Method for producing transglutaminase by bioconversion of kitchen waste leachate

Abstract

This invention discloses a method for the bioconversion of kitchen waste leachate to produce transglutaminase (TG enzyme), belonging to the field of microbial fermentation technology. Addressing the problem of high cost of industrial synthetic culture media, which restricts the large-scale application of TG enzyme, this invention uses *Streptomyces mobaraensis* CICC 11019 as the production strain. A kitchen waste leachate diluted with water at a volume ratio of 3:2 is used, with the addition of 2 g / L sodium acetate, 25 g / L soybean flour, 10 g / L corn bran flour, and 10 g / L peanut bran flour as the fermentation medium. After inoculum reactivation, seed culture, and fermentation medium preparation, fermentation is carried out at 28℃ and 150 rpm for 3 days. The resulting TG enzyme activity is 0.46-1.05 U / mL, an increase of 2.57-5.91 times compared to the untreated leachate. This invention transforms the complex and difficult-to-treat kitchen waste leachate into an economically valuable TG enzyme through *Streptomyces mobaraensis* fermentation. This enables the resource-based reuse and high-value transformation of kitchen waste, providing a feasible approach for the low-cost, large-scale application of TG enzymes in non-food sectors such as chemicals, textiles, leather, and bio-based materials. It also establishes a green biomanufacturing pathway that combines environmental governance significance with industrial utilization value.

Description

I. Technical Field

[0001] This invention relates to the field of transglutaminase (TG enzyme) fermentation technology, specifically to a method that uses kitchen waste leachate as the main bioconversion substrate and promotes the production of TG enzyme by Streptomyces mobaraensis through optimization methods such as dilution, supplementation of carbon and nitrogen sources and agricultural by-products, thereby converting difficult-to-treat kitchen waste leachate into enzyme preparations with economic added value and realizing the resource utilization and high-value utilization of kitchen waste. II. Background Technology

[0002] Transglutaminase (TG enzyme), as an important protein cross-linking enzyme, can catalyze acyl transfer reactions to create intramolecular and intermolecular covalent cross-links in proteins or peptides, thereby improving protein structure and function. Based on this characteristic, TG enzyme has broad application prospects in bio-based material construction, textile finishing, leather collagen modification, chemical biocatalysis, and chemical polymer modification. Furthermore, it can also be used as a candidate enzyme source in some biomaterials and medical excipient research scenarios. Current TG enzyme production typically relies on culture medium preparation and conventional liquid fermentation systems, with related processes focusing more on increasing enzyme yield, while neglecting the targeted bioconversion of complex organic wastes such as food waste and the recovery and utilization of high-value products. Deep liquid fermentation also suffers from inherent drawbacks such as large organic wastewater production and high energy consumption; while solid-state fermentation has certain advantages, traditional methods suffer from problems related to mass and heat transfer.

[0003] Leachate from food waste is rich in nutrients such as carbon, nitrogen, and minerals, making it a potentially inexpensive fermentation substrate. However, when using this leachate directly for fermentation to produce TG enzymes, the enzyme production efficiency is very low (typically below 0.2 U / mL) due to its complex composition, imbalanced carbon-nitrogen ratio, and the presence of inhibitory factors. -1 Meanwhile, food waste itself is a challenge for urban environmental management. If it can be converted into TG enzymes with industrial application value through microbial fermentation, it can be transformed from "waste awaiting treatment" into "usable raw materials for biomanufacturing." To address the above problems, this invention significantly improves the ability of Streptomyces to produce TG enzymes through optimized treatments such as diluting the food waste leachate, supplementing it with an exogenous carbon source (sodium acetate), and adding agricultural by-products (soybean flour, peanut bran flour, etc.). This allows the food waste leachate to form enzyme preparations with economic added value while being disposed of. III. Summary of the Invention

[0004] (I) Purpose of the Invention

[0005] This invention aims to develop a biotransformation method using kitchen waste leachate as the main substrate. Through Streptomyces fermentation, the complex and difficult-to-treat kitchen waste leachate is converted into transglutaminase (TG enzyme), which has economic added value. By diluting, supplementing with exogenous carbon, and adding agricultural byproducts, the fermentation system of the leachate is improved, thereby significantly enhancing the enzyme production capacity of Streptomyces. This solves the problem of low enzyme production efficiency when directly using leachate, realizing the resource-based reuse and high-value transformation of kitchen waste, turning waste into treasure, reducing the environmental burden of waste treatment, and forming a green biomanufacturing pathway that combines environmental governance significance with industrial utilization value.

