High-throughput screening method for aspergillus oryzae

Abstract

The application relates to a high-throughput screening method of Aspergillus oryzae, which comprises the following steps: (1) preparing an Aspergillus oryzae library; (2) embedding spore suspensions of Aspergillus oryzae contained in the Aspergillus oryzae library based on a droplet microfluidic cell sorting system, generating a plurality of droplets with a volume of 524-697 pL and collecting the droplets; (3) after the plurality of droplets formed in the step (2) are cultured at 28-32 DEG C for 7-8 h, glutamic acid pseudo-fluorescent protein sensors are injected into the droplets at a temperature of 15-17 DEG C by using a droplet injection chip, and the sensors are collected; (4) after the droplets in which the glutamic acid pseudo-fluorescent protein sensors are injected in the step (3) are reacted at 15-17 DEG C for 5-7 h to generate fluorescence signals, the droplets are sorted according to the fluorescence signal intensity by using a droplet sorting chip, and Aspergillus oryzae corresponding to 1‰ of the droplets with the lowest fluorescence signal intensity is screened. The method has high screening throughput, and realizes high-throughput screening of Aspergillus oryzae in a picoliter droplet system.

Description

Technical Field

[0001] This application relates to the field of microbial fermentation engineering technology, specifically to a high-throughput screening method for Aspergillus oryzae. Background Technology

[0002] Microbial strains are the foundation of the microbial fermentation industry, and high-performance strains are key to achieving economical and efficient production of bioproducts. When strains cannot meet the requirements of industrial production, high-quality and high-yield upgraded strains are generally obtained through strain selection and breeding to better serve industrial production. However, traditional breeding methods mainly rely on shaking flasks, solid plates, deep-well plates, and other culture methods, requiring periodic sampling and testing of relevant indicators to compare strain performance. This process is not only cumbersome and consumes a lot of manpower and resources, but also has low throughput, poor parallelism, and low breeding efficiency.

[0003] Aspergillus oryzae is a commonly used fermentation microorganism in traditional foods such as soy sauce and fermented bean paste. In solid-state fermentation, it primarily utilizes its secreted enzyme system to hydrolyze soybeans, wheat, and other grains, producing various amino acids, peptides, and reducing sugars. Solid-state fermentation systems offer excellent aeration, and Aspergillus oryzae grows primarily as aerial hyphae with numerous branching hyphae. Furthermore, the growing medium is made from natural materials, resulting in a rich variety of enzymes secreted during solid-state fermentation. Aspergillus oryzae can also grow in liquid environments; the earliest commercially available amylase was produced using liquid fermentation of this species. However, liquid fermentation has lower aeration than solid-state fermentation, and Aspergillus oryzae tends to form mycelial balls during the process. Additionally, the growing medium typically uses inducing agents, leading to a correlation between the secreted enzyme composition and the inducing agent, with higher yields of specific enzymes and lower levels of other enzyme components.

[0004] For the selection of superior microbial strains for soy sauce, solid-state fermentation is preferred for screening and evaluation to match the soy sauce fermentation system, allowing for targeted selection of Aspergillus oryzae strains suitable for solid-state fermentation. However, solid-state fermentation systems are complex and large-scale, resulting in long culture cycles and complicated operations, leading to extremely low breeding and screening efficiency. Liquid-state culture can achieve high-throughput culture of Aspergillus oryzae in a smaller system, while also facilitating standardized operations and improving screening efficiency. However, the enzyme system secreted by Aspergillus oryzae in liquid-state fermentation systems lacks comprehensiveness and cannot effectively reflect the actual function of the strain in solid-state fermentation systems.

[0005] The logic of strain selection is to enhance a specific characteristic of the strain while retaining other functions. This characteristic enhancement can be well characterized in a liquid fermentation system through the design of the culture medium. Therefore, in the design of the screening model, a liquid system can be used to characterize the advantageous characteristics of the selected strain, followed by a comprehensive evaluation of the strain's effects in traditional fermented products such as soy sauce through solid-state fermentation. This approach effectively combines the advantages of both solid-state and liquid-state fermentation with *Aspergillus oryzae*. However, to truly improve the efficiency of *Aspergillus oryzae* solid-state fermentation, it is necessary to miniaturize both the liquid and solid-state fermentation systems and ensure their stable operation; this is a problem that needs to be addressed.

[0006] Traditional solid-state fermentation takes place in an open or semi-open environment. Although Aspergillus oryzae is inoculated using a pure culture method, microorganisms in the environment will still participate in the solid-state fermentation process at different stages. If Aspergillus oryzae lacks resistance to contamination, other microorganisms in the environment will become the dominant species, inhibiting the normal growth of Aspergillus oryzae and leading to the failure of solid-state fermentation quality. Therefore, the resistance of Aspergillus oryzae to contamination is a key indicator for ensuring the stability of solid-state fermentation quality. Fast-growing Aspergillus oryzae can occupy the surface of raw materials more quickly, thereby inhibiting the growth of other microorganisms to a certain extent. Therefore, increasing the growth rate of Aspergillus oryzae can improve its resistance to contamination during the koji-making process. Currently, the assessment of the resistance to contamination of Aspergillus oryzae strains for solid-state fermentation is often the last step in the application of the strain. However, if the strain has poor resistance to contamination, it will not be able to be promoted and applied in industrialization, which will also lead to the failure of the entire strain breeding work.

[0007] The current common method for evaluating the contamination resistance of Aspergillus oryzae strains is to conduct antagonistic tests against common environmental microorganisms in solid-state fermentation. This method involves comparing the production speed of different strains by plating different strains, which is difficult to implement in a large-scale manner, resulting in low evaluation efficiency and a large workload. Therefore, a new method is needed to achieve a comprehensive evaluation of the contamination resistance of Aspergillus oryzae.

[0008] In recent years, the development of droplet microfluidics has brought new opportunities for high-throughput culture and screening of microorganisms. Droplet microfluidics refers to the establishment of a system for processing or manipulating extremely small volumes of liquid (10⁻⁶). -9 ~10 -15In liquid systems with volumes in the nanoliter (N) range, the dimensions of the channels and components are only tens to hundreds of micrometers in size. This allows for the formation of controllable, independent individual droplet microreactors for further manipulation. Biochemical reactions or detections within such small systems require only very small amounts of sample and reagents to obtain high-resolution, highly sensitive results, significantly reducing costs. Simultaneously, the nanoliter or even picoliter-level fluid volumes significantly improve reaction and analysis efficiency, thereby shortening the time required for detection and reaction. Compared to some traditional culture methods, droplet microfluidics offers significant advantages in system miniaturization, high throughput, and automated operation. Filamentous fungi such as Aspergillus oryzae require considerable space for hyphal elongation during growth, and the production and secretion of many products occur after the hyphae have grown to a certain extent. Due to this limitation, while picoliter-level droplet microfluidics are widely used in the screening of single-celled microorganisms such as bacteria and yeast, their application in filamentous fungi is still rarely reported. Furthermore, the selection of detection signals is a major challenge in the application of microfluidics.

