A method for preparing oyster juice based on mutual action of bacteria and enzymes and gradient variable temperature heat reaction
By using the synergistic process of bacterial-enzyme interaction and gradient temperature-controlled thermal reaction to prepare oyster sauce, the problems of flavor loss and low efficiency in traditional oyster sauce production have been solved. This method achieves efficient and stable flavor enhancement and cost reduction, producing oyster sauce with a rich aroma and mellow flavor.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHIMEIZHAI (YANGJIANG) FOOD CO LTD
- Filing Date
- 2026-05-18
- Publication Date
- 2026-07-03
AI Technical Summary
Traditional oyster juice production processes result in flavor loss, low production efficiency, and a limited range of flavors. Existing enzymatic hydrolysis technologies are costly and have unstable effects, and the Maillard reaction produces a limited variety of aroma compounds.
Oyster juice is prepared by combining bacterial-enzyme interaction with gradient temperature thermal reaction. By optimizing the fermentation enzyme production process of the strain and the synergistic effect of exogenous proteases, deep and targeted hydrolysis of oyster proteins is achieved. Combined with gradient temperature thermal reaction conditions, a rich library of flavor precursors is generated, and the flavor is enhanced by multi-component enzymatic hydrolysis and Maillard reaction.
This method produces high-quality oyster sauce with a rich aroma, mellow flavor, and smooth texture. It significantly increases the content of amino nitrogen, reduces volatile basic nitrogen, enriches characteristic aroma compounds, improves flavor complexity and stability, and reduces the cost of enzyme preparations.
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Figure CN122320185A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a method for preparing oyster sauce, and more particularly to a method for preparing oyster sauce based on the synergistic effect of bacterial-enzyme interaction and gradient temperature thermal reaction. Background Technology
[0002] Oyster sauce is a umami-rich condiment made primarily from oysters and is widely loved by consumers. Traditional oyster sauce production relies on prolonged heating and simmering to extract and concentrate the flavor compounds from the oysters. This method has several drawbacks: First, prolonged high-temperature simmering destroys some of the umami and nutrients in the oysters, such as amino acids and small peptides, resulting in flavor loss; second, the process is time-consuming and energy-intensive, leading to low production efficiency; finally, oyster sauce prepared using traditional methods has a limited flavor profile and lacks complex aroma layers, relying mainly on the natural umami of the oyster sauce itself and added seasonings.
[0003] To improve traditional processes, existing technologies have incorporated enzymatic hydrolysis and Maillard reaction flavoring techniques. For example, some existing solutions (such as patent publication number CN101564142A) disclose a method for preparing oyster sauce by stepwise enzymatic hydrolysis of oyster meat using exogenous proteases and finally adding salt. Compared to traditional boiling, this method improves protein utilization and flavor to a certain extent.
[0004] However, these existing technical solutions have the following drawbacks: First, directly adding commercial enzyme preparations is costly, and the enzyme activity may be affected by endogenous inhibitory substances in oyster pulp, resulting in unstable hydrolysis efficiency; second, the single enzyme cleavage site is limited, and the hydrolysis of proteins is not thorough enough, and the resulting large peptides are not conducive to the generation of aroma in the subsequent Maillard reaction; finally, the Maillard reaction used is mostly under a single isothermal condition, resulting in a limited variety and layering of aroma substances, and failing to form a complex and rich "oyster aroma" flavor profile.
[0005] Therefore, developing a new method to prepare oyster sauce with rich, full-bodied flavor and strong layering, which can optimize the interaction between bacteria and enzymes and the enzymatic hydrolysis process and have a synergistic effect with gradient temperature thermal reaction, has important industrial value. Summary of the Invention
[0006] To address the aforementioned technical problems, the present invention aims to provide a method for preparing oyster sauce based on the synergistic effect of bacterial-enzyme interaction and gradient temperature-dependent thermal reaction. This method optimizes the fermentation and enzyme production process of the microbial strain and works synergistically with exogenous proteases to achieve deep and targeted hydrolysis of oyster proteins, generating an ideal flavor precursor library rich in small molecule peptides and amino acids. Subsequently, gradient temperature-dependent thermal reaction conditions highly matched with this precursor library are designed to achieve targeted enhancement and regulation of flavor, ultimately producing a high-quality oyster sauce with a rich aroma, mellow umami flavor, smooth texture, and no unpleasant flavors.
