High-freshness fermented protein marinade and preparation method thereof

Abstract

The invention discloses a high-freshness fermented protein marinade and a preparation method thereof, and relates to the technical field of fermentation engineering. The method comprises the following steps: preparing a mushroom enzymolysis extracting solution, preparing a bacillus subtilis induced fermentation solution and a saccharomyces cerevisiae induced fermentation solution, and carrying out mixed fermentation. According to the invention, the efficient proteolysis capability of bacillus subtilis and the flavor nucleotide synthesis capability of saccharomyces cerevisiae are organically combined for the first time. The bacillus subtilis secretes high-activity neutral protease under the induction of a substrate, and mushroom protein is deeply hydrolyzed into small-molecule umami peptide; metabolizing the saccharomyces cerevisiae under the induction of the mushroom extract to generate 5 '; -nucleotides (GMP, IMP, etc.) and alcohol ester flavor substances. Peptide-nucleotide molecule level synergy is achieved in synergy fermentation of the two components, the palatable taste is mellow and mellow, the aftertaste is lasting, and the problems that in the prior art, the single enzymolysis flavor is thin, and physical compounding synergy is insufficient are solved.

Description

Technical Field

[0001] This invention relates to the field of fermentation engineering technology, specifically to a high-freshness fermented protein marinade and its preparation method. Background Technology

[0002] As consumers pay more attention to healthy eating, traditional high-sodium, high-MSG (monosodium glutamate) seasonings are gradually being replaced by new compound seasonings that are "low-sodium, high-umami, and natural." Among them, umami peptides, flavor nucleotides, and free amino acids produced using bio-fermentation technology have become the core direction of current seasoning research and development due to their natural taste, rich flavor, and nutritional functions.

[0003] Currently, the mainstream technical routes for preparing high-umami seasonings mainly include two categories: One approach is microbial enzymatic hydrolysis technology. For example, Chinese patent CN108783386A discloses a method for preparing seafood seasoning packets using heat-resistant Bacillus subtilis to assist in the enzymatic hydrolysis of aquatic proteins. This technology achieves efficient protein hydrolysis by having Bacillus subtilis cells and proteases work together on low-moisture aquatic proteins. However, this technology primarily focuses on the rapid degradation of proteins, producing mostly small peptides and amino acids, lacking the synthesis of complex flavor compounds (such as esters and higher alcohols), and does not involve synergistic effects with other fresh-producing strains, resulting in a product with a single layer of umami flavor, lacking richness and lingering aftertaste.

[0004] Secondly, there is yeast extract (YE). For example, Chinese patents CN115669906A and CN115944067A disclose low-sodium seasonings and brine seasonings rich in yeast extract, respectively. These technologies effectively solve the bitterness problem caused by low-sodium salt by physically combining yeast autolysate with enzymatically hydrolyzed animal protein and spices, and achieve a significant flavor-enhancing effect by utilizing the 5'-nucleotides abundant in yeast. However, the "enzymatically hydrolyzed animal protein" in existing technologies is usually a separately prepared raw material (mostly using commercial proteases for hydrolysis), and does not achieve metabolic coupling between Bacillus subtilis and yeast during fermentation. This physical compounding method not only increases production steps and costs, but also makes it difficult to achieve in-situ synergistic effects of peptides and nucleotides at the molecular level due to the separation of the sources of enzymatic hydrolysate and yeast extract.

[0005] Therefore, developing a high-freshness fermented protein marinade that is simple to process, low in cost, free of chemical inducer residues, and rich in flavor has significant application value and market prospects. Summary of the Invention

[0006] Therefore, the present invention provides a high-freshness fermented protein marinade and its preparation method to solve the problems in the prior art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: According to one aspect of the present invention, a method for preparing a high-freshness fermented protein marinade includes: Step 1: Preparation of mushroom enzymatic hydrolysis extract Select edible fungi, wash, dry, pulverize and sieve to obtain mushroom powder; mix the mushroom powder with water, adjust the pH to 5.0-6.0, add cellulase (0.2%-0.8%), and perform cellulase hydrolysis; after hydrolysis, adjust the pH to 6.5-7.0, add flavor protease (0.5%-1.0%), and continue protease hydrolysis; after hydrolysis, heat to 90-100℃ and hold for 10 minutes to inactivate the enzyme, cool and centrifuge, collect the supernatant, vacuum concentrate to a solid content of 10%-20%, pasteurize (80℃, 30 min) to obtain mushroom enzymatic extract; Step 2, Fermentation The mushroom enzymatic hydrolysis extract, Bacillus subtilis-induced fermentation broth, and Saccharomyces cerevisiae-induced fermentation broth were mixed in proportion, and after adjusting the pH, enzymatic hydrolysis was performed. During the enzymatic hydrolysis, the pH was controlled within the range of 6.5-7.0, and the amino nitrogen content was monitored. When the amino nitrogen reached 1.5-2.0 g / 100mL, the enzymatic hydrolysis was terminated. After the enzymatic hydrolysis was completed, the system was cooled and allowed to stand. Pasteurization was performed to obtain a high-freshness fermented protein marinade.

