Microbial fermentation real-time monitoring and molecular analysis method based on single-cell Raman spectrum technology

Abstract

The invention discloses a microbial fermentation real-time monitoring and molecular analysis method based on a single-cell Raman spectrum technology. The method comprises the following steps: firstly, preparing basic gold nanospheres which are microcosmically spherical and have the particle size distribution of 20-40 nm through a chloroauric acid oxidation reduction method, and characterizing the morphology of the basic gold nanospheres; secondly, the surface of the basic gold nanosphere is coated with a silicon dioxide thin layer through tetraethoxysilane hydrolysis and polymerization reaction, and the silicon dioxide functionalized gold nanosphere with the core-shell structure is prepared to serve as the microorganism living cell probe. The probe and different microbial cells are co-cultured, Raman fingerprint spectrums of the cells are sensitively captured, the single-cell Raman fingerprint spectrums are deeply analyzed by adopting a machine learning technology, and differential analysis (including strain difference, environment difference, metabolic capability difference and the like) is combined to determine the single-cell Raman fingerprint spectrums of the cells. Molecular indexes of specific expression of the microbial cells in different industrial production processes and different culture periods are screened out. The invention provides a universal tool for accurately realizing real-time monitoring and molecular analysis of microbial cells in different industrial processes, and has a wide industrial application prospect.

Description

Technical Field

[0001] The present invention belongs to the fields of functional materials, spectral detection and biomedicine, and specifically relates to a real-time monitoring and molecular analysis method of microbial fermentation based on single-cell Raman spectroscopy technology. Background Art

[0002] Biofermentation is the core and foundation of bioengineering, modern biotechnology and industrialization. With the rapid development of science and technology, people's living needs are increasing, the production scale of the fermentation industry is expanding, and the fermentation process is in urgent need of advanced control and continuous optimization to maximize production efficiency [Modern Industrial Economy and Informatization, 2023, 13(06), 107-109]. Immobilized fermentation technology is an emerging biotechnology in the field of biofermentation. This technology uses physical (adsorption method, embedding method) or chemical (covalent binding method, cross-linking method) methods to lock free industrial strains in a specific space to keep them active and reusable. Immobilized fermentation overcomes many shortcomings of traditional free fermentation processes in industrial production, such as complex process, long production cycle, low equipment utilization, easy inactivation of microbial strains and easy infection of foreign bacteria, and shows great application potential in the field of biofermentation [Process Biochem., 2016, 51, 1331-1337]. However, in immobilized fermentation based on physical or chemical methods, microbial cells are prone to mechanical damage, poor stability and autolysis, making it difficult to achieve truly large-scale and continuous industrial applications.

[0003] Biofilm immobilized fermentation technology is a new type of biological immobilized fermentation method based on the microbial clustering effect. After the cells are adsorbed on the carrier, they proliferate in large quantities to form a dense bacterial colony and then show the clustering effect. This effect enables physiological synergy between the members of the colony, thereby improving the metabolic activity and survival ability of the entire colony [Bioprocess, 2018, 16(03): 35-40]. Biofilm immobilized fermentation technology significantly improves the problems of mechanical damage and poor stability of microbial cells in the immobilized fermentation process based on physical or chemical methods. By significantly improving cell tolerance to maintain cell activity and achieve continuous fermentation, it has important industrial application prospects in many fields such as biofuels, bio-based chemicals, and enzyme preparations [Bioresource Technol., 2016, 219, 158-168.]. At present, a common problem in biofilm-based immobilized fermentation systems is that as fermentation conditions change dynamically, the chassis microbial cells as production units constantly respond to various stresses in the production process. Therefore, it is often difficult for strains to maintain maximum production capacity throughout the entire culture cycle, resulting in the inability to meet industrial needs in terms of economic benefits, which seriously hinders the further application and promotion of biofilm immobilized fermentation technology. Therefore, if the regularity of the response of microbial cells to stress during biofilm immobilization can be clarified, especially how the cell aggregation system maintains a high survival ability and metabolic activity in a complex environment, it will not only help to reveal the molecular mechanism of microbial clustering effect, but also provide important technical guidance for the directional transformation and screening of further industrial strains, thereby better promoting the development of biofilm immobilized fermentation technology.

