Obtenção do concentrado proteico de linhaça e sua aplicação na nutrição do jundiá

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(1)UNIVERSIDADE FEDERAL DE SANTA MARIA CENTRO DE CIÊNCIAS RURAIS PROGRAMA DE PÓS-GRADUAÇÃO EM ZOOTECNIA. Dirleise Pianesso. OBTENÇÃO DO CONCENTRADO PROTEICO DE LINHAÇA E SUA APLICAÇÃO NA NUTRIÇÃO DO JUNDIÁ. Santa Maria, RS 2018.

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(3) Dirleise Pianesso. OBTENÇÃO DO CONCENTRADO PROTEICO DE LINHAÇA E SUA APLICAÇÃO NA NUTRIÇÃO DO JUNDIÁ. Tese apresentada ao Curso de Doutorado do Programa de Graduação em Zootecnia, da Universidade Federal de Santa Maria (UFSM, RS), como requisito parcial para obtenção do título de Doutora em Zootecnia.. Orientadora: Profª Drª. Leila Picolli da Silva. Santa Maria, RS, Brasil 2018.

(4) __________________________________________________________ © 2018 Todos os direitos autorais reservados a Dirleise Pianesso. A reprodução de partes ou do todo deste trabalho só poderá ser feita mediante a citação da fonte. Endereço: Avenida Roraima, nº 1000, Bairro Camobi, Santa Maria, RS. CEP: 97105-900 Fone: (55) 3220-8365; E-mail: pianessodirleise@gmail.com.

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(7) AGRADECIMENTOS. A Deus, pela minha vida. Por me sustentar firme em cada dificuldade. Agradeço a minha família, meus pais Dirceu e Judith pela educação e apoio incondicional nestes 10 anos de formação. As minhas irmãs Cláudia Raquel e Denise, seus cônjuges, meus sobrinhos Natieli, Lucas e Sara, pela compreensão da minha ausência em muitos momentos e pelo incentivo que sempre me deram. Ao Paulo, que depois de colega se tornou meu esposo, pelo amor, dedicação e exemplo de profissional que és. E também a sua família, Inês, Celso, Joviane e Elizeu pelo incentivo durante nossa jornada acadêmica. À Universidade Federal de Santa Maria, Programa de Pós-Graduação em Zootecnia e ao Laboratório de Piscicultura que, por meio dos professores, funcionários e colaboradores proporcionam formação profissional de qualidade. À Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES), pela concessão da bolsa de Doutorado, código de financiamento 001. À professora Leila Picolli da Silva pela orientação, ensinamentos e acima de tudo, pelo exemplo e dedicação. Muito obrigada!!! Aos professores Rafael Lazzari e João Radünz Neto, muito obrigada pelos ensinamentos, incentivo e convivência. Aos colegas do Laboratório de Piscicultura pela amizade, vivência e colaboração. Somente com o auxílio de vocês este trabalho pôde ser realizado. As minhas colegas de graduação, que ao longo dessa trajetória se tornaram minhas irmãs, Pati e Taida, obrigada pelos momentos que passamos juntas, pelo apoio, com certeza nossa amizade foi fundamental para continuar diante das dificuldades. Ao Silvino, funcionário do Laboratório de Piscicultura, pelo auxílio nas análises. À Luiza Loebens pela realização das análises histológicas. Ao Marcos, pela ajuda na secretaria da Pós-graduação em Zootecnia. Aos professores membros da banca avaliadora, por colaborarem para aperfeiçoar cientificamente este trabalho. As empresas Selecta e Giovelli pela doação de ingredientes.. Agradeço todas as pessoas que contribuíram para que esta etapa de minha vida fosse alcançada. Muito obrigada!!!.

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(9) RESUMO. OBTENÇÃO DO CONCENTRADO PROTEICO DE LINHAÇA E SUA APLICAÇÃO NA NUTRIÇÃO DO JUNDIÁ AUTORA: Dirleise Pianesso ORIENTADORA: Leila Picolli da Silva. Este estudo teve como objetivo avaliar os efeitos de dietas contendo níveis crescentes de substituição da proteína da farinha de peixe (FP) pela proteína do concentrado proteico de linhaça (CPL) sobre o crescimento, deposição de nutrientes, índices somáticos, atividade de enzimas digestivas, respostas metabólicas e contagem de células caliciformes de jundiás (R. quelen). O CPL obtido através da metodologia do pH isoelétrico dos aminoácidos apresentou menor teor proteico (p<0,05) mas maior digestibilidade proteica (p<0,05) que a farinha de peixe. Posteriormente, o CPL foi incluído em níveis (0, 10, 20, 30 ou 40%) de substituição da proteína da FP nas dietas. Durante 60 dias experimentais, 500 jundiás, com peso médio inicial de 6,13 ± 0,97 g foram distribuídos em 20 tanques (70L), alimentados com cinco dietas experimentais em quatro repetições. Para análise estatística, os dados foram submetidos a teste de normalidade (Shapiro-Wilk), análise de variância (ANOVA), sendo as médias comparadas pelo teste de Tukey (5% de significância) e análise de correlação. Foram avaliados parâmetros de crescimento, índices somáticos, deposição corporal de nutrientes, atividades de enzimas digestivas tripsina e quimotripsina, bem como parâmetros metabólicos em plasma e fígado e a contagem de células caliciformes. A substituição da proteína da FP por CPL não influenciou significativamente (p>0,05) os parâmetros produtivos de deposição corporal e índices somáticos dos peixes. Houve correlação positiva (p<0,05) entre as variáveis de crescimento (ganho em peso, comprimento, taxa de crescimento específico, ganho em peso relativo e biomassa final) e o índice digestivo somátivo, mas, a conversão alimentar apresentou correlação negativa (p<0,05) com essa mesma variável. Quando a proteína da FP foi substituída por 30 ou 40% CPL nas dieta houve aumento (p<0,05) na atividade da enzima quimotripsina, diferindo dos que receberam a dieta com 0% CPL. Os níveis de albumina sérica foram superiores (p<0,05) nos peixes alimentados com a dieta em que foi substituído 20% da proteína da FP por CPL, em comparação aos tratamentos 10 e 30% CPL. Dietas contendo maiores níveis (30 ou 40%) de CPL em substituição a proteína da FP promoveram maior (p<0,05) concentração de aminoácidos livres no plasma. A substituição da proteína da FP por 30% CPL promoveu maior (p<0,05) concentração de proteína hepática diferindo do tratamento sem substituição da proteína da FP. A amônia hepática foi superior (p<0,05) nos peixes alimentados com a dieta 0% CPL, sem diferir dos tratamentos 10 e 40% CPL. Houve aumento na contagem de células caliciformes com a substituição de 30% da proteína da FP por CPL na dieta, quando comparada ao tratamento sem a adição do CPL (p<0,05). Dessa maneira, pode-se concluir que o CPL apresenta qualidade nutricional para substituir a proteína da farinha de peixe em até 40%, pois não altera o crescimento, eficiência de utilização da proteína e gordura para deposição corporal e pode promover efeitos benéficos na renovação celular intestinal.. Palavras-chave: Rhamdia quelen. Concentração proteica. Proteína vegetal. Linum uistatissimum L..

