LU502027B1 - Method for enzymatic hydrolysis of mussel cooking liquid - Google Patents

Abstract

This invention provides method for enzymatic hydrolysis of mussel cooking liquid that comprises the following steps: concentrating mussel cooking liquid to obtain mussel concentrated solution; 0.3-0.7g flavor protease was added to 100ml concentrated mussel solution, the pH was adjusted to 6.5-7.5, and the enzymolysis was carried out at 45-55℃ for 2-4 hours. This invention provides an enzymolysis product of mussel cooking liquid with unique flavor that is capable of being used as seasoning.

Description

Description LU502027 Method for enzymatic hydrolysis of mussel cooling liquid Technical field The invention relates to an enzymatic hydrolysis method of cooking liquid of aguatic products, particularly relates to method for enzymatic hydrolysis of mussel cooking guid, belonging to the field of seasoning products.

Background Mussels have high vield and strong growth ability, which is the main cultured shellfish in my country, having the yield that accounts for more than half of the world's yield, Al present, freezing or diving is the main processing method, In the process of mussel processing, about 1.5 tons of mussel cooking liquid will be produced for every ton of products manufactured. These mussel cooking liquid contain a lot of nutrients, if it is directly discharged into the water body, it would not only pollute the environment but also cause resources waste, Therefore, a method of using the mussel cooking liquid to develop natural mussel seasoning, which solves both the resources waste and the problem of environmental pollution, à generates good social and sconormie benefits by increasing the added value of shellfish processing products.

The invention of 2017100382485, "An Enzymatic Purification Process of Mussel Meat and Cooling Liquid" provides the following method: desaiting the water from cooking mussels to obtain concentrated solution, washing mussels, collecting and crushing meat, homogenizing them into homogensous slurry, and mbing the concentrated solution and mussel meat homogeneous slurry with a proper amount to form a mixiure, using complex protsase, Heating the mixture to enzymolysis liquid, filtering with ceramic membrane to obtain clear Bouid, separating the clear liquid with mermbrans io oblain interception liquid and permeation liquid, and the interception liquid is mussel polysaccharide liquid, decdorizing the permeate, decolorizing, and passing through the column to obtain refined filtrate; The filtrate is evaporated and concentrated under reduced pressure to obtain concentrated solution, and the concentrated solution is spray-dried to obtain mussel oligopeptide powder, Ethanol is added into the retentats,

stirred, and then the pracipiale Is separated and dried to oblain mussel IUGR 502027 polysaccharide.

Summary This invention provides method for enzymatic hydrolysis of mussel cooking liquid to obtain enzymolysis product with unique flavor that can be used as seasoning.

In order to solve the problem exist in the prior art, this invention provides method for gnzymatic hydrolysis of mussel cooking liquid described as followings: concentrating mussel cooking liquid to obtain mussel concentrated solution, C 3-0 79 flavor protsase was added to 100ml concentrated mussel solution, the pH was adjusted to 8.5.7.5, and the enzymolysis was carried out at 45-55°C for 2-4 hours.

The invention provides further improvement as following: adding 0.636 flavor protease into 100ml concentrated mussel solution, adjusting pH to 8.9, and hydrolyzing at 517 for 3.3h The invention provides further improvement as following: the mussel cooking liquid was heated at 80 and concentrated to 9-11% of the original volume to obtain mussel concentrated solution.

The invention provides further improvement as following: after the enzymolysis time is up, inactivating the enzyme (Smin in boiling Water bath, inactivate the enzyme), cooling to room temperature, and centrifuge, and the supernatant obtained by centrifugation is enzymolysis liquid.

in the invention, the response surface method is used to optimize the enzymolysis process of mussel cooking liquor.

1. Optimization of enzymatic hydrolysis process of mussel cooking liquor by response surface methodology: 11, Enzymolyais: The mussel cooking liquid was heated and concentrated at 80°C for 24 hours, which was 10% of the onginal volume, to obtain mussel concentrated solution. Fut four conical flasks into 100ml concentrated mussel solution, and add flavor proteass, trypsin, papain and compound protease into them respectively, and then carry out snzymolysis ON mussel cooking Hquor under the optimum enzymolysis conditions of the four proteases; As described in Table 1.

