Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Enzymatic production of a protein or polypeptide
Reexamination Certificate
1998-11-09
2002-04-16
Prats, Francisco (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Enzymatic production of a protein or polypeptide
C435S272000, C426S052000
Reexamination Certificate
active
06372452
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
This invention pertains to the technical field of the Agroalimentary and Medico-Pharmaceutical Industries and especially refers to human, animal and clinical nutrition.
More specifically, the invention provides a process to obtain vegetable peptones with a high degree of hydrolysis of great interest in the mentioned technical sectors.
STATE OF THE ART PRIOR TO THE INVENTION
The availability of different types of hydrolyzates in sufficiently large amounts and at reasonable prices would be of great importance and use for the preparation of special diets which, at present, are very expensive and hence, hardly used, although advisable from a medical standpoint.
Current societies and, particularly, those of developed countries, do not require random hydrolyzates [Parrado, J., Millán, F, Hernández-Pinzón, I., Bautista J and Machado A., 1993. Process Biochem., 28, 109-113; Ziajku S., Dzwolak W and Zubel J., 1994, Milk Science International, 49,7] aimed at general nutrition, culture of micro-organisms, additives for beverages, etc. Nowadays, hydrolyzates are required having well defined compositions and characteristics, either because they are not available on the market or because those existing are very expensive and need to be cheapened. Having hydrolyzates with a defined amino-acid composition [Parrado J., Millán F., Hernández-Pinzón., Bautista J and Machado A., 1993, J. Agr. Food Chem. 41, 1821-1825; Bautista J., Hernández-Pinzźn I., Alaiz M., Parrado J and Millán F., 1996, J. Agr. Food Chem. 44, 967-971] is very desirable, both for the nutrition industry and the medico-pharmaceutical ones [Furst P., 1992, Clin. Nutr. 10, 519-529]. Hence, for example, branched aminoacid (AAR) enrichened hydrolyzates, either with mixtures of free aminoacids or with peptides rich in AAR, have been successfully used in the treatment of various hepatic pathologies, especially encephalopathies, Grunggreiff K., Kleine F. D., Musie H. E., Diete U., Franke D., Klanck S., Page I., Kleine S., Losene B and Pfeiffer K. P., 1993, Z-Gaskoenterol, 31, 235-241; Rossi-Fanelli F., 1990, Adv. Exp. Med. Biol. 272, 227-233].
The production of proteic hydrolyzates from residues rich in proteins (meat flour) and non-conventional protein sources (vegetable proteins) [Anuario de Estadistica Agraria 1996, Edited by the Technical Department of the Ministry of Agriculture, Fisheries and Food; Boatright W. L., Hettiarachchy N. S., 1995 JAOLS, 72, 1445-1451; Paredes-López O., Ordorica-Falomir C., Olivares-Vázquez M. R., 1991 J. Food Sci. 53, 1396-1398] is a well established process in the food industry.
Enzymatic hydrolysis is a very efficient process which, moreover, may be performed under reasonable conditions of temperature, pH and pressure, such that the nutritional quality of the amino-acids is maintained practically un-changed, since the destruction or modification of certain amino-acid residues does not take place, different to the case of chemical hydrolysis. In enzymatic hydrolysis, hydralyzate neutralization is avoided and hence, the high content in salts which would limit the use of hydrolyzates in certain applications, for example, in diet foods and parenteral preparations.
In recent years, many enzymatic protein hydrolysis processes have been studied for their application at a commercial scale. The interest for these types of process is large and, although the production of simple proteic hydrylyzates is not a problem, the production of defined hydrolyzates, namely, well defined ones, as demanded by the market, continues to be a problem and represents the greatest challenge in this field. The latter type of hydrolyzate, if possible, should combine the following characteristics: (I) perfectly defined composition in relation to the amino-acids contained (aminogram), as well as their size distribution (% in peptides, oligo-peptides, tetra-tri-dipeptides and free aminoacids); (ii) absence of or a negligible bitter flavour and, (iii) be hypoalergenic.