[0006] (II) Technical Solution

[0007] To address the above problems, this invention first provides a culture medium formula for fermenting and producing transglutaminase (TG) using leachate from kitchen waste, which significantly improves the enzyme activity and yield of TG enzyme. Furthermore, this invention also provides a fermentation cultivation method that optimizes the leachate dilution ratio, the addition of exogenous carbon sources and agricultural by-products, enabling kitchen waste leachate to be stably converted into TG enzyme products with economic added value, and improving the resource utilization level of kitchen waste.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] Based on the first aspect of the present invention, a culture medium formulation is provided:

[0010] Subculture medium: The strain was subcultured using Gao's No. 1 medium. Composition of Gao's No. 1 medium (content / g / L) -1 The following mixture was prepared: 20.0g agar, 20.0g soluble starch, 0.5g K2HPO4, 0.24g MgSO4, 0.01g FeSO4·7H2O, 0.5g NaCl, and 0.1g KNO3. Dissolve in deionized water, adjust the pH to 7.2-7.4, sterilize at 121℃ for 30 minutes, pour into plates while hot, and use after the plates solidify.

[0011] Seed culture medium components (content / gL) -1 ): Glycerin 20.0, peptone 20.0, yeast extract 5.0, K2HPO4 2.0, KH2PO4 2.0, MgSO4·7H2O 2.0, dissolved in deionized water, heated and stirred until the solid components are completely dissolved, adjusted pH=7.0, and sterilized for later use.

[0012] Purified culture medium components (content / g L) -1 ): Glucose 40.0, Beef peptone 25.0, Corn steep liquor powder 10.0, K2HPO4 2.0, MgSO4·7H2O 2.0, NaCl 2.5.

[0013] Liquid fermentation culture medium for kitchen waste leachate: Take fresh kitchen waste and filter it through three layers of 100-mesh gauze to obtain the leachate. Dilute it with deionized water at a volume ratio of 3:2 (i.e., leachate volume ratio 60%), adjust the pH to 7.0, sterilize at 121℃ for 20 minutes, and cool before use. Further add exogenous carbon sources and agricultural by-products (content / g·L) to this medium. -1 ): Sodium acetate 2.0, soybean flour 25.0, corn bran flour 10.0, peanut bran flour 10.0.

[0014] It is worth noting that although the original leachate from kitchen waste is rich in nutrients such as carbon, nitrogen, and minerals, its enzyme production efficiency is very low when used directly. This invention significantly promotes the growth of Streptomyces and the secretion of TG enzymes by diluting the leachate to reduce osmotic pressure and the concentration of inhibitory factors, adding exogenous carbon sources to adjust the carbon-nitrogen ratio, and adding agricultural by-products to provide complex nutrients. The synergistic combination of these formulations not only greatly increases enzyme production but also achieves high-value utilization of kitchen waste. Based on a second aspect of this invention, a method for producing transglutaminase by fermentation using kitchen waste leachate is also provided, comprising the following steps:

[0015] Step (1): Strain revival

[0016] The *S. mobaraensis* strain used in this invention was purchased from the China Industrial Microbial Culture Collection Center (CICC). The strain number is CICC 11019. The purchased strain (lyophilized powder form) was activated and cultured according to the supplier's instructions. The specific procedures were as follows: The activated *S. mobaraensis* was propagated and cultured. Under aseptic conditions, the lyophilized powder was dissolved in 1 mL of sterile physiological saline. The bacterial suspension was then spread onto the surface of a Gao Shi No. 1 solid agar plate and incubated upside down in a 28°C incubator. After approximately 3-5 days, single colonies appeared on the plate. These single colonies were picked and streaked for purification, repeated 2-3 times to obtain a pure culture. The pure culture strain was then identified using 16S rDNA to confirm the purchased strain was correct. The strain was then preserved in the laboratory (stored in glycerol tubes at -80°C).