[0008] Furthermore, in step two, the preparation method of the Bacillus subtilis induced fermentation broth includes: inoculating Bacillus subtilis into an induction medium, incubating at 34-35℃ with shaking, centrifuging the fermentation broth at 4℃ and 10,000 rpm for 15 min, collecting the supernatant, and filtering the supernatant sequentially through 0.45μm and 0.22μm microporous membranes (PVDF material) for sterilization to obtain the Bacillus subtilis induced fermentation broth, which is stored at 4℃ for later use. The inoculum amount is 4-6% (v / v).

[0009] Furthermore, the induction culture medium comprises 2-4% soybean meal powder, 1-2% corn steep liquor, 1-2% soluble starch, 0.5-1.0 g / L CaCl2, 0.05-0.15 g / L MnSO4, and the remainder is water; the initial pH is 7.0-7.5.

[0010] Furthermore, in step two, the preparation method of the induced fermentation broth of *Saccharomyces cerevisiae* includes: inoculating *Saccharomyces cerevisiae* into an induction medium, first performing primary fermentation at 30°C under microaerobic conditions for 36-48 hours, then cooling to 8-15°C for post-ripening for 24-36 hours, centrifuging to remove the cells, collecting the supernatant to obtain the induced fermentation broth of *Saccharomyces cerevisiae*, and storing it at 4°C for later use. The inoculation amount is 5-8% (v / v).

[0011] Furthermore, the induction medium comprises malt extract and 5-10% (v / v) of mushroom enzymatic hydrolysis extract.

[0012] Furthermore, in step one, the mixing ratio of mushroom powder to water is 1:10-1:20.

[0013] Furthermore, in step one, the cellulose is enzymatically hydrolyzed at 50-55°C for 1.5-2 hours; the protein is enzymatically hydrolyzed at 45-50°C for another 1.5-4 hours.

[0014] Furthermore, in step two, the ratio of mushroom enzymatic hydrolysis extract, Bacillus subtilis-induced fermentation broth, and Saccharomyces cerevisiae-induced fermentation broth is 1:1:1 to 1:1:3.

[0015] Furthermore, in step two, the enzymatic hydrolysis temperature is controlled at 45-50℃, and the enzymatic hydrolysis is carried out with stirring for 2-4 hours. During this period, the pH is monitored regularly, and food-grade citric acid is used to maintain the pH within the range of 6.5-7.0. At the same time, the amino nitrogen content is monitored, and the enzymatic hydrolysis is terminated when the amino nitrogen reaches 1.5-2.0 g / 100mL. After the enzymatic hydrolysis is completed, the system is cooled to 28-30℃ and allowed to stand for 12-24 hours.

[0016] According to another aspect of the present invention, a high-freshness fermented protein marinade is provided, said marinade being prepared by the above-described method.

[0017] The high-freshness fermented protein marinade can be supplemented with low-sodium salt, spices and other auxiliary materials as needed, and then homogenized, spray-dried or directly packaged to make a paste or powdered finished marinade.

[0018] The present invention has the following advantages: This invention uses natural raw materials such as soybean meal powder, corn steep liquor, and mushroom extract for substrate induction, and uses food-grade calcium... 2+ Mn 2+ It replaces chemical inducers to enhance enzyme activity. There are no chemical inducer residues throughout the entire process, and the product meets clean labeling and food safety requirements.

[0019] This invention is the first to organically combine the highly efficient proteolytic ability of Bacillus subtilis with the flavor-enhancing nucleotide synthesis ability of Saccharomyces cerevisiae. Under substrate induction, Bacillus subtilis secretes highly active neutral proteases, deeply hydrolyzing mushroom proteins into small-molecule umami peptides; while Saccharomyces cerevisiae, under the induction of mushroom extracts, metabolizes to produce 5'-nucleotides (GMP, IMP, etc.) and alcohol esters for flavor. Through synergistic fermentation, both achieve peptide-nucleotide molecular-level synergy, resulting in a mellow, rich umami flavor with a long-lasting aftertaste, overcoming the problems of thin flavor from single enzymatic hydrolysis and insufficient synergistic effects from physical compounding in existing technologies.

