A method for producing gamma-aminobutyric acid by fermenting scallop skirt and application of product thereof

Abstract

The application discloses a method for producing high-yield gamma-aminobutyric acid by using scallop skirt edge fermentation and application of a product thereof. The method comprises the following steps: (1) constructing a recombinant Escherichia coli engineering strain overexpressing glutamate decarboxylase; (2) enriching the recombinant Escherichia coli by using an enzymatic hydrolysate of scallop skirt edge as a single nitrogen source nutrient medium; and (3) adding the enriched recombinant Escherichia coli into a substrate to perform biological fermentation and synthesize gamma-aminobutyric acid. The application initiatively uses scallop processing waste, i.e., skirt edge, as a single nitrogen source nutrient medium, and directionally releases endogenous nutrition flow highly matched with high-density propagation of Escherichia coli, so as to efficiently enrich the Escherichia coli. By finely adjusting the substrate matching, invalid residues and substrate inhibition are completely eliminated, only 5 h of catalysis is needed, the absolute yield of gamma-aminobutyric acid in the fermentation liquor reaches 19.42+0.46 g / L, and the substrate conversion rate is as high as 97.9+2.32%.

Description

Technical Field

[0001] This invention belongs to the field of high-value utilization technology of marine biomass resources, and relates to a method for high-yield γ-aminobutyric acid by fermentation of scallop skirts and the application of the product. Background Technology

[0002] Scallops are an important marine aquaculture species, with China producing over 1.8 million tons annually, according to statistics. However, during industrial processing, only 10%–16% of the adductor muscle, which accounts for a significant portion of the total weight, is considered a high-value food, while a staggering 84%–90% of the tissue (including the shell, viscera, and skirt) is discarded directly as a byproduct or waste. If these nitrogen-rich seafood wastes are not properly treated and are directly landfilled or dumped, they will not only lead to eutrophication and red tides in nearshore waters but also exacerbate greenhouse gas emissions, creating a heavy ecological burden. Scallop skirts are rich in protein and naturally contain glutamic acid, a precursor to various metabolisms, as well as unique trace elements that act as potential activators. They represent a highly promising and inexpensive source of carbon and nitrogen. Utilizing them in a green and high-value manner is a key strategy for promoting a circular bioeconomy.

[0003] When using microbial fermentation or biocatalysis to produce high-value-added bioactive molecules, constructing a high-density recombinant cell factory is a key factor determining the final yield. However, traditional recombinant E. coli fermentation is highly dependent on expensive commercially refined carbon and nitrogen sources (such as commercially available trypsin digests and yeast extracts), resulting in high fermentation costs. When attempting to use crude marine industrial and agricultural waste as an alternative substrate, conventional direct hydrolysis or single protease degradation processes lack targeted cleavage based on the metabolic preferences of E. coli, leading to a disordered nutrient stream composition and an excessively high proportion of large-molecule peptides. This not only prevents engineered strains from achieving efficient transmembrane absorption but also easily triggers metabolic imbalances and overflow inhibition, resulting in slow enrichment and propagation rates and low biomass accumulation density in the first stage, making it difficult to support the massive cell factory foundation required for subsequent high-intensity biotransformation.

[0004] Furthermore, γ-aminobutyric acid (GABA), as a naturally occurring non-protein amino acid, is a core signaling molecule for plants to cope with abiotic stresses such as salt stress. Exogenous application of GABA can significantly stimulate the osmotic regulation and antioxidant defense mechanisms of crops, alleviating crop growth inhibition caused by highly saline soils. However, GABA fermentation broth produced by existing technologies usually requires multiple downstream processes such as cumbersome chromatographic separation, desalination, and purification before it can be made into a commercial product, which further increases the application cost. If the crude fermentation broth is applied directly, the industrial and agricultural impurities or overflow byproducts remaining in the traditional culture medium often have antagonistic and toxic effects on plant germination. It is still impossible to achieve the actual agricultural physiological stress resistance efficacy of fermentation broth under the premise of "no purification and direct application".

