WO2008046993A2

WO2008046993A2 - New marine-originating biological product, method for producing the same and uses threof

Info

Publication number: WO2008046993A2
Application number: PCT/FR2007/001676
Authority: WO
Inventors: Estelle Le Bihan; Noussithé KOUETA
Original assignee: Universite De Caen Basse Normandie Umr Ifremer Physiologie Et Ecologie Des Mollusques Marins
Priority date: 2006-10-17
Filing date: 2007-10-15
Publication date: 2008-04-24
Also published as: FR2907129A1; WO2008046993A3

Abstract

The invention pertains to the field of chemistry and more precisely to that of biology. The invention specifically relates to the silage (or liquefaction) of the digestive organs of a cephalopod, such as cuttlefish, under the influence of endogen enzymes during the freeze-drying silage into a powdered product that can be preserved. The invention can be used in aquaculture for improving the growth rate of juvenile fish, in bacteriology for producing additives for culture media, for micro-organism culture media or in he food industry for compensating a protein deficit.

Description

"New biological product of marine origin, its process of obtaining and its uses"

The invention relates to the field of chemistry and more particularly to that of biology.

It more particularly relates to a means of recovery and / or recovery of animal waste for food, especially for animals.

It specifically relates to a process for ensiling or liquefying the digestive organs of a cephalopod under the influence of endogenous enzymes during silage.

The cuttlefish, is a cephalopod (Sepia officinalis or Sepia esculenta or Sepia pharaonis, and especially Sepia officinalis), which is abundant in the seas surrounding our country and is mainly fished in the basins of Cotentin.

As previously stated, (E. Le Bihan, A. Perrin and N. Koueta, Life and Environment 2006; 139-145), marine animals are marketed mainly in eviscerated form after freezing and this production constitutes a motive. important development for many coastal companies. These companies produce a large volume of waste, including skin and viscera.

The skin of cephalopods has already found a possibility of use because it contains a high proportion of polyunsaturated fatty acids. According to EP 1.027.833 it is possible to treat skin fragments with proteolytic enzymes to form an emulsion of the O / W emulsion type which contains the water-soluble components and especially the polyunsaturated fatty acids insoluble in the water. water. Among the components that are soluble in water are amino acids, oligoproteins with a molecular weight of less than 30,000 and proteins. This process already makes it possible to recover a significant waste and to eliminate a permanent risk of pollution of the environment by hydrolyzing cuttle-skin in this way.

However, this is not yet sufficient to achieve the disposal of cuttlefish waste and other cephalopods such as squid, octopus and other marine animals.

Studies have shown that the cutaneous digestive tract (golden cuttlefish) contains many enzymes and isoenzymes (Zeng X et al J Ocean Univ. Of China (Chem Abst 142 173513 (1997), Perrin A., 2004 ) that contribute to the process of autolysis.

It has also been shown that the enzymes present in the digestive gland of the cuttlefish [Sepia officinalis] contain forms activated by different metals such as Cu, Zn or Ag which make them substantially more active (Le Bihan E. et al., J. Exp. Mar. Biol Ecol 2004; 309, 47-66).

It has also been shown that the diet of experimental cuttlefish plays an important role in the secretion of digestive enzymes and in the growth of young cuttlefish (Perrin A et al., J. Exp. Mar. Biol Ecol 2004; 311 , 267-285)

Among the enzymes found in the digestive tract and especially in the digestive gland of cuttlefish and squid, proteases are mainly found (Hatate H. Suisan Daigakko Kenkyu Hokuku (1999) 47, 121-127, Perrin A., (2004), loc cit).

At the same time, unsaturated fatty acids, such as eicosapentaenoic acid and docosahexaenoic acid, have been identified in the cuttlefish viscera as evidenced by stationary phase chromatography and purification by passage over silica gel and alumina followed by characterization with nitrate. silver (Ikushim Y et al Japanese Patent JP 9008298 of 11/10/1990).

