BRPI1103789B1 - culture media for residual castor cake for simultaneous production of phytase and tanase enzymes, and detoxification of castor cake, by the microorganism paecilomyces variotii through solid fermentation, obtained enzymes and their uses - Google Patents

Abstract

RESIDUAL MAMMON PIE CULTURE MEDIA FOR SIMULTANEOUS PRODUCTION OF PHYTASE AND TANASE ENZYMES, AND DETOXIFICATION OF MAMMON PIE, BY THE MICROORGANISM PAECILOMYCES VARIOTII THROUGH SOLID FERMENTATION, EFFECTS. The present invention aims at the simultaneous production of tannase and phytase in residual castor cake in solid state fermentation with Paecilomyces variotii, in addition to decreasing the concentration of ricin after fermentation, thus promoting its detoxification. The use of enzymes in animal feed is known and is being well explored. The biggest difficulty in expanding the use of enzymes is still the cost of production. The use of residues for animal feed represents a viable alternative for this sector, as well as for the production of biocatalysts through solid fermentation. This invention determined optimal values in the constitution of the culture medium that uses residual castor cake for the production of phytase and tanase enzymes through solid fermentation by the microorganism Paecilomyces variotii in order to obtain the largest amount of enzyme produced and its greatest activities. Additionally, the present invention allows the detoxification of the residual castor cake, due to the degradation of ricin, its greatest toxic agent, and, thus, making it (...).

Description

FIELD OF THE INVENTION

The present invention aimed at determining parameters for production; simultaneous use of phytase and tanase enzymes through solid fermentation in residual castor cake using the fungus Paecilomyces variotii.

The use of enzymes in animal feed is known and is being well explored. The biggest difficulty in expanding the use of enzymes is still the cost of production. An alternative for this would be the use of these residues as a fermentation substrate for the production of enzymes.

Castor bean is an oilseed widely used for the production of biodiesel. The residual cake from oil extraction is very useful for fertilization and also rich in proteins, opening the possibility of its use as animal feed. This second application faces the problem of the presence of ricin, a toxic compound present in the cake, with the need to detoxify it before destiny as a feed, a fact observed through the use of the present invention.

Thus, the use of waste for animal feed represents a viable alternative for this sector, as well as for the production of biocatalysts through solid fermentation.

BACKGROUND OF THE INVENTION ENZYME PRODUCTION BY FERMENTATION IN SOLID STATE

Solid state fermentation (FES) provides the cultivation of microorganisms on solid substrates in the absence of a free aqueous phase (Pandey, A. Solid-state fermentation. Biochemistry Engeenering Journal 13, p. 81-84, 2003). However, the substrate must have adequate moisture to maintain the growth and metabolism of the microorganism, without exceeding the maximum water retention capacity by the matrix (Foong, CW; Janaun, J .; Krishnaiah, K .; Prabhakar, A. Effect of superficial air velocity on solid state fermentation of palm kernel cake in a lab scale fermenter using locally isolated strain (Industrial Crops and Products 30, p. 114-118, 2009).

The solid matrix used in the process can be both the source of nutrient and simply a support impregnated with nutrients suitable for the development of the microorganism (Pandey, 2003), (Nagao, N .; Matsuyama, T .; Yamamoto, H .; Toda, T A novel hybrid system of solid state and submerged fermentation with recycle for organic solid waste treatment (ProcessBiochemistry 39, p. 37-43, 2003). There are significant differences between the solid state and submerged fermentation (FS) processes. The main difference between FES and FS is the amount of free water in the culture medium. In FS, the amount of solids does not exceed 50 g / L, while in FES the content of sodium ranges from 20 to 70% of the total weight of the medium (Mitchell, DA; Losane, BK Definition, characteristic and potential. In: Doelie, H .; Mitchell, DA, Rols, CE Solid substrate cultivation (Elsevier Applied Science, p. 1-16,1992).

FES provides advantages over FS such as the use of simple and non-water-soluble culture media, composed of materials of plant origin, such as bran and husks of rice, wheat, corn and other cereals, requiring few additional nutrients in the medium. In addition, the cost of the fermentation medium can represent up to 30% of the total enzyme production. Thus, there is interest in the use of agro-industrial waste as a substrate, representing, in countries like Brazil, abundant and low-cost raw material (Gruncie, EBN; Gonçalves, AZ L; Pirota, RDPB; Balsalobre, MAA; Da Silva, RE Enzyme production by solidstate fermentation: application to animal nutrition Animal Feed Science and Technology 144, p. 1-22, 2008), (Pandey, 2003). Due to the low moisture content and the absence of free water in the medium, the likelihood of contamination by bacteria is reduced. The small amount of water used at FES also implies advantages, such as less reaction and higher product concentration (Nagao, 2003), (Mitchell, 1992) (Mohapatra, PK; Maity, C .; Rao, RS; Pati, BR ; Mondai, KC Tannase production by Bacillus licheniformis KBR6: Optimization of submerged culture conditions by Taguchi DOE methodology.Food Research International 42, p. 430-435, 2009; Singhania, RR; Patel, AK; Soccol, CR; Pandey, A Recent advances in solidstate fermentation (Biochemical Engineering Journal 44, p. 13-18, 2009).

Among the characteristics of FES, the low water activity of the solid culture medium influences the physiological aspects of microorganisms, such as their vegetative growth, sporulation, spore germination, enzyme production and enzymatic activity (Gruncie, 2008). FES may, in some cases, be more economically interesting in the production of enzymes. George and colleagues (George, S .; Raju, V .; Subramanian, TV; Jayaraman, K. Comparative study of protease production in solid substrate fermentation versus submerged fermentation. Bioprocess Engineering 16, p. 381-382, 1997) compared production protease between solid and submerged fermentation. The authors reported that for the same product yield, 100 mL of nutrients were used in FS and 1 g for FES.

ANIMAL FEED PRODUCTION USING AGRICULTURAL WASTE

The Brazilian economy is one of the most important economies in the world and is based mainly on agriculture. However, the large production of these agricultural materials generates large amounts of waste. In recent years there has been an increase in the attempt to make the use of these wastes more efficient, the disposal of which in the environment causes serious pollution problems (Soccol, CR; Vandenbergher, LPS Overview of applied solid-state fermentation in Brazil. Biochemical Engineering Journal 13, p 205-218, 2003).

Agro-industrial waste is widely produced by human, agricultural and industrial activity. Among the residues produced in significant quantities by the Brazilian industrial activity, we can mention: husk, straw and rice bran; wheat straw and bran; sugarcane bagasse; cassava leaf; orange pomace and castor bean pie (Schieber, A .; Stintzing, FC; Carle, R. By-products of plant food processing as a source of functional compounds - recent developments. Trends in Food Science ^ Technology 12, p. 401- 413, 2001); (Gririnha, 2008).

Most of these products have nutritional potential and thus have numerous possibilities for application, such as the production of microbial biomass for animal feed, that is, the use of these residues as substrates for the cultivation of microorganisms capable of biodegrading them to obtain nutrients for their development (Goel, G .; Puniya, AK; Aguilar, CN; Singh, K. Interaction of gut microflora with tannins in feeds. Naturwissenschaften 92, p. 497-503, 2005; Cao, L; Wang, W .; Yang , C .; Yang, Y .; Diana, J .; Yakupitiyage, A .; Luo, Z .; Li, D. Application of microbial phytase in fish feed.Enzyme and Microbial Technology 40, p. 497-507, 2007. ; Kuhad, R. G; Singh, A .; Tripathi, KK; Saxena, RK; Eriksson, KEL Microrganisms as an alternative source of protein. Nutrition Reviews 55, p. 65-75, 1997).

Generally, feeds are materials from organic sources in order to properly nourish the animal. The main characteristics of a ration are the energy availability, quantity of fibers (important for digestibility) and supplementary proteins, mainly essential amino acids (González-Martín, I .; Alvarez-Garcia, N .; Hernández-Andaluz, JL Instantaneous determination of crude proteins, fat and fiber in animal feeds using near infrared reflectancespectroscopy technology and remote reflectance fiber-optic probe (Animal Feed Science and Technology 128, p. 165-171, 2006); (Makkar, H. P. S. Review: Effects and fate of tannins in ruminant animals, adaptation to tannins, and strategies to overcome detrimental effects of feeding tannin-rich feeds. Small Ruminant Research 49, p. 241- 256, 2003).

Rations produced with agricultural by-products or residues from the food industries are frequently used, because there is a high availability of residues due to the high production of food (Bampidis, VA; Robinson, PH Citrus by-products as ruminant feeds: a review. Animal Feed Science and Technology 128, pp. 175-217, 2006). The use of this type of feed can bring some benefits to animals and, consequently, to the products obtained from them. An example of this is the higher productivity and quality of meat and milk (Vasta, V .; Nudda, A .; Cannas, A .; Lanza, M .; Priolo, A. Alternative feed resources and their effects on the quality of meat and milk from small ruminants, Animal Feed Science and Technology 147, pp. 223-246, 2008). However, these diets must be carefully formulated to ensure that antinutritional compounds, especially phytates and tannins, do not negatively interfere with the animal's health. These compounds can cause changes in the structure and composition of fatty acids in milk and also in meat (Hérvas, G .; Frutos, P .; Giráldez, FJ; Mantecón, AR; Del Pino, MCA; Effect of different doses of quebracho tannins extract on rumen fermentation in ewes (Animal Feed Science and Technology 109, pp. 65-78, 2003).