2) Centrifugation: after enzymolysais, inactivate the enzyme in boiling water bath 502027 Smin, then cool to room temperature, and centrifuge at 4000r/fmin for 10min: 3) Soreening of enzyme species: testing the centrifugal liquid obtained in step 2), and measuring its hydrolysis degree. The measured hydrolysis degree of flavor prolsass is the highest, and the flavor protisase is selected as the subsequent enzyme; à) Through single factor experiment of flavor proteases, the range of four influencing factors was determined, and the temperature, pH, time and enzyme dosage were optimized by response surface method on four factors and three levels. Taking the hydrolysis degree of mussel cooking liquid as the response value, the enzymatic hydrolysis process of mussel cooking guid Was optimized by using Design Expert 10.0 software.

Table 1. Enzymatic hydrolysis conditions of four enzyme preparations Enzymatic Types of enzymes PH Temperature’C | hydrolysis Additive amount time(h)

ERE 7 50 2 0.4 protease

KK

Note: The enzyme addition of 0.2% means that 0.39 of enzyme is added to 100m mussel concentrated solution, and so on for the rest.

Table 2. Level of test factors in response surface design Level Factor BE „} {i | X1 temperature CC? 45% 50 35 X2 pH ÈS 7 73 X3 Time (hi 2 3 4 X4 Additive amount of 0 enzymes {#6 2 {13 iS 0.7 5) Select samples with different degrees of hydrolysis from response surface test and select the best experimental conditions for flavor analysis, à Determination method of hydrolysis degree Determination of hydrolysis degree: refer to GBTH008.238-2003 formaldehyde potentiometric titration.

Degree of hydrolysis (DH) = amino acid nitrogen content in the cenirifuge/Lotai nitrogen content in the centrifuge.

3, Analysis of experimental results (1) the effect of the amount of enzyme on the degree of hydrolysis The added amourt of enzyme was controlled at 0.1%, 05%, 0.5%, 8.7% and 0.9%, and the hydrolysis temperature was 20°C, the time was 3h and the pH was pH? 0.

As Shown in Figure 1, with the increase of protease content, the degree of hydrolysis is on the rise, and when the amount of protease is 0.5%, the degree of hydrolysis tends to be stable. This is because the concentration of proteases is gradually saturated with the substrate, and adding too much protease will cause its self-hydrolysis, which will have a certain impact on the subsirate. Considering comprehensively, i is more appropriate to choose 0,3% ~ 0.7% of the amount of proteass.

(2) Effect of enzymolysis time on hydrolysis degree

The enzymolysis time was controlled at th, 2h, Sh, 4h and Sh, the hydrolysi® 502027 temperature was fixed at 50°C, pH was pH7,0, and the amount of enzyme was 0.5%.

As shown in Figure 2, with the increase of enzymoiysis time, the degree of hydrolysis showed a trend of first increasing and then decreasing, reaching the maximum value at the time of hydrolysis for 3h. Therefore, it is more appropriate to choose Z-4h for snzymolysis.

(2) Effect of pH on the degree of hydroiysis The pH was controlled at 6, 6.5, 7, 7.5 and 8, the hydrolysis temperature was fixed at 50°C, the amount of enzyme was 0.58%, and the time was 3h, | can be seen from Figures 3 that the degree of hydrolysis is the highest when the pH is 7, and the degree of hydrolysis above or below 7 will be lower than that of the opposite values, This is because gach enzyme has its optimum pH, and only in the right environment can the dissociated groups of the enzyme and substrate protein be in a dissociated state which is easy to combine and transform into products, otherwise the activity of the enzyme will be inhibited or even inactivated. Therefore, in the process of enzymatic hydrolysis, pH 65 ~ 7 5 is suitable.

(4) Effect of temperatures on hydrolysis degree The temperature was controlled at 40°C, 45°C, 50°C, 56°C, 60°C, the amount of immobilzed enzyme was 0.5%, the time was 3h, and the pH was oH7.0.

