Method for improving umami of protease hydrolysate

Abstract

The application discloses a method for improving umami of a proteolysis product, which comprises the following steps: when proteolysis is performed on a protein, an endoprotease is used in combination with an aminopeptidase APs8 to perform proteolysis, so as to obtain a proteolysis solution; the amino acid sequence of the aminopeptidase APs8 is shown as SEQ ID NO. 1; the protein is a protein derived from an animal or a plant, and is preferably an oyster protein; the endoprotease is selected from the group consisting of papain, bromelain and trypsin; and the application of the aminopeptidase APs8 in improving umami of a proteolysis product is also disclosed. The aminopeptidase APs8 is used in combination with the endoprotease to perform proteolysis on the protein, so that the degree of hydrolysis, umami value and ACE inhibitory activity of the protein can be improved significantly, and the method can be used for proteolysis, flavor improvement and function improvement of the oyster protein and other proteins. The research of the application provides technical support for high-value development of the oyster protein.

Description

Technical Field

[0001] This invention relates to a method for enhancing the umami flavor of protease hydrolysates, belonging to the field of food biotechnology. Background Technology

[0002] Oysters, as a marine biological resource rich in high-quality protein, have significant development value due to the presence of various bioactive peptides in their enzymatic hydrolysis products. However, conventional enzymatic hydrolysis of oyster protein suffers from problems such as insufficient degree of hydrolysis, noticeable bitterness in the products, and limited functional activity, which restricts its high-value utilization.

[0003] Aminopeptidases, as exopeptidases, can specifically cleave the N-terminal amino acids of peptide chains. When used in combination with endopeptidases, they can significantly improve the degree of protein hydrolysis, degrade bitter peptides, and release functional peptides. However, existing commercial aminopeptidases have shortcomings in oyster proteolysis, including poor substrate compatibility, low hydrolysis efficiency, and unsatisfactory flavor improvement. Therefore, screening for highly efficient aminopeptidases adapted to oyster proteins and clarifying their combination characteristics with endopeptidases is of great significance for improving the quality and functional activity of oyster proteolysis products. Summary of the Invention

[0004] In view of the above-mentioned prior art, the present invention provides a method for enhancing the umami flavor of protease hydrolysates, and also provides the application of aminopeptidase APs8 in enhancing the umami flavor of protease hydrolysates.

[0005] This invention is achieved through the following technical solution: A method for enhancing the umami flavor of protein hydrolysate is as follows: when hydrolyzing the protein, aminopeptidase APs8 is combined with an endopeptidase to obtain a protein hydrolysate; the amino acid sequence of the aminopeptidase APs8 is shown in SEQ ID NO.1.

[0006] Furthermore, the protein is derived from animals or plants, such as proteins derived from oysters, Antarctic krill, whiteleg shrimp, clams, hard clams, scallops, cod skin, tilapia skin, sea cucumbers, as well as spirulina protein, corn protein, soy protein, etc., with oyster protein being preferred.

[0007] Furthermore, the endopeptide is selected from any one or more of papain, bromelain, and trypsin. Even further, the endopeptide has an enzyme activity ≥200,000 U / g.

[0008] When the protein is oyster protein, the specific enzymatic hydrolysis method is as follows: take oyster meat, homogenize it to obtain oyster meat homogenate, add an appropriate amount of water as substrate solution; add endopeptidase to the substrate solution for enzymatic hydrolysis, and then add aminopeptidase APs8 to continue enzymatic hydrolysis to obtain oyster protein hydrolysate with an umami value of 10.97 or higher (when combined with bromelain, the umami value can reach 11.27).

[0009] Furthermore, the mass-to-volume ratio of the oyster meat homogenate to water is 1:3.

[0010] Furthermore, the amount of aminopeptidase APs8 and endopeptidase added is 1% of the oyster meat homogenate.

[0011] Furthermore, the parameters for enzymatic hydrolysis with endopeptidase were: 40℃ for 4 hours.

[0012] Furthermore, the parameters for adding aminopeptidase APs8 for further hydrolysis were: 40℃ for 4 hours.

[0013] Application of aminopeptidase APs8 in enhancing the umami flavor of proteolytic products.