[0017] Based on previous culture medium screening experiments in the laboratory, the strain exhibited optimal growth when subcultured using Gao's No. 1 medium. Gao's No. 1 medium was dissolved in deionized water, the pH was adjusted to 7.2-7.4, and the culture was sterilized at 121℃ for 30 minutes. The medium was then poured onto plates while still hot. After solidification, the strain was subcultured using the streak method and incubated at 28℃ for approximately one week. The aerial mycelia gradually turned greenish-gray, indicating that the spores were beginning to mature. At this stage, the microbial activity was strong and the culture could be used for fermentation inoculation. Subculture was performed every 30 days to maintain the strain's viability.

[0018] Step (2): Seed liquid preparation

[0019] The preparation method of seed culture medium is as follows: weigh each component according to the above seed culture medium formula, dissolve in deionized water, heat and stir until completely dissolved, adjust pH to 7.0, dispense into 250mL Erlenmeyer flasks (50mL per flask), plug with cotton plugs, sterilize at 121℃ for 20min, and cool for later use.

[0020] Select a plate that has grown for 7 days and has produced complete green-gray spores in good condition. Use a sterile inoculation spatula to scrape off 1 / 2 of the plate of spores and transfer it to a 250mL Erlenmeyer flask containing 50mL of seed culture medium.

[0021] After inoculation, the conical flask was placed in a constant temperature shaker at 28℃ and 150rpm for 48 hours. The seed culture should be uniformly turbid, indicating good bacterial growth and no signs of contamination. Seed culture OD 600 The value can generally reach 1.5-2.0, and the viable bacteria count is approximately 10. 8 A concentration of CFU / mL can be used as a seed culture for subsequent liquid fermentation inoculation.

[0022] Step (3): Preparation of liquid fermentation culture medium for kitchen waste leachate

[0023] Treatment and optimization of liquid fermentation medium for kitchen waste leachate: Leachate was diluted with deionized water at a volume ratio of 3:2 (leachate:deionized water) to reduce osmotic pressure and the concentration of inhibitory factors. After dilution, the pH was adjusted to 7.0 with 1 mol / L NaOH or HCl. Further exogenous carbon sources and agricultural byproducts were added to adjust the carbon-nitrogen ratio and provide nutrients: sodium acetate 2 g / L; soybean meal 25 g / L; corn bran meal 10 g / L; and peanut bran meal 10 g / L. Through optimization of fermentation conditions, the inoculum size for the seed culture was determined to be 10 mL.

[0024] Step (4): Inoculation and fermentation culture

[0025] The seed culture prepared in step (2) was inoculated into the liquid fermentation medium of kitchen waste leachate prepared in step (3) at an inoculation rate of 20% (volume ratio), that is, 10 mL of seed culture was inoculated for every 50 mL of medium. After inoculation, the conical flask was placed in a constant temperature shaker at 28℃ and the rotation speed was 150 rpm.

[0026] The optimal fermentation time is between 7.0 and 6.5, with a fermentation period of 3 days. This approach can both increase enzyme production and shorten the fermentation cycle.

[0027] Step (5): TG enzyme activity comparison

[0028] Using unoptimized raw kitchen waste leachate (undiluted, without any added carbon source, nitrogen source, or agricultural byproducts) as a control, its TG enzyme activity was approximately 0.16-0.19 U / mL. Fermentation using the optimized culture medium of this invention (diluted with deionized water at a volume ratio of 3:2, containing 2 g / L sodium acetate, 25 g / L soybean flour, 10 g / L corn bran flour, and 10 g / L peanut bran flour) resulted in TG enzyme activity reaching 0.46-1.05 U / mL, an increase of 2.57-5.91 times compared to the control. These results indicate that kitchen waste leachate, after dilution and compound nutrient regulation, can serve as an effective biotransformation substrate for Streptomyces fermentation to produce TG enzymes, transforming previously difficult-to-treat organic waste into enzyme preparations with application value and marketability.

[0029] (III) Beneficial Effects

[0030] 1. This invention achieves the bioconversion of food waste into enzyme preparations with economic added value. Using food waste leachate as the main fermentation substrate, the organic nutrients in the leachate are converted into TG enzymes through microbial fermentation. This changes the traditional treatment path of food waste, effectively replacing traditional synthetic culture media and simultaneously realizing the resource utilization of the leachate. Based on each ton of leachate, this process can directly save 1000-2000 yuan in traditional disposal costs. Combining the enzyme production revenue from fermentation and deducting the input of fermentation auxiliary materials, the comprehensive net benefit of each fermentation system can reach 1598-1877 yuan / ton. The fermentation system with added peanut bran powder has the best comprehensive benefit, with a net benefit of approximately 1877 yuan / ton; the system using only the basic leachate requires no additional auxiliary materials and has a comprehensive net benefit of 1598-1751 yuan / ton. This process achieves the harmless treatment of food waste leachate while simultaneously producing high-value-added enzyme preparations, thus combining environmental and economic benefits.

