Kelp and skipjack double-enzymolysis coupling Maillard aroma enhancement concentrated solution and preparation method thereof

Abstract

The invention belongs to the technical field of food seasoning processing, and particularly discloses a kelp and skipjack double-enzymolysis coupled Maillard aroma enhancement concentrated solution and a preparation method thereof. The preparation method comprises the following steps: rehydrating dried kelp powder, ground skipjack, euphausia superba powder and the like which are used as raw materials, and sequentially carrying out stepped double-enzymolysis, solid-liquid separation, Maillard reaction aroma enhancement, ultrahigh-pressure homogenization, concentration and blending, sterilization and filling. Rich flavor precursor substances such as amino acid, small molecule peptide and reducing sugar are efficiently released through a double-enzymolysis strategy, then rich and coordinated grilled fish and seafood characteristic flavor is generated through a controllable Maillard reaction, and finally the problem of product stability is solved through ultrahigh pressure homogenization. The concentrated solution prepared by the method is natural and mellow in flavor and uniform and stable in posture, can be widely applied to the fields of compound seasonings, leisure foods, prefabricated dishes and the like, and provides a new way for developing high-end natural seafood flavor products.

Description

Technical Field

[0001] This invention belongs to the field of food seasoning processing technology, and specifically discloses a kelp and bonito dual-enzyme hydrolysis coupled Maillard flavoring concentrate and its preparation method. Background Technology

[0002] Kelp and bonito, as marine biological resources with distinctive characteristics of Eastern cuisine, are rich in various flavor compounds, amino acids, nucleotides, and functional polysaccharides, making them ideal raw materials for preparing high-end natural seafood seasoning bases. Currently, seasoning products developed from these raw materials, such as bonito juice and kelp extract, while possessing a certain umami flavor, generally suffer from problems such as a single flavor profile, insufficient aroma intensity, and poor quality stability, failing to meet the modern food industry's pursuit of high quality, naturalness, and distinctive flavors.

[0003] Existing technologies mostly employ traditional processes such as single enzymatic hydrolysis or high-temperature extraction, which have significant limitations: First, while single enzymatic hydrolysis strategies (such as using only proteases) can release small peptides and amino acids, they are difficult to effectively break down the cell wall structure of kelp (such as cellulose and alginate), resulting in insufficient dissolution of flavor precursors and flavor polysaccharides in kelp, leading to resource waste and insufficient umami richness in the product; Second, conventional thermal reaction flavor enhancement (Maillard reaction) is mostly carried out at normal pressure and low temperature, resulting in low reaction efficiency, long reaction time, and insufficient control over the generation of key meat and roasted flavors, making it difficult to form a rich and harmonious complex seafood flavor profile.

[0004] A more prominent problem is the lack of effective methods for preserving and enhancing flavor compounds in existing processes. Direct Maillard reactions using enzymatic hydrolysates can easily lead to the loss of some volatile aroma components during prolonged heating; and the post-reaction products generally face technical bottlenecks such as oil oxidation, flavor loss, and poor physical stability (e.g., precipitation, stratification), severely impacting product shelf life and end-use performance. Furthermore, traditional techniques often rely on the addition of large amounts of chemically synthesized flavorings (such as ethyl maltol, furanone, etc.) or yeast extracts to compensate for insufficient flavor. This not only contradicts the consumer trend of "clean labels" but also distorts the product's flavor, resulting in a lack of naturalness and sophistication.

[0005] Therefore, the food industry urgently needs to develop a new green manufacturing technology that can efficiently and synergistically release the natural flavoring substances in kelp and bonito, and precisely control the Maillard reaction process to ultimately obtain a natural seafood flavoring concentrate with rich flavor, complex layers, and high stability. Summary of the Invention

[0006] To address the above-mentioned problems, this invention discloses a kelp and skipjack tuna dual-enzyme hydrolysis coupled Maillard flavoring concentrate and its preparation method.