[0020] This invention employs a two-step process: high-temperature enzymatic hydrolysis followed by low-temperature fusion. First, concentrated enzymatic hydrolysis is performed at 45-50℃, allowing proteases to efficiently generate umami peptides at the optimal temperature. Then, the temperature is lowered to 28-30℃ for static maturation, promoting the fusion of yeast flavor compounds and peptides. Compared to single-temperature fermentation, this method ensures both enzymatic hydrolysis efficiency and avoids the destruction of flavor compounds by high temperatures, achieving a dual optimization of peptide production efficiency and flavor complexity.

[0021] The Bacillus subtilis fermentation broth of this invention is sterilized by centrifugation and filtration through a 0.22μm microporous membrane, thoroughly removing live bacteria and retaining only the highly active protease solution for subsequent processing. This allows for precise control of the enzymatic hydrolysis time, preventing excessive hydrolysis, bitter peptide formation, and contamination by undesirable metabolites caused by the continued growth of residual live bacteria, thus ensuring stable and controllable product flavor. Attached Figure Description

[0022] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0023] The structures, proportions, sizes, etc. illustrated in this specification are only for the purpose of assisting those skilled in the art in understanding and reading the content disclosed herein, and are not intended to limit the conditions under which the present invention can be implemented. Therefore, they have no substantial technical significance. Any modifications to the structure, changes in the proportions, or adjustments to the size, without affecting the effects and objectives that the present invention can produce, should still fall within the scope of the technical content disclosed in the present invention.

[0024] Figure 1 This is a flow chart of the preparation process of a high-freshness fermented protein marinade provided in Embodiment 1 of the present invention; Figure 2 Sensory evaluation radar charts of different samples provided in Test Example 1 of this invention; Figure 3 This is a comparison chart of amino nitrogen and 5'-nucleotide content provided in Test Example 2 of the present invention; Figure 4 This is a stacked bar chart of peptide molecular weight distribution provided in Test Example 3 of the present invention. Detailed Implementation

[0025] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. 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.

[0026] Unless otherwise specified in the embodiments of this invention, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available products; different manufacturers and models of raw materials do not affect the implementation of the technical solution or the achievement of the technical effect of this invention.

[0027] Cellulase: Source leaf, S10042-100g, 400u / mg, biotechnology grade; Flavor protease: source leaf, S10153-100g, BR, 20u / mg; Both the cellulase and flavor protease are food-grade, and those skilled in the art can adjust the amount added according to the specific activity units of the selected enzyme preparation, while ensuring the enzymatic hydrolysis effect.

[0028] Bacillus subtilis: China General Microbiological Culture Collection Center CGMCC1.892; Saccharomyces cerevisiae: China Industrial Microbial Culture Collection Center (CICC) 30394; Bacillus subtilis induction medium: First, mix 30g of soybean meal powder with a small amount of water to form a paste, then add water, followed by 15g of corn steep liquor powder and 15g of soluble starch. Sterilize at 121℃ for 20 minutes to obtain the basic culture medium. Separately, dissolve 0.8g of CaCl2 and 0.1g of MnSO4 in water, then sterilize at 121℃ for 20 minutes to obtain the salt solution. Mix the basic culture medium and the salt solution, then add sterile distilled water to a final volume of 1000mL. Adjust the initial pH to 7.2 under aseptic conditions. Saccharomyces cerevisiae induction medium: 8% of the mushroom enzymatic hydrolysis extract obtained in step 1 of different examples was added to malt extract medium.

[0029] A method for preparing a high-freshness fermented protein marinade, the process flow diagram is as follows: Figure 1 As shown: 1. Preparation of mushroom enzymatic hydrolysis extract: Mix 50% shiitake mushrooms, 30% king oyster mushrooms, and 20% seafood mushrooms, wash, dry, and pulverize them through a 40-mesh sieve. Weigh 100g of mushroom powder and add 1500mL of water at a material-to-liquid ratio of 1:15 to make a slurry.

[0030] The pH was adjusted to 5.5 with citric acid, and 0.8 g (0.5%) of cellulase was added. Enzymatic hydrolysis was carried out at 55°C for 2 hours. Then, the pH was adjusted to 6.8, and 1.2 g (0.8%) of flavor protease was added. Enzymatic hydrolysis was carried out at 50°C for 3 hours. After hydrolysis, the temperature was raised to 95°C and held for 10 minutes to inactivate the enzyme. After cooling, the mixture was centrifuged (5000 rpm, 20 minutes), and the supernatant was collected. The supernatant was concentrated under vacuum to a solids content of 15%, and pasteurized (80°C, 30 minutes) to obtain approximately 800 mL of mushroom enzymatic hydrolysis extract.