[0014] Furthermore, the protein is derived from animal or plant sources, such as proteins from oysters, Antarctic krill, whiteleg shrimp, clams, hard clams, scallops, cod skin, tilapia skin, sea cucumbers, or spirulina protein, corn protein, and soy protein. Oyster protein is preferred.

[0015] Furthermore, in specific applications, it is combined with an endopeptide, wherein the endopeptide is selected from any one or more of papain, bromelain, and trypsin.

[0016] The present invention describes a method for enhancing the umami flavor of protein hydrolysates. This method employs a combination of aminopeptidase APs8 and an endopeptide to hydrolyze the protein. APs8 specifically cleaves the N-terminal hydrophobic amino acids of peptides produced by endopeptide hydrolysis, overcoming the bottleneck in peptide chain hydrolysis. When combined with bromelain, the degree of hydrolysis reaches 70.51%, a 73.4% improvement compared to commercial aminopeptidase groups. By degrading the N-terminal hydrophobic amino acid residues of bitter peptides, the bitterness value of the product is reduced, while umami-related amino acids (such as glutamic acid and aspartic acid) are released. The umami value of the bromelain-based combination increases from 10.17 to 11.27. Furthermore, the addition of APs8 enhances the ACE inhibitory activity of the protein hydrolysate, particularly when combined with papain, increasing the ACE inhibitory activity from 54.15% to 65.36%.

[0017] The method for enhancing the umami flavor of protease hydrolysates of the present invention can significantly improve the degree of protein hydrolysis, umami value and ACE inhibitory activity, especially the umami value, and can be used for the enzymatic hydrolysis, flavor improvement and functional enhancement of oyster protein and other proteins.

[0018] The aminopeptidase APs8 used in this invention is derived from *Stenotrophomonas maltophilia*, possessing advantages such as a broad substrate spectrum, high catalytic efficiency, and strong stability. Its combination with endopeptidase for oyster enzymatic hydrolysis effectively solves the bottleneck problems of traditional enzymatic hydrolysis processes, significantly improving protein hydrolysis capacity, flavor regulation, and functional activity. This research provides technical support for the high-value development of oyster proteins.

[0019] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description

[0020] Figure 1 Results of determination of the relative enzyme activity of aminopeptidase APs8 under different temperature conditions.

[0021] Figure 2 Results of determination of the relative enzyme activity of aminopeptidase APs8 under different pH conditions.

[0022] Figure 3 Results of oyster protein hydrolysis degree determination. Among them, boiled liquid represents treatment without the addition of any exogenous enzymes; blank control represents treatment without the addition of aminopeptidase; commercial aminopeptidase represents treatment with the addition of commercial aminopeptidase and endopeptidase (trypsin, papain, or bromelain); APs(Ag)-1 represents treatment with the addition of aminopeptidase APs1 and endopeptidase (trypsin, papain, or bromelain); APs(Sm)-8 represents treatment with the addition of aminopeptidase APs8 and endopeptidase (trypsin, papain, or bromelain).

[0023] Figure 4 Electronic tongue flavor radar diagram of the papain-related enzymatic digestion group. Among them, boiled liquid represents treatment without the addition of any exogenous enzymes; blank control represents treatment without the addition of aminopeptidase; papain + commercial aminopeptidase represents the combined use of commercial aminopeptidase and papain; papain + APs(Sm)-8 represents the combined use of aminopeptidase APs8 and papain; papain + APs(Ag)-1 represents the combined use of aminopeptidase APs1 and papain.

[0024] Figure 5Flavor radar diagram of the electronic tongue for bromelain-related enzymatic digestion groups. Among them, boiled liquid represents treatment without the addition of any exogenous enzymes; blank control represents treatment without the addition of aminopeptidase; bromelain + commercial aminopeptidase represents the combined use of commercial aminopeptidase and bromelain; bromelain + APs(Sm)-8 represents the combined use of aminopeptidase APs8 and bromelain; bromelain + APs(Ag)-1 represents the combined use of aminopeptidase APs1 and bromelain.