As shown in Figure 4, when the temperature is 40 ~ 50°C, the degree of hydrolysis is always on the rise. This is because before it is lower than is optimum temperature, the temperature is in a dominant position. With the increases of temperature, the molecular movement is violent, the contact opportunity between enzyme and substrate increases, the reaction raie accelerates, and the degree of hydrolysis increases. i reached the highest value at 50°C, and became stable at 50 ~ 55°C, but decreased obviously above 85°C. This is because the enzyme is an active protein. When the temperature is 160 high, its structure changes, the activity of the enzyme decreases, and the reaction raie decreases. Therefore, to sum up, X is more appropriate to choose 45 ~ 55°C as the enzymolysis temperature.

4. Optimization design of response surface

According to the Box-Benhnken design principle, a four-factor and three-level 502027 experimental design was carried oul by using the software of Design-Expert 8.0.6. Four factors, namely enzymolysis temperature (A), enzymolysis oH(B), enzymolysis time (C) and enzyme dosage (D), were taken as independent variables.

The hydrolysis degree {Y) of mussel cooking liquor was measured as the response vaiue, and the response surface analysis experiment was conducted, Ses the experimental analysis scheme and results.

Table 3, Response surface experimental analysis scheme and results LU502027 Number of Experiments Temperature(°C) pH Time(h) Additive amount of enzymes(%) Degree of Hydrolysis(%) i 45 7 J 03 134 2 30 7 3 LS 1358 3 35 3 3 LS 1342 4 35 7 3 8.7 Ruy = 53 #5 3 LS 133 & 45 7 3 83 FH 7 SQ 7 = 8.7 1355 8 50 335 3 {3 13,18 i si ès 3 Lt 13.3% 16 SQ * + 8.7 13,78 11 45 7 3 1.7 1381 {2 Sy 7 3 LS 168 14 50 7 3 iS 1386 18 MY 7 3 x Paw 16 35 ¥ + ÈS 13.56 17 Si 73 4 £3 13.234 18 43 FA 3 3 33H 19 45 ès 3 3.3 F356 ZU 45 Ÿ 4 {5 F338 3 56 7 3 3 1386 AS Su 7 4 0.3 13 2% ä0 63 4 8.3 LEG 4 50 63 3 {7 13,79 A 7 3 4.5 IL RE 26 35 7 3 3 135 3 54 6.3 à 33 {3.3 28 0 T3 3 0.7 £3.34 ès Mp TE 2 {LS 13

Regression equation vanance analysis With enzyme dosage, erzymolysis Ume 502027 enzymolysis temperature and enzymolysis pH as independent variables and hydrolysis degres of cooking liquor as response value, the quadratic polynomial model of nonlinear regression was fitted by using Design Expert 8.0.6 software. The prediction model is as follows: Y=+13 86+0 13K 1-017 X2+0 088K 3+0 Z0X4+0 11 X1X 2

0.027 XK 1K2+0 04851 X4+7 500E-003X2X3-0 11X2X4-0.030X3X4-0.22X1? 2 30X2° - 022X3° -0. 18X4? Variance analysis and significant comparison are shown in Table € Table 4 Variance Analysis of Hydrolysis Degree Regression Model Model Lis 14 13 31.53 < ABOUT Xi en | OI 15,68 AME X {5.34 à {34 28,39 SAT x3 £1.04 fd TAH HERA Xi ss à {48 150 SSH KIND £048 | ès 2.49 {LOR MINE TOI } TIER {23 $4403 Kis £08.03 } 8 10F03 0.61 $4478 NINA {ius 1 dôde 3.48 LOH KAR AOE 3 60-03 DIT 08 Ken Be? GET 33.03 < GN XF La A &3 23.68 sages X 0.3 t 03 15.25 $008 Pagidual error DIS 14 G43 Loss of aussi Kam EE 10 O1 1.38 Hadad Pare evror Den ds 4 13 The iota deviation 3.33 38 li can be sean from Figure 4 thai the mode! F<0 0001, indicating that tha response surface regression model is very significant: The missing term P = 0.4624 > 0.05 is not significant, which indicates that the regression equation can better fit the real respors® 502027 surface. From the four factors of X11, X2, X3, A4, the factors of X1, X2, X3, A4, X12, X2 à, X3 2, KA 2 have a significant effect on the results (P<0.05), but XTAZ, X2X3, X2X4, K2X4 have no significant effect on the results (P>0.05) By comparing the absolute value of the linear coefficient of the equation, the primary and secondary factors can be judged. The order of the influencing factors in this experiment is X3>X4>X2>ÄT.