[0004] At present, most of the research on biofilm immobilized fermentation systems focuses on carriers and bioreactors [Bioresource Technol., 2021, 340, 125700; Chem. Eng. J., 2023, 452.], and the fermentation performance of immobilized cells is further improved by designing advanced immobilized carriers or reactors, while there are few studies on microbial cells themselves. According to the survey, the commonly used microbial cell analysis technologies currently include mass spectrometry (MS), flow cytometry (FC), polymerase chain reaction (PCR), etc. Mass spectrometry [Science, 2006, 312, 212-217; Nature, 2003, 422, 198-207.] plays a significant role in the structural identification of biomolecules in microbial cells, especially the series of combined technologies developed based on mass spectrometry, such as liquid chromatography-mass spectrometry (HPLC-MS) and gas chromatography-mass spectrometry (GC-MS), which have higher resolution and can not only provide rich structural information of microbial molecules, but also accurately identify the modification sites of specific molecules, thereby achieving in-depth research on the "omics" level of microbial cells. However, the microbial cell analysis method based on mass spectrometry technology requires the extraction of biomacromolecules from cells before analysis, which inevitably damages the cells and makes them unusable for downstream analysis. Flow cytometry [J. Hazard. Mater., 2024, 461, 132656; Biotechniques, 2022, 72(4): 159-169.] is a single-cell analysis technology with multiplex detection capabilities. It can characterize microbial cells by multiple parameters, including protein expression levels, phenotypic characteristics, DNA content, and cell survival status, and achieve efficient and rapid analysis of a large number of microbial cell samples. However, flow cytometry is based on cell surface characteristic markers to achieve qualitative, positional or quantitative detection. Therefore, this technology is greatly affected by cell surface markers, often has the disadvantage of unstable results, and is prone to false positives or false negatives.Polymerase chain reaction (PCR) [Food Microbiol., 2017, 62, 282-288; Int. J. Food Microbiol., 2018, 270, 1-4.] is a highly sensitive and specific molecular tool for amplifying specific DNA fragments. The biggest feature of PCR technology is that it can greatly increase trace amounts of DNA. The test results are intuitive and easy to judge. It has a wide range of applications in the field of microorganisms. The analysis method based on PCR technology generally has a complicated operation process, and multiple experimental factors (such as primer design, amplification conditions, etc.) will affect the amplification effect, thereby affecting the accuracy of the final test results.

[0005] "Quality Evaluation of Industrial Microbial Strains - Raman Spectroscopy" (GB / T 38569-2020) is a national standard implemented on March 31, 2020. This standard specifies the method of evaluating the quality of industrial microbial strains by Raman spectroscopy, which is applicable to the quality consistency evaluation of bacteria, fungi, microalgae, etc. used in industrial fermentation relative to reference strains. However, the Raman signal intensity obtained by this evaluation method using Raman technology is low, and the information obtained is relatively limited; it only evaluates the quality consistency of the strain, and cannot "panoramic" monitor the dynamic changes of molecules in brewer's yeast cells during fermentation. Summary of the invention

[0006] Technical problem: Biofilm immobilized fermentation is a new type of microbial immobilized fermentation technology based on the microbial clustering effect. The present invention aims to address the common problems in the current application of biofilm immobilized fermentation, such as the dynamic changes in fermentation conditions, the strains constantly responding to various emergencies in the production process, making it difficult to maintain the maximum production capacity throughout the entire culture cycle, and other limitations. It provides a real-time monitoring and molecular analysis method for microbial fermentation based on single-cell Raman spectroscopy technology.

[0007] In order to solve the above problems, the technical solution adopted by the present invention is:

[0008] Real-time monitoring and molecular analysis of microbial fermentation based on single-cell Raman spectroscopy technology. Gold nanosphere probes are added to the microbial fermentation system for co-cultivation. Raman detection is performed after the cultivation to obtain high-quality single-cell Raman fingerprint spectra, monitor the dynamic changes of molecules in microbial cells during fermentation, and screen molecular indicators specifically expressed by microorganisms in different industrial production processes and different culture cycles.

[0009] The gold nanosphere probes are co-cultured with the microorganisms for 20-24 hours.

[0010] The concentration of gold nanosphere probes was 140-160 μg / L.

[0011] The microbial fermentation system includes a free fermentation system and an immobilized fermentation system.

[0012] The laser wavelength of Raman detection is 532nm, the objective lens is 50 times, and the detection range of Raman spectrum is 200-2000cm -1 .

[0013] The gold nanosphere probe is a core-shell structure, wherein the core is uniform spherical gold nanoparticles and the shell is a silicon dioxide polymer thin layer with a thickness of 5-10 nm.

[0014] The preparation of gold nanosphere probes includes the following steps:

[0015] (1) preparing a tetrachloroauric acid trihydrate solution with a concentration of 0.4-0.6 g / mL, adding a trisodium citrate aqueous solution with a concentration of 0.01 g / mL to the solution, and synthesizing by a "one-pot method" at a rotation speed of 800-1000 rpm to obtain basic gold nanospheres with a particle size distribution of 20-40 nm;

[0016] (2) At a rotation speed of 1600-2000 rpm, 8-12 mM tetraethoxysilane solution is added to step (1), and a 5-10 nm thick silica polymer thin layer is formed on the surface of the gold nanospheres through hydrolysis and polymerization reaction to obtain a gold nanosphere probe with a core-shell structure.

[0017] The microorganisms include but are not limited to Saccharomyces cerevisiae, bacteria, actinomycetes, algae and the like.

[0018] The analytical method proposed in the present invention takes Saccharomyces cerevisiae 1308 as an example to achieve comprehensive monitoring and comparison of different fermentation processes of Saccharomyces cerevisiae, explore its possible metabolic pathways, key biomedical proteins and regulatory mechanisms from the molecular and metabolic levels, and provide important technical guidance for the further targeted transformation and screening of bacterial strains, thereby better promoting the development of biofilm immobilization fermentation technology.