[0031] 2. Significantly improved enzyme production efficiency. Direct fermentation of raw kitchen waste leachate yields very low enzyme production efficiency (only 0.16-0.19 U / mL). This invention reduces osmotic pressure and inhibitory factor concentration through dilution, while simultaneously adding exogenous carbon sources (such as sodium acetate) and agricultural byproducts (such as soybean flour) to improve the microbial growth environment and mass transfer conditions. The optimized TG enzyme activity reaches 0.46-1.05 U / mL, an increase of 2.57-5.91 times compared to untreated leachate, and significantly superior to the effect of single optimization methods.

[0032] 3. Outstanding Environmental and Social Benefits. This invention uses kitchen waste leachate as a fermentation substrate to produce high-value-added TG enzymes. While obtaining industrial-grade enzymes, it also disposes of a large amount of organic waste, turning waste into treasure and reducing the environmental pressure of landfill or incineration. This aligns with the industrial orientation of circular economy and green biomanufacturing, possessing both significant environmental and social benefits.

[0033] 4. Broad application prospects. The TG enzyme produced by fermentation in this invention provides a high-quality raw material for related fields such as biomaterial cross-linking, leather processing, textile finishing, and pharmaceutical excipient synthesis. Furthermore, the successful implementation of this method provides a referable technical path for the fermentation production of high-value-added enzyme preparations (such as proteases, lipases, and cellulases) from other food waste or organic waste. IV. Description of the attached drawings

[0034] (I) Cultivation process flow chart

[0035] Figure 1 The entire process was demonstrated, from the activation, subculturing, and seed culture of S. mobaraensis strain (strain number CICC 11019), to the preparation of liquid fermentation medium for kitchen waste leachate (dilution treatment, addition of sodium acetate, soybean flour, corn bran flour, peanut bran flour, and mixed addition), and finally the detection of indicators such as enzyme activity, mycelial biomass, crude protein extraction, and nitrogen conversion rate.

[0036] (II) Comparison of TG enzyme activity

[0037] Figure 2 The figure shows a comparison of TGase enzyme activity under different treatments. This indicator is a key basis for measuring the enzyme production capacity and fermentation effect of *Streptomyces molybdenum*. The figure compares the test results of Examples 1-7, with Example 1 (100% kitchen waste leachate) showing the lowest TGase enzyme activity. Compared to Example 1, the enzyme activity of the diluted 60% kitchen waste leachate, as well as the groups with added sodium acetate, soybean flour, corn bran flour, peanut bran flour, and a mixture, all showed varying degrees of improvement, with the group adding soybean flour showing the most significant improvement. This demonstrates that supplementing the kitchen waste leachate system with nutritional additives can effectively increase TGase enzyme activity, fully showcasing the advantages of the improved formulation of this invention.

[0038] (III) Biomass Comparison Chart

[0039] Figure 3 This study compares the biomass of *Streptomyces mogulata* under different treatments. Mycelial dry biomass is a crucial indicator for assessing the growth status of *Streptomyces mogulata*. Example 1 (100% kitchen waste leachate) showed the lowest mycelial dry biomass among all groups, at only 12.198 g / L, indicating that the pure high-concentration leachate suffers from nutrient deficiency and accumulation of harmful substances, severely inhibiting the growth of *Streptomyces mogulata*. The other examples showed significant increases compared to Example 1. The addition of various auxiliary materials to the 60% kitchen waste leachate further improved the growth of the strain, with the addition of soybean powder showing the most significant effect on increasing mycelial biomass. Data comparison clearly demonstrates that adding suitable auxiliary materials can effectively improve the growth status of the bacteria, confirming the practical value of the optimized fermentation system using kitchen waste leachate of this invention.

