FAIDIGA, Izabel Cristina (Chácara São José, S/N° Bairro Sap, Caixa Postal 25 -000- Bariri- SP, 17250, BR)
1. Process for manufacturing enzymatic preparations obtained from bird feathers, characterized by comprising:
(a) preparing the chicken feathers by washing, drying and delipidation; (b) cultivation of microorganisms Bacillus subtilis strain AMR in a yeast extract medium;
(c) fermentation of feathers prepared from step (a) by microorganisms cultivated as in step (b), producing peptides and one or more enzymes selected from the group consisting of proteases, keratinases, amylases and cellu- lases.
2. Process according to claim 1 , characterized by additionally comprising the step of freeze-drying the product obtained in step (c) after removal of the microorganisms by centrifugation producing an enzymatic powder preparation. 3. Process according to claim 1 or 2, characterized wherein the delipidation of step (a) is carried out with chloroform solution: methanol (1 :1 v/v) for 1 h under agitation at 300 rpm at ambient temperature.
4. Process according to any of claims 1 to 3, characterized wherein after the step of delipidation in step (a), the feathers are dried overnight at 600C.
5. Process according to any of claims 1 to 4, characterized wherein the yeast extract medium of step (b) comprises yeast extract 0.5%, peptone 0.5%, KCI 2.0% and saccharose 2.0%.
6. Process according to any of claims 1 to 5, characterized wherein the cultivation of step (b) is carried out for 2-3 days at 28°C under constant agitation (300 rpm) and washed with saline (2x 3000rpm/20min).
7. Process according to any of claims 1 to 6, characterized wherein step (c) comprises the transfer of the microorganisms to medium PBS pH 7.0-8.0 (NaH2PO4 0.06M and K2HPO4 0.04M) with 1% chicken feathers, being cultivated for 5-7 days at 28°C under agitation at 300 rpm.
8. Enzymatic preparations, characterized by being obtainable by the process as defined in any of claims 1 to 7.
9. Use of enzymatic preparations as defined in claim 8, characterized by being for adding to the post-pellet of animal feed.
10. Use of enzymatic preparations as defined in claim 8, characterized by being for improving the digestibility of a feather meal. 11. Use of enzymatic preparations as defined in claim 8, characterized by being for assisting the digestion of feathers in digestors before they are heated to obtain a feather meal.
12. Use of enzymatic preparations as defined in claim 8, characterized by being for use as a capillary transformation agent. 13. Animal feed, characterized by containing enzymatic preparations as defined in claim 8.
14. Capillary transformation agent, characterized by containing enzymatic preparations as defined in claim 8.
The present invention refers to a process for manufacturing enzymatic preparations containing keratinases from birds feathers and has, among others aspects, applications in animal feed and in human hair. Background of the Invention and Related Prior Arts Over recent years, Brazil has significantly advanced in the field of aviculture, and is currently the world's largest exporter of chickens, accounting for 40% of world exports of the product and is the third largest producer on an increasing scale. By virtue of the expansion in the poultry industry, there has been a tremendous increase in the demand for raw materials for feed production and also in the production of agro-industrial residues, represented by discarded feathers (Moura.C.C. 2004. Farinha de penas e sangue em ragδes para sumos em crescimentos Soc. Bras. Zootecn. Vicosa.v 23:633-641 , Scapim .M. R., Loures, EG., Rostango, H. Cecon, P., Scapim, CA. 2003 Avaliagao nutricional de farinha de penas e de sangue para frangos de corte submetida a diferentes tratamentos termicos Scientiarum animal Sciences,v25, p. 91 -98). It has been estimated that in 2008, the sector produced between 700 and 800 thousand tons of feathers (5 to 7% of the weight of the birds), containing 90% of keratin.