[0009] Therefore, in order to address the challenge of Aspergillus oryzae growing in microdroplets, a high-throughput screening method for Aspergillus oryzae needs to be established in a picoliter-level droplet system. Summary of the Invention

[0010] Based on this, this application establishes a high-throughput screening method for Aspergillus oryzae with fast growth rate and good contamination resistance in a pi-level droplet system, thereby achieving high-throughput screening for contamination resistance first, followed by screening for high-yield characteristics, and thus improving the success rate of industrial application of selected strains.

[0011] The technical solution includes:

[0012] A high-throughput screening method for Aspergillus oryzae, the method comprising the following steps:

[0013] (1) Preparation of Aspergillus oryzae library;

[0014] (2) Based on the droplet microfluidic cell sorting system, the spore suspension of Aspergillus oryzae contained in the Aspergillus oryzae library is encapsulated using a droplet generation chip to generate and collect multiple droplets with a volume of 524~697pL.

[0015] (3) After the multiple droplets formed in step (2) are statically cultured at 28-32℃ for 7-8 hours, the glutamate fluorescent protein sensor is injected into each droplet using a droplet injection chip at 15-17℃ based on the droplet microfluidic cell sorting system and collected; and,

[0016] (4) After the glutamate fluorescent protein sensor is injected in step (3), the droplets react at 15~17℃ for 5~7h to generate a fluorescent signal. Based on the droplet microfluidic cell sorting system, the droplet sorting chip is used to sort the droplets according to the intensity of the fluorescent signal, and the Aspergillus oryzae corresponding to the 1‰ of the droplets with the lowest fluorescence signal intensity is selected.

[0017] In one embodiment, the method further includes:

[0018] (5) After demulsification of the droplets separated in step (4), glutaminase was induced and cultured using a microplate liquid flux culture method to screen for high-glutaminase-producing Aspergillus oryzae strains; and,

[0019] (6) The Aspergillus oryzae strains screened in step (5) are fermented using a device with a deep well plate and a solid-state flux culture method to screen for Aspergillus oryzae strains with strong resistance to contamination and high glutaminase production.

[0020] In one embodiment, in step (2), a spore suspension of the Aspergillus library is prepared by suspending and diluting the spores of the Aspergillus library in a microfluidic screening medium.

[0021] Optionally, the microfluidic screening medium comprises, by mass and volume percentage, the following components: 0.8-1.2% maltose syrup, 0.2-0.4% casein peptone, 0.8-1.2% glutamine, 2.7-3.3% potassium dihydrogen phosphate, 0.007-0.013% magnesium sulfate heptahydrate, 0.2-0.3% potassium chloride, and 4.5-5.5% glycerol.

[0022] In one embodiment, in step (2), droplets with a diameter of 50-55 μm and a volume of 524-697 pL are generated at a rate of 700-750 droplets / second.

[0023] In one embodiment, in step (3), the glutamate fluorescent protein sensor with a volume of 175-235 pL is injected into the droplet at a rate of 120-150 droplets / second.

[0024] In one embodiment, in step (4), the droplet sorting is performed at a rate of 130 to 160 droplets / s.

[0025] In one embodiment, step (5), the step of inducing glutaminase culture using a microplate liquid flux culture method to screen for high-glutaminase-producing Aspergillus oryzae strains, includes:

[0026] After droplet demulsification, a glutaminase-inducing medium was added to prepare a bacterial spore suspension, which was then inoculated into microplates for induction culture; and,

[0027] After induction culture, sterile physiological saline was added to each well, the supernatant was obtained, a glutamate fluorescent protein sensor was added, and fluorescence detection was performed after the reaction. Aspergillus oryzae strains that produce high levels of glutaminase were screened based on the fluorescence intensity.

[0028] In one embodiment, in step (5), perfluoro-1-octanol is added to the sorted droplets to perform the demulsification.

[0029] In one embodiment, the glutaminase induction medium comprises, by mass and volume percentage, the following components: 0.8-1.2% maltose syrup, 0.2-0.4% casein hydrolysate, 0.8-1.2% glutamine, 2.7-3.3% potassium dihydrogen phosphate, and 0.2-0.3% potassium chloride.

[0030] In one embodiment, the spore concentration in the bacterial spore suspension is 9-11 spores / mL, and 0.1-0.12 mL is inoculated into each well of a microplate and induced for 32-36 h at 28-32°C and 240-260 rpm.

[0031] In one embodiment, step (6), which involves using a device with a deep-well plate and a solid-state flux culture method for koji preparation and fermentation, and screening for Aspergillus oryzae strains with strong contamination resistance and high glutaminase production, includes:

[0032] The Aspergillus oryzae strain screened in step (5) was inoculated into a solid koji culture medium, and koji fermentation was carried out using a device containing a deep-well plate; wherein, the device containing the deep-well plate is a solid-state high-throughput culture device; and,

[0033] Aspergillus oryzae strains with strong resistance to contamination and high glutaminase production were screened by enzyme activity detection.

[0034] Optionally, the solid curing medium comprises soybeans, roasted wheat and water, wherein the mass ratio of soybeans to roasted wheat is 7:(2.8~3.2), and the water is 115~125% (w / w) of the sum of the masses of soybeans and roasted wheat.

[0035] Optionally, the solid-state flux culture device is used to carry out the fermentation of koji at a temperature of 28~32℃ for 40~44h.

[0036] Compared with traditional technologies, this application has the following advantages:

[0037] This application provides a method for germinating and culturing Aspergillus oryzae spores in 524-697 pL droplets based on droplet microfluidics. The droplets can be generated at a rate of 700-750 spores / s, injected at a rate of 120-150 spores / s, and sorted at a rate of 130-160 spores / s, thereby enabling high-throughput screening of fast-growing Aspergillus oryzae spores. Combined with microplate liquid-throughput culture and deep-well plate solid-throughput culture, this method enables high-throughput selection of Aspergillus oryzae strains with strong contamination resistance and high glutaminase activity, which can improve the success rate of industrial application of selected strains. Attached Figure Description

[0038] Figure 1 This shows the growth of Aspergillus oryzae spores in a droplet at different times. Detailed Implementation

[0039] To make the above-mentioned objectives, features, and advantages of this application more apparent and understandable, a detailed description of specific embodiments of this application is provided below. Many specific details are set forth in the following description to provide a thorough understanding of this application. However, this application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of this application. Therefore, this application is not limited to the specific embodiments disclosed below.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0041] The term “and / or” as used herein includes any and all combinations of one or more of the related listed items.

