BE1027237B1 - Process for the production of a reinforced gel-like myofibrillar protein - Google Patents

Abstract

The invention discloses a method for producing an amplified gel-like myofibrillary protein comprising the following steps: (1) producing a myofibrillary protein: (2) dissolving and concentrating the myofibrillar protein to obtain a concentrate; (3) add pyrogallic acid to the concentrate, mix homogeneously, and stand at RT to obtain a solution of mixed proteins; (4) vacuum packaging the mixed protein solution and treating it at an ultra high pressure; and (5) programmed heating and then rapid cooling of the material obtained in step (4) to obtain the highly gel-like myofibrillar protein. In the invention, the antioxidant ability of pyrogallic acid is used to reduce the degree of oxidation of the system of myofibrillar proteins, and ultra-high pressure treatment is used to change the internal structure of the myofibrillar proteins to form a regular three-dimensional network structure. The invention is suitable for controlling the quality of fish meat products in the field of food processing aids.

Description

Process for the production of a reinforced gel-like myofibrillary protein

TECHNICAL FIELD The invention relates to the production of a myofibrillar protein, in particular to a method for the production of a highly gel-like myofibrillar protein.

BACKGROUND ART Myofibrillar proteins are a type of salt-soluble protein with important biological functions, which make up the bulk of muscle fiber and account for 50% to 55% of the total content of proteins. These include the proteins myosin, actin, tropomyosin, troponin, etc. The myofibrillar proteins participate in muscle contraction and influence the tenderness of the meat as well as the functional properties of the meat product, such as: B. the gel formation ability, rheology, water retention, elasticity and texture. The gel-forming ability of the myofibrillar proteins is of great importance when treating the product of the fish mash type. Gel is to be understood as an elastic semi-solid which assumes a state between a solid and a liquid and has a regular appearance and a certain shape. The gelation of the proteins is caused by the cleavage of the non-covalent bonds and the change in the superficial structure of the proteins after regulation of the surrounding conditions or heating. In particular, the release of hydrophobic groups promotes the interaction between the proteins, so that the molecules of the proteins form a homogeneous gel with a network structure under polymerization.

The research showed that during the process of rinsing and depositing, the meat of mackerel was susceptible to oxidation, so that the content of carbonyl groups was significantly increased (Sylvie, Carolinep et al. 2015). In the case of the rainbow trout, the chemical action is like z. B. the hydrophobic power, the sulfur-sulfur bond, and the non-covalent bond, changed by the oxidation of the proteins, so that the configuration and the aggregation of the protein molecules changes (Herrera and Mackie 2004). The oxidation of the proteins in fish meat is also caused by the radical chain reaction, whereby the free radicals initially attacked the peptide structure with active side chains (Sante-Lhoutellier, Aubry et al. 2007), e.g. cysteine, tryptophan, and lysine, so that derivatives with carbonyl groups and the number of mercapto groups is reduced. This leads to a poorer gel formation ability, lower elasticity, and reduced water retention of the oxidized proteins in the fish meat.

CONTENT OF THE PRESENT INVENTION The object of the present invention is to overcome the disadvantages in the prior art and to provide a method for producing a strongly gel-like myofibrillary protein. In the present invention, the following technical solutions are used: 1) preparing a myofibrillary protein, adding a first buffer solution for separation to a crushed fish mash, homogenizing and centrifuging to separate the soluble proteins and obtain a precipitate; Add a second buffer solution for separation, homogenization, and filtering through cheesecloth

Centrifuge the solution, the precipitate being the myofibrillar protein. 2) Dissolving and concentrating the myofibrillar protein. Add the myofibrillary protein obtained in step 1) to a cold solution for dissolution, homogenization, standing at 0 ° C for 30 min, centrifugation, removal of the supernatant, and concentration with an ultrafiltration tube. 3) Antioxidation of myofibrillar protein, add pyrogallic acid to the myofibrillar protein obtained in step 2), mix homogeneously, and stand for 1-5 hours to obtain a mixed protein solution system. 4) Treatment at ultra high pressure vacuum packaging of the mixed protein solution obtained in step 3) and treatment at ultra high pressure of 100-600 MPa for 5-30mm. 5) thermally induced gelation place the product obtained in step (4) in a water bath at 20 ° C, programmed heating at a rate of 1 ° C / min to 85 ° C, maintaining the temperature of 85 ° C for 30 min, and After rapid cooling at 0 ° C, stand still for 12 hours to preserve the myofibrillar protein.