The four factors in the mode! are fixed pairwise, two of which are fixed at 0 level, and the submodel of the interaction of the other two factors on hydrolysis degree Y is obtained. Figure 5-10 shows the response surface diagram of the interaction of four factors on hydrolysis degree. Optimum condition verification test Response surface analysis showed that the optimum combination of the four influencing factors was enzymolysis temperature 51.08°C, oH6.87, enzymolysis time 3.28h, enzyme dosage 063%, and the predicted hydrolysis degree was 13.9805%. in order to test the accuracy of the prediction of the model the experiment was carried out under the optimum conditions of enzymolysis temperature 51°C, pHE. 9, enzymolysis time 3.2h and enzyme dosage

0.63%, and the hydrolysis degree was 14.01%, which was basically close to the predicted vaiue, indicating that the predicted value and the real value had a certain fit, further verifying the feasibility of the model.

The flavor substances of enzymatic hydrolysate were analyzed by gas chromatography-mass spectrometry.

That is to say, the present invention uses Design Expert 8.0.6 software and Box- Behnken response surface design method, On the basis of single factor experiment, four factors, namely enzymolysis time, enzymolysis temperature, enzymolysis pH and enzyme dosage, are selected as corresponding variables. Taking the optimum point of single factor experiment as the center, a horizontal valus around the optimum point is taken as the level of response surface, and the degree of hydrolysis is taken as the response value, and polynomial fitting regression is performed on the corresponding variables.

By adopting the method of the invention, the enzymolysis product of mussel cooking fiquor with unique flavor can be obtained, and can be used as a seasoning.

Brief Description Of The Figures LU502027 The specific embodiments of the present invention will be described in further detail with reference to the accompanying drawings.

Fig. 1 is a graph showing the effect of the amount of enzyme added on the degree of hydrolysis; Fig. & is the influence of enzymolysis time on hydrolysis degree; Fig. 2 is the influence of enzymoiysis pH on hydrolysis degree, Fig. 4 is the influence of enzymolysis temperature on hydrolysis degree: Fig. S is a response surface diagram of the interaction between enzvmoiysis temperature and enzymolysis pH Fig. 5 is 3 response surface diagram of the inisraction betwsen snzymolysis temperature and enzymolysis time; Fig. 7 is a response surface diagram of the interaction between enzymolysis temperature and enzyme dosage, Fig. 8 is a response surface diagram of the interaction between enzymaolysis pH and enzymoilysis time: Fig. 9 is à response surface diagram of the interaction between enzymatic hydrolysis pH and enzyme dosage; Fig. 10 is a response surface diagram of the interaction between enzymolysis ims and enzyme dosage.

Description of the present invention The invention will be further described with reference to the following specific gxarmples, but the scope of protection of the invention is not limited to this: The activity of favor proisase is about 8x10 4 Ug, and the flavor protease produced by Guangzhou Huagi Biological Co, Lid. can be selected, Examples 1-2 The method of enzymatic hydrolysis of mussel cooking liquor comprises the following steps in tum 1) The fresh mussel cooking liquid is heated at 60°C for 24 hours, and then concentrated to 10% of the original volume Io obtain mussel concentrated solution.

2) Add flavor protease into 100m! concentrated mussel solution, adjust the pH, and perform enzymolysis at 3 specific enzymolysis temperature for a corresponding time. The dosage of enzyme, enzymolysis temperature, enzymoiysis time and pH are shown in

Table 5 below. 0.3% enzyme dosage means adding 0.30 flavor proteases io 100m MUSSEL 502027 concentrated solution, and so on.

3) After the enzymolyeis time is up, inactivate the enzyme in boiling water bath for Grain, then cool to room temperature and centrifuge at 4000r/min for 10min; The supernatant obtained by centrifugation is enzymoiysis liquid. The flavor components and content of enzymatic hydrolysate were analyzed, and the results are shown in Table 6 below. The degree of hydrolysis is shown in Table 5.