[0042] In this application, if the unit of a data range is only followed by the right endpoint, it indicates that the units of the left and right endpoints are the same. For example, 800~850nm means that the units of the left endpoint "800" and the right endpoint "850" are both nm (nanometers).

[0043] One embodiment of this application provides a high-throughput screening method for Aspergillus oryzae with fast growth rate and good contamination resistance. The germination and hyphal culture of Aspergillus oryzae spores are realized in picoliter-level droplets with a volume of 524~697 pL. Based on the fact that picoliter droplets can generate 700~750 spores / second, inject 120~150 spores / second, and sort 130~160 spores / second, the method can screen out fast-growing Aspergillus oryzae spores in a high throughput.

[0044] Specifically, the method includes the following steps (1) to (4):

[0045] (1) Preparation of Aspergillus oryzae library.

[0046] In one specific example, the method for preparing the Aspergillus oryzae library includes at least one of purchasing, mutagenesis, and natural sampling.

[0047] In a specific example, the method for preparing the Aspergillus oryzae library is mutagenesis. Specifically, the Aspergillus oryzae library is prepared by mutagenesis of the starting strain of Aspergillus oryzae.

[0048] Optionally, the mutation includes plasma mutation.

[0049] In one specific example, the strain spores were induced to mutate under the conditions of 120W power, 135~145s time, and 10SLM gas flow rate.

[0050] (2) Based on the droplet microfluidic cell sorting system, the spore suspension of Aspergillus oryzae contained in the Aspergillus oryzae library is encapsulated using a droplet generation chip to generate and collect multiple droplets with a volume of 524~697pL.

[0051] In a specific example, Aspergillus oryzae spores from the Aspergillus oryzae library were suspended and diluted using a microfluidic screening medium to prepare an Aspergillus oryzae spore suspension. The concentration of the Aspergillus oryzae spore suspension was calculated based on the λ value in the Poisson distribution.

[0052] In a specific example, droplets with a diameter of 50-55 μm and a volume of 524-697 pL are generated at a rate of 700-750 droplets / second.

[0053] In a specific example, the microfluidic screening medium, by mass and volume percentage, comprises the following components: 0.8–1.2% maltose syrup, 0.2–0.4% casein peptone, 0.8–1.2% glutamine, 2.7–3.3% potassium dihydrogen phosphate, 0.007–0.013% magnesium sulfate heptahydrate, 0.2–0.3% potassium chloride, and 4.5–5.5% glycerol, with a pH of 7.0–7.5.

[0054] In one specific example, the microfluidic screening medium, by mass and volume percentage, comprises the following components: 1% maltose syrup, 0.3% casein peptone, 1% glutamine, 3.0% potassium dihydrogen phosphate, 0.01% magnesium sulfate heptahydrate, 0.2% potassium chloride, and 5% glycerol, with a pH of 7.0.

[0055] In this application, the microfluidic screening medium contains 4.5-5.5% glycerol to solve the problem of spores easily settling in aqueous solution, so that spores can be evenly distributed in the medium during the generation process.

[0056] (3) After the multiple droplets formed in step (2) are incubated at 28~32℃ for 7~8h, the glutamate fluorescent protein sensor is injected into each droplet and collected using a droplet injection chip at 15~17℃ based on the droplet microfluidic cell sorting system.

[0057] This application reveals that *Aspergillus oryzae*, when cultured in a specific liquid medium, preferentially utilizes amino acids as a rapid nitrogen source for spore germination and mycelial growth. Rapid spore growth and germination lead to rapid amino acid consumption. Therefore, the rate of amino acid consumption can be used as an important indicator of the early growth rate of *Aspergillus oryzae* in specific cultures. Furthermore, it was found that glutamic acid, an amino acid playing a crucial role in microbial energy metabolism, is preferentially utilized during the aforementioned *Aspergillus oryzae* growth process. By measuring changes in amino acid content, strains with rapid growth rates can be screened.

[0058] For amino acid determination, amino acid analyzers and liquid chromatography are commonly used. These methods have long testing cycles, complex sample pretreatment, and high costs, making it difficult to evaluate the growth characteristics of strains on a large scale. For the determination of single amino acids, glutamic acid, which has a umami flavor, has been extensively studied. Common detection methods include the SBA-40X bioenzyme electrode method and reagent kit methods. While these methods require less sample volume, have simpler pretreatment, and higher throughput compared to amino acid analyzers and liquid chromatography, they still cannot meet the high-throughput requirements (>10) for strain analysis. 5 The screening process generates a large number of samples, requiring high throughput. Fluorescent biosensors, developed and widely used in recent years against the backdrop of rapid advancements in microfluidics, are suitable for high-throughput screening. They can specifically bind to the analyte to excite fluorescent groups, enabling high-throughput detection through highly sensitive fluorescence signal detectors.

[0059] The microfluidic screening medium contains glutamate (derived from casein peptone). During the mycelial growth process after Aspergillus oryzae spore germination, the glutamate in the medium is consumed first, causing a decrease in the glutamate content. Therefore, after the injection of the glutamate-based fluorescent protein sensor reagent, the sensor can bind to the glutamate, thereby exciting a fluorescent signal. The faster the growth of the Aspergillus oryzae strain, the lower the fluorescence signal intensity in the droplet. Thus, by reverse screening the droplets with the lowest fluorescence intensity (1‰ of the droplets containing bacteria), fast-growing Aspergillus oryzae strains can be screened.

[0060] In one specific example, a glutamate-based fluorescent protein sensor with a volume of 175-235 pL is injected into a droplet at a rate of 120-150 droplets / second.

[0061] (4) After the glutamate fluorescent protein sensor is injected in step (3), the droplets react at 15~17℃ for 5~7h to generate a fluorescent signal. Based on the droplet microfluidic cell sorting system, the droplet sorting chip is used to sort the droplets according to the intensity of the fluorescent signal, and the Aspergillus oryzae corresponding to the 1‰ of the droplets with the lowest fluorescence signal intensity is selected.