The accumulation of feathers and their slow deterioration, forming sulphurous compounds that give off an extremely unpleasant odor, has spurred studies on keratinolytic microorganisms and the processes involved in biodegradation of keratin (Onifade, A A, Al-Sane, N A, Al- Musallam, AL- Zarban, S.1998. Review: Potentials for biotechnological applications of keratin-degrading microorganisms and their enzymes for nutriti- onal improvement of feathers and others keratins as livestock feed resources. Bioresource. Tecnology, 66: 1-19; Friedrich, J. Gradisar, H., Chaumont, J. P. 1999. Screening fungi for synthesis of keratinolytic enzymes. Letters Ap- plied Microbiology 38:127-130, Suntomsuk, W. & Suntornsuk, L.2003. Feather degrading by Bacillus FK 46 in submerged cultivation. Biosource Technology 86:239-243; Brandelli. A. 2008. Bacterial Keratinases: Useful Enzymes for Bioprocessing Agro-industrial Wastes and Beyond. Food Bioprocess Technol 1 :105-116). Countless works on the production of keratinases demonstrate that microorganisms like fungi and bacteria, for example, Aspergillus fumigatus (Santos, R.M.D.B., Almeida, J. and Felix, CR. 1995. Degrada- gao microbiana da queratina, Anais do ENZITEC 95, Organizacao, Institute de Quimica-UFRJ) and Chryseobacterium sp. (Riffel, A., Lucas, F.S., Heeb, P., Brandelli A. 2003. Characterization of a new keratinolytic bacterium that completely degrades native feather keratin. Archives of Microbiology 179: 258-265.), among others, are capable of degrading keratin "in natura" and have a potentially major use in biotechnological processes of feather degrading (Revisto por Bon, Elba, Vermelho, A B , Edit. Said, Suraia & C. L. R. Pietro, Rosemeire .2004. Enzimas como Agentes Biotecnolόgicos. Capftulo Editora Legis Summa, Ltda., Vermelho A.B, Nogueira de MeIo. Branquinha, M.H., Santos, A.L. d'Avila-Levy, CM. Couri, S, Bon.P.S. 2008 Enzymes pro- teoliticas: Aplicagόes Biotecnolόgicas em Enzymes em Biotecnologia: Pro- ducao, Aplicagδes e Mercado 2008 Bon, E. P. S. Corvo Vermelho, A.B, Paiva, CLA a Ferrara, M.A. Coelho.R.R.. Alencastro,R.B. Editora INTERCIENCIA; Vermelho A.B, Termignoni.C, Macedo.A.J., Brandelli, A. Elba P. S. Bone. P.S. 2008. Enzimas queratinolιticas:aplicagόes Biotecnolόgicas em Enzimas em Biotecnologia: Produgao, Aplicagόes e Mercado 2008 b. Bon, E. P.S. Corvo., Vermelho, A.B, Paiva, CLA a Ferrara, M.A. Coelho,R.R.. Alencas- tro.R.B. Editora INTERCIENCIA.).
In Brazil, most discarded feathers are transformed into feather meal. The conventional methods used for pre-treating the feathers are designed to make use of this residue in digestible animal feed are based on physical methods of grinding and cooking, at high temperatures and, this be- ing the case, are large consumers of energy and lead to the destruction of aminoacids, decreasing the biological value of the protein.
According to Naber et a/., 1961 (Effect of processing methods and aminoacid supplementation on dietary utilization of feather meal and protein by chicks. Poult. Sci.Savoy,40: 1234-1245) and Papadoulos, MC. 1985. (Effect of different processing conditions on aminoacid digestibility of feather meal determinated by chicken assay,Agricultura.64: 1729-1741), heat treat- ment alters the structure of the proteins and favors different types of links between proteins and substances such as fats and carbohydrates present in the meal. These new chemical links might compromise the availability of the aminoacids. In relation to the availability of feather meal aminoacids processed under pressure, Naber et al., 1961 (Effect of processing methods and aminoacid supplementation on dietary utilization of feather meal and protein by chicks. Poult. Sci.Savoy,40: 1234-1245) claimed that the processing methods caused significant variations in the nutritional value of the feather meals. Although cooking increases the availability of certain aminoacids, it destroys others, particularly unstable ones, under the effect of the heat. The feather meal contains a high content of crude protein. Over
85% to 90% of this protein is keratin, which, by virtue of its structure and large quantity of sulphurous aminoacids, has low solubility and high resistance to the action of enzymes, and should be hydrolyzed in order to be metabolized by animals (Santos, A. L. S., Gomes, A. V. C, Pessόa, M. F. Mostafa, S. & Curvello, F. A. 2006 Ni ' veis de inclusao de farinha de penas na dieta sobre o desempenho e caracteri ' sticas de carcaca de codornas para corte. Acta Scientiarum. Animal Sciences, 28 (1 ), 27-30). In this context, the hydrolysis of keratin by microbial keratinases is an advantageous option for the digestion of keratin, and being a natural method, non-thermal and without chemical additives, it represents an economic gain for the industry, besides using clean technology.
Processes biocatalyzed by enzymes pollute less. The characteristics of these industrial processes and the biodegradability presented by their effluents meet the requirements of ISO 9000 and ISO 14001 , which es- tablish standards for the quality of products and serve as guidelines for the production process characteristics, emphasizing lower energy consumption, low environmental impact and higher quality of the products. In this context, it is important to highlight that the enzymatic processing of raw materials results in products with greater added value.
