WO2013020189A1 - Media for the culture of waste castor-oil-plant cake for simultaneous production of the phytase and thanase enzymes, and detoxification of the castor-oil-plant cake, using the microorganism paecilomyces variotii and using solid fermentation, the enzymes obtained and the uses thereof - Google Patents

Abstract

The present invention relates to the simultaneous production of thanase and phytase from waste castor-oil-plant cake in solid-state fermentation with Paecilomyces variotii, and also the reduction in the concentration of castor oil after fermentation, thereby promoting the detoxification thereof. The use of enzymes in animal nutrition is known and is the subject of intense interest. The greatest difficulty in terms of wider use of enzymes is still the production cost. The use of waste for animal nutrition represents a viable alternative for this sector, and also for the production of biocatalysts by means of solid fermentation. The present invention determined optimum values for constitution of the culture medium using waste castor-oil-plant cake for production of the phytase and thanase enzymes by means of the solid fermentation of the microorganism Paecilomyces variotii, in such a manner as to produce the greatest possible quantity of enzyme and to achieve the greatest possible performance level thereof. In addition, the present invention enables the detoxification of waste castor-oil-plant cake on account of the degradation of castor oil, the greatest toxic agent thereof, thereby making the waste an ingredient that it is advantageous to use in animal feed.

Description

 "MEDIUM RESIDUAL PIE CULTURE FOR SIMULTANEOUS PRODUCTION OF THE PHYMASE AND TANASE ENZYMS, AND DETAXIFICATION OF THE MAMONA PIE BY PAECILOMYCES VARIOTII MICROORGANISM

 SOLID FERMENTATION, ENZYMS OBTAINED AND THEIR USES ". FIELD OF THE INVENTION

The present invention aimed to determine parameters for simultaneous production of phytase and tanase enzymes by solid fermentation in castor bean cake using the fungus Paecilomyces variotii.

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

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

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

BACKGROUND OF THE INVENTION Solid State Fermentation Enzyme Production

Solid state fermentation (FES) provides for 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 retention capacity of Water by 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 , pp. 114-118, 2009). The solid matrix used in the process can be both the nutrient source and simply a support impregnated with nutrients suitable for microorganism development (Pandey, 2003), (Nagao, N.; Matsuyama J.; Yamamoto, H .; Toda, T. A novel hybrid system of solid state and submerged fermentation with recycle for organic solid waste treatment (Process Biochemistry 39, pp. 37-43, 2003). There are significant differences between solid state fermentation 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 solids varies from 20 to 70% of the total weight of the medium (Mitchell, DA; Losane, BK Definition, characteristic and potential. In: Doelle, H., Mitchell, DA, Rols, CE Solid substrate cultivation (Elsevier Applied Science, pp. 1-16, 1992).

FES confers advantages over FS such as the use of simple, non-water soluble culture media composed of plant-based materials such as rice bran, wheat, maize and other cereals, requiring few additional nutrients in the medium. Additionally, the cost of the fermentation medium may represent up to 30% of the total enzyme production. Thus, there is interest in the use of agro-industrial residues as substrate, representing, in countries such as Brazil, abundant and low cost raw material (Graminha, EBN; Gonçalves, AZ L; Pirota, RDPB; Balsalobre, MAA; Da Silva, RE Enzyme production by solid-state fermentation: application to animal nutrition Animal Feed Science and Technology 144, pp. 1-22, 2008), (Pandey, 2003). Due to the low moisture content and the absence of free water in the medium, the likelihood of bacterial contamination is reduced. The small amount of water employed in FES also implies advantages such as lower 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 solid culture medium influences the physiological aspects of microorganisms, such as their vegetative growth, sporulation, spore germination, enzyme production and enzymatic activity (Graminha, 2008). FES may, in some cases, be economically more interesting in enzyme production. George et al. (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 of protease between solid and submerged fermentation. The authors reported that for the same product yield, 100 mL of nutrients in FS and 1 g in FES were used.

ANIMAL FEED PRODUCTION USING AGRICULTURAL WASTE

The Brazilian economy is one of the most important economies in the world and is mainly based 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 more efficient the use of these wastes whose disposal in the environment causes serious pollution problems (Soccol, CR; Vandenbergher, LPS). Biochemical Engineering Journal 13, p 205-218, 2003).

Agro-industrial waste is largely produced by human, agricultural and industrial activity. Among the residues produced in significant quantities by the Brazilian industrial activity, we can mention: rice husk, straw and bran; wheat straw and bran; sugarcane bagasse; cassava leaf; orange pomace and castor bean pie (Schieber, A.; Stintzing, F. C.; Carie, 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); (Graminha, 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, ie the use of these residues as substrates for cultivation of microorganisms capable of biodegrading them to obtain nutrients for their production. 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 Yang, Y; Diana, J.; Yakupitiyage, A.; Luo, Z; Li; D. Application of microbial phytase in fish feed Enzyme and Microbial Technology 40, pp. 497-507, 2007.; Kuhad, R. C. Singh, A. Tripathi, KK, Saxena, RK, Eriksson, KEL Microrganisms as an alternative source of protein Nutrition Reviews 55, 65-75, 1997).

Generally, rations are materials from organic sources for the purpose of properly nourishing the animal. The main characteristics of a diet are the energy availability, amount of fiber (important for digestibility) and supplementary proteins, mainly essential amino acids (González-artín, I .; Alvarez-García, N .; Hernández-Andaluz, JL. Instantaneous determination of crude proteins, fat and fiber in animal feeds using near infrared reflectance spectroscopy technology and remote reflectance fiber optic probe (Animal Feed Science and Technology 128, pp. 165-171, 2006); (Makkar, H. P. S. Review: Effects and Fate of Tannins in Animal Ruminants, Adaptation to Tannins, and Strategies to Overcome Detrimental Effects of Feeding Tannin-Rich Feeds. Small Ruminant Research 49, p. 241- 256, 2003).

Feeds produced with agricultural by-products or food industry residues are frequently used, this is because there is a high availability of residues due to high food production (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 yield 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, p. 223-246, 2008). However, these feeds should be carefully formulated to ensure that antinutritional compounds, especially phytates and tannins, do not adversely affect animal health. These compounds can cause changes in the structure and composition of milk and meat fatty acids (Herbs, G.; Fruits, 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 fish protein sources, thus eliminating animal proteins in feed formulation (Carter, CG; Hauler, RC. salmon, Salmo salar L Aquaculture 185, pp. 299-311, 2000). A study has shown that salmon (Hippoglossus hippoglossus L) needs a minimum protein concentration of 35% in their diet for their growth, requiring the use of agro-industrial residues that present this concentration, without the need for supplementation of other products (Arnason J. Imsland, AK; Gustavsson, A.; Gunnarsson, S.; Arnarson, I.; Reynisson, H.; Jónsson, AF; Smáradóttir, H.; Thorarensen, H. Optimum feed formulation for Atlantic halibut (Hippoglossus hippoglossus Aquaculture 291, pp. 188-191, 2009) There are diets supplemented with fermented shrimp residues that have a higher availability of essential amino acids than the diets containing unfermented residues (Narayan). , B .; Velappan, SP; Zituji, SP; Manjabhatta, SN Yield and chemical composition of fractions from fermented shrimp biowaste Waste Management & Research 28 , 64-70, 2009) MAMONA PRODUCTION

Castor bean, Ricinus communis, is grown in tropical regions for use of its oil, which is present in the seed, which is extensively used 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 beaver cake Animal Feed Science and Technology 120, p. 159-168, 2005); (Jones, DB Proteins of the bean beaver-their preparation, properties, and utilization. The Journal of The American OH Chemists Society, July, p. 247-251, 1947). The main producing countries are India with 60% of world production and China with 20%. Brazil in 2002 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 shift from energy sources via petroleum sources to vegetable oil, including castor oil.

