A NEW METHOD TO IMPROVE ENZYME HYDROLYSIS AND RESULTANT PROTEIN FLAVOR AND BIO-ACTIVITY OF FISH OFFCUTS

Abstract

The present invention relates to the use of turbine mixing during enzymatic hydrolysis of aquatic protein from species such as fish, aquatic mammals, crustaceans and/or mollusks, to obtain high quality aquatic protein hydrolysates, having very low oxidation, improved organoleptic profile and improved biological activity of interest, for human consumption and cosmetics. The turbine mixing can inhibit oxidation during hydrolysis, contribute to an increase in the bio-activity and decrease the bitter taste of the final product. The process can vary in starting material, pre-treatment, type and amount of enzyme, hydrolysis conditions, time, degree of hydrolysis and post-treatment.

Claims

1. A process for producing an aquatic protein hydrolysate with enzyme hydrolysis, comprising: a) subjecting a protein source material, water and an enzyme to turbine mixing to obtain enzymatic hydrolysis of the protein material; b) stopping the enzymatic hydrolysis by deactivating the enzyme under turbine mixing; and c) separating the obtained hydrolyzed aquatic peptide fraction from solid material.

2. The process of claim 1, wherein the turbine mixing takes place in a turbine mixing system incorporated into the reactor from the side or the top and can be fully or partially submerged in the reaction mass.

3. The process of claim 1, wherein the enzyme is selected from proteases from bacterial, fungal or marine species.

4. The process of claim 3, wherein the proteases are endo or exo proteases from Bacillus strains, Subtilisin, including Subtilisin from Bacillus licheniformis such as Alcalase, Protamex, Flavourzyme, Neutrase, Protease A Amano, Pescalase, Fromase Promod31 or Maxatase or mixtures thereof.

5. The process of claim 1, wherein the protein source material is selected from material from fish, including fish muscle, fish skin, fish viscera, fish bones, fish heads, other fish byproducts, and any combination thereof; aquatic mammals; crustaceans, including whole crustaceans, crustacean meat and crustacean shells and process byproducts; and mollusks.

6. The process of claim 1, wherein the protein source material is subjected to grinding or mincing in the presence of water, and utilizing the minced pulp in the hydrolysis reaction.

7. The process of claim 6, further comprising: adjusting the material prior to the hydrolysis to a protein content in the range of 0.1% to 30% w/v (protein/water); adjusting the said material to a pH in the range of 5 to 9; adjusting the mixture to a convenient temperature at which the selected enzyme(s) does not become heat inactivated, in the range 30 to 80 C.; allowing the enzymatic hydrolysis to proceed for a period in the range from about 10 minutes to 1 hour or until the degree of hydrolysis (% DH) has reached a desired value in the range 2 to 70% DH; and stopping the enzymatic hydrolysis by deactivating the enzyme.

8. The process of claim 1, wherein the separation of the hydrolyzed aquatic peptide fraction from solid material in step c) is carried out by concentration, and collecting said fraction.

9. The process of claim 8, further comprising drying said fraction.

10. The process of claim 1, wherein step b) is selected from: (i) raising the temperature of the said reaction mixture to a level not below 60 C., for 5 to 60 minutes, followed by cooling; or (ii) deactivating the employed enzyme by altering the pH to pH where said enzyme is deactivated, such as a pH below about 5 or above about 9.

11. The process according to claim 1, wherein the degree of hydrolysis is followed or measured in the final product.

12. The process according to claim 1, wherein separation of the protein hydrolysate is performed by filtration.

13. The process according to claim 1, wherein separation of the protein hydrolysate is performed by filtration using ultra filtration (UF) membranes, preferably with molecular weight cut-off selected from 30, 10, 5, 3 and 1 kDa.

14. The process according to claim 1, wherein separation of the protein hydrolysate is performed by centrifugation at a speed between 500 and 10000 G and elimination of the residue obtained.

15. The process according to claim 1, wherein the hydrolysed aquatic protein fraction is recovered by concentrating the fraction.

16. The process according to claim 1, wherein the hydrolysed aquatic protein fraction is recovered by drying the fraction.

17. The process according to claim 1, wherein the solid material separated in step c) is dried and sifted to produce bones having less than 1% w/w protein on the bone surface.

18. The process according to claim 17, wherein the dry sifting is carried out using a series of decreasing mesh size vibrating sieves.

19. An aquatic protein hydrolysate, obtainable by the process of claim 1.

20. The aquatic protein hydrolysate of claim 19, wherein the aquatic protein hydrolysate is in a form of a capsule; a dried form, including powder form, flakes, granules, pellets; a liquid; a semi-liquid; a suspension; an emulsion; or a syrup.

21. A method of preparing a composition comprising adding the aquatic protein hydrolysate according to claim 19 to the composition, wherein the composition is, a food product, a food supplement, pet food, animal feed, fish feed, fertilizer, cosmetic product, pharmaceutical preparation, nutraceutical preparation, or medicament.