The biotechnological processing of feathers for the production of animal feed, in contrast to chemical processing, offers advantages related to the non-destruction of essential aminoacids such as methionine, lysine and histidine, whose feathers already showed sub-optimum levels. Keratin is rich in sulphurate aminoacids such as cysteine and cystine (cysteine-cystine), as well as methionine. Studies show that the presence of these compounds in poultry food is important in the post-molt period (natural shedding of feathers) and also to increase egg laying, jointly with the protein content of the diet (Fi- lho JJ; J Vilar da Silva JH; Silva, EL; Ribeiro, MLG; Domiciano.T., Martins D; Rabello, CBV.2006 Exigencias nutricionais de metionina+cystine para poe- deiras semipesadas do inicio de produgao ate o pico de postura, R. Bras Zootec. vol.35 no.3 suppl.O Vigosa May/June 2006). Another advantage of the biotechnological method is that it does not form non-assimilable aminoa- cid derivatives (Bon, Elba, Vermelho, A B , Edit. Said, Suraia & C. L. R. Pie- tro, Rosemeire .2004. Enzymes como Agentes Biotecnolόgicos. Capitulo Editora Legis Summa, Ltda., Vermelho A.B, Nogueira de MeIo. Branquinha, M. H., Santos, A.L d'Avila-Levy, CM. Couri, S., Bon.P.S. 2008 a. Enzymes proteoliticas: Aplicacόes Biotecnolόgicas. Em Enzymes em Biotecnologia: Produgao, Aplicagόes E Mercado 2008 Bon, E. P. S. Corvo Vermelho, A.B, Paiva , CL.A a Ferrara, M.A. Coelho.R.R.. Alencastro.R.B. Editora INTER- CIENCIA.; Vermelho A.B, Termignoni.C, Macedo.A.J., Brandelli, A. Elba P. S. Bone.P.S. 2008. Enzymes queratinolιticas:aplicagόes Biotecnolόgicas. Em Enzymes em Biotecnologia: Produgao, Aplicagδes e Mercado 2008 b. Bon, E.P.S. Corvo., Vermelho, A.B, Paiva, CLA a Ferrara, M.A. Coelho.R.R.. A- lencastro.R.B. Editora INTERCIENCIA.). Brazil depends on the foreign market for enzymes used as animal feed additives. The list and table 1 below cite the main international industry suppliers of these enzymes and also some of the main proteases commercialized for the food industry. AB Enzymes - Germany Novozyme - Denmark Danisco - Denmark
BioResource International, Inc. - USA
Biocatalysts - UK
Adisseo - France
Bioresource - USA
Toyobo - Japan
Amano Enzyme Inc. - Japan
Nagase Biochemicals - Japan
Enzyme Development - USA
Gist-Brocades - The Netherlands
Alltech - USA
Table 1 Certain enzymes and/or enzymatic preparations containing commercialized proteases and keratinases
In animal feeds, these enzymes play important roles such as removing anti-nutritional factors, increasing the availability of nutrients, increasing the digestibility of non-starch polysaccharides and supplements in the production of endogenous enzymes in adult animals and in immature poultry and pigs.
The most relevant documents from the state of the art are cited below. Patent application PI0313288-9 discloses a method for breeding domestic poultry for slaughter, methods of boosting growth performance, improving the use efficiency of the food, enhancing the food digestion capacity, and decreasing the mortality of immature and developing animals that receive the animal feed. Methods of producing an extract from crude keratinase enzyme and animal feed supplements to obtain the same are also disclosed. The method in question includes the use of keratinase; however, it uses a concentrated and filtered supernatant, which presents purified keratinase from this supernatant.
In an entirely different manner, the present invention uses the crude supernatant centrifuged and freeze-dhed. Additionally, the method of obtaining the enzymes disclosed in patent application PI0313288-9 differs from the present invention by using a medium with soybean and other components. The present invention only uses chicken feathers with a buffer. Lastly, the end product obtained by the process of application PI0313288-9 is a keratinase, whereas the product obtained by the process of the present invention is a mixture of cellulases, amylases, other proteases, as well as keratinase.
European patent EP 0 670 681 discloses a method for maintaining animals on a diet containing keratin and food for animals comprising a source of carbohydrate and a source of proteins comprising keratin, in additi- on to a keratinase capable of hydrolyzing the keratin in an effective quantity to improve the digestion of keratin. The source of proteins containing keratin may also contain an enzyme of Bacillus licheniformis PWD- 1 to improve the digestion of keratin.
Differently to the present invention, the lysate of feathers conta- ins its own bacteria, that is, in this case the enzyme keratinase is together with the microorganisms in the biodigestor, hydrolyzing the feathers and, in a subsequent step, the bacteria are incorporated into the food. The present invention makes use of a hydrolyzed product with enzymes, and all the bacteria are removed from the mixture. Furthermore, the present invention uses Bacillus subtilis strain AMR instead of Bacillus licheniformis PWD-1 and the enzymatic preparation contains, in addition to keratinases, amylases, cellula- ses and other proteases. Therefore, it is concluded that the present method is superior as it does not need to incorporate the bacteria into the food and the enzymatic preparation is richer for having different classes of enzymes. This is also an important difference in terms of result obtained from the choice of strain. Each strain has different quantities and types of enzymes.
In turn, patent US 4.908.220 discloses an animal feed supplement made from partially hydrolyzed feathers, proteins and peptides cleaved from these partially hydrolyzed feathers and cells of Bacillus licheniformis PWD-1 ; and an animal feed comprising a source of carbohydrate and a source of proteins consisting essentially of partially hydrolyzed feathers, proteins and peptides cleaved from these partially hydrolyzed feathers and cells of Bacillus licheniformis PWD-1.