[0025] Figure 6 Electronic tongue flavor radar diagram of trypsin-related enzymatic digestion group. Among them, boiled liquid represents treatment without the addition of any exogenous enzymes; blank control represents treatment without the addition of aminopeptidase; trypsin + commercial aminopeptidase represents the combined use of commercial aminopeptidase and trypsin; trypsin + APs(Sm)-8 represents the combined use of aminopeptidase APs8 and trypsin; trypsin + APs(Ag)-1 represents the combined use of aminopeptidase APs1 and trypsin.

[0026] Figure 7 Results of ACE inhibitory activity assay. The boiled solution represents the treatment without any exogenous enzymes; the blank control represents the treatment without aminopeptidase; the commercial aminopeptidase represents the treatment with commercial aminopeptidase and endopeptidase (trypsin, papain, or bromelain); APs(Ag)-1 represents the treatment with aminopeptidase APs1 and endopeptidase (trypsin, papain, or bromelain); and APs(Sm)-8 represents the treatment with aminopeptidase APs8 and endopeptidase (trypsin, papain, or bromelain). Detailed Implementation

[0027] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0028] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0029] Example 1: Selection and Heterologous Expression of Aminopeptidase The inventors' laboratory has discovered a natural aminopeptidase, APs8, from *Stenotrophomonas maltophilia*, with its amino acid sequence shown in SEQ ID NO.1 and its encoding gene nucleotide sequence shown in SEQ ID NO.2. This invention attempts to utilize APs8 to enzymatically hydrolyze oyster protein and then combine it with an endopeptide to investigate its compatibility with oyster protein and whether it can enhance the umami value of the oyster protein hydrolysate.

[0030] The APs8 gene was amplified from the genome of Stenotrophomonas maltophilia, and a pET-28a(+) recombinant vector was constructed and transformed into Escherichia coli BL21(DE3). The recombinant engineered bacteria were inoculated into LB medium (containing 50 μg / mL kanamycin) and cultured at 37℃ and 200 rpm until OD200. 600 The concentration of isopropyl-β-D-thiogalactoside (IPTG) was 0.6–0.8 mM, and the mixture was induced and cultured at 16 °C and 200 rpm for 16 h.

[0031] The bacterial cells were collected by centrifugation, and the supernatant was collected by sonication and centrifugation. The supernatant was purified by affinity purification with Ni-NTA resin, and the target protein was eluted with 80 mM imidazole buffer. The protein was concentrated and desalted by ultrafiltration to obtain pure enzyme solution. The enzyme solution was lyophilized to obtain APs8 enzyme powder (enzyme activity ≥20,000 U / g) and stored in a desiccator for later use.

[0032] Meanwhile, the aminopeptidase APs1 developed by the inventor's laboratory (described in CN 118879668 A, its amino acid sequence is shown in SEQ ID NO.3) and a commercially available aminopeptidase (enzyme activity 20,000 U / g) were selected as control enzymes. Papain (200,000 U / g), bromelain (200,000 U / g), and trypsin (200,000 U / g), commonly used in the food industry, were selected as endopeptides.

[0033] Example 2 Enzymatic Properties Analysis The optimal reaction temperature for aminopeptidase APs8 was determined by measuring enzyme activity within different temperature ranges (10–60 °C). The relative enzyme activity under each temperature condition was calculated, with the highest enzyme activity defined as 100%.

[0034] The optimal reaction pH for aminopeptidase APs8 was determined by measuring its activity in different buffers ranging from pH 4.0 to 10.0 (the pH buffers used included: 50 mM citrate buffer (pH 4.0–6.0), 50 mM phosphate buffer (pH 6.0–8.0), 50 mM Tris-HCl buffer (pH 8.0–9.0), and 50 mM Gly-NaOH buffer (pH 9.0–10.0). The relative enzyme activity under each pH condition was calculated with the highest enzyme activity defined as 100%.