[0007] The technical solution adopted by this invention to solve the problem is: a method for preparing oyster sauce based on the synergistic effect of bacterial-enzyme interaction and gradient temperature thermal reaction, comprising the following steps: S1. Raw material pretreatment: Select fresh oysters, clean and remove the shells, wash the oyster meat and set aside. Cook the oyster meat in water, filter and separate to obtain oyster water liquid; mix the cooked oyster meat with an appropriate amount of water and blend to obtain oyster paste liquid for later use.
[0008] S2. Enzymatic hydrolysis by bacterial-enzyme interaction: Adjust the pH of the oyster broth to 6.0-7.5, inoculate with protease-producing bacteria, and ferment at 35-40℃ for 48-60 hours. After fermentation, add exogenous protease for enzymatic hydrolysis for 3-7 hours. After hydrolysis, rapidly raise the temperature to above 90℃ and maintain it for 10-15 minutes to sterilize and inactivate the enzymes. Then cool to room temperature to obtain oyster hydrolysate.
[0009] S3. Gradient temperature change thermal reaction: Add 1%-5% of reducing sugar by mass to the obtained oyster enzymatic hydrolysate, mix well, adjust the pH value to neutral, react at 70-90℃ for 2-7 hours, raise the temperature to 100-120℃ and react for 3-7 hours, cool after the reaction is completed to obtain oyster juice.
[0010] As a further improvement to the above technical solution, in step S2, the fermentation culture includes a static treatment and a stirring treatment. The static treatment lasts for 4-48 hours, and the stirring treatment involves stirring for 10-30 minutes every 6-12 hours during the fermentation culture.
[0011] As a further improvement to the above technical solution, in step S2, the bacterial strain is one or a mixture of two or more of Bacillus subtilis, Aspergillus oryzae, Aspergillus niger, Bacillus licheniformis, and Bacillus amyloliquefaciens in any proportion.
[0012] As a further improvement to the above technical solution, in step S2, the inoculation amount of the strain is 1%-5% of the oyster broth mass.
[0013] As a further improvement to the above technical solution, in step S2, the protease is one or a mixture of two or more of alkaline protease, neutral protease, papain, EF108AN enzyme, and FF104AN enzyme in any proportion.
[0014] As a further improvement to the above technical solution, in step S2, the amount of protease added is 0.1%-0.5% of the oyster broth mass.
[0015] As a further improvement to the above technical solution, in step S3, the reducing sugar is one or a mixture of two or more of D-fructose, D-galactose, D-xylose, D-ribose, glucose, lactose, and maltose in any proportion.
[0016] The beneficial effects of this invention are: 1. This invention creatively adopts a process route of "bacterial-enzyme interaction and hydrolysis + stepwise gradient temperature change thermal reaction". During the fermentation process, the endonuclease produced is responsible for efficiently cleaving the peptide bonds inside the protein, generating a large number of small molecule peptides and amino acids, providing sufficient precursor substances for the gradient temperature change thermal reaction, and is more likely to combine with reducing sugars in the gradient temperature change thermal reaction; the exonuclease can hydrolyze the hydrophobic terminal peptide bonds, reduce the production of bitter peptides, and improve the flavor of the product. The subsequent gradient temperature change thermal reaction generates basic aroma substances at low temperature, and then reacts deeply at high temperature to form complex and rich aroma compounds with roasted meat aroma, nutty aroma and other aromas. The two complement each other and jointly construct the rich taste and aroma profile of oyster sauce.
[0017] 2. This invention significantly improves the overall quality of the final product through the synergistic effect of bacterial-enzyme interaction hydrolysis and gradient temperature-dependent thermal reaction. It effectively increases the amino nitrogen content and reduces the volatile basic nitrogen content. In terms of flavor optimization, it reduces the fishy smell of 1-octen-3-ol while enriching characteristic aroma compounds such as n-octanal and 3-ethyl-2,5-methylpyrazine, effectively reducing the fishy smell of oyster sauce and enhancing the product's flavor. Ultimately, it achieves a simultaneous improvement in sensory evaluation and flavor stability. Oyster sauce prepared using this method exhibits significantly superior umami and aroma compared to products produced using traditional methods, with a strong flavor profile and a long-lasting aftertaste. Furthermore, the use of bacterial-enzyme interaction hydrolysis reduces enzyme preparation costs and improves the stability and efficiency of enzymatic hydrolysis. Attached Figure Description
[0018] The present invention will be further explained and described below with reference to the accompanying drawings and specific embodiments.
[0019] Figure 1 The amino nitrogen content is shown in the examples and comparative examples; Figure 2 The volatile basic nitrogen content is shown in the examples and comparative examples; Figure 3 Sensory evaluation radar charts for both examples and comparative models; Figure 4 A table comparing the content of characteristic flavor compounds in the examples and comparative examples; Figure 5 This is a process flow diagram of the present invention. Detailed Implementation
[0020] In all embodiments of the present invention, unless otherwise emphasized, temperature and pressure are at normal temperature and pressure. Unless otherwise specified, the equipment can be used according to conventional settings.