Superiorly, the preparation of the present invention does not pre- sent feather remains, merely aminoacids and soluble peptides. Similarly to that set forth above, these feather lysates do not exist in the mixture of the present invention, in which the feathers are 100% solubilized. The remains of feathers are harmful to the feed because they are not digestible. Additionally, the present invention uses Bacillus subtilis strain AMR instead of Bacillus licheniformis PWD-1.
Patent US 4.959.311 discloses a method of degrading keratin material, comprising the steps of combining the keratin material with Bacillus licheniformis PWD-1 to form a fermenting medium and ferment the medium for a sufficient time to degrade the material. In this patent, the enzyme kera- tinase is purified and its characteristics are described.
Differently to this US patent, the present invention does not use purified enzymes, but rather a mixture containing keratinases, other proteases, amylases and cellulases. The preparation of the present invention includes the peptides and the aminoacids obtained by feather hydrolysis. The commercialization of purified enzymes is hard to incorporate into tons of animal feed. Additionally, the invention uses Bacillus subtilis strain AMR instead of Bacillus licheniformis PWD-1.
Therefore, in an inventive and unexpected manner, the present invention solves the technical problems referred to above.
With the increase in prices of the components for animal feed, enzymes represent an alternative to curb increasing production costs. Enzymatic complexes began to be used frequently in Brazil in the year 2008.
Including additives can lead to savings of up to 3% in food costs, besides reducing the fattening time for poultry. If the birds used to take an average of
40 days to weigh 1.7 Kg, today, thanks to the use of additives, this weight can be achieved on the 34 th day. The housing time is another element to reduce production costs.
Keratinases and other peptidases have acquired major biotech- nological importance due to their ever-increasing industrial applications. They are currently used by the pharmaceuticals industry, in medicines and cosmetics, by the detergents industry, the food industry and the leather in- dustry as depilators. They are also used to treat fabrics in the textile industry and in bioremediation and composting processes.
The world enzymes market is in the order of four billion dollars, of which 2.2 billion dollars is the market for industrial enzymes (technical enzymes, and for the food and animal feed industry). Data compiled by the Ministry of Science and Technology confirm that Brazil lags behind in this field, where the use of catalysis prevails in detriment to biocatalysis. Hence, the Brazilian external market in 2005 was valued at 147.2 million dollars, of which 86% accounts for imports, indicating a technological and strategic disadvantage in terms of production and use of these catalysts in the country. Peptidases represent 60% of world sales of enzymes, of which 40% is microbial in origin. The production of enzymatic complexes as additives in animal feed grows each year as a consequence of the increase in animal production. On the international market, there are Versazyme™ and Valkerase™, produced by BioResource International Inc., in North Carolina, USA. These technological products are concentrates of keratinases of Bacillus lichenifor- mis PWD-1 , which are used to be incorporated into post pellet of poultry feed to assist in digesting the keratin present in the feed. These products have major application in industries that incorporate feather meal into the feed.
Another area of application is in cosmetics, including facial peelings, leather depilation, in the pharmaceutics and textile industry and in the production of detergents, where these keratinases can have major application. One example is Arazyme® produced by Insect Biotech Co., Ltd, In Korea.
The other residues produced in the process comprise bacteria cells and a small quantity of undigested feathers (5%) that can be used in compost as bioactives. Summary of the Invention
The present invention refers to the process for manufacturing enzymatic preparations obtained from bird feathers, comprising the preparation of chicken feathers by washing with detergent or water, drying and delipidation; the cultivation of microorganisms Bacillus subtilis strain AMR in a yeast extract medium; the fermentation of feathers by microorganisms, producing peptides and one or more enzymes selected from the group consisting of proteases, keratinases, amylases and cellulases.
An optional step consists of freeze-drying the product obtained after removing the microorganisms by centrifugation producing an enzymatic powder preparation. Delipidation can be carried out with any delipidation solution, for example, a chloroform solution: methanol (1 :1 v/v) for 1 h under agitation at 300 rpm at ambient temperature. After this step, the feathers are optionally dried overnight at 60 0 C.
The yeast extract medium may comprise yeast extract 0.5%, peptone 0.5%, KCI 2.0% and saccharose 2.0%.
The cultivation can be carried out for 2-3 days at 28°C under constant agitation (300 rpm) and washing can be with saline (2x 3000rpm/20min).
The fermentation of the feathers may comprise the transfer of microorganisms for the medium PBS pH 7.0-8.0 (NaH 2 PO 4 0.06M and K 2 HPO 4 0.04M) with 1% of chicken feathers, being cultivated for 5-7 days at 28°C under agitation at 300 rpm.
The present invention also refers to enzymatic preparations obtainable by the process mentioned above and, additionally, to the use thereof for addition to the post-pellet of animal feed, to improve the digestibility of a feather meal and to assist the digestion of feathers in digestors prior to heating to obtain a feather meal.