[0019] Taking Saccharomyces cerevisiae as an example, the present invention is based on the real-time monitoring and molecular analysis method of microbial fermentation based on single-cell Raman spectroscopy technology, such as Fig.10 As shown, the specific steps are as follows:

[0020] Step 1: Prepare a gold nanosphere solution by redox reaction: First, prepare a tetrachloroauric acid trihydrate solution, stir and heat to boiling in a 100°C oil bath; then quickly add a trisodium citrate solution, continue heating and stirring; after the reaction is completed, naturally cool to room temperature to synthesize a gold nanosphere solution with a particle size of 20-40nm;

[0021] Step 2: forming a thin layer of silicon dioxide polymer on the surface of the gold nanospheres through hydrolysis and polymerization of tetraethoxysilane;

[0022] Step 3: Construction of free and immobilized fermentation systems of brewer's yeast, and evaluation of the fermentation status; cotton towels were added as carriers during the immobilized fermentation process, and the old culture medium was poured out after a single fermentation, and new culture medium was added for continuous fermentation, and the OD was measured. 600 , glucose content and ethanol production to assess the fermentation status;

[0023] Step 4: adding the core-shell gold nanosphere probe prepared in step 2 to the free and immobilized fermentation systems constructed in step 3, taking samples and performing Raman detection to obtain high-quality single-cell Raman fingerprint spectra;

[0024] Step 5: Analyze the Raman fingerprint spectrum to monitor the dynamic changes of molecules in the Saccharomyces cerevisiae cells during the fermentation process. Discover new genes and proteins related to yeast cell proliferation and metabolic regulation.

[0025] Preferably, in step 4, the Raman detection laser wavelength is 532 nm, the objective lens is 50 times, and the Raman spectrum detection range is 200-2000 cm -1 .

[0026] Step 5: Analyze the Raman fingerprint spectrum, which mainly includes two parts:

[0027] ① Through machine learning models and verification research, the differential signal peaks of different cell samples are screened out from the complex Raman molecular fingerprint spectrum of brewer's yeast single cells to achieve accurate distinction of brewer's yeast cell phenotypes; ② The Raman characteristic peaks of the screened differential signal peaks are attributed to clarify the differences and correlations in molecular expression between different phenotypes of brewer's yeast.

[0028] According to the monitoring needs, samples are taken for Raman detection, and the obtained Raman fingerprint spectrum can reflect the state of the molecules at the sampling time point. We monitor yeast in real time from multiple dimensions such as different fermentation time points, different fermentation rounds, and different fermentation methods, realizing "panoramic" monitoring of the dynamic changes of molecules in brewer's yeast cells during the fermentation process.

[0029] The present invention co-cultures the gold nanosphere probes with core-shell structures with different microbial cells, sensitively captures the Raman fingerprints of the cells, and uses machine learning technology to deeply analyze the Raman fingerprints of single cells, and then combines differential analysis (including strain differences, environmental differences, metabolic capacity differences, etc.) to screen out the molecular indicators specifically expressed by microbial cells in different industrial production processes and different culture cycles. This invention provides a universal tool for accurately realizing real-time monitoring and molecular analysis of microbial cells in different industrial processes, and has broad industrial application prospects.

[0030] The method can be directly used to monitor brewer's yeast cells, guide the modification of the fermentation process, and improve the fermentation efficiency or product yield.

[0031] The core-shell gold nanosphere probes were co-cultured with Saccharomyces cerevisiae cells for 20-24 hours at a probe concentration of 140-160 μg / L. After the co-culture, the Saccharomyces cerevisiae cells that had taken up the probes were placed under a Raman spectrometer for Raman detection to obtain high-resolution single-cell Raman spectra. Machine learning technology was then used to screen out differential Raman peaks and attribute them, clarifying the differences and correlations in molecular expression between different phenotypes of Saccharomyces cerevisiae, and discovering new genes and proteins related to Saccharomyces cerevisiae cell proliferation and metabolic regulation.

[0032] The probe used in the present invention is a core-shell gold nanosphere. On the surface of the gold nanosphere, a silica sphere film with good polydispersity is synthesized by ammonia catalysis of TEOS, and the core-shell gold nanosphere with good stability and good dispersibility is prepared by the Stober method. We apply it to the field of industrial microorganisms, greatly improving the value and practical utility of the probe, forming a unique technical advantage, especially in the signal enhancement of brewer's yeast cells.

[0033] The present invention applies the prepared core-shell gold nanosphere probe to the field of brewer's yeast fermentation. It can be well taken up by yeast cells, enhance the Raman signal intensity in the brewer's yeast cells, and achieve ultra-sensitive enrichment of multiple biomolecule information in the cells, including nucleic acids, proteins, lipids, etc.

[0034] The method proposed in the present invention innovatively applies core-shell gold nanospheres to the field of immobilized fermentation of Saccharomyces cerevisiae, uses Raman spectroscopy technology to "panoramic" monitor the dynamic changes of molecules in Saccharomyces cerevisiae cells during the fermentation process, and analyzes the constructed high-quality Saccharomyces cerevisiae single-cell molecular fingerprint map to discover new genes and proteins related to yeast cell proliferation and metabolic regulation. This not only lays a theoretical foundation for the study of the molecular mechanism of biofilm immobilized fermentation, but also provides a universal analytical tool for the whole-omics molecular analysis of other types of industrial microbial cells, which has broad industrial application prospects.