The residual castor bean cake represents half the weight of the seed and has an amount of 34 - 36% protein (Gowda, N.; S.; Pai; DT; Srinivas, RB; Bharadwaj, U. ; Sridhar, M .; Satyanarayana, M.L; Prasad, CS; Ramachandra, KS; Sampath, K. Evaluation of beaver (Ricinus communis) seed cake in the total mixed ration for sheep.Journal of the Science of Food and Agriculture 89 , pp. 216-220, 2008); (Anandan, 2005); 20% fiber, 0.7% calcium; 0.8% phosphorus and 4% ether extract (Souza, R. M. Effect of detoxified castor bean meal on swine hematological values. Master's dissertation. Federal University of Minas Gerais, Minas Gerais, 1979). Nevertheless, castor bean cake cannot be used as a protein source because of its toxicity and is 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-οχο-3-pyridinecarbonitrile (C ₈ H _{8 2} 02) present in castor bean cake with low toxicity (Beltrão, NOR). ; Lima, RLS Application of castor oil as a source of energy: Biodiesel In: Azevedo, DMP; Beltrão, NOR (Ed.) Agribusiness of castor in Brazil 2 * Edition, Embrapa Technological Information, Brasília-DF, p 395-416, 2007).

The formerly known CB-1A (Castor Bean Allergen) allergen complex, currently referred to as Albumin 2S is also present in nuts and other seeds such as Maranhão nuts, hazelnuts, mustard and cotton (Breiteneder, H.; Radauer, C. A classification of plant food allergens Journal of Allergy and Clinical Immunology 113, p. 821-30, 2004). The presence of this toxin has been known for many years, however, detoxification treatments applied to the cake are generally inefficient (Anandan, 2005).

Ricin (CAS No: 9009-86-3) is one of the most potent phytotoxins known and is 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 receptor-binding B-chain described by Stirpe et al. (Stirpe, F.; Batelli, MG. Ribosome-inactivating proteins: progress and problems. Cellular and Molecular Life Sciences 63, p. 1850-1866, 2006), which can be viewed in Rutenber's Appendix 1 (Rutenber, E.; Robertus, JD Structure of Ricin B-chain at 2.5 Â resolution. Proteins: Structure, Func and Genetics 10, pp. 260-269, 1991).

The A-chain removes an adenine residue in a ribosomal RNA loop region, with this modification, these ribosomes cannot support protein synthesis, as can be seen in Figure 1. This inactivation can be performed in proportion of one ricin to every 2,000 ribosomes every minute, a rate 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, pp. 281-288, 1975).

The heat-resistant ricin could easily be inactivated by any cooking or autoclaving process (Anandan, 2005), but this process is not viable for industrial scale application due to the high cost. To solve this problem, a detoxification via fermentation could be performed (Anandan, 2005); (Godoy, M. G. Microbial lipase production and simultaneous detoxification of agroindustrial tailings. Master's Dissertation, IQ-UFRJ - RJ, 2009), a fact accomplished by the present invention.

TANINES AND TANASE Tannins are a group of water-soluble, high molecular weight phenolic compounds capable of precipitating proteins and binding to metals (chelators). These compounds complex with cellulose, pectin and starch making them insoluble. Hydrolyzable tannins, such as elagitanines and galotanines, and condensed tannins, also named proanthocyanidins (Gross, GG From lignins to tannins: forty years of enzyme studies on the biosynthesis of phenolic compounds. -3031, 2008; Waghorn, 2008).

Hydrolyzable tannins (Figure 2) are joined by ester bonds between gallic acid groups 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 esterified gallic acids generally with glucose in their hydroxyl groups (galotanines or elagitanines) (Battestin, V; Matsuda, LK; Macedo, GA. Sources and applications of tannins and tanases 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, pp. 1479-1492, 2007). They are present in large quantities in foods, 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.; AggarwaI, NK; Kumar, K.; Kumar, A. Tannase production by Aspergillus fumigatus MA under solid-state fermentation World Journal of Microbiology and Biotechnology 24, pp. 3023-3030, 2008). These compounds inhibit the growth of many microorganisms by complexing with proteins. These characteristics are highly antinutritional 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 niger ATCC 16620. Bioresource Technology 96, pp. 1223-1228, 2005).

The negative effect of tannins on animal nutrition is due to their ability to bind to macromolecules, decreasing 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 uptake in ruminants, yielding higher growth rate and milk yield (Belmares, R .; Contreras-Esquivel, J Rodríguez-Herrera, R.; Colonel, AR; Aguilar, CN Microbial production of tannase: an enzyme with potential use in the food industry (Lebensmittel-Wissenschaft und-Technologie 37, p. 857-864, p. 2004); (Min, BR; Barry, TN; Attwood, GT; McNabb, WC The effect of condensed tannins on nutrition and health of fed fresh temperate ruminants: a review. Animal Feed Science and Technology 106, p. 3-19, 2003 ).

Tanase (EC 3.1.1.20) or tannin-acylhydrolase (TAH) catalyzes the hydrolysis of gallic acid ester bonds (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, pp. 384-390, 2007); (Trevino-Cueto, B .; Luis, M .; Contreras-Esquivel, J. C; Rodriguez, 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 can also be produced by bacteria of the genus Bacillus, Corynebacterium, Klebsiela, Streptococcus and Selenomonas.

Vegetables also produce tannase to accelerate the fruit ripening process (Aissam, H.; Errachidi, F.; Penninckx, MJ; Merzouki, M .; Benlemlih, M. Production of tannase by Aspergillus niger HA37 Growing on tannic acid and olive Mill waste aters. World Journal of Microbiology & Biotechnology 21, p. 609-614, 2005); (Batra, A.; Saxena, RK Potential tannase producers from the Aspergillus and Penicillium genera. Process Biochemistry 40, p. 1553-1557, 2005); (Hamdy, HS; 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, CN; Gutiérrez-Sanchez, G. Review: sources, properties, applications and potential uses of tanninacyl hydrolase. Food Science and Technology International 7, pp. 373-382, 2001b); (Deschamps, AM; Otuk, G.; Lebeault, JM Production of tannase and degradation of chestnut tannin by bacteria. Journal of Fermentation Technology 61, p. 55-59, 1983); (Rodrigues, THS; Dantas, MAA; Pinto, GAS; Gonçalves, LRB Tannase production by solid-state fermentation of cashew apple bagasse. Applied Biochemistry and Biotechnology 136-140, p. 675-688, 2007); (Rodriguez, H .; Rivas, B .; Gomez-Cordovés, C.; Muz, 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, DL 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, JS; Dutta, JR; Nanda, RK; Banerjee, R. Strain Improvement for Tannase Production from the Cultivation 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 nutrients contained in it, such as the breakdown of antinutritional factors. At the same time, it reduces costs for feed improvement, as this enzyme can be produced via fermentation (Battestin, 2004; Graminha 2009). There are studies using the action of tannase produced by Paecilomyces variotii in broom sorghum grains on antinutritional factors, in this case tannins. In these studies a reduction in tannins was verified in the samples treated with tannase, phosphorus increase, digestibility improvement and phosphorus excretion decrease compared to raw sorghum (Schons, PF Detanification and dephytinization of sorghum grains (Sorghum bicolor) by tanase and phytase and biological study. Master's Dissertation - UNICAMP-FEA, 2009).