[0005] To address the numerous shortcomings of existing technologies, this invention proposes a closed-loop biorefining pathway: "marine waste treatment - high-performance cell factory enrichment - bioactive molecule transformation - modern green agricultural application." This invention utilizes a multi-site cleavage strategy with compound enzymes to directionally transform scallop skirts into a high-performance single nitrogen source substrate specifically for *E. coli*. Through systematic optimization of the first-stage engineered bacteria enrichment and growth process and the second-stage precise matching biotransformation, it not only achieves unconventional high-density propagation and cell accumulation of engineered bacteria but also realizes an extreme substrate conversion rate and unconventional physiological synergistic effects in downstream wheat applications, developing a low-cost, high-efficiency, and eco-friendly plant stress-resistance biostimulant. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a method for high-yield γ-aminobutyric acid (GABA) fermentation using scallop skirts and its agricultural application. The core innovation of this invention lies in its pioneering use of scallop processing waste skirts as a single nitrogen source nutrient substrate, directionally releasing an endogenous nutrient flow highly compatible with the high-density propagation of recombinant bacteria, thereby achieving efficient enrichment and extraordinary accumulation of engineered bacteria in a single stage. This invention not only realizes the high-value resource utilization of marine waste but also significantly reduces the culture material cost of recombinant E. coli and provides a highly efficient plant salt-tolerant biostimulant with physiological resistance efficacy surpassing that of chemically purified products under the premise of direct application without purification.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows: a method for high-yield production of γ-aminobutyric acid (GABA) by fermentation of scallop skirts and its application, comprising the following steps: (1) Constructing a recombinant Escherichia coli engineered strain overexpressing glutamate decarboxylase; (2) The recombinant Escherichia coli was enriched using the enzymatic hydrolysate of scallop skirt as a single nitrogen source nutrient substrate; the scallop skirt was enzymatically hydrolyzed using a special compound enzyme for animal protein hydrolysis at a temperature of 40-60 ℃ and a time of 10-18 h; the carbon-nitrogen ratio of the culture system was controlled at 1:3-5:1. (3) The enriched recombinant Escherichia coli was added to the substrate for bio-fermentation to synthesize γ-aminobutyric acid.

[0008] Preferably, in step (3), the substrate includes monosodium glutamate and glutamic acid, and the bio-fermentation system also contains pyridoxal coenzyme phosphate and isopropyl-β-D-thiogalactoside as an inducer; wherein the final concentration of pyridoxal coenzyme phosphate is 0.05-0.4 mM, the final concentration of the inducer is 0.01-0.4 mM, the amount of monosodium glutamate added is 20-30 g / L, the amount of glutamic acid added is 10-15 g / L, and the fermentation catalysis time is 4-8 h.

[0009] Preferably, the present invention also provides the application of the fermentation product containing γ-aminobutyric acid obtained by the method in the preparation of plant stress stimulants.

[0010] Preferably, the plant stress stimulant is a crude extract containing fermentation supernatant that does not require purification, used to alleviate salt stress in plants; the plant is wheat, which is used for seed soaking treatment.

[0011] Preferably, the fermentation supernatant is diluted to a final concentration of γ-aminobutyric acid of 0.2~10 mM.

[0012] Through the above technical solution, the present invention has the following beneficial effects: 1. Achieved unconventional high-density propagation and biomass accumulation of engineered bacteria in the first stage: This invention targets the specific metabolic preferences of recombinant E. coli, utilizing a specialized complex enzyme for animal protein hydrolysis to directionally break down the skirt protein structure, avoiding the technical defects of conventional direct hydrolysis or single protease hydrolysis, which releases excessively high proportions of large-molecule peptides and results in poor transmembrane absorption and utilization. Through global optimization of growth temperature, enzymatic hydrolysis time, and initial carbon-nitrogen ratio using response surface methodology, the metabolic overflow inhibition easily caused by conventional marine waste substrates was completely eliminated. After 8 hours of fermentation, the actual cell density jumped significantly to 3.34±0.17×10⁻⁶. 9 CFU / mL provides an extremely strong high-performance cell population basis for subsequent efficient biocatalysis.

[0013] 2. High fermentation yield and substrate conversion rate under precise two-stage matching control: Based on the engineered cells with high-density enrichment in the skirt substrate in the first stage, the second stage precisely controls the substrate amount within a precise matching range of 22 g / L monosodium glutamate and 11 g / L glutamate, achieving a precise balance between the precursor substrate supply rate and the high concentration of decarboxylase catalytic capacity of the cells. Only 5 hours of saturated catalytic reaction is required to achieve a GABA yield of 10-20 g / L in the fermentation broth, with complete elimination of precursor substrate residues. The substrate decarboxylation conversion rate of the feedstock reaches 95-99.9%, demonstrating extremely high catalytic economy.