[0007] To achieve the above objectives, the present invention includes the following technical solutions: A method for preparing a concentrated kelp and skipjack tuna double-enzyme hydrolysis coupled Maillard flavoring agent includes the following steps: (1) Raw material preparation and rehydration: Mix the following raw materials by weight: 15-30 parts dried kelp powder, 10-20 parts bonito flakes / powder, 5-15 parts Antarctic krill powder, 5-10 parts shiitake mushroom powder, 3-8 parts dried scallop extract, 8-15 parts glucose, 3-8 parts xylose, 0.5-2 parts L-cysteine ​​hydrochloride, 1-4 parts L-alanine, 0.5-1.5 parts methionine, and 100-200 parts deionized water. Stir and rehydrate at 50-60℃ for 30-60 minutes to form a uniform slurry. (2) Stepwise double enzymatic hydrolysis: (2a) First stage animal proteolytic hydrolysis: Adjust the pH of the slurry obtained in step (1) to 6.5-7.5 and the temperature to 50-55℃, add endopeptidase, and control the amount of enzyme added to be 0.05%-0.3% of the total weight of the slurry; (2b) Second stage of plant polysaccharide and flavor enhancement enzymatic hydrolysis: Adjust the pH of the system to 4.5-5.5 and the temperature to 55-60℃. First, add cellulase, controlling the amount of enzyme added to be 0.03%-0.1% of the total weight of the slurry. At the same time, add exopeptidase, controlling the amount of enzyme added to be 0.02%-0.1% of the total weight of the slurry. Enzymatic hydrolysis for 1-2 hours. (2c) Enzyme inactivation: rapidly heat the enzyme hydrolysate to 95-100℃, maintain for 10-15 minutes, and then cool to room temperature; (3) Solid-liquid separation: Filter the enzyme hydrolysate after enzyme inactivation in step (2c) through a 100-200 mesh sieve and collect the clear enzyme hydrolysate; (4) Maillard reaction flavoring: Add 2%-5% of the total weight of yeast extract and 1%-3% of fish oil to the enzymatic hydrolysate obtained in step (3), adjust the pH to 7.5-8.5 with sodium hydroxide solution, place it in a reaction vessel, and react for 30-90 minutes at 110-125℃ and 0.15-0.25MPa pressure; (5) Ultra-high pressure homogenization: The Maillard reaction solution obtained in step (4) is subjected to ultra-high pressure homogenization treatment 1-2 times at 60-80℃ and 50-80MPa pressure; (6) Concentration and blending: The homogenized liquid from step (5) is vacuum concentrated at -0.08~-0.10MPa and 60-70℃ until the soluble solids content is 40-60%; then 0.1%-0.5% of edible salt and 0.01%-0.05% of disodium 5'-ribonucleotide (I+G) by weight of the concentrated liquid are added and stirred evenly to obtain the flavor-enhancing concentrated liquid.

[0008] Furthermore, the preparation method of the above-mentioned kelp and skipjack tuna double-enzyme hydrolysis coupled Maillard flavoring concentrate is characterized in that step (6) is followed by step (7): (7) Sterilization and filling: The prepared concentrate is sterilized by ultra-high temperature instantaneous sterilization at 130-140℃ for 5-10 seconds; after cooling to 85-90℃, it is aseptically hot-filled to obtain the kelp and bonito double-enzyme hydrolysis coupled Maillard flavoring concentrate.

[0009] Furthermore, in the above preparation method, in step (1), the Antarctic krill powder is a powder made from Antarctic krill after defatting and drying, with a protein content of not less than 60% and a fat content of not more than 15%.

[0010] Furthermore, in the above preparation method, in step (2a), the amount of the endopeptidase added is 0.1%-0.2% of the total weight of the slurry.

[0011] Furthermore, in the above preparation method, in step (2a), the endopeptide is one or a combination of two of papain or neutral protease.

[0012] Furthermore, in the above preparation method, in step (2b), the exopeptidase is either a flavor protease or an aminopeptidase.

[0013] Furthermore, in the above preparation method, in step (2b), while adding cellulase and exopeptidase, alginate lyase is added, and the amount of enzyme added is controlled to be 0.01%-0.05% of the total weight of the slurry.

[0014] The present invention also discloses a kelp and skipjack tuna double-enzyme hydrolysis coupled Maillard flavoring concentrate, which is prepared by any of the above preparation methods.

[0015] Furthermore, the above-mentioned flavoring concentrate is a uniform brownish-red emulsion that does not separate or precipitate after standing for 30 days. It has a rich and mellow complex aroma of grilled fish, smoked fish, and seafood, and its amino acid nitrogen content is not less than 0.8g / 100g.

[0016] This invention also discloses the application of the above-mentioned flavor-enhancing concentrate in the preparation of compound seasonings, instant noodle sauce packets, pre-made vegetable soup bases, snack food seasonings, or pet food palatability enhancers.

[0017] Compared with the prior art, the present invention has the following outstanding advantages: 1. Significant Flavor Enhancement: The step-by-step dual-enzyme hydrolysis process can more effectively hydrolyze proteins and polysaccharides in different raw materials, providing a richer and more balanced supply of flavor precursors such as amino acids, small peptides, and reducing sugars for the subsequent Maillard reaction. Compared with traditional single-enzyme hydrolysis, it helps to form a richer roasted aroma, meaty aroma, and a more mellow seafood base flavor.

[0018] 2. Improved product stability: By introducing an ultra-high pressure homogenization step, the physical stability of the concentrate system is effectively improved, reducing the possibility of stratification and sedimentation during storage, extending shelf life, and making it easier to use.

[0019] 3. More efficient use of raw materials: The process design takes into account the characteristics of both animal and plant-based raw materials. In particular, the polysaccharide enzymatic hydrolysis promotes the destruction of kelp cell walls and the release of contents, thereby improving the comprehensive utilization rate of raw materials.

