High-throughput screening method for lactic acid bacteria by droplet microfluidic coupling lactic acid oxidase fluorescence detection

Abstract

The application belongs to the technical field of microorganism screening, and specifically provides a high-throughput screening method for strains by droplet microfluidic coupling lactic acid oxidase fluorescence detection. The high-throughput screening method for high-yield lactic acid strains of Streptococcus thermophilus based on droplet microfluidic coupling lactic acid oxidase fluorescence detection specifically comprises the following steps: 1) construction of a droplet microfluidic screening system; 2) establishment of a droplet lactic acid content detection system; and 3) screening of high-yield lactic acid strains of Streptococcus thermophilus. The method can be used to directionally screen high-yield lactic acid strains of Streptococcus thermophilus, and has the characteristics of efficient and specific screening, and has important application value in the preparation of fermented milk.

Description

Technical Field

[0001] This invention belongs to the field of microbial screening technology, and specifically relates to a high-throughput screening method for strains using droplet microfluidic coupling with lactate oxidase fluorescence detection. Background Technology

[0002] In the dairy, probiotic, and fermented food industries, *Streptococcus salivarius* subsp. *thermophilus* (… Streptococcus salivarius subsp. thermophilus Lactic acid (LSA) is a crucial core strain in the fermentation agent, and its acid-producing capacity directly determines fermentation efficiency, product flavor, texture, and shelf life. Therefore, rapidly and efficiently screening for superior LSA-producing strains is a key step in the industry's continuous optimization and cost reduction. Currently, industrial and laboratory screening of high-acid-producing strains mainly relies on traditional methods, including plate titration, direct measurement of fermentation broth acidity with a pH meter, and indirect or direct quantification using microplate-based spectrophotometry. While these methods are widely used, they have inherent and insurmountable limitations. First, their screening throughput is extremely low, relying on extensive manual operations and discrete sample processing, making it difficult to handle massive mutant libraries constructed through mutagenesis or engineering. Second, the process is time-consuming, typically requiring several days of cultivation to obtain detectable colonies or sufficient fermentation broth, resulting in a long screening cycle. A more fundamental drawback is that these methods cannot perform early, non-destructive phenotypic identification at the single-clonal level. Traditional screening, based on colony or population cultures, is easily affected by low-acid-producing individuals in the population, resulting in "high-yielding strains" that are actually mixed populations with questionable genetic purity and stability. Furthermore, conventional pH testing is susceptible to interference from other metabolic organic acids, lacking specificity; while some colorimetric methods may have sensitivity limitations, making it difficult to detect trace amounts of lactic acid secreted by microorganisms in the early stages of growth, thus failing to achieve dynamic, early productivity assessment.

[0003] In recent years, droplet microfluidics has brought revolutionary prospects to high-throughput screening of microorganisms, enabling the encapsulation of individual microorganisms in microdroplets for isolated culture and analysis at the single-cell level. However, existing technologies mostly focus on screening based on fluorescent protein reporter genes or universal metabolic indicators, lacking direct and highly sensitive quantitative methods for lactate, a specific terminal small molecule metabolite. Deeply integrating the high-throughput and single-cell advantages of microfluidics with specific and sensitive detection technologies for lactate to construct an integrated screening platform remains a pressing technical challenge in this field. Summary of the Invention

[0004] This invention aims to provide a high-throughput screening method for high-lactic acid-producing strains of Streptococcus salivarius subsp. thermophilus based on droplet microfluidics and lactate oxidase coupled fluorescence detection. Its core technology lies in constructing a complete technical closed loop that integrates a droplet microfluidic screening system, a highly specific and sensitive fluorescence detection system, and intelligent signal analysis and sorting logic, thereby achieving efficient, accurate, and automated screening of high-lactic acid-producing strains of Streptococcus salivarius subsp. thermophilus.

[0005] The objective of this invention is achieved through the following technical solution: In a first aspect, the present invention provides a high-throughput screening method for high lactic acid-producing strains of Streptococcus salivarius subsp. thermophilus based on droplet microfluidics and lactate oxidase coupled fluorescence detection, characterized by comprising the following steps: (1) construction of a droplet microfluidic screening system; (2) establishment of a lactate oxidase coupled fluorescence detection system; and (3) high-throughput screening and collection of high lactic acid-producing strains.

[0006] Further, in step (1), a mixed system containing a mixed sample of Streptococcus salivarius thermophilus subsp., M17 lactose medium, F1 stabilizer (fluorocarbon surfactant, 10%) and AF405 fluorescent reagent is used to prepare microdroplets using a droplet generator, and the single-strain encapsulation rate λ is controlled to be 0.05 to 0.2.

