FR2835703A1 - Production of oil and protein hydrolyzate for use, e.g., in foodstuffs from marine source such as filleting discards or fish heads and tail comprises enzymatic hydrolysis at specified temperature - Google Patents

Abstract

A method of obtaining an oil and a protein hydrolyzate from a marine source of proteinic tissues such as filleting discards, fish heads and tails, shellfish or whole fish of low commercial value comprising enzymatic hydrolysis at no more than 60 degreesC to give an aqueous mixture of hydrolyzates and a fatty phase comprising the oil, phase separation, and stopping of the hydrolysis in the aqueous phase. An Independent claim is included for the protein hydrolyzate and oil obtained by the claimed method.

Description

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 The subject of the present invention is a process for obtaining an oil and a protein hydrolyzate from a marine source of protein tissues, such as thread deviations, fish heads or bones, shells or whole fish of low commercial value; it also relates to the oil and the protein hydrolyzate obtained by implementing this process.

 Different more or less complex processes have been used or are still used for the extraction of oil from fish such as sardines, herring, menhadens, tuna, salmon, to name only the most used marine sources.

 These methods are based on relatively old techniques described in particular in British patents Nos. 199,057,438 056,590,638 and 1,061,028.

 All these techniques are based on a heat treatment in water of the raw material (whole fish or thread deviations) up to a temperature above 95 C, treatment which leads to the coagulation of the proteins of this material; a possible addition of soda, mineral acid or organic solvents is likely to improve the extraction yields.

 The product of this heat treatment comprises an aqueous phase containing a coagulate of proteins and oil which are separated by centrifugation, the protein coagulate being most often intended for animal feed as fishmeal.

 The above processes have various drawbacks resulting from the heat treatment and the possible use of solvents: degradation of the original organoleptic and aromatic qualities of the raw material treated; deterioration of the nutritional quality of the oil.

 A cold oil extraction process is also known, but it especially concerns the de-oiling of fish flesh after freezing, without however treating the discrepancies (FR-2 757 021).

 In addition, low value raw materials

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 commercial, such as thread deviations, fish bones and heads and certain types of fish, are currently little valued while they represent a significant protein potential in the sector of seafood processing industries.

 The object of the present invention is to overcome the drawbacks of the aforementioned known methods and to develop any marine source of protein tissue. To this end, it relates to a process in accordance with that defined in the first paragraph of this description and which is characterized in that it comprises: (a) the enzymatic hydrolysis of said marine source of protein tissues at a temperature of at most 60 C, for example in the range 45 to 55 C to obtain an aqueous reaction mixture comprising a protein hydrolyzate and an oil, (b) separating said mixture into an aqueous phase comprising said protein hydrolyzate and a fatty phase comprising said oil, and (c) stopping said enzymatic hydrolysis within said aqueous phase comprising the protein hydrolyzate.

 The enzymatic hydrolysis proposed according to the invention leads to the rapid destructuring of the protein tissues from the marine source and thus to the release of the oil from the adipose tissues contained in said protein tissues.

 As this process does not use solvents and is implemented at relatively low temperatures, it is not likely to alter the organoleptic and aromatic qualities of the products obtained, nor their nutritional value.

 In addition, it is perfectly suited to the treatment of materials of low commercial value and therefore makes it possible to develop both the oil and the proteins contained in these materials.

 The enzymatic hydrolysis of the process according to the invention is preferably carried out in the presence of one or more proteases and at a pH at which the action of this or these protease (s) is optimal.

 As proteases, one or more enzymes can be chosen from the group consisting of endopeptidases from

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 Bacillus subtilis (optimum pH: 7.0-7.5), endopeptidases from Bacillus licheniformis (optimum pH: 8.0-8.5), endopeptidases from Bacillus amyloliquefaciens and complex endoproteases from Aspergillus oryzae (optimum pH: 6.0-6.5).