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(11) ABSTRACT. OBTAINING THE LINSEED PROTEIN CONCENTRATE AND ITS APPLICATION IN NUTRITION OF SILVER CATFISH. AUTHOR: Dirleise Pianesso ADVISOR: Leila Picolli da Silva. The objective of this study was to evaluate the effects of diets containing increasing levels of fish meal (FM) protein replacement by linseed protein concentrate (LPC) on growth, nutrient deposition, somatic indexes, digestive enzymes activity, metabolic responses and counting of goblet cell in silver catfish (R. quelen). The LPC obtained by means of the isoelectric pH of the amino acids presented lower protein content (P<0.05) but higher protein digestibility (P<0.05) than fish meal. Subsequently, the LPC was included in levels (0, 10, 20, 30 or 40%) of substitution of the FM protein in the diets. During 60 experimental days, 500 jundias, with initial mean weight of 6.13 ± 0.97 g were distributed in 20 tanks (70L), fed five experimental diets in four replicates. For statistical analysis, the data were submitted to normality test (Shapiro-Wilk), variance analysis (ANOVA), and the means were compared by Tukey test (5% significance) and correlation analysis. Were evaluated growth performance, somatic indexes, body nutrient deposition, trypsin and chymotrypsin digestive enzyme activities, as well as metabolic parameters in plasma and liver besides, goblet cell counts. The replacement of FM protein by LPC did not significantly influence (P>0.05) the productive parameters of body deposition and somatic indexes the fish. There was a positive correlation (P<0.05) between the growth variables (weight gain, length, specific growth rate, relative weight gain and final biomass) and the somatic digestive index, but feed conversion apparent presented a negative correlation (P<0.05) with this same variable. When the FM protein was replaced by 30 or 40% LPC in the diet, there was an increase (P<0.05) in the activity of the chymotrypsin enzyme, differing from fish those fed the 0% LPC diet. Serum albumin levels were higher (P<0.05) in fish fed with the diet in which 20% of the FM protein was replaced by LPC, compared to treatments 10 and 30% LPC. Diets containing higher levels (30 or 40%) of LPC in substitution of the FM protein promoted a higher (P<0.05) concentration of free amino acids in plasma. Replacement of FM protein by 30% LPC promoted a higher (P<0.05) concentration of hepatic protein differing from treatment without substitution of FM protein. Hepatic ammonia was higher (P<0.05) in fish fed the 0% LPC diet, without differing from treatments 10 and 40% LPC. There was an increase in the goblet cell count with the replacement of 30% of the FM protein by LPC in the diet, when compared to the treatment without the addition of LPC (P<0.05). Thus, it can be concluded that LPC presents nutritional quality to replace fish meal protein in up to 40%, as it does not alter the growth, efficiency of protein and fat utilization for body deposition and may promote beneficial effects on cell renewal the intestinal.. Keywords: Rhamdia quelen. Protein concentration. Vegetable protein. Linum uistatissimum L..

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(13) LISTA DE ILUSTRAÇÕES ARTIGO 1 - LINSEED MEAL PROTEIN: ALTERNATIVE TO FISH MEAL IN PRACTICAL DIETS FOR SILVER CATFISH Figure 1 −. Amino acid composition of fish meal and linseed protein concentrate.................................................................................................... 48. ARTIGO 2 - NUTRITIONAL ASSESSMENT OF LINSEED MEAL (Linum usitatissimum L.) PROTEIN CONCENTRATE IN FEED OF SILVER CATFISH Figure 1 − Figure 2 −. Nutritional composition of linseed protein concentrate (LPC) and fish meal (FM).............................................................................................................. 75 Effect of increasing levels of LPC in the diet on goblet cell count (cells/g) in silver catfish.............................................................................................. 76.

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(15) LISTA DE TABELAS ARTIGO 1 - LINSEED MEAL PROTEIN: ALTERNATIVE TO FISH MEAL IN PRACTICAL DIETS FOR SILVER CATFISH Table 1 − Table 2 − Table 3 − Table 4 −. Nutritional composition of linseed protein concentrate (LPC) and fish meal (mean ± SD).................................................................................................. Formulation, proximate analyses and amino acids profile of experimental diets used during feeding trial....................................................................... Productive performance and somatic index of silver catfish of different experimental groups fed experimental diets.................................................. Digestive enzymes activity (mean ± SD) in silver catfish of different experimental groups fed different levels of linseed protein concentrate (LPC)............................................................................................................. 43 44 46. 47. ARTIGO 2 - NUTRITIONAL ASSESSMENT OF LINSEED MEAL (Linum usitatissimum L.) PROTEIN CONCENTRATE IN FEED OF SILVER CATFISH Table 1 − Table 2 − Table 3 − Table 4 −. Formulation and proximal composition of experimental diets (natural matter)........................................................................................................ Productive indexes, nutrient deposition and survival of silver catfish fed with increasing levels of LPC in the diet in substitution of fish meal............................................................................................................ Plasma biochemistry values for silver catfish of different experimental groups fed different experimental diets....................................................... Hepatic biochemistry values for silver catfish of different experimental groups fed different experimental diets........................................................ 70. 72 73 74.

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(17) LISTA DE APÊNDICES APÊNDICE A – ARTIGO 3 .................................................................................................. 85.

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(19) LISTA DE ANEXOS ANEXO A - NORMAS DA REVISTA AQUACULTURE NUTRITION ....................... 110 ANEXO B - NORMAS DA REVISTA ANIMAL FEED SCIENCE AND TECHNOLOGY .............................................................................................. 112 ANEXO C - ETAPAS DA OBTENÇÃO DO CONCENTRADO PROTEICO DE LINHAÇA (CPL) ............................................................................................ 121 ANEXO D – DIETAS E INSTALAÇÕES EXPERIMENTAIS ....................................... 122.

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(21) SUMÁRIO. 1 INTRODUÇÃO ............................................................................................................. 21 1.1 OBJETIVOS ................................................................................................................... 23 1.1.1 Objetivo geral ................................................................................................................. 23 1.1.2 Objetivos específicos ...................................................................................................... 23 2 ARTIGO 1 - LINSEED MEAL PROTEIN: ALTERNATIVE TO FISH MEAL IN PRACTICAL DIETS FOR SILVER CATFISH ........................................................ 25 ABSTRACT .................................................................................................................... 26 INTRODUCTION .......................................................................................................... 26 MATERIALS AND METHODS .................................................................................... 28 RESULTS ....................................................................................................................... 33 DISCUSSION ................................................................................................................. 34 ACKNOWLEDGEMENTS ............................................................................................ 38 REFERENCES................................................................................................................ 38 3 ARTIGO 2 - NUTRITIONAL ASSESSMENT OF LINSEED MEAL (Linum usitatissimum L.) PROTEIN CONCENTRATE IN FEED OF SILVER CATFISH 49 ABSTRACT .................................................................................................................... 50 INTRODUCTION .......................................................................................................... 51 MATERIALS AND METHODS .................................................................................... 52 RESULTS ....................................................................................................................... 56 DISCUSSION ................................................................................................................. 58 ACKNOWLEDGMENTS .............................................................................................. 62 REFERENCES................................................................................................................ 63 4 DISCUSSÃO GERAL................................................................................................... 77 5 CONCLUSÕES GERAIS ............................................................................................. 81 REFERÊNCIAS ............................................................................................................ 82 APÊNDICE A – ARTIGO 3 ......................................................................................... 85 ANEXO A - NORMAS DA REVISTA AQUACULTURE NUTRITION ............. 110 ANEXO B - NORMAS DA REVISTA ANIMAL FEED SCIENCE AND TECHNOLOGY.......................................................................................................... 112 ANEXO C – ETAPAS DA OBTENÇÃO DO CONCENTRADO PROTEICO DE LINHAÇA (CPL) ........................................................................................................ 121 ANEXO D – DIETAS E INSTALAÇÕES EXPERIMENTAIS ............................. 122.