[0020] 4. In line with the clean label trend: This method mainly relies on enzymatic hydrolysis and thermal reaction to construct flavor, reducing the dependence on exogenous chemically synthesized flavorings, and making product labels simpler. Attached Figure Description

[0021] Figure 1 Results of determination of dual enzymatic hydrolysis efficiency and flavor precursor generation - amino nitrogen (g / 100g); Figure 2 Results of dual enzymatic hydrolysis efficiency and flavor precursor generation: Percentage of short peptides <1000Da (%); Figure 3 Results of determination of dual enzymatic hydrolysis efficiency and flavor precursor generation - reducing sugar content (g / 100g); Figure 4 Comparison of relative contents of Maillard reaction flavor compounds (μg / kg). Detailed Implementation

[0022] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. The amount of enzyme preparations added in the present invention is calculated as a weight percentage (w / w) of the total weight of the slurry. Those skilled in the art can adapt the dosage within the percentage range given in the present invention according to the recommended dosage of the enzyme preparation product used. When using products with particularly high enzyme activity, the amount added can be reduced accordingly.

[0023] Table 1: Raw Material List ; Example 1 A concentrated flavoring agent for kelp and skipjack tuna with dual enzymatic hydrolysis coupled with Maillard, prepared by the following steps: (1) Raw material preparation and rehydration: Accurately weigh 15kg of dried kelp powder, 10kg of bonito flakes, 5kg of Antarctic krill powder, 5kg of shiitake mushroom powder, 3kg of dried scallop extract, 8kg of glucose, 3kg of xylose, 0.5kg of L-cysteine ​​hydrochloride, 1kg of L-alanine, and 0.5kg of methionine, and stir with 100kg of deionized water at 55℃ for 45 minutes to form a uniform slurry.

[0024] (2) Stepwise double enzymatic hydrolysis: (2a) First stage animal enzymatic hydrolysis: Adjust the pH of the slurry to 7.0, the temperature to 52°C, add papain, and control the amount of enzyme added to be 0.15% of the total weight of the slurry. Hydrolyze for 3 hours. (2b) Second stage of plant polysaccharide and flavor enhancement enzymatic hydrolysis: Adjust the pH of the system to 5.0 (use food-grade acidulants (such as citric acid, lactic acid or hydrochloric acid solution) to adjust the pH of the enzymatic hydrolysis system), set the temperature to 58°C, add cellulase, and control the amount of enzyme added to 0.05% of the total weight of the slurry; at the same time, add flavor protease, and control the amount of enzyme added to 0.05% of the total weight of the slurry; enzymatic hydrolysis for 1.5 hours; (2c) Enzyme inactivation: The enzyme hydrolysate is rapidly heated to 98°C, held for 12 minutes, and then cooled to 25°C; (3) Solid-liquid separation: The enzyme hydrolysate after enzyme inactivation is centrifuged (4000 rpm, 15 minutes), and the supernatant is collected to obtain the clear enzyme hydrolysate; (4) Maillard reaction flavoring: Add 3% of yeast extract and 2% of fish oil by weight to the enzymatic hydrolysate, adjust the pH to 8.0 with sodium hydroxide solution, place it in a reaction vessel, and react for 60 minutes at 120℃ and 0.2MPa pressure; (5) Ultra-high pressure homogenization: The Maillard reaction solution was subjected to ultra-high pressure homogenization twice at 70℃ and 60MPa pressure; (6) Concentration and preparation: The homogenized liquid was vacuum concentrated at -0.09 MPa and 65°C until the soluble solids content was 50%; then 0.3% of the total weight of the concentrated liquid was added to edible salt and 0.03% of disodium 5'-ribonucleotide (I+G), and stirred evenly; (7) Sterilization and filling: The prepared concentrate is sterilized by ultra-high temperature instantaneous sterilization (135°C, 8 seconds), cooled to 88°C and then aseptically filled to obtain the flavor-enhancing concentrate.

[0025] Example 2 A concentrated flavoring agent for kelp and skipjack tuna with dual enzymatic hydrolysis coupled with Maillard, prepared by the following steps: (1) Raw material preparation and rehydration: Accurately weigh 22kg of dried kelp powder, 15kg of bonito flakes, 10kg of Antarctic krill powder, 7kg of shiitake mushroom powder, 5kg of dried scallop extract, 12kg of glucose, 5kg of xylose, 1.2kg of L-cysteine ​​hydrochloride, 2.5kg of L-alanine, and 1kg of methionine. Mix with 150kg of deionized water at 50℃ for 60 minutes to form a uniform slurry.