[0031] 2. Preparation of Bacillus subtilis-induced fermentation broth: Bacillus subtilis slant culture was inoculated into seed culture medium (same as induction culture medium) and cultured at 34℃ and 220 rpm for 18 h to obtain the seed culture. A 5% inoculum was then added to the induction culture medium, in 50 mL / 250 mL Erlenmeyer flasks, and cultured at 34℃ with shaking at 220 rpm for 32 h. After incubation, the fermentation broth was centrifuged at 4℃ and 10,000 rpm for 15 min, and the supernatant was collected. The supernatant was then filtered through 0.45 μm and 0.22 μm PVDF microporous membranes for sterilization to obtain sterile Bacillus subtilis induced fermentation broth (hereinafter referred to as Bacillus subtilis induced fermentation broth), which was stored at 4℃. The neutral protease activity was measured to be 450 U / mL.

[0032] 3. Preparation of brewing yeast induced fermentation broth: Saccharomyces cerevisiae slant culture was inoculated into malt extract liquid medium and cultured at 28℃ for 24 h to obtain the seed culture. An 8% inoculum was then inoculated into induction medium (200 mL / 500 mL Erlenmeyer flask), and incubated statically at 30℃ (microaerobic) for 48 h, followed by post-ripening at 10℃ for 24 h. After incubation, the culture was centrifuged (5000 rpm, 15 min) to remove the cells, and the supernatant was collected to obtain the induced fermentation broth of Saccharomyces cerevisiae, which was stored at 4℃. The total 5'-nucleotide content was determined to be 0.19 g / L (Folin-Ciocalteu method).

[0033] 4. Co-fermentation Take 600 mL of mushroom enzymatic hydrolysis extract, 300 mL of Bacillus subtilis-induced fermentation broth, and 300 mL of Saccharomyces cerevisiae-induced fermentation broth (volume ratio 2:1:1), mix thoroughly, and adjust the pH to 6.5. Place the mixture in a fermenter, heat to 48℃, and stir for enzymatic hydrolysis. During this process, automatically titrate with food-grade citric acid (10% solution) to maintain the pH at 6.5-7.0. Take samples every 30 minutes to determine the amino nitrogen content (formaldehyde titration method). After 3 hours of enzymatic hydrolysis, the amino nitrogen reaches 1.85 g / 100 mL, and sensory evaluation shows no bitterness. Immediately cool to 30℃. Continue to stand for 16 hours to allow flavor integration. After completion, heat to 80℃ and maintain for 30 minutes for pasteurization to obtain approximately 1100 mL of basic high-freshness fermentation broth.

[0034] 5. Finished marinade Take 1000mL of basic fermentation liquid, add 120g of low sodium salt (sodium chloride: potassium chloride = 7:3), 40g of white sugar, and 10g of compound spices. After homogenization, spray dry to obtain about 280g of powdered high-freshness fermented protein marinade.

[0035] Example 2 The mushroom raw material in step 1 was changed to shiitake mushrooms, and the rest of the operation was the same as in Example 1. Results: amino nitrogen 1.65g / 100mL, total 5'-nucleotides 0.31g / L (mainly GMP), sensory umami was good, but the body was slightly inferior to that in Example 1.

[0036] Example 3 Except for mixing the mushroom enzymatic hydrolysis extract, Bacillus subtilis-induced fermentation broth, and Saccharomyces cerevisiae-induced fermentation broth at a volume ratio of 1:1:1, the rest was the same as in Example 1. Results: Amino nitrogen 1.72 g / 100 mL, total 5'-nucleotides 0.41 g / L, good sensory umami flavor, but peptide content slightly lower than in Example 1, and umami intensity slightly lower.

[0037] Example 4 Mushroom enzymatic hydrolysis extract, Bacillus subtilis-induced fermentation broth, and Saccharomyces cerevisiae-induced fermentation broth were mixed in a volume ratio of 1:1:3. The enzymatic hydrolysis temperature was controlled at 45℃, and the time was extended to 4 hours to ensure that the amino nitrogen reached 1.9 g / 100mL. The temperature was then lowered to 28℃ and allowed to stand for 12 hours. Other steps were the same as in Example 1. Results: Amino nitrogen 1.86 g / 100mL, total 5'-nucleotides 0.55 g / L, significantly increased 5'-GMP content in the product, rich aroma of fermented rice and fruit, suitable for poultry marinating.