[0035] Beneficial effects: Compared with the existing industrial microbial cell analysis technology, the present invention has the following advantages:

[0036] First, compared with existing analysis techniques such as mass spectrometry, flow cytometry, and PCR, the microbial immobilization fermentation research method developed in the present invention based on Raman technology has the advantage of being minimally disturbed by the detection environment and being suitable for directly monitoring changes in strains in complex industrial systems.

[0037] Secondly, the core-shell gold nanosphere probe described in the present invention is synthesized by a "one-pot method" with a simple and high yield; the shell is prepared by hydrolysis and polymerization of tetraethoxysilane, which is highly controllable, and the prepared silica-functionalized gold nanoparticles have good stability and dispersibility. The prepared core-shell gold nanosphere probe is applied to the fermentation of brewer's yeast, and it can be well taken up by yeast cells, and a high-resolution single-cell Raman fingerprint spectrum can be obtained, realizing ultra-sensitive enrichment of multiple biomolecular information in cells, including nucleic acids, proteins, lipids, etc.

[0038] The method constructed by the present invention introduces silica-coated gold nanoparticles into the field of brewer's yeast fermentation, uses Raman technology to monitor the dynamic changes of molecules in brewer's yeast cells during the fermentation process, screens molecular indicators specifically expressed by yeast cells in different industrial production processes and different culture cycles, and discovers new genes and proteins related to yeast cell proliferation and metabolic regulation, which can realize the accurate analysis and typing of various low-abundance biological molecules in various industrial microbial cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The dynamic light scattering image and transmission electron microscopy characterization image of the basic gold nanospheres prepared in Example 1, the scale bar is 50 nm.

[0040] Figure 2 (a) and (b) the UV-visible absorption spectrum of the core-shell gold nanospheres prepared in Example 2.

[0041] Figure 3 In order to measure the Raman signal enhancement effect (a), signal stability (b) and sample repeatability (c) of the core-shell gold nanospheres prepared in Example 2, the representative Raman reporter molecule 4-NTP was taken as an example.

[0042] Figure 4 The stability of the core-shell gold nanospheres prepared in Example 2 in the culture medium.

[0043] Figure 5 To determine the biocompatibility of the core-shell gold nanospheres prepared in Example 2.

[0044] Figure 6 To measure the cellular internalization of the core-shell gold nanospheres prepared in Example 2.

[0045] Figure 7 Schematic diagram (a) and status evaluation diagram (b) of yeast cells in free fermentation in Example 10 and Implementation Case 11.

[0046] Figure 8Schematic diagram (a) and status evaluation diagrams (b, c) of the immobilized fermented yeast cells in Examples 12 and 13.

[0047] Fig. 9 Single-cell Raman spectra of yeast cells and gold nanospheres with core-shell structures after co-culture in free fermentation (black) and immobilized fermentation systems (red and blue) in Example 11 and Example 13.

[0048] Fig.10 Flow chart of real-time monitoring and molecular analysis of yeast cell fermentation based on single-cell Raman spectroscopy. DETAILED DESCRIPTION

[0049] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0050] Real-time monitoring and molecular analysis of microbial fermentation based on single-cell Raman spectroscopy. This method mainly includes three core links:

[0051] ① First, core-shell gold nanospheres were prepared as cell probes to ultrasensitively enrich low-abundance biomolecule information in microbial cells of different fermentation modes;

[0052] ② Secondly, establish a stable and high-quality universal method for Raman expression profiles of microbial cells, which can be used to collect Raman expression profiles of microbial cells in different fermentation modes;

[0053] ③Finally, machine learning is combined with Raman spectroscopy to conduct in-depth analysis and mining of a large number of microbial single-cell Raman expression profiles obtained in the experiment, and to screen out the differential molecules of cells with different fermentation methods.

[0054] In the following examples, Saccharomyces cerevisiae 1308 is used as an example to illustrate the method of the present invention. Saccharomyces cerevisiae 1308 is from the China Industrial Microbiological Culture Collection Center (numbered CICC1308).

[0055] Example 1: Preparation of basic gold nanospheres

[0056] 10 μL of tetrachloroauric acid (III) trihydrate solution (HAuCl4·3H2O, 0.5 g / mL) was added to 50 mL of pure water and heated to boiling under stirring in an oil bath at 100°C. Then 0.3 mL of freshly prepared trisodium citrate solution (TSC, 0.01 g / mL) was quickly added, and the solution began to change from light yellow to purple red. The solution was heated and stirred for 8 minutes, and then naturally cooled to room temperature to obtain gold nanospheres with a particle size of about 30 nm. The solution was stored at 4°C as a basic solution for later use. The basic gold nanosphere material obtained by the above synthesis method showed that its microscopic morphology was regular spherical, and the dynamic light scattering results showed that the average particle size of the material was about 30 nm ( Figure 1 ).

