Produção de L-Lisina por processos fermentativos e desenvolvimento de produto para nutrição animal

Orientador : Prof. Dr. Carlos Ricardo SoccolTese (doutorado) - Universidade Federal do Paraná, Setor de Tecnologia, Programa de Pós-Graduação em Engenharia de Bioprocessos e Biotecnologia. Defesa: Curitiba, 13/08/2014Inclui referênciasÁrea de concentração: Biotecnologia agroalimentarResumo: A economia de mercado dos tempos atuais exige altas produções a baixos custos, e normalmente a relação custo-benefício determina a viabilidade ou a inviabilidade de um negócio. Para a agroindústria não é diferente. Quando se busca a criação de bovinos, suínos, aves, ovinos, dentre outros para o abate, em geral o objetivo é engordar os animais em um curto espaço de tempo e com a maior produção de proteína possível. Há mais de 50 anos o homem domina a técnica de produção de aminoácidos a partir de fermentações, e produtos a base de aminoácidos essenciais e limitantes ao crescimento são agregados às rações para acelerar o crescimento e ganho de peso dos animais. A L-Lisina é um dos 3 aminoácidos essenciais mais importantes para as fases iniciais do crescimento, e seu mercado, no Brasil é dominado por multinacionais. Os produtos mais recorrentes são vendidos em uma forma purificada, normalmente em forma de pó ou de cristais. O objetivo principal deste trabalho foi o de desenvolver um processo viável técnica e economicamente, tendo como produto final uma farinha rica em L-Lisina, e cujo suporte seja um resíduo, subproduto ou coproduto da agroindústria nacional. Inicialmente cepas de Corynebacterium glutamicum foram reativadas e selecionadas, de acordo com seu potencial de produção de L-Lisina. A cepa ATCC 21799 apresentou os melhores resultados, produzindo inicialmente 1,7g/l de L-Lisina, quando cultivada em meio a base de caldo nutriente, em shaker, a 120 rpm, temperatura de 30 oC e pH inicial de 7,0. Após otimização da composição do meio de cultivo, e com fontes mais baratas de nitrogênio e carbono (sulfato de amônio como fonte inorgânica de nitrogênio e melaço de cana como fonte de carbono), a produção atingiu 7,8 g/l. Após geração e seleção de mutantes pelo método clássico da luz ultravioleta, a produção atingiu 9,3g/l. Na etapa seguinte, o caldo fermentado rico em L-Lisina foi impregnado em matrizes sólidas secas, moídas e classificadas (farelo de trigo, casca de soja, bagaço de cana, polpa cítrica e resíduo de malte cervejeiro) e então submetido a ciclos de secagem e reimpregnações. As farinhas resultantes apresentaram conteúdos de até 13,% de L-Lisina em massa seca, e uma análise econômica preliminar mostrou que tais produtos possuem potencial de mercado, desde que os rendimentos da etapa fermentativa sejam maximizados. Neste caso, as farinhas desenvolvidas teriam custo variando entre R$0,24 e R$ 0,33 para alimentar um suíno por dia, enquanto que os produtos atualmente comercializados teriam um custo de R$0,45.Abstract: The nowadays market economy requires high productions at low costs, and usually the cost-benefit ratio determines the viability or non-viability of a business. For agribusiness is no different. When cattle, pigs, poultry, sheep, among others are raised for slaughter, the general goal is to fatten the animals in a short time and with the greatest possible protein production. For over 50 years, human mastered the technique of production of amino acids from fermentation, and products based on essential and limiting amino acids for growth are aggregated to feed to accelerate growth and weight gain of the animals. L-Lysine is one of the three most important amino acids essential for the early stages of growth, and its market in Brazil is dominated by multinationals. The most frequent products are sold in a purified form, normally in the form of powder or crystals. The main objective of this work was to develop a technically and economically viable process, whose final product is a flour rich in L-Lysine, and whose support is a waste, a byproduct or a coproduct of the national agribusiness. Initially, strains of Corynebacterium glutamicum were reactivated and selected according to their potential for production of L-Lysine. The ATCC 21799 strain showed the best results, initially producing 1.7 g/l of L-Lysine, when growth in a medium based in nutrient broth in a shaker at 120 rpm, 30 oC and initial pH of 7.0. After optimization of the composition of the medium, and with cheaper sources of carbon and nitrogen (ammonium sulphate as inorganic nitrogen source and cane molasses as carbon source), production reached 7.8 g/l. After generation and selection of mutants by the classical method of ultraviolet light, production reached 9,3g/l. In the next stage, the fermented broth rich in L-Lysine was impregnated in dried, milled and classified solid matrices (wheat bran, soybean hulls, sugar cane bagasse, citrus pulp and brewer spent grain) and then subjected to cycles of drying and reimpregnation. The resulting flours presented contents up to 13$ 0.24 and R$0.33 to feed one pig per day, while the currently marketed products have a cost of R$ 0.45