[0014] 3. Excellent agricultural application results and green direct application without purification: The GABA fermentation supernatant obtained by this invention can be applied directly without complicated downstream processes such as chromatographic separation and desalting purification. In alleviating the high salt stress of wheat under 100 mM NaCl, the unpurified crude liquid not only contains a high concentration of biogenic GABA, but also naturally retains endogenous small molecule peptides, oligopeptides and residual trace metabolites in the skirt enzymatic hydrolysate, which produce a strong physiological synergistic effect in regulating plant systems. It significantly outperformed the positive control salicylic acid (PC group) and the control group of commercial grade pure GABA at the same concentration in improving the growth phenotypes of wheat seedlings, such as fresh weight, dry weight, germination index and population vigor index. At the same time, it made a comprehensive breakthrough in the efficacy of clearing lipid peroxidation MDA accumulation, leapfrogging the primary defense SOD activity and optimizing the overall demodulation of antioxidant enzyme spectrum (POD, CAT) stress response, accelerating the return of wheat to normal germination and growth physiological homeostasis, and successfully opening up a closed-loop ecological cycle path of "marine waste treatment - high-performance cell enrichment - biocatalytic transformation - modern green agricultural application". Attached Figure Description

[0015] Figure 1 This is a flowchart of the method for high-yield γ-aminobutyric acid fermentation using scallop skirts according to the present invention; Figure 2 The following are SDS-PAGE identification (a) and HPLC detection of GABA for recombinant Escherichia coli provided in Example 1 of this invention; wherein, in a: A: E. coli BL21(DE3) B: E. coli BL21(DE3)-pET28a-EC (before induction) C: E. coli BL21(DE3)-pET28a-EC (after induction); Figure 3 The effects of different enzymatic hydrolysis pretreatment conditions provided in Example 2 of this invention on cell density after 8 hours of fermentation are shown; wherein, a. different enzyme types; b. different enzymatic hydrolysis times; c. different enzyme-to-substrate ratios; d. different enzymatic hydrolysis temperatures; e. different solid-liquid ratios; f. different C / N ratios. Figure 4 The response surface plot shows the effect of different enzymatic hydrolysis pretreatment conditions provided in Example 3 of the present invention on cell density after 8 hours of fermentation; where a is enzymatic hydrolysis temperature & enzymatic hydrolysis time; b is enzymatic hydrolysis temperature & C / N ratio; c is enzymatic hydrolysis time & C / N ratio. Figure 5 The effect of pyridoxal coenzyme phosphate (PLP) concentration on GABA conversion rate provided in Example 4 of this invention; Figure 6 The effect of isopropyl-β-D-thiogalactoside (IPTG) concentration on GABA conversion rate provided in Example 5 of this invention; Figure 7The response surface methodology for the optimized biotransformation of GABA by *E. coli* provided in Example 6 of this invention is shown below; where a. final IPTG concentration & final PLP concentration; b. total feed concentration & final PLP concentration; c. total feed concentration & final IPTG concentration. Figure 8 The relationship between GABA conversion rate and the substrates monosodium glutamate (MSG) and glutamate under optimized conditions provided in Example 7 of this invention; wherein, a. the relationship with substrate concentration; b. the relationship with reaction time; Figure 9 The effects of GABA skirt fermentation supernatant soaking on the growth indicators of wheat seed germination under salt stress provided in Example 8 of the present invention are as follows: a. wet weight; b. dry weight; c. germination potential; d. germination rate; e. germination index; f. vigor index; CK: water-soaked seeds germinate in water, NC: water-soaked seeds germinate in brine, PC: salicylic acid-soaked seeds germinate in brine; Figure 10 The effects of GABA skirt fermentation supernatant soaking on the germination of wheat seeds under salt stress provided in Example 8 of this invention are shown in the following figures: CK: water-soaked seeds germinate in water, NC: water-soaked seeds germinate in brine, and PC: salicylic acid-soaked seeds germinate in brine. The values ​​represent: a. MDA content; b. SOD activity; c. POD activity; and d. CTA activity. Detailed Implementation

[0016] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments.

[0017] Example 1: Construction of recombinant engineered Escherichia coli strains and establishment of HPLC detection method for products. The overall process is as follows: Figure 1 As shown.

[0018] Recombinant plasmid construction The gene sequence of glutamate decarboxylase from *E. coli* (Gene ID: 946058) was retrieved from the NCBI database. After codon optimization targeting the codon bias of *E. coli*, the gene was cloned into the overexpression plasmid pET28a, with the insertion position located 6 bp downstream of the ribosome binding site (RBS) of the T7 promoter, and the recombinant plasmid pET28a-EC (sequence listing SEQ ID NO.1) was constructed.

[0019] Transformation and Identification of Engineered Strains The recombinant plasmid pET28a-EC was transformed into... E. coli After BL21(DE3), stress selection was performed using LB medium containing kanamycin. Subsequently, whole cells after induction were analyzed by SDS-PAGE.