[0057] Example 2: Preparation of core-shell gold nanospheres

[0058] Take 10 mL of the basic gold nanoparticle solution prepared above, add 40 mL of anhydrous ethanol, then add 0.7 mL of ammonia water (28%) to the suspension, and stir the mixture for 5 minutes. Then add 10 mL of tetraethoxysilane (Ethylsilicate, TEOS) ethanol solution (10 mM) to the suspension, continue stirring for 15 minutes, centrifuge the solution at 7000 rpm for 10 minutes after stirring, wash it with ultrapure water 3 times, and finally redisperse it in 10 mL of ultrapure water to obtain gold nanospheres with a core-shell structure, which are stored at 4°C for later use. The maximum absorption peaks of the ultraviolet absorption spectrum of the material before and after synthesis are at 527 nm and 528 nm, respectively ( Figure 2 a), zeta potential shows that the zeta potential value of the material changed from -18.5mV before synthesis to -8.72mV, indicating the successful preparation of core-shell structured gold nanospheres ( Figure 2 b).

[0059] Example 3: The Raman signal enhancement effect of the core-shell gold nanospheres prepared in Example 2 was measured. 2 mL of the core-shell gold nanosphere solution was ultrasonically dispersed for 3 minutes using an ultrasonic cleaner, and then placed in a low-speed refrigerated centrifuge and centrifuged at 1500 rpm for 5 minutes to remove the precipitated particles. After the centrifugation, 1 mL of the supernatant was taken into a centrifuge tube for later use.

[0060] Weigh 1 mg of 4-nitrothiophenol (4-NTP), add it to 1 mL of anhydrous ethanol, use an ultrasonic cleaner to sonicate for 5 min to fully dissolve the solid, and prepare a 1 mg / mL NTP alcohol solution. Use a pipette to draw 10 μL of the solution, add it to the above 1 mL of gold nanosphere solution, use an ultrasonic cleaner to sonicate for 5 min to fully mix it, and let it stand in the dark for 2 h.

[0061] Use a pipette to take 10 μL of the mixed solution after standing for 2 hours, drop it on a glass slide, and air-dry it at room temperature away from light. Then take a small amount of NTP powder and place it on the glass slide, and use a Raman spectrometer to perform Raman detection on the air-dried sample and 4-NTP powder. Figure 3 As shown in a, 4-NTP powder has no obvious Raman signal, while the air-dried sample shows multiple obvious Raman characteristic peaks, indicating that the material can greatly enhance the Raman signal. The specific Raman detection parameter settings are shown in Table 1.

[0062] Table 1 Raman detection parameters

[0063]

[0064] Example 4: Determination of the signal stability of the core-shell gold nanospheres prepared in Example 2

[0065] Take 2 mL of the core-shell gold nanosphere solution, use an ultrasonic cleaner to ultrasonically disperse it for 3 minutes, then put it into a low-speed refrigerated centrifuge and centrifuge it at a speed of 1500 rpm for 5 minutes to remove large particles. After the centrifugation, take 1 mL of the supernatant into a centrifuge tube for later use.

[0066] Weigh 1 mg of 4-nitrothiophenol (4-NTP), add it to 1 mL of anhydrous ethanol, use an ultrasonic cleaner to sonicate for 5 min to fully dissolve the solid, and prepare a 1 mg / mL NTP alcohol solution. Use a pipette to draw 10 μL of the solution, add it to the above 1 mL of gold nanosphere solution, use an ultrasonic cleaner to sonicate for 5 min to fully mix it, and let it stand in the dark for 2 h.

[0067] Use a pipette to take 10 μL of the mixed solution after standing for 2 hours, drop it on a glass slide, and air-dry it at room temperature away from light. Use a Raman spectrometer to perform Raman detection on the air-dried sample. Randomly select 5 different detection positions for each sample. Figure 3 As shown in b, the Raman spectra at five different detection positions all have Raman characteristic peaks with almost the same intensity and position, indicating that the material has good signal stability. (The detection conditions are the same as the Raman detection conditions in Example 3).

[0068] Example 5: Determination of the sample repeatability of the core-shell gold nanospheres prepared in Example 2

[0069] 2 mL of core-shell gold nanosphere solution was taken from each of the five batches, and ultrasonically dispersed for 3 min using an ultrasonic cleaner. The solution was then placed in a low-speed refrigerated centrifuge and centrifuged at 1500 rpm for 5 min to remove large particles. After the centrifugation, 1 mL of the supernatant was taken from each centrifuge tube for later use.

[0070] Weigh 1 mg of 4-nitrothiophenol (4-NTP), add it to 1 mL of anhydrous ethanol, use an ultrasonic cleaner to sonicate for 5 min to fully dissolve the solid, and prepare a 1 mg / mL NTP alcohol solution. Use a pipette to draw 10 μL of the solution, add it to the above 1 mL of gold nanosphere solution, use an ultrasonic cleaner to sonicate for 5 min to fully mix it, and let it stand in the dark for 2 h.