 The enzymes will preferably be selected according to the nature of the raw material to be hydrolyzed and the functional and / or nutritional properties desired for the protein hydrolyzate to be obtained.

 Thus, for example, the use of endopeptidases from Bacillus subtilis leads to the production of insoluble collagenous tissues, while endopeptisases from Bacillus licheniformis makes it possible to obtain a protein hydrolyzate perfectly soluble in cold water.

 In accordance with the invention, the enzymatic hydrolysis is advantageously controlled until a degree of hydrolysis is obtained (percentage of the hydrolyzed peptide bonds relative to the total number of peptide bonds) of at most 10%.

 Under these conditions, the development of bitterness is avoided which would be an obstacle to the use of the protein hydrolyzate obtained, in food formulation.

 The control of the enzymatic hydrolysis is preferably carried out by adjusting the pH of the reaction mixture to maintain it substantially at the value for which the action of the enzyme or enzymes used is optimal.

 On this subject, it will be specified that during hydrolysis, there is generation of carboxyl function resulting from the cleavage of the peptide bonds and therefore reduction of the pH of the reaction medium and therefore slowing down or even stopping of hydrolysis.

 To maintain the pH at the desired value, it is therefore necessary to neutralize the reaction medium, that is to say these carboxyl functions, with a base, in particular an aqueous sodium hydroxide solution; this solution will preferably be 2-4 M to limit the dilution of the reaction mixture.

 It will be noted that simultaneously with the above-mentioned neutralization, there is neutralization of the fatty acids released, which improves the quality of the oil.

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dans laquelle : - B = volume (en ml) de solution aqueuse de base ajoutée, - NB = normalité de ladite solution aqueuse de base, - MP = masse de protéines (en g) présente dans le milieu réactionnel (azote total x 6,25), - htot = nombre total de liaisons peptidiques de la matière première (en méq. /g de protéines) - x = degré de dissociation moyen des fonctions amines libérées par l'hydrolyse :

où pK représente le pK moyen des fonctions oc-amines libérées durant l'hydrolyse. The relationship between the degree of hydrolysis (DH) of the marine source to be treated and the consumption of basic solution is given by the following formula (1):

in which: - B = volume (in ml) of basic aqueous solution added, - NB = normality of said basic aqueous solution, - MP = mass of proteins (in g) present in the reaction medium (total nitrogen x 6, 25), - htot = total number of peptide bonds of the raw material (in meq. / G of proteins) - x = average degree of dissociation of the amine functions released by hydrolysis:

where pK represents the average pK of the oc-amine functions released during the hydrolysis.

 According to Steinhardt and Beychok (1964), the pK values at 250 C of the groups (-COOH) and (-NH3 +) are estimated respectively at 3.1-3, 6 and 7.5-7, 8.

 It will also be noted that the htot parameter in formula (1) above can be determined from the amino acid composition of the raw material. For marine products, the htot is around 8.6 meq. / g protein.

 Once the various parameters of formula (1) have been determined, it is easy to calculate the value B, that is to say the volume of aqueous base solution to be added to achieve the desired degree of hydrolysis.

 Other techniques for measuring the degree of hydrolysis can be used, such as for example a dosing method

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 colorimetric of the primary amine groups released during hydrolysis or else a determination of total nitrogenous materials and soluble nitrogenous materials (Process Biochem., 1994; 29: 257-262); according to the results of these assays, the hydrolysis is continued to advance by adjusting the pH of the reaction mixture as indicated above.

 According to another characteristic of the invention, the protein content of the reaction mixture is preferably adjusted to a value of 7 to 15% by weight, in particular from 7 to 8% by weight, by addition of water.

 Advantageously, the above-mentioned separation (b) comprises a centrifugation preferably carried out under an inert atmosphere and at a temperature of 30-400 C.

 This centrifugation operation can in particular be implemented in a centrifuge-sludge separator.