[0021] It should be understood that these descriptions are merely exemplary and are not intended to limit the scope of the invention.
[0022] Reference Figure 1-5 A method for preparing oyster sauce based on the synergistic effect of bacterial-enzyme interaction and gradient temperature thermal reaction includes the following steps: S1. Pre-treatment: Wash the fresh oyster meat, cook it in water, filter and separate it to obtain oyster boiling liquid; mix the cooked oyster meat with an appropriate amount of water and then blend it to obtain oyster paste for later use.
[0023] S2. Enzymatic hydrolysis by bacterial-enzyme interaction: Adjust the pH of the oyster broth to 6.0-7.5, inoculate with protease-producing bacteria, ferment and culture at 35-40℃ for 48-60 hours, and add exogenous protease for enzymatic hydrolysis for 3-7 hours. After enzymatic hydrolysis, quickly raise the temperature to above 90℃ and maintain it for 10-15 minutes to sterilize and inactivate the enzyme, and then cool to room temperature to obtain oyster enzymatic hydrolysate.
[0024] S3. Gradient temperature change thermal reaction: Add 1%-5% of reducing sugar by mass to the obtained oyster enzymatic hydrolysate, mix well, adjust the pH value to neutral, react at 70-90℃ for 2-7 hours, raise the temperature to 100-120℃ and react for 3-7 hours, cool after the reaction is completed to obtain oyster juice.
[0025] In some embodiments, in step S2, the fermentation culture includes a static treatment and a stirring treatment. The static treatment lasts for 4-48 hours, and the stirring treatment involves stirring once every 6-12 hours during the fermentation culture, with each stirring lasting 10-30 minutes.
[0026] In some embodiments, in step S2, the bacterial strain is one or a mixture of two or more of the following in any proportion: Bacillus subtilis (CCTCC AB 130410), Aspergillus oryzae (CICC 2053), Aspergillus niger (CPCC 400524), Bacillus licheniformis (CGMCC 1.265), and Bacillus amyloliquefaciens (SICC 1.1021).
[0027] In some embodiments, in step S2, the inoculation amount of the microbial strain is 1%-5% of the mass of the oyster broth.
[0028] In some embodiments, in step S2, the protease is one or a mixture of two or more of the following in any proportion: alkaline protease, neutral protease, papain, EF108AN enzyme (a compound of various food-grade proteases with different functions, purchased from Angel Enzyme Preparations (Yichang) Co., Ltd.), and FF104AN enzyme (a compound protease derived from Aspergillus oryzae and Bacillus subtilis, purchased from Angel Enzyme Preparations (Yichang) Co., Ltd.).
[0029] In some embodiments, in step S2, the amount of protease added is 0.1%-0.5% of the oyster broth mass.
[0030] In some embodiments, in step S3, the reducing sugar is one or a mixture of two or more of D-fructose, D-galactose, D-xylose, D-ribose, glucose, lactose, and maltose in any proportion.
[0031] In some embodiments, the oyster hydrolysate from step S2 is centrifuged to obtain the supernatant. Compared with not centrifuging, not centrifuging can retain more flavor substances and solids.
[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be noted that, unless otherwise specified, the embodiments and features described herein can be combined with each other.
[0033] Example 1: S1. Pretreatment: Wash the fresh oyster meat, cook it in water, filter and separate to obtain oyster boiling liquid; mix the cooked oyster meat with an appropriate amount of water at a ratio of 1:2 (w / w) and then blend to obtain oyster paste for later use.
[0034] S2. Enzymatic hydrolysis by bacterial-enzyme interaction: Adjust the pH of the oyster broth to 6.5, inoculate it with 3% of Bacillus subtilis (CCTCC AB 130410) bacterial suspension and 2% of Aspergillus oryzae (CICC 2053) spore suspension, and incubate at 35℃ for 12 hours (i.e., static treatment). Then, stir for 20 minutes every 8 hours (i.e., stirring treatment). The total fermentation time is 48 hours. After fermentation, add 0.3% FF104AN enzyme for 7 hours of enzymatic hydrolysis. After the enzymatic hydrolysis is completed, quickly raise the temperature to 95℃ and hold for 10 minutes to sterilize and inactivate the enzyme. After cooling to room temperature, centrifuge and collect the supernatant to obtain oyster enzymatic hydrolysate.