[0007] Furthermore, the microdroplets prepared in step (1) have a particle size of 40-60 μm. The droplets are placed in an incubator at 40-45°C for static incubation, with a relative humidity of 40%-60% maintained in the incubator, and the incubation time is controlled at 20-30 h.

[0008] Furthermore, in step (2), a droplet fusion instrument is used to pair and fuse the cultured bacterial droplets with the detection reagent droplets in a microfluidic channel to construct an isolated two-stage enzymatic reaction system.

[0009] Furthermore, the detection reagent droplets contain Amplex Red, lactate oxidase (LOX), horseradish peroxidase (HRP), F1 stabilizer, and Tris-HCl buffer.

[0010] Furthermore, the concentrations of each component in the test reagent are as follows: Amplex Red 40–350 μM, lactate oxidase (LOX) 8–120 mg / L, horseradish peroxidase (HRP) 0.3–6 U / mL, fluorocarbon surfactant F1 stabilizer 0.05–0.6%, and incubation time in the dark 0.5–6 h.

[0011] Furthermore, the optimal detection reagent composition is 200 μM Amplex Red, 50 mg / L LOX, 3 U / mL HRP and 0.5% F1 stabilizer. After the reaction, the fluorescence signal is detected through the PE fluorescence channel (excitation wavelength 480-495 nm, emission wavelength 570-590 nm).

[0012] Further, in step (3), the PE fluorescence intensity of the fused droplets is detected by flow cytometry or droplet sorting instrument. Multi-dimensional sorting parameters such as fluorescence intensity threshold, droplet diameter and CV value are set. High fluorescence positive droplets with the highest fluorescence intensity of 0.1% to 0.3% are selected for precise sorting and collected into a 96-well plate, with each well corresponding to a single reaction unit. Preferably, the droplet diameter range is 40 to 60 μm and the CV value is <6%.

[0013] Furthermore, the method also includes a step of validating the strains obtained by sorting: quantitatively detecting lactic acid production using an aminoantipyrine-vanillic acid colorimetric system or high performance liquid chromatography, and inoculating the screened strains into milk base to verify the acid production capacity of fermented milk.

[0014] Furthermore, the verification step includes: (4) Screen the high lactic acid-producing Streptococcus thermophilus subsp. strains from step (3), verify the lactic acid produced by the aminoantipyrine and vanillic acid detection system, and test the fermented milk of the selected Streptococcus thermophilus subsp. strains to verify the lactic acid production capacity of Streptococcus thermophilus subsp. strains.

[0015] Furthermore, the steps for verifying the generated lactic acid using the aminoantipyrine and vanillic acid detection system are as follows: 1) The strains obtained in step (3) were inoculated into M17-lactose liquid medium at 2% (v / v) and cultured at 42 ±1℃ for 12 h. The supernatant was removed and the sample was filtered through a 0.22 μm filter to obtain the test sample. 2) A mixed detection system was formed using 0.2 U / mL peroxidase, 0.5 mM 4-aminoantipyrine, and 2 mM vanillic acid; 3) Add 100 μL of the sample to be tested to the reaction system, mix immediately, and incubate in a 37℃ water bath for 10–15 min to oxidize lactic acid to generate H2O2 and catalyze the colorimetric reaction; 4) Measure the absorbance at 505 nm using a spectrophotometer, with the reaction solution without sample as a blank control; 5) Calculate the actual concentration of lactic acid in the sample based on the pre-established standard curve (known lactic acid concentration vs. absorbance / fluorescence intensity).

[0016] Furthermore, the sorted *Streptococcus salivarius* subsp. *thermophilus* strains were tested in fermented milk to clarify the steps involved in lactic acid production: 1) Heat pure milk to 90~100℃ and maintain for 5 minutes to obtain milk base; 2) The strains obtained in step (3) were inoculated into M17-lactose liquid medium at 2% (v / v) and cultured at 42 ±1℃ for 12 h to complete the activation of the strains; 3) The activated strains were serially diluted 10 times according to the national standard "Food Safety Standard - Microbiological Examination of Food - Lactic Acid Bacteria Examination" (GB / 4789.35-2016), and cultured at 42 ± 1℃ for 36-48 h. The number of microorganisms in the fermentation broth was measured to determine the number of strains in the stationary phase, and the amount added to the milk base was calculated. 4) According to 1×10 7 CFU / mL was added to the milk base, and fermentation was carried out to obtain the fermentation substrate; 5) The lactic acid content in the fermentation substrate was detected using the external standard method.