[0035] The results of the determination of the relative enzyme activity of aminopeptidase APs8 under different temperature conditions are as follows: Figure 1 As shown, the relative enzyme activity of aminopeptidase APs8 under different pH conditions is as follows: Figure 2 As shown. By Figure 1 and Figure 2 It is evident that the optimal reaction temperature for aminopeptidase APs8 is 45℃, and the optimal reaction pH is Tris-HCl buffer at pH 8.0. Furthermore, enzyme activity is significantly limited under weakly acidic conditions. The relative enzyme activity is highest between 40 and 45℃, exceeding 80% of the maximum activity. However, when the temperature exceeds 45℃, the enzyme activity rapidly decreases, reaching only 0.23% of the maximum activity at 55℃. In buffers with pH 9.0 and 10.0, the relative enzyme activity drops below 30%, indicating that a highly alkaline environment inhibits enzyme catalysis.

[0036] Example 3: Enzymatic hydrolysis of oyster protein (1) Pretreatment of oyster protein After removing the shells from fresh Pacific oyster meat, soak it repeatedly in 4°C ice water for 30 minutes each time to desalinate it, then rinse it with distilled water and drain it. Use a homogenizer to grind the oyster meat into a homogenate, and refrigerate it at -20°C for later use to avoid protein denaturation affecting the enzymatic hydrolysis effect.

[0037] (2) Compound enzymatic hydrolysis process Take 5.00 g of frozen oyster meat homogenate, add 15 mL of distilled water at a mass-to-volume ratio of 1:3, and stir well to obtain the substrate solution.

[0038] Five experimental groups were designed (excluding the control group, the other four groups were further divided into three subgroups), as follows: Control group (boiling solution group): No exogenous enzymes were added, and the reaction was stopped by water bath at 40℃ for 8 h; the reaction was stopped by boiling water bath for 10 minutes.

[0039] Blank control group: Papain, trypsin, and bromelain (0.05 g each) were added and enzymatically hydrolyzed at 40°C for 8 h; the reaction was terminated by boiling in a water bath for 10 minutes.

[0040] Invention Group (APs8 group): Papain, trypsin, and bromelain (0.05g each) were added, and the mixture was enzymatically hydrolyzed at 40℃ for 4 h. Then, 0.05g of APs8 enzyme powder (prepared in Example 1) was added, and the enzymatic hydrolysis was continued for another 4 h. The reaction was terminated by boiling in a water bath for 10 minutes.

[0041] Commercial aminopeptidase group: Papain, trypsin, and bromelain (0.05 g each) were added and enzymatically hydrolyzed at 40°C for 4 h. Then, 0.05 g of commercial aminopeptidase was added and enzymatic hydrolysis was continued for another 4 h. The reaction was terminated by boiling in a water bath for 10 minutes.

[0042] Aminopeptidase APs1 group: Papain, trypsin, and bromelain (0.05 g each) were added and enzymatically hydrolyzed at 40°C for 4 h. Then, 0.05 g of aminopeptidase APs1 was added and enzymatic hydrolysis was continued for another 4 h. The reaction was terminated by boiling in a water bath for 10 minutes.

[0043] After the reaction, cool to room temperature, centrifuge at 8000 r / min for 10 min, collect the supernatant, which is the oyster protein hydrolysis product, and store it in a -20℃ refrigerator for later use.

[0044] Example 4 Detection of enzymatic hydrolysis products The o-phthalaldehyde (OPA) method was used, with Ser as the standard to construct a standard curve. The free amino content and degree of hydrolysis were calculated by measuring the absorbance at 340 nm. The AAE (umami), CO0 (bitter), and AE1 (astringent) sensors of the SA402B Plus-EX electronic tongue were used to detect the flavor indicators of the enzymatic hydrolysate. Based on the ACE2 inhibitor screening kit, the inhibition rate was determined by fluorescence spectrophotometry (excitation wavelength 325 nm, emission wavelength 393 nm).

[0045] (1) Determination of degree of hydrolysis The degree of hydrolysis of the oyster protease hydrolysate obtained in Example 3 was determined, and the results are as follows: Figure 3 As shown in the figure, in the enzymatic hydrolysis experiments of oyster protein by different proteases, the degree of hydrolysis was generally higher than that of the boiled broth group without any exogenous enzymes when papain, bromelain, and trypsin were added, demonstrating the effectiveness of exogenous enzymes in optimizing the hydrolysis of oyster protein. The degree of hydrolysis reached its highest level when bromelain was used, indicating that bromelain can effectively degrade oyster protein. The enzymatic hydrolysis group with bromelain combined with aminopeptidase APs8 had the highest degree of hydrolysis, reaching 70.51%, which was 1.73 times and 1.47 times that of the commercial aminopeptidase group (40.66%) and the aminopeptidase APs1 group (47.91%), respectively.