In step 1) in the above process, the first buffer solution for separation has a pH of 6.9 - 7.1 and contains 0.08 - 0.12 M KCI, 1.8 - 2.2 mM MeCl, 0.8 - 1.2 mM EGTA, 0.4 - 0.6 mM DTT and 8 - 11 mM K, HPO ,. The second buffer solution for separation has a pH value of 5.9 - 6.1 and contains 0.08 - 0.12 M NaCl and 0.8 - 1.2 mM NaN :. The homogenization in step (1) is carried out under the condition of 7000-10000rpm for 3-8 minutes.

Centrifugation is carried out under the conditions of 7000-9000 rpm at 4-8 ° C. for 10-30 minutes.

and where in step 2) the solution for dissolution is a solution with 0.5 - 1.0 M NaCl and a pH of 7.0 - 8.0. In step 3), the concentration of added pyrogallic acid is 6 µmol / g of proteins - 300 µmol / g of proteins.

In step 4), the evacuation takes 15 - 60 s for vacuum packaging, the thermal packaging 1.0 - 3.0 s; the pressure in the ultra-high pressure treatment is 100-600 MPa, and the pressure will hold for 5-30 minutes.

The invention has the following advantageous aspects: In the invention, the antioxidant ability of pyrogallic acid is used to reduce the degree of oxidation of the system of myofibrillar proteins, and treatment under ultra-high pressure is used to change the internal structure of the myofibrillar proteins and thus a to form regular three-dimensional network structure. The invention is suitable for controlling the quality of fish meat products in the field of food processing aids.

BRIEF DESCRIPTION OF THE DRAWING Figure 1 shows a flow diagram of the present invention.

FIG. 2 shows the SEM images (Scanning Electron Microscopy) of the strongly gel-like myofibrillar proteins that were obtained under various treatment conditions according to exemplary embodiments 1 to 4 of the invention.

DETAILED DESCRIPTION The technical solutions of the present invention are described and illustrated in more detail by means of the specific exemplary embodiments in connection with the figures.

Example 1 Referring to Figure 1, a method for producing an amplified gel-like myofibrillary protein can be seen, comprising the following steps: (1) Adding a first buffer solution for separation to a minced fish mash, homogenizing and centrifuging to obtain the soluble proteins to separate and obtain a first precipitate; Adding a second buffer solution to the first precipitate for separation, homogenizing, filtering through three layers of cheesecloth to obtain a liquid, and centrifuging to obtain a second precipitate; wherein the first buffer solution for separation has a pH of 7.0 and contains 0.1 MKCI, 2 mM MgCh, 1 mM EGTA, 0.5 mM DTT and 10 mM K, HPO-; and the second buffer solution for separation has a pH of 6.0 and 0.1 M NaCl and 1 mM NaN; includes. In this step, homogenization is carried out under the condition of 8000 rpm for 5 minutes and centrifugation under the condition of 8000 rpm at 4 ° C. for 15 minutes.

(2) adding a cold buffer solution for dissolution to the second precipitate, homogenizing, standing at 0 ° C for 30 minutes, removing the supernatant after centrifugation, and concentrating with an ultrafiltration tube to obtain a concentrate having a concentration of 20 mg / mL; where the solution for dissolution is a solution with 0.6 M NaCl and a pH of 8.0. In this step, centrifugation is carried out under the conditions of 8000 rpm at 4 ° C. for 15 minutes.

(3) Add pyrogallic acid to the concentrate, mix homogeneously, and stand at RT for 2 hours to obtain a solution of mixed proteins; the concentration of pyrogallic acid in the concentrate being 300 µmol / g of proteins.

(4) Vacuum packaging of the mixed protein solution for 30 seconds, thermal packaging of the vacuum packaging for 2.5 seconds, and treatment at an ultra-high pressure of 400 MPa for 15 minutes. (5) Place the material obtained in the step (4) in a water bath at 20 ° C, heat at a rate of 1 ° C / min to 85 ° C, hold the

Temperature for 30 minutes, then immediately standing still at 0 ° C for 12 hours in order to preserve the strongly gel-like myofibrillar protein.

Example 2

(1) adding a first separation buffer solution to a minced fish mash, homogenizing and centrifuging to separate the soluble proteins and obtain a first precipitate; Add a second buffer solution to the first precipitate for separation, homogenize, filter through three layers of cheesecloth to preserve one

Liquid, and centrifuge to obtain a second precipitate; wherein the first buffer solution for separation has a pH of 7.0 and contains 0.1 MKCI, 2 mM MgCh, 1 mM EGTA, 0.5 mM DTT and 10 mM K, HPO-; and the second buffer solution for separation has a pH of 6.0 and 0.1 M NaCl and 1 mM NaN :; includes.