[0038] Comparative Example 1 Without Bacillus subtilis-induced fermentation broth, the mushroom enzymatic extract and Saccharomyces cerevisiae-induced fermentation broth were mixed at a 2:1 ratio (with an equal volume of water added), and the mixture was directly incubated at 30°C for 24 hours. The remaining procedures were the same as in Example 1. Results: Amino nitrogen was only 0.58 g / 100 mL, 5'-nucleotides were 0.38 g / L, and sensory evaluation showed a bland umami flavor, lacking richness and meatiness.

[0039] Comparative Example 2 Without brewer's yeast-induced fermentation broth, only mushroom enzymatic hydrolysis extract and Bacillus subtilis-induced fermentation broth were mixed at a 2:1 ratio (with an equal volume of water added), otherwise the same as in Example 1. Results: Amino nitrogen approximately 1.80 g / 100mL; total 5'-nucleotides: <0.10 g / L; umami flavor was relatively pronounced, but the flavor was thin, lacking richness and aftertaste, similar to commercially available ordinary enzymatic hydrolyzed seasonings.

[0040] Comparative Example 3 The Bacillus subtilis-induced fermentation broth was directly mixed with mushroom enzymatic hydrolysis extract and Saccharomyces cerevisiae-induced fermentation broth without centrifugation or filtration for co-fermentation, with the rest of the process the same as in Example 1. Results: During enzymatic hydrolysis, the pH continuously rose to 8.2, and the amino nitrogen reached 2.1 g / 100 mL in 2.5 h. However, a distinct ammonia and bitter taste appeared, and the sensory score was low (with a strong bitter taste), indicating that residual live bacteria caused excessive hydrolysis and produced off-flavors.

[0041] Comparative Example 4 Take 3g of commercially available yeast extract (source leaf, V34630-500g) and mix it with 100mL of mushroom enzymatic hydrolysis extract prepared in Example 1. Add low-sodium salt and other ingredients directly to make a marinade. Sensory evaluation: The umami flavor is relatively obvious, but it lacks the richness and aftertaste brought by fermentation. The umami flavor is relatively "thin" and is significantly different from the product of this invention.

[0042] Comparative Example 5 The mushroom enzymatic hydrolysis extract, Bacillus subtilis-induced fermentation broth, and Saccharomyces cerevisiae-induced fermentation broth were mixed in a 2:1:1 ratio and directly fermented at a constant temperature of 30℃ for 24 hours, without undergoing the 45-50℃ enzymatic hydrolysis stage. The rest of the process was the same as in Example 1. Results: Amino nitrogen was only 1.12 g / 100 mL, peptide yield was significantly lower than in Example 1, and sensory umami flavor was insufficient, indicating low protease activity and inadequate umami peptide formation at low temperatures.

[0043] Comparative Example 6 Bacillus subtilis was replaced with a strain containing IPTG (1 mmol / L) and Cd. 2+ The culture was induced in LB medium (50 μmol / L), and the fermentation broth was treated as in Example 1 (filtration sterilization), followed by co-fermentation. Results: Although the enzyme activity in the fermentation broth reached 520 U / mL, the product tested positive for IPTG residue (0.02 mg / kg) and Cd. 2+The residue (0.05 mg / kg) does not meet food standards, and sensory evaluation indicates a slight metallic taste. This suggests that while chemical induction can increase enzyme activity, it poses safety risks and flavor defects, making it unsuitable for food production.

[0044] Comparative Example 7 Prepared separately: Solution A: Use only mushroom enzymatic hydrolysis extract + Bacillus subtilis induced fermentation broth, enzymatic hydrolysis at 48℃ to the endpoint, and then inactivate the enzyme.

[0045] Solution B: Use only mushroom enzymatic hydrolysis extract + brewer's yeast-induced fermentation broth, ferment at 30℃ for 24 hours to inactivate enzymes.

[0046] Sterilized solutions A and B were mixed in the same ratio as in Example 1 (2:1:1 conversion), otherwise the same as in Example 1. Results: Amino nitrogen approximately 1.80 g / 100 mL; total 5'-nucleotides approximately 0.42 g / L; umami flavor was pronounced, but lacked richness, the flavor was "two-layered," lacking a cohesive and rounded feel. Comparative Example 8 In step 4, the reaction was continuously stirred at 48°C until the amino nitrogen content reached the target. Then, the temperature was directly increased to inactivate the enzyme, omitting the step of "cooling down to 28-30°C and standing for 12-24 hours". Other steps were the same as in Example 1. Results: Amino nitrogen 1.86 g / 100 mL; total 5'-nucleotides: 0.38 g / L; umami flavor was direct but harsh, lacking richness and aftertaste.