[0020] The results of the induced expression identification of the recombinant engineered strain are as follows: Figure 2 As shown in Figure a. From Figure 2 As shown in Figure a, compared with the wild-type strain (lane A) and the uninduced strain (lane B), the recombinant strain (lane C) exhibited a distinct recombinant protein-specific target band at 52.6 kDa, indicating that the recombinant E. coli engineered strain overexpressing glutamate decarboxylase was successfully constructed. The correctly identified strain was mixed with 40% sterile glycerol at a 1:1 volume ratio and stored at -80 ℃ for later use.

[0021] III. High-performance liquid chromatography (HPLC) detection and characterization of the product γ-aminobutyric acid (GABA) To enable accurate qualitative and quantitative analysis of biotransformation products, a pre-column derivatization high-performance liquid chromatography (HPLC) method was established for detection. Preparation of derivatization reagent: Weigh 0.1 g of o-phthalaldehyde and dissolve it in 1 mL of acetonitrile. Add 130 µL of β-mercaptoethanol, and then adjust the volume to 10 mL with borate buffer at pH 10.2. Store in the dark for later use.

[0022] Sample derivatization: The fermentation broth to be tested was centrifuged at 10,000 rpm for 10 min, and the fermentation supernatant was filtered through a 0.22 µm microporous membrane. The filtered fermentation supernatant was mixed with the derivatization reagent at a volume ratio of 4:1, and the mixture was subjected to precise pre-column derivatization at room temperature for 2 min before injection. The single injection volume was 10 µL.

[0023] Chromatographic conditions were controlled as follows: A Shim-pack GIST C18 5 µm 150 mm × 4.6 mm column was used. Mobile phase A consisted of 8 g of crystalline sodium acetate dissolved in ultrapure water and diluted to 1 L, followed by the addition of 220 mL of triethylamine. The pH was adjusted to 7.2 ± 0.02 with glacial acetic acid, and then 5 mL of tetrahydrofuran was added. The mixture was then filtered to remove gas. Mobile phase B was chromatographically pure methanol. Isocratic elution was performed at a volume ratio of 50:50 between mobile phase A and mobile phase B. The system flow rate was controlled at 1 mL / min, the column temperature was controlled at 40 °C, and the UV detection wavelength was set to 338 nm.

[0024] The HPLC chromatograms of the biotransformation solution and the standard are as follows: Figure 2 As shown in b. From Figure 2 As shown in b, under the above chromatographic conditions, a strong absorption peak appeared in the fermentation broth sample at a retention time of 3.7 min. The retention time and peak shape of this peak were completely consistent with the γ-aminobutyric acid (GABA) standard, indicating that the target conversion product accumulated in the fermentation broth of this invention is γ-aminobutyric acid (GABA).

[0025] Example 2: Enzymatic pretreatment of scallop skirt by a compound enzyme specifically for animal protein hydrolysis 1. The scallop skirts, a waste product from scallop processing, are washed with clean water and dried in a 60°C oven until constant weight. The dried skirts are then pulverized using a high-speed pulverizer, passed through a 300-mesh sieve, and the powder is collected for later use.

[0026] 2. Prepare a substrate solution by suspending the skirt-shaped powder in deionized water at a material-to-liquid ratio of 20% (w / v). Add a commercially available complex enzyme for animal protein hydrolysis to the system, controlling the enzyme dosage to 2% (enzyme to substrate mass ratio). Place the system in a water bath at the optimal catalytic temperature of 50 °C for continuous enzymatic hydrolysis for 14 h. After enzymatic hydrolysis, centrifuge and collect the supernatant to obtain the skirt-shaped complex enzymatic hydrolysate. Use the prepared skirt-shaped complex enzymatic hydrolysate as the core nitrogen source matrix to prepare the initial fermentation medium. Add D-anhydrous glucose to adjust the initial carbon-to-nitrogen ratio (C / N) to 1:1, and add 1% (w / v) NaCl. Inoculate with the recombinant Escherichia coli constructed in Example 1 and ferment for 8 h.

[0027] The actual cell density results after 8 hours of fermentation under the treatment of a special compound enzyme for animal protein hydrolysis are shown in the attached figure. Figure 3 As shown in group E. Figure 3 It can be seen that the actual measured bacterial density reached 1.85 × 10⁻⁶. 9 The CFU / mL concentration achieved efficient propagation and proliferation of engineered bacteria on scallop skirt waste substrate.