[0035] S3. Gradient temperature change thermal reaction: Add 3% by mass of 1:1 compound D-fructose and D-galactose to the obtained oyster enzymatic hydrolysate, mix well, adjust the pH value to 7.0, react at 80℃ for 2 hours, raise the temperature to 100℃ and react for 3 hours, and cool to room temperature with ice water after the reaction to obtain Example 1.
[0036] Example 2: S1. Pretreatment: Wash the fresh oyster meat, cook it in water, filter and separate to obtain oyster boiling liquid; mix the cooked oyster meat with an appropriate amount of water at a ratio of 1:1.5 (w / w) and then blend to obtain oyster paste for later use.
[0037] S2. Enzymatic hydrolysis by bacterial-enzyme interaction: Adjust the pH of the oyster broth to 7.0, inoculate it with 4% of Aspergillus niger (CPCC400524) spore suspension and 3% of Bacillus licheniformis (CGMCC 1.265) bacterial suspension by weight of the oyster broth, and incubate at 40℃ for 24 hours. Then, stir for 30 minutes every 12 hours for a total fermentation time of 60 hours. After fermentation, add 0.2% EF108AN enzyme for 5 hours of enzymatic hydrolysis. After the enzymatic hydrolysis is completed, quickly raise the temperature to 95℃ and hold for 10 minutes to sterilize and inactivate the enzyme. After cooling to room temperature, centrifuge and collect the supernatant to obtain the oyster enzymatic hydrolysate.
[0038] S3. Gradient temperature change thermal reaction: Add 5% by mass of 2:1 compound D-fructose and D-galactose to the obtained oyster enzymatic hydrolysate, mix well, adjust the pH value to 7.0, react at 70℃ for 2 hours, raise the temperature to 110℃ and react for 4 hours, and cool to room temperature with ice water after the reaction is completed to obtain Example 2.
[0039] Example 3: S1. Pretreatment: Wash the fresh oyster meat, cook it in water, filter and separate to obtain oyster boiling liquid; mix the cooked oyster meat with an appropriate amount of water at a ratio of 1:2.5 (w / w) and then blend to obtain oyster paste for later use.
[0040] S2. Enzymatic hydrolysis by bacterial-enzyme interaction: Adjust the pH of the oyster broth to 6.0, and inoculate it with 3% of Bacillus subtilis (CCTCC AB 130410) bacterial suspension, 5% of Bacillus amyloliquefaciens (SICC 1.1021) bacterial suspension, and 3% of Aspergillus oryzae (CICC 2053) spore suspension. After static culture at 35℃ for 8 hours, start stirring for 15 minutes every 6 hours, with a total fermentation time of 50 hours. After fermentation, add 0.4% alkaline protease for 3 hours of enzymatic hydrolysis. After the enzymatic hydrolysis is completed, quickly raise the temperature to 95℃ and hold for 10 minutes to sterilize and inactivate the enzyme. After cooling to room temperature, centrifuge and collect the supernatant to obtain the oyster enzymatic hydrolysate.
[0041] S3, Gradient temperature change thermal reaction: Add 1% by mass of D-fructose and D-galactose in a 3:1 ratio to the obtained oyster enzymatic hydrolysate, mix well, adjust the pH to 7.0, react at 70°C for 2 hours, raise the temperature to 100°C and react for 3 hours, and cool to room temperature with ice water after the reaction to obtain Example 3.
[0042] Example 4: S1. Pretreatment: Wash the fresh oyster meat, cook it in water, filter and separate to obtain oyster boiling liquid; mix the cooked oyster meat with an appropriate amount of water at a ratio of 1:2 (w / w) and then blend to obtain oyster paste for later use.
[0043] S2. Enzymatic hydrolysis by bacterial-enzyme interaction: Adjust the pH of the oyster broth to 6.8, inoculate it with 3% of Bacillus licheniformis (CGMCC 1.265) bacterial suspension and 3% of Bacillus subtilis (CCTCC AB 130410) bacterial suspension by weight of the oyster broth, and incubate at 40℃ for 10 hours. Then, stir for 25 minutes every 10 hours, for a total fermentation time of 54 hours. After fermentation, add 0.5% papain for 5 hours of enzymatic hydrolysis. After the enzymatic hydrolysis is completed, quickly raise the temperature to 95℃ and hold for 10 minutes to sterilize and inactivate the enzyme. After cooling to room temperature, centrifuge and collect the supernatant to obtain the oyster enzymatic hydrolysate.