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(23) 21. 1 INTRODUÇÃO. A aquicultura apresentou crescimento anual de 5,8% durante o período 2001-2016 (FAO, 2018), principalmente impulsionada pela produção de peixes, que representou 92,5% (47,5 milhões de toneladas) da aquicultura mundial. No Brasil, o crescimento da piscicultura foi de 8% em 2017 (ANUÁRIO PEIXE BR, 2018), o qual deve seguir em ritmo crescente nos próximos anos. Esta expansão produtiva é acompanhada pelo aumento na demanda de rações, em geral formuladas com percentuais elevados de farinha de peixe, fonte proteica considerada como padrão das dietas, em função de seu elevado valor biológico (PORTZ; FURUYA, 2012). Este cenário aponta alguns entraves futuros quanto à disponibilidade e custo desse ingrediente, demonstrando a necessidade de conduzirmos estudos em busca de alternativas para o uso da farinha de peixe na matriz produtiva atual e futura. Muitas pesquisas têm se dedicado à avaliação da substituição da farinha de peixe por fontes proteicas vegetais, como ingredientes alternativos e sustentáveis na alimentação aquicola (CABRAL et al., 2011). Obviamente, esta substituição exige cautela para evitar competitividade desnecessária por fontes usadas como base dietética em outras espécies zootécnicas consolidadas (aves e suínos), como o farelo de soja, que sem dúvida é reconhecido por sua qualidade proteica, mas uma ‘commodity’, que tem seu preço vinculado ao dólar. Em determinados períodos esse ingrediente tem seu custo elevado, acarretando sérios problemas à cadeia produtiva, pois os eventuais substitutos ocasionam, na maioria das vezes, transtornos para indústria e produtores, como atrasos na terminação dos animais, redução do ganho em peso, aumento na conversão alimentar e modificações metabólicas como gliconeogênese, indicando uso ineficiente da proteína para o crescimento. Sendo assim, há necessidade de desenvolver tecnologias apropriadas para obtenção de ingredientes protéicos nutricionalmente eficientes para arraçoamento de peixes, a partir de espécies vegetais adaptadas ao cultivo em distintas regiões do País, mas ainda pouco exploradas na nutrição de não ruminantes. A linhaça (Linum uistatissimum L.) é uma cultura de inverno, com potencial para ser utilizada na rotação de culturas no RS. É empregada a anos como ingrediente alimentar funcional na dieta humana, devido a suas propriedades como elevado teor de ácidos graxos essenciais, fibra alimentar, lignanas e compostos fenólicos (BERGLUND; ZOLLINGER, 2007; HALL; TULBEK; XU, 2006). O principal produto da linhaça é o óleo, mas após o beneficiamento da semente é gerado um resíduo nutricionalmente rico com elevado conteúdo de fibra e proteína, denominado farelo de linhaça. Este coproduto ainda é pouco explorado, sendo majoritariamente destinado à alimentação de animais ruminantes, mas com.

(24) 22. severa restrição de uso para não ruminantes devido aos fatores antinutricionais intrínsecos (mucilagens). O fracionamento da linhaça (óleo, proteína, fibras) agrega valor ao produto e cria diferentes possibilidades para seu uso na nutrição humana e animal, colaborando para fomentar ainda mais o seu cultivo. Os ingredientes vegetais apresentam desvantagens nutricionais em relação à farinha de peixe que podem reduzir a eficiência de utilização dos nutrientes, e ainda provocar o aparecimento de lesões no sistema digestório (KROGDAHL et al., 2010). Segundo Gatlin III et al. (2007), tais ingredientes devem apresentar composições nutricionais adequadas, como baixos teores de fibra, amido, especialmente carboidratos não-solúveis e antinutrientes, além de conteúdo de proteína elevado, perfil favorável de aminoácidos, alta digestibilidade de nutrientes e palatabilidade. Ou seja, o principal entrave para o uso das fontes vegetais na nutrição de peixes não está relacionado somente a qualidade nutricional, mas principalmente, a carência de informações e tecnologias para obtenção, separação e aplicação dos diferentes coprodutos. As características nutricionais das fontes protéicas vegetais podem ser otimizadas através de estratégias e técnicas específicas (químicas, físicas ou biológicas), tais como aquelas que visam a concentração de proteínas vegetais. A concentração proteica promove melhorias na composição nutricional e limita os efeitos negativos dos compostos antinutricionais, aumentando o uso de fontes vegetais na aquicultura e reduzindo a dependência de fontes tradicionais (GATLIN III et al., 2007). Os concentrados proteicos vegetais são fontes alternativas e factíveis para a alimentação de peixes, em relação ao uso das fontes in natura (DENG et al., 2006), pois em geral apresentam qualidade nutricional superior (YUE; ZHOU, 2008). As técnicas para obtenção de concentrados proteicos podem modificar o perfil aminoácidico da matéria prima e diminuir a concentração de antinutrientes e inibidores enzimáticos. Entretanto, o rendimento de extração de proteínas é limitado, pois são estruturas muito complexas, heterogêneas e estão associadas a outros compostos. Além disso, a extração e concentração podem ser obtidas pela aplicação de várias técnicas, usadas de forma individual ou associada (químicas, físicas, enzimáticas), pois em uma matéria prima existem formas proteicas solúveis facilmente carreadas pela água, formas insolúveis estruturadas e formas fortemente ligadas à polissacarídeos (LINDEN; LORIENT, 1996). No caso de proteínas vegetais, que estão fortemente ligadas a compostos indigestíveis pelos monogástricos, busca-se separá-las ou isolá-las destes antinutrientes (celulose, lignina, polifenóis, entre outros), aumentando a acessibilidade das enzimas digestivas ao alimento, o.

(25) 23. que se reflete positivamente na redução da poluição ambiental devido a melhor digestão e absorção dos nutrientes (LINDEN; LORIENT, 1996). Por ser uma espécie nativa de hábito alimentar onívoro e apresentar características desejáveis para produção, o jundiá (Rhamdia quelen) têm sido alvo de estudos no Sul do Brasil. Pesquisas desenvolvidas para avaliar a inclusão de concentrados proteicos vegetais indicam que há potencial de substituição parcial da proteína de origem animal por concentrados proteicos de girassol, crambe (LOVATTO et al., 2014) e concentrado de farelo de semente de abóbora (LOVATTO et al, 2016; LOVATTO et al, 2015) na dieta dessa espécie. Nesse sentido, são desafios para aquicultura avaliar fontes proteicas alternativas regionais como a linhaça, agregar valor nutricional, tecnológico e comercial a coprodutos agroindustriais, além de, viabilizar a redução dos custos e contribuir para a nutrição de peixes, aliando as demandas das diferentes cadeias produtivas.. 1.1 OBJETIVOS. 1.1.1 Objetivo geral. Estudar métodos físico/químicos para obtenção do concentrado proteico de linhaça (CPL) e avaliar sua inclusão como substituto da farinha de peixe na alimentação do jundiá com ênfase no crescimento, atividade de enzimas digestivas e respostas metabólicas dos peixes.. 1.1.2 Objetivos específicos  Aplicar e otimizar técnicas de concentração proteica em farelo de linhaça demucilado;  Caracterizar o concentrado proteico de linhaça quanto a sua composição nutricional, perfil aminoácidico e digestibilidade in vitro da proteína;  Determinar os efeitos da inclusão do CPL sobre o desempenho produtivo, índices somáticos, composição e deposição corporal de nutrientes, parâmetros bioquímicos plasmáticos e hepáticos, atividade das enzimas protease ácida, tripsina e quimotripsina e sobre a contagem de células caliciformes. O presente trabalho de doutorado faz parte do projeto “Alternativas de nutrientes e compostos bioativos: estudo do fracionamento da linhaça para nutrição de peixes” (registro n° 043216), proposto com vistas à obtenção de frações concentradas de proteína, fibra solúvel e.

(26) 24. fibra insolúvel de linhaça (Apêndice A). Nesta tese estão descritas a obtenção e caracterização do concentrado proteico de linhaça (CPL) e sua avaliação biológica em peixes. Os resultados estão apresentados na forma de artigos científicos. O Artigo 1 traz a comparação nutricional entre o CPL e a farinha de peixe e trata também da inclusão desse ingrediente na dieta e a avaliação da resposta nutricional de jundiás (parâmetros produtivos, índices somáticos e atividades de enzimas digestivas). No Artigo 2 são apresentados e discutidos os resultados sobre o teor proteico e a digestibilidade in vitro da proteína do CPL em relação a farinha de peixe, bem como, as respostas metabólicas (peso final, sobrevivência, deposição de nutrientes, bioquímica plasmática e hepática e contagem de células caliciformes) promovidas pela inclusão do CPL na dieta de jundiás. No Apêndice A se encontra o Artigo 3, onde estão descritos os métodos testados para obtenção e otimização do CPL e das demais frações da linhaça, bem como, a caracterização nutricional dessas frações..