Table 5 Additive amount of Degree of enzymes(%) Hydrolysis(%) 6 10.3 45 3 8.5 113.11 9 0.63 Si 23.3 6.5 14.01 ; Table 5 Retention i | Aroma component Sample 1 Sample 2 Sample 3 time (min)

7.67 2-Methylpyrazine 0.22 0.27 0.28

10.75 2,5-Dimethylpyrazine 21.36 29.06 27.63

13.87 2-ethyl-5-methylpyrazine 1.76 1.99 2.1

14.1 2,3,5-trimethylpyrazine 3.05 - 3.61

15.52 2-Methyl-5-isopropylpyrazine 0.6 0.95 0.66 17 2-Ethyl-3,6-dimethylpyrazine 12.24 15.09 17.71

17.49 2,3-Dimethyl-5-cthylpyrazine - 0.17 -

17.58 2,3,5,6-Tetramethylpyrazine - 0.27 -

17.65 2-Methyl-5-propyl-pyrazine 0.15 - 0.16

17.74 2-Ethanyl-3,5-dimethylpyrazine 0.13 - 0.13

19.93 2,3-Diethyl-5-methylpyrazine 0.17 0.25 0.29

20.06 3,5-Dicthyl-2-methyl-pyrazine 0.46 0.3 0.36

20.22 3,5-Dimethyl-2-propylpyrazine 0.47 0.44 0.50

20.36 2-ethyl-3,5,6-trimethylpyrazine 0.52 0.59 0.66

21.13 5-sec-butyl-2,3-dimethylpyrazine - 0.13 0.20 2,5-dimethyl-3-(2-methylpropyl) LUS02027

21.42 i 0.32 0.46 0.60 pyrazine

21.62 3,3-Dimethyl-2-(2-methylpropyl) _ _ 0.64 pyrazine

23.53 2-Methyl-6-(3-methyl-butyl)-pyrazine - 0.29 0.14 2,5-dimethyl-3-(2-

24.9 . 0.23 0.34 0.46 methylbutyl)pyrazine 2,5-Dimethyl-3-(3-

25.34 ; 1.49 1.86 4.22 methylbutyl)pyrazine

34.17 2-butyl-3,5-dimethylpyrazine 0.33 0.38 0.45 Total 435 52.84 60.8 Nitrogenous compounds

1.43 2-(aziridine-1-yl)ethylamine - 0.25 0.29

1.52 Trimethylamine 1.3 1.69 1.55

16.13 Decanenitrile 3.6 - -

17.88 Allylcyclohexylamine - - - Sum 5.94 2.98 3.09 Aldehydes

2.86 3-Methylbutyraldehyde 0.4 0.34 0.35

2.97 2-methylbutanal 0.23 0.22 0.2

8.69 3-furfural - 0.1 -

11.77 3-Methylthiopropanal - - 0.15 Retention ; | Aroma component Sample 1 Sample 2 Sample 3 time (min)

13.47 Benzaldehyde 2.23 1.03 1.63 azabicyclo[3.2.1]octane-8-

16.38 - - 2.96 carbaldehyde

17.26 Nonanal 4.63 4.76 6.22

17.52 Phenylacetaldehyde 0.38 - 0.62

21.05 cis-7-tetradecenal - 0.21 -

23.81 (Z)-2-decenal 2.14 1.90 2.54

27.53 Dodecaldehyde 03

26.5 2-Hydroxy-4-methylbenzaldehyde - 0.16 - Sum 10.01 8.72 14.67 Alcohol compounds