[0028] Example 3: Optimization of basic growth conditions for high-density cell accumulation in the first stage Based on the single-factor experiment in Example 2, a central composite response surface methodology was designed with enzymatic hydrolysis temperature (A), enzymatic hydrolysis time (B), and carbon-to-nitrogen ratio (C) as independent variables, and the actual cell density of the engineered bacteria after 8 hours of fermentation as the response value. The central level for enzymatic hydrolysis temperature was set at 50 °C, the central level for enzymatic hydrolysis time at 16 h, and the central level for the initial carbon-to-nitrogen ratio of the culture medium at 3:1. Mathematical analysis and process prediction were performed based on the regression model. The three-dimensional response surface diagram under the multi-factor interaction effect is attached. Figure 4 As shown. By Figure 4 It was found that the linear effects of the carbon-to-nitrogen ratio (C) and enzymatic hydrolysis time (B) significantly influenced cell density, with the C-to-nitrogen ratio having the most significant effect and being the dominant factor affecting cell growth. Response surface methodology analysis predicted the optimal basic growth conditions corresponding to the maximum response value as follows: enzymatic hydrolysis temperature 53 ℃, enzymatic hydrolysis time 14 h, and initial C-to-nitrogen ratio 2.5:1. Validation experiments showed that under these globally optimal multi-factor conditions, after 8 h of fermentation, the actual cell density reached 3.34 ± 0.17 μg / L. 9 CFU / mL.

[0029] Example 4: Process optimization of Escherichia coli biotransformation of GABA In the high-density fermentation broth of recombinant *E. coli* obtained in Example 3, the second stage of biotransformation was initiated. To investigate the single-factor dose-effect of the inducer IPTG concentration, after pre-induction with different concentrations of IPTG for 16 h, coenzyme PLP with a final concentration of 0.2 mM and initial substrates of 30 g / L monosodium glutamate and 15 g / L glutamate were added to the fermentation system, and the decarboxylation catalytic reaction was maintained for 8 h. The results of the single-factor tests are attached. Figure 6 As shown. By Figure 6 It can be seen that as the concentration of IPTG is gradually increased, the conversion rate of GABA rises rapidly; when the IPTG concentration reaches 0.1 mM, the conversion rate reaches its highest point, which is 53.8 ± 2.23%. After that, if the IPTG concentration is further increased, the conversion rate begins to show a gradual downward trend.

[0030] To investigate the single-factor dose-effect of exogenous coenzyme PLP, the fermentation broth was pre-induced with IPTG at a final concentration of 0.2 mM for 16 h, followed by the addition of sodium glutamate (30 g / L) and glutamate (15 g / L) to maintain the decarboxylation reaction for 8 h. The effect of exogenous PLP concentration on GABA conversion is shown in the attached figure. Figure 5 As shown. By Figure 5 It was found that in the control group (0 mM) without additional PLP, the system maintained a background conversion rate of 44.4%. With the gradual increase of exogenous PLP concentration, the conversion rate showed a significant positive dose-dependent effect; when the PLP concentration increased to 0.2 mM, the conversion rate reached its highest value (approximately 59.79%), and thereafter, further increasing the PLP concentration to 0.4 mM resulted in a clear plateau effect in the conversion rate, which no longer continued to increase significantly.

[0031] Based on the above single-factor experiments, using the biomass of recombinant E. coli accumulated at high density, a central composite response surface design was conducted with coenzyme PLP concentration (A), inducer IPTG concentration (B), and total supplemental nutrient concentration (C) as independent variables. The resulting three-dimensional response surface diagram of the multi-factor interactions is attached. Figure 7 As shown. Through mathematical analysis and validation using a response surface regression model, the optimal conversion process conditions were obtained as follows: final concentration of coenzyme PLP 0.39 mM, final concentration of inducer IPTG 0.16 mM, and total feed concentration 34.33 g / L (including 24.5 g / L of added glucose and 9.8 g / L of added skirt hydrolysate based on the dry weight of the skirt). Fermentation was validated within this optimized control range, and the actual measured substrate conversion rate reached 69.3 ± 2.58%.

[0032] Combining the optimal basic growth parameters of the first stage with the optimized transformation parameters of the second stage, a two-stage fine-controlled fermentation was performed. In the first stage (0–8 h), the culture was continuously carried out at 37 ℃ for 8 h to rapidly accumulate engineered cell biomass. After the first stage fermentation, the process switched to the second stage, where the feed-in nutrient substrate optimized by the response surface methodology was added to the fermentation broth in a single injection, followed by induction with 0.16 mM IPTG for 16 h, and then addition of 0.39 mM coenzyme PLP. Based on this regulation, to completely eliminate inhibition from high-concentration substrates, the amount of precursor substrate added was precisely matched as follows: a single injection of 22 g / L monosodium glutamate and 11 g / L glutamate (…). Figure 8 (a) Maintain the decarboxylation catalytic reaction for 8 h.