• Preparation of Instant Teas: one of the most consumed beverages in the world, for its aroma, taste and mainly medicinal effects. In the preparation of these teas, which are often in icy form, it produces an insoluble precipitate. This precipitate is the polyphenol complex, which is the main problem detected. Tanase, added to the product process, catalyzes the breakdown of ester bonds, which eventually cleaves insoluble compounds, decreasing their turbidity and increasing their quality, as 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, pp. 215-260, 1997); (Khokhar, S.; Magnusdottir, S. G. M. Total phenol, catechin, and caffeine contents of commonly consumed teas in the United Kingdom. Journal of Agricultural and Food Chemistry 50, p. 565-570, 2002).

• Brewing: Beers feature polyphenolic compounds from malt. Thus tanase cleaves these compounds and decreases their turbidity, making them a product acceptable to the market (Battestin, 2004).

• Gallic Acid Production: used mainly in the pharmaceutical industries, such 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, pp. 296-302, 2001a).

• Antioxidant Production: 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 a tannase from Paecilomyces variotii. Food Chemistry 108, p. 228-233, 2008).

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

One of the most studied topics about TAH concerns 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 pH stability 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; optimal 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 growing 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, GA 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, SS; Banerjee, R .; Pandey, A.; Szakacs, G. Purification, Characterization and some studies on secondary structure of tannase from Aspergillus awamori nakazawa. Process Biochemistry 40, p. 3251-3254, 2005); (Sharma, S.; Agarwal, L.; Saxena, RK Statistical optimization for tannase production from Aspergillus niger under submerged fermentation. Indian Journal of Microbiology 47, pp. 132-138, 2007); (Sharma, S.; Agarwal, L.; Saxena, RK Purification, immobilization and Characterization of tannase from Penicillium variable. Bioresource Technology 99, p. 2544-2551, 2008).

RIBBONS AND PHYTASE

Phytic acid / W / ^{'o-lnositol-l,} 2,3,4,5,6-hexakisphosphate (Figure 5) is a cyclic alcohol derived from glucose 6 - phosphate groups attached to each carbon of the glycoside molecule. Among the compounds grouped in phosphorylates, phytic acid is the most abundant in vegetables, especially in seeds as it has a storage function of the phosphorus group to obtain energy (Raboy, V. Molecules of interest: myo-lnositol-1, 2). 3,4,5,6-hexakisphosphate (Phytochemistry 64, pp. 1033-1043, 2003). The presence of excess phytate pollutes the environment and also disturbs the diet of monogastric animals. Phytate acts as an antinutrient by binding to proteins, amino acids and lipids and chelating minerals such as calcium, iron, zinc and magnesium thus forming insoluble salts (Honsson, SJ; Davis, RP). Phytase-hydrolysing enzyme by some fungi Enzyme and Microbial Technology 5, pp. 377-343, 1983). In addition, it interacts with digestive enzymes reducing their activities, influencing digestion and impairing the utilization of vitamins.

Although phytate serves as the primary 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 phosphorus nutritional needs (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) . Unused phytate phosphorus is excreted as an environmental pollutant in intensive farming areas (Lei, X. G .; Porres, J.M. Phytase Enzyme, Applications and Biotechnology. Biotechnology Letters 25, p. 1787-1794, 2003).

The enzyme phosphohydrolase catalyzes the phosphate and phytic acid hydrolysis (Figure 6) to inorganic phosphate and myo-inositol phosphate derivatives. Phytases are classified as histidine acid phosphatases (Histidine Acid Phosphatases - PAHs), 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-phosphohydrolase) originates mainly from the microbial pathway and 6-phytase is derived from plants. To obtain microbial phytase, the fungi are the most researched, among them we can mention the genera Penicillium, Aspergillus and Mucor. Among the yeasts, the genus Saccharomyces stands out (Dvoráková, J. Phytase: Sources, preparation and exploitation. Microbiological Folia 43, Issue 4, p.323-338, 1998); (Ies, EF; Macedo, GA Progressive screening of thermostable yeasts for phytase product. 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, M. ;; Szakacs, G.; Pandey, A. Comparison of phytase product on wheat bran and oilcakes in solid-state fermentation by Mucor racemosus (Bioresource Technology 97, pp. 506-511, 2006).

Phytase supplementation in animal feed increases phosphorus bioavailability in monogastric animals, which consequently reduces phosphorus pollution in the environment. The enzyme also prevents: chelation of phytic acid with metal ions, protein binding, lipids and carbohydrates, thus increasing their nutrition in the feed (Vats, 2004).

Research in the area of phytase enzyme production is important not only for the investigation of a poorly exploited source of phytase, but also for 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 growing medium, by-products from the processing of cassava starch supplemented with nitrogen source for solid state fermentation with Aspergillus niger for phytase production. A maximum yield of 6.73 UA / g of dry matter was obtained. The enzyme showed residual activity of 4.71 UA / g at 75 ° C for 30 minutes, which would support in processing in the feed industry (Hong, K .; Ma, Y .; Li. M. Solid-state fermentation of phytase from cassava dregs. Applied Biochemistry and Biotechnology 91-93, p. 777-785, 2001).

Pie from coconut oil extraction was used to produce extracellular phytase via solid state fermentation with Rhizopus oligosporus. 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, pp. 251-260, 2002).

ANIMAL FEED ENZYMES

Enzymes are currently used in numerous industrial products and processes, while new application areas are being added due to their effectiveness and economy in their actions, especially in reducing energy use, for reaction activation and water quantity ( 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 the action of bromelain on meat, a protease that increases the tenderness of the product, making it more interesting for consumption. Enzymes can also increase nutrient availability, especially in animal feed such as xylanase and beta-glucanase which are used in cereals that aid in nutrient digestibility in monogastric animals which, unlike ruminants, are unable to fully hydrolyze the nutrients. plant foods, 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 agroindustrial residues economically favors the formulation of rations, however these residues must be supplemented with enzymes that hydrolyze compounds to increase their nutritional value, such as the antinutritional factors already mentioned for tannins and phytates. Thus, to extract maximum nutritional value, exogenous enzymes are added to foods or residues for better absorption in monogastric animals (Pariza, MW; Cook, M. Determining the safety of enzymes used in animal feed. Regulatory Toxicology and Pharmacology 56, 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 various plant species and structures. However, this phosphorus is not available to monogastrics, since they do not have enzymes that hydrolyze 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 Poult Science Journal 57, p. 109-126, 2001).

There is also supplementation of enzyme complexes in animal feed, such as carbohydrate addition, represented by xylanase, beta-glucanase and cellulase made in wheat feed, which showed improved growth and apparent increase in nutrient digestibility 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 The in vitro protease additive increases fermentation of alfalfa diets by mixed ruminal microorganisms. Journal of Animal Science 87, p.1097-1105, 2009) verified the action of protease in base feed of vegetable components for ruminants. They concluded that this enzyme acts by removing structural proteins from the plant cell wall and consequently it provides a greater amount of nutrients for digestibility in the ruminant microbiota.

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 feed additive animal through the 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, enabling its application in animal feed, the present invention, in turn uses the microorganism Paecilomyces variotii to Simultaneous production of phytase and tanase enzymes in castor bean residual cake.

BRIEF DESCRIPTION OF THE INVENTION

For biodiesel production, castor oil stands out among oilseeds. In addition to oil, castor bean after extraction produces cake that is of great interest for organic fertilization. The pie also has a high protein concentration, 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 have been emerging to detoxify castor bean cake and use it in animal feed. Among these techniques, solid state fermentation has shown promise in detoxification of the cake, and simultaneously can 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 castor bean cake.

According to the established fermentation kinetics, it was determined that the castor bean cake medium showed the highest activity at 48 and 72 hours for tanase and phytase production, 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 substrate.

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

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

BRIEF DESCRIPTION OF THE FIGURES Figure 1: Ribosomal RNA "loop" and Ricin 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).

FigureS: Chemical Structure of Phytic Acid described by Raboy et al. (2003).