[0026] (2) Stepwise double enzymatic hydrolysis: (2a) First stage animal enzymatic hydrolysis: Adjust the pH of the slurry to 6.8, the temperature to 53°C, add neutral protease, control the amount of enzyme added to be 0.18%, and hydrolyze for 2.5 hours; (2b) Second stage of plant polysaccharide and flavor enhancement enzymatic hydrolysis: Adjust the pH of the system to 5.2 and the temperature to 57℃, add cellulase, and control the enzyme addition amount to 0.06%; at the same time, add aminopeptidase, and control the enzyme addition amount to 0.06%; and add alginate lyase, and control the enzyme addition amount to 0.03%; enzymatic hydrolysis for 1 hour; (2c) Enzyme inactivation: rapidly heat the enzyme hydrolysate to 100°C, maintain for 10 minutes, and then cool to 25°C; (3) Solid-liquid separation: The enzyme hydrolysate after enzyme inactivation is filtered through a 200-mesh sieve and the filtrate is collected; (4) Maillard reaction flavoring: Add 4% of the total weight of yeast extract and 1.5% of fish oil to the enzymatic hydrolysate, adjust the pH to 8.2 with sodium hydroxide solution, place it in a reaction vessel, and react for 75 minutes at 118℃ and 0.18MPa pressure; (5) Ultra-high pressure homogenization: The Maillard reaction solution was subjected to ultra-high pressure homogenization once at 65℃ and 70MPa. (6) Concentration and preparation: The homogenized liquid was vacuum concentrated at -0.085 MPa and 68°C until the soluble solids content was 45%; then 0.4% of the total weight of the concentrated liquid was added to edible salt and 0.02% of disodium 5'-ribonucleotide (I+G), and stirred evenly; (7) Sterilization and filling: The prepared concentrate is sterilized by ultra-high temperature instantaneous sterilization (138°C, 6 seconds), cooled to 90°C and then aseptically filled to obtain the flavor-enhancing concentrate.

[0027] Example 3 A concentrated flavoring agent for kelp and skipjack tuna with dual enzymatic hydrolysis coupled with Maillard, prepared by the following steps: (1) Raw material preparation and rehydration: Accurately weigh 30kg of dried kelp powder, 20kg of bonito flakes, 15kg of Antarctic krill powder, 10kg of shiitake mushroom powder, 8kg of dried scallop extract, 15kg of glucose, 8kg of xylose, 2kg of L-cysteine ​​hydrochloride, 4kg of L-alanine, and 1.5kg of methionine. Mix with 200kg of deionized water at 60℃ for 30 minutes to form a uniform slurry.

[0028] (2) Stepwise double enzymatic hydrolysis: (2a) First stage animal enzymatic hydrolysis: Adjust the pH of the slurry to 7.2, the temperature to 55℃, add a compound protease (papain and neutral protease mixed in a 1:1 ratio), control the amount of enzyme added to be 0.22%, and hydrolyze for 4 hours; (2b) Second stage of plant polysaccharide and flavor enhancement enzymatic hydrolysis: Adjust the pH of the system to 4.8 and the temperature to 60℃, add cellulase, and control the enzyme addition amount to 0.08%; at the same time, add flavor protease, and control the enzyme addition amount to 0.08%; and add alginate lyase, and control the enzyme addition amount to 0.04%; enzymatic hydrolysis for 2 hours; (2c) Enzyme inactivation: rapidly heat the enzyme hydrolysate to 95°C, maintain for 15 minutes, and then cool to 25°C; (3) Solid-liquid separation: The enzyme hydrolysate after enzyme inactivation is centrifuged (5000 rpm, 10 minutes) and the supernatant is collected; (4) Maillard reaction flavoring: Add 5% of the total weight of yeast extract and 3% of fish oil to the enzymatic hydrolysate, adjust the pH to 7.8 with sodium hydroxide solution, place it in a reaction vessel, and react for 45 minutes at 125℃ and 0.22MPa pressure; (5) Ultra-high pressure homogenization: The Maillard reaction solution was subjected to ultra-high pressure homogenization twice at 75℃ and 50MPa pressure; (6) Concentration and preparation: The homogenized liquid was vacuum concentrated at -0.095 MPa and 62°C until the soluble solids content was 55%; then 0.2% of the total weight of the concentrated liquid was added to edible salt and 0.04% of disodium 5'-ribonucleotide (I+G), and stirred evenly; (7) Sterilization and filling: The prepared concentrate is sterilized by ultra-high temperature instantaneous sterilization (132°C, 10 seconds), cooled to 85°C and then aseptically filled to obtain the flavor-enhancing concentrate.

[0029] Comparative Example 1 Process Comparison: No double enzymatic hydrolysis step A flavoring liquid, the preparation method of which is basically the same as that in Example 2, the only difference being: The enzymatic hydrolysis process in step (2) is omitted. After the raw material is rehydrated, the Maillard reaction in step (4) is carried out directly (the reaction time is extended to 120 minutes), and the subsequent steps are the same.

[0030] Comparative Example 2 Process Comparison: No Second-Stage Enzymatic Hydrolysis A flavoring liquid, the preparation method of which is basically the same as that in Example 2, the only difference being: Only the first stage of enzymatic hydrolysis in step (2a) is performed. After completion, the enzyme is directly inactivated, and the second stage of enzymatic hydrolysis in step (2b) is not performed. Subsequent steps are the same.

[0031] Comparative Example 3 Process Comparison: No Maillard Reaction Step A flavoring liquid, the preparation method of which is basically the same as that in Example 2, the only difference being: The Maillard reaction in step (4) is omitted. After solid-liquid separation, the enzymatic hydrolysate is directly subjected to ultra-high pressure homogenization in step (5) and subsequent steps.