[0062] In a specific example, the droplet sorting is performed at a rate of 130-160 droplets / s.

[0063] Specifically, the laser power was set to 100mW, the gain voltage to 4.2~4.5V, the electric field strength to 60~64, the sorting time to 50~80μs, and the screening threshold was adjusted to screen out the 1‰ bacterial droplets with the lowest fluorescence signal intensity.

[0064] Glutamic acid is an important umami substance, and its level determines the umami flavor of traditional fermented foods such as soy sauce. In the fermentation of soy sauce and other traditional foods, glutamic acid is mainly formed by the breakdown of glutamine by glutaminase secreted by *Aspergillus oryzae* at a certain growth stage. Therefore, screening *Aspergillus oryzae* strains that produce high levels of glutaminase is an important means of enhancing the umami flavor of traditional fermented foods such as soy sauce. *Aspergillus oryzae* glutaminase secretion is only induced after a certain amount of free amino acids have been utilized in the culture system. Therefore, glutaminase induction occurs in the middle to late stages of *Aspergillus oryzae* growth.

[0065] Therefore, after screening for fast-growing strains in the microdroplet system, the selected strains are further induced to produce high-glutaminase using high-micro-system well plates to screen for high-glutaminase-producing Aspergillus oryzae strains. Then, the high-glutaminase-producing strains are further fermented in a solid-state microreactor, which can ultimately achieve the screening of Aspergillus oryzae with high contamination resistance and high glutaminase production.

[0066] An embodiment of this application also provides a high-throughput screening method for Aspergillus oryzae with strong anti-contamination ability and high glutaminase production, the method including the above steps (1) to (4), and further including steps (5) to (6):

[0067] (5) After the droplets separated in step (4) are demulsified, glutaminase is induced and cultured using microplate liquid flux culture method to screen out Aspergillus oryzae strains that produce high glutaminase.

[0068] In a specific example, the induction culture of glutaminase using a microplate liquid flux culture method to screen for high-glutaminase-producing Aspergillus oryzae strains includes the following steps a~b:

[0069] Step a: After the droplets break the emulsion, glutaminase induction medium is added to prepare a bacterial spore suspension, which is then inoculated into microplates for induction culture.

[0070] Specifically, the spore concentration in the bacterial spore suspension was 9-11 spores / mL, and 0.1 mL was inoculated into each well of a microplate and induced to grow at 28-32℃ and 240-260 rpm for 32-36 h.

[0071] Step b: After induction culture, add 50-60 μL of sterile physiological saline to each well, shake to mix, obtain the supernatant, add a glutamate fluorescent protein sensor, and detect the fluorescence signal after the reaction. Screen for Aspergillus oryzae strains that produce high levels of glutaminase based on the fluorescence signal intensity. After glutaminase is produced through induction culture, it decomposes glutamine to obtain glutamate; strains with relatively high fluorescence intensity are selected.

[0072] Specifically, the volume ratio of the supernatant to the glutamate fluorescent protein sensor is 1:(0.9-1.1).

[0073] Specifically, fluorescence detection is performed using an ELISA reader. More specifically, the method for fluorescence detection using an ELISA reader is as follows: set the excitation wavelength to 485 / 515nm and the emission wavelength to a gain of 50, and detect the fluorescence signal at room temperature.

[0074] Specifically, strains with fluorescence intensity more than 10% higher than the unmutated Aspergillus oryzae starting strain were selected.

[0075] Optionally, the unmutated Aspergillus oryzae originating strain is the unmutated Hu Niang 3.042 strain.

[0076] The preparation process for the spore suspension of the unmutated Aspergillus oryzae starting strain was as follows: Unmutated Aspergillus oryzae starting strain spores were dispersed and suspended in a microfluidic screening medium, and the concentration was adjusted to be consistent with the above-mentioned Aspergillus oryzae spore suspension. Except for the absence of microfluidic operation, the Aspergillus oryzae spores underwent the same culture temperature and culture time as the microfluidic-operated Aspergillus oryzae spores.

[0077] Specifically, perfluoro-1-octanol is added to the sorted droplets to demulsify them.

[0078] Specifically, the glutaminase induction medium, by mass and volume percentage, comprises the following components: 0.8–1.2% maltose syrup, 0.2–0.4% casein hydrolysate, 0.8–1.2% glutamine, 2.7–3.3% potassium dihydrogen phosphate, and 0.2–0.3% potassium chloride, with a pH of 7.0–7.5.

[0079] Specifically, the glutaminase induction medium, by mass and volume percentage, comprises the following components: 1% maltose syrup, 0.3% casein hydrolysate, 1% glutamine, 3% potassium dihydrogen phosphate, and 0.2% potassium chloride, with a pH of 7.5.

[0080] (6) The Aspergillus oryzae strains screened in step (5) were fermented using a device with a deep well plate and a solid-state flux culture method to screen for Aspergillus oryzae strains with strong resistance to contamination and high glutaminase production.

[0081] Specifically, the fermentation process for koji making, using a device with deep-well plates and a solid-state flux culture method, involves screening for Aspergillus oryzae with strong contamination resistance and high glutaminase production, including the following steps S1~S2:

[0082] Step S1: Inoculate the Aspergillus oryzae strain selected in step (5) into the solid koji culture medium and carry out koji fermentation using a solid-state throughput culture device (CN 107446804A).

[0083] Specifically, the solid curing medium comprises soybeans, roasted wheat and water, with a mass ratio of soybeans to roasted wheat of 7:(2.8~3.2), and the water is 115~125% (w / w) of the total mass of soybeans and roasted wheat.

[0084] Optionally, the solid curing medium comprises soybeans, roasted wheat and water, wherein the mass ratio of soybeans to roasted wheat is 7:3, and the water is 120% (w / w) of the total mass of soybeans and roasted wheat.

[0085] Specifically, the solid-state flux culture device is used to carry out the fermentation of koji at a temperature of 28~32℃ for 40~44h.

[0086] Step S2: Screening for Aspergillus oryzae strains with strong resistance to contamination and high glutaminase production by enzyme activity detection.

[0087] Specifically, the glutaminase activity of the koji obtained from solid-state fermentation was tested, and Aspergillus strains with glutaminase activity 20% higher than the unmutated Aspergillus oryzae starting strain were screened out by enzyme activity.

[0088] The high-throughput screening method for Aspergillus oryzae in this application first uses picosecond microfluidics to perform high-throughput screening for growth rate and contamination resistance in the early stage of Aspergillus oryzae hyphae growth, before they grow large enough to puncture droplets. Then, microplates are used to screen for high-yield characteristics of the product. This avoids the difficulty of Aspergillus oryzae hyphae requiring a large space for growth in microdroplets, which could puncture the droplets.