Figure 6: Phytic Acid Hydrolysis described by Dvoráková et al. (1998).

Figure 7: Minimum concentration required to inhibit cell growth, considering the percentage of living cells in relation to the amount of protein in castor bean cake extract.

Figure 8: Percentage of growth of cells in fresh pie and at each fermentation time (24, 8 and 72 hours).

BRIEF DESCRIPTION OF ANNEXES Annex 1: Structure of Ricina described by Rutenber et al. (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 saline volume and relative humidity, (b) as a function of relative humidity and tannic acid, and ( c) as a function of tannic acid and saline volume.

Annex 4: SDS-PAGE 12% protein extract from each sample tested.

* M = Bookmark; R = purified Ricin; IN = fresh castor bean pie; 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.

** Molecular Mass of each Marker chain.

DETAILED DESCRIPTION OF THE INVENTION

WASTE CHARACTERIZATION

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

For this characterization, the castor bean cake must first be crushed and subjected to a 1.68 mm 10 mesh sieve size separation process.

A) pH determination:

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 potentiometer. For its use as a culture medium for enzyme production, the residual castor bean cake must have a pH of 5.95, ranging from 0.04 to plus or minus. B) Water Percentage (Karl-Fischer):

To determine the amount of water present In 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 Automotive exhaust particulate properties of environmental significance (Environmental Science and Technology 279, pp. 457-1086, 1975).

This methodology is based on the oxidation of S0 ₂ by l ₂ , which make up Karl Fischer's reagent, in the presence of water, as illustrated in the following chemical equation: l ₂ + S0 ₂ + H ₂ 0 -> 2HI + H ₂ S0 ₄

This methodology was performed by volumetric titration that directly provides the sample water percentage, and it is necessary to supply the sample mass added in the titration flask. The amount of water in the sample should be approximately 4.78%, plus or minus 0.07%.

C) Humidity:

To determine the moisture content of the sample, incubate 0.5 g for 24 hours in an oven at 105 ° C and weigh at regular intervals until these samples reach constant weight.

For the castor bean residual cake to be used for optimum phytase and tanase production, it must have a moisture content of 6.7%, in relation to the total sample weight, with a variation of 0.16% more or less, as shown in Table 1.

Table 1: pH, amount of water and relative humidity of castor bean cake in natura. Castor Pie

 Average Test pH 5.95 ± 0.04

 Water (%) 4.78 ± 0.07

Humidity (%) 6.7 ± 0.16

FERMENTATIVE PROCESS

A) Microorganism

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

After incubation, a cell suspension with a homogenizer should be performed, resulting in a concentration of 9 x 10 ⁶ cells / mL.

B) Fermentation Medium 1 part of the residue (castor bean cake) to 1 part of the saline solution (g / L) consisting of: 1.0 KH2PO4 should be mixed in the g / mL ratio. 2.0 of NH4 NO3; 0.2 MgS0 ₄ .7H ₂ 0; 0.02 CaCl ₂ .2H ₂ 0; 0.004 MNCI ₂ .4H ₂ 0; 0.002 of Na ₂ Mo0 ₄ .2H ₂ 0 and 0.0025 of FeS0 ₄ .7H ₂ 0; and 10% tannic acid. Then the mixing vessel should be autoclaved at 121 ° C for 15 minutes.

After being cooled to room temperature, the spore suspension of Paecilomyces variotii is added to the Potato Dextrose Agar (PDA - OXOID - CM0139) culture medium with a 0.2% tannic acid supplement. 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 tanase and phytase enzymes in castor bean cake, the correct ratio of tannic acid to saline concentration in the enzyme production medium must be maintained.

Phytase: The optimal incubation period of the microorganism has been determined.

Paecilomyces variotii for phytase production is 72 hours, with a slight decrease until 96 hours, when the production value becomes constant and decreases after 120 hours, as shown in Table 2.

Table 2: Evaluation of fermentation kinetics for phytase activity in castor bean pie.

Phytase (U / mL)

 Incubation Time (hours) Average *

^'24 to 12.28 ± 0.28

 48 9, 19 ± 0.03 b

 72 49.79 ± 0.16 c

 13.89 ± 1.53 to

* Means followed by the same letter do not differ by Tukey's test (p> 0.05) at the 5% probability level. Additionally, it was determined that, in relation to relative humidity (RH%), the range in which the highest enzymatic activity is obtained is between 85 and 90% of humidity present in the medium incubation site, with an optimum percentage of 90%.

Values above 90% cause an imbalance between temperature and humidity in the Climatic Chamber, disfavoring the optimal environment for enzymatic production by Paecilomyces variotii. Lower concentrations of moisture in relation to the above range produce less phytase, as can be seen in Annex 2. The addition of tannic acid to the culture medium with castor bean cake is not necessary as it does not interfere with the production of this enzyme. In addition, phytic acid supplementation, which is generally used for phytase production, is not required, resulting in more economically viable production. The percentage of saline volume in relation to the total weight of the medium is between 25 and 28%, with the optimum concentration of 25%. Smaller percentages of solution volume inhibit the growth of the microorganism.

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

Tanase:

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

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

Annex 3 indicates that the range in which the highest enzymatic activity would be obtained would be between 84 to 90% relative humidity (RH%), with an optimum humidity percentage of 90%.

Values above 90% cause an imbalance between humidity and temperature in the Climatic Chamber. Air humidity conditions less than 84% decrease enzymatic activity.

The activity of the tannase enzyme is higher at concentrations between 4.6 and 6% of tannic acid, with optimal enzymatic activity at 4.6% and higher tannic acid concentrations decrease the tannic activity. For the concentration of saline volume in relation to the total weight of the medium, the range of 25 to 33% would stimulate enzyme production, with optimum concentration of 25%. Lower saline concentrations prevent the growth of the microorganism in the medium and the increase of saline in the culture medium diminished the tannic activity.

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 can 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 climate chamber for the medium optimized for castor bean tannase production.

Relative Humidity of Cultivation Medium (%)

Sample * Incubation Time ** Initial Moisture Final Moisture TM 48ii 48 hours ^' 25% 27% * TM 48h = castor bean cake medium with 4.6% tannic acid weight to total medium weight (p / w) and 25% of the saline volume in relation to the total weight of the medium (v / w).

Thus, to optimize the conditions for the production of tanase, in 10 g of medium, would be: 3 mL of SS (%); 0.46 g AT (%); 6.54 g castor bean cake incubated at 90% RH. Thus obtaining an activity of 700 U / mL or 2800 U / g substrate in the fermentation by 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 enzyme production, this time being used in the experimental design step.

Using castor bean cake as substrate, the best fermentation time for enzymatic production of tanase was after 48 hours, as shown in Table 4.

Table 4: Evaluation of fermentation kinetics for tannin activity in castor bean pie.

Tanase (U / mL)

 Incubation Time (hours) Average *

 24 162 ± 10.2 a

 48 428 ± 10.1

 72 249 ± 16.9 c

 96 211 ± 17.3 c

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

After 48 hours of incubation the enzyme 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. Phytase (U / mL)

 Incubation Time (hours) Average *

24 ^'12 .28 ± 0.28 a

 48 9.19 ± 0.03 b

 72 49.79 ± 0.16 c

 13.89 ± 1.53 to

* 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 on the enzyme production medium were analyzed. The central rotational composite design (DCCR) technique was used, presenting 8 factor points (pf), 6 axial points (pa) and 3 central points (pc) totaling 17 trials (2 ³ pf + 6 pa + 3 pc = 17 ). The 3 independent variables were: relative humidity () in Climatic Chamber (New Ethics Model 420 / CLDTS 300); tannic acid concentration, which was expressed as a percentage of the total weight of the medium (w / w); and the saline volume, which was expressed as a percentage of the total weight of the medium (v / w). The dependent variables (responses) were tanase and phytase enzymatic activity.