(27) 25. 2 ARTIGO 1 Linseed meal protein: Alternative to fish meal in practical diets for silver catfish*. 1 2 3. Dirleise Pianesso1**, Fernanda Rodrigues Goulart1, Taida Juliana Adorian1, Patrícia Inês. 4. Mombach1, Joziane Soares de Lima1, Thaís Soares dos Santos1, Bruno Bianchi Loureiro1,. 5. Leila Picolli da Silva1. 6 7. 1. Department of Animal Science, Federal University of Santa Maria, Santa Maria, Rio Grande. 8. do Sul. Av. Roraima nº 1000, Cidade Universitária, Bairro Camobi, Santa Maria – RS,. 9. Brazil. CEP: 97105-900.. 10 11. Corresponding author.. 12. (**) Dirleise Pianesso. 13. Departamento de Zootecnia. 14. Universidade Federal de Santa Maria. 15. Av. Roraima nº 1000, Cidade Universitária, Bairro Camobi. 16. CEP: 97105-900 – Santa Maria, RS, Brazil. 17. Phone: 55 (55) 3220-8365. 18. Fax: 55 (55) 3220-8240. 19. E-mail: pianessodirleise@gmail.com. 20 21. Keywords: Growth. Digestive enzyme activity. Rhamdia quelen. Nutritional comparison.. 22. Linseed protein concentrate. Linum uistatissimum L.. *. Artigo submetido à revista Aquaculture Nutrition (em revisão).

(28) 26. 23. Abstract. 24. An study 60‐ days feeding trial was conducted to evaluate the efficiency of substitution. 25. of fish meal (FM) protein by of linseed protein concentrate (LPC) in the diet of silver catfish. 26. (Rhamdia quelen) (6.13 g). Five isonitrogenous and isoenergetic experimental diets were. 27. formulated to replace the FM protein by LPC at the levels of 0, 10, 20, 30 or 40%. Diets were. 28. offered to silver catfish up apparent satiation. LPC had a lower protein content compared to. 29. FM, but in vitro results showed higher digestibility of LPC protein than FM. There were no. 30. significant differences (P > 0.05) in the growth, digestive somatic index and intestinal quotient. 31. of fish fed different levels of LPC. The activity of the digestive enzymes did not differ among. 32. the treatments, except for chymotrypsin, which was higher (P <0.05), the silver catfish fed with. 33. diets in which LPC replaced 30 or 40% of FM protein. LPC can substitute up 40% of the protein. 34. the FM in diet for silver catfish, without adverse effects in growth performance the specie.. 35 36. 1. Introduction. 37. Aquaculture has grown exponentially worldwide, and Brazil has been appointed as one. 38. of the main producers of Latin America in the next years, with an increase of 104% by 2025. 39. (FAO 2016). It is expected that such expansion will result from incentives to cultivation of. 40. native fish species, which will increase demands for qualitatively adequate and economically. 41. viable ingredients for aquafeed formulations. Fish meal is a protein ingredient traditionally used. 42. in aquafeeds, but its high cost, high nutritional variability, supply oscillations and low. 43. environmental sustainability encourage the development of alternative feedstuffs.. 44. By-products of terrestrial animals (meat and bone meal, blood meal, feather meal) are. 45. sources of protein seen with great potential to replace fish meal in aquaculture (Naylor et al.. 46. 2009), because it presents good crude protein concentration (45-65%). However, for nutritional. 47. and palatability characteristics, the use of fish oil and fish meal is still prioritized in aquafarming.

(29) 27. 48. (Naylor et al. 2009). In Brazil, fish meal is a scarce feedstock and it is made from fish industry. 49. residues, resulting in a product below international standards and with wide nutritional. 50. variability, high mineral content, lipid rancidity and proteins degradation (Teixeira et al. 2006).. 51. Moreover, a product with such characteristics causes a greater environmental pollution. 52. (Tsukamoto & Takahashi, 1992). Currently a wide range of plants such as barley, canola, maize,. 53. cotton, soybeans and wheat are used in aquaculture; however, the inclusion of alternative. 54. sources depends on the nutritional requirements of species, prices and specific environmental. 55. regulations of production systems (Naylor et al. 2009). Soybeans, for example, have. 56. experienced drastic price increases in recent years and have been largely used as a protein. 57. source for ruminant and monogastric animal production. Proposals for a new aquafeed matrix. 58. should seek for economically viable, environmentally friendly ingredients with high nutritional. 59. value (Gatlin III et al. 2007) and with sufficient availability to meet the regional demands,. 60. which will enhance sustainability and local economy.. 61. Linseed (Linum uistatissimum L. – family Linaceae) is used in winter crops rotation in. 62. southern Brazil because it does not have phytosanitary limitations as other forage species (oat,. 63. wheat, triticale) usually grown. In Brazil, according to the Brazilian Institute of Geography and. 64. Statistics - IBGE (2010), approximately 16.000 tons of linseed were produced in 2010,. 65. production led by Rio Grande do Sul state.. 66. After extracting the oil, which is the main product of this culture, linseed powder. 67. remains as a residue, which is composed of approximately 35% crude protein (Baldanzi et al.,. 68. 1988), but with limited use in the nutrition of monogastric animals due to its high content of. 69. soluble fibers (mucilage), which increases the food viscosity and reduce nutrients digestion and. 70. absorption. To optimize the use of this feedstuff, which is largely available in the region, it is. 71. necessary the application of technologies to minimize its undesirable characteristics, such as. 72. those of protein concentration, which usually enhance the nutritional value and digestibility of.

(30) 28. 73. in natura sources, increasing the possibilities of including this ingredient in aquaculture diets. 74. (Lovatto et al. 2016; Lovatto et al. 2015; Yue & Zhou, 2008).. 75. It should be noted that omnivorous fish, such as silver catfish (Rhamdia quelen), has. 76. shown good adaptability to the utilization of plant protein concentrates in diets (Lovatto et al.. 77. 2016; Lovatto et al. 2015; Lovatto et al. 2014; Tyska et al. 2013), with similar or higher. 78. performance than fish meal. In southern Brazil, it is expected that silver catfish cultivation will. 79. grow significantly in the coming years due to its great adaptability to large scale cultivation.. 80. The aim of this study was to evaluate the effect of partial replacement of fish meal. 81. protein with linseed protein concentrate on the growth, somatic index and in the digestive. 82. activities measurement of silver catfish.. 83 84. 2. Materials and methods. 85 86. 2.1 Comparing the nutrients of LPC versus in FM. 87. 2.1.1 Feed ingredients: chemical composition and in vitro protein digestibility. 88. Linseed protein concentrate (LPC) was obtained at the Fisheries Laboratory of the. 89. Federal University of Santa Maria, RS, Brazil. Firstly, the linseed mucilage was extracted. 90. (Goulart et al., 2012) and after drying and milling, the linseed meal was degreased (hexane. 91. solvent). Subsequently, the protein was concentrated in laboratory scale by the extraction and. 92. precipitation of the protein at the isoelectric pH, following a method described by Smith et al.. 93. (1946) and Lovatto et al. (2016).. 94. For the study of the nutritional composition, samples of LPC and fish meal (FM) (fish. 95. filleting waste meal - Oreochromis niloticus) were analyzed for determination of dry matter. 96. (105 °C for 24 hours), ash (550 °C for 6 hours) and crude protein (CP), where generated. 97. nitrogen (N) was multiplied by 6.25 to obtain CP according to the methods outlined by AOAC.