[0017] Furthermore, the final concentration of the F1 stabilizer in the screening system is 0.1% to 0.6%, and the final concentration of the AF405 fluorescent reagent in the screening system is 400 to 800 nM.

[0018] Furthermore, the droplet generation chip is fabricated using a plastic injection molding process. After droplet generation, it is first incubated at a constant temperature of 42℃ for 25h±2h, and then droplet fusion and detection are performed.

[0019] Secondly, the application of the high-throughput screening method for high lactic acid-producing strains of Streptococcus salivarius based on droplet microfluidics and lactate oxidase coupled fluorescence detection described in the first aspect in screening high lactic acid-producing Streptococcus salivarius strains.

[0020] Furthermore, the method can also serve as a general high-throughput metabolic screening platform, and can be extended to the screening of high-producing organic acid strains of other acid-producing microorganisms; preferably, the microorganisms include lactic acid bacteria, yeast, and bifidobacteria.

[0021] Thirdly, the method described in the first aspect is provided for use in the preparation of high-yield strains of Streptococcus salivarius thermophilus or fermented dairy products.

[0022] Fourthly, the method described in the first aspect is provided for application in constructing a high-throughput metabolic phenotype screening platform for microorganisms or optimizing the breeding process of fermentation strains.

[0023] Fifthly, a droplet microfluidic screening system is provided, the system comprising: (1) Droplet generation module: The screening system was constructed by mixing a mixed Streptococcus salivarius thermophilic subspecies YH, M17 lactose medium, F1 stabilizer (fluorocarbon surfactant, 10%) and AF405 fluorescent reagent, and then preparing 50 ± 2 μm water-in-oil microdroplets using a droplet generator to control the encapsulation of a single bacterial cell in the droplet, i.e., the encapsulation rate λ=0.1.

[0024] (2) Droplet generation and incubation module: After the droplets are generated in the chip, they are incubated at a constant temperature of 42 ℃ for 25 h (pure water is added to the incubator to maintain a relative humidity of 50% to complete the process of bacterial growth and lactic acid secretion).

[0025] (3) Droplet fusion and detection module: In the detection channel, the droplet containing the target strain is precisely fused with the droplet pre-embedded with fluorescent detection reagents (including lactate oxidase, horseradish peroxidase, fluorescent substrate, etc.) by the droplet fusion instrument to realize the in-situ reaction initiation.

[0026] (4) Sorting and collection module: Combined with flow cytometer or sorter, high-yield lactic acid droplets are identified and physically separated in real time according to preset fluorescence intensity threshold and droplet size parameters, and collected into 96-well plate for subsequent verification and preservation.

[0027] Sixthly, a lactate oxidase-coupled fluorescence detection system is provided: the core of this system is a fluorescence reaction based on enzyme cascade amplification. In microdroplets embedding single *Streptococcus salivarius* subspecies *Thermophilus* cells, we introduced a coupled enzyme system composed of lactate oxidase and horseradish peroxidase, and added a fluorescent substrate. Its working principle is as follows: Lactate oxidation: Under the catalysis of lactate oxidase (LOX), L-lactate secreted by bacteria is oxidized by molecular oxygen (O2) to produce pyruvate and hydrogen peroxide (H2O2). This reaction provides the key substrate H2O2 for subsequent fluorescence generation. The reaction formula is as follows:

[0028] Fluorescence generation reaction: Horseradish peroxidase (HRP) uses H₂O₂ generated in the first step of the reaction as an oxidant to catalyze the oxidation of the non-fluorescent substrate Ampliflu™ Red (AUR), generating Resorufin, a product with strong fluorescent properties. By measuring the fluorescence intensity of Resorufin using a fluorescence detection system, the amount of H₂O₂ generated can be indirectly reflected, thus establishing a quantitative correlation between lactic acid concentration and fluorescence intensity. The reaction formula is as follows:

[0029] The advantages of this system are: (1) High specificity: lactate oxidase is highly specific to lactate, which greatly reduces the interference of other metabolites (such as acetic acid and propionic acid); (2) High sensitivity: the enzyme-catalyzed cascade reaction realizes the secondary amplification of the signal, which can detect the trace amount of lactate secreted by bacterial growth; (3) Compatibility: all reaction components can be pre-mixed in the aqueous phase of the droplet, which can be seamlessly integrated with microfluidic operation.

[0030] Key aspects of this system include: (1) Optimization of reagent composition and concentration: Amplex Red (final concentration 50–300 μM), lactate oxidase (LOX, final concentration 10–100 mg / L), horseradish peroxidase (HRP, final concentration 0.5–5 U / mL) and F1 stabilizer (final concentration 0.1–0.5%) were precisely prepared in the detection droplets.