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 tannic acid concentrations were tested in order to reduce the costs of the production medium.

The amount of salt added in the fermentation medium is described in the Detailed Description in the Fermentation Process item. In this study, the volume of water added to the fermentation medium was evaluated. The range of water volume The amount added was determined according to the maximum absorption capacity of the substrate, without presenting free water in the medium, according to Table 6. The actual values used in the planning tests are presented in Table 7 and the tests matrix in Table 8. Table 7: Values used in DCCR for three factors.

Levels

 Variables * -1.68 -1 0 1 1.68

RH (%) 60 66 75 84 90 volume SS (%) 25 31 40 49 55

TA (%) 4.6 6 8 10 11.4

* AT (%) = Tannic Acid Concentration (w / w); SS volume (%) = Saline volume (v / p); RH (%) = Percent Relative Humidity of Air.

Table 8: Design matrix containing coded variable values.

Coded Levels of Non-Diodic Effects of Variables Independent Variables * Independent *

Et ¾ ¾ ^■ vetam ©: 8S (%) AT (%)

1 -1 -1 -1 66 31 6

2 +1 -1 -1 84 31 6

3 -1 +1 -1 6 49 6

4 +1 +1 -1 84 49 6

5 -I -1 +1 66 31 10

6 +1 -I +1 84 31 10

7 -! +1 +1 66 49 10

8 +1 +1 +1 8 49 10

 -1.68 0 0 60 40 8

10 + im 0 0 90 40 8

11 0 -1.68 0 75 25 8

12 0 +1.68 0 75 55 8

13 0 0 75 40 4.6

14 0 0 +1.68 75 40 11,

15 0 0 0 75 40 S

16 0 0 © 75 40 8

17 Q 0 0 75 40 8

18 0 0 0 75 40 8

* xl = RH (%) =% relative humidity; x2 = SS volume (%) =% saline volume to total weight (v / w); x3 = TA (%) =% of tannic acid weight in relation to total weight (w / w). According to Table 6, the maximum water capacity that the castor cake absorbed in 10 grams was 11 mL of water, ie, in a medium with castor cake and water, 55% of the weight of the medium was represented by water. .

Table 6: Castor Pie Water Absorption Capacity. Water Absorption Capacity

 Sample Mass * Absorbed Water Volume Moisture (¾)

 10 g 1 1 ul 55

* Weight of castor bean pie posted to assess water absorbing capacity in its medium.

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

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

A) DCCR for Phytase Production

The central rotational composite design was used to evaluate the response or dependent variable of phytase enzymatic activity. The assays were incubated with the microorganism for 72 hours, which was the time with the highest phytase activity. The independent variables studied were: relative air humidity, tannic acid concentration and the percentage of saline volume added in relation to the medium. Table 9 shows the matrix of the independent variables under study, with real and coded values, and the phytase dependent variable during 72 hours of incubation.

Analyzing the activity values obtained in Table 9, there is an increase in the enzymatic activity, ranging from 1.97 U / mL for test 3 (66% relative humidity, 49% of the volume of Saline solution in relation to total weight and 6% tannic acid) to a maximum activity of 42.01 U / mL for test 2 (84% relative humidity, 31% saline volume and 6% tannic acid).

Table 9: DCCR 2 ³ matrix and the response of phytase enzymatic activity after 72 hours of incubation.

Coded Values Real Values Answer

 Phytase

Test'; ¾ UR <%) SS volume (%)

 (U / mL) l -1 -1 -1 66 31 6 17.4

2 1 -1 -1 84 31 6 42.01

3 -1 1 -1 66 49 6 1 _T 97

4 1 1 -1 84 49 6 8.8

5 -1 -1 1 66 31 10 10.69

6 1 -1 1 84 31 10 28.32

7 -1 1 1 66 49 10 2.51

S 1 1 84 49 10 4.96

9 -1.68 0 0 60 40 s 2, 1

10 1,668 0 90 40 8 37,84

11 Q -1.08 0 75 25 s 29.19

12 ϋ 1.68 0 75 55 8 538

13 0 0 -1.68 75 40 4.6 6.91

14 0 0 1.68 75 40 11.4 7.18

15 0 0 0 75 ^'40 8 4.81

16 0 0 § 75 40 S 5.58

17 0 0 0 75 40 8 4.62 xl = RH (%) = percentage of relative air humidity; x2 = SS volume (%) =% Saline volume to total weight (v / p); x3 = TA (%) =% of tannic acid weight in relation to total weight (w / w).

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

With the results of Table 10, it was found that the linear and quadratic variables of AT (%) (tannic acid concentration) and interaction UR (%) with AT (%) and RH (%) with TA (%) were not significant, being evaluated for Residues at Source of Variation, presenting pvalue equal to 0.17, 0.67, 0.36 and 0.18, respectively. Thus, only the variables Mean, RH (%) (L) and (Q), SS volume (%) (L) and (Q) and interaction RH (%) and SS volume (%) were evaluated for Source Regression. ANOVA variation.

Table 10: Results of Regression Coefficient, Standard Error, t, p and Confidence Limit in optimizing culture medium components (relative humidity, saline volume and tannic acid) on phytase activity.

Coefficient Limit Limit

 Error

 of t (7) p-value Trust Default Trust

 Regression - $> 0, <Hs + 90,%

Average * 5.06 2.37 2.13 0.0703 0.56 9.54

(1) RH (%) {L) * 8.14 1.11 7.31 0.0001 6.02 10.24

RH (%> (<¾ * 5.16 1.23 4.21 0.0039 2.84 7.48

(2) SS volume (%) (L> * -8.80 1.11 -7.91 0.0001 -10.91 -6.69 SS volume (%) (Q) * 4/16 1.23 3 .39 0.0115 1.83 6.47

(3) AT <%) (L) -1.70 1.11 -1.53 0.1701 -3.81 0.40

TA (%) (Q) 0.54 1.23 0.44 0.6742 -1.78 2.85

1L x 2L * -4.12 1.46 -2.83 0.0253 -6.87 -1.36

HL x 3L -L42 1.46 -0.98 0.3614 -4.17 1.33

2L x 3L 2, 14 1.46 1.47 0.1851 -0.61 4.8

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

Analysis of variance (ANOVA) is shown in Table 11.

Table 11: Analysis of variance in the study of the effect of culture medium components (RH (%), SS volume (%) and TA (%)) on phytase activity. Sum Source Average Degrees

 Quadratic Variation Quadratic Freedom

 Regression 2514 5 503 25.8 0.00001

Waste 214 11 20

 Lack of Adjustment 213.5 9 23.72 91.2

 Pure ro 0.517 2 0.26

 Total 2727.6 16

R2 = 0.92 Ftab (0.1.5; 11) = 2.45 Ftab (0.1.9; 2) = 9.38

For the correlation coefficient (R) the value obtained was 0.96, for the determination coefficient (R ² ) 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 Misadjustment obtained from ANOVA was 91.2 (9.72 times higher than the Ftabeled value = 9.38) and indicated a misadjustment of the model larger than indicated by the tabulated value. The Regression F value obtained was 25.8 (10.53 times higher than the Ftabeled value = 2.45), indicating that the model for phytase activity can be considered statistically significant at 90% level. confidence.

These results can be considered satisfactory and sufficient, allowing to obtain a coded model that describes the answers as a function of the analyzed variables. From the validation of the study parameters, we obtained the quadratic polynomial model that represents the enzymatic activity behavior (1): 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. Annex 2 shows the effects of the components relative humidity and percentage of saline volume in relation to the total weight of the medium on the production of tanase by Paecilomyces variotii.