[0040] (iv) Nitrogen Conversion Rate Chart for Kitchen Waste

[0041] Figure 4 This is a comparison chart of nitrogen conversion rates in food waste. Nitrogen conversion rate is a core parameter for measuring the efficiency of *Streptomyces molybdenum* in utilizing nitrogen sources in food waste leachate. The chart shows the comparative data for Examples 1-7. Example 1 (100% food waste leachate) has a nitrogen conversion rate of only 21%, the lowest among all groups. All other groups show significant improvements compared to Example 1, with Example 4 (60% leachate + soybean powder) achieving a nitrogen conversion rate of 57%, demonstrating the most significant improvement. The data comparison clearly shows that the addition of auxiliary materials to the food waste leachate system in this invention can significantly improve nitrogen source utilization efficiency, reflecting the significant advantages of this invention in optimizing fermentation processes and resource utilization. V. Detailed Implementation Methods

[0042] The present invention will be further illustrated below with specific embodiments. These embodiments are merely illustrative and do not limit the scope of the invention.

[0043] Unless otherwise specified, the raw materials and reagents used in the following embodiments of the present invention can be obtained commercially.

[0044] The analytical and testing methods used in the following embodiments of the present invention are as follows:

[0045] (I) TG enzyme activity assay method:

[0046] 1. Reagents:

[0047] Reagent A: Mix 0.03 mol / L Na-CBZ-Gln-Gly, 0.1 mol / L hydroxylamine hydrochloride, 0.01 mol / L cysteine ​​hydrochloride, and 0.2 mol / L Tris-HCl buffer at pH 6.0 in a volume ratio of 1:1:1:2. Adjust the pH to 6.0 with 1 mol / L HCl during the preparation process.

[0048] Reagent B: Prepare 3 mol / L hydrochloric acid, 12% trichloroacetic acid, and 5% FeCl3 separately, and then mix them in a volume ratio of 1:1:1.

[0049] 2. Plotting the standard curve:

[0050] Standard solutions of transglutaminase (TGT) with concentrations of 0, 0.0625 μmol / mL, 0.1250 μmol / mL, 0.1875 μmol / mL, 0.2500 μmol / mL, and 0.3750 μmol / mL were prepared using Sigma-Aldrich (T5398). 500 μL of reagent A was mixed with 200 μL of each TGT standard solution in a 1.5 mL centrifuge tube, with three replicates for each concentration. The mixture was incubated at 37.0℃ ± 0.5℃ for 10 min, and the reaction was terminated by adding 500 μL of reagent B. The reaction solution with a TGT concentration of 0 served as a blank. The absorbance of each dilution was measured at 525 nm using a microplate reader. A standard curve was plotted with TGT concentration on the ordinate and absorbance of the dilution on the abscissa. A linear regression equation was obtained using the least squares method, with a correlation coefficient of 0.9994.

[0051] Sample determination: Take 200 μL of appropriately diluted crude enzyme solution and proceed as described above. Calculate the amount of hydroxamic acid produced based on the standard curve. Enzyme activity unit (U / mL) is defined as the amount of enzyme required to catalyze the production of 1 μmol of hydroxamic acid per minute at 37℃ and pH 6.0. Perform three replicates for each batch of samples, and take the mean ± standard deviation.

[0052] (II) Biomass Measurement Methods

[0053] 1. Filtration and separation: Take 10 mL of the fermented culture medium into a centrifuge tube, centrifuge at 8000 r / min and 4℃ for 10 min, and the precipitate obtained is mycelium.

[0054] 2. Washing the mycelium: Gently rinse the mycelium with an appropriate amount of PBS solution (pH 7.0) to remove impurities such as culture medium components and metabolic products attached to the surface of the mycelium. The washing process should be gentle to prevent the mycelium from falling off and being lost. Generally, wash 2-3 times, using only enough distilled water to just cover the mycelium each time.

[0055] 3. Freeze-drying: Centrifuge tubes containing mycelium were frozen at -20°C for 12 hours, then transferred to -80°C and frozen for 15 minutes. They were then quickly placed in a vacuum freeze dryer and freeze-dried for 12 hours until constant weight was achieved. The weight was then recorded as m1. The centrifuge tubes were then washed and dried in a drying oven for 12 hours, followed by cooling for 1 hour. This cooling and weighing process was repeated until the difference between two weighings was less than 0.002 g. At this point, constant weight was considered achieved, and the final weight was recorded as m2.