In aquaculture, some diets formulated with soybean meal are interesting as alternative sources to protein sources of fish, thus eliminating animal proteins in the formulation of the feed (Carter, CG; Hauler, RC Fish meal replacement by plant meals in extruded feeds for Atlantic salmon, Salmo saiar L Aquaculture 185, pp. 299-311, 2000). A study showed that salmon (Hippoglossus hippoglossus L) needs a minimum concentration of 35% of proteins in its food for its growth, being necessary the use of agroindustrial residues that present this concentration, without the need of supplementation of other products (Arnason , J .; Imsland, AK; Gústavsson, A .; Gunnarsson, S .; Arnarson, I .; Reynisson, H .; Jónsson, AF; Smáradóttir, H .; Thorarensen, H. Optimum feed formulation for Atlantic halibut (Hippoglossus hippoglossus L: Minimum protein content in diet for maximum growth. Aquaculture 291, p. 188-191, 2009) There are diets supplemented with fermented shrimp residues, which showed a greater availability of essential amino acids in relation to the diet containing unfermented residue (Narayan , B .; Velappan, SP; Zituji, SP; Manjabhatta, SN Yield and chemical composition of fractions from fermented shrimp biowaste. L / Vαste Management & Research 28, p. 64-70, 2009).

MAMMON PRODUCTION

Castor bean, Ricinus communis, is grown in tropical regions to use its oil, which is present in the seed, which is used extensively for industrial and medicinal purposes (Anandan, S .; Kumar, GKA; Ghosh, J .; Ramachandra, KS Effect of different physical and chemical treatments on detoxification of ricin in castor cake (Animal Feed Science and Technology 120, p. 159-168, 2005); (Jones, D. B. Proteins of the beaver bean-their preparation ,, properties, and utilization. The Journal of The American Oil Chemists' Society, July, p. 247-251, 1947). The main producing countries are India, with 60% of world production and China, with 20%. In 2002, Brazil produced 37,000 tons of oil, representing only 7.5% of world production (Savy Filho, A. Mamona: Agricultural Technology. Campinas. SP: EMOPI, 2005). But this scenario is changing with the gradual substitution of obtaining energy from petroleum sources for vegetable oil, including castor oil.

Residual castor oil cake, residue from oil extraction, represents half the weight of the seed and has an amount of 34 - 36% protein (Gowda, NKS; Pal, DT; Srinivas, RB; Bharadwaj, U .; Sridhar, M .; Satyanarayana, M. L; Prasad, CS; Ramachandra, KS; Sampath, K. Evaluation of castor (Ricinus communis) seed cake in the total mixed ration for sheep. Journal of the Science of Food and Agriculture 89, p. 216 -220, 2008); (Anandan, 2005); 20% fiber, 0.7% calcium; 0.8% phosphorus and 4% ether extract (Souza, R. M. Effect of detoxified castor meal on hematological values of swine. Master's Dissertation. Federal University of Minas Gerais, Minas Gerais, 1979). Despite this, castor bean cake cannot be used as a source of protein due to its toxicity, being generally used as an organic fertilizer (Jones, 1947). Among the toxins present are ricinin, albumin 2S and ricin.

Ricinin is a toxic alkaloid called 1,2-dihydro-4-methoxy-1-methyl-2-oxo-3-pyridinocarbonitrile (CgHgNzC ^), present in castor bean cake and of low toxicity (Beltrão, NEM; Lima, RLS Castor oil application as an energy source: Biodiesel. In: Azevedo, DMP; Beltrão, NEM (Ed.). Castor agro-business in Brazil. 23 Edition, Embrapa Technological Information, Brasília-DF, p. 395- 416,2007 ).

The allergenic complex formerly known as CB-1A (Castor Bean Allergen), currently referred to as Albumin 2S, is also present in nuts and other seeds such as Brazil nut, hazelnut, mustard and cotton (Breiteneder, H .; Radauer, C. A classification of plant food allergens.

Allergy and Clinical Immunology 113, p. 821-30, 2004). The presence of this toxin has been known for many years, however, the treatments applied to the pie with regard to detoxification are generally inefficient (Anandan, 2005).

Ricin (CAS No: 9009-86-3) is one of the most potent phytotoxins known, being classified as a type 2 ribosome inactivating protein (RIP), which are heterodimers composed of two chains joined by disulfide bridges: chain A , enzymatically active; and the B chain, a ligand of receptors, described by Stírpe and collaborators (Stírpe, F .; Batelli, MG Ribossome-inactivating proteins: progress and problems. Cellular and Molecular Life Sciences 63, p. 1850-1866, 2006), which can be viewed in Appendix 1, by Rutenber (Rutenber, E .; Robertus, JD Structure of ricin B-chain at 2.5 Â resolution. Proteins: Structure, Funcion and Genetics 10, p. 260- 269,1991).

The A chain removes an adenine residue in a "loop" region of the ribosomal RNA, with this modification, these ribosomes cannot support protein synthesis, as can be seen in Figure 1. This inactivation can be performed in the proportion of a ricin to every 2,000 ribosomes every minute, a speed at which the cell cannot keep up (Olsnes, S .; Fernandez-Puemtes, C .; Carrasco, L; Vazquez, D. Ribosome inactivation by the toxin lectins abrin and ricin. Kinetics of the enzimic activity of the A-chain toxin (European Journal of Biochemistry, v. 60, p. 281-288,1975).

Because ricin is thermosensitive, it could easily be inactivated by any cooking or autoclaving process (Anandan, 2005), but this process is not viable for application on an industrial scale due to the high cost. To solve this problem, a detoxification via fermentation could be performed (Anandan, 2005); (Godoy, M. G. Production of microbial lipase and simultaneous detoxification of agro-industrial waste. Master's Dissertation, IQ-UFRJ - RJ, 2009), fact accomplished by the present invention.

TANINOSIS TANASE

Tannins are a group of phenolic compounds with high molecular weight, soluble in water, capable of precipitating proteins and binding to metals (chelators). These compounds are complexed with cellulose, pectin and starch making them insoluble. They are classified in two groups, hydrolyzable tannins, such as elagitanines and galotanins, and condensates, also named by proanthocyanidins (Gross, GG From lignins to tannins: forty years of enzyme studies on the biosynthesis of phenolic compounds. Phytochemistry 69, p. 3018 -3031, 2008; Waghorn, 2008).

Hydrolyzable tannins (Figure 2) are joined by ester bonds between groups of gallic acid and glucose residue through esterase bonds and depsidase bonds (Mueller-Harvey, I. Analysis of hydrolysable tannins. Animal Feed Science and Technology 91, p. 3 -20, 2001). The basic unit (monomer) of these tannins are polyols, which are gallic acids esterified generally with glucose in their hydroxyl groups (galotanins or elagitanines) (Battestin, V .; Matsuda, L K .; Macedo, GA Sources and applications of tannins and tannins in food, Food and Nutrition 15, pp. 63-72 2004); (Gross, 2008).

Condensed tannins (Figure 3) are more widely distributed than hydrolysables in the plant kingdom, they are condensed due to their compact structure (Mutabaruka, R .; Hairiah, K .; Cadisch, G. Microbial degradation of hydrolysable and condensed tannin polyphenol- protein complexes in soils from different land-use histories (Soil Biology & Biochemistry 39, p. 1479-1492, 2007). They are present in large quantities in food, they can contain from 2 to 50 flavonoid units. Condensates are resistant to hydrolysis, due to the absence of ester and depsidic bonds (Battestin, 2004); (Gross, 2008).

Tannins are often distributed in different parts of plants such as seeds, flowers, bark and leaves. They occur naturally in the secondary metabolism of vegetables and have been considered the fourth most abundant constituent, after cellulose, hemicellulose and lignin (Manjit; Yadav, A .; Aggarwal, NK; Kumar, K .; Kumar, A. Tannase production by Aspergillus fumigatus MA under solid-state fermentation (World Journal of Microbiology and Biotechnology 24, p. 3023-3030, 2008). These compounds inhibit the growth of many microorganisms by complexing with proteins. These characteristics are highly anti-nutritional and prevent the use of plants rich in tannins for animal feed (Sabu, A .; Pandey, A .; Daud, MJ; Szakacs, G. Tamarind seed powder and palm kernel cake: two novel agroresidues for the production of tannase under solid state fermentation by Aspergillus nigerATCC 16620. Bioresource Technology 96, p. 1223-1228,2005).

The negative effect of tannins on animal nutrition is due to their ability to bind to macromolecules, reducing the absorption of these components. However, low levels of tannin concentration (40 g / kg dry matter) in the feed have shown an increase in nitrogen assimilation in ruminants, yielding a higher growth rate and milk production (Belmares, R .; Contreras-Esquivel, J .; Rodríguez-Herrera, R .; Coronel, AR; Aguilar, CN Microbial production of tannase :. an enzyme with potential use in food industry. Lebensmittel-Wissenschaft und-; Technologic 37, p. 857-864, p. 2004) ; (Min, BR; Barry, TN; Attwood, GT; McNabb, WC The effect of condensed tannins on the nutrition and health of ruminants fed fresh temperate forages: a review. Animal Feed Science and Technology 106, p. 3-19, 2003 ).

Tannase (EC 3.1.1.20) or tannin-acyl hydrolase (TAH) catalyzes the hydrolysis of ester linkages of gallic acid (Figure 4) in hydrolyzable tannin molecules (Kumar, R .; Sharma, J .; Singh, R. Production of tannase from Aspergillus rubber under solid-state fermentation using jamun (Syzygium cumini} leaves. Microbiological Research 162, p. 384-390, 2007); (Trevino-Cueto, B .; Luis, M .; Contreras-Esquivel, JC ; Rodríguez, R .; Aguilera, A .; Aguilar, CN Gallic acid and tannase accumulation during fungal solid state culture of a tannin-rich desert plant (Larrea tridentata Cov.). Bioresource Technology 98, p. 721-724, 2007) This enzyme is produced by some filamentous fungi, mainly of the species: Aspergillus, Penicillium, Fusarium and Trichoderma, but it can also be produced by bacteria of the genus Bacillus, Corynebacterium, Klebsíela, Streptococcus and Selenomonas.

