WO2010102362A1 - Procédé de fabrication de préparations enzymatiques obtenues à partir de plumes d'oiseaux, préparations enzymatiques ainsi faites, leur utilisation, aliments pour animaux et agent de transformation capillaire - Google Patents

Procédé de fabrication de préparations enzymatiques obtenues à partir de plumes d'oiseaux, préparations enzymatiques ainsi faites, leur utilisation, aliments pour animaux et agent de transformation capillaire Download PDF

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WO2010102362A1
WO2010102362A1 PCT/BR2010/000063 BR2010000063W WO2010102362A1 WO 2010102362 A1 WO2010102362 A1 WO 2010102362A1 BR 2010000063 W BR2010000063 W BR 2010000063W WO 2010102362 A1 WO2010102362 A1 WO 2010102362A1
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feathers
enzymatic
enzymatic preparations
enzymes
process according
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PCT/BR2010/000063
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English (en)
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Izabel Cristina Faidiga
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Serraria União De Bariri Ltda.
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    • 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)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
    • A23J1/10—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from hair, feathers, horn, skins, leather, bones, or the like
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/14—Pretreatment of feeding-stuffs with enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/20—Animal feeding-stuffs from material of animal origin
    • A23K10/26—Animal feeding-stuffs from material of animal origin from waste material, e.g. feathers, bones or skin
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/142—Amino acids; Derivatives thereof
    • A23K20/147—Polymeric derivatives, e.g. peptides or proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/189—Enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/65—Collagen; Gelatin; Keratin; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
    • A61K8/981—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of mammals or bird
    • A61K8/985—Skin or skin outgrowth, e.g. hair, nails
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q5/00—Preparations for care of the hair

Definitions

  • 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.
  • 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, Pess ⁇ a, M. F. Mostafa, S. & Curvello, F. A. 2006 Ni ' veis de effetao de farinha de penas na dieta sobre o desempenho echaracteri ' sticas de carcaca de codornas para corte. Acta Scientiarum. Animal Sciences, 28 (1 ), 27-30).
  • 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.
  • the biotechnological processing of feathers for the production of animal feed 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.
  • biotechnological method 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 Biotecnol ⁇ gicos. 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the enzyme kera- tinase is purified and its characteristics are described.
  • 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.
  • the present invention solves the technical problems referred to above.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • MALDI-TOF Microx 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 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 ⁇ -cyan-4- hydroxycya- nic acid (5 ⁇ g/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).
  • the samples were applied (20-30 ⁇ L) 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).
  • 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).
  • 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).
  • 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).
  • 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 ⁇ m, followed by filtration in membrane with pores of 0.22 ⁇ m.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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%.
  • 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.
  • 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
  • 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.

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  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention porte sur un procédé de fabrication de préparations enzymatiques obtenues à partir de plumes d'oiseaux, sur des préparations enzymatiques pouvant être obtenues par le procédé mentionné ci-dessus et, de plus, sur leur utilisation pour l'addition à des post-granulés d'aliments pour animaux afin d'améliorer la digestibilité d'une farine de plume et d'aider à la digestion des plumes dans des digesteurs avant qu'elles ne soient chauffées pour obtenir une farine de plume. L'invention porte également sur des aliments pour animaux et sur un agent de transformation capillaire.
PCT/BR2010/000063 2009-03-09 2010-03-09 Procédé de fabrication de préparations enzymatiques obtenues à partir de plumes d'oiseaux, préparations enzymatiques ainsi faites, leur utilisation, aliments pour animaux et agent de transformation capillaire WO2010102362A1 (fr)

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BRPI0900748-2A BRPI0900748A2 (pt) 2009-03-09 2009-03-09 processo para fabricação de preparados enzimáticos obtidos de pena de aves, preparados enzimáticos assim fabricados, uso dos mesmos, ração animal e agente de transformação capilar
BRPI0900748-2 2009-03-09

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WO2010102362A1 true WO2010102362A1 (fr) 2010-09-16

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CN102517235A (zh) * 2011-12-27 2012-06-27 湖南农业大学 一种枯草芽孢杆菌
CN104798981A (zh) * 2015-03-17 2015-07-29 齐鲁工业大学 一种利用碱性蛋白酶/角蛋白酶制备羽毛蛋白粉的方法
EP2776570A4 (fr) * 2011-11-07 2016-04-20 Mars Inc Protéines alimentaires et procédés pour la production
US10420783B2 (en) 2013-06-20 2019-09-24 Mars, Incorporated Performance food product

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2776570A4 (fr) * 2011-11-07 2016-04-20 Mars Inc Protéines alimentaires et procédés pour la production
US9943089B2 (en) 2011-11-07 2018-04-17 Mars, Incorporated Food protein ingredient and methods for producing
CN102517235A (zh) * 2011-12-27 2012-06-27 湖南农业大学 一种枯草芽孢杆菌
US10420783B2 (en) 2013-06-20 2019-09-24 Mars, Incorporated Performance food product
US10980823B2 (en) 2013-06-20 2021-04-20 Mars, Incorporated Performance food product
CN104798981A (zh) * 2015-03-17 2015-07-29 齐鲁工业大学 一种利用碱性蛋白酶/角蛋白酶制备羽毛蛋白粉的方法

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