B) DCCR for Tanase Production Table 12 shows the matrix of the independent variables under study, with actual, coded values and the tanase dependent variable after 48 hours of fermentation.

Analyzing the values of tanasic activity obtained. Table 12 shows an increase in activity, ranging from 104 U / mL in test 12 (75% relative humidity, 55% added saline volume to total medium weight and 8% 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: OCCR 2 ³ matrix and the response of tanase enzymatic activity after 48 hours of fermentation.

Coded Values Actual Values Answer

Tanase

LiR tests) SS volume (¾) AT f / e)

 (U / mL)

1 -1 -1 -1 66 31 6 384

 2 I -1 84 31 6 573

3 -I 1 -1 66 49 6 124

 4 1 I -1 84 49 6 216

5 -1 -1 1 66 31 10 199

 6 1 -1 1 84 31 10 206

 7 -1 1 1 66 49 10 218

S 1 1 1 84 49 10 204

9 -IM 0 0 60 40 s 120

10 1.68 0 0 90 40 8 430

 11 0 -L6S 0 75 25 8 35S

12 0 1.68 0 75 55 8 104

13 0 0 -1.68 · 75 40 4.6 386

 14 0 0 1.68 75 40 1L4 172

 15 0 0 0 75 40 8 326

U 0 0 0 75 40 8 302

17 0 0 0 75 40 8 288 xl = RH (%) = relative humidity (%); x2 = SS volume (%) =% Saline volume to total weight (v / p); x3 = TA (%) =% of tannic acid weight in relation to total weight (w / w).

Table 13 presents the regression coefficients, standard error, t, p-value and confidence limit of the variables and their interactions, in response to tannase activity with a 90% statistical confidence limit (p <0.10).

Table 13: Results of Regression Coefficient, Standard Error, t, p and Confidence Limit in optimizing culture medium components (relative humidity, saline volume and tannic acid) on tanase activity.

Coefficient Limit Limit

 Error

 of t (7) p-Yalor Confidence Standard Confidence

Regression -90,% +90 ₉ ο

Average * 305 25 12.4 0.000005 258 352

(1) RH (%) (L) * 58 12 5.0 0.001504 36 80

I R (%) (Q) - 13 -0.7 0.489433 -33 15

(2) volume SS (%) (L> * -75 12 -6.5 0.000333 -97 -53 SS volume (° / o) (Q) * -25 13 -1, 0.091974 -49 -0.7

(3) AT (%) (L> *. -61 12 -5.3 0.001181 -83 -39

TA (%) (Q) -8 13 -0.6 0.555943 -32 16

1L x 2L -15 15 -1.0 0.361548 -43 14

1L x 3L * -36 15 -2.4 0.048719 -65 -7

2L X 3L * 79 15 5.2 0.001194 51 108

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

With the results of Table 5, the variables RH (%) quadratic (relative humidity), AT (%) quadratic (tannic acid concentration) and interaction RH (%) and SS volume (%) were not significant, showing p -value 0.48, 0.55 and 0.36, respectively. Thus, only the variables RH (%) (L), SS volume (%) (L), SS volume (%) (Q), AT (%) (L), interaction UR (%) with AT (%) and SS volume interaction (%) with TA (%) were evaluated for Source of Variation Regression in ANOVA. Analysis of variance (ANOVA) is shown in Table 14.

Table 14: Analysis of variance in the study of the effect of culture medium components (RH (%), SS volume (%) and TA (%)) on tanase activity.

Sum Fout Average Degrees

 Quadratic Variation Quadratic Freedom

 Regression 240329 6 40055 253 0.00001

Waste 15826 10 1583

 Lack of Adjustment 15086.9 8 1886 5, 1 1

 Pure Error 738.7 2 369

 Total 256154.5 16

R 2 = 0.94 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 (R ² ) was 0.94, indicating a good correlation between the values obtained by the experiment and those predicted by the model. The F-value of Non-Adjustment obtained from ANOVA was 5.11 (1.83 times lower than the value of Ft = 9.37), resulting in a lack of model adjustment lower than indicated by the tabulated value. . The regression F value obtained was 25.3 (10.2 times higher than the Ftabeled value = 2.46), indicating that the model for tannase activity can be considered statistically significant at 90% level. confidence.

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

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. Annex 3 (a), (b) and (c) illustrate the effects of the components relative humidity, tannic acid concentration and percentage of volume of saline in relation to the total weight of the medium in the production of tanase by Paecilomyces variotii.

Example 3; 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 on filtration was called crude solid extract. The filtrate was centrifuged at 7100 xg for 30 minutes at 4 ° C and was then called crude enzyme extract (Lekha, PK; Lonsane, BK). , pp. 215-260, 1997). The enzyme extract was used to determine the activity of the tanase and phytase enzymes. 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 as phenolic compounds, hydrolysable tannins, condensates, evaluation of the presence of ricin and determination of the detoxification process.

A) Total Phenol Extraction The following solvents were used for the extraction process, in the proportion of 50% solvent and 50% water: Ethyl Acetate, Acetone and Methanol. It was also tested with the 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 150 rpm shaking 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 Follin-Ciocaulteau technique (Singleton, VL Rossi) was used. AJ Colorimetry of total phenolics with phosphomolybdic phosphotungstic acid reagents. American Journal of Enology and Viticulture 20, p. 144-158, 1965) for determination of phenols present in the sample. This method is based on the reduction of phosphomolybdic and phosphotungstic acid by the phenol hydroxyls producing a blue color, quantified by spectrophotometry at 760 nm.

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

C) Determination of Hydrolyzable Tannins The methodology of Brune and collaborators (Brune, Hallberg, L.

Skânberg, A. Determination of iron-binding phenolic groups in foods. Journal of Food Science 56, p. 128-131, 1991) and sample extraction was performed as indicated for the total phenols. An iron (III) ammonium sulphate solution called FAS was used. The solution consisted of: 89% 0.1 M acetate buffer - pH 4.4; 10% 1% gum arabic solution; 1% 5% ammonium iron (III) sulphate solution. 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 followed by a tannic acid calibration curve according to the methodology described.

D) Determination of Condensed Tannins

The methodology of Prince and co-workers was employed (Price, M.L. Van Scoyoc, S. Butler, LG. Critical evaluation of the vanillin reaction as an assay for tannin in sorghum grain. Journal of Agricultural and Food Chemistry 26, p. 1214 - 1218, 1978), being specific for flavonoids and dihydroxychalconas having single bonding at positions 2 and 3 and a free beta ring hydroxyl. 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 to form a red compound.

Sample extraction was performed as in hydrolysable phenols and tannins. A vanillin solution containing: 1 part vanillin solution (1% vanillin dissolved in methanol) and 1 part HCI solution (8% HCl in methanol) was used.

The method was performed by adding 1 mL of the study sample and 5 mL of the vanillin solution over 5 minutes, and 1 mL of the vanillin solution was placed every 1 minute. The blank consisted of 1 mL of distilled water and 5 mL of vanillin solution, then the reaction was conducted for 20 minutes and finally read at a spectrophotometer at 500 nm. The course of the reaction was followed by a catechin calibration curve according to the methodology described. D) Results

Table 15 shows the Total Phenol concentrations 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 bean cake. Table 15: Concentration of Total Phenols in unfermented castor bean cake in different types of solvents.

Total Phenols (mg / g sample)

 Solvent * Average

Ethyl Acetate 42.93 ± 1.75

 Acetone 46.99 ± 1.55

Methanol 32.25 ± 2.41

Hexane 3.28 ± 0.37 * Acetate = 1: 1 Ethyl Acetate and Distilled Water; Acetone = 1: 1 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 presented the highest 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 food samples, especially vegetables. But the above results indicate that the best solvent for phenolic compounds extraction are those with polarity, which is not the case with Hexane because it is a nonpolar 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 their contents before and after the castor bean cake fermentation.