[0056] 4. Calculate biomass: Biomass (based on mycelial dry weight, g / L) is calculated using the formula: Biomass = (m1 - m2) × 100.

[0057] (III) Methods for determining nitrogen conversion rate

[0058] 1. Sample Preparation. Three replicate fermentation groups were set up. Waste samples were thoroughly mixed before fermentation. Three samples were frozen and stored for determining total liquid nitrogen (N1) before fermentation. Three replicates of inoculated bacteria were prepared and lyophilized for determining bacterial nitrogen (N2) before fermentation.

[0059] 2. Sample processing after fermentation. After fermentation, the mixture was centrifuged at 8000 rpm and 4°C for 10 min to separate the cell precipitate and supernatant. The supernatant was mixed with an equal volume of anhydrous ethanol at 4°C, allowed to stand at low temperature, and then centrifuged at 10000 rpm and 4°C for 10 min to obtain the crude enzyme precipitate. The cells and crude enzyme were first frozen for 1 h, and then freeze-dried under vacuum for 24 h. The dry weight of the cells after fermentation was calculated using EP tubes to determine the nitrogen content N3, and the dry weight of the crude enzyme was used to determine the nitrogen content N4.

[0060] 3. Nitrogen Content Calculation and Unit Conversion. The nitrogen mass fraction (%N) of each sample was determined using an elemental analyzer. Individual sample nitrogen content: Nitrogen content is the dry weight nitrogen mass fraction of the sample. The unified conversion unit is g / L: Nitrogen content / Fermentation system volume.

[0061] Nitrogen conversion rate calculation formula: Nitrogen conversion rate = Post-fermentation microbial nitrogen - Pre-fermentation microbial nitrogen + Crude enzyme nitrogen / Pre-fermentation total liquid nitrogen

[0062] Note: All nitrogen content units in the formula are g / L, and the volume is the total volume of the fermentation system.

[0063] The present invention can be implemented as follows:

[0064] (I) Example 1

[0065] Control - Untreated leachate: Liquid fermentation medium for kitchen waste leachate was sterilized at 121℃ for 20 minutes. After cooling, 10 mL of seed culture was inoculated into 50 mL of medium. After inoculation, the conical flask was placed in a constant temperature shaker at 28℃ and a rotation speed of 150 rpm. Fermentation lasted for 3 days.

[0066] Dilution treatment: Liquid fermentation culture medium for kitchen waste leachate: Kitchen waste leachate is diluted with deionized water at a volume ratio of 3:2, sterilized at 121℃ for 20 minutes, and inoculated after cooling.

[0067] (II) Example 2

[0068] Dilution treatment: The liquid fermentation medium for kitchen waste leachate was diluted with deionized water at a volume ratio of 3:2, sterilized at 121℃ for 20 minutes, and after cooling, 10mL of seed culture was inoculated into 50mL of medium. After inoculation, the conical flask was placed in a constant temperature shaker at 28℃ and a rotation speed of 150rpm for 3 days of fermentation.

[0069] (III) Example 3

[0070] Dilution + Sodium Acetate: Liquid Fermentation Medium for Kitchen Waste Leachate. The kitchen waste leachate was diluted with deionized water at a volume ratio of 3:2, and 2 g / L of sodium acetate was added. The mixture was sterilized at 121℃ for 20 minutes. After cooling, 10 mL of seed culture was inoculated into 50 mL of medium. After inoculation, the conical flask was placed in a constant temperature shaker at 28℃ and a rotation speed of 150 rpm for 3 days of fermentation.

[0071] (iv) Example 4

[0072] Dilution + Soybean Powder: Liquid Fermentation Culture Medium for Kitchen Waste Leachate. The kitchen waste leachate was diluted with deionized water at a volume ratio of 3:2. 25 g / L of soybean powder was added, and the mixture was sterilized at 121℃ for 20 minutes. After cooling, 10 mL of seed culture was inoculated into 50 mL of culture medium. After inoculation, the conical flask was placed in a constant temperature shaker at 28℃ and a rotation speed of 150 rpm for 3 days of fermentation.

