Mateus G. Godoy

Production of an acidic and thermostable lipase of the mesophilic fungus Penicillium simplicissimum by solid-state fermentation.

The production of a lipase by a wild-type Brazilian strain of Penicillium simplicissimum in solid-state fermentation of babassu cake, an abundant residue of the oil industry, was studied. The enzyme production reached about 90 U/g in 72 h, with a specific activity of 4.5 U/mg of total proteins. The crude lipase showed high activities at 35-60 degrees C and pH 4.0-6.0, with a maximum activity at 50 degrees C and pH 4.0-5.0. Enzyme stability was enhanced at pH 5.0 and 6.0, with a maximum half-life of 5.02 h at 50 degrees C and pH 5.0. Thus, this lipase shows a thermophilic and thermostable behavior, what is not common among lipases from mesophilic filamentous fungi. The crude enzyme catalysed the hydrolysis of triglycerides and p-nitrophenyl esters (C4:0-C18:0), preferably acting on substrates with medium-chain fatty acids. This non-purified lipase in addition to interesting properties showed a reduced production cost making feasible its applicability in many fields.

Lipase production and Penicillium simplicissimum morphology in solid‐state and submerged fermentations

A comparative study of Penicillium simplicissimum morphology and lipase production was performed using solid-state (SSF) and submerged (SmF) fermentation. SSF was carried out on babassu cake as culture medium and SmF on a semi-synthetic medium and a medium based on suspended babassu cake grains. Yield of product on biomass, specific activity and conidia production were 3.3-, 1.3- and 2-fold higher in SSF. In SmF, the type of fungus growth differed according to the medium. Using the semi-synthetic medium, the fungus formed densely interwoven mycelial masses without conidia production, whereas using the babassu-based medium the fungus formed free mycelia and adhered to the surfaces of the grains, producing conidia. The results show that babassu cake induces conidiation in SmF. In SSF, the fungus not only grew on the surface of the grains, producing conidia abundantly, but also effectively colonized and penetrated the babassu particles. The high conidia production and lipase productivity in SSF may be related to the low availability of nutrients or to other stimuli associated with this type of fermentation. Thus, the high production of the thermostable P. simplicissimum lipase, using a non-supplemented, low-cost agro-industrial residue as the culture medium, demonstrates the biotechnological potential of SSF for the production of industrial enzymes.

Simultaneous allergen inactivation and detoxification of castor bean cake by treatment with calcium compounds

Ricinus communis L. is of great economic importance due to the oil extracted from its seeds. Castor oil has been used for pharmaceutical and industrial applications, as a lubricant or coating agent, as a component of plastic products, as a fungicide or in the synthesis of biodiesel fuels. After oil extraction, a castor cake with a large amount of protein is obtained. However, this by-product cannot be used as animal feed due to the presence of toxic (ricin) and allergenic (2S albumin) proteins. Here, we propose two processes for detoxification and allergen inactivation of the castor cake. In addition, we establish a biological test to detect ricin and validate these detoxification processes. In this test, Vero cells were treated with ricin, and cell death was assessed by cell counting and measurement of lactate dehydrogenase activity. The limit of detection of the Vero cell assay was 10 ng/mL using a concentration of 1.6 × 105 cells/well. Solid-state fermentation (SSF) and treatment with calcium compounds were used as cake detoxification processes. For SSF, Aspergillus niger was grown using a castor cake as a substrate, and this cake was analyzed after 24, 48, 72, and 96 h of SSF. Ricin was eliminated after 24 h of SSF treatment. The cake was treated with 4 or 8% Ca(OH)2 or CaO, and both the toxicity and the allergenic properties were entirely abolished. A by-product free of toxicity and allergens was obtained.

Impact of extraction parameters on the recovery of lipolytic activity from fermented babassu cake.

Enzyme extraction from solid matrix is as important step in solid-state fermentation to obtain soluble enzymes for further immobilization and application in biocatalysis. A method for the recovery of a pool of lipases from Penicillium simplicissimum produced by solid-state fermentation was developed. For lipase recovery different extraction solution was used and phosphate buffer containing Tween 80 and NaCl showed the best results, yielding lipase activity of 85.7 U/g and 65.7 U/g, respectively. The parameters with great impacts on enzyme extraction detected by the Plackett-Burman analysis were studied by Central Composite Rotatable experimental designs where a quadratic model was built showing maximum predicted lipase activity (160 U/g) at 25°C, Tween 80 0.5% (w/v), pH 8.0 and extraction solution 7 mL/g, maintaining constant buffer molarity of 0.1 M and 200 rpm. After the optimization process a 2.5 fold increase in lipase activity in the crude extract was obtained, comparing the intial value (64 U/g) with the experimental design (160 U/g), thus improving the overall productivity of the process.