[0033] The biocatalytic reaction kinetics curves and extreme conversion rate tracking diagrams of this invention under the above-mentioned finely matched regulation and substrate conditions are attached. Figure 8 As shown. (From the appendix) Figure 8 The tracking data in section b shows that at 5 h of catalytic reaction, the supply of precursor substrate and the catalytic capacity of intracellular decarboxylase reached a precise equilibrium, and the system completely entered the saturation plateau phase. Further extending the reaction time to 8 h did not increase the yield further. At the 5-h catalytic endpoint, the absolute yield of GABA in the fermentation broth reached 19.42 ± 0.46 g / L (…). Figure 8 (Solid line in b), and the precursor substrate residue was completely eliminated, with the biodecarboxylation conversion rate of the raw material reaching 97.9±2.32% ( Figure 8 (b-dashed line). After the reaction was completed, the fermentation broth was centrifuged at 10000 r / min for 10 min to remove the cells. The supernatant was collected and filtered through a 0.45 μm filter membrane to obtain the crude fermentation broth of highly bioactive GABA that does not require purification.

[0034] Example 5: Application of the GABA crude fermentation broth of the present invention in inducing salt tolerance activity in wheat seeds The supernatant from the skirt fermentation of GABA obtained in the above examples was diluted with deionized water to prepare a series of experimental treatment solutions containing effective GABA concentrations of 0.2 mM, 1.5 mM, 3 mM, 5 mM, and 10 mM. Plump and uniformly sized wheat seeds of Jimai 22 were selected, surface-sterilized with 1% sodium hypochlorite for 10 min, and washed. The seeds were then placed in each treatment solution at a material-to-liquid ratio of 1:5 (w / v) and soaked in the dark at 20 ℃ for 12 h. After soaking, the wheat seeds were placed in petri dishes lined with sterile gauze (30 seeds per dish), and 30 mL of 100 mM NaCl solution was added to construct a high-salt stress environment. Simultaneously, a water blank control (CK group), a saline negative control (NC group), a 0.2 mM salicylic acid positive control (PC group), and control groups containing equal doses of pure GABA at various concentrations were set up. The plants were placed in an artificial climate chamber and cultivated continuously for 8 days, with water replenished daily to maintain their initial weight. On the 8th day, growth indicators and antioxidant biochemical indicators were measured.

[0035] The results of growth and biochemical index measurements are shown in the attached figures. Figure 9 and attached Figure 10 As shown, high salt stress (NC group) significantly inhibited wheat seedling growth, with seedling fresh weight and dry weight decreasing by 40.47% and 46.15% respectively compared to the control group, and germination potential and germination rate were severely impaired. Treatment with the fermentation supernatant of this invention effectively alleviated the growth inhibition caused by salt stress. At 5 mM and 10 mM, the seedling fresh weight of the fermentation broth group was significantly higher than that of the salicylic acid (PC) group, and significantly better than that of the same concentration of pure GABA solution at 0.2 mM, 1.5 mM, and 5 mM. Regarding germination potential and germination rate, all fermentation broth groups showed significant recovery, with the germination potential of the 5 mM fermentation broth significantly higher than that of the same concentration of pure GABA group. For germination index and vigor index, the 3–10 mM fermentation broth group was significantly better than the PC group and the corresponding pure GABA group, with the vigor index at 10 mM being 47.87% higher than that of the PC group.

[0036] Regarding biochemical defense indicators, high salt stress led to a sharp increase of approximately 69.02% in malondialdehyde (MDA) content in leaves. After treatment with fermentation supernatant, the MDA content in leaves showed a significant dose-dependent decrease, recovering to near the level of the salt-free control at 10 mM. Simultaneously, the fermentation supernatant significantly activated superoxide dismutase (SOD) activity in the 0.2–5 mM range, making it significantly higher than the NC, PC, and pure GABA groups. Furthermore, the extremely high levels of POD and CAT activation under high salt stress showed a significant downward trend after fermentation supernatant treatment, with POD activity at 5 mM and 10 mM and CAT activity at 3 mM significantly lower than the corresponding pure GABA groups. These results demonstrate that in the unpurified skirt fermentation broth, biogenic GABA and endogenous peptides and residual small molecule metabolites produced by skirt enzymatic hydrolysis exhibit a significant synergistic effect, with inducing resistance exceeding that of traditional salicylic acid reagents and pure chemical GABA alone.

[0037] Comparative Example 1: Control group pretreated with a single type of neutral protease Except in step two of Example 2, during the matrix enzymatic hydrolysis process, the "animal protein hydrolysis-specific complex enzyme" is replaced with an equal mass of conventional single-type neutral protease (i.e., Figure 3 Except for Group D of a), the substrate concentration (20%), enzyme dosage (2%), hydrolysis temperature (50 °C), hydrolysis time (14 h), and fermentation culture were exactly the same as in Example 2.