[0071] Use a pipette to take 10 μL of the mixed solution after standing for 2 hours, drop it on a glass slide, and air-dry it at room temperature away from light. Use a Raman spectrometer to perform Raman detection on the air-dried samples. Five different detection positions are randomly selected for each batch of samples. Figure 3 c, 1137cm -1The Raman peak signal intensity at 100 nm was calculated, and the RSD value of the Raman signal of the air-dried sample was 0.80%, indicating that the material has good sample repeatability. (The detection conditions are the same as the Raman detection conditions in Example 3).

[0072] Example 6: Determination of the stability of the core-shell gold nanospheres prepared in Example 2

[0073] Different concentrations of core-shell gold nanospheres (0, 25, 50, 100, 150, 200, 300, 400, 500 μg / L) prepared in Example 2 were added to YPD medium and fermentation medium, respectively, and photographs were taken under visible light after 24 hours.

[0074] Different concentrations of core-shell gold nanospheres (0, 25, 50, 100, 150, 200, 300, 400, 500 μg / L) prepared in Example 2 were added to YPD medium and fermentation medium, respectively. The ultraviolet absorption of the solution was characterized using a UV-visible spectrophotometer, and a quartz cuvette was used as a sample detection container.

[0075] Culture medium formula:

[0076] ①YPD medium: 22g / L glucose monohydrate, 10g / L yeast powder, 20g / L peptone. Sterilize at 115℃ for 20min.

[0077] ② Fermentation medium formula: 220g / L glucose monohydrate, 4g / L peptone, 4g / L ammonium sulfate, 3g / L yeast powder, 3g / L potassium dihydrogen phosphate, 0.5g / L anhydrous magnesium sulfate, 0.05g / L zinc sulfate heptahydrate, 0.05g / L ferrous sulfate heptahydrate. Sterilize at 115℃ for 20min for use.

[0078] The results are as follows Figure 4 As shown, the UV absorption curves of the prepared core-shell gold nanospheres in the growth medium (a) and the fermentation medium (b) have no obvious changes or fluctuations in the entire wavelength range, indicating that the material has good stability in the culture medium.

[0079] Example 7: Determination of the biocompatibility of the core-shell gold nanospheres prepared in Example 2

[0080] The PI staining method was used to investigate the effects of different concentrations of core-shell gold nanospheres on yeast cell viability. Activated Saccharomyces cerevisiae cells were first added to the required wells of a 96-well plate, and then YPD culture medium containing different concentrations of core-shell gold nanospheres (0, 25, 50, 100, 150, 200, 300, 400, 500 μg / L) was added, and incubated at 30°C for 20 hours. Then 100 μL of PI detection working solution was added to each well, and the well was placed in a 30°C environment and incubated in the dark for 30 minutes. Five replicate wells were set for each concentration. A laser confocal microscope was used to observe the number of dead cells and total cells at a wavelength of 545 nm. The results are shown in the figure. Figure 5 As shown, within the concentration range of 0-500 μg / L, the number of dead cells (red) was small, indicating that the material had good biocompatibility.

[0081] Example 8: Determination of the cellular internalization of the core-shell gold nanospheres prepared in Example 2

[0082] First, the core-shell gold nanospheres were labeled with fluorescein isothiocyanate (FITC), and a 1.0 mM FITC stock solution was prepared in anhydrous ethanol. 10 μL of the prepared FITC solution was added to 10 mL of the core-shell gold nanosphere solution under stirring, and the mixture was equilibrated at room temperature in the dark. Then, in order to test whether the core-shell gold nanospheres were internalized in Saccharomyces cerevisiae cells, 100 μL of DiD dye was added to the Saccharomyces cerevisiae suspension containing FITC-labeled core-shell gold nanospheres and incubated in the dark for 1 hour. After incubation, the cells were observed under a fluorescence microscope. The results are shown in Figure 2. Figure 6 As shown, the strong green fluorescence signal (FITC-AuNPs) and red fluorescence (DiD) were almost completely distributed in the cells, indicating that the prepared core-shell gold nanospheres were internalized in Saccharomyces cerevisiae cells.

[0083] Example 9: Cultivation of Saccharomyces cerevisiae cells and construction of different fermentation systems

[0084] Taking Saccharomyces cerevisiae 1308 as an example, the process is as follows:

[0085] Strain activation: Take out the frozen glycerol tube strain from the -80℃ refrigerator, thaw it on ice, then immediately dip the glycerol bacterial solution with a sterile inoculation loop on the sterilized clean bench, draw four zones on the YPD solid culture medium plate, seal it with sealing film, and place it in a 30℃ incubator for about 36 hours. After the culture is completed, store it in a 4℃ refrigerator.

[0086] Seed culture: In a clean bench, scrape all the colonies on the plate into YPD medium (100 mL) and culture in a shaker at 30°C and 220 rpm for 18-20 h.

[0087] Free fermentation system: After the seed culture is completed, the seed liquid (20 mL) is inoculated into the sterilized fermentation medium (200 mL) at a 10% inoculation rate, and fermented in a shaker at 35°C and 220 rpm. The fermentation is stopped when the glucose is exhausted.

[0088] Pretreatment of cotton towels: Wash a whole piece of cotton towel in pure water, place it in a drying oven at 60°C to dry, and cut the towel after drying, ensuring that each towel weighs 8g after cutting.