 The stopping step (c) of the method according to the invention is carried out by inativation / denaturation of the hydrolysis enzymes, preferably by injection of water vapor into the aqueous phase comprising the protein hydrolyzate, so that that the temperature of the aqueous phase comprising the protein hydrolyzate is equal to or greater than 900 ° C. for 5 to 20 minutes.

 If necessary, the process according to the invention also comprises an operation for neutralizing (d) the reaction mixture obtained in step (a), this operation (d) being carried out before operation (b). The purpose of this neutralization is to neutralize the free fatty acids present in the fatty phase, these being capable of leading to the rancidity of this phase. Said neutralization therefore makes it possible to obtain a high quality oil.

 Advantageously, the method according to the invention also comprises a step of prior grinding (e) of the marine source to obtain fragments with a size of 0.5 to 5 cm; this facilitates the stirring of the reaction mixture and increases the contact surface with the hydrolysis enzymes and therefore reduces the duration of the hydrolysis reaction.

 If necessary, it is also possible to provide a

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 filtration operation (f) of the reaction mixture before operations (b) and (d), in order to remove any solid matter (bones, bones, non-hydrolyzed protein tissues) present in the aqueous phase.

 According to the invention, after the operation (c) of stopping the enzymatic hydrolysis, the protein hydrolyzate can be cooled preferably to a temperature of 4 ° C., in an inert atmosphere, this cooling being able to be followed by a concentration, atomization or lyophilization operation, depending on the potential uses of the hydrolyzate.

 As for the fatty phase containing the oil, it is advantageously cooled to a temperature of 4 to 12 ° C. and stored under an inert atmosphere (for example nitrogen) in order to avoid oxidation reactions.

 Thanks to the process described above, it is possible to carry out a rapid extraction of the oil contained in the protein tissues of a marine source and this, under managed conditions (relatively low temperature and relatively short duration) and with yields. completely comparable to those of previous extraction methods using solvents.

 Furthermore, due to the aforementioned gentle conditions and the absence of the use of any solvent, the oil and the protein hydrolyzate obtained by the process according to the invention are of very high quality and therefore find their application in particular. in animal and human food formulation, cosmetology and nutraceuticals.

 The process which has just been described can be schematized as follows:

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Source marine protéique broyage addition d'eau enzymes v contrôle du réacteur themiostaté degré d'hydrolyse sous agitation filtration neutralisation."résidus phase aqueuse protéique + phase lipidique centrifugation huile hydrolysat protéique + t refroidissement traitement thermique (injection de vapeur) stockage sous atmosphère inerte refroidissement refroidissement lyophilisation concentration atomisation

Marine protein source grinding addition of water enzymes v control of the thermostatic reactor degree of hydrolysis with stirring filtration neutralization. "Residues aqueous protein phase + lipid phase centrifugation oil protein hydrolyzate + t cooling heat treatment (steam injection) storage under inert atmosphere cooling cooling lyophilization concentration atomization

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An example of implementation of the latter will be given below, by way of illustration of the present invention; it involves obtaining oil and salmon protein hydrolyzate.

 100 kg of salmon heads and tails are subjected to mechanical grinding, in order to obtain a substrate consisting of fragments in size and preferably between 1 and 5 cm. Then, the substrate is diluted with water at 10-80 C, in a mass ratio of 0.5 to 3.0, for example 1.1.

 The mixture obtained is brought to the optimum temperature of action of the hydrolysis enzyme used described below, ie 55 C. Then the pH of the mixture is adjusted to the optimum action value of said enzyme, ie pH = 8.0, using a 4M sodium hydroxide solution with stirring. The hydrolysis enzyme is then added, in this case a protease sold by the company NOVO under the brand name ALCALASE 2.4 L, with an enzyme / substrate ratio of 0.1 to 10% (0.3% in the present example ).

 The degree of hydrolysis is controlled by progressive addition of a 4M aqueous sodium hydroxide solution, in order to maintain the pH at a value substantially equal to 8.0.

 The degree of hydrolysis is followed by one or other of the techniques described above.