[0089] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0090] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0091] In this embodiment, the droplet microfluidic cell sorting system is the DREM Cell droplet microfluidic cell sorter from Wuxi Yuanqing Tianmu Biotechnology Co., Ltd. The droplet generation chip is the DREM Cell large droplet generation chip from Wuxi Yuanqing Tianmu Biotechnology Co., Ltd. The droplet injection chip is the DREM Cell large droplet injection chip from Wuxi Yuanqing Tianmu Biotechnology Co., Ltd. The droplet sorting chip is the DREM Cell large droplet sorting chip from Wuxi Yuanqing Tianmu Biotechnology Co., Ltd. The fluorescent protein sensor is the iGLuSnFR glutamate fluorescent protein sensor reagent from Wuxi Yuanqing Tianmu Biotechnology Co., Ltd., which specifically binds to glutamate to excite fluorescent groups.

[0092] Example 1: Changes in glutamate levels during the growth of Aspergillus oryzae in microfluidic screening medium

[0093] Spores were collected from the mature slant of Aspergillus oryzae strain Hu Niang 3.042 and added to sterile physiological saline to prepare a solution with a concentration of 10. 7 A spore suspension of 1 spore / ml was prepared. 1 mL of the spore suspension was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of microfluidic selection medium in a clean bench. The flask was then placed in a shaker at 30℃ and 200 rpm. For the first 10 hours of culture, samples were taken every 2 hours to measure the glutamate content in the medium. Subsequently, samples were taken at 24 h, 48 h, 72 h, 104 h, and 120 h to measure the glutamate content. Glutamate detection was performed using the Jinan Yanhe Biotechnology SBA-40X biosensor analyzer.

[0094] The microfluidic screening medium formulation (w / v) is: 1% maltose syrup, 0.3% casein peptone, 1% glutamine, 3% potassium dihydrogen phosphate, 0.01% magnesium sulfate heptahydrate, 0.2% potassium chloride, 5% glycerol, and the remainder is water, pH 7.0.

[0095] The results are shown in Table 1, indicating that in the microfluidic screening medium, Aspergillus oryzae preferentially utilizes amino acids (using glutamic acid as an indicator), and grows and germinates quickly while consuming amino acids rapidly.

[0096] Table 1. Changes in glutamate levels during the growth of Aspergillus oryzae in microfluidic screening medium.

[0097]

[0098] Example 2: Growth of Aspergillus oryzae spores in droplets

[0099] (1) Preparation of Aspergillus oryzae spore suspension:

[0100] Spores were collected from the mature slant of *Aspergillus oryzae* strain 3.042 and transferred to a microfluidic screening medium. The required spore suspension concentration was calculated based on the Poisson distribution λ = 0.3, and a concentration of 5.7 × 10⁻⁶ was prepared. 5 A spore suspension of 1 spore per mL.

[0101] (2) Droplet formation:

[0102] Using a droplet microfluidic cell sorting system and a droplet generation chip, channel 1 is connected to droplet generation oil and channel 2 is connected to spore suspension. The pressure of channel 1 is set to 200 and the pressure of channel 2 is set to 250 on the control software. Droplets with a diameter of 50 μm and a volume of 524 pL are generated at a rate of 750 droplets / second and collected in a Teflon tube with an inner diameter of 0.56 mm.

[0103] The microfluidic screening medium formulation (w / v) is: 1% maltose syrup, 0.3% casein peptone, 1% glutamine, 3% potassium dihydrogen phosphate, 0.01% magnesium sulfate heptahydrate, 0.2% potassium chloride, 5% glycerol, and the remainder is water, pH 7.0.

[0104] (3) Droplet observation and spore growth record:

[0105] The collected droplets were incubated statically in a 30°C incubator. At different time points, a suitable amount of droplets was poured from the Teflon collection tube into a Nexcelom Cellometer SD100 cell counting chamber. The cells were observed and their lengths (including spore and hyphal lengths, measured as the longest observed cell length) were determined under an optical microscope. The results are shown in the figure. Figure 1 As shown in Table 2, the results indicate that when the culture time exceeds 8 hours, some hyphae have grown to a considerable length and can puncture the droplets. Therefore, in this application, the culture time is selected as 7-8 hours to screen for growth rates in the early stage of hyphae growth before they grow long enough to puncture the droplets, thereby increasing throughput.

[0106] Table 2. Growth of Aspergillus oryzae in droplets at different times.

[0107]

[0108] Example 3: Picoliter-level droplet microfluidic screening model experiment for Aspergillus oryzae strains with strong contamination resistance

[0109] Two Aspergillus oryzae strains, Hu Niang 3.042 and ZA195 (proposed taxonomic name Aspergillus oryzae was deposited on May 18, 2021, at the Guangdong Provincial Microbial Culture Collection Center, accession number GDMCC NO: 61675, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou), with different known growth rates, contamination resistance, and colony morphologies, were used in a model experiment to screen for Aspergillus oryzae strains with strong contamination resistance using microfluidic droplet chromatography. ZA195 showed a faster growth rate and stronger contamination resistance than Hu Niang 3.042.

[0110] (1) Preparation of mixed spore suspension of Aspergillus oryzae strains:

[0111] Spores were collected from mature slant cultures of *Aspergillus oryzae* strains Hu Niang 3.042 and ZA195 and transferred to microfluidic screening medium. The required spore suspension concentration was calculated based on Poisson distribution λ = 0.3, and the concentration prepared was 5.7 × 10⁻⁶. 5 A spore suspension of 1 spore per mL was prepared. Then, equal volumes of the spore suspensions from the two strains were mixed to prepare a mixed spore suspension.

[0112] The microfluidic screening medium formulation (w / v) is: 1% maltose syrup, 0.3% casein peptone, 1% glutamine, 3% potassium dihydrogen phosphate, 0.01% magnesium sulfate heptahydrate, 0.2% potassium chloride, 5% glycerol, and the remainder is water, pH 7.0.

[0113] (2) Droplet formation:

[0114] Using a droplet microfluidic cell sorting system and a droplet generation chip, channel 1 is connected to droplet generation oil and channel 2 is connected to spore suspension. The pressure of channel 1 is set to 200 and the pressure of channel 2 is set to 250 on the control software. Droplets with a diameter of 50 μm and a volume of 524 pL are generated at a rate of 750 droplets / second and collected in a Teflon tube with an inner diameter of 0.56 mm.

