G E M E N T S The authors wish to thank Avipal Alimentos S.A., for supplying the mechanically deboned chicken meat and Conselho Nacional de Desenvolvimento Científico e Tecnológico for sponsoring this study. C O N C L U S I O N Proximate analyses revealed that the spraydried mechanically deboned chicken meat protein hydrolysate consisted of proximately 62% protein and very low lipid content, characteristics that are usually desired for food preparations with special ends such as diets for weight control, enteral nutrition, among others. Biological parameters indicated that protein hydrolysate, a lowcost product, presented very good nutritional properties, despite its low content of methionine and cystine, the limiting amino acids. Protein hydrolysate could be used in industrial preparations and specialized adult nutritional products and supplements and, when supplemented with the limiting amino acids, its use could be tested in infant formulas. C O L L A B O R A T O R S D.M. ROSSI participated in all the stages of the study. J.G. VENZKE helped with the animal experiments and enzymatic hydrolysis of the mechanically deboned chicken meat. S.H. FLÔRES cosupervised the study, following all the stages. M.A.Z. AYUB supervised and coordinated the project and wrote the manuscript. R E F E R E N C E S 1. Associação Brasileira de Exportadores de Frango. Produção mundial de carne de frango [acesso Sept]. Disponível em: http:// mercadomundial/php . 2. Negrão CC, Mizubuti IY, Morita MC, Colli C, Ida EI, Shimokomaki M. 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