Process 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, comprising: a) subjecting an aquatic protein source material, water and at least one enzyme to turbine mixing with an axial flow pattern to enzymatically hydrolyze the aquatic protein source material; b) stopping the enzymatic hydrolysis by deactivating the at least one enzyme under turbine mixing with the axial flow pattern to obtain a hydrolyzed aquatic peptide fraction and solid material; and c) separating the hydrolyzed aquatic peptide fraction from the solid material to obtain the aquatic protein hydrolysate.

2. The process of claim 1, wherein the turbine mixing takes place in a turbine mixing system incorporated into a reactor from the side or the top.

3. The process of claim 1, wherein the at least one enzyme comprises proteases from bacterial, fungal or marine species.

4. The process of claim 1, wherein the aquatic protein source material comprises fish material, aquatic mammals, crustaceans, or mollusks, or any combination of the foregoing.

5. The process of claim 1, further comprising grinding or mincing the aquatic protein source material in the presence of water to produce ground or minced pulp, and utilizing the ground or minced pulp in the enzymatic hydrolysis.

6. The process of claim 5, wherein the protein content of the aquatic protein source material in water prior to enzymatic hydrolysis is in the range of 0.1% to 30% w/v of protein/water; wherein enzymatic hydrolysis is performed at a pH in the range of 5 to 9; wherein enzymatic hydrolysis is performed at a temperature in the range of 30 C. to 80 C. at which the at least one enzyme does not become heat inactivated; and wherein enzymatic hydrolysis proceeds 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 of 2% to 70% DH.

7. The process of claim 1, wherein the hydrolyzed aquatic peptide fraction is separated from the solid material by concentrating and collecting the hydrolyzed aquatic peptide fraction.

8. The process of claim 7, further comprising drying the separated hydrolyzed aquatic peptide fraction.

9. The process of claim 1, wherein the enzyme is deactivated at a temperature not below 60 C., for 5 to 60 minutes, followed by cooling.

10. The process according to claim 1, wherein the degree of enzymatic hydrolysis is followed or measured in the aquatic protein hydrolysate.

11. The process according to claim 1, wherein the hydrolyzed aquatic peptide fraction is separated from the solid material by filtration.

12. The process according to claim 11, wherein the filtration is performed using an ultra filtration membrane.

13. The process according to claim 1, wherein the hydrolyzed aquatic peptide fraction is separated from the solid material by centrifugation.

14. The process according to claim 1, further comprising concentrating the separated hydrolyzed aquatic peptide fraction to obtain the aquatic protein hydrolysate.

15. The process according to claim 1, further comprising drying the separated hydrolyzed aquatic peptide fraction to obtain the aquatic protein hydrolysate.

16. The process of claim 1, wherein the aquatic protein source material comprises fish muscle, fish skin, fish viscera, fish bones, fish heads, other fish byproducts, or any combination thereof.

17. The process of claim 4, wherein the crustaceans is whole crustaceans, crustacean meat, crustacean shells, crustacean byproducts, or any combination thereof.

18. The process of claim 1, wherein the at least one enzyme is deactivated at a pH below about 5 or above about 9.

19. The process of claim 12, wherein the ultrafiltration membrane has a molecular weight cut-off between 1 kDa and 30 kDa.

20. The process of claim 13, wherein centrifugation is performed at a speed between 500 and 10000 G.

21. The process of claim 1, wherein the at least one enzyme is a mixture of endo and exo proteases.

22. The process of claim 21, wherein the at least one enzyme is a mixture of endo and exo proteases from Bacillus strains.

23. The process of claim 21, wherein the at least one enzyme is a mixture of endo and exo proteases from Bacillus licheniformis.

24. The process of claim 3, wherein the at least one enzyme is Subtilisin.

Description

DETAILED DESCRIPTION OF THE DRAWINGS

(1) 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) (%).

(2) 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.

(3) 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.

(4) 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.

(5) Initiation: LH.fwdarw.L.

(6) Propagation: L.+O.sub.2.fwdarw.LOO. LOO.+LH.fwdarw.LOOH+L.

(7) Termination: LOO.+LOO..fwdarw. LOO.+L..fwdarw. non-radical products L.+L.7.fwdarw.

(8) 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.

(9) 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).

(10) 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.

(11) 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).

(12) 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

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

(14) 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.

(15) 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.

(16) 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.

(17) 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.

(18) 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

(19) The Effect of Turbine Mixing Versus Normal Agitation on Organoleptic Performance in Enzyme Hydrolyzed Salmon Protein Hydrolysate Powder

(20) 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

(21) 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

(22) 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.

(23) 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.

(24) 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.

(25) 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