[0031] (2) Reaction conditions control: After adding AUR-HRP, incubate under light conditions for 2–5 h to make the fluorescence intensity of PE (PE channel, Ex / Em≈ 488 / 585 nm) have a good linear relationship with the lactic acid concentration.

[0032] (3) Sensitivity enhancement mechanism: By utilizing the enzyme cascade amplification effect, trace amounts of lactic acid molecules are converted into detectable strong fluorescent signals to meet the precise quantitative requirements of low-concentration lactic acid in the early fermentation stage.

[0033] (4) Specificity guarantee: Lactate oxidase has high substrate specificity for lactate, which can effectively distinguish other organic acids and avoid false positive signals caused by cross-reaction.

[0034] This system not only achieves highly sensitive quantification of lactic acid production, but also directly reflects the metabolic capacity of the strain through the intensity of the fluorescence signal, providing a reliable basis for subsequent sorting.

[0035] Seventhly, an intelligent signal analysis and sorting logic is provided to screen high-yield strains, mainly including: (1) Signal acquisition and preprocessing: Flow cytometry was used to simultaneously acquire multidimensional parameters such as PE fluorescence intensity, droplet diameter, and morphological integrity of the droplets. The raw data were standardized to eliminate background noise and non-specific signal interference.

[0036] (2) Threshold setting and sorting gate design: Setting the fluorescence intensity threshold: Based on the fluorescence intensity distribution of lactic acid standard droplets of different concentrations, the fluorescence intensity of PE is set to be ≥ 2.0 times, that is, the fluorescence intensity of the 10 mg / mL LA group is more than twice that of the 1 mg / mL group.

[0037] (3) Set a CV value threshold (coefficient of variation < 5%) to ensure signal stability and repeatability of the same batch of droplets.

[0038] (4) Set a droplet diameter threshold (50μm ± 2 μm) to exclude abnormal signals caused by droplet fusion failure or breakage.

[0039] (5) The injection volume is 20µL~1.5 mL, the sorting speed is 100~1000 droplets / s, and the droplets with the highest fluorescence intensity of the top 0.2% are selected as "high-yield lactic acid droplets" for precise sorting.

[0040] (6) Sorting and collection: The sorted droplets are physically separated and collected into a 96-well plate by a microfluidic sorting valve, with each well corresponding to a single droplet, so as to realize the preservation of strains at the single-clone level and subsequent verification.

[0041] (7) Verification and iterative optimization: After culturing the selected strains, their high lactic acid production phenotype was confirmed by using an aminoantipyrine and vanillic acid detection system based on the concentration of hydrogen peroxide produced.

[0042] This sorting logic not only improves screening efficiency (sorting speed of approximately 1000 droplets / second), but also significantly reduces the cost of manual intervention, ensuring the accuracy and repeatability of screening results.

[0043] Eighthly, the system described in the fifth aspect is provided for the application of Streptococcus thermophilus subsp. salivarius in verifying high lactic acid production capacity in dairy products.

[0044] The beneficial effects of this invention include: This invention, through the synergistic effect of the three core technical modules mentioned above, achieves high-throughput screening of high-yield lactic acid bacteria strains of *Streptococcus thermophilus* subsp. *salivarius*, from single-cell encapsulation to in-situ metabolic detection and intelligent sorting. Compared with traditional methods (such as plate titration, pH meter measurement, and microplate spectrophotometry), this invention has the following significant advantages: High throughput: Tens of thousands of droplets can be screened in a single batch, increasing efficiency by dozens of times; High sensitivity: capable of detecting lactic acid production down to the mg / mL level; High specificity: The lactate oxidase system avoids interference from other organic acids; High degree of automation: Signal analysis and sorting are fully programmable; Low cost and short cycle: No complicated instruments or a lot of manual operation are required, and the screening cycle is shortened.

[0045] This invention is not only applicable to the screening of high-yield lactic acid bacteria strains of Streptococcus salivarius thermophilus, but can also be used as a general platform to extend to the high-throughput metabolic screening of other acid-producing microorganisms (such as lactic acid bacteria, yeast, etc.), and has broad application prospects and industrialization value. Attached Figure Description

[0046] Figure 1 Real-time image generation of droplets; Figure 2 The conditions of YH mixed samples after incubation in bright field and fluorescence field; Figure 3 Droplet pairing and fusion of real-time images; Figure 4 Fluorescence intensity of a 10mg and 50mg mixed droplet on a sorting instrument; Figure 5 The appearance of 1 mg and 10 mg mixed droplets under bright field and PE200 fluorescence microscopes; Figure 6 The fluorescence intensity of the YH mixed sample under 405 and PE excitation light in a droplet sorter after incubation; Figure 7 Standard curve and color reaction of the 4-aminoantipyrine and vanillic acid detection system; Figure 8 Comparison of lactic acid production of screened strains after large-scale culture with YH sample; Figure 9 Screening strains for lactic acid content in fermented milk. Detailed Implementation