[0089] Immobilized fermentation system: After the seed culture is completed, the seed liquid (20 mL) is added to 200 mL of fermentation medium with a cotton towel carrier (8 g) at a 10% inoculation rate, and fermented in a shaker at 35°C and 220 rpm. After one round of fermentation is completed, the old medium in the shake bottle is poured out and an equal volume of fresh fermentation medium is added for a new round of fermentation. Thereafter, the above steps are repeated for a total of five rounds of fermentation.

[0090] Example 10: Collecting Raman fingerprints of yeast cells in the free fermentation system in Example 9

[0091] First, the status of yeast cells in the free fermentation system was evaluated according to the free fermentation method in Example 9. Samples were taken at 0, 4, 8, 12, and 22 hours of fermentation, with a sampling volume of 3 mL each time. The samples were pretreated as soon as possible and the OD was measured. 600 The fermentation status was evaluated by the following methods: (19-fold dilution), ethanol yield (centrifugation at 8000 rpm for 1 min, supernatant diluted 100 times) and residual sugar (centrifugation at 8000 rpm for 1 min, supernatant diluted 100 times). The results are shown in Table 1. Figure 7 As shown in Figure 2, the glucose content gradually decreased with the increase of fermentation time, and the ethanol yield gradually increased with the increase of fermentation time. Finally, the ethanol yield reached about 80g / L, and the OD 600 The value of also gradually increases with the increase of fermentation time, indicating that the cell density is increasing. All of the above results show that the state of free fermentation is normal. Secondly, use a pipette to take 10 μL of samples taken at different time points, drop them on a glass slide, and air-dry them naturally at room temperature away from light. The air-dried samples are placed under a confocal Raman spectrometer for Raman spectroscopy detection. The detection conditions are shown in Table 2.

[0092] Table 2 Raman detection parameters

[0093]

[0094]

[0095] Example 11: Co-culture of the core-shell gold nanospheres prepared in Example 2 with the brewer's yeast in Example 9 for free fermentation and collection of Raman fingerprints

[0096] First, a co-culture experiment of core-shell gold nanospheres and Saccharomyces cerevisiae was conducted. According to the free fermentation method in Example 9, after the seed solution was inoculated into the fermentation medium, 26 mL of core-shell gold nanosphere solution was added, and fermentation was carried out in a shaker at 35 ° C and 220 rpm. The fermentation was stopped after the glucose was exhausted. Samples were taken at 0, 4, 8, 12, and 22 hours of fermentation, with a sampling volume of 3 mL each time, and pre-treated as soon as possible to measure OD 600 The fermentation status was evaluated by the ethanol yield (centrifugation at 8000 rpm for 1 min, the supernatant was diluted 100 times) and the residual sugar content (centrifugation at 8000 rpm for 1 min, the supernatant was diluted 100 times). The results are as follows Figure 7 As shown in Figure 2, the glucose content gradually decreased with the increase of fermentation time, and the ethanol yield gradually increased with the increase of fermentation time. Finally, the ethanol yield reached about 80g / L, and the OD 600 The value of also gradually increased with the increase of fermentation time, indicating that the cell density is increasing. All of the above results show that the state of free fermentation is normal. Secondly, the single-cell Raman spectra of the co-culture system of core-shell gold nanospheres and brewer's yeast cells were collected. 10 μL of samples taken at different time points were taken with a pipette and dropped on a glass slide. They were naturally air-dried at room temperature and away from light. The air-dried samples were placed under a confocal Raman spectrometer for Raman spectroscopy detection. The typical single-cell Raman spectra are shown in Figure 1. Fig. 9 The detection conditions are the same as the Raman detection conditions in Example 10.

[0097] Example 12: Collecting Raman fingerprints of yeast cells in the immobilized fermentation system in Example 9

[0098] According to the immobilized fermentation method in Example 9, samples were taken after each round of fermentation. The sampling volume was 3 mL each time, and pretreatment was performed as soon as possible to measure OD 600 The fermentation status was evaluated by ethanol yield (centrifugation at 8000 rpm for 1 min, and the supernatant was diluted 100 times) and pH value. Then the old culture medium was discarded, the cotton towel was taken out, and the lines of the cotton towel carrier were gently removed with tweezers. The cotton towel was gently washed twice in PBS buffer and placed in a centrifuge tube for later use. The towel was then eluted with PBS buffer and the OD of the eluate was measured. 600 , the results are as follows Figure 8 As shown in the figure, the ethanol yield at the end of each round of fermentation was about 80 g / L, the pH value was about 3, and the OD of the free phase was 600 As the fermentation rounds increased, the OD of the eluate gradually decreased.600 On the contrary, it gradually increases with the increase of fermentation rounds. These results all indicate that the state of immobilized fermentation is normal. Then, 10 μL of the sample taken at the end of fermentation was taken with a pipette, dropped on a glass slide, and air-dried naturally at room temperature in the dark. The air-dried sample and the line rinsed with PBS were placed under a confocal Raman spectrometer for Raman spectroscopy detection. The detection conditions were the same as the Raman detection conditions in Example 10.