[0044] S3. Gradient temperature change thermal reaction: Add 4% by mass of D-fructose and D-galactose in a 2:1 ratio to the obtained oyster enzymatic hydrolysate, mix well, adjust the pH to 7.0, react at 80℃ for 2 hours, raise the temperature to 110℃ and react for 4 hours, and cool to room temperature with ice water after the reaction to obtain Example 4.
[0045] Example 5: S1. Pretreatment: Wash the fresh oyster meat, cook it in water, filter and separate to obtain oyster boiling liquid; mix the cooked oyster meat with an appropriate amount of water at a ratio of 1:1.8 (w / w) and then blend to obtain oyster paste for later use.
[0046] S2. Enzymatic hydrolysis by bacterial-enzyme interaction: The pH of the oyster slurry was adjusted to 7.2, and 4% of the oyster slurry mass of Aspergillus oryzae (CICC2053) spore suspension was inoculated. After static culture at 35℃ for 24 hours, the mixture was stirred for 30 minutes every 12 hours during fermentation, with a total fermentation time of 55 hours. After fermentation, 0.1% neutral protease was added for enzymatic hydrolysis for 4 hours. After the enzymatic hydrolysis was completed, the temperature was rapidly raised to 95℃ and held for 10 minutes to sterilize and inactivate the enzyme. After cooling to room temperature, the supernatant was collected by centrifugation to obtain the oyster enzymatic hydrolysate.
[0047] S3. Gradient temperature change thermal reaction: Add 2% by mass of D-fructose and D-galactose in a 1:2 ratio to the obtained oyster enzymatic hydrolysate, mix well, adjust the pH to 7.0, react at 75°C for 2 hours, raise the temperature to 115°C and react for 3 hours, and cool to room temperature with ice water after the reaction to obtain Example 5.
[0048] Comparative Example 1: S1. Pretreatment: Wash the fresh oyster meat, cook it in water, filter and separate to obtain oyster boiling liquid; mix the cooked oyster meat with an appropriate amount of water at a ratio of 1:2 (w / w) and then blend to obtain oyster paste for later use.
[0049] S2. Enzymatic hydrolysis by bacterial-enzyme interaction: Adjust the pH of the oyster slurry to 6.5, inoculate with 3% of the oyster slurry mass of Bacillus subtilis (CCTCC AB 130410) bacterial suspension, and incubate at 37℃ for 24 hours without stirring during fermentation. After fermentation, rapidly raise the temperature to 95℃ and maintain it for 10 minutes to sterilize and inactivate enzymes. After cooling to room temperature, centrifuge and collect the supernatant to obtain oyster enzymatic hydrolysate.
[0050] S3, Gradient temperature change thermal reaction: Add 3% D-fructose to the obtained oyster enzymatic hydrolysate, mix well, adjust the pH to 7.0, react at 100℃ for 5 hours, and cool to room temperature after the reaction to obtain Comparative Example 1.
[0051] The comparative example used a single strain of bacteria for fermentation without the addition of exogenous proteases, and the thermal reaction was carried out under a single constant temperature condition. Compared with Example 1 of the present invention, its amino nitrogen content was significantly reduced and its sensory score was significantly lower. This directly proves the necessity of "bacteria + enzyme" interaction and gradient temperature thermal reaction for improving hydrolysis efficiency and enriching flavor.
[0052] Comparative Example 2: S1. Pretreatment: Wash the fresh oyster meat, cook it in water, filter and separate to obtain oyster boiling liquid; mix the cooked oyster meat with an appropriate amount of water at a ratio of 1:2 (w / w) and then blend to obtain oyster paste for later use.
[0053] S2. Enzymatic hydrolysis by bacterial-enzyme interaction: Adjust the pH of the oyster slurry to 7.0, inoculate with 5% of the oyster slurry mass of Aspergillus niger spore suspension, and incubate at 35℃ for 48 hours. During fermentation, stir for 30 minutes every 12 hours. After fermentation, rapidly raise the temperature to 95℃ and hold for 10 minutes to sterilize and inactivate enzymes. After cooling to room temperature, centrifuge and collect the supernatant to obtain oyster enzymatic hydrolysate.
[0054] S3, Gradient temperature change thermal reaction: Add 3% D-galactose to the obtained oyster enzymatic hydrolysate, mix well, adjust the pH to 7.0, react at 100℃ for 5 hours, and cool to room temperature after the reaction to obtain Comparative Example 2.
[0055] Comparative Example 2 changed the strain (Aspergillus niger), but still did not add exogenous protease and the thermal reaction was a single isothermal process. The results were similar to those of Comparative Example 1, which further illustrates that no matter what protease-producing strain is used, the lack of exogenous enzyme synergy and gradient temperature process cannot achieve the efficient hydrolysis and flavor optimization described in this invention. This proves that the synergistic effect of this invention is universal rather than dependent on a specific strain.