[0038] The actual cell density of *E. coli* was measured after 8 hours of fermentation, and the experimental comparison results are attached. Figure 3 As shown in group D of a. From Figure 3 It can be seen that the bacterial accumulation level in the group treated with a single type of neutral protease was significantly low, and its actual bacterial density failed to exceed 0.96 × 10⁻⁶. 9 The CFU / mL concentration was significantly lower than that of the complex enzyme group (Group E) in Example 2.

[0039] Comparative Example 2: Control group pretreated with a single type of trypsin Except in step two of Example 2, where the "animal protein hydrolysis-specific complex enzyme" is replaced with an equal mass of conventional single-type trypsin (i.e., Figure 3 Except for Group B of Example a), the substrate concentration (20%), enzyme dosage (2%), hydrolysis temperature (50 °C), hydrolysis time (14 h), and fermentation culture were completely identical to those in Example 2. The actual cell density of *E. coli* was measured after 8 h of fermentation, and the experimental comparison results are shown in the attached figure. Figure 3 As shown in group B of a. From Figure 3It can be seen that the bacterial accumulation level in the group treated with a single type of trypsin was low, with an actual bacterial density of only 0.77 × 10⁻⁶. 9 The concentration was around CFU / mL, significantly lower than the accumulation effect of the complex enzyme group (Group E) in Example 2.

[0040] Comparative Example 3: Control group pretreated with a single type of papain Except in step two of Example 2, during the matrix enzymatic hydrolysis process, the "animal protein hydrolysis-specific complex enzyme" was replaced with an equal mass of conventional single-type papain (i.e., Figure 3 Except for Group C of a), the substrate concentration (20%), enzyme dosage (2%), hydrolysis temperature (50 °C), hydrolysis time (14 h), and fermentation culture were completely identical to those in Example 2. The actual cell density of *E. coli* was measured after 8 h of fermentation, and the experimental comparison results are attached. Figure 3 As shown in group C of a. From Figure 3 It can be seen that the bacterial accumulation level in the group treated with a single type of papain was also significantly low, and the actual bacterial density failed to exceed 1.05 × 10⁻⁶. 9 The CFU / mL ratio was significantly lower than the high-density propagation effect of the complex enzyme group (Group E) in Example 2.

[0041] Comparative Example 4: Control experiment on the effect of enzymatic digestion temperature on cell enrichment density Under the conditions of maintaining a hydrolysis time of 14 h, an initial carbon-to-nitrogen ratio (C / N) of 1:1, a substrate concentration of 20%, and an enzyme dosage of 2%, the hydrolysis temperature was compared within the group using a single-factor gradient method. Figure 3 As shown in section d, when the enzymatic hydrolysis temperature increases from 40 ℃ to 45 ℃, the cell density increases from 1.54 × 10⁻⁶. 9 CFU / mL rose slowly to 1.58 × 10⁻⁶ 9 CFU / mL; reaching 1.91 × 10⁻⁶ at 50 °C. 9 CFU / mL; when the temperature was further increased to 60 ℃, the cell density was 1.93 × 10⁻⁶. 9 CFU / mL. Intragroup comparisons showed that while single-factor temperature adjustment improved cell density, even at the optimal single-factor level of 60 ℃, the cell density was only 1.93 × 10⁻⁶. 9 The CFU / mL is still far lower than the cell density of 3.34 × 10⁻⁶ after global optimization of the response surface methodology in Example 3. 9 The CFU / mL result confirms that a single-dimensional adjustment cannot achieve full utilization of nutrient flow and high-density enrichment of bacterial cells.

[0042] Comparative Example 5: Control experiment on the effect of carbon-nitrogen ratio (C / N) on bacterial cell enrichment density Under the conditions of maintaining a hydrolysis time of 14 h, a hydrolysis temperature of 50°C, a substrate concentration of 20%, and an enzyme dosage of 2%, the carbon-to-nitrogen ratio (C / N) was compared within groups using a single-factor gradient method. Figure 3 As shown in f, as the C / N ratio increases from 1:3 to 3:1, the cell density exhibits a trend of first increasing and then decreasing: when the C / N ratio is 1:3, the cell density is only 1.47±10. 9 CFU / mL; reaching 2.55±10 at a C / N ratio of 2:1. 9 CFU / mL; the peak value of 3.15 × 10⁻⁶ CFU / mL was reached in the single-factor experiment at a C / N ratio of 3:1. 9 CFU / mL; however, when the carbon-to-nitrogen ratio was too high, reaching 5:1, the cell density dropped sharply to 0.92 × 10⁻⁶. 9 CFU / m. This in-group data not only reveals the extreme sensitivity of nutrient ratios to bacterial growth, but also shows that even under the optimal single-factor condition of a 3:1 carbon-to-nitrogen ratio, the results (3.15 × 10⁻⁶) are significantly different. 9 The CFU / mL ratio was still lower than the cell density optimized by response surface methodology in Example 3 (3.34 × 10⁻⁶). 9 The CFU / mL result strongly demonstrates the crucial role of the synergistic interaction of multiple factors in improving fermentation yield.