[0047] Test Example 1 The sensory quality of the high-freshness fermented protein marinades prepared in Examples 1-4 and Comparative Examples 1-8 of this invention was evaluated and compared with commercially available similar products. The results are shown in Table 1.

[0048] Test method: The judging panel consists of 10 professionally trained food sensory evaluators (5 men and 5 women, aged 25-45).

[0049] Sample preparation: The marinades prepared in each example and comparative example were mixed into a 5% salt solution (based on the marinade) according to a unified formula, heated to 80°C and held for 5 minutes, and then cooled to about 40°C for blind sample numbering.

[0050] Evaluation Criteria: A 10-point scoring system was used to score the umami intensity, richness, aftertaste persistence, flavor harmony, and overall acceptability. Scoring Criteria: 9-10 points: Excellent, distinctive features, no defects; 7-8 points: Good, distinctive features, slight shortcomings; 5-6 points: Average, features are identifiable, but there are obvious defects; 3-4 points: Poor, features are vague, defects are serious; 1-2 points: Very poor, unacceptable.

[0051] Control sample: Commercially available enzymatic hydrolyzed seasoning (Angel brand, deodorizing and flavor-enhancing powder, purchased from Taobao), prepared using the same method.

[0052] Sensory evaluation radar charts of different samples as follows Figure 2 As shown.

[0053] Table 1 Sensory Evaluation Scoring Results Note: Comparative Example 6 did not undergo sensory evaluation testing because it was found to contain chemical inducer residues and therefore did not meet food safety standards.

[0054] From Table 1 and Figure 2 It can be seen that the sensory scores of Examples 1-4 of the present invention are significantly higher than those of the comparative examples and the commercially available control. Among them, Example 1 (best overall balance) and Example 4 (high nucleotide content, unique flavor) performed best. Comparative Example 3 had the lowest score due to bitterness; although the physicochemical indicators of Comparative Example 7 (physical mixing) were similar, its flavor harmony was significantly lower than that of Example 1, which was co-fermented in situ, proving the necessity of "co-fermentation".

[0055] Test Example 2 Key physicochemical indicators of the basic high-freshness fermentation broth prepared in each example and comparative example (all subsequent samples were sterilized) were determined, including amino nitrogen content, total 5'-nucleotide content, soluble solids content, and pH value. The results are shown in Table 2.

[0056] Test method: Amino nitrogen content: determined by formaldehyde titration (GB 5009.235-2016).

[0057] Total 5'-nucleotides: High performance liquid chromatography (HPLC) was used, referring to GB 5413.40-2016.

[0058] Soluble solids: determined using an Abbe refractometer (20℃).

[0059] pH value: measured directly using a pH meter.

[0060] The comparison chart of amino nitrogen and 5'-nucleotide content is shown below. Figure 3 As shown.

[0061] Table 2 Results of Physicochemical Indicators From Table 2 and Figure 3It can be seen that the amino nitrogen and 5'-nucleotide contents in Examples 1 and 4 are at relatively high levels, indicating that the dual-strain synergistic fermentation can effectively produce umami peptides and flavor nucleotides. In Comparative Example 3, excessive hydrolysis due to residual live bacteria resulted in an abnormally high amino nitrogen content and pH, leading to product deterioration. Comparative Examples 1, 2, 5, 7, and 8 all showed deficiencies in their indicators, consistent with the sensory evaluation scores.

[0062] Test Example 3 The molecular weight distribution of peptides in each basic high-umami fermentation broth sample was analyzed, especially the proportion of small molecule umami peptides (<1000Da).

[0063] Test method: High-performance gel filtration chromatography (HPGPC) was used. The sample was filtered through a 0.45 μm filter membrane before injection. The chromatographic column was a TSKgel G2000SWXL (7.8 × 300 mm), the mobile phase was acetonitrile-water-trifluoroacetic acid (45:55:0.1, v / v), the flow rate was 0.5 mL / min, and the detection wavelength was 220 nm.

[0064] Peptide molecular weight distribution stacked bar chart as follows Figure 4 As shown.