[0047] The following detailed embodiments further illustrate the concept and technical effects of the present invention to fully understand its purpose, features, and effects. Unless otherwise specified, all methods described are conventional methods. Unless otherwise specified, all materials are available from publicly available commercial sources. The illustrative embodiments and descriptions of the present invention are used to explain the invention and do not constitute an undue limitation thereof. It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0048] For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0049] The mixed sample YH containing *Streptococcus thermophilus* involved in the embodiments of this invention were all strains independently isolated and screened by the Key Laboratory of Functional Dairy Products jointly established by the Ministry of Education and Beijing Municipality.

[0050] The culture medium and lysis buffer formulations involved in the embodiments of this invention include: The M17 medium formula is as follows: 2.5 g casein peptone; 2.5 g meat peptone; 5.0 g soybean peptone; 2.5 g yeast extract; 5.0 g beef extract; 19.0 g β-glycerophosphate; 0.25 g magnesium sulfate; 0.5 g ascorbic acid; 5.0 g lactose; 12.75 g agar, adjusted to pH 7.2±0.2.

[0051] M17-lactose-based medium is obtained by adding 2% (m / v) lactose to M17 medium.

[0052] The detection methods involved in the embodiments of the present invention include: Indirect detection of lactic acid content using an aminoantipyrine and vanillic acid assay system: (1) Vanillic acid is dissolved in DMSO to ensure that the concentration of DMSO in the final reaction system is 3% (v / v).

[0053] (2) Preparation of the predicted mixture: The concentration of lactate oxidase was 0.5 U / mL, the concentration of horseradish peroxidase was 0.4 U / mL, the final concentration of 4-aminoantipyrine was 0.3 mM, the final concentration of vanillic acid dissolved in DMSO was 0.15 mM, the concentration of DMSO in the final reaction system was 3% (v / v), and the volume was adjusted to 3 mL with disodium hydrogen phosphate buffer. (3) Prepare a series of lactic acid standard solutions of known concentrations (0, 100, 200, 300, 400, 500, 600 μM); (4) Add 50 μL of standard lactic acid and the sample to be tested, then add 100 μL of prediction mixture, cover the plate, and gently shake for 30 s using an ELISA plate shaker to ensure that the liquid in the well is fully mixed. (5) Place the reaction plate at 37°C and incubate in the dark for 20 min. Set the detection wavelength to 500-510 nm on the microplate reader and read the absorbance value of each well. Calculate the lactic acid content according to the standard curve to obtain the lactic acid concentration in the original milk sample.

[0054] Example 1: Construction of a Droplet Microfluidic Screening System

[0055] (1) Take a basic culture medium system containing the target microbial strain and dilute the microorganism to an appropriate concentration (phase A). Prepare droplets with uniform particle size (50 ± 2 μm) using a droplet generation chip. (2) After the droplets are generated in the chip, they are incubated at a constant temperature of 42 ℃ for 25 h (pure water is added to the incubator to maintain a relative humidity of 50%). (3) In the detection channel, droplets containing the target strain are precisely fused with droplets pre-embedded with fluorescent detection reagents (including lactate oxidase, horseradish peroxidase, fluorescent substrate, etc.) using a droplet fusion instrument. The results are as follows Figure 1 As shown, the droplet generation chip is made of plastic and manufactured using an injection molding process, which ensures stable operation, ease of use, and reliable performance. Figure 2 The results, as shown by fluorescence microscopy, revealed a significant difference in fluorescence signal between the droplets corresponding to the fused YH strain and the blank droplets, with the droplets exhibiting intact morphology and no breakage or aggregation. This confirms that the fluorescence signal distinguishability and droplet quality of the YH strain droplets meet the requirements for use in a droplet sorting instrument.

[0056] Example 2: Establishment and Optimization of Droplet Detection System

[0057] (1) As shown in Table 1, add phase A reagent containing 10-50 mg / L lactic acid standard to the droplet fusion apparatus and incubate at 42°C for 25 h; (2) As shown in Table 1, the B phase test reagent is precisely prepared with Amplex Red (final concentration 50–300 μM), lactate oxidase (LOX, final concentration 10–100 mg / L), horseradish peroxidase (HRP, final concentration 0.5–5 U / mL) and F1 stabilizer (final concentration 0.1–0.5%) to form the test reagent.