In this step, homogenization is carried out under the condition of 8000 rpm for 5 minutes and centrifugation under the condition of 8000 rpm at 4 ° C. for 15 minutes. (2) Adding a cold solution to the second precipitate for dissolution, homogenizing, standing at 0 ° C for 30 minutes, removing the

Supernatant after centrifugation, and concentration with an ultrafiltration tube to obtain a concentrate having a concentration of 20 mg / mL; where the solution for dissolution is a solution with 0.6 M NaCl and a pH of 8.0.

In this step, that becomes

Centrifugation carried out under the conditions of 8000rpm at 4 ° C for 15min. (3) Vacuum packaging of the concentrate for 30 seconds, thermal packaging of the vacuum packaging for 2.5 seconds, and treatment at an ultra-high pressure of 400 MPa for 15 minutes. (4) Place the material obtained in step (3) in a water bath at 20 ° C, heat at a rate of 1 ° C / min to 85 ° C, hold the temperature for 30 minutes, and then immediately stand still at 0 ° C for 12 hours to receive the product.

Example 3 (1) Adding a first buffer solution for separation to a minced fish mash, homogenizing and centrifuging to separate the soluble proteins and obtain a first precipitate; Adding a second buffer solution to the first precipitate for separation, homogenizing, filtering through three layers of cheesecloth to obtain a liquid, and centrifuging to obtain a second precipitate; wherein the first buffer solution for separation has a pH of 7.0 and contains 0.1 MKCI, 2 mM MgCh, 1 mM EGTA, 0.5 mM DTT and 10 mM K; HPO; and the second buffer solution for separation has a pH of 6.0 and 0.1 M NaCl and 1 mM NaN; includes. In this step, homogenization is carried out under the condition of 8000 rpm for 5 minutes and centrifugation under the condition of 8000 rpm at 4 ° C. for 15 minutes.

(2) adding a cold solution for dissolution to the second precipitate, homogenizing, standing at 0 ° C for 30 minutes, removing the supernatant after centrifugation, and concentrating with an ultrafiltration tube to obtain a concentrate having a concentration of 20 mg / mL; where the solution for dissolution is a solution with 0.6 M NaCl and a pH of 8.0. In this step, centrifugation is carried out under the conditions of 8000 rpm at 4 ° C. for 15 minutes.

(3) Add pyrogallic acid to the concentrate, mix homogeneously, and stand at RT for 2 hours to obtain a solution of mixed proteins; the concentration of pyrogallic acid in the concentrate being 300 µmol / g of proteins.

(4) Place the solution of mixed proteins in a water bath at 20 ° C, heat at a rate of 1 ° C / min to 85 ° C, hold the temperature for 30 min, and then immediately stand still at 0 ° C for 12 hours, to get the product. Example 4 (1) adding a first buffer solution for separation to a crushed fish mash, homogenizing and centrifuging to separate the soluble proteins and obtain a first precipitate; Adding a second buffer solution to the first precipitate for separation, homogenizing, filtering through three layers of cheesecloth to obtain a liquid, and centrifuging to obtain a second precipitate; wherein the first buffer solution for separation has a pH of 7.0 and contains 0.1 MKCI, 2 mM MgCh, 1 mM EGTA, 0.5 mM DTT and 10 mM K, HPO-; and the second buffer solution for separation has a pH of 6.0 and 0.1 M NaCl and 1 mM NaN; includes. In this step, homogenization is carried out under the condition of 8000 rpm for 5 minutes and centrifugation under the condition of 8000 rpm at 4 ° C. for 15 minutes.

(2) adding a cold buffer solution for dissolution to the second precipitate, homogenizing, standing at 0 ° C for 30 minutes, removing the supernatant after centrifugation, and concentrating with an ultrafiltration tube to obtain a concentrate having a concentration of 20 mg / mL; where the solution for dissolution is a solution with 0.6 M NaCl and a pH of 8.0. In this step, centrifugation is carried out under the conditions of 8000 rpm at 4 ° C. for 15 minutes. (3) Place the concentrate obtained in step (2) in a water bath at 20 ° C, heat at a rate of 1 ° C / min to 85 ° C, hold the temperature for 30 min, and then immediately stand still at 0 ° C for 12 hours to receive the product. It can be seen from FIG. 2 that in Example 1, in which the myofibrillar proteins were obtained by means of the pyrogallic acid in combination with the treatment under ultra high pressure, the gel of proteins has small pores, fine particles and a regular, neat network. It is therefore better than the other examples in terms of the property of the gel.