Vegetables also produce tannins to accelerate the fruit ripening process (Aissam, H .; Errachidi, F .; Penninckx, M. J .; Merzouki, M .;

Benlemlih, M. Production of tannase by Aspergillus niger HA37 growing on tannic acid and olive Mill waste waters. World Journal of Microbiology & Biotechnology 21, p. 609-614, 2005); (Batra, A .; Saxena, R. K. Potential tannase producers from the genera Aspergillus and Penicillium. Process Biochemistry 40, p. 1553-1557, 2005); (Hamdy, H. S .; Purification and characterization of a newly isolated stable long-life tannase produced by Fusarium subglutinans - Wollenweber and Reinking. Journal of Pharmaceutical Innovation 3, p. 142-151, 2008); (Aguilar, C. N .; Gutiérrez-Sánchez, G. Review: sources, properties, applications and potential uses of tannin acyl hydrolase. Food Science and Technology International 7, p. 373-382, 2001b); (Deschamps, A. M .; Otuk, G .; Lebeault, J. M. Production of tannase and degradation of chestnut tannin by bacteria. Journal of Fermentation Technology 61, p. 55-59,1983); (Rodrigues, T. H. S .; Dantas, M. A. A .; Pinto, G. A. S .; Gonçalves, L. R. B. Tannase production by solid-state fermentation of cashew apple bagasse. Applied Biochemistry and Biotechnology 136-140, p. 675-688, 2007); (Rodríguez, H .; Rivas, B .; Gómez-Cordovés, C .; Munoz, R. Characterization of tannase activity in cell-free extracts of Lactobacillus plantarum CECT 748T. International Journal of Food Microbiology 121, p. 92-98, 2008); (Van de Lagemaat, J .; Pyle, D. L Modeling the uptake and growth kinetics of Penicillium glabrum in a tannic acid-containing solid-state fermentation for tannase production. Process Biochemistry 40, p. 1773-1782, 2005); (Purohit, J. S .; Dutta, J. R .; Nanda, R. K .; Banerjee, R. Strain improvement for tannase production from coculture of Aspergillus foetidus and Rhizopus oryzae. Bioresource Technology 97, p. 795-801, 2006); (Belmares, 2004).

Tanase has numerous applications such as: • Animal Feed: the use of enzymes in feed makes it possible to increase the assimilation of the nutrients contained in it, such as the breakdown of antinutritional factors. At the same time it reduces the costs for improving the feed, since this enzyme can be produced via fermentation (Battestin, 2004; Gruncie 2009). There are studies that used the action of tannins, produced by Paecilomyces variotii, on broom sorghum grains on antinutritional factors, in this case tannins. In these studies, a reduction in tannins, an increase in phosphorus, an improvement in digestibility and a decrease in phosphorus excretion, compared to raw sorghum (Schons, PF Detanification and defitinization of sorghum grains) were found in samples treated with tannase. tannase and phytase and biological study. Master's Dissertation - UNICAMP-FEA, 2009). • Preparation of Instant Teas: one of the most consumed drinks in the world, for its aroma, flavor and mainly medicinal effects. In the preparation of these teas, which are often in ice form, it produces an insoluble precipitate. This precipitate is the polyphenol complex, which is the main problem detected. Tannase, added to the product process, 'catalyzes the breakdown of ester bonds, which ends up cleaving insoluble compounds, decreasing their turbidity and increasing their quality, since the enzyme releases phenolic and volatile compounds (Lekha, PK; Lonsane, BK Production and application of tannin acyl hydrolase: State of the art Advances in Applied Biochemistry and Microbiology 44, p. 215-260, 1997); (Khokhar, S .; Magnusdottir, S. G. M. Total phenol, catechin, and caffeine contents of teas commonly consumed in the United Kingdom. Journal of Agricultural and Food Chemistry 50, p. 565-570,2002). • Brewing: beers contain polyphenolic compounds from malt. Thus, tanase cleaves these compounds and reduces their turbidity, making it an acceptable product for the market (Battestin, 2004). • Production of Gallic Acid: used mainly in the pharmaceutical industries, as in the synthesis of trimethoprim, antibacterial agent and sulfonamide (Aguilar, CN; Augur C .; Favela-Torres, E .; Viniegra-González, G. Production of tannase by Aspergillus niger Aa-20 in submerged and solid-state fermentation: influence of glucose and tannic acid (Journal of Industrial Microbiology & Biotechnology 26, p. 296-302, 2001a). • Production of Antioxidants: Tanase cleaves polyphenolic compounds resulting in compounds such as epigallocatechin, epicatechin and gallic acid, which are molecular structures with antioxidant capacity (Battestin, V .; Macedo, GA; De Freitas, VAP Hydrolysis of epigallocatechin gallate using tannase from Paecilomyces variotii, Food Chemistry 108, pp. 228-233, 2008).

TAH can be obtained from several sources, such as from animals (gut of ruminants), plants (leaves, fruit peel, branches) and mainly from microorganisms, since its production is more stable and abundant, compared to other sources. In addition, microorganisms can be genetically manipulated to improve the enzyme and its production (Battestin, V .; Macedo, G. A. Tannase Production by Paecilomyces variotii. Bioresource Technology 98, p. 1832-1837, 2007b); (Aguilar, CN; Rodríguez, R .; Gutiérrez-Sánchez, G .; Augur, C .; Favela-Torres, E .; Prado-Barragan, LA; Ramirez-Coronel, A .; Contreras-Esquivel, JC Microbial tannases: advances and perspectives Applied Microbiology and Biotechnology 76, p. 47-59, 2007).

One of the most studied topics on TAH refers to the chemical properties of this enzyme, but the mechanism of action and regulation is not yet fully understood. The fungal TAH enzyme is a glycoprotein with a stability pH in the range of 3.5 and 8.0; optimal pH of 5.5 and 6.0; stability temperature in the range of 30 and 60 ° C; optimum temperature between 30 and 40 ° C; isoelectric point of 4.0 and 4.5 and molecular mass between 186 and 300 kDa. These properties vary according to the type of microorganism and cultivation conditions of the strain used. TAH is inhibited by Cu2 +, Zn + 2, Fe + 2, Mn + 2 and Mg + 2, being inactivated by EDTA, 2-mercaptoethanol, sodium thioglycolate, magnesium and calcium sulfate and ofenanthroline (Aguilar, 2001a); (Aguilar, 2007); (Battestin, V .; Macedo, G. A. Purification and biochemical characterization of tannase from a newly isolated strain of Paecilomyces variotii. Food Biotechnology 21, p. 207-216, 2007a); (Belmares, 2004); (Mahapatra, K .; Nanda, RK; Bag, S. 5 .; Banerjee, R .; Pandey, A .; Szakacs, G. Purification, characterization and some studies on secondary structure of tannase from Aspergillus awamori nakazawa. Process Biochemistry 40 , pp. 3251-3254, 2005); (Sharma, S .; Agarwal, L; Saxena, R. K, Statistical optimization for tannase production from Aspergillus niger under submerged fermentation. Indian Journal of Microbiology 47, p. 132-138, 2007); (Sharma, S .; Agarwal, L; Saxena, R. K. Purification, immobilization and characterization of tannase from Penicillium variable. Bioresource Technology 99, p. 2544-2551, 2008),

FITATES AND PHYTASE

Phytic acid, / W / o-lnositol-l, 2,3,4,5,6-hexachisphosphate (Figure 5), is a cyclic alcohol derived from glucose with 6 phosphate groups attached to each carbon of the glycosidic molecule. Among the compounds grouped in phosphorylates, phytic acid is the most abundant in vegetables, mainly in seeds since it has a storage function for the phosphorus group for energy (Raboy, V. Molecules of interest: myo-lnositol-l, 2, 3,4,5,6-hexakisphosphate, Phytochemistry 64, p. 1033-1043, 2003). The presence of excess phytate pollutes the environment as well as disrupting the diet of monogastric animals. Phytate acts as an antinutrient by binding to proteins, amino acids and lipids and chelating minerals like calcium, iron, zinc and magnesium, thus forming insoluble salts (Howson, SJ; Davis, RP Production of phytase-hydrolysing enzyme by some fungi. Enzyme and Microbial Technology 5, pp. 377-343, 1983), In addition, it interacts with digestive enzymes reducing its activities, influencing digestion and impairing the use of vitamins.

Although phytate serves as the main source of energy and phosphorus for seed germination, bound phosphorus is poorly available for monogastric animals, so inorganic phosphorus, a non-renewable and expensive mineral, is added to pig diets, fish and poultry to meet their nutritional needs for phosphorus (Vats, P .; Banerjee, UC Production studies and catalytic properties of phytases (myo-inositolhaxakisphosphate phosphohydrolases): an overview. Enzyme and Microbial Technology 35, p. 3-14, 2004) . The phytate phosphorus that is not used is excreted becoming an environmental pollutant in areas of intensive farming (Lei, X. G .; Porres, J. M. Phytase enzimology, applications and biotechnology. Biotechnology Letters 25, p. 1787-1794,2003).