[0115] (3) Injection of the fluorescent protein sensor:

[0116] After being incubated at 30°C for 7 hours, the generated droplets were cooled to 4°C for 30 minutes to slow spore growth. The room temperature was set to 15-17°C. Using a droplet microfluidic cell sorting system and a droplet injection chip, channel 1 was connected to the droplet generation oil, channel 2 to the generated droplets after static incubation, and channel 3 to 4°C pre-cooled iGLuSnFR glutamate fluorescent protein sensor reagent. The control software was set to pressure 200 for channel 1, 245 for channel 2, and 200 for channel 3, injecting 175 pL of glutamate fluorescent protein sensor reagent into each droplet at a rate of 150 droplets / second. The injected droplets were collected in Teflon tubing with an inner diameter of 0.56 mm.

[0117] (4) Droplet sorting

[0118] Droplets injected with the fluorescent protein sensor were allowed to stand at 15-17°C for 6 hours. With the room temperature set at 15-17°C, a droplet microfluidic cell sorting system and a droplet sorting chip were used. Channel 1 was connected to the droplet sorting oil, and channel 2 was connected to the droplets injected with the fluorescent protein sensor and allowed to stand. The pressure in channel 1 was adjusted to 225, and the pressure in channel 2 to 150, with a sorting rate of 150 droplets / second. The laser power was set to 100mW, the gain voltage to 4.25V, the electric field strength to 60, the sorting time to 50μs, and the screening threshold to 2.8, selecting the 1‰ of bacterial droplets with the lowest fluorescence intensity. The sorted droplets were collected from the channel outlet furthest from the electrode into centrifuge tubes, while the channel outlet closer to the electrode was connected to a waste pipe.

[0119] (5) Plate culture verification

[0120] Inside a clean bench, an equal volume of perfluoro-1-octanol was added to the sorted droplets, and the mixture was shaken for 2 minutes to break the emulsion. The emulsion was then diluted with sterile saline to a bacterial concentration of 100 cells / mL. The diluted bacterial suspension was then spread onto PDA plates, with 0.1 mL of the suspension spread on each plate.

[0121] The formulation of the PDA plate is (w / v): 30% potato extract powder, 2% glucose, 1.5% agar, 0.01% chloramphenicol, and the remainder is water, with natural pH.

[0122] The coated PDA plates were placed in a 32℃ incubator and incubated for 48 hours. Strains were identified and counted based on colony morphology. 87.4% of the colonies exhibited ZA195 morphology, demonstrating that the epidermal-upgraded droplet microfluidic screening method achieved a high screening rate for highly resistant Aspergillus oryzae strains.

[0123] Example 4: Picoliter-level droplet microfluidic screening for fast-growing mutant strains

[0124] (1) Construction of a mutant library by mutagenesis of Aspergillus oryzae spores:

[0125] Spores were collected from the mature slant of Aspergillus oryzae strain Hu Niang 3.042 and added to sterile physiological saline to prepare a solution with a concentration of 10. 7 A spore suspension of 10 μL / mL was prepared. The spore suspension was spread onto a slide, and mutagenesis was induced using an ARTP-II mutagenizer with the following conditions: power 120 W, time 140 s, gas flow rate 10 SLM. After mutagenesis, the spores on the slide were washed with 1 ml of microfluidic selection medium to obtain a mutant library.

[0126] The sterile saline solution was a 0.9% (w / v) sodium chloride solution with 0.01% (w / v) Tween 80 added.

[0127] The microfluidic screening medium formulation (w / v) is: 1% maltose syrup, 0.3% casein peptone, 1% glutamine, 3% potassium dihydrogen phosphate, 0.01% magnesium sulfate heptahydrate, 0.2% potassium chloride, 5% glycerol, and the remainder is water, pH 7.0.

[0128] (2) Droplet formation:

[0129] Based on the mutant library, the required spore suspension concentration was calculated according to the Poisson distribution λ=0.3, and the spore suspension was diluted to 4.3×10⁻⁶ using microfluidic screening medium. 5 The droplet microfluidic cell sorting system and droplet generation chip were used. Channel 1 was connected to droplet generation oil, and channel 2 was connected to spore suspension. The pressure was set to 200 for channel 1 and 280 for channel 2 on the control software. Droplets with a diameter of 55 μm and a volume of 697 pL were generated at a rate of 700 droplets / second and collected in a Teflon tube with an inner diameter of 0.56 mm. Droplet generation continued for 1 hour, and a total of 2.52 × 10⁻⁶ droplets were collected. 6 A droplet.

[0130] (3) Injection of the fluorescent protein sensor:

[0131] After being incubated at 30°C for 7 hours, the generated droplets were cooled to 4°C for 30 minutes to slow spore growth. The room temperature was set to 15-17°C. Using a droplet microfluidic cell sorting system and a droplet injection chip, channel 1 was connected to the droplet generation oil, channel 2 to the statically incubated droplets, and channel 3 to pre-cooled iGLuSnFR glutamate fluorescent protein sensor reagent at 4°C. The control software was set to pressures of 180 for channel 1, 220 for channel 2, and 190 for channel 3, injecting 235 pL of glutamate fluorescent protein sensor reagent into each droplet at a rate of 125 droplets / second. The injected droplets were collected in Teflon tubing with an inner diameter of 0.56 mm. The total injection time for all droplets was 5.6 hours.

[0132] (4) Droplet sorting:

[0133] Since the reaction time was calculated from the moment the droplets were injected, the droplets were allowed to stand at 15-17°C for 0.5 hours after injection. The first droplets injected had completed the reaction and were ready for sorting. The room temperature was set to 15-17°C. Using a droplet microfluidic cell sorting system and a droplet sorting chip, channel 1 was connected to the droplet sorting oil, and channel 2 was connected to droplets that had reacted for 6 hours after being injected with the fluorescent protein sensor. The pressure in channel 1 was adjusted to 230, and the pressure in channel 2 to 150, with a sorting rate of 130 droplets / second. The laser power was set to 100mW, the gain voltage to 4.2V, the electric field strength to 62, the sorting time to 80μs, and the screening threshold to 3.1, selecting the 1‰ of bacterial droplets with the lowest fluorescence intensity. The sorted droplets were collected from the channel outlet furthest from the electrode into centrifuge tubes, while the channel outlet closest to the electrode was connected to a waste tube. The total sorting time for all droplets was 5.4 hours.