[0043] Comparative Example 6: Control group with low added concentration of pyridoxal coenzyme phosphate In a parallel experiment exploring the effect of coenzyme PLP concentration on GABA conversion, the final concentration of exogenous coenzyme pyridoxal phosphate (PLP) was controlled and adjusted to 0.05 mM (i.e., Figure 5 (The 0.05 mM group in the example) and the remaining basic fermentation operation process and conversion parameters are the same as in Example 4.

[0044] After the reaction was completed, the conversion rate of the precursor substrate at this concentration group was measured, and the corresponding results are shown in the attached figure. Figure 5 The 0.05 mM group is shown in the figure. (From...) Figure 5 Internal data comparison shows that the conversion rate exhibits a significant positive dose-dependent effect with increasing exogenous PLP concentration. When the PLP concentration is low (0.05 mM), the fermentation broth only shows a conversion rate of approximately 50%, significantly lagging behind and lower than [previous level]. Figure 5 The highest substrate conversion rate (approximately 60%) was achieved when the PLP concentration was increased to the saturation addition levels of 0.2 mM and 0.4 mM used in this invention.

[0045] Comparative Example 7: Low Concentration IPTG Inducer Control Group In a parallel experiment exploring the effect of IPTG concentration on GABA conversion, the final concentration of IPTG was controlled and adjusted to 0.01 mM (i.e., Figure 6(The 0.01 mM group in the example) and the remaining basic fermentation operation process and conversion parameters are the same as in Example 4.

[0046] After the reaction was completed, the decarboxylation conversion rate of the precursor substrate at this concentration group was measured, and the corresponding results are shown in the attached figure. Figure 7 The 0.01 mM group is shown in the figure. (From...) Figure 6 Internal data comparison revealed that the conversion rate was extremely low in the control group (0 mM) without additional inducer supplementation. Furthermore, when IPTG was added and the final concentration was adjusted to a low 0.01 mM, the final biological decarboxylation conversion rate was only about 44% due to the weakened heterologous expression initiation ability of the target decarboxylase, significantly lagging behind and lower than [previous value]. Figure 6 The highest conversion rate achieved in this stage (53.8 ± 2.23%) was reached when the IPTG concentration was further increased and optimized to 0.1 mM.

Claims

1. A method for high-yield γ-aminobutyric acid (GABA) fermentation using scallop skirts, characterized in that, Includes the following steps: (1) Constructing a recombinant Escherichia coli engineered strain overexpressing glutamate decarboxylase; (2) The recombinant Escherichia coli was enriched using the enzymatic hydrolysate of scallop skirt as a single nitrogen source nutrient substrate; the scallop skirt was enzymatically hydrolyzed using a special compound enzyme for animal protein hydrolysis at a temperature of 40-60 ℃ and a time of 10-18 h; the carbon-nitrogen ratio of the culture system was controlled at 1:3-5:

1. (3) The enriched recombinant Escherichia coli was added to the substrate for bio-fermentation to synthesize γ-aminobutyric acid.

2. The method according to claim 1, characterized in that, In step (3), the substrate includes monosodium glutamate and glutamic acid. The bio-fermentation system also contains pyridoxal coenzyme phosphate and isopropyl-β-D-thiogalactoside as an inducer. The final concentration of pyridoxal coenzyme phosphate is 0.05-0.4 mM, and the final concentration of the inducer is 0.01-0.4 mM. The amount of monosodium glutamate added is 20-30 g / L, the amount of glutamic acid added is 10-15 g / L, and the fermentation catalysis time is 4-8 h.

3. The application of the fermentation product containing γ-aminobutyric acid obtained by the method described in claim 1 or 2 in the preparation of plant stress stimulants.

4. The application according to claim 3, characterized in that, The plant stress-relieving stimulant is a crude extract containing fermentation supernatant that does not require purification, used to alleviate salt stress in plants; the plant is wheat, which is used for seed soaking treatment.

5. The application according to claim 4, characterized in that: The fermentation supernatant was diluted to a final concentration of γ-aminobutyric acid of 0.2-10 mM.

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