The enzyme phosphohydrolase acid catalyzes the hydrolysis of phosphate and phytic acid (Figure 6) to inorganic phosphate and myo-inositol phosphate derivatives. Phytases are classified as histidine acid phosphatases (Histidine Acid Phosphatases - HAPs), a subclass of phosphatases (Vats, 2004). There is another classification based on the position of the first phosphate to be hydrolyzed, named 3-phytase (EC3.1.3.8) and 6-phytase (EC3.1.3.26), of which 3-phytase (myo-inositol-hexakisphosphate-3 -phosfohydrolase) originates mainly via microbial and 6-phytase is derived from plants. To obtain microbial phytase, fungi are the most researched, among them we can mention the genera PenicilHum, Aspergillus and Mucor, Among the yeasts stands out the genus Soccharomyces (Dvoráková, J. Phytase: Sources, preparation and exploitation. Folia Microbiológica 43 , Issue 4, p.323-338, 1998); (Ries, E. F .; Macedo, G. A. Progressive screening of thermostable yeasts for phytase production. Food Science and Biotechnology 18, p. 655-660, 2009). By the fermentative process, phytase can be produced using low-cost substrates such as oilseed cake (Roopesh, K .; Ramachandran, S .; Nampoothiri, KM; Szakacs, G .; Pandey, A. Comparison of phytase production on wheat bran and oilcakes in solid-state fermentation by Mucor racemosus (Bioresource Technology 97, pp. 506-511, 2006).

Supplementation of phytase in animal feed increases the bioavailability of. phosphorus in monogastric animals, which consequently reduces pollution 'gives phosphorus to the environment. The enzyme also prevents: chelation of phytic acid with metal ions, binding of proteins, lipids and carbohydrates, thus increasing your nutrition in the diet (Vats, 2004).

Research in the phytase enzyme production area is highlighted not only by the investigation of a little explored phytase source but also by the search for phytases with desirable characteristics for the animal feed industry, mainly related to thermostability, low pH stability and cost of the fermentation process.

Hong and colleagues used cassava residues as a culture medium, by-products from the processing of cassava starch, supplemented with a nitrogen source for solid state fermentation with Aspergillus niger for the production of phytase. A maximum yield of 6.73 AU / g of dry matter was obtained. The enzyme showed residual activity of 4.71 AU / g at 75 ° C for 30 minutes, which would support processing in the feed industry (Hong, K .; Ma, Y .; Lt, M. Solid-state fermentation of phytase from cassava dregs (Applied Biochemistry and Biotechnology 91-93, p. 777-785,2001).

Pie from the extraction of coconut oil was used to produce phytase extrace I u la r via solid state fermentation with Rhizopus oligosporus. The maximum enzymatic production of 14.29 AU / g of dry substrate after 96 hours of incubation without nutrient supplementation (Sabu, A .; Sarita, S .; Pandey, A .; Bogar, B .; Szakacs, G .; Soccol , CR Solid-state fermentation for production of phytase by Rhizopus oligosporus (Applied Biochemistry and Biotechnology 102-103, p. 251-26Q, 2002).

ANIMAL FEED ENZYMES

Enzymes are currently used in numerous products and industrial processes, at the same time new areas of application are being added, due to their efficiency and economy in their actions, mainly in reducing the use of energy, to activate the reaction and the amount of water ( Kirk, O .; Borchert, TV; Fuglsang, CC Industrial enzyme applications. Current Opinion in Biotechnology 13, pp. 345-351, 2002).

The use of enzymes as additives in foods is also well known, such as, for example, the action of bromelain in meat, a protease that increases the tenderness of the product, making it more interesting for consumption. Enzymes can also increase the availability of nutrients, especially in animal feeds, such as xylanase and beta-glucanase, which are used in cereals that help the digestibility of nutrients in monogastric animals, which, unlike ruminants, are unable to fully hydrolyze the nutrients. foods of plant origin, mainly cellulose and hemicellulose (Polizeli, MLTM; Rizzatti, ACS; Monti, R .; Terenzi, HF; Jorge, JA; Amorim, DS Xylanases from fungi: properties and industrial applications. Applied Microbiological Biotechnologic 67, p. 577-591,2005).

The use of agro-industrial residues favors the formulation of feed economically, however these residues must be supplemented with enzymes that hydrolyze compounds to increase their nutritional value, such as the anti-nutritional factors already mentioned for tannins and phytates. Thus, to extract maximum nutritional value, exogenous enzymes are added to food or waste for better absorption to monogastric animals (Pariza, MW; Cook, M. Determining the safety of enzymes used in animal feed. Regulatory Toxicology and Pharmacology 5ft p. 332 -342, 2010).

In recent years, the focus has been on the use of organic phosphorus for food, stored in phytic acid, which is present in several species and structures of plants. However, this phosphorus is not available to monogastric- • V's, as they do not contain enzymes that hydrolyze the phytate and release phosphorus. Thus, supplementation of the phytase enzyme in the feed assists in animal nutrition (Lei, X. G .; Stahl, C. H. Nutritional benefits of phytase and dietary determinants of its efficacy. Journal of Applied Animal Research 17, p. 97-112, 2000); (Kies, A. K .; Van Hemert, K. H. F .; Saber, W. C. Effect of phytase on protein and amino acid digestibility and energy utilization. World's Poult Science Journal 57, p. 109-126, 2001).

There is also the supplementation of enzyme complexes in animal feed, such as the addition of carbohydrates, represented by xylanase, beta-glucanase and cellulase made in wheat-based feed, which showed an improvement in growth and an apparent increase in the digestibility of nutrients in pigs (Emiola, IA; Opapeju, FO; Slominski, BA; Nyachoti, CM Growth performance and nutrient digestibility in pigs fed wheat distillers dried grains with solublesbased diets supplemented with a multicarbohydrase enzyme. Journal of Animal Science 87, p. 2315-2322, 2009 ).

Colombatto et al. (Colombatto, D .; Beauchemin, KA A protease additive increases fermentation of alfalfa diets by mixed ruminal microorganisms in vitro. Journal of Animal Science 87, p.1097-1105, 2009) verifies the action of protease in feed to based on vegetable components, for ruminants. They concluded that this enzyme acts by removing structural proteins from the plant cell wall and consequently provides a greater amount of nutrients for digestibility in the microbiota of ruminants.

Nuero and Reyes (Nuero, OM; Reyes, F .; Enzymes for animal feeding from Penicillium chrysogenum mycelial wastes from penicillin manufacture. Letters in Applied Microbiology 34, p. 413- 416, 2002) verified multi-enzyme production for use as a feed additive animal through Penicillium chrysogenum .. The microorganism produced enzymes such as: tannase, lipase, invertase and beta-1, 3-glucanase, with enzymatic activities comparable to the commercial one and thus, allowing its application in animal feed, the present invention, by its instead uses the microorganism Paecilomyces variotii for simultaneous production of phytase enzymes and tannins in residual castor cake.

BRIEF DESCRIPTION OF THE INVENTION

For biodiesel production, castor oil stands out among oilseeds. In addition to the oil, the castor after extraction produces the pie that is of great interest for organic fertilization. The pie also has a high concentration of proteins, which brings possibilities for its use as an ingredient for animal nutrition. However, this application faces problems in the presence of toxins, especially ricin, a protein that prevents protein synthesis in the cells of animals that ingest them. Currently, several techniques are emerging to detoxify the castor bean cake and use it in animal feed. Among these techniques, solid state fermentation has shown promise in detoxifying the pie, and can simultaneously be used to produce enzymes of biotechnological interest, increasing its commercial value.

The present invention aimed at optimizing the simultaneous production of phytase and tanase enzymes using the fungus Paecilomyces variotii through solid state fermentation in residual castor cake.

According to the established fermentation kinetics, it was determined that the medium containing castor bean cake showed greater activity in 48 and 72 hours for the production of tannase and phytase, respectively. The best conditions for tanase production were: 90% relative humidity, 25% solution.

saline and 4.6% tannic acid, obtaining an enzymatic activity of 2800 U / g of substrate.

In turn, for the production of phytase enzyme, the best conditions were: 90% relative humidity of the environment and 25% saline, obtaining an enzymatic activity of 280 U / g of substrate.

During the fermentative process, the ricin concentration was lower with the use of the fungus Paecilomyces variotii. This result was confirmed by the gel electrophoresis tests and the biological test.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: "Loop" of the ribosomal RNA and Ricina's depurination site, described by 'Stirpe et al. (2006).

Figure 2: Chemical structure of the Hydrolyzable Tannin described by Battestin et al. (2004).

Figure 3; Example of the Chemical Structure of Condensed Tannin described by Battestin et al. (2004).

Figure 4: Tannic Acid Hydrolysis described by Battestin et al. (2004).

Figure5: Chemical structure of Phytic Acid described by Raboy and collaborators (2003).

Figure 6; Hydrolysis of Phytic Acid described by Dvoráková and collaborators (1998).

Figure 7: Minimum concentration required to inhibit cell growth, considering the percentage of live cells in relation to the amount of proteins in the castor bean pie extract.

Figure 8: Percentage of cell growth in fresh pie and at each fermentation time (24.48 and 72 hours).

BRIEF DESCRIPTION OF ANNEXES

Annex 1: Structure of Ricina described by Rutenber and collaborators (1991).

Annex 2: Response Surface and Contour Curve for Phytase Activity (U / mL)

Annex 3: Response Surface and Contour Curve Tanase Activity (U / mL): (a) as a function of the volume of saline and relative humidity, (b) as a function of relative humidity and tannic acid and ( c) as a function of tannic acid and volume of saline solution.

Annex 4: SDS-PAGE 12% of the protein extract of each sample tested. 1 M = Marker; R = purified ricin; IN = Castor Pie in natura; Au = Autoclaved Castor Pie; 24 = Fermented Castor Pie for 24 hours; 48 = Fermented Castor Pie for 48 hours; 72 = Fermented Castor Pie for 72 hours. 2 * Molecular Mass of each Marker chain.

DETAILED DESCRIPTION OF THE INVENTION WASTE CHARACTERIZATION

Prior to the use of residual castor cake as a culture medium for the production of the enzymes tanase and phytase, this residue must be characterized for certification as to its characteristics, to obtain optimal amounts of the enzymes, from the smallest possible amount of culture medium. .

For this characterization, first the castor bean cake must be crushed and subjected to a process of granulometric separation in 10 mesh sieves, with 1.68 mm.