It should be clear to the person skilled in the art that the technical effects the same or similar to the above examples can nevertheless be obtained and should also be included in the scope of the invention, as long as the technical solutions of the invention change within the following range: A method for producing a Reinforced gel-like myofibrillary protein comprises the following steps: (1) Adding a first buffer solution for separation to a minced fish mash, homogenizing and centrifuging to make the soluble

To separate proteins and obtain a first precipitate; Adding a second buffer solution to the first precipitate for separation, homogenizing, filtering and centrifuging to obtain a second precipitate; wherein the first buffer solution for separation has a pH of

6.9-7.1 and 0.08-0.12 M KCI, 1.8-2.2 mM MgCl, 0.8-1.2 mM EGTA, 0.4-0.6 mM DTT and 8-11 mM K, HPO- «; whereby the second buffer solution for separation has a pH value of 5.9 - 6.1 and 0.08 -

0.12 M NaCl and 0.8-1.2 mM NaN; includes; (2) adding a cold solution to the second precipitate for dissolution, homogenizing, standing at 1-2 ° C for 25-40 minutes, taking out the supernatant after centrifugation, and concentrating with an ultrafiltration tube to obtain a concentrate; wherein the buffer solution for dissolution is a solution with 0.5-0.7 M NaCl and a pH of 7.9-8.1; (3) Add pyrogallic acid to the concentrate, mix homogeneously, and stand at RT for 1-5 hours to obtain a solution of mixed proteins; wherein the concentration of pyrogallic acid in the concentrate is 6u - 300 µmol / g of protein; (4) vacuum packaging the mixed protein solution and treating at an ultra high pressure of 100-600 MPa for 5-25 minutes; and (5) placing the material obtained in step (4) in a water bath at 18-22 ° C, heating at a rate of 0.8-1.2 ° C / min to 82-86 ° C, holding the temperature for 25-40 min , and then immediately standing still at 0 ° C for 10-13 hours in order to preserve the reinforced gel-like myofibrillary protein. the homogenization in step (1) is carried out under the condition of 7000-10000rpm for 3-8 minutes. centrifugation in steps (1) and (2)

is carried out under the conditions of 7000 - 9000 rpm at 4 - 8 ° C for 10 - 30 minutes.

Only the preferred examples of the invention have been described above, which are not intended to limit the scope of the invention.

All equivalent changes and modifications made according to the patent scope of the invention and the contents of the description are intended to be within the scope of the invention.

Claims (1)