The patents existing to obtain proteic hydrolyzates, including both the chemical approach (use of acids and bases) and the enzymatic one (use of enzymes), are general and do not indicate the substrates used nor, in the case of enzymatic hydrolysis, the enzymes employed and of course, do not include the details (Patent 489358, No. 2278/79. Industrial Property Register. Nestlé).
DETAILED DESCRIPTION OF THE INVENTION
This invention, as indicated in the title, refers to a process to obtain vegetable peptones with a high degree of hydrolysis.
More specifically, in this report, the process to obtain enzymatic hydrolyzates of vegetable proteins with a high degree of hydrolysis (GH 50%) is described, specifying, the substrate(s) used [sunflowers, rape seeds, chickpeas, lentils, lupins and grape refuse] and the enzymes employed, including their commercial name. The process is the basis to obtain what has been called “customised hydrolyzates”.
The main purpose is to include the preparation of the substrates, which in all cases implies the elimination of the polyphenols. The elimination of the latter, present in all vegetable protein isolates, is necessary because these substances generally inhibit the enzymatic activity of the proteases, such that their elimination is absolutely necessary for the efficient operation of the process.
The second important purpose is the optimization of the hydrolysis process whose treatment with more than one protease, is based on the resistance to hydrolysis of the allergic “epitopes” present in proteins. For this reason, in an initial treatment, the proteins are hydrolyzed with a) some endoprotease(s) with non-specific activity(ies), such that in a second step, once a practically constant degree of hydrolysis has been reached (12-15% depending on the substrate), a mixture of more specific endoproteases and exoproteases are added together (resulting in degrees of hydrolysis ≧50%), such that the probabilities of destroying the allergenic “epitopes” is almost total, besides eliminating the possible bitter flavor of some hydrolyzates. The proteolytic enzymes that have been used in each substrate are: ALKALASE™, KERASE™ and FLAVOURZYME™ for sunflower proteins; L-600 and FLAVOURZYME™ for rape seed; goglet and FLAVOURZYME™ for chickpeas, lentils and lupinus proteins; alkalase and kerase for grape refuse proteins. The hydrolyzates are concentrated and micronized to give a dry product.
Below, the characteristics and specifications of the aforementioned proteolytic enzymes used in this invention are indicated:
ALCALASE™ enzyme preparation: more specifically, ALCALASE™ 2.4 L, is a bacterial protease, being highly effective and specifically developed for the hydrolysis of all types of proteins. It is products at from a selected strain of Bacillus Licheniformes. It is an endopeptidase with an approximate molecular weight of 27,300 daltons. It is easily soluble in water at all concentrations and its density is 1.18 g/ml. It has an activity of 2.4 Anson Units per gram (Au/g), optimum pH of 8 and optimum temperature of 50° C.-60° C. It is available in the liquid form and is available from the company, Novo Nordisk Bioindustrial S.A.
FLAVOURZYME™ enzyme preparation: FLAVOURZYME™ more specifically, FLAVOURZYME™ 1,000 MG, is produced by the fermentation of Aspergillus oryzae and contains endoprotease and exopeptidase activity. The molecular weight differs from the different endoproteases, since it is an endo- and exo-protease complex. It is easily soluble in water, has an activity of 1,000 LAPU/g, optimum pH of 7 and optimum temperature of 50° C. It comes in its 35granulated form and is available from Novo Nordisk Bioindustrial S.A.
KERASE™ enzyme preparation: this is a microbial enzyme and is obtained from Streptomyces Fradiae. It is a mixture of endo- and exo-peptidase, which behave like serin protease and is stable in the presence of oxygen. It is non-toxic, free from antibiotics and bacterial contamination. It is easily soluble in water, has an activity of 10−
Bautista Palomas Juan
Millan Rodriguez Francisco
Olias Jimenez Jose Manuel
Consejo Superior de Investigaciones Cientificas
Ladas & Parry
Prats Francisco
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