A) Determination of pH:

5 ml of deionized water should be added to 0.5 g of sample, the mixture was vigorously stirred. After 10 minutes, the pH of the supernatant is measured in a pot.

For its use as a culture medium for the production of enzymes, the castor bean cake must have a pH of 5.95, with a variation of 0.04 more or less.

B) Percentage of Water (Karl-Fischer):

To determine the amount of water present In the residual castor bean cake, it is suggested to use methodology for samples with low amount of free water, such as cereals, fruit peel, vegetable residues and grains, such as that developed by Laitinen (Laitnen, HA Boyer, KW Automobile exhaust particulates properties of environmental significance (Environmental Science and Technology 279, pp. 457-1086, 1975).

This methodology is based on the oxidation of SO2 by 12, which constitute Karl Fischer's reagent, in the presence of water, as illustrated in the following chemical equation. I2 + SO2 + H2O -> 2HI + H2SO4

This methodology was performed by volumetric titration that directly provides the percentage of water in the sample, being necessary to supply the sample mass added in the titration flask.

The amount of water in the sample should be approximately 4.78%, with a variation of 0.07% more or less.

C) Humidity:

To determine the humidity of the sample, an incubation of 0.5 g for 24 hours in an oven at 105 "C should be performed, and weighing should be done at regular intervals until these samples reach constant weight.

For the castor bean cake to be used for optimal phytase and tannase production, it must have a moisture content of 6.7%, in relation to the total weight of the sample, with a variation of 0.16% more or less, as can be seen in Table 1. Table 1: pH, amount of water and relative humidity of castor cake in naturo.

FERMENTATIVE PROCESS A) Microorganism

The microorganism Paecilomyces variotii should be kept in Potato Dextrose Agar (PDA - OXOID - CM0139) with a 0.2% tannic acid supplement (Tanal B - Prozyn - BioSolutions) and incubated in an oven at 30 ° C for 72 hours.

After incubation, a cell suspension with a homogenizer should be performed, resulting in a concentration of 9 x 106 cells / mL at the end

B) Fermentation medium

In the proportion of g / ml, 1 part of the residue (residual castor bean cake) should be mixed to 1 part of the saline solution (g / L) composed of: 1.0 KH2PO4; 2.0 of NH4NO3; 0.2 MgSO4.7H2O; 0.02 CaCl2.2H2O; 0.004 MnCI2.4H2O; 0.002 Na2Mo04.2H20 and 0.0025 FeSO4.7H2O; and 10% tannic acid. Then, the container in which the mixture was made must be autoclaved at 121 ° C for 15 minutes.

After being cooled to room temperature, the spore suspension of Paecilomyces variotii is added to the culture medium Potato Dextrose Agar (PDA - OXOID - CM0139) with a supplement of 0.2% tannic acid. After inoculation, the containers should be incubated in an oven at 30 ° C for 120 hours.

C) Enzyme Production Optimization

To optimize the production of the enzymes tanase and phytase in the residual castor cake, it is necessary to maintain the correct ratio between the concentration of tannic acid and saline in the enzyme production medium.

Phytase:

It was determined that the optimal incubation period for the microorganism Pαecilomyces vαriotii for phytase production is 72 hours, suffering a small drop until 96 hours, when the production value becomes constant and falls after 120 hours, as we can check in Table 2. Table 2: Evaluation of fermentation kinetics for phytase activity in Castor Pie.

It is not necessary to add tannic acid to the culture medium with residual castor cake, as it does not interfere with the production of this enzyme. Additionally, phytic acid supplementation is not necessary, which is generally used for phytase production, resulting in a production with greater economic viability.

The percentage of the volume of the saline solution in relation to the total weight of the medium is between 25 and 28%, with the optimal concentration being 25%. Lower percentages of the solution volume inhibit the growth of the microorganism.

Thus, it was determined that the best conditions for the production of phytase, in 10 g of medium, would be: 2.5 mL of SS (%); 7.5 g of castor cake, incubated at 90% RH. Thus, an activity of 70 U / mL or 280 U / g of substrate was obtained in the fermentation by the P. variotii strain.

Tanase:

To optimize the production of the enzyme tanase, the concentration of tannic acid and the percentage of the volume of saline solution added in relation to the medium were also determined.

The optimization of fermentation kinetics, in order to obtain the largest amount of tannase in the shortest possible time, showed that for the production of this enzyme, the optimal fermentation time is 48 hours

Appendix 3 indicates that the range in which the highest enzyme activity would be obtained would be between 84 to 90% relative humidity (UR%), with an optimal moisture percentage of 90%.

Values above 90% cause an imbalance between humidity and temperature in the Climate Chamber. Air humidity conditions below 84% decrease enzyme activity.

The activity of the enzyme tanase is higher in concentrations between 4.6 to 6% of tannic acid, with optimal enzyme activity in 4.6% and higher concentrations of tannic acid decrease the tannic activity.

For the concentration of the volume of the saline solution in relation to the total weight of the medium, the range of 25 to 33% would stimulate the production of the enzyme, with an optimal concentration of 25%. Lower concentrations of the saline solution prevent the growth of the microorganism in the medium and the increase of the saline solution in the culture medium decreased the tannase activity.

* TM 48h = meio de cultivo de torta de mamona com 4,6% do peso do ácido tânico em relação ao peso total do meio (p/p) e 25% do volume da solução salina em relação ao peso total do meio (v/p).The relative humidity of the medium present before fermentation was 25% and after incubation in the Chamber there is a small increase of 2%, resulting in 27%. However, when the Chamber is used at 90% relative humidity, it is possible to maintain the humidity present in the culture medium, with no loss of water from the medium to the air, resulting in a balance of the water present in the medium with the air, as can be seen in Table 3. Table 3: Relative humidity of the culture medium before and after incubation in the climatic chamber for the medium optimized for the production of tannins in castor cake.

* TM 48h = castor bean culture medium with 4.6% of the tannic acid weight in relation to the total weight of the medium (w / w) and 25% of the volume of the saline solution in relation to the total weight of the medium (v /P).

Thus, to optimize the conditions for the production of tanase, in 10 g of medium, it would be: 3 mL of SS (%); 0.46 g of AT (%); 6.54 g of castor cake, incubated at 90% RH. Thus, an activity of 700 U / mL or 2800 U / g of substrate was obtained in the fermentation by the P. variotii strain.

EXAMPLES Example 1: FERMENTATION KINETICS

In order to define the fermentation kinetics, the enzymatic activities of tanase and phytase were determined at different times: 24, 48, 72 and 96 hours.

Thus defining the best fermentation time of the medium for the production of enzymes, this time being used in the experimental design stage.

*Médias seguidas de mesma letra não diferem entre si pelo teste de Tukey (p>0,05) ao nível de 5% de probabilidade.Using castor bean cake as a substrate, the best fermentation time for enzymatic production of tanase was after 48 hours, as shown in Table 5. Table 4: Evaluation of fermentation kinetics for tannin activity in castor bean cake.

* Means followed by the same letter do not differ by Tukey's test (p> 0.05) at the 5% probability level.

*Médias seguidas de mesma letra não diferem entre si pelo teste de Tukey (p>0,05) ao nível de 5% de probabilidade.After 48 hours of incubation, the enzymatic activity decreased to 249 U / mL after 72 hours and remained statistically constant. For phytase enzyme, the best incubation time for its production was after 72 hours, after 96 hours the activity decreased to 13.89 U / mL, as shown in Table 5. Table 5: Evaluation of fermentation kinetics for phytase activity in Castor bean pie.

* Means followed by the same letter do not differ by Tukey's test (p> 0.05) at the 5% probability level.

Example 2: EXPERIMENTAL DESIGN

In order to optimize the production of tanase and phytase enzymes in castor bean cake, the influence of tannic acid concentration and saline volume in the enzyme production medium was analyzed. The central rotational composite design (DCCR) technique was used, with 8 factorial points (mp), 6 axial points (mp) and 3 central points (mp) totaling 17 trials (23 mp + 6 bp + 3 pc = 17) . The 3 independent variables were: relative humidity (%) in a Climatic Chamber (Nova Ética-Modelo 420 / CLDTS 300); tannic acid concentration, which was expressed as a percentage of the total weight of the medium (w / w); and the volume of the saline solution, which was expressed as a percentage of the total weight of the medium (v / p). The dependent variables (responses) were enzyme activity of tannase and phytase.

The concentration of tannic acid (%) used in this study was determined according to Battestin et al. (2007b), where the maximum tanase production was obtained using 8 to 15% tannic acid. For the present work, lower concentrations of tannic acid were tested in order to reduce the costs of the production medium. ,

* AT (%) = Concentração de Ácido Tânico (p/p); volume SS {%) = volume da Solução Salina (v/p); UR (%) = Porcentagem da Umidade Relativa do Ar. Tabela 8: Matriz do delineamento contendo valores codificados das variáveis.

* xl= UR (%) = % umidade relativa do ar; x2 = volume SS (%) = % do volume da solução salina em relação ao peso total (v/p); x3 = AT (%) = % do peso do ácido tânico em relação ao peso total (p/p).The amount of salt added in the fermentation medium is described in the Detailed Description, in the item Fermentation Process. In this study, the volume of water added to the fermentation medium was evaluated. The range of water volume added was determined according to the maximum absorption capacity of the substrate, without having free water in the medium, as shown in Table 6. The actual values used in the planning tests are shown in Table 7 and the matrix of the tests in Table 8. Table 7: Values used in the DCCR for three factors.

* AT (%) = Tannic Acid Concentration (w / w); volume SS (%) = volume of saline solution (v / w); RH (%) = Percentage of Relative Air Humidity. Table 8: Design matrix containing coded values of variables.

* xl = UR (%) =% relative humidity; x2 = volume SS (%) =% of the volume of the saline solution in relation to the total weight (v / w); x3 = AT (%) =% of the tannic acid weight in relation to the total weight (w / w).