Furthermore, the enzymatic preparations of the present invention have use as capillary transformation agents. The present invention also provides an animal feed and a capillary transformation agent. Detailed Description of the Invention
The enzymatic preparations of the present invention contain enzymes such as keratinases and other proteases, as well as cellulases and amylases. The preparations also contain peptides obtained from the keratin of chicken feathers.
The present invention refers to two main embodiments: animal feed and capillary transformation. Embodiment 1 - Animal feed
The enzymatic preparations can be added to the post-pellet of animal feed or be used to improve the digestibility of feathers and feather meal or assist in the digestion of feathers in digestors before they are heated to obtain the feather meal. The preparations can also be used in the post- pellet of feed in general, due to the variety of enzymes.
The enzymatic preparations are obtained by cultivating microorganisms with strains isolated from Brazilian poultry industries. These strains and bacilli kind were isolated from Brazilian poultry and avian granges and were deposited in culture collections at the Oswaldo Cruz Foundation - Brazil. This is the first time that these strains have been used.
The feathers are fermented by the bacilli under optimal pH conditions, temperature and salts, producing highly digestible peptides and enzy- mes such as proteases, keratinases, amylases and cellulases. The product is freeze-dried after removal of the microorganisms by centrifugation producing an enzymatic powder preparation.
The enzymatic method preserves all the aminoacids of the feather. What is more, it uses feathers from the Brazilian poultry industry as agro-industrial residue, being beneficial for recycling this material as it pollutes the environment owing to its difficult and slow decomposition.
The present invention has demonstrated, by way of tests with trypsin and pancreatin, that these enzymes increase the digestibility of the feather meal and the feathers "in natura". Accordingly, with the use of the enzymatic preparation, the feather meal becomes more digestible and can be used in feed for any breeding animal.
Moreover, the present invention transforms a feather meal into a product with greater digestibility. Many poultry industries prefer to sell it to feed manufacturers and not to add it to their own poultry feed. This is a me- thod to recycle discarded chicken feathers.
The microorganisms used are highly keratinolytic and proteolytic, capable of hydrolyzing feathers producing peptides and aminoacids. The present invention used strains of Bacillus sp, which have been used in industrial processes since 1960 and are GRAS ("Generally Regarded As Safe"). The method basically generates, besides proteases, keratinases and other enzymes, the keratin hydrolyzed products which enrich the product with ami- noacids and peptides.
Therefore, in an altogether innovative manner, the present invention uses highly productive native Brazilian strains of enzymatic complexes which differ qualitatively and/or quantitatively from the state of the art. The preparation is enriched with keratin hydrolyzed products that are not separa- ted from the end mixture and the preparation can be used to increase the digestibility both of the feathers in natura in fermentors and the feather meal in biofermentors.
Additionally, the methodology in question has a mixture of enzymes that helps degrade the keratin, protein, starch and cellulose. The enzy- mes can be commercialized in isolation or in enzymatic complexes, and the mixture has the main classes of enzymes with a large quantity of proteases and keratinases.
The present invention uses microorganisms as direct producers of biocatalyst (enzyme) in the production system of enzymes and the keratin hydrolyzed products. The use of microorganisms in the production systems of enzymes is advantageous because the production time is shortened, so it was easy to obtain large populations of microorganisms and consequently, enzyme, and also obtain a control of the production in all phases. Other advantages are that the microorganisms can be genetically improved, increa- sing the quantity and/or quality of the biocatalyst produced, also always allowing high activity strains to be selected.
The growth medium of the present invention is low cost due to the abundance of raw materials in Brazil (agro-industrial residues from the poultry industry). It is therefore a method that still has the advantage of po- tentially making use of one of the main residues generated by Brazilian industrial activities. Methodology 1. Preparing the chicken feathers
The feathers used in the culture medium were white chicken feathers washed with detergent under running water, dried at 60 0 C and delipida- ted with chloroform: methanol (1 :1 v/v) for 1 h under agitation at 300 rpm at ambient temperature. The delipidation was carried out in a 4L beaker with 1/3 of the volume of same in feathers with 1 L of chloroform solution: methanol 1 :1 (v/v). Next the feathers were removed and dried overnight at 60 0 C. The feathers were added whole to the culture medium as main source of carbon and nitrogen. 2. Microorganism and culture conditions
To obtain the keratin hydrolyzed products, the Bacillus subtilis s- train AMR was used. It was isolated by the laboratory from agro-industrial residues of the poultry industry RICA and currently deposited at the culture collection of Oswaldo Cruz Foundation - Brazil under registration # 1266. This microorganism was chosen because of its intense keratinolytic activity for chicken feathers.
