Biological evaluation of mechanically deboned chicken meat protein hydrolysate

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

ORIGINAL ORIGINAL

Biological evaluation of mechanically deboned chicken meat protein hydrolysate¹

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

Daniele Misturini Rossi^I; Simone Hickmann Flôres^I; Janaína Guimarães Venzke^II; Marco Antonio Záchia Ayub^I

^IUniversidade Federal do Rio Grande do Sul, Instituto de Ciência e Tecnologia de Alimentos. Av. Bento Gonçalves, 9500, 91501-970, Porto Alegre, RS, Brasil. Correspondência para/Correspondence to: D.M. Rossi. E-mail: <[e-mail]>

^IIUniversidade Federal do Rio Grande do Sul, Departamento de Medicina Interna. Porto Alegre, RS Brasil

ABSTRACT

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 spray-drier. Three groups (n=6) of male Wistar rats received diets containing casein, mechanically deboned chicken meat protein hydrolysate and a protein-free diet. The rats were randomly assigned to individual cages with controlled temperature (22ºC) 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.

RESUMO

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 (22ºC), 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.

INTRODUCTION

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 2007¹. It is reckoned that at least 20% of chicken fresh-cut carcasses are transformed into Mechanically Deboned Chicken Meat (MDCM), which could be used in processed meats, such as meat emulsion, paste meat and chicken nuggets². MDCM is a by-product 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 carcasses³.

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 needs⁴. 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 properties⁵. 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 properties⁶. 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 diets⁷.

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 ^8-10. Considerable attention has been given to the use of MDCM as an ingredient in the manufacture of sausages and other meat products¹¹, especially considering its nutritional quality^2,12,13. However, few studies have 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.

METHODS

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 RC-5B, Du Pont Instruments, USA) and concentrated to 20% of solids in a rotavap (Labortora, Heidolph 400, Germany). The hydrolysate was spray-dried in a laboratory spray-dryer (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 anti-humectant agent.

The samples of MDCM and protein hydrolysate were collected and analyzed for moisture, ash, total fat¹⁴ and crude protein content (N x 6. 25) by the Kjeldahl method¹⁴.

Young, 21-23 days old, white male Wistar rats, weighing 40.0-55.0g, 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. Protein-free 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 method¹⁴.

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 HPLC-amino acid analyzer. Tryptophan was analyzed according to Spies¹⁷.

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 Sgarbieri¹⁸: TD = ((Ni - NF₁ - NF₂) /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 protein-free diet.

NPR = weight gain of test group + weight loss of protein-free 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.

RESULTS AND DISCUSSION

Proximate chemical composition

The chemical composition of mechanically deboned chicken meat and its spray-dried 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 off-products 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 exclusion of the insoluble protein fraction by centrifugal separation²⁰. Spray-dried 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 spray-drying process.

Nutritional indices

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 107.08g of feed, gained less weight than those fed protein hydrolysate (total consumption of 100.96g 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 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 3.07. 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 protein²⁴. 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 (2-5-year-olds), the results indicated that MDCM protein hydrolysate contained adequate amounts of lysine, treonine, isoleucine and histidine²⁵. 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 acids¹⁸. 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 L-methionine 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 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.

CONCLUSION

Proximate analyses revealed that the spray-dried 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 low-cost 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.

ACKNOWLEDGEMENTS

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.

COLLABORATORS

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 co-supervised the study, following all the stages. M.A.Z. AYUB supervised and coordinated the project and wrote the manuscript.