*Peso da torta de mamona postado para avaliar a capacidade de absorção de água em seu meio.According to Table 6, the maximum water capacity that the castor cake absorbed in 10 grams was 11 mL of water, that is, in a medium with castor cake and water, 55% of the medium weight was represented by water . Table 6: Water Absorption Capacity of Castor Pie.

* Weight of castor bean cake posted to assess the water absorption capacity in your environment.

With the results of the planning, it was possible in the STATISTCA 7.0 software to determine the regression coefficients for the response of interest, calculated ANOVA to analyze the possibility of constructing the response equation as a function of the statistically significant regression coefficients and also the response surface. , with a significance level of 10% (p-value <0.1).

The first design was performed considering the dependent variable enzyme activity of phytase after 72 hours of fermentation and the second design for the dependent variable enzyme activity of tanase after 48 hours of fermentation.

A) DCCR for Phytase Production

The central rotational composite design was used to assess the response or dependent variable on enzyme activity of phytase. The assays were incubated with the microorganism for 72 hours, which was the time that showed the highest phytase activity. The independent variables studied were: the relative humidity of the air, the concentration of tannic acid and the percentage of the volume of saline solution added in relation to the medium.

Table 9 shows the matrix of the independent variables under study, with real and coded values, and the dependent variable phytase during 72 hours of incubation.

xl = UR (%) = porcentagem da umidade relativa do ar; x2 = volume SS (%) = % do volume da Solução Salina em relação ao peso total (v/p); x3 = AT (%) = % do peso do ácido tânico em relação ao peso total (p/p).Analyzing the activity values obtained in Table 9, there is an increase in enzyme activity, varying from 1.97 U / mL for test 3 (66% relative humidity, 49% of the air volume) Saline solution in relation to total weight and 6% tannic acid) up to a maximum activity of 42.01 U / mL, for test 2 (84% relative air humidity, 31% of the volume of saline solution and 6% of tannic acid). Table 9: DCCR 23e matrix the response of phytase enzyme activity after 72 hours of incubation.

xl = UR (%) = percentage of relative air humidity; x2 = volume SS (%) =% of the volume of the Saline Solution in relation to the total weight (v / w); x3 = AT (%) =% by weight of tannic acid in relation to total weight (w / w).

Table 10 presents the values of the regression coefficients, t and p-value for 10 to evaluate which are the variables and their interactions, statistically significant, above 90% (p <0.10).

* parâmetros estatisticamente significativos a 90% de nível de confiança. L = parâmetro linear; Q= parâmetro quadrático.With the results of Table 10, it was found that the linear, and quadratic variables of AT (%) (tannic acid concentration) and UR (%) interaction with AT (%) and UR (%) with AT (%) did not were significant, being evaluated for Residues at Source dç Variação, presenting pvalue equal to 0.17, 0.67, 0.36 and 0.18, respectively. Thus, only the variables Mean, UR (%) (L) and (Q), SS volume (%) (L) and (Q) and UR interaction (%) and SS volume (%) were evaluated for Source Regression. Variation in ANOVA. Table 10: Results of the Regression Coefficient, Standard Error, t, p and Limit of Confidence in the optimization of the components of the culture medium (relative humidity, volume of saline and tannic acid) in phytase activity.

* statistically significant parameters at 90% confidence level. L = linear parameter; Q = quadratic parameter.

R2=0,92 Ftab(0,l;5;ll)=2,45 Ftab(0,l;9;2)=9,38The analysis of variance (ANOVA) is shown in Table 11. Table 11: Analysis of variance in the study of the effect of the components of the culture medium (UR (%), volume SS (%) and AT (%)) on phytase activity .

R2 = 0.92 Ftab (0. 1.5; ll) = 2.45 Ftab (0. 1; 9; 2) = 9.38

For the correlation coefficient (R) the value obtained was 0.96, for the determination coefficient (R2) it was 0.92, indicating a satisfactory correlation between the values obtained by the experiment and those predicted by the model. The F value of the lack of adjustment obtained from ANOVA was 91.2 (9.72 times greater than the value of Ftabelado = 9.38) and indicated a lack of adjustment of the model greater than that indicated by the tabulated value. The F value of Regression obtained was 25.8 (10.53 times greater than the value of Ftabelado = 2.45), indicating that the model for phytase activity can be considered statistically significant at 90% level of confidence.

These results can be considered satisfactory and sufficient, allowing to obtain a coded model that describes the responses according to the analyzed variables. From the validation of the study parameters, the quadratic polynomial model that represents the behavior of the enzymatic activity (1) was obtained:

Phytase (U / mL) = 5.74 + 8.14 * (UR) + 5 * (UR) 2 -8.8 * (SS volume) + 4 * (SS volume) 2 - 4.12 * (UR) * (SS volume) (1)

The quadratic polynomial model was used to construct the response surfaces and contour curves. Appendix 2 shows the effects of the components relative humidity of the air and percentage of the volume of the saline solution in relation to the total weight of the medium, in the production of tanase by Paecilomyces variotii.

B) DCCR for Tanase Production

Table 12 shows the matrix of the independent variables under study, with real, coded values and the dependent variable tanase after 48 hours of fermentation.

xl = UR (%) - umidade relativa do ar (%); x2 = volume SS (%) = % do volume da Solução Salina em relação ao peso total (v/p) ; x3 = AT (%) = % do peso do ácido tânico em relação ao peso total (p/p).Analyzing the values of tannase activity obtained. Table 12 shows an increase in activity, varying from 104 U / mL in test 12 (75% relative air humidity, 55% of the volume of saline added in relation to the total weight of the medium and 8% of tannic acid supplemented in the medium) to 573 U / mL in test 2 (84% relative air humidity, 31% saline volume and 6% tannic acid). Table 12: DCCR 23 matrix and the enzyme activity response of the tanase after 48 hours of fermentation. Encoded Values Actual Values Answer

xl = UR (%) - relative humidity (%); x2 = volume SS (%) =% of the volume of the Saline Solution in relation to the total weight (v / w); x3 = AT (%) =% by weight of tannic acid in relation to total weight (w / w).

* parâmetros estatisticamente significativos a 90% de nível de confiança. L = parâmetro linear; Q = parâmetro quadrático.Table 13 shows the regression coefficients, standard error, t, p-value and confidence limit of the variables and their interactions, in response to the activity of the tanase with a statistical confidence limit of 90% (p <0.10). Table 13: Results of the Regression Coefficient, Standard Error, t, p and Limit of

* statistically significant parameters at 90% confidence level. L = linear parameter; Q = quadratic parameter.

With the results of Table 5, the variables UR (%) quadratic (relative humidity), AT (%) quadratic (tannic acid concentration) and interaction UR (%) and volume SS (%) were not significant, presenting p -value equal to 0.48, 0.55 and 0.36, respectively. Thus, only the variables UR (%) (L), volume SS (%) (L), volume SS (%) (Q), AT (%) (L), interaction UR (%) with AT (%) and volume SS interaction (%) with TA (%) were evaluated for Regression at the Source of Variation in ANOVA.

R2=O,94 Ftab(0,l;6;10)=2,46 Ftab(0,l;8;2)=9,37The analysis of variance (ANOVA) is shown in Table 14. Table 14: Analysis of variance in the study of the effect of the components of the culture medium (UR (%), volume SS (%) and AT (%)) on the tanase activity .

R2 = O, 94 Ftab (0, 1, 6, 10) = 2.46 Ftab (0, 1, 8, 2) = 9.37

For the correlation coefficient (R) the value obtained was 0.97, for the determination coefficient (R2) it was 0.94, indicating a good correlation between the values obtained by the experiment and those predicted by the model. The F-value of the lack of adjustment obtained from ANOVA was 5.11 (1.83 times less than the value of Ftabelado = 9.37), resulting in a lack of adjustment of the model less than that indicated by the tabulated value. . The F value of the Regression obtained was 25.3 (10.2 times greater than the value of Ftabelado = 2.46), indicating that the model for the tanase activity can be considered statistically significant at 90% level of confidence.

These results can be obtained by a coded model that describes the responses according to the variables analyzed. From the validation of the study parameters, the quadratic polynomial model that represents the behavior of the enzymatic activity (2) was obtained:

Tanase (U / mL) = 288 + 58.24 * (UR) -75.21 * (SS volume) -20.93 * (SS volume) 2 - 60.77 * (AT) -36 * (UR) * (AT) + 79.25 * (SS volume) * (AT) (2)

The quadratic polynomial model was used to construct the response surfaces and contour curves. Appendix 3, (a), (b) and (c), illustrates the effects of the components relative humidity of the air, concentration of tannic acid and percentage of the volume of the saline solution in relation to the total weight of the medium, in the production of tanase by Paecilomyces variotii.

Example3: ENZYMATIC EXTRACTION AND ANALYTICAL DETERMINATIONS

After fermentation, the enzyme was extracted by adding 4 parts of acetate buffer pH 5.5 - 0.02 M to 1 part of the fermentation medium in each Erlenmeyer. The flasks were shaken at 200 rpm for 1 hour (Battestin, 2007b). The solution was filtered through gauze and the extract retained in the filtration was called crude solid extract. The filtrate was centrifuged at 7100 xg for 30 minutes at 4 ° C, and was then called a crude enzyme extract (Lekha, PK; Lonsane, BK Production and application of tannin acyl hydrolase: State of the art. Advances in Applied Biochemistry and Microbiology 44 , pp. 215-260, 1997). The enzymatic extract was used to determine the activity of the enzymes tanase and phytase. The crude solid extract was used for analytical determinations of total phenols, condensed tannins, hydrolyzed tannins and detoxification.