The levels of Total Phenols and Hydrolyzable Tannins of the samples, fresh castor bean cake and optimized and fermented culture medium for phytase production (after 72 hours of fermentation) are presented in Table 16.

Table 16: Concentration of Total Phenols and Hydrolyzable Tannins before and after fermentation.

Total Phenols and Hydrolyzable Tannins (mg / g sample)

 Samples * FT TH

TM 28.14 ± 2.16 3.34 + 0.19

 TM F72h 14.03 ± 5.4 0.52 ± 0.18

* TM = fresh castor bean pie; TM F72h = optimized and fermented culture medium for phytase production (after 72 hours of fermentation). The results showed that the fermentation of castor bean cake by Paecilomyces variotii after 72 hours of incubation decreased the concentration of total phenols as well as hydrolysable tannins. Probably the microorganism produced enzymes that hydrolyzed these compounds, such as tannase, which has the ability to cleave hydrolyzable tannins. For these samples, insufficient concentrations of condensed tannins were not detected, probably not sufficient amounts of samples were placed in the extraction process to determine the condensed tannins.

No studies were found in the recent literature that involved the determination of phenolic compounds in castor bean cake, as these residues are not yet of interest in the production of animal feed and consequently there are no studies to analyze compounds of functional interest to the animal.

Xu et al. (Xu, L; Diosady, LL Rapid method for total phenolic acid determination in rapeseed / canola female. Food Research International 30, pp. 571-564, 1997) quantified the presence of phenolic compounds in canola, a seed used for extraction of its oil for the biofuels sector. An extraction step was carried out in which the sample underwent acid extraction of acetone followed by alkaline hydrolysis, then acidified, then extracted with ethyl ether and ethyl acetate and finally evaporated and dissolved in methanol. . This extraction was performed to obtain three types of phenols: free, esterified and insoluble, favoring a larger range of compounds identified in the colorimetric analysis. According to the results, the canola showed 1683 milligrams of phenolic compounds per 100 grams of sample.

According to Kozlowska and colleagues (Kozlowska, H .; Naczk,.; 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 found that castor bean cake presented a composition of phenolic compounds more expressive in relation to canola, presenting advantage over its phenolic composition for future use as animal feed. Example 4: EVALUATION OF SOLID FERMENTATION PURPOSE DETAXIFICATION

A) SDS-PAGE Ricin Detection

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

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 castor cake (Annex 4).

First, extraction was made by adding phosphate buffered saline (PBS) pH 7.2 in a 1: 4 ratio (1 g solid residue to 4 mL buffer) in the residue which was stirred for 3 hours, then centrifuged at 14000 rpm for 15 minutes at room temperature. Protein quantification was determined for the determination of ricin in the sample (Bradford, MM A rapid and sensitive method for the quantification 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 μί of the supernatant to 40 æl buffer containing Sodium Dodecyl Sulfate (SDS) and β-mercaptoethanol, totaling 120 μί. Of this total, 25 μί were applied to run the 12% SDS-PAGE gel (10x10x0.1 cm). After the run, proteins were fixed and stained for 1 hour with 0.1% Coomassie-Brilliant-Blue solution in methanol / acetic acid / water (45/10/45, v / v / v). After this 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 where ricin appears in the gel are in the range between 31.3 and 38.2 kDa and are also visualized in the run with purified ricin. The samples of fresh castor bean cake and autoclaved castor bean cake presented the two bands of ricin in the gel, showing that the methodology is valid for identification of this compound in protein extract samples. 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 compound hydrolysis. In the 48 and 72 hour fermentation samples, the bands where ricin are 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 minimal protein detection in SDSPAGE by Coomassie Blue is 10 g / mL sample, lower concentrations are hardly detectable by the method (Anandan, 2005); (Godoy, 2009) and (Schagger, H. Tricine-SDS-PAGE. Nature Protocols 1, pp. 16-22, 2006).

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

B) Toxic Activity Test in Cell Culture

The MTT test was used according to Mosmann 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, type RAW 264.7 cells were cultured in DMEM glasshouse at 37 ° C in the presence of C0 ₂ in its atmosphere. For 100 μί of cell culture, a volume of 100 μί MTT (5 mg / mL in PBS) and sample extract were added to microplate wells. Each microplate was incubated in the oven for 24 hours. After incubation, the microplates were read in a spectrophotometer at 540 nm absorbance.

The assay was conducted using the ricin obtained as described in the extraction item to determine the minimum dose to induce cell culture death. For this, different dilutions of protein made in PBS pH 7.0: 100 were used. μ§ / ιτιί, 50 μg / mL, 10 μ8 / ιηί, 1 μg / 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 live cell cultures to verify its viability in the presence of fresh and fermented castor bean cake.

According to Figure 7, the minimum concentration required for cell death to occur was 1 g / mL cake extract. Lower concentrations of cell viability in the culture medium were close to 100% and thus did not interfere with its growth. From these values, the second test was performed with extracts of fermented material with 1 μg / mL at different times (24, 48 and 72 hours of fermentation). According to Figure 8, during the fermentation process, in its time progress, the cell viability was increasing and after 72 hours of fermentation presented approximately 100% of the cell viability. Thus, probably the microorganism Paecilomyces variotii 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 climate chamber for the optimum medium for phytase production in castor bean cake.

Relative Humidity of Cultivation Medium (%)

 Sample * Incubation Time ** Initial Humidity Final Humidity

TM 72h 72 hours 27% ^: 27%

* TM 72h = castor bean cake culture medium with 25% of saline volume in relation to the total weight of the medium (v / p).

** 90% Relative Humidity Climate Chamber Incubation at 30 ° C.

Claims

1. Culture medium for phytase enzyme production by the microorganism Paecilomyces variotii, characterized in that it is from castor bean residual cake.

2. Culture medium for production of the phytase enzyme by the microorganism Paecilomyces variotii characterized by its residual castor bean cake and saline solution;

Culture medium for production of the phytase enzyme by the microorganism Paecilomyces variotii according to Claim 2, characterized in that the saline solution is composed in grams per liter of 1 part KH ₂ P0; 2 parts of NH NO ₃ ; 0.2 parts MgSO ₄ .7H ₂₀ ; 0.02 shares of CaCl ₂ .2H ₂ 0; 0.004 shares of MNCI ₂ .4H ₂ 0; 0.002 parts of Na ₂ Mo0 ₄ .2H ₂ 0 and 0.0025 parts of FeS0 ₄ .7H ₂ 0.

Culture medium for production of the phytase enzyme by the microorganism Paecilomyces variotii according to Claim 2, characterized in that it does not contain phytic acid.

Culture medium for production of the phytase enzyme by the microorganism Paecilomyces variotii according to claim 2, characterized in that it preferably contains no tannic acid.

Culture medium for production of the phytase enzyme by the microorganism Paecilomyces variotii according to Claim 2, characterized in that it contains from 25 to 28% of saline in relation to the total weight of the medium.

Culture medium for production of the phytase enzyme by the microorganism Paecilomyces variotii according to Claim 6, characterized in that it preferably contains 25% saline in relation to the total weight of the medium.

Culture medium for production of the phytase enzyme by the microorganism Paecilomyces variotii according to Claim 2, characterized in that it preferably contains 75% of the total weight of the castor bean cake.

Phytase enzyme characterized by being produced by the microorganism Paecilomyces variotii by solid fermentation.

10. Phytase enzyme characterized by being produced by the microorganism Paecilomyces variotii by solid fermentation in residual castor bean cake.