[0134] Example 5: Preliminary screening of high-glutaminase-producing Aspergillus oryzae mutant strains using microplate liquid flux culture

[0135] (1) Dilution and dispensing of droplets into microplates after demulsification:

[0136] In a clean bench, an equal volume of perfluoro-1-octanol was added to the 1‰ fluorescent droplets collected after droplet sorting in Example 4, and the mixture was shaken for 2 min to break the emulsion. Then, an appropriate amount of glutaminase induction medium was added to dilute the spore suspension to a cell concentration of 10c CFU / mL. All diluted spore suspensions were then aliquoted into sterile 96-well plates, 0.1 mL per well. Additionally, 0.1 mL of unmutated *Hu Niang* 3.042 spore suspension at a cell concentration of 10c CFU / mL was added to one well of each plate. A total of 665 wells were aliquoted into 7 96-well plates after droplet demulsification. After sealing with a film, the plates were incubated in a shaker at 30°C and 250 rpm for 36 h.

[0137] Obtaining the Huniang 3.042 spore suspension: Unmutated Huniang 3.042 spores were dispersed and suspended in a microfluidic screening medium, and the concentration was adjusted to be consistent with that of the mutant library spore suspension. Except for not performing microfluidic operations, the spores underwent the same culture temperature and culture time as the mutant library spores that underwent microfluidic operations.

[0138] The glutaminase induction medium formula is (w / v): 1% maltose syrup, 0.3% casein hydrolysate, 1% glutamine, 3% potassium dihydrogen phosphate, 0.2% potassium chloride, and the remainder is water, pH 7.5.

[0139] (2) Detection of glutamic acid in Aspergillus oryzae culture medium:

[0140] In a clean bench, using a pipette, add 50 μL of sterile physiological saline to each well of a 96-well plate after the culture in step (1). After shaking to mix, carefully aspirate 50 μL of mycelium-free supernatant culture medium from each well into a new black 96-well plate. Add 50 μL of iGLuSnFR glutamate fluorescent protein sensor reagent to each well, gently shake to mix, and incubate at room temperature in the dark for 2 hours. Subsequently, fluorescence detection is performed on a microplate reader. The excitation and emission wavelengths are set to 485 / 515 nm, with a gain of 50. Fluorescence signal detection is performed at room temperature. After detection, 43 wells with fluorescence signal intensity more than 10% higher than that of the unmutated Hu Niang 3.042 were selected.

[0141] Example 6: Deep-well plate solid-state flux culture and secondary screening

[0142] In Example 5, 43 wells with fluorescence signal intensity more than 10% higher than that of the unmutated Hu Niang 3.042 were selected. Mycelial balls were picked from the corresponding Aspergillus oryzae culture wells in a clean bench and inoculated into solid culture medium. Solid fermentation was carried out using the device described in the patent "A Solid-State Throughput Culture Device and Its Application" (CN 107446804A) in accordance with Example 2. One mycelial ball was inoculated into each well. In addition, each set of devices had one well for inoculating the mycelial balls of the unmutated Aspergillus oryzae starting strain. The entire culture device was placed in a constant temperature environment of 30°C for 44 hours.

[0143] The glutaminase activity of the koji obtained from solid-state culture was detected. The method for detecting glutaminase activity in the koji was based on the reference "Optimization of Glutaminase Activity Determination Conditions in Soy Sauce Koji" (Zou Minjuan, 2013). Four Aspergillus oryzae mutant strains with glutaminase activity more than 20% higher than that of Aspergillus oryzae Hu Niang 3.042 were screened by enzyme activity, as shown in Table 3.

[0144] Table 3. The percentage increase in glutaminase activity of the four Aspergillus oryzae mutant strains compared to Hu Niang 3.042.

[0145]

[0146] Example 7: Validation of contamination resistance of the screened Aspergillus oryzae mutant strains

[0147] (1) Preparation of despore-forming filtrate:

[0148] Take 50g of soy sauce koji produced at maturity, add 100g of sterile distilled water, place it in a horizontal shaker and shake for 10 minutes. After removing it, filter the insoluble matter with a sterile filter cloth. Filter the filtrate with a microfiltration membrane with a pore size of 2μm to remove Aspergillus oryzae spores while retaining turbidity-related microorganisms.

[0149] (2) Mixed coating plate:

[0150] Four Aspergillus oryzae strains (A-D3, C-H6, D-F2, F-G1, and Hu Niang 3.042) screened from deep-well plate solid-state flux culture in Example 6 were activated by inoculation onto slant plates. Spores were then collected from the slant plates and suspended in sterile physiological saline to prepare a concentration of approximately 1×10⁻⁶. 3 A spore suspension of 1 spore / mL was prepared, and then mixed with the despore-free koji filtrate at a volume ratio of 1:9. After shaking well, 0.1 mL of the mixture was spread onto koji culture medium plates. After spreading, the plates were incubated at 30°C for 48 h, and the diameter of Aspergillus oryzae colonies was measured. The results are shown in Table 4. The four Aspergillus oryzae mutant strains screened showed stronger resistance to contamination than Hu Niang 3.042.

[0151] The preparation method of the koji culture medium is as follows: Weigh 25 parts of the koji produced at the end of soy sauce production, add 225 parts of sterile water and shake evenly, let stand in a 4°C refrigerator for 30 min for extraction, then filter with sterile filter cloth to obtain filtrate, and filter with a 0.22 μm needle filter to obtain sterile koji clear liquid, add 1.5% agar powder and boil to dissolve, then pour into sterile petri dishes and let cool naturally for later use.

[0152] Table 4. Comparison of mycelial growth between the starting strain and the screened Aspergillus oryzae mutant strain co-cultured with other microorganisms.

[0153]

[0154] Example 8: Validation of the quality of koji-making and fermented crude oil from the screened Aspergillus oryzae mutant strain.

[0155] Four Aspergillus oryzae mutant strains (A-D3, C-H6, D-F2, and F-G1) screened from deep-well plate solid-state flux culture in Example 6 were activated by transfer to slant culture and then expanded into Erlenmeyer flasks. After that, small-scale koji making and fermentation were carried out. (The Erlenmeyer flask culture medium was prepared from wheat, defatted soybeans, and other raw materials, and the koji making culture medium was prepared from soybeans, wheat, and other raw materials. The raw material ratio, watering, cooking, and fermentation parameters were all based on the published book "Soy Sauce Science and Brewing Technology".)