The bacillus was cultivated in a yeast extract medium (Figure 1 ) (yeast extract 0.5%, peptone 0.5%, KCI 2.0% and saccharose 2.0%;) for 2 days at 28°C under constant agitation (300 rpm) to obtain cellular mass and washed with saline (2x 3000rpm/20min) to remove the components from the medium before being inoculated in the medium containing 1% of feathers. Next the cells were transferred to the medium PBS pH 8.0 (NaH 2 PO 4 0.06M and K 2 HPO 4 0.04M) with 1 % of chicken feathers (prepared according to the procedure described in the prior item) and supplemented with 0.01% of yeast extract. The sample was cultivated in this medium for 5-6 days at 28°C under agitation at 300 rpm (Figure 2). At the end of the fermentation of the bacillus in the medium containing feathers as main source of carbon and nitrogen, the supernatant was withdrawn by centrifugation at 4,000 rpm for 20 minutes. 3. Analysis of the peptides present in the culture medium using MALDI-TOF
MALDI-TOF (Matrix assisted laser desorption/ionization - Time of flight) analyses were performed to detect peptides in the culture superna- tant (obtained according to the prior item) generated by hydrolysis of feathers by the peptidases of the B.subtilis AMR. The supernatant containing 4.99 mg/mL of proteins dosed using the Lowry method (Lowry et al. 1951 ), was partially purified in ZipTip C18 O ZipTip C18 was balanced with a solution of acetonitrile (ACN) 100% followed by washing with trifluoroacetic acid (TFA) 0.1%. After this process, the peptides were fixed in ZipTip C18 resin, washed with TFA 0.1 % to remove the salts, phosphates and/or DMSO which blur the reading. Elution was performed with 0.1% of TFA in ACN 50%. The purified samples were incorporated to the matrices of α-cyan-4- hydroxycya- nic acid (5μg/mL in TFA 0.1 % in ACN 50%) 1 :1. The mixture was then applied on the plate for analysis by MALDI-TOF. The MALDITOF analysis revealed fragments with molecular mass of 800-1100 Dalton in the crude supernatant of the cultures (Figure 3).
4. Zymography with gelatin substrate and keratin To the concentrated supernatants was added the buffer of the sample for peptidase [buffer Tris-HCI 0.32M, pH 6.8; glycerol 48% (v/v); sodium dodecyl sulfate 8% (w/v) and bromophenol blue 0.06% (w/v)] in the proportion 6:4 (60 μL of the sample for 40 μL of the buffer of the sample for peptidase). The samples were applied (20-30 μL) to polyacrylamide gel (La- emmli, 1970) at 12.5% containing 1% (w/v) of gelatin or co-polymerized keratin (Heussen & Dowdle, 1980), and subjected to a voltage of 170V for 2.5 hours at 4 Q C. Thereafter, the gels were washed with Triton X-100 2.5 % (v/v) twice for 15 minutes under agitation (70 rpm/ mim) to remove the SDS. Next, the gels were incubated for 48 hours at 37-C in a buffer of citric acid pH 5.0 (48.5 mL of citric acid 0.1 M and 51.5 mL of Na 2 HPO 4 0.2M). To detect the proteolytic activity, the gels were colored with coomassie blue [5 mL of stock solution (coomassie blue 2% w/v); 4 mL of acetic acid; 20 mL of methanol and 11 mL of distilled water] overnight and discolored with methanol solution: acetic acid: water (50:10:40 v/v/v), under agitation, until the degradation bands appeared (fig. 4).
5. Presence of other enzymes
The enzymatic preparation was also analyzed for the presence of other enzymes. Amylases and cellulases were detected. The method used the incorporation of the substrates on petri plates containing mei simbels without other sources of protein and starch. The proteases analyzed in addition to the keratinases also showed specificity for casein and gelatin (Figure 5).
6. Enzymatic degradation of the feather meal by enzymatic preparation
The microorganism B. subtilis strain AMR, was cultivated in a yeast extract medium for 48 hours under agitation (300rpm) at ambient temperature to obtain cellular mass. After this period, the cells were washed twi- ce with sterile saline (3000 rpm/ 20 min) and inoculated in the optimized media for feathers (1.5% of feathers, pH 8.0, MgSO 4 , MnCI 2 and CaCI 2 , in the concentrations of 0.5 and 1 mM) or feather meal (2.0% feather meal pH 7.0) for 6 days under ideal conditions (constant agitation, 300 rpm, at ambient temperature). At the end of incubation, the fermentation medium was centrifu- ged (3000 rpm/ 20min) and the culture supernatant (crude enzymatic preparation) was clarified in a membrane with pores of 0.45 μm, followed by filtration in membrane with pores of 0.22 μm. Added to the crude enzymatic extract was sodium azide (0.05%) and distributed in Erlenmeyer flasks contai- ning 2, 4, 6, 8 and 10% of feathers or feather meal (proportional to the volume of enzymatic extract used) autoclaved. The flasks were kept at ambient temperature under constant agitation (300rpm) for 24, 48, 42 and 96 hours. At the end of the enzymatic hydrolysis, the remaining feathers or feather meal were dehydrated (60 0 C/ 72 h) and weighed to evaluate loss of mass, besi- des having been submitted to the digestibility assay in vitro. The supernatant from the reaction mixture, collected daily, was evaluated for its soluble protein concentration.