Ultimately, E. Le Bihan et al, Food Chemistry 98 (2006) 39-51 showed that cephalopods are an important economic resource for global fisheries. Lower Normandy is the largest cephalopod producing region in France. The cuttlefish thus produced is intended to be exported in frozen form to the countries of the Mediterranean Basin and to Japan. Traditionally, viscera have been considered as waste without practical use except for a small part. However, the cuttlefish viscera are an important part of the cuttlefish mass (15 to 25%). As a result, this type of residue represents a significant commercial loss due to the lack of recovery of such a residue.

The use of lipids, which represent 10% of viscera, makes cephalopod viscera may be the basis of autolysates of marine products to produce lipids and easily digestible peptides, which can be used as an ingredient in aquaculture.

The process of post-mortem autolysis of the viscera results in the release of the endogenous enzymes so that the degree of autolysis and the degree of hydrolysis of the amide functions, determine the degree of hydrolysis of the peptide bonds and ultimately the molecular weight proteins and peptides on which the duration of the enzymatic reaction depends. The level of enzymatic activity, the pH evolution during autolysis, the influence of Trichloroacetic acid on the level of soluble proteins were also determined (Le Bihan E. et al., 2006, Food Chemistry, 98, 39-51).

The invention therefore consists in subjecting the cuttlefish viscera, and in particular Sepia officinalis, to partial or complete autolysis by the endogenous enzymes to form lysates containing soluble proteins, oil aggregates rich in polyunsaturated fatty acids, proteins , carbohydrates.

This autolysis process is also characterized by the fact that the pH of the medium is kept below 6 during the process by addition of an aliphatic carboxylic acid such as propionic acid.

The autolysis process is also defined by the fact that the level of soluble proteins in trichloroacetic acid varies. He is produces a breakdown of lysosomes and thereby the amount of soluble proteins in trichloroacetic acid is increased.

The invention is further characterized by a decrease in the level of proteins of high molecular weight and in particular of molecular weight greater than 20 kDa while the level of low molecular weight proteins (<6.5 kDa) remains unchanged during storage.

In addition, it was determined that during the autolysis of the viscera, the specific activity of amylase increases significantly (p <0.05) and then decrease, as a result of the degradation of the enzyme and its instability in storage conditions.

The activity of carboxypeptidases A 'and B' present in the viscera increased significantly (p <0.05) compared to the viscera of live animals. Thus, under the conditions of autolysis, the carboxypeptidases must be considered as very stable under the experimental conditions used.

The specific activity of chymotrypsin was found to be significantly higher compared to the activity determined in live cuttlefish. However, this increase tends to decrease as a result of degradation occurring during mill processing.

Autolysis is mainly due to high levels of acid proteases (cathepsins), chymotrypsin and trypsin in the digestive system of carnivorous animals.

An important phenomenon during autolysis is the release of digestive proteases due to the breakdown of zymogen vesicles present in the cells, which induces significantly increased alkaline protease activity.

During silage, the C22: 6 - 3 fatty acid content increases while the level of C20: 5 - 3 fatty acids decreases. Nevertheless, the overall content of polyunsaturated fatty acids remains high. Ultimately, the cuttlefish viscera after autolysis contain on average:

86-87g - per 100 g of protein dry weight 12 g per 100 g of dry weight of fat and 2.1 g per 100 g of dry weight of carbohydrates

The freezing of the cuttlefish induces a significant lowering of the content of proteins, lipids and carbohydrates in the viscera. However, the relative proportions of these different constituents remain similar during storage at -2O ⁰ C.

The following Tables I and II show the differences in compositions of the cuttlefish viscera after different treatments during storage.

The silages of cuttlefish viscera are then lyophilized to ensure better preservation and facilitate their use. The addition of an aliphatic organic acid to the silage (such as propionic acid) and the addition of an antioxidant (such as vitamin E, terbutylparacresol or ethoxiquin) also facilitate autolysis and conservation.

After biochemical analysis of the various constituents, the lyophilized silages according to the invention were tested in vivo to determine their suitability for use in aquaculture.