Claims 1. A method for producing a reinforced gel-like myofibrillary protein, characterized in that the method comprises the following steps: (1) adding a first buffer solution for separation to a minced fish mash, homogenizing and centrifuging to separate the soluble proteins and a first To receive precipitation; Adding a second buffer solution to the first precipitate for separation, homogenizing, filtering and centrifuging to obtain a second precipitate; wherein the first buffer solution for separation has a pH of 6.9-7.1 and 0.08-0.12 MKCI, 1.8-2.2 mM MgCh, 0.8-1.2 mM EGTA, 0.4-0.6 mM DTT and 8-11 mM K, HPO- «; whereby the second buffer solution for separation has a pH value of 5.9 - 6.1 and 0.08 - 0.12 M NaCl and 0.8-1.2 mM NaN; includes; (2) adding a cold solution to the second precipitate for dissolution, homogenizing, standing at 1-2 ° C for 25-40 minutes, taking out the supernatant after centrifugation, and concentrating with an ultrafiltration tube to obtain a concentrate; where the solution for dissolution is a solution with 0.5-0.7 M NaCl and a pH of 7.9 -8. (3) Add pyrogallic acid to the concentrate, mix homogeneously, and stand at RT for 1-5 hours to obtain a solution of mixed proteins; wherein the concentration of pyrogallic acid in the concentrate is 6u - 300 µmol / g of proteins; (4) vacuum packaging the mixed protein solution and treating at an ultra high pressure of 100-600 MPa for 5-25 minutes; and (5) placing the material obtained in step (4) in a water bath at 18-22 ° C, heating at a rate of 0.8-1.2 ° C / min to 82- 86 ° C, hold the temperature for 25-40 min, and then immediately stand still at 0 ° C for 10-13 hours to obtain a gel of the reinforced gel-like myofibrillar proteins. 2. The method for producing according to claim 1, characterized in that the first buffer solution for separation has a pH of 7.0 and contains 0.1 M KCI, 2 mM MgCl ,, 1 mM EGTA, 0.5 mM DTT and 10 mM K, HPO4 . 3. The method of production according to claim 1, characterized in that the second buffer solution for separation has a pH of 6.0 and 0.1 M NaCl and 1 mM NaN; includes. 4. The method for producing according to claim 1, characterized in that, wherein the solution for dissolution is a solution with 0.6 M NaCl and a pH of 8.0. 5. The method for producing according to claim 1, characterized in that the homogenization in step (1) is carried out under the condition of 7000-10,000 rpm for 3-8 minutes. 6. The method for producing according to claim 1, characterized in that the centrifuging in steps (1) and (2) is carried out under the conditions of 7000-9000 rpm at 4-8 ° C for 10-30 minutes. 7. The method of manufacturing according to claim 1, characterized in that step (4) comprises: vacuum packaging the solution of mixed proteins and treatment at an ultra high pressure of 400 MPa for 15 min. 8. The method of manufacture according to claim 1, characterized in that step (5) comprises: placing the material obtained in step (4) in a water bath at 20 ° C, heating at a rate of 1 ° C / min to 85 ° C, hold the temperature for 30min, and then immediately Stand at 0 ° C for 12 hours to obtain a gel of the strongly gel-like myofibrillary protein. 9. A method for producing a reinforced gel-like myofibrillary protein comprising 1) producing a myofibrillary protein: adding a first buffer solution for separation to a minced fish mash, homogenizing and centrifuging to separate the soluble proteins and obtain a precipitate; Adding a second buffer solution for separation, homogenization, filtering through cheesecloth and centrifugation of the solution, the precipitate being the myofibrillar protein; 2) Dissolving and concentrating the myofibrillar protein: adding the myofibrillary protein obtained in step 1) to a cold solution for dissolution, homogenization, standing at 0 ° C for 30 min, centrifugation, removal of the supernatant, and concentration with an ultrafiltration tube; 3) Antioxidation of myofibrillar protein: adding pyrogallic acid to the myofibrillar protein obtained in step 2), mixing homogeneously, and standing for 1-5 hours to obtain a mixed protein solution system; 4) treatment at ultra high pressure vacuum packaging of the mixed protein solution obtained in step 3) and treatment at ultra high pressure of 100-600 MPa for 5-30 minutes; wherein in step 1) in the method the first buffer solution for separation 0.08 - 0.12 M KCI, 1.8 - 2.2 mM MgCh, 0.8 - 1.2 MM EGTA, 0.4 - 0.6 mM DTT and 8 - 11 mM K, HPO4 and has a pH of 6.9 - 7.1, and the second buffer solution for separation 0.08 - 0.12 M NaCl and Contains 0.8 - 1.2 mM NaN3 and has a pH value of 5.9 - 6.1; and where in step 2) the solution for dissolution is a solution with 0.5 - 1.0 M NaCl and a pH of 7.0 - 8.0. 10. A method for producing a reinforced gel-like myofibrillary protein according to claim 9, characterized in that the method further comprises: 5) thermally induced gel formation: placing the product obtained in step (4) in a water bath at 20 ° C, programmed heating at a Rate from 1 ° C / min to 85 ° C, hold the temperature at 85 ° C for 30 min, and stand after rapid cooling at 0 ° C for 12 hours to obtain the myofibrillary protein. 11. The method for producing a reinforced gel-like myofibrillary protein according to claim 9 or 10, characterized in that in step 1) the homogenization under the condition of 7000-10000rpm for 3-8 min and the centrifugation under the conditions of 7000-9000rpm at 4 - 8 ° C for 10 - 30 minutes. 12. The method for producing a strongly gel-like myofibrillar protein according to claim 9 or 10, characterized in that in step 3) the concentration of the added pyrogallic acid is 6 µmol / g of proteins - 300 µmol / g of proteins. 13. A method for producing a reinforced gel-like myofibrillary protein according to claim 9 or 10, characterized in that in step 4) in vacuum packaging the evacuation takes 15 - 60 s, the thermal packaging takes 1.0 - 3.0 s, the pressure in the ultra-high pressure treatment 100 - 600 MPa and the pressure will hold for 5 - 30 minutes.