[0065] Table 3. Molecular weight distribution of peptides From Table 3 and Figure 4 It can be seen that the proportion of small molecule peptides <1000 Da in Examples 1 and 4 is as high as 80% or more, indicating that the enzymatic hydrolysis is sufficient and conducive to the perception of umami. Comparative Example 1, due to the lack of Bacillus subtilis-induced fermentation broth, has a high proportion of large molecule peptides and insufficient hydrolysis; Comparative Example 3, due to excessive hydrolysis, has an abnormally high proportion of peptides <500 Da, but this is accompanied by bitterness; Comparative Example 5, due to low-temperature enzymatic hydrolysis, has a relatively low proportion of small molecule peptides. The results show that the process of the present invention can effectively produce umami peptides within the ideal molecular weight range. The molecular weight distribution of Comparative Example 6 (chemically induced) is very close to (or even slightly higher than) that of Example 1, indicating that although chemical induction can improve efficiency, it does not improve the quality distribution of peptides and brings safety risks. The molecular weight distribution of Comparative Example 8 (without low-temperature ripening) is almost identical to that of Example 1. The proportion of small molecule peptides in Comparative Examples 4 and 7 is lower than that of Example 1, and there is a slightly higher proportion of large molecule residues, indicating that whether commercially available extracts are used directly or mixed after stepwise fermentation, it is impossible to achieve the ideal peptide structure formed by in-situ co-fermentation.

[0066] Test Example 4 The composition and content of free amino acids in each basic high-umami fermentation broth sample were determined, especially the ratio of umami amino acids (glutamic acid, aspartic acid) and sweet amino acids (glycine, alanine, serine, proline).

[0067] Table 4. Content of major free amino acids (mg / 100mL) As shown in Table 4, Examples 1 and 4 had the highest total amount of umami amino acids (Glu+Asp) and were also rich in sweet amino acids, forming a good taste buffer. This is highly consistent with the "richness" and "harmony" in the sensory evaluation. Although Comparative Example 3 had the highest total amount of umami amino acids, due to uncontrolled hydrolysis, a large number of bitter peptides were produced (see Table 3, where the proportion of <500 Da peptides is as high as 70.2%), leading to a taste imbalance. The umami perception was masked by bitterness, which is completely consistent with the sensory score (Table 1) of only 4.0 points for umami intensity. This indicates that simply pursuing a high content of amino acids cannot bring a good umami experience; the reasonable distribution of peptides and the synergy of nucleotides are key. Comparative Example 1 had an extremely low content of free amino acids due to incomplete hydrolysis.

[0068] Test Example 5 The marinade of Example 1 of this invention was applied to marinate chicken, and its effects on chicken water retention, tenderness, saltiness perception and flavor enhancement were evaluated and compared with commercially available marinade (Angel, fishy and lingering flavor powder, purchased from Taobao).

[0069] Test method: Marinating: Take fresh chicken breast and cut it into 5cm×3cm×1cm slices, 100g per serving. Add 5g of the powdered marinade prepared in each example and comparative example (by weight of marinade). The control group added an equal amount of commercially available marinade (Angel brand, fishy and flavor-removing powder, purchased from Taobao). The blank group did not add any marinade. Marinate at 4℃ for 2 hours.

[0070] Cooking: Place the marinated meat slices in a steamer and steam over high heat for 10 minutes. Remove and cool to room temperature.

[0071] Water retention test: Weigh the meat before and after cooking and calculate the cooking loss rate (cooking loss rate = (raw meat weight - cooked meat weight) / raw meat weight × 100%). The higher the water retention, the lower the cooking loss rate.

[0072] Tenderness determination: Shear force was measured using a texture analyzer (TA-XT Plus) with an HDP / BSK bladeset probe and a test speed of 2 mm / s. The maximum shear force (N) was recorded, and the smaller the value, the more tender the meat.

[0073] Table 5 Evaluation of the marinating effect on chicken Note: Comparative Example 6 did not meet food safety standards due to the detection of chemical inducer residues, and therefore no taste and related indicator evaluation tests were conducted.

[0074] As shown in Table 5, the marinades prepared in Examples 1 and 4 significantly reduced the cooking loss rate of chicken, improved water retention, and greatly reduced shear force, resulting in more tender meat. This is attributed to the excellent water-holding and tenderizing effects of the large number of small molecule peptides (<1000 Da) produced in this invention. The comparative products, lacking specific small molecule peptide profiles or exhibiting off-flavors, did not perform as well as those of this invention.

[0075] Test Example 6 The food safety indicators of the marinade of the present invention (Example 1) were tested.

[0076] Test method: IPTG residues: The detection limit was 0.001 mg / kg using liquid chromatography-mass spectrometry (LC-MS / MS).

[0077] Cd 2+ Residue: Atomic absorption spectrophotometry (GB 5009.15-2014) was used.

[0078] Microbiological indicators: Total bacterial count, coliform bacteria, Salmonella, and Staphylococcus aureus were tested according to the GB 4789 series standards.

[0079] Heavy metal indicators: Lead, arsenic, mercury, etc. are tested according to GB 2762.