[0058] (3) Reaction condition control: A phase droplets and B phase detection reagents were added to the droplet fusion instrument at the same time and incubated at 37°C in the dark for 3 h. Then, the droplets were further sorted and analyzed by the droplet sorter (PE channel, Ex / Em=488nm / 585nm).

[0059] Table 1 Droplet detection system

[0060] Under the same incubation conditions and consistent detection parameters, droplet reaction systems using 10 mg / mL and 50 mg / mL lactic acid as substrates, respectively, yielded the following results: Figure 3 The results show that phases A and B were fused using droplet fusion technology; fluorescence detection results are as follows. Figure 4 The results showed that the PE fluorescence intensity ratio was ≥2.0 (i.e., the fluorescence intensity of the 50 mg / mL LA group was more than twice that of the 10 mg / mL group). This result indicates that the fluorescence signals corresponding to different substrate concentrations are significantly distinguishable, and the system signal resolution meets the core requirements for microbial strain sorting (i.e., the ability to distinguish strains with different lactic acid production capabilities through differences in fluorescence intensity).

[0061] Furthermore, as shown in Table 2, the fluorescence monitoring system was optimized using 1 mg and 10 mg lactic acid. Under the experimental conditions and detection parameters, the droplet fluorescence signals of 1 mg / mL and 10 mg / mL lactic acid standards were detected, and the results are as follows. Figure 5The results showed a statistically significant difference in PE fluorescence intensity between the two groups, and the signal discrimination met the requirements for quantitative detection. This confirms that the optimized system can effectively distinguish between lactic acid concentrations as low as 1 mg / mL and 10 mg / mL. Compared to the unoptimized system, the detection sensitivity was significantly improved.

[0062] Table 2 Optimized Fluorescence Monitoring System for Lactic Acid

[0063] Example 4: Screening of high-yield lactic acid bacteria strains

[0064] (1) As shown in Table 3, take the basic culture medium system (phase A) containing the target microbial strain and prepare droplets (50 μm) with uniform particle size using microfluidic chip technology.

[0065] (2) Transfer the droplets to a sealed incubation container of the droplet fusion instrument and incubate at a constant temperature of 42 °C for 25 h (add pure water to the incubator to maintain a relative humidity of 50%).

[0066] (3) Inject AUR-HRP fluorescent detection reagent (phase B) into the target droplet, and then place the fused droplet at 37℃ (4) in the dark for 3 hours to allow the lactic acid produced by the strain's metabolism to fully react with the detection system.

[0067] (5) After incubation, take a 1 μL droplet sample and place it under a fluorescence microscope for quality detection. Simultaneously collect white light imaging (to observe the integrity of the droplet morphology) and fluorescence imaging (Ex / Em = 488 nm / 585 nm, PE channel) of the droplet. Combine the imaging results to determine whether the droplet meets the requirements for use on the sorting instrument.

[0068] (6) On-machine sorting: For droplets that have passed quality inspection, optical detection conditions (excitation wavelength 488nm, emission wavelength 585nm) are set based on the parameters optimized in the pre-experiment. The fluorescence signal and scattered light signal of the droplets are collected in real time, and the flow stability and signal repeatability of the droplets are monitored simultaneously. The positive threshold of PE fluorescence intensity is further set, and positive droplets with high fluorescence intensity are selected for precise sorting.

[0069] (7) Printing culture: Using the instrument’s precise printing function, the sorted positive droplets are collected one by one into a 96-well plate containing sterile basic culture medium. Each well corresponds to a single positive droplet. After sealing, the plate is placed in a 42 ℃ incubator for later use in the isolation, purification and functional verification of the strain.

[0070] Table 3 Screening system for high lactic acid production strains

[0071] The results are as follows Figure 6The fluorescence signal distribution results showed that the PE fluorescence intensity of the vast majority of droplets was concentrated in the range of 1800-2200, which is consistent with the fluorescence signal distribution of blank droplets without lactic acid production, and was therefore identified as a negative droplet population. Further, a PE fluorescence intensity ≥2800 was set as the positive threshold, and the top 0.2% of high-fluorescence droplets were selected as positive droplets.