[0032] Comparative Example 4 Process Comparison: No Ultra-High Pressure Homogenization Step A flavoring liquid, the preparation method of which is basically the same as that in Example 2, the only difference being: The ultra-high pressure homogenization process in step (5) is omitted. The Maillard reaction solution is directly concentrated in step (6).

[0033] Comparative Example 5 Raw material comparison: No key Maillard reaction substrate - reducing sugar A flavoring liquid is prepared in the same way as in Example 2, but the raw materials do not contain glucose and xylose.

[0034] Comparative Example 6 Raw material comparison: Sulfur-free amino acid precursors A flavoring liquid is prepared in the same way as in Example 2, but the raw materials do not contain L-cysteine ​​hydrochloride and methionine.

[0035] Comparative Example 7 Ingredient Comparison: No complex umami base A seasoning liquid is prepared in the same way as in Example 2, but the raw materials do not contain Antarctic krill powder and scallop extract.

[0036] Test Example 1 Determination of dual enzymatic hydrolysis efficiency and flavor precursor production Experimental objective: To verify the synergistic effect of the stepwise dual-enzymatic hydrolysis process in the efficient release of flavor precursors (amino acids, small peptides, and reducing sugars).

[0037] Test group: Example 2 (complete double enzymatic hydrolysis process).

[0038] Control group: Comparative Example 1 (no enzymatic hydrolysis), Comparative Example 2 (enzymatic hydrolysis only).

[0039] Test method: Amino nitrogen content: The amino nitrogen content in the enzymatic hydrolysate was determined by the formaldehyde value method in GB5009.235-2016 "Determination of Amino Nitrogen in Food" to measure the degree of protein hydrolysis and the amount of amino acids released.

[0040] Peptide molecular weight distribution: The molecular weight distribution of peptides in the enzymatic hydrolysate was determined by gel permeation chromatography, and the percentage of short peptides with a molecular weight less than 1000 Da was calculated.

[0041] Reducing sugar content: The content of reducing sugars produced by polysaccharide hydrolysis in the enzymatic hydrolysate was determined by direct titration, referring to GB5009.7-2016 "Determination of reducing sugars in food".

[0042] The experimental results are shown in Table 2 and Figures 1-3 .

[0043] Table 2 Results of determination of double enzymatic hydrolysis efficiency and flavor precursor production.

[0044] ; Conclusion: The stepwise dual-enzymatic hydrolysis process (protease + polysaccharide enzyme) employed in this invention exhibits a significant synergistic effect. Compared with no hydrolysis and single enzymatic hydrolysis, it achieves the highest yield of amino nitrogen and short peptides, indicating the most thorough protein hydrolysis. More importantly, through the second-stage polysaccharide hydrolysis (cellulase and alginate lyase), a large amount of reducing sugars (Example 2, 3.2 g / 100 g) were successfully released from raw materials such as kelp, providing ample flavor precursors for the subsequent Maillard reaction, which is impossible to achieve with a single protease hydrolysis process.

[0045] Test Example 2 Maillard reaction products and aroma profile analysis Experimental objective: To evaluate the effect of the coupled Maillard reaction on the enhancement of the types and contents of flavor compounds and the overall aroma characteristics of the final product.

[0046] Test group: Example 2 (complete process).

[0047] Control group: Comparative example 3 (no Maillard reaction).

[0048] Test method: Volatile flavor compounds analysis: Headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) was used to analyze the volatile flavor components in the samples. Qualitative analysis was performed by comparison with a standard mass spectrometry library, and semi-quantitative analysis was conducted using the internal standard method.

[0049] Key flavor compounds: Focus on the characteristic products of the Maillard reaction, such as sulfur-containing compounds (e.g., furanthiol, mercaptofuran), pyrazines, aldehydes and ketones, and calculate their relative contents.

[0050] Electronic nose analysis: An electronic nose sensor array is used to collect and analyze the overall aroma profile of the sample, and principal component analysis is used to distinguish the overall flavor characteristics of different samples.

[0051] The experimental results are shown in Table 3 and Figure 4 .

[0052] Table 3. Comparison of relative contents of key Maillard reaction flavor compounds (μg / kg) ; Conclusion: The Maillard reaction step is crucial for forming the core flavor of the product. Following the Maillard reaction, the product produces a large number of key meaty, roasted, and nutty flavor compounds, especially with a dramatic increase in the content of sulfur-containing compounds and pyrazines—characteristic flavors completely absent in a single enzymatic hydrolysate. Electronic nose principal component analysis showed that the aroma profiles of Example 2 and Comparative Example 3 were completely separated, with Example 2 exhibiting significantly higher response values ​​on the sensors representing "roasted" and "meaty" aromas. This demonstrates that the present invention successfully imparts a rich, full-bodied, and harmonious complex seafood flavor to the product through the Maillard reaction.