The main objective of the analytical determinations was to evaluate the concentrations of compounds of nutritional value in the pre-fermented and post-fermented medium such as phenolic compounds, hydrolyzable, condensed tannins, evaluation of the presence of ricin and determination of the detoxification process.

A) Extraction of Total Phenols

The following solvents were used for the extraction process, in the proportion of 50% of the solvent and 50% of water: Ethyl Acetate, Acetone and Methanol. It was also tested with Hexane solvent in its anhydrous form (100%). 200 mg of the sample was weighed in 10 ml of the solvent, then incubated at room temperature with shaking at 150 rpm for 120 minutes. After homogenization, they were centrifuged at 1320 xg at 5 ° C for 15 minutes and analyzed by determination of Total Phenols (Naczk, M .; Shahidi, F. Extraction and analysis of phenolics in food. Journal of Chromatography A 1054, p. 95-111 , 2004); (Schons, 2009).

B) Determination of Total Phenols

The Follin-Ciocaulteau technique (Singleton, V. L Rossi, JAJ Colorimetry of total phenolics with phosphomolybdic phosphotungstic acid reagents. American Journal of Enology and Viticulture 20, p. 144-158,1965) was used to determine the phenols present in the sample . This method is based on the reduction of phosphomolybdic and phosphotungstic acid by the hydroxyls of the phenols, producing a blue color, quantified by spectrophotometry at 760 nm.

In a 50 mL Erlenmeyer, 1 mL of the Follin-Ciocaulteau reagent, 10 mL of distilled water, 1 mL of the sample were added and left to stand for 3 minutes. 8 ml of sodium carbonate (7.5%) were added and the reaction took place for 2 hours in a dark place. After 2 hours, the sample was read on a spectrophotometer at an absorbance of 760 nm. The blank was composed of all the constituents of the reaction, except for the sample. The course of the reaction was accompanied by a calibration curve with gallic acid, according to the methodology described.

C) Determination of Hydrolyzable Tannins

The methodology of Brune and collaborators (Brune, M. Hallberg, L. Skânberg, A. Determination of iron-binding phenolic groups in foods. Journal of Food Science 56, p. 128-131, 1991) was used, and the extraction of samples were performed as indicated for total phenols. A solution of iron (III) sulfate and ammonium, called FAS, was used. The solution consisted of: 89% 0.1 M acetate buffer solution - pH 4.4; 10% 1% gum arabic solution; 1% solution of iron (III) sulfate and 5% ammonium. In a test tube, 2 ml of the study sample and 8 ml of the FAS solution were added. After 15 minutes of reaction, the absorbance at 578 nm was read. The blank was performed by replacing the sample with distilled water. The reaction course was accompanied by a calibration curve with tannic acid, according to the methodology described.

D) Determination of Condensed Tannins

The methodology of Prince et al. (Price, ML Van Scoyoc, S. Butler, LG A critical evaluation of the vanillin reaction as an assay for tannin in sorghum grain was used. Journal of Agricultural and Food Chemistry 26, p. 1214-1218 , 1978), being specific for flavonoids and dihydroxychhalcones that have a simple bond in positions 2 and 3 and a free hydroxyl in the beta ring. Vanillin is protonated in an acidic solution, resulting in a weak electrophilic carbocation, which reacts with the aromatic ring at positions 6 or 8, this compound is immediately dehydrated forming a red compound.

Sample extraction was performed as with hydrolysable phenols and tannins. He used a vanillin solution containing: 1 part for the vanillin solution (1% vanillin dissolved in methanol) and 1 part for the HCI solution (8% HCI in methanol).

The method was carried out with the addition of 1 ml of the study sample and 5 ml of the vanillin solution for 5 minutes, and every 1 minute, 1 ml of the vanillin solution was placed. The blank consisted of 1 ml of distilled water and 5 ml of the vanillin solution, then the reaction was conducted for 20 minutes and finally the reading was made on a spectrophotometer at 500 nm. The reaction course was accompanied by a catechin calibration curve, according to the methodology described.

D) Results

*Acetato = 1:1 de Acetato de Etila e Água destilada; Acetona = 1:1 de Acetona e Água destilada; Metanol = 1:1 de Metanol e Água destilada; Hexano = somente Hexano.Table 15 shows the concentrations of Total Phenols in each gram of sample extracted by four different solvents diluted 1: 1 with distilled water, except for Hexane: Ethyl Acetate, Acetone and Methanol, in unfermented castor cake. Table 15: Concentration of Total Phenols in unfermented castor cake in different types of solvents.

* Acetate = 1: 1 Ethyl Acetate and distilled water; Acetone = 1: 1 of Acetone and distilled water; Methanol = 1: 1 Methanol and distilled water; Hexane = Hexane only.

The results show that the solvent Acetone diluted 1: 1 with distilled water showed a higher concentration of phenols, with an average of 47 milligrams per gram of sample.

There is no consensus on the use of a solvent to extract phenolic compounds in most samples in the food area, mainly in vegetables. But the results above indicate that the best solvent for the extraction of phenolic compounds are those that have polarity, which does not occur with Hexane because it is a supporting solvent and does not interact with polar compounds, only with supports.

For better extraction of Total Phenols and also for Hydrolyzable and Condensed Tannins, the Acetone solution was used to analyze its contents before and after the fermentation of the castor cake.

*TM = Torta de Mamona in natura; TM F72h = meio de cultivo otimizado e fermentado para produção de fitase (após 72 horas de fermentação).The contents of Total Phenols and Hydrolyzable Tannins in the samples, castor cake in natura and optimized and fermented culture medium for phytase production (after 72 hours of fermentation) are shown in Table 16. Table 16: Concentration of Total Phenols and Tannins Hydrolyzable before and after fermentation.

* TM = Castor Pie in natura; TM F72h = optimized and fermented culture medium for phytase production (after 72 hours of fermentation).

The results showed that the fermentation of the castor bean cake by Pαecilomyces vαriotii after 72 hours of incubation decreased the concentration of total phenols and also of hydrolyzable tannins. Probably the microorganism produced enzymes that hydrolyzed these compounds, such as tannins, which have the ability to cleave hydrolyzable tannins. For these samples, not enough concentrations of condensed tannins were detected, probably not enough samples were posted in the extraction process to determine the tannins. condensed.

Research involving the determination of phenolic compounds in castor bean cake was not found in the recent literature, since these residues are not yet of interest in the production of animal feed and, consequently, there are no studies for analysis of compounds of functional interest to the animal.

Xu and collaborators {Xu, L; Diosady, L. L Rapid method for total phenolic acid determination in rapeseed / canola meals. Food Research Internationa! 30, p. 571- 574,1997) quantified the presence of phenolic compounds in canola, a seed used to extract its oil for the biofuels sector. An extraction step was carried out in which the sample underwent an acid extraction of acetone followed by an alkaline hydrolysis, then underwent an acidification, then an extraction of ethyl ether with ethyl acetate and finally by evaporation. methanol. This extraction was carried out in order to obtain three types of phenols: free, esterified and insoluble, favoring a wider range of compounds identified in colorimetric analysis. According to the results, canola had 1683 milligrams of phenolic compounds per 100 grams of sample.

According to Kozlowska et al. (Kozlowska, H .; Naczk, M .; Shahihi, F .; Zadernowski, R. Phenolic acids and tannins in rapeseed and canola. In Canola and Rapeseed: Production, Chemistry, Nutrition and Processing Technology, ed. F Shahidi, AVI Book, New York, NY, pp. 193-210, 1991) the concentration of phenolic acids in canola meal is in the range of 6.4 to 12.8 grams per kg of sample.

From these results we verified that the castor bean cake presented a composition of phenolic compounds more expressive in relation to canola, showing an advantage in relation to its phenolic composition for future use as animal feed.

Example 4: EVALUATION OF THE DETOXIFICATION OF THE MAMMONA PIE BY SOLID STATE FERMENTATION A) Detection of ricin by SDS-PAGE

To evaluate the detoxification of castor bean cake via solid state fermentation by Paecilomyces variotii, the presence of ricin was identified by SDS-PAGE. The ricin had 2 chains which, when cleaved by the SDS and run on the polyacrylamide gel, have two bands, one called chain A with 32 kDa and the other chain B with 34 kDa (Anandan, 2005).

The protein extract of the fermented product was evaluated at different incubation times, between 24 and 72 hours, comparing it with purified ricin, fresh castor and autoclaved cake (Annex 4).

First, extraction was carried out with the addition of phosphate buffered saline (PBS) pH 7.2 in the proportion 1: 4 (1 g of solid residue to 4 ml of buffer) in the residue that was kept under stirring for 3 hours, then subjected to centrifugation at 14000 rpm for 15 minutes at room temperature. To determine ricin present in the sample, protein quantification was performed (Bradford, MM A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Analytical Biochemistry 72, p. 248, 1976) to obtain a standard concentration for all samples. He added 80 pL of the supernatant to 40 pL of buffer containing Sodium Dodecyl Sulfate (SDS) and β-mercaptoethanol, totaling 120 pL. Of this total, 25 pL was applied to run the 12% SDS-PAGE gel (10x10x0, 1 cm). After the run, the proteins were fixed and stained for 1 hour with Coomassie-Brilliant-Blue 0.1% solution in methanol / acetic acid / water (45/10/45, v / v / v). After that time, the gel was bleached with a solution containing methanol / acetic acid / water (45/10/45, v / v / v).