Experimental trials of juvenile molluscs [Sepia officinalis], fish [Dicentrarchus labrax] and crustacean larvae (Penaeus japonicus) were conducted to test the impact of these enrichments on larvae and juveniles. The animals are placed in experimental breeding for 40 days in the case of cuttlefish, 4 months for bars and 60 days for shrimps. They received feed enriched in silage (1 to 10% of the diet). Feed enrichment tests with ensilages thus prepared showed a strong improvement in the growth rate of juveniles cuttlefish, bars and Japanese shrimp. Survival was not affected by feeding fortification during the experiments.

The use of silage can also be useful in the production of culture media supplement (isolated cells of marine molluscs or bacteria) and the regulation of physiological phenomena. For this reason, the effect of silage on viability and cellular physiological activity, was tested on isolated cells of the digestive gland of cuttlefish. The experiments have shown that the addition of silage in the culture medium allows a significant increase in cell viability and provokes a stimulation of their physiological activity (secretion of digestive enzymes). Comparison of the effects of silage with growth factors (IGF, EGF, insulin) on isolated cells of the cutaneous digestive gland shows similarities.

In addition, the addition of silage to the culture medium of abalone haemocytes increases the adhesion of cells and the number of cytoplasmic extensions thereof. Silage stimulates immune defenses in abalone haemocytes in primary culture. Thus, the ensilages LBBMA4 and LBBMA25 show a stimulation of the lysosomal activities (stimulation of 176% and 329% respectively), of phenoloxidase (stimulation of 110% and 120% respectively) and anti-protease (inhibition of 35% and 35% respectively against 19 for the control) compared with that assayed in the control hemocyte culture medium.

Finally, the use of silage, as a culture medium alone, for microorganisms shows that bacteria and yeasts can develop further on this type of culture medium. This makes it possible to consider the possibility of replacing the peptones currently used with silages of cuttlefish viscera with a result at least comparable. Silage because of its high protein content is still used in food.

The following examples explain the invention without limiting it. The figures provided below are subject to variations depending on seasonality. Example I

Silage manufactured at 4 ⁰ C

25 liters of mixed viscera (viscera and cuttle-fish eyes) are milled in a roller mill. The grind is placed in an acid resistant plastic container (polypropylene). 1.5% w / v is added to the juice. of propionic acid and then 250 mg / l of an antioxidant. The mixture is mixed until homogeneous and is then incubated at ^{40 °} C. for 50 days. The silage juice is freeze-dried and the lyophilsate is distributed in sealed containers protected from moisture.

Silage composition after lyophilization:

Silage manufactured at 4 ⁰ C:

(LBBMA4)

pH: 5.29 ± 0.05

Soluble TCA protein (mg / ml) 399.5 + 16

Total protein (g / 10C) g dry weight) 84.61 ± 3

HMW carbohydrates (g / 100 g dry weight) 1.45 + 0.01

LMW carbohydrates (g / 100 g dry weight) 1, 19 + 0.005

Total carbohydrates (g / 100 g dry weight) 2.63 + 0.016

Total fat (g / 100 g dry weight) 8.5 + 0.4

Peptides <6.5 kDa (%): 80.7 ± 0.4

Digestibility (%): 89.53 ± 2.15

Composition in amino acids (mg / g of 240 ± 9 dry weight)

TABLE I: Composition of silage after freeze drying Example II

Silage manufactured at 25 ⁰ C

The same procedure was used as in Example I but the cuttlefish viscera were incubated at 25 ° C for 50 days. The silage juice is dried by lyophilization under the same conditions as in Example I.

The silage composition after lyophilization obtained under these conditions was determined. It should be noted that the ensilage product obtained under these conditions has a substantially higher amino acid content and a statistically higher digestibility coefficient (95.59 ± 3, 11 against 89.53 ± 2.15). The content of peptides of low molecular weight (<6.5 kDa) and total lipids is also statistically higher. It therefore appears that silage made at 25 ° C has a significantly higher nutritive value.