(31) 29. 98. (1995). The residual fat was extracted and quantified by the cold-water extraction method. 99. (Bligh & Dyer, 1959). Total insoluble and soluble dietary fiber was also determined in LPC. 100. (AOAC 1985). The analysis of calcium and phosphorus included the phase of minerals. 101. digestion and quantification by atomic absorption spectrometry for calcium and in the visible. 102. region for phosphorus (Tedesco et al. 1995).. 103. For determination of the hydration and fat binding capacities, water or oil was added to. 104. the samples (at 1:20 w/v) and allowed to stand for 24 hours. Subsequently, the samples were. 105. centrifuged (1300xg for 10 min), the supernatant fraction was removed and the hydration and. 106. fat-binding capacities were calculated from the weight difference between the wet and dry. 107. sample (Wang & Kinsella, 1976).. 108. The amino acid content of the ingredients (Figure 1) were extracted with hydrochloric. 109. acid (6 N) for 24 h (0.3 mg sample in 9 ml HCl) and then derivatized with phenyl isothiocyanate.. 110. The derivatized samples were separated by high performance liquid chromatography (Model. 111. P4000-Thermo Fisher Scientific, Waltham, MA) in reverse phase with UV detection at 254 nm,. 112. based on the methods described by White et al. (1986).. 113. The in vitro protein digestibility testing was carried out based on Mauron (1973), with. 114. modifications outlined by Dias et al. (2010). The method is based on the digestion of the sample. 115. by pepsin (1:10.000, Nuclear) and pancreatic (Sigma, São Paulo, Brazil) enzymes. This method. 116. for determination of in vitro digestibility of protein results from the relation between the total. 117. nitrogen of the sample, the nitrogen digested, the nitrogen produced by the autodigestion of the. 118. enzymes and the nitrogen originally soluble in the sample. The analysis starts with the addition. 119. of 100 mg of defatted sample, 10 ml of HCl 0.1N and 4 mg of pepsin (under stirring at 37 °C. 120. for 1 hour). Subsequently, the pH was adjusted to 7.0 (NaOH 0.4 N) and 20 mg of pancreatin. 121. was added (dilution in sodium phosphate buffer 0.1M pH 8.5) and the samples were kept at 37. 122. °C under agitation for 3 hours. Afterwards, the reaction is stoped with trichloroacetic acid (final.

(32) 30. 123. concentration of 5% acid in each tube) and an aliquot (2 ml) is withdrawn to determine the total. 124. nitrogen. Then, the remainder was centrifuged (16700xg /10 min) for analysis of the digested. 125. nitrogen (2 ml). For each test sample, a blank follows, without addition of the enzymatic. 126. solutions (soluble nitrogen). To determine the nitrogen coming from enzymatic autodigestion,. 127. a tube that receives only pepsin and pancreatin solutions follows. The nitrogen content in each. 128. fraction was determined by the micro Kjeldahl method. To compare the digestibility of the. 129. samples, casein (Synth, 90% purity) was adopted as the standard.. 130 131. 2.2 Comparing growth of silver catfish fed with LPC and FM-based diets. 132. 2.2.1 Preparation of experimental diets. 133. Linseed protein concentrate (LPC) was assessed as a replacement for FM. The FM used. 134. is composed of residues from filleting of tilapia (Oreochromis niloticus) and was purchased. 135. from Copisces, Toledo, Brazil. Before use it was sieved through 590 μm. The experimental. 136. diets were formulated considering the percentage of crude protein as well as the amino acid. 137. profile obtained in the LPC, the required level of 370 g kg-1 of crude protein as defined by. 138. Meyer & Fracalossi (2004), 13.4 MJ of digestible energy kg-1 and essential amino acid (Montes-. 139. Girao & Fracalossi, 2006). The experimental diets were based on the replacement of increasing. 140. levels (0, 10, 20, 30 or 40%) of FM protein for LPC protein.. 141. Diets were pelletized, and to this end the ingredients were ground, weighed and mixed. 142. manually. Afterwards, oil and water (cold) were added to obtain pellets (4 mm), which were. 143. dried (55 °C) in an air circulation oven (MA035; Marconi, Brazil) for 24 hours.. 144. The content of ash, dry matter, crude protein (AOAC 1995), fat (Bligh & Dyer, 1959),. 145. total insoluble and soluble dietary fiber, (AOAC 1985), and hydration capacity (Wang &. 146. Kinsella, 1976), was determined in the diets.. 147.

(33) 31. 148. 2.2.2 Fish and experimental conditions. 149. All procedures involving the experimental fish were carried out in conformity with the. 150. guidelines approved by the Committee of Ethics and Animal Wellbeing of the Federal. 151. University of Santa Maria, protocol number 8015120816. Before the beginning of the. 152. experimental period a group of fish (1200 fish) was acclimated to the experimental system for. 153. 15 days receiving commercial feed (36% crude protein) to apparent satiation. From this group. 154. a total of 500 fish with mean initial body mass of 6.13 ± 0.97 g and total length of 8.74 ± 0.48. 155. cm (mean ± standard deviation) were used. The fish were randomly distributed into 20 tanks,. 156. in a total of five treatments and four replications. Each 70-L capacity tank received 25 fish. The. 157. tanks were connected to a water recirculation system with two biological filters (1000 W. 158. electrical resistance) and a 500 L water tank.. 159. The experiment duration was 60 days. In this period, the experimental diets were offered. 160. to apparent satiation three times a day (08:30 a.m., 1 p.m. and 5 p.m.). The tanks were cleaned. 161. twice a day (7 a.m. and 3 p.m.) to remove faecal matter and uneaten feeds.. 162. Throughout the experimental period, water quality parameters were they remained as. 163. follows: temperature: 25.6±1.49 °C; dissolved oxygen: 7.10±0.70 mg L-1; pH: 7.5±0.24 units;. 164. total ammonia: 0.30±0.11 mg L-1; nitrite: 0.2±0.17 mg L-1; alkalinity: 33.6±13.60 mg L-1 of. 165. CaCO3; and hardness: 34.40±18.45 mg L-1 of CaCO3. All parameters remained appropriate for. 166. the silver catfish cultivation (Baldisserotto & Silva, 2004).. 167 168. 2.2.3 Growth parameters and digestive indexes of fish. 169. To determine the fish growth, two biometrics were performed, one at the beginning and. 170. the other at the end of the experimental period. Feeding was suspended for 24 hours prior to. 171. sampling. During biometrics, the animals were anesthetized with benzocaine (100 mg L-1). The. 172. total length (cm) and body mass (g) were measured using an ictiometer and digital scale..

(34) 32. 173. Subsequently, the following data were calculated: total biomass= which consisted of the sum. 174. of the fish body mass of each experimental unit; relative weight gain (RWG %) = (weight gain. 175. / initial body mass) × 100; specific growth rate (% day-1): SGR [(ln final body mass – ln initial. 176. body mass)/60]*100; feed conversion ratio (FCR) = feed offered (g)/ weight gain (g).. 177. At the beginning and end of the experiment, three fish per tank (12 per treatment) were. 178. euthanized with benzocaine overdose (10%, ≥250 mg L-1) according to the American. 179. Veterinary Medical Association (2013). The fish was eviscerated, and the digestive tract and. 180. the fat that wrapped the tissue were removed. After being weighed and measured, they were. 181. stored in micro tubes (frozen at -20 ºC) for subsequent analyses. The following measures were. 182. calculated: digestive somatic index (DSI= (Digestive tract weight / body weight)*100) and the. 183. intestinal quotient (IQ= length of the digestive tract / total length of the fish).. 184 185. 2.3 Investigating digestive enzyme activity of silver catfish fed with LPC and FM-based diets. 186. The stomach and a 10-cm portion of the fish’s anterior intestine were removed. 187. (measured with ictiometer after removal of stomach) and frozen (-20 °C) to assess the activity. 188. of acid protease, trypsin and chymotrypsin digestive enzymes. The tissues was dissected in Petri. 189. dishes to remove any remaining content in the stomach and intestine and then were. 190. homogenized; The tissues homogenization was performed with buffer (Tris 0.02 M / phosphate. 191. 0.01 M, pH 7.5 in 50% glycerol) at 1:20 tissue:buffer ratio, using Turrax tissue homogenizer. 192. (Marconi, Brazil, MA 102). The homogenized tissues were centrifuged at 1200xg for 10 min,. 193. and the supernatants were utilized as enzymes source.. 194. The activity of acid protease was measured in the stomach using casein as substrate. 195. according to the method of Hidalgo et al. (1999). The assay was performed using a 0.2 M KCl. 196. buffer at pH 1.8, and the samples were incubated at 30 ◦C for 40 min. The reaction was. 197. terminated with 15% TCA, and absorbance was recorded at 280 nm. The activity of trypsin and.