[0053] Test Example 3 Product stability and application performance testing Experimental objective: To verify the effect of ultra-high pressure homogenization process on improving the physical stability of the product and to evaluate its flavor performance in practical applications.

[0054] Test group: Example 2 (homogenized under ultra-high pressure).

[0055] Control group: Comparative example 4 (without ultra-high pressure homogenization).

[0056] Test method: Stability test: The two groups of products were placed under accelerated test conditions (40℃ constant temperature chamber) and observed for 30 days. On day 0, day 15, and day 30, the presence of stratification, sedimentation, or oil ring phenomena was recorded, and the centrifugal sedimentation rate was measured.

[0057] Cold solubility and flavor release: 0.5g of the concentrate was added to 100mL of 60℃ warm water, and the dissolution and dispersion rate and the clarity of the solution were observed after stirring. A sensory evaluation team was hired to assess the release rate and persistence of its umami and aroma intensity (out of 5).

[0058] The experimental results are shown in Table 4.

[0059] Table 4. Results of product stability and application performance tests.

[0060] ; Conclusion: Ultra-high pressure homogenization significantly improved the physical stability of the product. The product in Example 2 remained homogeneous with extremely low sedimentation after 30 days of accelerated storage, while Comparative Example 4 showed severe stratification and sedimentation. Furthermore, the homogenized product exhibited excellent cold solubility, rapidly dispersing in warm water, and its flavor compounds were released more quickly and fully, resulting in a higher sensory evaluation score. This demonstrates that this step is crucial for ensuring product shelf life and end-application performance.

[0061] Test Example 4 Verification of the necessity of the entire process and raw material compatibility and sensory evaluation Experimental objective: To comprehensively evaluate the decisive role of the complete process, key steps, and core ingredient compatibility of this invention on the sensory quality of the final product, and to conduct a comprehensive comparison with products lacking any key steps or ingredients.

[0062] Test group: Example 1, Example 2, Example 3 (complete process).

[0063] Control group: Comparative Example 1 (no double enzymatic hydrolysis), Comparative Example 2 (no second-stage enzymatic hydrolysis), Comparative Example 3 (no Maillard reaction), Comparative Example 4 (no ultra-high pressure homogenization), Comparative Example 5 (no reducing sugar), Comparative Example 6 (no sulfur-containing amino acids), Comparative Example 7 (no complex umami substrate).

[0064] Test method: Ten trained sensory evaluators conducted blind evaluations of each sample's color, aroma intensity, aroma quality (harmony, realism), taste (umami intensity, richness, off-flavors), and form (uniformity, stability) using a 1-9 point preference rating system.

[0065] The experimental results are shown in Table 5.

[0066] Table 5. Sensory evaluation results of the necessity of all elements of process and raw materials (average score) ; Note: The superscripts in the table are explained as follows: a) dull color; b) obvious fishy smell of kelp; c) slight sedimentation; d) only salty and raw material taste, without characteristic roasted aroma; e) severe stratification after standing, resulting in a significant drop in score; f) almost no Maillard reaction color and caramel or roasted aroma; g) severely lacking key characteristic aromas such as meaty and sulfurous aromas; h) significantly insufficient umami intensity and fullness, resulting in a thin mouthfeel.

[0067] in conclusion: The excellent and stable high scores of Examples 1-3 demonstrate the universality and reliability of the process parameters of the present invention, with Example 2 being the most preferred solution.

[0068] The scoring results of Comparative Examples 1-4 confirm the necessity of each process step: enzymatic hydrolysis is the foundation, Maillard reaction forms the core flavor, and ultra-high pressure homogenization ensures application stability.

[0069] The scoring results of comparison ratios 5-7 strongly demonstrate the synergistic effect of the core ingredient formulation: Comparative Example 5 (without reducing sugar) scored extremely low in color, aroma intensity, and quality, proving that reducing sugars (glucose, xylose) are essential substrates for the Maillard reaction to produce an appealing brown color and rich caramel and roasted flavor; they are indispensable.

[0070] Comparative Example 6 (without sulfur-containing amino acids) showed serious defects in aroma intensity and quality, proving that sulfur-containing amino acids (L-cysteine ​​hydrochloride, methionine) are the core precursors for generating key meat aroma and sulfur-containing flavor compounds such as furanthiols, and their absence resulted in a fatal shortcoming in the product's flavor profile.

[0071] Comparative Example 7 (without complex umami substrate) scored significantly lower in umami intensity and richness, proving that the addition of Antarctic krill powder and scallop extract is not a simple additive, but may achieve a synergistic effect of umami flavor by providing a richer combination of nucleotides (IMP, GMP) and amino acids, which greatly enhances the fullness and persistence of the flavor.

[0072] Application Example 1 Application in high-end compound seasoning sauces Application Purpose: To demonstrate the advantages of using the concentrated liquid of this invention as the core flavor component in replacing chemical seasonings and creating a "clean label" high-end compound seasoning sauce.

[0073] Application method: Develop a Japanese-style teriyaki sauce.