According to Annex 4, the bands in which the ricin appears in the gel are in the range between 31.3 and 38.2 kDa, and are also visualized in the run with the purified ricin. The samples of fresh castor cake and autoclaved castor cake '. presented the two ricin bands on the gel, showing that the methodology is valid for identifying this compound in samples of protein extract. The autoclaving process, at 121 ° C for 15 minutes, did not destroy the ricin chains. In the sample of protein extract fermented after 24 hours, the presence of ricin is poorly visible, demonstrating a probable initiation of the hydrolysis of the compound. In the samples of 48 and 72 hours of fermentation, the bands where the ricin is located are completely absent, possibly showing that the microorganism Paecilomyces variotii hydrolyzed the ricin, making possible a detoxified and viable cake for use in animal feed. The minimum detection of proteins in SDSPAGE by Coomassie Blue is 10 pg / mL of sample, lower concentrations are hardly detectable by the method (Anandan, 2005); (Godoy, 2009) and (Schãgger, H. Tricine-SDS-PAGE. Nature Protocols 1, p. 16-22, 2006).

An alternative would be to use the silver dye to detect compounds with lower concentrations.

B) Toxic Activity Test in Cell Culture

The MTT test was used according to Mosmann's methodology (Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Meth. 65, 55-63). During the tests, RAW 264.7 cells were grown in DMEM in greenhouses at 37 ° C in the presence of CO2 in their atmosphere. For 100 pL of cell culture, a volume of 100 pL of MTT (5 mg / mL in PBS) and sample extract were added in microplate wells. Each microplate was incubated in the greenhouse for 24 hours. After incubation, the microplates were read on a spectrophotometer at 540 nm absorbance.

The assay was conducted using the ricin obtained as described in the extraction item, in order to determine the minimum dose to induce death in the cell culture. For this, different dilutions of the protein made in PBS pH 7.0: 100 were used

Ug / mL, 50 pg / mL, 10 pg / mL, 1 pg / mL, 100 ng / mL, 50 ng / mL, 10 ng / mL, 1 ng / mL, 100 pg / mL, 50 pg / mL and 10 pg / ml. Then, the minimum inhibitory concentration of the extract on the cells was used to evaluate the fermented substrate at 24, 48 and 72 hours of fermentation.

Through the results obtained by electrophoresis analysis, the samples were tested in cultures of live cells to verify its viability in the presence of fresh and fermented castor cake.

* TM 72h = meio de cultivo de torta de mamona com 25% do volume da solução salina em relação ao peso total do meio (v/p). ** Incubação em Câmara Climática a 90% de Umidade Relativa do Ar a temperatura de 30°C.According to Figure 7, the minimum concentration required for cell death to occur was 1 pg / mL of pie extract. Lower concentrations the cell viability in the culture medium was close to 100% and thus did not interfere with its growth. From these values, the second test was performed with extracts of the fermented material with 1 pg / mL at different times (24, 48 and 72 hours of fermentation). According to Figure 8, during the fermentation process, in its progression of time, cell viability increased, after which after 72 hours of fermentation it presented approximately 100% of cell viability. Thus, probably the microorganism Pαecilomyces vαriotii hydrolyzed the possible toxic compounds (ricin) in the medium and favored the growth of the cells under study. Table 17: Relative humidity of the culture medium before and after incubation in the climatic chamber for the optimum medium for the production of phytase in castor cake.

* TM 72h = castor bean culture medium with 25% of the volume of saline in relation to the total weight of the medium (v / w). ** Incubation in a Climatic Chamber at 90% Relative Humidity at a temperature of 30 ° C.

Claims (39)

1. Culture medium for the production of phytase enzyme by the microorganism Paecilomyces variotii characterized by being made up of residual castor cake and saline solution; 2. Culture medium for the production of phytase enzyme by the microorganism Paecilomyces variotii, according to claim 1, characterized in that the saline solution is composed in grams per liter of 1 part of KH2PO4; 2 parts of NH4NO3; 0.2 parts of MgSO4.7H2O; 0.02 parts of CaCl2.2H2O; 0.004 parts of MnCI2.4H2O; 0.002 parts of Na2MoO4.2H2O and 0.0025 parts of FeSO4.7H2O. 3. Culture medium for the production of phytase enzyme by the microorganism Paecilomyces variotii, according to claim 1, characterized in that it does not contain phytic acid. 4. Culture medium for the production of phytase enzyme by the microorganism Paecilomyces variotii, according to claim 1, characterized in that it preferably does not contain tannic acid. 5. Culture medium for the production of phytase enzyme by the microorganism Paecilomyces variotii, according to claim 1, characterized by containing from 25 to 28% of saline solution, in relation to the total weight of the medium. 6. Culture medium for the production of phytase enzyme by the microorganism Paecilomyces variotii, according to claim 5, characterized in that it preferably contains 25% of saline solution, in relation to the total weight of the medium. 7. Culture medium for the production of phytase enzyme by the microorganism Paecilomyces variotii, according to claim 1, characterized in that it preferably contains 75% of the total weight made up of the residual castor cake. 8. Process for the production of phytase enzyme, characterized by being made by the microorganism Paecilomyces variotii through solid fermentation. Process for the production of phytase enzyme, according to claim 8, characterized in that it is in residual castor cake. Process for the production of phytase enzyme according to claims 8 and 9, characterized in that it is carried out as described in claims 1 to 7. Process for the production of phytase enzyme according to claims 8 to 10, characterized in that it is preferably obtained after 72 hours of incubation. 12. Process for the production of phytase enzyme, according to claims 8 to 11, characterized in that between 85 and 90% of ambient relative humidity, preferably 90%, is obtained. 13. Culture medium for the production of enzyme tanase by the microorganism Paecilomyces varioti characterized by being made up of residual castor bean cake, saline solution and tannic acid; 14. Culture medium for production of the enzyme tanase by the microorganism Paecilomyces variotii, according to claim 13, characterized in that the saline solution is composed in grams per liter of 1 part of KH2PO4; 2 parts of NH4NO3; 0.2 parts of MgSO4.7H2O; 0.02 parts of CaCl2.2H2O; 0.004 parts of MnCl2.4H2O; 0.002 parts of Na2MoO4.2H2O and 0.0025 parts of FeSO4.7H2O. 15. Culture medium for production of enzyme tanase by the microorganism Paecilomyces variotii, according to claim 13, characterized by containing 4.6 to 6.0% of tannic acid, in relation to the total weight of the medium. 16. Culture medium for production of the enzyme tanase by the microorganism Paecilomyces variotii, according to claim 15, characterized in that it preferably contains 4.6% of tannic acid in relation to the total weight of the medium. 17. Culture medium for production of the enzyme tanase by the microorganism Paecilomyces variotii, according to claim 13, characterized by containing from 25 to 33% of saline solution, in relation to the total weight of the medium. 18. Culture medium for production of the enzyme tanase by the microorganism Paecilomyces variotii, according to claim 17, characterized in that it preferably contains 25% of saline solution, in relation to the total weight of the medium. 19. Culture medium for production of enzyme tanase by the microorganism Paecilomyces variotii, according to claim 13, characterized in that it preferably contains 65.4% of residual castor cake. 20. Process for the production of enzyme tanase, characterized by being made by the microorganism Paecilomyces variotii through solid fermentation. 21. Process for producing the enzyme tanase, according to claim 20, characterized in that it is in residual castor cake. 22. Process for producing the tanase enzyme according to claims 20 and 21, characterized in that it is produced as described in claims 13 to 19. 23. Process for producing the tanase enzyme according to claims 20 to 22, characterized in that it is obtained preferably after 48 hours of incubation. 24. Process for producing the enzyme tanase, according to claims 20 to 23, characterized in that it is obtained in an environment with relative humidity between 84 and 90%, preferably 90%. 25. Culture medium for simultaneous production of phytase and tanase enzymes by the microorganism Paecilomyces variotii characterized by being made up of residual castor cake and saline solution with or without tannic acid. 26. Culture medium for simultaneous production of phytase and tanase enzymes by the microorganism Paecilomyces variotii, according to claim 25, characterized in that the saline solution is composed in grams per liter of 1 part of KH2PO4; 2 parts of NH4NO3; 0.2 parts of MgSO4.7H2O; 0.02 parts of CaCl2.2H2O; 0.004 parts of MnCI2.4H2O; 0.002 parts of Na2MoO4.2H2O and 0.0025 parts of FeSO4.7H2O. 27. Culture medium for simultaneous production of phytase and tanase enzymes by the microorganism Paecilomyces variotii, according to claim 25, characterized in that it does not contain phytic acid. 28. Culture medium for simultaneous production of phytase and tanase enzymes by the microorganism Paecilomyces variotii, according to claim 25, characterized by containing tannic acid from 4.6 to 6% tannic acid. 29. Culture medium for simultaneous production of phytase and tanase enzymes by the microorganism Paecilomyces variotii, according to claim 25, characterized by containing from 25 to 33% of saline solution, in relation to the total weight of the medium. 30. Culture medium for simultaneous production of phytase and tanase enzymes by the microorganism Paecilomyces variotii, according to claim 29, characterized in that it preferably contains 25% of saline solution, in relation to the total weight of the medium. 31. Culture medium for simultaneous production of phytase and tanase enzymes by the microorganism Paecilomyces variotii, according to claim 25, characterized by containing from 65.4 to 75% of the total weight made up of the residual castor cake. 32. Use of the phytase enzyme obtained according to claims 1 to 7, characterized by being for food supplementation. 33. Use of the phytase enzyme obtained according to claims 1 to 7, characterized by being for supplementation in animal feed. 34. Use of the tanase enzyme obtained according to claims 13 to 19, characterized by being for food supplementation. 35. Use of the tanase enzyme obtained according to claims 13 to 19, characterized for being for supplementation in animal feed. 36. Use of the phytase enzyme obtained according to claims 25 to 31, characterized by being for food supplementation. 37. Use of the phytase enzyme obtained according to claims 25 to 31, characterized by being for supplementation in animal feed. 38. Use of the tanase enzyme obtained according to claims 25 to 31, characterized by being for food supplementation. 39. Use of the tanase enzyme obtained according to claims 25 to 31, characterized by being for supplementation in animal feed

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