Description

DETAILED DESCRIPTION OF THE DRAWINGS

[0060] FIG. 1 illustrates oxidation according to the TBARS assay (mol malondialdehydes (MDA)/kg sample) during formation of the different salmon protein hydrolysates at various turbine/mixing speeds during hydrolysis; SPH-N (normal mixing), SPH-T (turbine mixing) measured as a function of degree of hydrolysis (DH) (%).

[0061] FIG. 2 shows a plot of mean scores of odour and taste attributes of SPH-N and SPH-T powder at 20% degree of hydrolysis.

[0062] FIG. 3 shows the CACO-2 in vitro cell based increase in iron uptake property for SPH-N and SPH-T at various degrees of hydrolysis.

[0063] The process of auto-oxidation and development of rancidity in food is characterized by a free radical chain mechanism proceeding via initiation, propagation, and termination stages.

[0064] Initiation: LH.fwdarw.L.

[0065] Propagation: L.+O.sub.2.fwdarw.LOO. [0066] LOO.+LH.fwdarw.LOOH+L.

[0067] Termination: LOO.+LOO..fwdarw. [0068] LOO.+L..fwdarw. non-radical products [0069] L.+L.7.fwdarw.

[0070] Highly unstable free radicals and hydroperoxides are formed that destroy bio-active peptides and small organic vitamins and help to develop off flavors in the resultant protein hydrolysate powders. Most aquatic species are high in polyunsaturated fatty acids and contain pro-oxidants such as hemoglobin and iron. These muscle constituents interact largely during enzymatic hydrolysis processing and the resultant off taste and odor are carried over into the final aquatic protein hydrolysate powder. Thus, the reaction conditions during enzymatic hydrolysis have been shown to demonstrate a major impact on oxidation. The culprit compounds, such as ketones, aldehydes and alcohols are formed at a steady rate during enzymatic hydrolysis due to the ideal conditions of slightly acid pH, 60 C. temperature and aqueous medium. They then bind to proteins and peptides and form insoluble lipid-protein complexes which leads to the off taste and odor.

[0071] In order to measure the progress of oxidation as a variant during various mixing techniques, it was necessary to follow the transformation and/or formation of reactants, intermediates and products. Since many of these compounds are very unstable, and since they are differently affected by the presence of oxygen, pro-oxidants and antioxidants, we used the universal TBARS method to measure oxidation in all its forms. TBARS has been found to be a very good indicator of lipid oxidation in seafood products and is often well correlated with sensory tests. As can be seen from FIG. 1, the turbine mixing of fish byproducts results in significantly less oxidation for the same degree of hydrolyzed protein hydrolysate. This can be attributed to both the higher rate of enzymatic hydrolysis for the turbine mixing system (as shown below in example 1) as well as the lack of pulling into solution of head space gases particularly oxygen and oxidatively produced volatile free radicals which occurs significantly during normal mixing and which is very minimized in turbine mixing. The dissolved oxygen and oxidatively produced free radicals increase the rate of oxidation of the fish protein hydrolysate product in solution for normal mixed protein hydrolysate powder (SPH-N) versus turbine mixed protein hydrolysate powder (SPH-T).

[0072] The reduction in TBARS value leads to an improvement in organoleptic profile as shown in FIG. 2. Several taste and odour profiles were measured during the test which was scored from 0 to 100. Rancid fish odor and taste and fishy, sour and bitter taste profiles improved significantly for turbine mixed (SPH-T) versus normal mixed (SPH-N) comparably hydrolyzed protein powder.

[0073] Finally, the turbine mixed protein hydrolysate powder also exhibited improved bio-activity as measured by an in vitro CACO-2 cell assay for measuring iron uptake. As can be seen in FIG. 3, the turbine mixed protein hydrolysate powder (SPH-T) has significantly better iron uptake ability as compared to the normal mixed protein hydrolysate powder (SPH-N).

[0074] The features of the invention mentioned above as well as others, will emerge more clearly from a reading of the following description of an example embodiment, the said examples being intended to be illustrative and non-limiting.

EXAMPLES

Example 1

[0075] The Effect of Turbine Mixing Versus Normal Agitation on Oxidation Levels (TBARS) in Enzyme Hydrolyzed Salmon Protein Hydrolysate Powder

[0076] Salmon backbones and heads separated and ground after filleting of whole salmon, are subjected to protein hydrolysis using a papain protease extract. The only variable in the experimentation is the method of mixing employedturbine versus normal agitation using a stirring rod and paddle agitator.