The ensilages made according to the technique of Example I and Example II are used as a dietary supplement in animal nutrition, for the stimulation of growth for young animals, for the stimulation and maturation of the digestive system of juveniles or larvae, for the increase of the digestibility of such foods and again as an immunostimulant.

TABLE II

Silage composition after lyophilization TABLE III

Stability of silage products Since 0 to 12 months after production

Silage manufactured at 4 ° C Silage made at 25 ° C:

Time (months): 12 8 12 12 8 12 Salmonella: Absence Absence Entero-bacteria: Absence Absence

Dioxin (WHO-2.57 pg / g 2.27 pg / g Toxic EQuivalent of dry matrix

PCB (WHO-2.56 pg / g 2.32 pg / g ToxicEquivalent of dry matrix

PAH (WHO-1.88 μg / kg 2.62 μg / kg ToxicEQuivalent of dry matrix

Dioxin + PCB 5.13 μg / g 4.59 μg / g (WHO-

ToxicEQuivalent of dry matrix

Benzo-pyrene (WHO -0.09 μg / kg 0.15 μg / ki ToxicEquivalent of dry matrix

Peptides <6.5kDa (%): 80.7 84.8 85.6 86.4 89.3 91.4 90.91 91.5 pH: 5.29 5.18 5.14 5.12 5.94 5.61 5.56 5.5

± 0.05 ± 0.2 ± 0.1 ± 0.2 ± 0.05 ± 0.1 ± 0.06 ± 0.1

Soluble protein 399.5 374 383 374 404 379 367 367 in acid ± 16 ± 18 ± 11 ± 8.7 ± 13 ± 9 ± 14 ± 12 Trichloroacetic (in mg / ml) TABLE IV

Fatty acid composition of silage products at 4 and 25 ° C

Table IV: Composition in fatty acids of viscera resulting from ensiling (%

The composition of the silage corresponds to the dosages carried out on a single silage.

Claims

1. A method of ensiling (or liquefying) the digestive organs of a cephalopod under the influence of endogenous enzymes and enzymes possibly made during silage, to obtain a nutrient-rich autolysate juice that it is dehydrated by physical means to obtain a powdery product which is preserved in the absence of moisture.

2. A silage process according to claim 1 wherein the endogenous enzymes made during silage consist essentially of an amylase.

3. Silage process according to claim 1 and claim 2 wherein the endogenous enzymes made during silage consist of carboxypeptidases A 'and B'.

4. A silage process according to claim 3, wherein the autolysis is also due to the action of acid proteases, chymotrypsin and trypsin present in the digestive system of carnivorous animals.

A silage method according to claim 2 and claim 3, wherein the silage produces a release of digestive proteases as a result of the breaking of the zymogen vesicles.

6. Silage process according to claim 1, wherein the digestive organs of cephalopod are those of the cuttlefish.

7. Silage process according to one of the preceding claims wherein the cephalopod is Sepia Officinalis.

8. A method of silage according to one of the preceding claims wherein there is added to the silage product an organic aliphatic acid to maintain the pH between 5 and 6.

A silage process according to one of the preceding claims wherein an antioxidant is added to the silage juice to promote preservation.

10. Freeze-dried cephalopod silage products according to claim 1 characterized by a high total protein content of at least 63.90% of dry product, a lipid content greater than 8 g% of dry product and a content of carbohydrates of the order of 2 g% of dry product.

The cephalopod silage product of claim 10 wherein the protein content is in the range of 86 to 87 g per 100 g dry weight.

The cephalopod silage product of claim 10 wherein the lipid content is in the range of 12 g per 100 g dry weight.

13. Use of silage-enriched foods according to claim 10, in particular for rearing juveniles of molluscs, or fish and crustacean larvae.

14. Use of silage products according to claim 10 for producing or manufacturing supplements of culture media.

15. Use of the silage products according to claim 10 in the culture of abalone haemocytes, with a view to increasing the adhesion of the cells and the number of cytoplasmic extensions of the cells.

16. Use of silage products according to claim 10 in food because of their high protein content.