[0056] Comparative Example 3: S1. Pretreatment: Wash the fresh oyster meat, cook it in water, filter and separate to obtain oyster boiling liquid; mix the cooked oyster meat with an appropriate amount of water at a ratio of 1:2 (w / w) and then blend to obtain oyster paste for later use.
[0057] S2. Enzymatic hydrolysis via bacterial-enzyme interaction: Adjust the pH of the oyster broth to 5.5, inoculate with a 3% (by weight) suspension of Bacillus licheniformis (CGMCC 1.265) from the oyster broth, and incubate statically at 45°C for 48 hours without stirring during fermentation. No exogenous proteases are added after fermentation. After fermentation, rapidly raise the temperature to 95°C and maintain for 10 minutes to sterilize and inactivate the enzymes. After cooling to room temperature, centrifuge and collect the supernatant to obtain the oyster enzymatic hydrolysate.
[0058] S3, Gradient temperature change thermal reaction: Add 3% D-fructose to the obtained oyster enzymatic hydrolysate, mix well, adjust the pH to 7.0, react at 100℃ for 5 hours, and cool to room temperature after the reaction to obtain Comparative Example 3.
[0059] Although Comparative Example 3 used a protease-producing strain, the fermentation conditions (pH deviating from the optimal range, no stirring) were not optimized, and no exogenous protease or gradient temperature step was added, resulting in unsatisfactory hydrolysis. This indicates that relying solely on the strain's own enzyme production is insufficient; optimization of fermentation conditions and the addition of exogenous protease are both indispensable, forming the core of the bacterial-enzyme interaction.
[0060] Comparative Example 4: S1. Pretreatment: Wash the fresh oyster meat, cook it in water, filter and separate to obtain oyster boiling liquid; mix the cooked oyster meat with an appropriate amount of water at a ratio of 1:2 (w / w) and then blend to obtain oyster paste for later use.
[0061] S2. Enzymatic hydrolysis by bacterial-enzyme interaction: 0.2% of FF104AN enzyme by weight of oyster slurry was added directly to the oyster slurry. After static culture at 50℃ for 4 hours, the temperature was rapidly raised to 95℃ and held for 10 minutes to inactivate the enzyme. After cooling to room temperature, the supernatant was collected by centrifugation to obtain oyster enzymatic hydrolysate.
[0062] S3, Gradient temperature change thermal reaction: Add 3% D-xylose to the obtained oyster enzymatic hydrolysate, mix well, adjust the pH to 7.0, react at 100℃ for 5 hours, and cool to room temperature after the reaction to obtain Comparative Example 4.
[0063] Comparative Example 4 used only exogenous proteases without microbial fermentation. Although the amino nitrogen content was higher than that of Comparative Examples 1 to 3, its flavor complexity and body were far inferior to Example 1. This fully demonstrates that there is a significant synergistic effect between the endonucleases produced by microbial fermentation and the exogenous proteases. The interaction between microorganisms and enzymes is not only an additive effect of enzyme activity, but more importantly, it optimizes the spectrum of small peptides and amino acids in the hydrolysis products, providing a more ideal library of flavor precursors for subsequent gradient temperature-varying thermal reactions.
[0064] The physicochemical properties and sensory evaluation of Examples 1, 2, 3, 4, 5, Comparative Examples 1, 2, 3, and 4 obtained by the above method were tested, and the results are as follows: Figure 1-3 As shown.
[0065] Specifically, the physicochemical indicators were tested as follows: Amino acid nitrogen: The method was followed according to GB 5009.235-2016 "National Food Safety Standard - Determination of Amino Acid Nitrogen in Food". The results are as follows: Figure 1 As shown. Sensory evaluation method: Twenty professional evaluators conducted blind tests on oyster sauce samples processed in different ways, evaluating umami intensity, umami persistence, fishy intensity, metallic intensity, bitterness intensity, rancidity intensity, body, viscosity, particle size, and transparency. Each dimension was scored out of 5 points; the higher the score, the stronger the performance in that dimension. The results are shown below. Figure 2 As shown.