Algumas aplicações da Inteligência Artificial em Biotecnologia

The present work is a revision about neural networks. Initially presents a little introduction to neural networks, fuzzy logic, a brief history, and the applications of Neural Networks on Biotechnology. The chosen sub-areas of the applications of Neural Networks on Biotechnology are, Solid-State Fermentation Optimization, DNA Sequencing, Molecular Sequencing Analysis, Quantitative Structure-Activity Relationship, Soft Sensing, Spectra Interpretation, Data Mining, each one use a special kind of neural network like feedforward, recurrent, siamese, art, among others. Applications of the Neural-Networks in spectra interpretation and Quantitative Structure-activity relationships, is a direct application to Chemistry and consequently also to Biochemistry and Biotechnology. Soft Sensing is a special example for applications on Biotechnology. It is a method to measure variables that normally can’t be directly measure. Solid state fermentation was optimized and presenting, as result, a strong increasing of production efficiency.O presente trabalho é uma revisão sobre redes neurais. Inicialmente apresenta uma breve introdução a redes neurais, lógica difusa, um breve histórico, e aplicações de Redes Neurais em Biotecnologia. As subáreas escolhidas para aplicação das redes neurais são, Otimização da Fermentação no Estado-Sólido, Sequenciamento de DNA, Análise Molecular Sequencial, Relação Quantitativa Strutura-Atividade, Sensores inteligentes, Interpretação de espectros, Mineração de Dados, sendo que cada um usa um tipo especial de rede neural, tais como feed forward, recorrente, siamesa, art, entre outros. Aplicações de Redes Neurais em interpretação de espectros e Relação Quantitativa Estrutura-Atividade, como uma aplicação direta à química e consequentemente também para a Bioquímica e Biotecnologia. Os sensores Inteligentes são um exemplo especial de aplicação em Biotecnologia. É um método de medir variáveis que normalmente não podem ser medidas de forma direta. Fermentações no Estado-sólido foram otimizadas e, apresentaram como resultado um forte aumento do rendimento na produção final

Development of an L-Lysine Enriched Bran for Animal Nutrition via Submerged Fermentation by Corynebacterium glutamicum using Agroindustrial Substrates

L-Lysine is an essential aminoacid added as supplement for animal feed. The aim of this work was to produce an L-Lysine enriched bran using Brazilian agroindustrial byproducts. Both the raw material costs and purification steps were minimized. Firstly, medium composition for the growth of Corynebacterium glutamicum ATCC 21799 was optimized targeting enhanced L-Lysine production - salt, vitamins and nitrogen sources concentrations were tested and selected. Next, UV mutant strains were generated and the best producers were used in formulated media using sugarcane molasses. It was reached a production of 9.3 g/L of L-Lysine with the optimized formulated media. This L-Lyisne rich broth was then impregnated and cyclically reimpregnated in pre-treated solid matrixes (sugarcane bagasse, citrus pulp, brewer spent grain, soybean husk and wheat bran). After processing, it was generated enriched brans with significant amounts of L-Lysine (13.8%, 7.0%, 8.9%, 5.9% and 8.4%, respectively), which has an interesting market potential for animal feed

Statistical Optimization of Laccase Production and Delignification of Sugarcane Bagasse by Pleurotus ostreatus in Solid-State Fermentation

Laccases are oxidative enzymes related to the degradation of phenolic compounds, including lignin units, with concomitant reduction of oxygen to water. Delignification is a necessary pretreatment step in the process of converting plant biomass into fermentable sugars. The objective of this work was to optimize the production of laccases and to evaluate the delignification of sugarcane bagasse by Pleurotus ostreatus in solid-state fermentation. Among eight variables (pH, water activity, temperature, and concentrations of CuSO4, (NH4)2SO4, KH2PO4, asparagine, and yeast extract), copper sulfate and ammonium sulfate concentrations were demonstrated to significantly influence laccase production. The replacement of ammonium sulfate by yeast extract and the addition of ferulic acid as inducer provided increases of 5.7- and 2.0-fold, respectively, in laccase activity. Optimization of laccase production as a function of yeast extract, copper sulfate, and ferulic acid concentrations was performed by response surface methodology and optimal concentrations were 6.4 g/L, 172.6 μM, and 1.86 mM, respectively. Experimentally, the maximum laccase activity of 151.6 U/g was produced at the 5th day of solid-state fermentation. Lignin content in sugarcane bagasse was reduced from 31.89% to 26.36% after 5 days and to 20.79% after 15 days by the biological treatment of solid-state fermentation

Statistical Optimization of Laccase Production and Delignification of Sugarcane Bagasse by Pleurotus ostreatus in Solid-State Fermentation

Laccases are oxidative enzymes related to the degradation of phenolic compounds, including lignin units, with concomitant reduction of oxygen to water. Delignification is a necessary pretreatment step in the process of converting plant biomass into fermentable sugars. The objective of this work was to optimize the production of laccases and to evaluate the delignification of sugarcane bagasse by Pleurotus ostreatus in solid-state fermentation. Among eight variables (pH, water activity, temperature, and concentrations of CuSO 4 , (NH 4 ) 2 SO 4 , KH 2 PO 4 , asparagine, and yeast extract), copper sulfate and ammonium sulfate concentrations were demonstrated to significantly influence laccase production. The replacement of ammonium sulfate by yeast extract and the addition of ferulic acid as inducer provided increases of 5.7-and 2.0-fold, respectively, in laccase activity. Optimization of laccase production as a function of yeast extract, copper sulfate, and ferulic acid concentrations was performed by response surface methodology and optimal concentrations were 6.4 g/L, 172.6 M, and 1.86 mM, respectively. Experimentally, the maximum laccase activity of 151.6 U/g was produced at the 5th day of solid-state fermentation. Lignin content in sugarcane bagasse was reduced from 31.89% to 26.36% after 5 days and to 20.79% after 15 days by the biological treatment of solid-state fermentation