[0074] The formulation of this invention is as follows: concentrated liquid from Example 2 of this invention (25%), brewed soy sauce (20%), mirin (15%), white sugar (12%), apple puree (10%), starch (8%), and water (10%).

[0075] Comparative formulation (commercial standard): commercially available bonito broth (10%), brewed soy sauce (20%), mirin (15%), white sugar (12%), apple puree (10%), starch (8%), water (20%), monosodium glutamate (2%), I+G (0.5%), caramel color (1.5%), soy sauce flavoring (1%).

[0076] The application effects are shown in Table 6.

[0077] Table 6 Application in high-end compound seasoning sauces ; Conclusion: The concentrate of this invention successfully replaces monosodium glutamate, I+G, flavorings and caramel color, providing sufficient umami, body aroma, roasted aroma and color on its own, achieving a perfect unity of "clean label" and "superior flavor", and significantly improving the product's premium pricing power.

[0078] Application Example 2 Application in seasoning powders for snack puffed foods Application objective: To verify the adsorption, persistence and resistance to loss of flavor when the concentrated liquid of the present invention is applied to snack foods after being powdered.

[0079] Application method: Mix the concentrated liquid from Example 2 with maltodextrin (1:3) and spray dry to prepare flavor powder. Used for coating potato chips.

[0080] The process of this invention is as follows: After the potato slices are fried, a layer of edible oil (accounting for 2% of the weight of the potato slices) is immediately sprayed on them, and then the flavor powder of this invention (accounting for 3% of the weight of the potato slices) is evenly sprinkled on them.

[0081] Comparative process: Potato chips of the same weight are deep-fried, then sprayed with cooking oil and sprinkled with commercially available bonito flavoring powder (3% of the weight of the potato chips, the main ingredients of which are salt, monosodium glutamate, white sugar, bonito powder, yeast extract, maltodextrin and flavoring).

[0082] Table 7 shows a comparison of the application effects.

[0083] Table 7 Application of Seasoning Powder in Snack Puffed Foods ; Conclusion: The product of this invention has a flavor derived from the enzymatic hydrolysis and Maillard reaction of real ingredients, and the flavor substances are more tightly bound to the matrix. Therefore, the flavor is more durable, more resistant to high-temperature processing, and has a more stable shelf life, which is significantly better than commercially available seasoning powders that are mainly formulated with flavorings and monosodium glutamate.

[0084] Application Example 3 Application Purpose: To demonstrate how the concentrated liquid of this invention, as the core of the soup base, can quickly form a rich soup color and flavor, simplifying the production process of pre-made dishes.

[0085] Application method: Develop a pre-mixed liquid for "Oden soup base".

[0086] The formula of this invention is as follows: 1 kg of water, 40 g of the concentrated liquid from Example 2 of this invention, appropriate amounts of salt and sugar for seasoning, and boil.

[0087] Comparative formula (traditional process simulation): 1kg water, add 10g dried kelp (soak for 30 minutes and then remove), 15g bonito shavings (boil for 30 seconds and then filter), and then add salt, sugar, MSG and soy sauce for seasoning.

[0088] Table 8 shows a comparison of application effects. Table 8 Comparison of Application Effects ; Conclusion: The product of this invention provides a "foolproof" high-quality soup base solution for the pre-cooked food and catering industries, which greatly improves production efficiency, stability and standardization, solves many pain points of traditional cooking processes, and has extremely high commercial application value.

[0089] Summary of Implementation Examples: Through the above system tests, the product of this invention embodiment demonstrates the following advantages: 1. High enzymatic hydrolysis efficiency: The amino nitrogen content of the double enzymatic hydrolysate reaches 0.85g / 100g, the proportion of short peptides with a molecular weight of less than 1000Da is as high as 78.5%, and the reducing sugar content reaches 3.2g / 100g, which are 10.6 times, 5.2 times and 8 times that of the unhydrolyzed solution, respectively, proving that the flavor precursors are fully released.

[0090] 2. Abundant Flavor Compounds: After Maillard reaction, the total number of key volatile flavor compounds in the product reached 68, an increase of 94% compared to the unreacted sample (35 compounds). Among them, key meat and roasting aroma compounds such as 2-methyl-3-furanthiol (15.6 μg / kg) and 2,5-dimethylpyrazine (42.3 μg / kg) have increased significantly from non-existent to present (with an increase of over 1800%).

[0091] 3. Good product stability: After ultra-high pressure homogenization, the initial centrifugal sedimentation rate of the product is only 0.5%. After accelerated storage at 40℃ for 30 days, the sedimentation rate only rises to 1.2%, which is far lower than that of the unhomogenized sample (25.5%). It also has good cold solubility (<5 seconds).

[0092] 4. Leading Sensory Evaluation: In blind taste tests, the overall acceptance score of the product of this invention was as high as 8.9 out of 9. It significantly outperformed all comparative samples (scoring range 4.9-7.2) and commercially available similar products (7.7) in terms of umami intensity (9.0), aroma intensity (8.9), and aroma quality (8.8), quantitatively demonstrating its superior sensory quality.

