Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/14—Fungi; Culture media therefor

Definitions

• 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.
• 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.
• animal feed residues represents a viable alternative for this sector, as well as for the production of biocatalysts through solid fermentation.
• Solid state fermentation 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).
• 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).
• FS submerged fermentation
• 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.
• agro-industrial residues 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).
• FES 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.
• Agro-industrial waste is largely produced by human, agricultural and industrial activity.
• rice husk, straw and bran 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).
• 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-art ⁇ n, I .; Alvarez-Garc ⁇ a, 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.
• 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.
• 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%.
• 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.
• castor bean cake cannot be used as a protein source because of its toxicity and is generally used as an organic fertilizer (Jones, 1947).
• toxins present are ricinin, albumin 2S and ricin.
• Ricinin is a toxic alkaloid called 1,2-dihydro-4-methoxy-1-methyl-2- ⁇ -3-pyridinecarbonitrile (C 8 H 8 2 02) present in castor bean cake with low toxicity (Beltr ⁇ o, NOR). ; Lima, RLS Application of castor oil as a source of energy: Biodiesel In: Azevedo, DMP; Beltr ⁇ o, NOR (Ed.) Agribusiness of castor in Brazil 2 * Edition, Embrapa Technological Information, Bras ⁇ lia-DF, p 395-416, 2007).
• CB-1A Cosmetic Bean Allergen
• Albumin 2S 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 Maranh ⁇ o 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).
• 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 ⁇ resolution. Proteins: Structure, Func and Genetics 10, pp. 260-269, 1991).
• RIP ribosome inactivating protein
• 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.
• 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.
• 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.
• 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).
• tannins on animal nutrition is due to their ability to bind to macromolecules, decreasing the absorption of these components.
• 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 Rodr ⁇ guez-Herrera, R.; Jr, AR; Aguilar, CN Microbial production of tannase: an enzyme with potential use in the food industry (Lebensstoff-Wissenschaft und-Technologie 37, p. 857-864, p.
• 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.
• 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.
• 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.
• 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).
• 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.
• microorganisms can be genetically engineered for enzyme improvement and production (Battestin, V; Macedo, G. A. Tannase Production by Paecilomyces variotii. Bioresource Technology 98, p.
• 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.
• 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.
• 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.
• 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).
• 3-phytase EC3.1.3.8
• 6-phytase EC3.1.3.26
• 3-phytase myo-inositol-hexakisphosphate
• 6-phytase 6-phytase
• 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).
• 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).
• 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).
• 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).
• 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.
• 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.
• 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 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).
• 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.
• the castor bean cake must first be crushed and subjected to a 1.68 mm 10 mesh sieve size separation process.
• 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%.
• the castor bean residual cake 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
• 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.
• PDA - OXOID - CM0139 Potato Dextrose Agar
• tannic acid supplement Teanal B - Prozyn - BioSolutions
• a cell suspension with a homogenizer should be performed, resulting in a concentration of 9 x 10 6 cells / mL.
• 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.
• 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%.
• 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.
• 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%.
• the chamber when 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.
• Table 4 Evaluation of fermentation kinetics for tannin activity in castor bean pie.
• Example 2 EXPERIMENTAL DESIGN
• DCCR central rotational composite design
• 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 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.
• 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.
• Table 8 Design matrix containing coded variable values.
• 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.
• 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.
• Table 9 DCCR 2 3 matrix and the response of phytase enzymatic activity after 72 hours of incubation.
• 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).
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• Table 14 Analysis of variance in the study of the effect of culture medium components (RH (%), SS volume (%) and TA (%)) on tanase activity.
• 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)
• 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.
• 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.
• 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.
• Acetone solution was used to analyze their contents before and after the castor bean cake fermentation.
• Table 16 Concentration of Total Phenols and Hydrolyzable Tannins before and after fermentation.
• 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.
• 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.
• 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).
• 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.
• the presence of ricin is poorly visible, demonstrating a probable initiation of compound hydrolysis.
• 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).
• 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).
• type RAW 264.7 cells were cultured in DMEM glasshouse at 37 ° C in the presence of C0 2 in its atmosphere.
• a volume of 100 ⁇ 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.
• different dilutions of protein made in PBS pH 7.0: 100 were used. ⁇ / ⁇ , 50 ⁇ g / mL, 10 ⁇ 8 / ⁇ , 1 ⁇ g / mL, 100 ng / mL, 50 ng / mL, 10 ng / mL, 1 ng / mL, 100 pg / mL, 50 pg / mL, and 10 pg / mL.
• 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.
• 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.
• the second test was performed with extracts of fermented material with 1 ⁇ g / mL at different times (24, 48 and 72 hours of fermentation).
• the cell viability was increasing and after 72 hours of fermentation presented approximately 100% of the cell viability.
• 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.
• TM 72h castor bean cake culture medium with 25% of saline volume in relation to the total weight of the medium (v / p).

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