[0066] from Figure 1 As can be seen, the amino nitrogen content of the oyster juice prepared in the example groups was significantly higher than that in the comparative group (P < 0.05), which directly proves the high efficiency of bacterial-enzyme hydrolysis. Example 1 had the highest amino nitrogen content. The increase in amino nitrogen content in Example 1 is mainly attributed to the high enzyme activity of the bacteria during the enzyme production stage of bacterial fermentation. This enzyme activity more effectively catalyzes the cleavage of peptide bonds in oyster proteins, generating oligopeptides, polypeptides, and free amino acid molecules. This verifies the synergistic effect of optimized parameters, effectively promoting protein degradation and the generation of flavor precursors. Other example groups and the comparative group had relatively low amino nitrogen content due to parameter deviations, such as single or mismatched bacterial strains, insufficient fermentation time, unsuitable pH or temperature, or the high cost of using commercial enzyme preparations, resulting in insufficient protein degradation.
[0067] from Figure 2As can be seen, the TVB-N content of the oyster sauce prepared in each embodiment of the present invention is between 0.0123 and 0.0175 g / 100ml, which is significantly lower than that of all comparative examples (p < 0.05). Among them, the TVB-N value of Example 1 is the lowest (0.0123 g / 100ml), which is about 57%, 59%, and 53% lower than that of Comparative Examples 1, 2, and 3, respectively, and about 32% lower than that of Comparative Example 4 (single exogenous enzymatic hydrolysis). Comparative Examples 1 to 3, because they only used microbial fermentation without adding exogenous proteases, or the fermentation conditions were improper (such as pH deviation, lack of stirring), resulted in a high degree of protein putrefaction, with TVB-N values as high as 0.0264 to 0.0302 g / 100ml; although Comparative Example 4 added exogenous proteases, it lacked the synergistic effect of endonucleases produced by microbial fermentation, and its TVB-N value (0.0182 g / 100ml) was still significantly higher than that of all examples. The above data fully demonstrates that the synergistic process of "bacterial-enzyme interaction + gradient temperature thermal reaction" of this invention can effectively inhibit the excessive degradation of proteins and the generation of amines, significantly reduce the volatile basic nitrogen content of oyster juice, and thus improve the freshness and quality of the product.
[0068] from Figure 3 As can be seen, the oyster sauce prepared using Example 1 has better flavor and texture, with a rich taste, strong oyster aroma, and freshness without any fishy smell. This is mainly due to the optimized bacterial-enzyme interaction enzymatic hydrolysis process providing richer and more balanced flavor precursors (peptides and amino acid profiles of specific molecular weights) for the gradient temperature-variable thermal reaction, which, combined with the step-by-step gradient temperature-variable thermal reaction, jointly creates an excellent flavor profile. In contrast, the oyster sauce prepared using other example groups and comparative groups showed varying degrees of decline in overall sensory qualities because the bacterial-enzyme interaction enzymatic hydrolysis effect or the gradient temperature-variable thermal reaction conditions were not optimal. For example, Comparative Example 1 may lack umami, Comparative Example 2 may have a single flavor profile, Comparative Example 3 may have developed off-flavors due to poor fermentation, and Comparative Example 4, while having acceptable umami, may not have the same flavor complexity and body as the bacterial-enzyme hydrolysate.
[0069] from Figure 4As can be seen, all embodiments of the present invention can efficiently enrich aroma-enhancing substances such as n-octanal, 3-ethyl-2,5-methylpyrazine, 2-pentylfuran, phenylacetaldehyde, and trans-2-nonenal, while significantly reducing the content of fishy-smelling substances 1-octen-3-ol and hexanal. Taking Example 1 as an example, its n-octanal content (156.3 μg / kg) is approximately 5.5 times that of Comparative Example 1 and 2.5 times that of Comparative Example 4; its 3-ethyl-2,5-methylpyrazine content (89.6 μg / kg) is approximately 7.2 times that of Comparative Example 1 and 2.5 times that of Comparative Example 4; while the fishy-smelling substance 1-octen-3-ol (4.2 μg / kg) is only 18% of Comparative Example 1 and 27% of Comparative Example 4. Although the aroma indicators of Examples 2 to 5 are slightly lower than those of Example 1, they are still significantly better than all comparative examples. Comparative Examples 1 to 3, lacking the synergistic effect of exogenous proteases, had a limited peptide and amino acid profile in their fermentation products, resulting in fewer and lower concentrations of aroma compounds generated during subsequent thermal reactions. Comparative Example 4, although containing exogenous proteases, lacked the deep protein cleavage by endonucleases produced during microbial fermentation, leading to suboptimal flavor precursors. Its aroma-enhancing substance content was significantly lower than the example group, while the residual amount of fishy-smelling substances was noticeably higher. These data confirm that this invention, through the synergistic effect of microbial-enzyme interaction and gradient temperature-dependent thermal reactions, can directionally enrich characteristic aroma compounds and inhibit the formation of fishy-smelling substances, achieving a significant improvement in oyster sauce flavor. The method of this invention can effectively reduce the content of the fishy-smelling substance 1-octen-3-ol, while simultaneously enriching characteristic aroma compounds such as n-octanal and 3-ethyl-2,5-methylpyrazine, which is crucial for forming a complex flavor system.