 When the desired level is reached, the reaction mixture is filtered through a sieve in a tank to remove solids and non-hydrolyzed protein tissue.

 The filtrate obtained is then subjected to a centrifugation operation on a separator of the centrifuge-sludge separator type at a centrifugation temperature of 50 C; this results in the separation of an oil and a protein hydrolyzate.

 The oil is immediately packaged under an inert atmosphere.

 As for the protein hydrolyzate, it is brought to a temperature of 95 ° C. for 20 minutes, by injection of living water vapor, to interrupt the hydrolysis process.

 The protein hydrolyzate thus treated is then cooled, then frozen before processing to bring it into a form suitable for its use.

Claims (16)

1. Process for obtaining an oil and a protein hydrolyzate from a marine source of protein tissues, such as thread deviations, fish heads or bones, shellfish or whole fish of low commercial value, characterized in that it comprises: (a) the enzymatic hydrolysis of said marine source of protein tissues at a temperature of at most 60 C, to obtain an aqueous reaction mixture comprising a protein hydrolyzate and an oil , (b) separating said mixture into an aqueous phase comprising said protein hydrolyzate and a fatty phase comprising said oil, and (c) stopping said enzymatic hydrolysis within said aqueous phase comprising protein hydrolyzate.  2. Method according to claim 1, characterized in that the enzymatic hydrolysis is carried out in the presence of one or more proteases and at a pH at which the action of this or these proteases is optimal.  3. Method according to claim 2, characterized in that said proteases are chosen from the group consisting of endopeptidases of Bacillus subtilis, endopeptidases of Bacillus licheniformis, endopeptidases of Bacillus amyloliquefaciens and complex endoproteases of Aspergillus oryzae.  4. Method according to any one of claims 1 to 3, characterized in that the enzymatic hydrolysis is controlled until a degree of hydrolysis of at most 10% is obtained.  5. Method according to claim 4, characterized in that the enzymatic hydrolysis is controlled by adjusting the pH of the reaction mixture to maintain it substantially at the value for which the action of the protease or proteases is optimal.  6. Method according to claim 5, characterized in that said adjustment is carried out by neutralization with a base, preferably an aqueous sodium hydroxide solution, of the reaction mixture. <Desc / Clms Page number 10>  7. Method according to any one of the preceding claims, characterized in that the enzymatic hydrolysis is carried out at a temperature in the range from 45 to 550 C.  8. Method according to any one of the preceding claims, characterized in that the protein content of the reaction mixture is adjusted to a value of 7 to 15% by weight by addition of water.  9. Method according to any one of the preceding claims, characterized in that the separation (b) comprises a centrifugation.  10. Method according to any one of the preceding claims, characterized in that the stopping (c) is carried out by injection of water vapor into the aqueous phase containing the protein hydrolyzate, so that the temperature of said aqueous phase comprising the protein hydrolyzate is equal to or greater than 900 C for 5 to 20 minutes.  11. Method according to any one of the preceding claims, characterized in that it comprises, if necessary, an operation of neutralization (d) of the reaction mixture obtained by operation (a), this operation of neutralization being brought into work before the operation (b).  12. Method according to any one of the preceding claims, characterized in that it further comprises a grinding operation (e) of said marine source of protein tissues to obtain fragments with a size of 0.5 to 5 cm. .  13. Method according to any one of the preceding claims, characterized in that it further comprises a filtration operation (f) of the reaction mixture to remove any solid matter from the aqueous phase, before operations (b) and (d).  14. Method according to any one of the preceding claims, characterized in that it further comprises an operation of cooling to 40 C of the aqueous phase and from 4 to 12 C of the fatty phase, in an atmosphere of inert gas.  15. Method according to any one of the preceding claims, characterized in that it further comprises a <Desc / Clms Page number 11>  concentration, atomization or lyophilization operation of said protein hydrolyzate.  16. Protein hydrolyzate and oil obtained by implementing the method according to any one of claims 1 to 15.