[0073] (V) Example 5

[0074] Dilution + Peanut Bran Powder: Liquid Fermentation Culture Medium for Kitchen Waste Leachate. The kitchen waste leachate was diluted with deionized water at a volume ratio of 3:2. 10 g / L of peanut bran powder was added, and the mixture was sterilized at 121℃ for 20 minutes. After cooling, 10 mL of seed culture was inoculated into 50 mL of culture medium. After inoculation, the conical flask was placed in a constant temperature shaker at 28℃ and a rotation speed of 150 rpm for 3 days of fermentation.

[0075] (vi) Example 6

[0076] Dilution + Corn Bran Powder: Liquid Fermentation Culture Medium for Kitchen Waste Leachate. The kitchen waste leachate was diluted with deionized water at a volume ratio of 3:2. 10 g / L of corn bran powder was added, and the mixture was sterilized at 121℃ for 20 minutes. After cooling, 10 mL of seed culture was inoculated into 50 mL of culture medium. After inoculation, the conical flask was placed in a constant temperature shaker at 28℃ and a rotation speed of 150 rpm for 3 days of fermentation.

[0077] (VII) Example 7

[0078] Full Formula - Dilution + Mixing Addition: Liquid Fermentation Culture Medium for Kitchen Waste Leachate. Dilute the kitchen waste leachate with deionized water at a volume ratio of 3:2. Add 2g / L sodium acetate, 25g / L soybean flour, 10g / L peanut bran flour, and 10g / L corn bran flour. Sterilize at 121℃ for 20 minutes. After cooling, inoculate 10mL of seed culture into 50mL of culture medium. After inoculation, place the conical flask in a constant temperature shaker at 28℃ and a rotation speed of 150rpm for 3 days of fermentation.

Claims

1. A method for bioconversion of kitchen waste leachate to produce transglutaminase, characterized in that, include: Using widely available and inexpensive kitchen waste leachate and agricultural by-products as fermentation substrates, Streptomyces mobaraensis was used to efficiently produce transglutaminase (TG enzyme) through fermentation. Strain reactivation: The strain was reactivated using Gao's No. 1 medium. Gao's No. 1 medium was dissolved in deionized water, the pH was adjusted to 7.2-7.4, and the medium was sterilized at 121℃ for 30 minutes. The medium was then poured onto plates while still hot. After solidification, the strain was subcultured using the streak method and placed in a 28℃ incubator until the aerial mycelia gradually turned greenish-gray. This indicates that the spores have begun to mature. At this point, the microbial activity is strong and the strain can be used for fermentation inoculation. Seed culture preparation: Seed culture medium components (content / g / L) -1 ): Glycerin 20.0, peptone 20.0, yeast extract 5.0, K2HPO4 2.0, KH2PO4 2.0, MgSO4·7H2O 2.0, dissolved in deionized water, heated and stirred until the solid components were completely dissolved, adjusted to pH 7.0, and sterilized for later use. Select plates with good growth status after 7 days of growth, producing greenish-gray spores. Scrape half of the spores from the plate with an inoculation spatula and inoculate them into a 250mL Erlenmeyer flask containing 50mL of seed culture medium. Incubate at 28℃ and 150rpm in a constant temperature shaker for 48 hours to obtain the fermentation seed culture for subsequent liquid fermentation inoculation. Preparation of liquid fermentation medium for kitchen waste leachate: Fresh kitchen waste was taken and filtered through three layers of 100-mesh gauze to obtain the kitchen waste leachate. The liquid fermentation medium for the kitchen waste leachate was treated as follows: deionized water was added at a volume ratio of 3:2 to dilute it, thereby reducing osmotic pressure and the concentration of inhibitory factors; sodium acetate 2 g / L was added to adjust the carbon-nitrogen ratio; soybean flour 25 g / L, corn bran flour 10 g / L, and peanut bran flour 10 g / L were added to provide balanced nutrition. The inoculum volume for fermentation was 10 mL. Culture environment control: The temperature was controlled at 28℃ throughout the process, the shaking speed was 150 rpm, and the fermentation pH was between 6.5 and 7.

0. The optimal fermentation time was 3 days, which can improve enzyme yield and shorten the fermentation cycle. After fermentation was completed, the product was measured.

2. The method for transglutaminase fermentation using kitchen waste leachate and agricultural by-products as a culture medium according to claim 1, characterized in that, The *Streptomyces molina* strain was purchased from the China Industrial Microbial Culture Collection Center (CICC), accession number CICC 11019. The purchased strain (lyophilized powder) was activated and cultured according to the instructions, and the strain was then preserved in the laboratory. 16S rDNA identification of the strain confirmed that the purchased strain was correct.