[0070] In summary, this invention establishes optimal reaction conditions for a combined enzymatic hydrolysis-thermal reaction process using a multi-microbial enzyme interaction and a Maillard dual-stage heating system. The synergistic effect of these two processes enhances the deodorization and aroma enhancement effects of oysters and their processed products. Optimized microbial fermentation precisely controls the peptide composition of the oyster hydrolysate, providing a highly efficient substrate for the gradient temperature-varying thermal reaction. Optimized Maillard processes reduce residual bitter peptides, thereby improving product flavor. The multi-microbial combination efficiently decomposes oyster proteins into small-molecule peptides and amino acids, significantly improving the bioavailability of the hydrolysate. This makes the treated hydrolysate more readily bind with reducing sugars during the gradient temperature-varying thermal reaction, significantly improving the flavor characteristics of oyster juice.
[0071] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. All equivalent structural transformations made using the contents of the present invention's specification and drawings under the inventive concept of the present invention, or direct or indirect applications in other related technical fields, are included within the patent protection scope of the present invention.
Claims
1. A method for preparing oyster juice based on the synergistic effect of microbial enzyme interaction and gradient temperature heat reaction, characterized in that, Includes the following steps: S1. Raw material pretreatment: Select fresh oysters, clean and remove the shells, wash the oyster meat and set aside. Boil the oyster meat in water, filter and separate to obtain oyster boiling liquid; mix the cooked oyster meat with an appropriate amount of water and blend to obtain oyster paste liquid for later use. S2. Enzymatic hydrolysis by bacterial-enzyme interaction: Adjust the pH of the oyster broth to 6.0-7.5, inoculate with protease-producing bacteria, and ferment at 35-40℃ for 48-60 hours. After fermentation, add exogenous protease for enzymatic hydrolysis for 3-7 hours. After hydrolysis, rapidly raise the temperature to above 90℃ and maintain it for 10-15 minutes to sterilize and inactivate the enzymes. Then cool to room temperature to obtain oyster hydrolysate. S3. Gradient temperature change thermal reaction: Add 1%-5% of reducing sugar by mass to the obtained oyster enzymatic hydrolysate, mix well, adjust the pH value to neutral, react at 70-90℃ for 2-7 hours, raise the temperature to 100-120℃ and react for 3-7 hours, cool after the reaction is completed to obtain oyster juice.
2. The method for preparing oyster sauce based on the synergistic effect of bacterial-enzyme interaction and gradient temperature-dependent thermal reaction as described in claim 1, characterized in that: In step S2, the fermentation culture includes a static treatment and a stirring treatment. The static treatment lasts for 4-48 hours, and the stirring treatment involves stirring for 10-30 minutes every 6-12 hours during the fermentation culture.
3. The method for preparing oyster sauce based on the synergistic effect of bacterial-enzyme interaction and gradient temperature-dependent thermal reaction as described in claim 1, characterized in that: In step S2, the bacterial strain is one or a mixture of two or more of Bacillus subtilis, Aspergillus oryzae, Aspergillus niger, Bacillus licheniformis, and Bacillus amyloliquefaciens in any proportion.
4. The method for preparing oyster sauce based on the synergistic effect of bacterial-enzyme interaction and gradient temperature-dependent thermal reaction as described in claim 1, characterized in that: In step S2, the inoculation amount of the bacterial strain is 1%-5% of the oyster broth mass.
5. The method for preparing oyster sauce based on the synergistic effect of bacterial-enzyme interaction and gradient temperature-dependent thermal reaction as described in claim 1, characterized in that: In step S2, the protease is one or a mixture of two or more of the following in any proportion: alkaline protease, neutral protease, papain, EF108AN enzyme, and FF104AN enzyme.
6. The method for preparing oyster sauce based on the synergistic effect of bacterial-enzyme interaction and gradient temperature-dependent thermal reaction as described in claim 1, characterized in that: In step S2, the amount of protease added is 0.1%-0.5% of the oyster broth mass.
7. The method for preparing oyster sauce based on the synergistic effect of bacterial-enzyme interaction and gradient temperature-dependent thermal reaction as described in claim 1, characterized in that: In step S3, the reducing sugar is one or a mixture of two or more of D-fructose, D-galactose, D-xylose, D-ribose, glucose, lactose, and maltose in any proportion.