Chapter 12 – Agricultural Residues as Animal Feed: Protein Enrichment and Detoxification Using Solid-State Fermentation

Abstract Feed is one of the main costs in animal breeding and several studies have been carried out aimed at reducing these costs. An economically interesting alternative is the use of agro-industrial residues in the final formulation of the animal feed. However, many of these residues have properties that may hinder or make it unfeasible for this purpose, such as the presence of toxic or antinutritional compounds or inadequate levels of essential amino acids. In this context, solid-state fermentation (SSF) presents itself as a promising alternative in the use of these residues as a culture medium making its use feasible in animal feed. This chapter addresses the removal of toxic and antinutritional compounds and protein enrichment of residues through SSF. In addition, the utilization of agro-industrial residues for the production of enzymes by SSF are discussed; for example, xylanases, cellulases, amylases, tannases, and phytases. These enzymes can be used in animal feed in order to improve the bioavailability of certain nutrients.

Solid-state fermentation of co-products from palm oil processing: Production of lipase and xylanase and effects on chemical composition

Abstract The palm oil industry generates large amounts of lignocellulosic co-products. Palm kernel cake (PKC) and palm pressed fibre (PPF) have nutritional limitations as ingredients in animal feed, and are therefore little used. Solid-state fermentation (SSF) is one alternative treatment to improve the nutritional value of these co-products and to increase their possible use, through the production of enzymes such as lipases and xylanases. These enzymes can reduce the contents of undesirable compounds, such as lipids, and degrade some components of the fibres to improve the digestibility of these co-products. The fungi Aspergillus niger, Aspergillus oryzae and Aspergillus awamori were able to grow in PKC/PPF (40/60 w/w) culture medium by SSF, and to produce xylanase and lipase. A. niger showed the highest lipase activity (20.7 U g−1) at 72 h. A. awamori higher xylanase activity than the other fungi at all culture periods, reaching a maximum activity of 134.2 U g−1 at 72 h. The unfermented co-products contained 7.49% lipids and 7.38% non-fibrous carbohydrates (NFC). Lipase produced by these fungi during SSF reduced the lipid content by 36%, 40% and 45% for A. oryzae, A. awamori and A. niger, respectively. The production of xylanases by SSF probably increased the NFC contents by up to 64%. Fermented solids with A. oryzae and A. awamori had the highest levels of NFC, 20.3% and 13.94%, respectively, which improved the nutritional value of these co-products.

Fermentação em Estado Sólido da torta de mamona (Ricinus communis) para obtenção de ração animal

Introducao: A torta de mamona, produzida a partir da extracao do oleo das sementes, e um coproduto rico em nutrientes, porem possui compostos toxicos e antinutriconais, como a ricina e acido fitico respectivamente. A destoxificacao pode ser realizada por um processo biologico denominado Fermentacao em Estado Solido (FES), o qual utiliza um substrato como nutriente e suporte para o crescimento de microrganismos, como o fungo filamentoso Penicillium simplicissimum. Essa torta pode ser valorada e utilizada como um componente de racao animal. Para tal aplicacao, torna-se imprescindivel analisar a composicao quimica-nutricional da torta bem como verificar a presenca de compostos antinutricionais. Metodos: A determinacao de proteinas das amostras foi obtida pelo metodo Kjeldahl, utilizando acido sulfurico concentrado e a extracao da fracao lipidica foi realizada em aparelho de Soxhlet, com eter de petroleo como solvente de extracao. As analises de Fibras em Detergente Neutro (FDN) e Fibras em Detergente Acido (FDA) foram quantificadas pelo metodo de Van Soest. O teor acido fitico, um antinutriente, quelante de minerais, foi determinado por cromatografia de troca anionica. Resultados: Os altos teores proteicos foram observados em 24h de fermentacao (49%). Os lipidios foram avaliados e a torta in natura (23,1g de lipideos/100g de torta) obteve uma reducao de 98% ao final da fermentacao em 96h. Os resultados obtidos de FDN para as amostras fermentadas e in natura se mantiveram em media em torno de 64%, enquanto que para FDA em media 52,2%. Os valores obtidos do teor de acido fitico ficaram abaixo de 5,8g/100g, para todas as amostras analisadas. Conclusoes: Os resultados obtidos apontam para um possivel uso da torta como componente de racao para ruminantes, devido a altos valores proteicos, baixo teor de acido fitico e tambem pela reducao significativa de lipidios apos a fermentacao pois apesar de serem fonte de energia sao passiveis de rancificacao.