[0099] Example 13: Co-cultivation of the core-shell gold nanospheres prepared in Example 2 and the brewer's yeast in Example 9 for immobilized fermentation and collection of Raman fingerprints

[0100] First, a co-culture experiment of core-shell gold nanospheres and Saccharomyces cerevisiae was carried out. According to the immobilized fermentation method in Example 9, after the seed solution was inoculated into the fermentation medium, 26 mL of core-shell gold nanosphere solution was added and fermented in a shaker at 35°C and 220 rpm. After each round of fermentation, samples were taken, each sample volume was 3 mL, and pre-treated as soon as possible to measure OD 600 The fermentation status was evaluated by ethanol yield (centrifugation at 8000 rpm for 1 min, and the supernatant was diluted 100 times) and pH value. Then the old culture medium was discarded, the cotton towel was taken out, and the lines of the cotton towel carrier were gently removed with tweezers. The cotton towel was gently washed twice in PBS buffer and placed in a centrifuge tube for later use. The towel was then eluted with PBS buffer and the OD of the eluate was measured. 600 , the results are as follows Figure 8 As shown in the figure, the ethanol yield at the end of each round of fermentation was about 80 g / L, the pH value was about 3, and the OD of the free phase was 600 As the fermentation rounds increased, the OD of the eluate gradually decreased. 600 On the contrary, it gradually increases with the increase of fermentation rounds. These results all indicate that the state of immobilized fermentation is normal. Secondly, collect the single-cell Raman spectra of the co-culture system of core-shell gold nanospheres and brewer's yeast cells. Use a pipette to take 10 μL of the sample taken at the end of fermentation, drop it on a glass slide, and air-dry it naturally at room temperature away from light. The air-dried samples and the lines rinsed with PBS are placed under a confocal Raman spectrometer for Raman spectroscopy detection. The typical single-cell Raman spectrum is shown in Fig. 9 The above steps were repeated for the remaining four fermentations. The detection conditions were the same as the Raman detection conditions in Example 10. The characteristic peaks were assigned to the different single-cell Raman spectra in Example 11, Example 12, and Example 13, and the results are shown in Table 3.

[0101] Table 3 Attribution of Raman characteristic peaks

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Claims

1. A method for real-time monitoring and molecular analysis of microbial fermentation based on single-cell Raman spectroscopy, characterized in that: Gold nanosphere probes are added to the microbial fermentation system for co-cultivation. Raman detection is performed after the cultivation to obtain high-quality single-cell Raman fingerprint spectra, monitor the dynamic changes of molecules in microbial cells during the fermentation process, and screen molecular indicators specifically expressed by microorganisms in different industrial production processes and different culture cycles.

2. The method for real-time monitoring and molecular analysis of microbial fermentation based on single-cell Raman spectroscopy according to claim 1, characterized in that: The gold nanosphere probes are co-cultured with the microorganisms for 20-24 hours.

3. The method for real-time monitoring and molecular analysis of microbial fermentation based on single-cell Raman spectroscopy according to claim 1, characterized in that: The concentration of gold nanosphere probes was 140-160 μg / L.

4. The method for real-time monitoring and molecular analysis of microbial fermentation based on single-cell Raman spectroscopy according to claim 1, characterized in that: The microbial fermentation system includes a free fermentation system and an immobilized fermentation system.

5. The method for real-time monitoring and molecular analysis of microbial fermentation based on single-cell Raman spectroscopy according to claim 1, characterized in that: The laser wavelength of Raman detection is 532 nm, the objective lens is 50 times, and the detection range of Raman spectrum is 200-2000 cm -1 .

6. The method for real-time monitoring and molecular analysis of microbial fermentation based on single-cell Raman spectroscopy according to claim 1, characterized in that: The gold nanosphere probe is a core-shell structure, wherein the core is uniform spherical gold nanoparticles and the shell is a thin layer of silicon dioxide polymer with a thickness of 5-10 nm.

7. The method for real-time monitoring and molecular analysis of microbial fermentation based on single-cell Raman spectroscopy according to claim 1, characterized in that: The microorganisms are saccharomyces cerevisiae, bacteria, actinomycetes and algae.

8. The method for real-time monitoring and molecular analysis of microbial fermentation based on single-cell Raman spectroscopy according to claim 6, characterized in that: The preparation of gold nanosphere probes includes the following steps: (1) preparing a 0.4-0.6 g / mL tetrachloroauric acid trihydrate solution, adding a 0.01 g / mL trisodium citrate aqueous solution to the solution, and synthesizing the basic gold nanospheres with a particle size distribution of 20-40 nm by a "one-pot method" at a rotation speed of 800-1000 rpm; (2) At a rotation speed of 1600-2000 rpm, add 8-12 mM tetraethoxysilane solution to step (1), and form a 5-10 nm thick silica polymer layer on the surface of the gold nanospheres through hydrolysis and polymerization reactions to obtain a core-shell structured gold nanosphere probe.

9. The method for real-time monitoring and molecular analysis of microbial fermentation based on single-cell Raman spectroscopy according to claim 1, characterized in that: The specifically expressed molecular indicators include nucleic acids, proteins, and lipids.

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