[0046] The above results indicate that by rationally selecting and combining enzymes, the processing quality of oysters can be effectively improved, demonstrating significant potential for industrial application. In the experimental groups using papain and trypsin, the enzymatic hydrolysis effect of aminopeptidase APs8 was generally superior to the other two groups of aminopeptidases, further indicating that aminopeptidase APs8 can effectively enhance the degree of hydrolysis of oyster proteins.

[0047] (2) Flavor analysis The oyster protein hydrolysate obtained in Example 3 was measured using an electronic tongue, and the results are as follows: Figure 4 , Figure 5 , Figure 6 As shown in the figure. The results indicate that the overall differences in bitterness and astringency among the enzymatic hydrolysates were relatively small. Figure 4 It is evident that in the papain group, the bitterness value decreased from 8.00 in the boiled liquid to 7.45, while the astringency value remained within the range of 7.72–8.04. Figure 5 It is evident that the umami value increased from 10.17 in the boiled liquid to 11.27 in the combination of bromelain and aminopeptidase APs8, indicating that the addition of aminopeptidase APs8 may have a positive effect on improving umami. Similarly, from Figure 6 As can be seen, the bitterness and astringency values ​​of the trypsin group did not change significantly, while the umami value increased significantly to 10.97. In contrast, the positive control group with added commercial aminopeptidase and aminopeptidase APs1 did not show a significant change in umami. This indicates that aminopeptidase APs8 can enhance the umami value of the proteolytic products, which is an advantage that other aminopeptidases do not possess (currently reported aminopeptidases have not been shown to have a significant effect on enhancing umami).

[0048] (3) ACE inhibitory activity assay The ACE inhibitory activity of the oyster protease hydrolysate obtained in Example 3 was determined, and the results are as follows: Figure 7 As shown. By Figure 7 As can be seen, the papain-based enzymatic digests exhibited a high overall ACE inhibition rate. Among them, the combined papain and aminopeptidase APs8 digests showed the highest ACE inhibition activity, reaching 65.36%. This indicates that aminopeptidase APs8 may promote the synthesis of ACE-inhibiting peptides, thereby improving the overall inhibitory effect. The ACE inhibition activities of commercial aminopeptidase and aminopeptidase APs1 digests were both lower than the blank control group without added aminopeptidase, showing relatively low inhibitory capacity. This result further highlights the superiority of aminopeptidase APs8 when used in combination with the three endopeptidases, effectively enhancing both the degree of hydrolysis and the ACE inhibition capacity of the digests.

[0049] Example 5: Study on the effect of aminopeptidase APs8 in enhancing the umami flavor of protease hydrolysates The above experiments show that aminopeptidase APs8 can enhance the umami value of oyster proteolytic products. This experiment will conduct further comparisons.

[0050] The aminopeptidases APs1, APs2, and APs3 identified by the inventor's laboratory (the amino acid sequence of aminopeptidase APs2 is shown in SEQ ID NO.4, and the amino acid sequence of aminopeptidase APs3 is shown in SEQ ID NO.5) were expressed, purified, and prepared into enzyme powder using conventional methods. They were then combined with papain, bromelain, and neutral protease to study the changes in flavor of oyster hydrolysate with and without the addition of aminopeptidase, following the method described in Example 3.

[0051] The results showed that adding aminopeptidases APs1, APs2, and APs3 reduced the bitterness of oyster protein hydrolysate, but did not increase the umami value, a stark contrast to the results of aminopeptidase APs8. Considering the results in Example 4 where commercially available aminopeptidases failed to enhance the umami flavor of oyster hydrolysate, it can be concluded that not all aminopeptidases can improve the umami flavor of protein hydrolysate. Compared to traditional aminopeptidases, aminopeptidase APs8 has unparalleled advantages in enhancing umami flavor.