[0080] Test results: IPTG residue: Not detected (<0.001 mg / kg); Cd 2+ Residue: Not detected (<0.005 mg / kg); Total bacterial count: <10 CFU / g; Coliform bacteria: <3 MPN / g; Pathogenic bacteria: Not detected; Lead: 0.02 mg / kg (far below the limit specified in GB 2762); Total arsenic: 0.01 mg / kg; Mercury: Not detected.

[0081] The marinade of this invention has no chemical inducer residues, and its microbial and heavy metal indicators meet the national food safety standards, making it safe for use in food processing.

[0082] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing a high-freshness fermented protein marinade, characterized in that, The method includes: Step 1: Preparation of mushroom enzymatic hydrolysis extract Select edible fungi, wash, dry, pulverize and sieve to obtain mushroom powder; mix the mushroom powder with water, adjust the pH to 5.0-6.0, add cellulase, and perform cellulase hydrolysis; after hydrolysis, adjust the pH to 6.5-7.0, add flavor protease, and continue protease hydrolysis; after hydrolysis, heat to 90-100℃ to inactivate the enzyme, cool and centrifuge, collect the supernatant, and vacuum concentrate to a solid content of 10%-20% to obtain mushroom enzymatic hydrolysis extract; Step 2, Fermentation The mushroom enzymatic hydrolysis extract, Bacillus subtilis-induced fermentation broth, and Saccharomyces cerevisiae-induced fermentation broth were mixed in proportion, and after adjusting the pH, enzymatic hydrolysis was performed. During the enzymatic hydrolysis, the pH was controlled within the range of 6.5-7.0, and the amino nitrogen content was monitored. When the amino nitrogen reached 1.5-2.0 g / 100mL, the enzymatic hydrolysis was terminated. After the enzymatic hydrolysis was completed, the system was cooled and allowed to stand. Pasteurization was performed to obtain a high-freshness fermented protein marinade.

2. The method for preparing a high-freshness fermented protein marinade according to claim 1, characterized in that, In step two, the preparation method of Bacillus subtilis induced fermentation broth includes: inoculating Bacillus subtilis into an induction culture medium, culturing at 34-35℃ with shaking, centrifuging, collecting the supernatant, filtering to remove bacteria, and obtaining Bacillus subtilis induced fermentation broth.

3. The method for preparing a high-freshness fermented protein marinade according to claim 2, characterized in that, The induction culture medium comprises 2-4% soybean meal powder, 1-2% corn steep liquor, 1-2% soluble starch, 0.5-1.0 g / L CaCl2, 0.05-0.15 g / L MnSO4, and the remainder is water; the initial pH is 7.0-7.

5.

4. The method for preparing a high-freshness fermented protein marinade according to claim 1, characterized in that, In step two, the preparation method of the induced fermentation broth of Saccharomyces cerevisiae includes: inoculating Saccharomyces cerevisiae into an induction medium, first fermenting at 30°C under microaerobic conditions for 36-48 hours, then cooling to 8-15°C for post-ripening for 24-36 hours, centrifuging to remove the cells, and collecting the supernatant to obtain the induced fermentation broth of Saccharomyces cerevisiae.

5. The method for preparing a high-freshness fermented protein marinade according to claim 4, characterized in that, The induction medium consists of malt extract and 5-10% (v / v) of mushroom enzymatic hydrolysis extract.

6. The method for preparing a high-freshness fermented protein marinade according to claim 1, characterized in that, In step one, the ratio of mushroom powder to water is 1:10 to 1:

20.

7. The method for preparing a high-freshness fermented protein marinade according to claim 1, characterized in that, In step one, the cellulose is enzymatically hydrolyzed at 50-55℃ for 1.5-2 hours; the protein is enzymatically hydrolyzed at 45-50℃ for another 1.5-4 hours.

8. The method for preparing a high-freshness fermented protein marinade according to claim 1, characterized in that, In step two, the ratio of mushroom enzymatic hydrolysis extract, Bacillus subtilis-induced fermentation broth, and Saccharomyces cerevisiae-induced fermentation broth is 1:1:1 to 1:1:

3.

9. The method for preparing a high-freshness fermented protein marinade according to claim 1, characterized in that, In step two, the enzymatic hydrolysis temperature is controlled at 45-50℃, and the enzymatic hydrolysis is carried out by stirring for 2-4 hours. After the enzymatic hydrolysis is completed, the system is cooled to 28-30℃ and left to stand for 12-24 hours.

10. A high-freshness fermented protein marinade, characterized in that, The marinade is prepared by any one of the methods described in claims 1-9.

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