(35) 33. 198. chymotrypsin alkaline protease was determined in the intestine. Trypsin was determined with. 199. N-p-Tosyl-l-arginine methyl ester hydrochloride (TAME) as substrate, and the extracts were. 200. incubated (25◦C) in 2-ml buffer (0.2 M Tris/0.01 CaCl2) at pH 8.1. Chymotrypsin activity was. 201. determined with N-benzoyl-l-tyrosine ethyl ester (BTEE) as substrate, and the extracts were. 202. incubated in 1-ml buffer (0.1 M Tris/0.1 CaCl2) at pH 7.8. The enzymes trypsin and. 203. chymotrypsin were dosed in duplicate, and their activities were recorded at 247 and 256 nm,. 204. respectively, according to Hummel (1959).. 205 206. 2.4 Statistical data analysis. 207. Data were subjected to analysis of variance (ANOVA) and normality test of residuals. 208. (Shapiro-Wilk). For the biological assay data, the third order regression was tested. However,. 209. there was no adjustment of the studied variables to the regression models. The data and the. 210. variables that are present in the item results (subtitle 3.2) were subjected also to the correlation. 211. analysis. The means were compared by the Tukey test at 5% probability and are expressed as. 212. mean ± standard deviation. The SAS statistical package was used to perform the statistical. 213. analysis of the data.. 214 215. 3. Results. 216 217. 3.1 Comparing the nutrients of LPC versus FM. 218. The LPC protein content was 10.7% lower compared to the FM protein (Table 1). The. 219. lipid content was higher in the LPC. It was found a higher concentration of dry matter, ash and. 220. total phosphorus in FM. Calcium content was 76.6% higher in FM. The hydration capacity was. 221. similar in the evaluated ingredients. A higher fat-binding capacity was found in FM. The in. 222. vitro digestibility of protein of LPC was 8% higher than the FM (Table 1)..

(36) 34. 223. There was an increase of 34, 37, 50 and 30% for essential amino acid concentration. 224. arginine, isoleucine, phenylalanine and valine, respectively, in the LPC in comparison to FM.. 225. Histidine concentration was similar in both ingredients tested (Figure 1).. 226 227. 3.2 Comparing growth of silver catfish fed with LPC and FM - basead diets. 228. There were no significant effects of increasing levels of FM protein substitution with. 229. LPC on weight gain, total biomass, length, RWG (relative weight gain), SGR (specific growth. 230. rate), AFC (apparent feed conversion), digestive somatic index (DSI) and intestinal quotient. 231. (IQ) (Table 3). The fish growth variables (weight gain, length, SGR) exhibited positive. 232. correlation (P = 0.01; r= 0.53) with the digestive somatic index (DSI). The correlation was also. 233. positive (P = 0.05; r = 0.44) between RWG and DSI. Total biomass was also positively. 234. correlated with DSI (P = 0.002; r = 0.64). On the other hand, for apparent feed conversion. 235. (AFC) there was a negative correlation (P < 0.05; r = -0.59) with DSI.. 236 237. 3.3 Activity of digestive enzymes of silver catfish fed with LPC. 238. There were no significant effects of increasing levels of LPC in replacement of FM. 239. protein on the acid protease and trypsin activity of the fish (Table 4). The chymotrypsin activity. 240. was higher in fish fed with 30 and 40% replacement of the FM protein by LPC than in silver. 241. catfish fed with the diet without LPC (Table 4).. 242 243. 4. Discussion. 244. The average protein content in the linseed meal is 29%, which can increase to 33% when. 245. physicochemical methods for reduction of its mucilage can be used (Goulart et al. 2012). When. 246. applying the protein concentration to the demucilated and defatted meal, we obtained a 60%. 247. rise in protein content, resulting in an ingredient with 532.4 g kg-1 crude protein. For crambe.

(37) 35. 248. and sunflower meals, Lovatto et al. (2017) observed that the isoelectric pH extraction (pHi). 249. method was the most efficient in terms of the yield and crude protein content of the final protein. 250. concentrates. According to the same authors the extraction by isoelectric pH led to 69.20% and. 251. 56.80% increases in crude protein content for the crambe and sunflower protein concentrates,. 252. respectively. Although fish meal (FM) exhibited a higher crude protein content (596.5 g/kg). 253. than the LPC (532.4 g/kg), the in vitro digestibility of this plant ingredient (889.8 g/kg) was. 254. higher, equaling its biological value to the reference animal source (824.5 g/kg). Qualitatively,. 255. the protein concentration allowed to achieve an ingredient with a similar amino acid profile to. 256. that of the FM for most of the essential amino acid.. 257. The higher contents of dry matter, ash, calcium and phosphorus of FM are due to their. 258. nature, predominantly from tilapia (Oreochromis niloticus) filleting waste. The higher lipid. 259. content of LPC results from the technique used in the defatting of the initial feedstock (linseed. 260. cake), which enabled only a 65% removal of total fat, resulting in LPC with 127.5 g of lipids. 261. kg-1. It should be noted that the lipids present in the linseed are of high quality, since 73% of. 262. them consist of polyunsaturated fat acids, 18% of monounsaturated and 9% of saturated fat. 263. acids (Tarpila et al. 2005).. 264. The ingredients hydration capacity provides key information on the processing. 265. technology and feeds stability. Higher values indicate a need for more water addition to the. 266. feedstuffs mixture, but it can also be indicative of a lower buoyance time of the extruded feeds.. 267. The origin of the studied protein ingredients did not have an influence on the samples hydration. 268. capacity nor did it cause alterations in the water absorption capacity of the experimental diets.. 269. The fat-binding capacity of the ingredients influences the absorptive speed and the stability of. 270. lipids binding to the other nutrients of the mixture, influencing food stability and nutrients. 271. absorption, although FM has exhibited a higher fat-binding capacity than LPC, the difference. 272. between the feedstuffs was not sufficient to affect the fat absorption of the experimental.

(38) 36. 273. diets.The productive efficiency of the fishes was not affected by replacing FM with increasing. 274. LPC levels in the diet, which was expected due to the good results of the in vitro digestibility. 275. trial. Protein concentrates of other plant sources have also indicated a viable use for silver. 276. catfish nutrition. Lovatto et al. (2015) observed that pumpkin seed protein concentrate can. 277. replace up to 50% of FM for the same fish species, without affecting adversely the animals’. 278. performance.. 279. According to Baldisserotto (2009), the fishes can change the structure and absorptive. 280. capacity of their digestive system with changes in the carbohydrate and dietary fiber contents.. 281. In our study, the similar chemical composition of the experimental diets led to no changes in. 282. the somatic indexes DSI and IQ between the treatments.. 283. As expected, there was a positive correlation between the DSI and the fishes specific. 284. growth variables since higher DSI indicates a larger contact area between the food and the. 285. digestive tract favoring digestion and nutrients absorption and being reflected by a higher. 286. growth rate. The negative correlation between DSI and AFC is also explained by the utilization. 287. of the ingested feed, where the smaller digestive area diminishes the nutrients assimilation, thus. 288. affecting adversely the fish performance.. 289. The increasing addition of LPC to the diets of silver catfish did not cause any alteration. 290. in the acid protease and trypsin enzymes activity but enhanced the chymotrypsin activity. In an. 291. attempt to improve the digestive utilization of the ingredients, fish adapt their digestive. 292. processes, changing the enzymatic secretion and profile, the absorption and transport of. 293. nutrients (Honorato et al. 2011; Baldisserotto 2009). In sources with higher dietary fibers. 294. contents and potentially antinutritional compounds (phenols, alkaloids, tannins, etc.), such. 295. adaptations are frequently reported in literature. Santigosa et al. (2008) report that replacing. 296. fish meal with plant protein sources (corn gluten meal, wheat gluten, extruded peas and. 297. rapeseed meal) can change the activity of protease, chymotrypsin and trypsin enzymes in trout.