[0072] Example 5: Screening strains for lactic acid content detection

[0073] This implementation case uses a mixed sample of YH and eight sorted strains Y1-Y8 to indirectly detect lactic acid content using an aminoantipyrine and vanillic acid detection system. The specific operation is as follows: (1) The selected Y1-Y8 strains were added to 200 μL of M17-lactose-based medium and cultured at 42℃ for 25 h; (2) Centrifuge at 10000×g for 1 min, collect the supernatant, and then filter it through a 0.22μm filter membrane to obtain the sample to be tested; (3) Preparation of the predicted mixture: the concentration of lactate oxidase was 0.5 U / mL, the concentration of horseradish peroxidase was 0.4 U / mL, the final concentration of 4-aminoantipyrine was 0.3 mM, the final concentration of vanillic acid dissolved in DMSO was 0.15 mM, the concentration of DMSO in the final reaction system was 3% (v / v), and the volume was adjusted to 3 mL with disodium hydrogen phosphate buffer. (4) Prepare a series of lactic acid standard solutions of known concentrations (0, 100, 200, 300, 400, 500, 600 μM); (5) Add 50 μL of standard lactate and disodium hydrogen phosphate buffer to dilute the sample to be tested 150 times, then add 100 μL of prediction mixture, cover the plate, and gently shake for 30 s using an ELISA plate shaker to ensure that the liquid in the well is fully mixed. (6) Place the reaction plate at 37°C and incubate in the dark for 20 min. Set the detection wavelength to 500-510 nm on the microplate reader and read the absorbance value of each well. Calculate the lactic acid content according to the standard curve to obtain the lactic acid concentration in the original milk sample.

[0074] The results are as follows Figure 7 The standard curve, y = 0.0049x + 0.0827, has an R² of 0.9995, indicating a highly significant linear relationship. This method, using measured absorbance values, can be used for accurate quantification of lactic acid content. Lactic acid production was determined using the validated quantitative detection method described above. The results are as follows... Figure 8The results showed that the lactic acid production of the YH mixed sample was 394.86 μM, and all eight candidate strains Y1-Y8 screened had the ability to synthesize lactic acid. Among them, the lactic acid production of seven strains was significantly increased to varying degrees compared with the control strain YH. The lactic acid production of strain Y7 was the highest, with a lactic acid production of 449.96 μM, which was about 14.0% higher than the control.

[0075] Example 6: Determination of lactic acid content in fermented milk

[0076] In this embodiment, the nine strains of *Streptococcus salivarius* thermophilic subspecies described in Example 5 were inoculated into fermented milk base, and the lactic acid content of the fermented milk was determined to verify the acid-producing capacity of different *Streptococcus salivarius* thermophilic subspecies. The specific method is as follows: Pure milk is heated to 95°C and held for 5 minutes to obtain a milk base; 1. Use 2% ( v / v Inoculate into M17-lactose liquid medium and incubate at 42 ± 1℃ for 12 h; 2. The strains were serially diluted 10-fold according to the national standard "Food Safety Standard - Microbiological Examination of Food - Lactic Acid Bacteria Examination" (GB / 4789.35-2016), and cultured at 42 ± 1℃ for 36-48 h. The number of microorganisms in the fermentation broth was measured to determine the number of strains in the stationary phase, and the amount added to the milk base was calculated.

[0077] 3. According to 1×10 7 CFU / mL was added to the milk base and fermented for 3 hours to obtain fermented milk. The acidity (°T) of the strain was measured. Three biological replicates were set up for each experiment.

[0078] 4. Accurately weigh 0.2 g of fermented milk into a 10 ml centrifuge tube, add 4 mL of 0.01 mol / L sulfuric acid, shake thoroughly to mix, centrifuge the mixture at 5000 rpm for 15 min, take the supernatant and filter it through a 0.22 μm filter membrane to obtain the sample to be tested.

[0079] 2. Detection of lactic acid: Prepare lactic acid standard solutions with mass concentrations of 0 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L and 8 g / L, and plot a standard curve with the concentration of the standard solution on the x-axis and the peak area on the y-axis.

[0080] 3. A Wonda Sil C18-WR (4.6×250 mm, 5 μm) column dedicated to organic acids was used with 0.1 M disodium hydrogen phosphate as the mobile phase, a flow rate of 600 μL / min, a column temperature of 32 ℃, an injection volume of 10 μL, and a detection wavelength of 214 nm. The lactic acid content in the sample to be tested in step 1 was determined by the external standard method.

[0081] The results are as follows Figure 9 As shown, after 3 hours of fermentation, the lactic acid content in the mixed sample YH was 5.44 g / L. The droplets Y1-Y7 selected from this sample, after fermentation, produced fermented milk with lactic acid contents of 5.59, 5.77, 5.36, 5.62, 6.05, 5.80, 6.31, and 6.07 g / L, respectively. The lactic acid production of these seven strains was higher than that of the mixed sample YH, demonstrating the high lactic acid production capacity of the strains.