[0052] Example 6: Study on the use of aminopeptidase APs8 to enhance the umami flavor of other protein hydrolysates Based on Example 3 above, aminopeptidase APs8 was applied to improve the flavor of other protein hydrolysates. The selected protein raw materials were Antarctic krill, whiteleg shrimp, four-cornered clam, hard clam, scallop, cod skin, tilapia skin, sea cucumber, spirulina, corn protein, and soy protein.

[0053] Results: Compared with the group without aminopeptidase, the umami flavor of each protein hydrolysate increased by 12.5%, 9.43%, 13.2%, 6.55%, 10.01%, 8.27%, 5.44%, 3.25%, 7.98%, 8.44%, and 8.56% after the addition of aminopeptidase APs8. This indicates that aminopeptidase APs8 has a universal effect in improving the umami flavor of protein hydrolysates.

[0054] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. A method for enhancing the umami flavor of protease hydrolysates, characterized in that: When performing enzymatic hydrolysis on the protein, an aminopeptidase APs8 and an endopeptidase were used in combination to obtain a protein hydrolysate; the amino acid sequence of the aminopeptidase APs8 is shown in SEQ ID NO.

1.

2. The method for enhancing the umami flavor of protease hydrolysates according to claim 1, characterized in that: The protein is derived from animals or plants.

3. The method for enhancing the umami flavor of protease hydrolysates according to claim 2, characterized in that: The protein is derived from oysters, Antarctic krill, whiteleg shrimp, four-cornered clams, hard clams, scallops, cod skin, tilapia skin, sea cucumbers, or spirulina protein, corn protein, and soy protein.

4. The method for enhancing the umami flavor of protease hydrolysates according to claim 1, characterized in that: The endopeptide is selected from any one or more of papain, bromelain, and trypsin.

5. The method for enhancing the umami flavor of protease hydrolysates according to claim 1, characterized in that, The protein is oyster protein. The specific enzymatic hydrolysis method is as follows: take oyster meat, homogenize it to obtain oyster meat homogenate, add an appropriate amount of water as substrate solution; add endopeptidase to substrate solution for enzymatic hydrolysis, and then add aminopeptidase APs8 to continue enzymatic hydrolysis to obtain oyster protein hydrolysate with an umami value of 10.97 or higher.

6. The method for enhancing the umami flavor of protease hydrolysates according to claim 5, characterized in that: The mass-to-volume ratio of oyster meat homogenate to water is 1:3; the amount of aminopeptidase APs8 and endopeptidase added is 1% of the oyster meat homogenate; the parameters for hydrolysis with endopeptidase are: 40℃ for 4 hours; the parameters for further hydrolysis with aminopeptidase APs8 are: 40℃ for 4 hours.

7. Application of aminopeptidase APs8 in enhancing the umami flavor of proteolytic products, wherein the amino acid sequence of aminopeptidase APs8 is shown in SEQ ID NO.

1.

8. The application of aminopeptidase APs8 according to claim 7 in enhancing the umami flavor of proteolytic products, characterized in that: The protein is derived from oysters, Antarctic krill, whiteleg shrimp, four-cornered clams, hard clams, scallops, cod skin, tilapia skin, sea cucumbers, or spirulina protein, corn protein, and soy protein.

9. The application of aminopeptidase APs8 according to claim 7 in enhancing the umami flavor of proteolytic products, characterized in that: In specific applications, it is combined with an endopeptide; the endopeptide is selected from any one or more of papain, bromelain, and trypsin.

10. The application of aminopeptidase APs8 according to claim 7, 8, or 9 in enhancing the umami flavor of proteolytic products, characterized in that: The protein is oyster protein. In specific applications, oyster meat is taken, homogenized to obtain oyster meat homogenate, and an appropriate amount of water is added as a substrate solution. An endopeptidase is added to the substrate solution for enzymatic hydrolysis, and then aminopeptidase APs8 is added for further enzymatic hydrolysis to obtain an oyster protein hydrolysate with an umami value of 10.97 or higher.

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