(39) 37. 298. (Oncorhynchus mykiss) and sea bream (Spaurus aurata). Lovatto et al. (2016) found a higher. 299. trypsin activity in animals fed with diets containing pumpkin seed meal, which was pointed as. 300. an attempt of the body to increase the protein absorption of this source. However, when. 301. concentrating the protein, the authors observed that the adaptive effects diminished. Lovatto et. 302. al. (2014) did not find changes either in the trypsin activity of silver catfish fed with crambe. 303. and sunflower concentrates in replacement to 25 and 50% fish meal. It was concluded that in. 304. the protein concentration process, dietary fiber contents and antinutritional factors were. 305. decreased, diminishing the adaptive effects of the animals’ digestive tract.. 306. Chymotrypsin is a proteolytic enzyme that hydrolyzes of peptide bonds adjacent formed. 307. for aromatic amino acids (Riegel 2012). The increasing addition of LPC to the diets caused. 308. increased concentration of aromatic amino acid phenylalanine and tyrosine. This explains the. 309. higher activity of chymotrypsin in the LPC diets. It should also be noted that fish meal is. 310. composed of proteins that require more time to be hydrolyzed due to the structure in which the. 311. fibers are arranged. On the other hand, the LPC proteins, for having undergone a chemical. 312. process, are formed by smaller, rapidly hydrolyzed peptides. Zambonino-Infante et al. (1997). 313. report that higher rates of smaller peptides (di/tri peptides) in the diet can also contribute to a. 314. higher activity of chymotrypsin.. 315. Our results show that the linseed protein concentration by isoelectric pH provided a. 316. concentrate with the same biological value as FM. This ingredient was used to replace up to. 317. 40% of the fish meal without causing adverse effects on growth, digestive indexes or hydrolytic. 318. efficiency of silver catfish’s digestive enzymes. As linseed is a feedstuff largely available in the. 319. region, obtaining a protein concentrate from this source is viable and holds potential to boost. 320. the development of the two production chains, linseed and silver catfish.. 321 322.

(40) 38. 323. Acknowledgements. 324. The authors would like to thank the National Council for Technological Development. 325. (CNPq) for granting a research productivity scholarship (Leila Picolli da Silva) – Process. 326. number 307757/2015-3; to the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. 327. - Brasil (CAPES) - Finance Code 001 by granting a doctorate scholarship (Dirleise Pianesso);. 328. and to Giovelli & Cia Ltda for the linseed courtesy provided.. 329 330. References. 331. AOAC (1985). Association of analytical chemists. Total dietary fiber in foods – enzimatic-. 332. gravimetric method – first action. Journal Association of Official Analytical Chemists. 68,. 333. 399.. 334 335 336 337. AOAC (1995). Official Methods of Analysis. Association of Official Analytical Chemists, Washington, DC. AVMA (2013). Association American Veterinary Medical. Guidelines on Euthanasia, Schaumburg, IL, USA.. 338. Baldanzi, G. Baier A.C., Floss, E.L., Manara, W., Manara, N.T.F., Veiga, P. & Tarragó, M.F.S.. 339. (1998). As lavouras de inverno: cevada – tremoço – linho - lentilha. Editora Globo, Rio de. 340. Janeiro, BR.. 341 342. Baldisserotto, B. (2009). Fisiologia de peixes aplicada à piscicultura 2° ed. Universidade Federal de Santa Maria, Santa Maria, BR.. 343. Baldisserotto, B. & Silva, L.V.F. (2004). Qualidade da água. In: Baldisserotto, B. & Radünz. 344. Neto, J. (eds.), Criação do jundiá. Vol. 1, pp. 73–94. Universidade Federal de Santa Maria,. 345. Santa Maria.. 346 347. Bligh, E.G. & Dyer, W.J. (1959). Rapid method of total lipid extraction and purification. Journal of Physiology and Biochemistry, 37, 911–917..

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(45) 43. 441. Table 1 Nutritional composition of linseed protein concentrate (LPC) and fish meal (mean ±. 442. SD) Nutrient. LPC. Fish meal. ..................(g kg-1 of natural matter).............. Crude protein. 532.4 ± 0.33. 596.5 ± 0.96. Lipid. 127.5 ± 0.18. 102.5 ± 0.36. Dry matter. 936.2 ± 0.14. 942.9 ± 0.29. Ashes. 24.4 ± 0.05. 251.1 ± 0.17. 13.2. 56.4. 5.7 ± 0.07. 25.7 ± 0.05. Total dietary fiber. 291.0 ± 5.43. NA. Soluble fiber. 212.0 ± 5.25. NA. Insoluble fiber. 78.8 ± 0.17. NA. Hydration capacity (g water/g). 3.04 ± 0.07. 3.03 ± 0.14. Fat-binding capacity (g fat/g). 0.74 ± 0.02. 2.18 ± 0.20. Digestibility of protein. 889.8 ± 1.65. 824.5 ± 0.21. Calcium Phosphorus. 443. Crude protein, lipid, dry matter, ashes, hydration capacity and fat-binding capacity are. 444. expressed as means ± standard deviation of three measurements (n = 3). The other variables n. 445. = 2. Calcium was determined in a sample..

(46) 44. 446. Table 2 Formulation, proximate composition and amino acid composition of experimental diets. 447. used during feeding trial (60 days) of silver catfish. 448. .....................Levels of LPC1........................ 0% 10% 20% 30% 40% -1 Diet formulation (g kg ) 2 Fish meal 408.8 367.8 326.4 285.3 245.0 LPC 0 45.8 91.6 137.4 183.2 3 SPC 60% 200 200 200 200 200 4 Corn starch 210.3 211.0 206.8 205.0 203.1 Soybean oil 27.4 25.6 26.0 25.3 24.2 5 Vitamin Mineral Mix 35 35 35 35 35 Dicalcium phosphate 0 10 15 10 30 Calcitic limestone 0 5 7 6 13 Salt 5 5 5 5 5 6 BHT 0.1 0.1 0.1 0.1 0.1 L- methionine 4.7 4.8 5.1 5.2 5.2 Cellulose7 108.7 89.9 82 85.7 56.2 Vitamin C 0.5 0.5 0.5 0.5 0.5 -1 Proximate composition of experimental diets (g kg ) Crude Protein 381.1 381.6 378.6 379.6 378.6 8 13.4 13.4 13.4 13.4 Calculated energy (MJ/kg) 13.4 Lipids 74.2 74.7 75.7 76.8 79.8 Dry matter 958.8 968.3 958 957.2 958.9 Ash 136.3 142 136.7 121.9 135.1 9 Calcium 23.9 26.4 26.7 23.4 29.1 Total phosphorus9 12.1 13.3 13.4 11.7 14.7 Total dietary fiber 290 259 275 273 241 Soluble fiber 92 49 66 40 34 Insoluble fiber 198 210 210 230 233 Hydration capacity (g water) 2.18 2.32 2.32 2.26 2.23 Fat CC (g fat) 1.08 1.08 1.04 1.05 1.0 Essential amino acid composition (g kg-1)9 27.8 28.6 29.5 30.5 Arginine 26.9 7.3 7.3 7.4 7.4 Histidine 7.2 Isoleucine 13.5 13.9 14.3 14.7 15.2 Leucine 24.1 24.2 24.2 24.3 24.4 Lysine 22.6 22.1 21.6 21.2 20.7 13.7 13.7 13.7 Methionine + Cysteine 13.7 13.7 Phenylalanine 14.6 15.1 15.6 16.2 16.7 13.9 13.9 13.8 13.8 Threonine 14 Tyrosine 9.5 9.6 9.8 9.9 9.9 Valine 15.4 15.8 16.2 16.6 17.0 1 Levels (0, 10, 20, 30 or 40%) of replacement of fish meal (FM) protein for linseed protein. 449. concentrate (LPC).. Ingredients.