3. The method for transglutaminase fermentation using kitchen waste leachate and agricultural by-products as a culture medium according to claim 1, characterized in that, The composition (content / g / L) of Gao's No. 1 culture medium -1 The following mixture was prepared: 20.0g agar, 20.0g soluble starch, 0.5g K2HPO4, 0.24g MgSO4, 0.01g FeSO4·7H2O, 0.5g NaCl, and 1.0g KNO3. Dissolve in deionized water, adjust the pH to 7.2-7.4, sterilize at 121℃ for 30 minutes, pour into plates while hot, and use after the plates solidify.

4. The method for transglutaminase fermentation using kitchen waste leachate and agricultural by-products as a culture medium according to claim 1, characterized in that, The soluble starch needs to be dissolved separately by heating in a water bath before being mixed with other ingredients; otherwise, the seed liquid will be uneven.

5. The method for glutamine transaminase fermentation using kitchen waste leachate and agricultural by-products as a culture medium according to claim 1, characterized in that, The seed culture is shaken at 150 rpm. Too low a speed will result in insufficient dissolved oxygen, uneven mass transfer, cell aggregation, and pH and metabolic shift. Too high a speed will result in impaired shear force, excessive dissolved oxygen, increased foaming, abnormal metabolism, and increased energy consumption.

6. The method for transglutaminase fermentation using kitchen waste leachate and agricultural by-products as a culture medium according to claim 1, characterized in that, The kitchen waste is taken from a kitchen waste treatment plant, and after being crushed and compressed, it is filtered through three layers of 100-mesh gauze in the laboratory to obtain kitchen waste leachate.

7. The method for glutamine transaminase fermentation using kitchen waste leachate and agricultural by-products as a culture medium according to claim 1, characterized in that, The volume ratio of the kitchen waste leachate to deionized water is 3:

2.

8. The method for transglutaminase fermentation using kitchen waste leachate and agricultural by-products as a culture medium according to claim 1, characterized in that, The soybean flour, corn bran flour, and peanut bran flour need to be dried, thoroughly ground in a pulverizer, and then added after passing through a 20-mesh sieve.

9. The method for glutamine transaminase fermentation using kitchen waste leachate and agricultural by-products as a culture medium according to claim 1, characterized in that, The fermentation raw materials, namely kitchen waste leachate, soybean flour, corn bran flour and peanut bran flour, are inexpensive. Based on each ton of leachate, this process can directly save 1,000-2,000 yuan in traditional disposal costs. After combining the enzyme production revenue from fermentation and deducting the input of fermentation auxiliary materials, the comprehensive net benefit of each group of fermentation systems can reach 1,598-1,877 yuan / ton.

10. The method for glutamine transaminase fermentation using kitchen waste leachate and agricultural by-products as a culture medium according to claim 1, characterized in that, The direct fermentation efficiency of untreated raw kitchen waste leachate for enzyme production is very low (only 0.16-0.19 U / mL). By diluting the leachate to reduce osmotic pressure and the concentration of inhibitory factors, and by adding exogenous carbon sources (such as sodium acetate) and agricultural byproducts (such as soybean flour), the microbial growth environment and mass transfer conditions are improved. The optimized transglutaminase activity can reach 0.46-1.05 U / mL, an increase of 2.57-5.91 times compared to the untreated leachate.

11. The method for producing transglutaminase by fermentation of kitchen waste leachate according to claim 1, characterized in that, The fermentation temperature was 28℃, the fermentation time was 3 days, and the pH naturally decreased and remained between 6.5 and 7.0 during the fermentation process.

12. The method for producing transglutaminase by fermentation of kitchen waste leachate according to claim 1, characterized in that, The fermentation of kitchen waste leachate to produce transglutaminase is an environmentally friendly process that produces industrial enzymes while simultaneously disposing of organic waste, meeting the requirements of circular economy and green biomanufacturing.

13. The method for producing transglutaminase by fermentation of kitchen waste leachate according to claim 1, characterized in that, The produced transglutaminase is suitable for applications in biomaterial cross-linking, protein modification, leather processing, textile finishing, and industrial enzyme preparation; it can also be used as an auxiliary enzyme source in the preparation or research of pharmaceutical excipients as needed.

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