Phytase enzyme characterized in that it is produced by the microorganism Paecilomyces variotii by solid fermentation in culture medium as described in claims 1 to 8.

12. Phytase enzyme produced by the microorganism Paecilomyces variotii by solid fermentation in residual castor bean cake, preferably obtained after 72 hours of incubation.

Phytase enzyme produced by the microorganism Paecilomyces variotii by solid fermentation in culture medium as described in claims 1 to 8, which is preferably obtained after 72 hours of incubation.

Phytase enzyme produced by the microorganism Paecilomyces variotii by solid fermentation in castor bean residual cake characterized by being obtained in between 85 and 90% of ambient relative humidity.

Phytase enzyme produced by the microorganism Paecilomyces variotii by solid fermentation in residual castor bean cake characterized by being preferably obtained in an environment with 90% relative humidity.

16. Culture medium for production of tannase by the microorganism Paecilomyces variotii, characterized in that it is from castor bean residual cake.

17. Culture medium for the production of enzyme tanase by the micro-organism Paecilomyces varioti, which consists of castor bean cake, saline and tannic acid;

Culture medium for the production of enzyme tase by the microorganism Paecilomyces variotii according to claim 17, characterized in that the saline solution is composed in grams per liter of 1 part KH ₂ P0 ₄ ; 2 parts NH ₄ NO ₃ ; 0.2 parts MgSO ₄ .7H ₂₀ ; 0.02 shares of CaCl ₂ .2H ₂ 0; 0.004 parts MnCl ₂ .4H ₂ 0; 0.002 parts of Na ₂ Mo0 ₄ .2H ₂ 0 and 0.0025 parts of FeS0 ₄ .7H ₂ 0.

Culture medium for production of enzyme tase by the microorganism Paecilomyces variotii according to claim 17, characterized in that it contains from 4.6 to 6.0% tannic acid in relation to the total weight of the medium.

Culture medium for production of enzyme tase by the microorganism Paecilomyces variotii according to claim 19, characterized in that it preferably contains 4.6% tannic acid in relation to the total weight of the medium.

Culture medium for the production of enzyme tasease by the microorganism Paecilomyces variotii according to claim 17, characterized in that it contains from 25 to 33% of saline in relation to the total weight of the medium.

Culture medium for the production of enzyme tasease by the microorganism Paecilomyces variotii according to claim 21, characterized in that it preferably contains 25% saline in relation to the total weight of the medium.

Culture medium for production of enzyme tase by the microorganism Paecilomyces variotii according to claim 17, characterized in that it preferably contains 65.4% of castor bean cake.

24. Tannase enzyme characterized by being produced by the microorganism Paecilomyces variotii by solid fermentation.

25. Tannase enzyme characterized by being produced by the microorganism Paecilomyces variotii by solid fermentation in residual castor cake.

Enzyme tannase characterized by being produced by the microorganism Paecilomyces variotii by solid fermentation in culture medium as described in claims 16 to 23.

27. Enzyme tannase produced by Paecilomyces variotii microorganism by solid fermentation in residual castor bean cake, which is preferably obtained after 48 hours of incubation.

Enzyme tannase produced by Paecilomyces variotii by solid fermentation in culture medium as described in claims 16 to 23, which is preferably obtained after 48 hours of incubation.

An enzyme tase produced by Paecilomyces varíotii by solid fermentation in culture medium as described in claims 16 to 23, characterized in that it is obtained in an environment with relative humidity between 84 and 90%.

Tase enzyme produced by the microorganism Paecilomyces varíotii by solid fermentation in culture medium as described in claims 16 to 23 characterized in that it is preferably obtained in an environment with relative humidity of 90%.

31. Culture medium for the simultaneous production of the phytase and tanase enzymes by the microorganism Paecilomyces varíotii, characterized by its residual castor bean cake.

32. Culture medium for the simultaneous production of phytase and tanase enzymes by the micro-organism Paecilomyces varíotii characterized by its castor bean cake and saline solution;

A culture medium for the simultaneous production of the phytase and tanase enzymes by the micro-organism Paecilomyces varíotii according to Claim 32, characterized in that the saline solution is composed in grams per liter of 1 part KH ₂ P0 ₄ ; 2 parts NH ₄ NO ₃ ; 0.2 parts MgS0 .7H ₂ 0; 0.02 shares of CaCl ₂ .2H ₂ 0; 0.004 shares of MNCI ₂ .4H ₂ 0; 0.002 parts Na ₂ Mo0 ₄ .2H ₂ 0 and 0.0025 parts of FeS0 ₄ .7H ₂ 0.

A culture medium for the simultaneous production of the phytase and tanase enzymes by the microorganism Paecilomyces varíotii according to claim 32, characterized in that it does not contain phytic acid.

A culture medium for the simultaneous production of the phytase and tanase enzymes by the microorganism Paecilomyces variotii according to claim 32, characterized in that it contains tannic acid of 4,6 to 6% tannic acid.

Culture medium for the simultaneous production of the phytase and tanase enzymes by the microorganism Paecilomyces varíotii according to claim 32, characterized in that it contains from 25 to 33% of saline in relation to the total weight of the medium.

A culture medium for the simultaneous production of the phytase and tanase enzymes by the microorganism Paecilomyces variotii according to claim 36, characterized in that it preferably contains 25% saline in relation to the total weight of the medium.

A culture medium for the simultaneous production of the phytase and tanase enzymes by the microorganism Paecilomyces variotii according to claim 32, which comprises from 65,4 to 75% of the total weight of castor bean cake.

Phytase enzyme characterized in that it is produced by the microorganism Paecilomyces variotii by solid fermentation in culture medium as described in claims 31 to 38.

Phytase enzyme produced by the microorganism Paecilomyces variotii by solid fermentation in culture medium as described in claims 31 to 38, which is preferably obtained after 72 hours of incubation.

Phytase enzyme produced by the microorganism Paecilomyces variotii by solid fermentation in culture medium as described in claims 31 to 38 characterized in that it is obtained in an environment with relative humidity between 85 and 90%.

Phytase enzyme produced by the microorganism Paecilomyces variotii by solid fermentation in culture medium as described in claims 31 to 38 characterized in that it is preferably obtained in an environment with relative humidity of 90%.

Enzyme tanase characterized by being produced by the microorganism Paecilomyces variotii by solid fermentation in culture medium as described in claims 31 to 38.

Enzyme tanase produced by the microorganism Paecilomyces variotii by solid fermentation in culture medium as described in claims 31 to 38, which is preferably obtained after 48 hours of incubation.

Tase enzyme produced by the microorganism Paecilomyces variotii by solid fermentation in a culture medium as described in claims 31 to 38 characterized in that it is obtained in an environment with relative humidity between 84 and 90%.

Enzyme tannase produced by the microorganism Paecilomyces variotii by solid fermentation in culture medium as described in claims 31 to 38 characterized in that it is preferably obtained in an environment with relative humidity of 90%.

Use of the phytase enzyme obtained according to claims 1 to 8, characterized in that it is for food supplementation.

Use of the phytase enzyme obtained according to claims 1 to 8, characterized in that it is for supplementation in animal feed.

Use of the enzyme tanase obtained according to claims 17 to 23, characterized in that it is for food supplementation.

Use of the enzyme tanase obtained according to claims 17 to 23, characterized in that it is for supplementation in animal feed.

Use of the phytase enzyme obtained according to claims 31 to 38, characterized in that it is for food supplementation.

Use of the phytase enzyme obtained according to claims 31 to 38, characterized in that it is for supplementation in animal feed.

Use of the enzyme tanase obtained according to claims 31 to 38, characterized in that it is for food supplementation.

Use of the enzyme tanase obtained according to claims 31 to 38, characterized in that it is for supplementation in animal feed.