The previously autoclaved feathers were used as substrate of the enzymatic degradation. The maximum concentration of proteins was noted after the fourth day, when feathers were used at 4% (8,416 mg/mL), as shown in Figure 6. In the reaction mixture with feathers at 5%, the concentration of proteins after 24 hours was 7.178 mg/mL, twice higher than the concentration of proteins from the enzymatic extract (3.55mg/ml_).
The same experiment was conducted to evaluate the enzymatic degradation of feather meal, using as enzymatic extract the supernatant of B. subtilis AMR cultivated in the presence of this substrate. The dosage of proteins of the supernatant of the reaction mixture showed that, over the days, the concentration of proteins increased (Figure 7), with a maximum of 16.665 mg/mL on the third day in the reaction mixture containing 5% of feather meal and keeping it constant, which could indicate the inhibition of the enzymes (or part thereof) by the product. In this concen- tration of feather meal (5%), the initial concentration of proteins was 3.153mg/ml_ and after 24 hours of degradation, the concentration rose to 11.232mg/ml_. High concentrations of proteins were also achieved after 4 days of reaction, when feather meal at 3 and 4% (15.735 ± 0.444 and 15.575 ± 0.151 mg/mL, respectively) were used. With 1 and 2% of feather meal as substrate of the reaction, the increase in the concentration of proteins was not so evident (Figure 7).
As the concentration of feather meal increased in the reaction mixture, the weight loss of the meal at the end of the process was lower. In the reaction mixture containing 1% of feather meal, the weight loss (degrada- tion yield) was 42%, a significantly high percentage, considering that the microbial degradation is about 70% (Figure 8). The feathers weighed after 4 days of incubation showed a weight loss of up to 13.25±0.58 % and 11.96 ± 2.39% (in the mixture containing 4% and 1% of feathers, respectively). In all the concentrations of feathers used in the reaction mixture, the weight loss was about 11 % (Figure 8).
7. Determination of digestibility in vitro
Feathers or feather meal (0.02g), before and after microbial fermentation, was put into tubes containing 1.5 ml_ of HCI 0.1 M containing 0.15 mg of pepsin, followed by incubation at 37 5 C for 3h. The suspension was neutralized with NaOH 0.5M and treated with 0.4mg of pancreatin in 0.75 mL of a phosphate buffer containing 0.005M of sodium azide, and the mixture incubated for 24h at 37 5 C. After incubation, these samples were treated with 1 ml_ of TCA 10% and centrifuged at 9000 rpm for 5 minutes. The protein of the supernatant was measured by the Lowry et al. method (1951 ) and the digestibility was calculated by the rate of protein in the supernatant and protein in the sample. The digestibility (degradation with pepsin and pancreatin) of feathers increased when 1% of feathers was used as reaction substrate (0.0421) in relation to the control feathers, without treatment (control - 0.0314) (Figure 9). It was noted that the digestibility of this feather meal slightly decreased (Figure 9), there being no significant differences between the figures noted for the different concentrations of feather meal used (Figure 7). Enzymatic degradation of the feathers could be carried out to obtain keratin hydrolyzed products, but since the feather degradation yield is low (Figure 8) and the remaining feathers did not become more digestible, it was concluded that the keratinases and other peptidases produced by the B. subtilis are not efficient in feather degradation and that the presence of the microorganism is necessary to degrade this substrate. Nevertheless, the enzymatic digestion of the feather meal proved promising in obtaining proteins. The optimization of parameters like pH and temperature, as well as the addition of reducing agents may increase degradation of the meal. 8. Aminoacids of the feather meal after fermentation in vivo by the microor- ganisms
The aminoacid composition of the feather meal fermented by the microorganism in vivo was analyzed and it was demonstrated that the aminoacids are released due to enzymatic action. Table 2
Table 2 - aminoacids released by the feather meal enzyme after fermentation Embodiment 2 - Capillary transformation
Another advantage offered by the present invention is the fact that the cosmetics industry can establish an important link with the poultry industry to make use of these residues, saving them from being incorporated into animal feed or polluting the environment.
Therefore, another use of the enzymatic preparation of the present invention is as a capillary transformation agent. The enzymes are able to cleave disulphide bridges when used with other transformation agents su- ch as thioglycolate, which enhance their use. The result is a decrease in hair waviness, also in comparison with the effect of hair straightening.
The most common types of straightening are temporary (brushing), progressive (progressive blow-dry and relaxation lasting from one to three months) and definitive (known as 'Brazilian' or 'Japanese' hair straigh- tening / thermal reconditioning - lasting up to one year). All have their own agents for each specific need. These agents used in capillary transformation are chemicals.
Hair can basically be transformed by way of two processes: relaxing and perming. Both use solutions with chemical agents that reduce the disulphide bridges of the cystines of the hair keratin, precisely in the hair cortex.
After washing, the hair is treated with other chemical agents to oxidize the cysteines back into cystines in the new hair form (smooth: rela- xing or curly: perming). The most well-known and widely used substances in beauty salons are sodium hydroxide, calcium hydroxide, lithium, guanidine and ammonia. There are other components such as ethalonamine, derived from ammonium thioglycolate, a most common component in hair straighte- ners, which have a greater straightening power.