8.9 Furfuryl alcohol 2.37 3.24 3.24

11.3 1-Octen-3-ol 1.15 1.24 1.35

20.6 Maltitol 0.11 - 0.19

27.52 2-Methyl-undecanol 0.13 0.31 0.25

28.99 Dodecanol - - 1.48

30.17 3,7,11-Trimethyl-1-dodecanol 0.12 - 0.1

28.81 Tridecanol 1.1 1.77 0.33

30.16 2-Methyl-1-hexadecanol - - - LUS02027

34.86 2-(Dodecane)ethanol 0.34 - 0.36 Sum 5.32 6.56 7.30 Ketones

2.36 2,3-Butanedione 0.30

16.33 2-nonanone 1.50 0.25 1.30

20.12 2- hydroxy -3,5,5- trimethylcyclohex - _ 0.33 0.24 1- en -1- one

28.36 Methylchromone - 0.75 - Sum 1.50 1.33 1.84 Esters

2.57 Formyl acetate 0.14 0.17 0.22

11.02 Methylcyclopentene carboxylate 0.5 1.45 0.93 Sum 0.64 1.62 1.15 Aromatic compounds

4.73 Toluene - - -

9.13 Methoxyphenoxime 0.3 0.41 0.7

22.34 1-methylene-1H-indene - - -

26.18 2-Methylnaphthalene 0.12 - -

29.25 2-Methyl-7-octadecyne - 0.1 -

36.69 2,3-Drhydro-1,1 3-trimethy1-3 -phenyl- 0.51 _ _ 1H-indene Sum 0.93 0.51 0.7 Phenolic compounds

30.64 2,4-Di-tert-butylphenol 2.19 6.17 4.66 Sum 2.19 6.17 4.66 Others

2.41 Vinyl ether 0.16 - -

10.8 4,6-Dimethylpyrimidine - - -

11.41 2-Acetylfuran - 0.11 0.1

17.26 2,3-Dihydrobenzo[b]thiophene - - -

18.36 2-Acetylpyrrole 0.31 0.38 0.4

20.8 2-Acetyl-2-thiazoline - 0.18 -

28.55 2-Amino-4-hydroxyquinoline hydrate 0.49 - 0.60

29.82 5,7-Dimethyl-1H-indazole 0.24 0.48 0.25 Sum 1.20 1.15 1.35 From the aspect of flavor, the number of useful pasks detected in sample No Bis the highest, and the contents of aldehydes and pyrazines, which play an important role in the flavor of mussel cooking Hauor, are higher than other samples, From the point of view 2Us02027 hydrolysis degree, the hydrolysis degree of No 9 sample measured by formaldehyde titration is also the highest, so it is considered that No. © sample is the best sample.

Comparative Embodiment 1: The flavor protease in Embodiment 9 was changed into the compound protease as described in 2014 7100382495, and hydrolysis was carried out under the optimal enzymolysis conditions; The rest is the same as Embodiment 9 The final result is that the total content of pyrazines is 51.1%, and the total content of aldehydes is 3.2%.

Comparative Embodiment 2-1: The preparation method of mussel concentrated solution in Embodiment 9 was changed to: The fresh mussel cooking liguid was healed at 80°C and concentrated to 20% of the original volume to obtain mussel concentrated solution À 200ml of mussel concentrated solution A was used instead of 100mi of mussel concentrated solution, so that the effective amount was the same; The amount of flavor protease is the same as that in Embodiment 8; The rast is the same as Example 9.

The final result is that the hydrolysis degree is 13.56%, the total content of pyrazines is 54%, and the total content of aldehydes is only 4.7%.

Finally, it should be noled that the above list is only a few specific embodiments of the present invention, Obviously, the present invention is noi limited to the above embodiments, but there are many variations. All modifications that can be directly derived or associated by those skilled in the art from the disclosure of the present invention should be regarded as the scope of protection of the present invention,

Claims (4)

Claims LU502027

1. Method for enzymatic hydrolysis of mussel cooking liquid is characterized in concentrating mussel cooking liquid to obtain mussel concentrated solution; 03-079 favor protease was added to 100m concentrated mussel solution, the pH was adiusisd to 65-76, and the enzymolysis was carried out at 45-55°C for 2-4 hours, à Method for enzymatic hydrolysis of mussel cooking liquid is characterized in adding 0.63g flavor protease into 100ri concentrated mussel solution, adiusting pH to

6.9, and hydrolyzing at 51°C for 23h.

3. Method for enzymatic hydrolysis of mussel cooking liquid is characterized in the mussel cooking liquid was heated at 60°C and concentrated is 8-11% of the original volume to obtain mussel concentrated solution.

4. Method for enzymatic hydrolysis of mussel cooking liquid is characterized in inactivating the enzyme is inactivated when the enzymolysis is finished, then cooling it to room temperature for centrifuging, and the supernatant obtained by centrifugation is enzymolysis liquid.