[0156] The pH value, neutral protease, and glutaminase levels in the fermented koji were measured, and the results are shown in Table 5. The total acid, amino nitrogen, glutamic acid, and reducing sugar levels in the fermented crude oil were also measured, and the results are shown in Table 6. The method for detecting glutaminase activity in the koji material was based on the reference "Optimization of Glutaminase Activity Determination Conditions in Soy Sauce Koji" (Zou Minjuan, 2013), and the methods for detecting other physicochemical indicators were based on the reference "ARTP Mutagenesis Technology for Breeding High-Glutaminase-Producing Aspergillus Strains" (Zhou Qiyang et al., 2019).

[0157] The results showed that the glutaminase activity of the four Aspergillus oryzae mutant strains A-D3, C-H6, D-F2, and F-G1 in the fermentation koji was higher than that of the original strain Hu Niang 3.042, and the glutamic acid content of the fermented crude oil was also higher than that of the original strain Hu Niang 3.042.

[0158] Table 5. Koji-making indicators for starting strains and screened Aspergillus oryzae mutants

[0159]

[0160] Note: Relative activity is based on the enzyme activity of the starting strain Hu Niang 3.042 (1.0), and the enzyme activity of other strains is their corresponding multiple.

[0161] Table 6. Indicators of crude oil fermented from starting strains and screened Aspergillus oryzae mutants.

[0162]

[0163] Example 9: Comparison of the effectiveness of the high-throughput screening method for Aspergillus oryzae of this application with other screening methods applied to filamentous fungi.

[0164] Table 7 compares the effectiveness of the high-throughput screening method for Aspergillus oryzae described in this application with other screening methods applied to filamentous fungi.

[0165] Table 7 compares the effectiveness of the high-throughput screening method for Aspergillus oryzae described in this application with other screening methods applied to filamentous fungi.

[0166]

[0167] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0168] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A high-throughput screening method for Aspergillus oryzae, characterized in that, The method includes the following steps: (1) Preparation of Aspergillus oryzae library; (2) Based on the droplet microfluidic cell sorting system, the spore suspension of Aspergillus oryzae contained in the Aspergillus oryzae library is encapsulated using a droplet generation chip to generate and collect multiple droplets with a volume of 524 to 697 pL. (3) After the multiple droplets formed in step (2) are statically cultured at 28–32°C for 7–8 h, the glutamate fluorescent protein sensor is injected into each droplet using a droplet injection chip at 15–17°C and collected, based on a droplet microfluidic cell sorting system; and... (4) The droplets injected with the glutamate fluorescent protein sensor in step (3) react at 15-17℃ for 5-7 hours to generate a fluorescent signal. Based on the droplet microfluidic cell sorting system, the droplet sorting chip is used to sort the droplets according to the intensity of the fluorescent signal, and the Aspergillus oryzae corresponding to the 1‰ of the droplets with the lowest fluorescence signal intensity is selected. In step (2), the spores of the Aspergillus oryzae library are suspended and diluted in a microfluidic screening medium to prepare a spore suspension of the Aspergillus oryzae library; The microfluidic screening medium comprises, by mass and volume percentage, the following components: 0.8–1.2% maltose syrup, 0.2–0.4% casein peptone, 0.8–1.2% glutamine, 2.7–3.3% potassium dihydrogen phosphate, 0.007–0.013% magnesium sulfate heptahydrate, 0.2–0.3% potassium chloride, and 4.5–5.5% glycerol.

2. The method according to claim 1, characterized in that, The method further includes: (5) After demulsification of the droplets separated in step (4), glutaminase was induced and cultured using a microplate liquid flux culture method to screen for high-glutaminase-producing Aspergillus oryzae strains; and, (6) The Aspergillus oryzae strains screened in step (5) are fermented using a device with a deep well plate and a solid-state flux culture method to screen for Aspergillus oryzae strains with strong resistance to contamination and high glutaminase production.

3. The method according to claim 1 or 2, characterized in that, In step (2), droplets with a diameter of 50-55 μm and a volume of 524-697 pL are generated at a rate of 700-750 droplets / second.

4. The method according to claim 1 or 2, characterized in that, In step (3), the glutamate fluorescent protein sensor with a volume of 175-235 pL is injected into the droplet at a rate of 120-150 droplets / second.

5. The method according to claim 1 or 2, characterized in that, In step (4), the droplet sorting is performed at a rate of 130 to 160 droplets / s.

6. The method according to claim 2, characterized in that, In step (5), the process of inducing glutaminase production using a microplate liquid flux culture method and screening for high-glutaminase-producing Aspergillus oryzae strains includes: After droplet demulsification, a glutaminase-inducing medium was added to prepare a bacterial spore suspension, which was then inoculated into microplates for induction culture; and, After induction culture, sterile physiological saline was added to each well, the supernatant was obtained, a glutamate fluorescent protein sensor was added, and fluorescence detection was performed after the reaction. Aspergillus oryzae strains that produce high levels of glutaminase were screened based on the fluorescence intensity.

7. The method according to claim 2 or 6, characterized in that, In step (5), perfluoro-1-octanol is added to the sorted droplets to perform the demulsification.

8. The method according to claim 6, characterized in that, The glutaminase induction medium comprises, by mass and volume percentage, the following components: 0.8–1.2% maltose syrup, 0.2–0.4% casein hydrolysate, 0.8–1.2% glutamine, 2.7–3.3% potassium dihydrogen phosphate, and 0.2–0.3% potassium chloride. The spore concentration in the bacterial spore suspension is 9–11 spores / mL. 0.1–0.12 mL is inoculated into each well of a microplate and induced for 32–36 h at 28–32 °C and 240–260 rpm.

9. The method according to claim 2, characterized in that, In step (6), the process of using a device with a deep-well plate and a solid-state flux culture method for koji making and fermentation, and screening for Aspergillus oryzae strains with strong contamination resistance and high glutaminase production includes: The Aspergillus oryzae strain screened in step (5) was inoculated into a solid koji culture medium, and koji fermentation was carried out using a device containing a deep-well plate; wherein, the device containing the deep-well plate is a solid-state high-throughput culture device; and, Aspergillus oryzae strains with strong resistance to contamination and high glutaminase production were screened by enzyme activity assay.

10. The method as described in claim 9, characterized in that, The solid curing medium comprises soybeans, roasted wheat and water, wherein the mass ratio of soybeans to roasted wheat is 7:(2.8-3.2), and the water is 115-125% (w / w) of the sum of the masses of soybeans and roasted wheat.

11. The method as described in claim 9, characterized in that, The koji-making fermentation was carried out in a solid-state flux culture device at a temperature of 28–32°C for 40–44 hours.

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