In Brazil, the formula of some progressive blow-drying include the controversial product formol (formaldehyde), but in a percentage authorized by ANVISA (National Health Surveillance Agency). The formol vapors are highly aggressive to mucuses, the eyes and respiratory organs, besides being potentially allergenic and carcinogenic. Excessive use thereof can cause skin irritation. In this case, although there are no scientific studies on straightening mechanisms, it is known that aminoacids and peptides of keratin are used, besides the formol and heat, forming a film that covers the hair. Research carried out by the Adolf o Lutz Institute revealed that 52% of the samples evaluated available on the Brazilian market had a concentration 10 times higher than the limit of 0.2g of formol, permitted by health legislation.
The enzymatic method proposed in the present patent application is pioneer, considering that there is as yet no method on the world market that uses enzymes in capillary straightening processes. The keratinase enzyme cleaves the disulphide bridges of the hair allowing the hair to be molded in straight form with the assistance of heat. In the preparation in question, besides the keratinase enzyme, there are other proteases, namely, all from the class of serines and peptidases, which help the product to penetrate, aminoacids and peptides from keratin of chicken feathers, which help to recompose the protein losses. The feather hydrolyzed products used in this hair straightening help to recompose the cuticle improving the softness and sheen of the capillary fiber. Culture means and cultivation
The substrates used were whole chicken feathers as the sole or main source of carbon and nitrogen. The feathers were previously washed with detergent, rinsed exhaustively under running water and the lipids removed with a solution of chloroform: methanol (1 :1 v/v) under agitation (300rpm/1 h). The microorganism used, a strain of Bacillus subtilis, was cultivated in a yeast extract medium (yeast extract 0.5%, peptone 0.5%, KCI 2.0% and saccharose 2.0%) for 72 hours with agitation (300rpm) to obtain cellular mass and washed with saline (2x 3000rpm/20min) for removal of the components from the medium before being inoculated in the media of feathers at 1%. (Medium PBS-chicken feathers: contains phosphate buffer (NaH 2 PO 4 0.06M and K 2 HPO 4 0.04M) pH 7.2 with 1% of feathers). It was cultivated in this medium for 7 days at ambient temperature. At the end of this time, the culture medium was centrifuged (3000rpm/20min) to remove the cells and the supernatant was f reeze-dried.
This freeze-dried product contains the enzymes and the peptides and aminoacids of keratin that were used in this study on hair straightening. Previously certain tests were performed with the freeze-dried product such as zymographies to confirm the presence of enzymes and MALDI TOF to de- termine the presence of peptides, as respectively shown in Figures 10, 11 and 12. Product application
The freeze-dried product was dissolved in a phosphate buffer (NaH 2 PO 4 0.06M and K 2 HPO 4 0.04M) pH 7.2, the same buffer used in the culture means. The solution was then applied to locks of commercial human hair and the product reacted for 20 minutes. After washing, the lock was submitted to a hotplate establishing the straight format of the capillary fiber. The experiment was repeated 3 times for each different batch of the freeze- dried product. Evaluation
The results were evaluated by sensorial analyses and hydration measures, showing surprising and highly positive results.
There is nothing similar to the use of enzymes as a capillary transformation agent described in the state of the art. Thus far all methods of straightening or decreasing the waves are chemicals, without exception. Detailed Description of the Drawings Figure 1 : Electronic microscopy of Bacillus subtilis AMR in medium contai- ning chicken feathers.
Figure 2: Cultivation medium of B subtilis AMR, showing the near complete degradation of the feathers (90-95%) after five days of cultivation. Figure 3: MALDI-TOF of the culture supernatant of B. subtilis, AMR in medi- urn of feathers after 12Oh of cultivation, showing the peptides derived from the keratin of feathers.
Figure 4: Zymography with gelatin and keratin from the Bacillus subtillis supernatant in feathers demonstrating the presence of proteolytic and kerati- nolytic enzymes. Figure 5: Presence of gelatinases, caseinases, cellulases and amylases in different strains of Bacillus SP.
Figure 6: Concentration of proteins of the reaction mixture of the enzymatic degradation of feathers (sterile). Figure 7: Concentration of proteins of the reaction mixture of the enzymatic degradation of feather meal.
Figure 8: Degradation yield of feathers and feather meal submitted to enzymatic degradation.
Figure 9: Digestibility in vitro of feathers (sterile) and feather meal submitted to enzymatic degradation. Figure 10: Disulphide bridges of cystine of the keratin.
Figure 11 : Zymography of the keratinolytic enzymes (A) and proteases (B) present in the enzymatic extract (freeze-dried).
Figure 12 A: MALDI TOF showing the presence of peptides obtained from the keratin of feathers from the medium that was hydrolyzed by the kerati- nolytic enzymes and by the proteases of the microorganism.
Figure 12 B: Keratin from feathers (standard), not hydrolyzed showing a peak of 9,000 and 10,000 m/z which corresponds to intact keratin.