(47) 45. 450. 2. Fish filleting waste meal (Oreochromis niloticus), Copisces, Toledo, PR.. 451. 3. Soy protein concentrate (60% crude protein, X.SOY-200), Selecta, Goiânia, MG.. 452. 4. Qualimax, Liotécnica, Embu, SP.. 453. 5. Composition of vitamin and mineral mixture (kg/product): folic acid 997.50 mg, pantothenic. 454. acid 9975.00 mg, biotin 159.60 mg, cobalt 39.90 mg, copper 2800.00 mg, ethoxyquin 24.78. 455. g, iron 19.62 g; iodine 120.00 mg, manganese 5200.00 mg, niacin 19.95 g, selenium 119.70. 456. mg, vitamin A 1995000 UI, vitamin B1 4987.50 mg, vitamin B12 5985.00 mg, vitamin B2. 457. 4987.50 g, vitamin B6 4987.50 mg, vitamin C 70.00 g; vitamin D3 198000.05 UI; vitamin. 458. E 19950.00 UI; vitamin K 997.50 mg, zinc 28.00 g. Nutron- Cargill®, SP, Brazil.. 459. 6. Butyl hydroxy toluene (BHT).. 460. 7. Microcrystalline cellulose, Synth®, Diadema, SP, Brazil.. 461. 8. Digestible energy calculated according to ingredient analysis = [(crude protein × 5640 kcal/kg. 462. × 0.9) + (fat × 9510 kcal/kg × 0.85) + (Carbohydrates soluble in neutral detergent × 4110. 463. kcal/kg ×0.50)] (Jobling, 1983).. 464. 9. Calculated by analyzing ingredients..

(48) 46. 465 Table 3 Productive performance and somatic index of silver catfish fed with increasing levels of replace fish meal (FM) protein for linseed protein 466 concentrate (LPC) in the diets Variables2. ------------------- Levels of LPC (%)1-----------------------. ANOVA. 0. 10. 20. 30. 40. p-value. 43.69 ± 7.54. 35.87 ± 5.59. 42.83 ± 12.03. 41.90 ± 7.78. 38.91 ± 13.83. 0.786. 906.69 ± 250.67. 887.65 ± 153.40. 1055.16 ± 276.0. 990.42 ± 218.39. 940.94 ± 340.71. 0.886. 16.87 ± 0.74. 15.78 ± 0.62. 16.76 ± 1.32. 16.62 ± 0.87. 16.17 ± 1.55. 0.600. 712.77 ± 122.97. 585.15 ± 91.22. 698.78 ± 196.32. 683.56 ± 127.0. 634.79 ± 225.62. 0.786. SGR. 3.48 ± 0.27. 3.20 ± 0.22. 3.42 ± 0.41. 3.41 ± 0.27. 3.26 ± 0.51. 0.769. AFC. 1.26 ± 0.45. 1.06 ± 0.07. 1.08 ± 0.07. 1.04 ± 0.13. 1.13 ± 0.17. 0.647. Weight gain (g) Total biomass (g) Length (cm) RWG (%). Somatic index DSI. 3.06 ± 0.42. 2.99 ± 0.36. 3.26 ± 0.39. 3.46 ± 0.46. 3.27 ± 0.35. 0.090. IQ. 1.12 ± 0.15. 1.21 ± 0.18. 1.06 ± 0.15. 1.21 ± 0.19. 1.15 ± 0.22. 0.224. 467 Means ± standard deviation (n=4) compared by Tukey’s test at 5% level of significance. 1 Treatments: 0, 10, 20, 30 or 40% of replacement of fish 468 meal (FM) protein for linseed protein concentrate (LPC). 2 RWG: Relative weight gain; SGR: Specific growth rate; AFC: Apparent feed conversion; 469 DSI: Digestive somatic index; and IQ: intestinal quotient..

(49) 47. 470. Table 4 Activity of digestive enzymes of silver catfish fed with increasing levels of replace fish. 471. meal (FM) protein for linseed protein concentrate (LPC) in the diets1 Digestive enzymes2 Levels of LPC (%)1. ----Stomach ----. ---------------- Intestine----------------. Acid Protease. Trypsin. Chymotrypsin. 0. 45.69 ± 20.57. 8.77 ± 2.84. 7582 ± 1861.0 b. 10. 45.46 ± 19.14. 8.63 ± 1.73. 8927 ± 1008.70 ab. 20. 60.98 ± 21.02. 9.01 ± 2.16. 8797 ± 2059.70 ab. 30. 32.58 ± 23.55. 9.93 ± 3.44. 10448 ± 2928.45 ª. 40. 45.87 ± 24.73. 10.22 ± 3.30. 10374 ± 3056.54 ª. 0.062. 0.551. 0.031. ANOVA p-value 472. Means ± standard deviation compared by Tukey test at 5% level of significance (n = 12).. 473. 1. 474. concentrate (LPC). 2Acid Protease = μg tyrosine hydrolyzed/min/mg protein; Trypsin = µmol. 475. TAME hydrolyzed/min/mg protein; Chymotrypsin = µmol BTEE hydrolyzed/min/mg protein.. Treatments: 0, 10, 20, 30 or 40% of replacement of fish meal (FM) protein for linseed protein.

(50) 48. 476. Figure 1 Amino acid composition (%) of fish meal and linseed protein concentrate 7 6 LPC. Fish meal. %. 5 4 3 2 1 0. Essential amino acid. 477.

(51) 49. 3 ARTIGO 2. 1. Nutritional assessment of linseed meal (Linum usitatissimum L.) protein concentrate in feed. 2. of silver catfish*. 3 4 5 6 7 8. D. Pianessoa**, T.J. Adoriana, P.I. Mombacha, M.O. Dalcina, L. Loebensb, Y.B. Tellesa, S.S.. 9. Roballoa, N.M. Lovattoa, L.P. Silvaa. 10 a. 11. Department of Animal Science, Federal University of Santa Maria, Av. Roraima nº 1000, Cidade Universitária, Bairro Camobi, Santa Maria – RS, Brazil. CEP: 97105-900.. 12 13 14. b. 15. Westphalen, Linha 7 de Setembro, BR 386, s/n, Zona Rural, Frederico Westphalen – RS,. 16. Brazil. CEP: 98400000.. Federal Institute of Education, Science and Technology Farroupilha, Campus Frederico. 17 18 19. ** Corresponding author. Tel: 55 (55) 3220-8365; Fax: 55 (55) 3220-8240; EM:. 20. pianessodirleise@gmail.com. *. Artigo submetido à revista Animal Feed Science and Technology.

(52) 50. 21. Abstract. 22. Linseed protein concentrate (LPC) was produced in the laboratory and subsequently a. 23. feed assay was performed to evaluate the replace of increasing levels of fish meal protein for. 24. LPC on growth, nutrient utilization, metabolic responses and goblet cells of silver catfish. 25. (Rhamdia quelen). Five isoproteic and isocaloric diets were formulated with 0, 100, 200, 300. 26. or 400 g/kg replace of fish meal protein for LPC. Each diet was randomly distributed to. 27. quadruplicate groups of 25 fish (initial average weight of 6.13 g) per tank, totaling 20 tanks.. 28. The crude protein of LPC was lower (P<0.05) than that of fish meal. However, its in vitro. 29. digestibility was higher (P<0.05). Fish fed with LPC presented the same growth and nutrient. 30. utilization (P>0.05) than animals submitted to the 0 g/kg LPC diet. Diets of 300 and 400 g/kg. 31. LPC replacing fish meal protein provided higher (P<0.05) free amino acid content in plasma.. 32. Hepatic protein was higher (P<0.05) in the 300 g/kg LPC treatment, differing from the 0 g/kg. 33. LPC diet. Hepatic ammonia was higher (P<0.05) in fish submitted to 0 g/kg LPC diet, differing. 34. from 200 and 300 g/kg treatments. Fish fed with 300 g/kg LPC presented more (P<0.05) goblet. 35. cells, differing from the 0 g/kg LPC group. LPC presents equivalent nutritional quality and can. 36. replace fish meal protein by up to 400 g/kg without causing metabolic and histological injuries. 37. that affect growth and nutrient utilization.. 38 39. Keywords: replace fish meal, vegetable protein, goblet cells, metabolism, Rhamdia quelen. 40 41. Abbreviations: FW, final weight; BPD, body protein deposition; BFD, body fat deposition;. 42. PRC, protein retention coeficiente; Free AA, free amino acids; ALT, alanine aminotransferase;. 43. ALF, alkaline phosphatase; HSI, Hepatosomatic index.. 44 45.