Received on: 13/12/2007

Final version resubmitted on: 12/8/2008

Approved on: 9/2/2009

• 1. Associação Brasileira de Exportadores de Frango. Produção mundial de carne de frango. 2007 [acesso 2007 5 Sept]. Disponível em: <http://www.abef.com.br/estatisticas/mercadomundial/mercadomundial/php>
• 2. Negrão CC, Mizubuti IY, Morita MC, Colli C, Ida EI, Shimokomaki M. Biological evaluation of mechanically deboned chicken meat protein quality. Food Chem. 2005; 90(4):579-83. doi:10.10 16/j.foodchem.2004.05.017.
• 3. Froning GW. Mechanical deboned of poultry and fish. New York: Advances in Food Research Academic Press; 1981.
• 4. Clemente A. Enzymatic protein hydrolysates in human nutrition. Trends Food Sci Technol. 2000; 11(7):254-62. doi:10.1016/S0924-2244(01)00007-3.
• 5. Xia SH, Wang Z, Xu SY. Characteristics of Bellamya purificata snail foot protein and enzymatic hydrolysates. Food Chem. 2007, 101(3); 101:1188-96. doi:10.1016/j.foodchem.2006.03.031.
• 6. Mahmoud MI. Physicochemical and functional properties of protein hydrolysates in nutritional products. Food Technol. 1994; 48(10):89-5.
• 7. Frokjaer S. Use of hydrolysates for protein supplementation. Food Technol. 1994; 48(10):86-8.
• 8. Clemente A, Vioque J, Sanchéz-Vioque R, Pedroche J, Bautista J, Millán F. Protein quality of chickpea (Cicer arietinum L.) protein hydrolysates. Food Chem. 1999; 67(3):269-74. doi:10.1016/S0308-8146(99)00130-2.
• 9. Simpson BK, Nayeri G, Yalayan V, Ashie INA. Enzymatic hydrolysis of shrimp meat. Food Chem. 1998; 61(1-2):131-8. doi:10.1016/S0308-8146(97)00131-3.
• 10. Guérard F, Dufossé L, De La Broise D, Binet A. Enzymatic hydrolysis of proteins from yellowfin tuna (Thunnus albacares) wastes using Alcalase. J Mol Catal B: Enzym. 2001; 11(4-6):1051-59. doi: 10.1016/S1381-1177(00)00031-X.
• 11. Mielnik MB, Aaby K, Rolfsen K, Ellekjaer MR, Nilsson A. Quality of comminuted sausages formulated from mechanically deboned poultry meat. Meat Sci. 2002; 61(1):73-4. doi:10.1016/S0309-1740(01)00167-X.
• 12. Essary EO. Moisture, fat, protein and mineral content of mechanically deboned poultry meat. J Food Sci. 1979; 44(4):1070-3. doi:10.1111/j.1365-2621.1979.tb03449.
• 13. Tanaka MCY, Shimokomaki M. Collagen types in mechanically deboned chicken meat. J Food Biochem. 1996; 20(3):215-25. doi:10.1111/j.1745-4514.1996.tb00552.
• 14. Association of Official Analytical Chemists. Official methods of analysis. Washington (DC): AOAC; 1990.
• 15. Reeves PG, Forrest HN, Fahey GCJ. AIN-93 Purified diets for laboratory rodents: Final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J Nutr. 1993; 123(11):1939-51.
• 16. Spakman DH, Stein WH, Moore S. Automatic recording apparatus for use in the chromatography of amino acids. Anal Chem. 1958; 30(7):1190-206. doi: 10.1021/ac60139a006.
• 17. Spies JR. Determination of tryptophan in proteins. Anal Chem. 1967; 39(10):1412-5.
• 18. Sgarbieri VC. Proteínas em alimentos protéicos: propriedades, degradações, modificações. São Paulo: Varela; 1996.
• 19. Sogi DS, Bhatia R, Garg SK, Bawa AS. Biological evaluation of tomato waste seed meals and protein concentrate. Food Chem. 2005; 89(1):53-6 doi: 10.1016/j.foodchem.2004.01.083.
• 20. Shahidi F, Han XQ, Synowiecki J. Production and characteristics of protein hydrolysates from capelin (Mallotus villosus). Food Chem. 1995; 53(3):285-93. doi:10.1016/0308-8146(95)93934-J.
• 21. Hamid A, Bakar J, Bee GH. Nutritional quality of spray dried protein hydrolysate from Black Tilapia (Oreochromis mossambicus) Food Chem. 2002; 78(1):69-4. doi:10.1016/S0308-8146(01)00380-6.
• 22. Soares LHB, Marques DN, Albuquerque PM, Ayub MAZ. Influence of some commercial proteases and enzymatic associations on the hydrolytic solubilization of deboned poultry meat proteins. Food Sci Technol Int. 2000; 6(4):301-6. doi: 10.1177/108201320000600404.
• 23. Rebeca BD, Peña-Vera MT, Díaz-Castañeda M. Production of fish protein hydrolysates with bacterial proteases; yield and nutritional value. J Food Sci. 1991; 56(2):309-14. doi: 10.1111/j.1365-2621.1991.tb05268.
• 24. Rangel A, Saraiva K, Schwengber P, Narciso MS, Domont GB, Ferreira ST, et al Biological evaluation of a protein isolate from cowpea (Vigna unguiculata) seeds. Food Chem. 2004; 87(4):491-9. doi:10.1016/j.foodchem.2003.12.023.
• ²⁵
  Food and Agriculture Organization. Energy and protein requirements. Report of a joint FAO/WHO/UNU Expert Consultation. World Health Organization. Geneve: World Health Organization; 1985. Technical Report Series 724.

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; 2007. Sponsored by Conselho Nacional de Desenvolvimento Científico e Tecnológico (Process number 505861/2004-6)

Publication Dates

• Publication in this collection
  22 Mar 2010
• Date of issue
  Dec 2009

History

• Accepted
  09 Feb 2009
• Received
  13 Dec 2007
• Reviewed
  12 Aug 2008