[0093] The above are merely a few preferred embodiments of the present invention, described in a relatively specific and detailed manner, but they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. A method for preparing a concentrated kelp and skipjack tuna double-enzyme hydrolysis coupled Maillard flavoring agent, characterized in that, Includes the following steps: (1) Raw material preparation and rehydration: Mix the following raw materials by weight: 15-30 parts dried kelp powder, 10-20 parts bonito flakes / powder, 5-15 parts Antarctic krill powder, 5-10 parts shiitake mushroom powder, 3-8 parts dried scallop extract, 8-15 parts glucose, 3-8 parts xylose, 0.5-2 parts L-cysteine ​​hydrochloride, 1-4 parts L-alanine, 0.5-1.5 parts methionine, and 100-200 parts deionized water. Stir and rehydrate at 50-60℃ for 30-60 minutes to form a uniform slurry. (2) Stepwise double enzymatic hydrolysis: (2a) First stage animal enzymatic hydrolysis: Adjust the pH of the slurry obtained in step (1) to 6.5-7.5, the temperature to 50-55℃, add endopeptidase, control the amount of enzyme added to be 0.05%-0.3% of the total weight of the slurry, and hydrolyze for 2-4 hours; (2b) Second stage of plant polysaccharide and flavor enhancement enzymatic hydrolysis: Adjust the pH of the system to 4.5-5.5 and the temperature to 55-60℃. First, add cellulase, controlling the amount of enzyme added to be 0.03%-0.1% of the total weight of the slurry. At the same time, add exopeptidase, controlling the amount of enzyme added to be 0.02%-0.1% of the total weight of the slurry. Enzymatic hydrolysis for 1-2 hours. (2c) Enzyme inactivation: rapidly heat the enzyme hydrolysate to 95-100℃, maintain for 10-15 minutes, and then cool to room temperature; (3) Solid-liquid separation: The enzyme hydrolysate after enzyme inactivation in step (2c) is filtered through a 100-200 mesh sieve and the clear enzyme hydrolysate is collected; (4) Maillard reaction flavoring: Add 2%-5% of the total weight of yeast extract and 1%-3% of fish oil to the enzymatic hydrolysate obtained in step (3), adjust the pH to 7.5-8.5 with sodium hydroxide solution, place it in a reaction vessel, and react for 30-90 minutes at 110-125℃ and 0.15-0.25MPa pressure; (5) Ultra-high pressure homogenization: The Maillard reaction solution obtained in step (4) is subjected to ultra-high pressure homogenization treatment 1-2 times at 60-80℃ and 50-80MPa pressure; (6) Concentration and blending: The homogenized liquid from step (5) is vacuum concentrated at -0.08~-0.10MPa and 60-70℃ until the soluble solids content is 40-60%; then 0.1%-0.5% of edible salt and 0.01%-0.05% of disodium 5'-ribonucleotide (I+G) by weight of the concentrated liquid are added and stirred evenly to obtain the flavor-enhancing concentrated liquid.

2. The method for preparing a concentrated kelp and skipjack tuna double-enzyme hydrolysis coupled Maillard flavoring agent according to claim 1, characterized in that, Step (6) is followed by step (7): (7) Sterilization and filling: The prepared concentrate is sterilized by ultra-high temperature instantaneous sterilization at 130-140℃ for 5-10 seconds; after cooling to 85-90℃, it is aseptically hot-filled to obtain the kelp and bonito double-enzyme hydrolysis coupled Maillard flavoring concentrate.

3. The preparation method according to claim 1, characterized in that, In step (1), the Antarctic krill powder is a powder made from Antarctic krill after defatting and drying, with a protein content of not less than 60% and a fat content of not more than 15%.

4. The preparation method according to claim 1, characterized in that, In step (2a), the amount of the endopeptidase added is 0.1%-0.2% of the total weight of the slurry.

5. The preparation method according to claim 1, characterized in that, In step (2a), the endopeptide is one or a combination of two of papain or neutral protease.

6. The preparation method according to claim 1, characterized in that, In step (2b), the exopeptidase is either a flavor protease or an aminopeptidase.

7. The preparation method according to claim 1, characterized in that, In step (2b), while adding cellulase and exopeptidase, alginate lyase is added, and the amount of enzyme added is controlled to be 0.01%-0.05% of the total weight of the slurry.

8. A concentrated extract of kelp and skipjack tuna with dual enzymatic hydrolysis coupled Maillard flavoring, characterized in that, It is prepared by the preparation method described in any one of claims 1-7.

9. The flavor-enhancing concentrate according to claim 8, characterized in that, The concentrate is a uniform brownish-red emulsion that does not separate or precipitate after standing for 30 days. It has a rich and mellow aroma of grilled fish, smoked fish, and seafood, and its amino acid nitrogen content is not less than 0.8g / 100g.

10. The use of the flavor-enhancing concentrate as described in claim 8 or 9 in the preparation of compound seasonings, instant noodle sauce packets, pre-made soup bases, snack food seasonings, or pet food palatability enhancers.

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