[0077] 1 kg of salmon backbone and head is ground into smaller pieces using a Waring blender such that the resultant pieces are between 5 mm and 4 cm in size. 100 g of this material is added into a 1 liter jacketed glass reactor and 200 ml of warm water at 60 C. is added. The resultant mass is warmed back to 60 C. using hot water in the jacket. For production of the Normal agitated protein hydrolysate powder (SPH-N), the reactor is equipped with a stirring rod attached to a motor at the top end and a 4 paddle stirrer at the bottom end, inch (1.27 cm) from the bottom of the reactor. For production of the Turbine agitated protein hydrolysate powder (SPH-T), the reactor is equipped with a turbine mixer which is either entirely or partially from the side or the top of the reactor, immersed in the reaction mixture.

[0078] Agitation is started and maintained at 50 RPM for the normal agitator and at full vortex speed for the turbine agitator and 1 g of the papain protease extract is added into the reactor. The reaction is stirred and approximately 10 ml of material is extracted from the reactor at designated times and centrifuged at 6000 RPM to separate the mass into solid, water and oil layers. The water layer was extracted with a pipette and dried to a powder in a lyophilizer and the degree of hydrolysis versus TBARS values were determined using methods well described in the art, at different degrees of hydrolysis and plotted as shown in FIG. 1.

[0079] Table 1 below also shows a direct comparison of time versus degree of hydrolysis for SPH-N and SPH-T revealing the much quicker hydrolysis time for turbine mixed hydrolysis reactions.

TABLE-US-00001 TABLE 1 Time vs. Degree of Hydrolysis for SPH-N and SPH-T Time % DH - SPH-N % DH SPH-T 5 minutes 4 9 10 minutes 7 17 15 minutes 12 26 20 minutes 17 31 25 minutes 23 38

Example 2

[0080] The Effect of Turbine Mixing Versus Normal Agitation on Organoleptic Performance in Enzyme Hydrolyzed Salmon Protein Hydrolysate Powder

[0081] The lyophilized dried salmon protein hydrolysate powders from both mixing methods SPH-N and SPH-T were analyzed by a panel of experts for organoleptic properties against six descriptors on a rising scale of 10-100 used by the marine industry for its products and the results plotted in FIG. 2.

Example 3

[0082] The Effect of Turbine Mixing Versus Normal Agitation on Bioactivity Performance as Measured by CACO-2 Cell Uptake Levels by Enzyme Hydrolyzed Salmon Protein Hydrolysate Powder

[0083] Intestinal cell cultures, like Caco-2 cell lines have gained in popularity as an in-vitro model of iron absorption. The human colon carcinoma cell line, Caco-2, is grown on microporous membranes in bifurcated chambers and the cells differentiated spontaneously into bipolar enterocytes that exhibit many of the characteristics of normal epithelial cells. (microvilli, tight inter-cellular junctions and border associated enzymes). The cells grow differentiated so that the apical pole extends into the upper chamber and the basal lateral pole is exposed to the lower chamber. The study can then measure iron uptake from the apical chamber, transport into the cell and secretion into the basal chamber. These cells have iron transport kinetics supporting both a saturable and non-saturable iron transport pathway, similar to observations in human and animal intestines. It should be noted that only the extrinsic added iron (10 mol/L) was used to measure the iron uptake in this experiment since no accurate way is available to determine intrinsic iron uptake and hence the values shown represent the minimum uptake that would have occurred in each digest which is a similar assumption as made in human studies.

[0084] A modified form of the commercially available 24 well Caco-2 assay kit from Celsis In vitro Technologies was used in this assay. The Celsis kit was pre-plated with Caco-2 cells with Corning Transwell filters. These Caco-2 cultures are considered acceptable for transport studies and meet the transepithelial electrical resistance (TEER) criteria of 1000 ohms. Uptake of iron was studied with Caco-2 cells grown on permeable membrane supports for 16 days, by which time cells are fully differentiated. At time zero, 1.5 ml of the different protein solutions (1%-32%) and .sup.59Fe (10 mol/L) were added to the apical chambers of the inserts. The plates were covered and incubated at 37 C. in a shaking water bath for 120 minutes. To evaluate uptake of 59Fe by the different protein solutions (1%-32%), the membranes were removed, gently washed with PBS and placed in the scintillation vials. Five ml of liquid scintillation cocktail was added to each scintillation vial and radioactive counts were measured on a Beckman LS 6500 multipurpose liquid scintillation counter. Uptake of iron by cell monolayers was expressed as nmol/well. Three wells were examined per treatment and experiments were repeated three times to give n=9 wells per treatment.

[0085] Table 2 and FIG. 3 show the results of CACO-2 iron uptake for SPH-N and SPH-T powder at various degree of hydrolysis powders.

TABLE-US-00002 TABLE 2 CACO-2 Iron Uptake Values for SPH-N and SPH-T Iron uptake Iron uptake % DH (nmol/cell) SPH-N (nmol/cell) SPH-T 0 0.021 0.019 4 0.035 0.041 8 0.055 0.073 15 0.136 0.155 26 0.139 0.167