[0082] In summary, this invention achieves high-throughput screening of high-lactic acid-producing strains of *Streptococcus thermophilus* through the synergistic effect of three core technical modules. This method possesses significant advantages such as high throughput, high sensitivity, good specificity, high degree of automation, and short cycle time.

[0083] Finally, it should be noted that the above description is only used to illustrate the technical solutions of the present invention and is not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention.

Claims

1. A high-throughput screening method for high-lactic acid-producing strains of *Streptococcus thermophilus* based on droplet microfluidics and lactate oxidase-coupled fluorescence detection, characterized in that, Includes the following steps: (1) Construction of a droplet microfluidic screening system; (2) Establishment of a lactate oxidase-coupled fluorescence detection system; (3) High-throughput screening and collection of high-yield lactic acid strains.

2. The method according to claim 1, characterized in that, In step (1), a mixture of samples containing *Streptococcus thermophilus* subsp. *salivarius*, M17 lactose medium, F1 stabilizer containing fluorocarbon surfactant, and AF405 fluorescent reagent was used to prepare microdroplets using a droplet generator. The single-strain encapsulation rate λ was controlled to be 0.05–0.

2. The microdroplets prepared in step (1) had a particle size of 40–60 μm. The droplets were placed in an incubator at 40–45 °C for static incubation, with a relative humidity of 40%–60% maintained in the incubator, and the incubation time was controlled to be 20–30 h.

3. The method according to claim 2, characterized in that, In step (2), a droplet fusion instrument is used to pair and fuse the cultured bacterial droplets with the detection reagent droplets in a microfluidic channel to construct an isolated two-stage enzymatic reaction system; the detection reagent droplets contain Amplex Red, lactate oxidase (LOX), horseradish peroxidase (HRP), F1 stabilizer and Tris-HCl buffer.

4. The method according to claim 3, characterized in that, The concentrations of each component in the test reagent are as follows: Amplex Red 40–350 μM, lactate oxidase (LOX) 8–120 mg / L, horseradish peroxidase (HRP) 0.3–6 U / mL, F1 stabilizer 0.05–0.6%, and incubation time in the dark is 0.5–6 h.

5. The method according to claim 4, characterized in that, In step (3), the PE fluorescence intensity of the fused droplets is detected by flow cytometer or droplet sorter. Multi-dimensional sorting parameters such as fluorescence intensity threshold, droplet diameter of 40-60 μm and CV value <6% are set. High fluorescence positive droplets with the highest fluorescence intensity of 0.1%-0.3% are selected for precise sorting and collected into a 96-well plate, with each well corresponding to a single reaction unit.

6. The method according to claim 5, characterized in that, The method also includes a step of validating the strains obtained by sorting: quantitatively detecting lactic acid production using an aminoantipyrine-vanillic acid colorimetric system or high performance liquid chromatography, and inoculating the screened strains into milk base to verify the acid production capacity of fermented milk.

7. A droplet microfluidic screening system, characterized in that, The system includes: (1) Droplet generation module: The screening system was constructed by mixing a mixed Streptococcus salivarius thermophilic subspecies YH, M17 lactose medium, F1 stabilizer and AF405 fluorescent reagent, and then preparing 40-60 μm water-in-oil microdroplets using a droplet generator to control the encapsulation of a single bacterial cell in the droplet, i.e. the encapsulation rate λ=0.05-0.2; (2) Droplet generation and incubation module: After the droplets are generated in the chip, they are incubated at a constant temperature of 40-45℃; (3) Droplet fusion and detection module: In the detection channel, droplets containing the target strain and droplets pre-embedded with fluorescent detection reagents are precisely fused together by a droplet fusion instrument to achieve in-situ reaction initiation; (4) Sorting and collection module: Combined with flow cytometer or sorter, high-yield lactic acid droplets are identified and physically separated in real time according to preset fluorescence intensity threshold and droplet size parameters, and collected into 96-well plate for subsequent verification and preservation.

8. The system according to claim 7, characterized in that, The fluorescent detection reagent droplets contain AmplexRed, lactate oxidase (LOX), horseradish peroxidase (HRP), fluorocarbon surfactant F1 stabilizer, and Tris-HCl buffer.

9. The system according to claim 7 or 8, characterized in that, The system also includes a module for validating the strains, which uses an aminoantipyrine-vanillic acid colorimetric system or high performance liquid chromatography to quantitatively detect lactic acid production, and inoculates the screened strains into milk base to verify the acid production capacity of fermented milk.

10. The application of the method according to any one of claims 1 to 6 or the system according to any one of claims 7 to 9 in a high-throughput metabolic phenotype screening platform for microorganisms or in an optimized fermentation strain breeding process.

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