Method for improving single-cell Raman sorting efficiency of grease microorganisms in fermented grains and application

Abstract

The invention belongs to the technical field of wine brewing, and relates to a method for improving single-cell Raman sorting efficiency of grease microorganisms in fermented grains, which comprises the following steps: fermented grain microorganism extraction: taking fermented grains to prepare a plate bacterial colony; a plate bacterial colony is collected and subjected to fermentation culture in a culture medium, the culture medium is formed by mixing an IM culture medium and a JP culture medium, and the mixing volume ratio of the IM culture medium to the JP culture medium is 1: (3-5); and performing Raman spectrum detection. Fermentation culture is carried out through a culture medium formed by mixing an IM culture medium and a JP culture medium, fermented grain microbial oil performance expression is increased, fermented grain microbial single-cell groups tend to show oil production characteristics, target bacteria are rapidly and accurately screened out from a large number of microorganisms with complex varieties in fermented grains, and the yield of the fermented grains is improved. Interference of non-target microorganisms in the fermented grains is reduced, the sorting accuracy is improved, and rapid and accurate detection and sorting of grease microorganism single cells in the fermented grains are achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of winemaking, and relates to a method for improving the efficiency of single-cell Raman sorting of oil microorganisms in wine mash and its application. Background Art

[0002] The production process of Maotai-flavor liquor is an open natural fermentation process. Relying on the rich flora system in the brewing process, combined with its unique brewing processes such as multiple cooking, fermentation, and wine extraction, it has formed rich flavor characteristics and taste. Among them, the microorganisms involved in the brewing of Maotai-flavor liquor are diverse and complex. At the same time, the normal growth and metabolism of core brewing microorganisms play a decisive role in the quality and yield of liquor. However, traditional microbial detection and sorting methods are often inefficient and cumbersome to operate, and it is difficult to meet the needs of modern industrial production. Therefore, the development of an efficient and accurate method for detecting and sorting fermented mash microorganisms is of great significance for improving the production quality inspection of Moutai liquor.

[0003] In the prior art, the separation and screening of microorganisms usually adopts the dilution coating method, microscopic observation method or molecular biology technology. Although these methods can achieve the separation and identification of target bacteria to a certain extent, they have the problems of complex operation, time-consuming and labor-intensive, and low accuracy. Especially in the production process of Moutai, due to the wide variety of microorganisms in the fermented grains, the separation and screening of target strains are more difficult.

[0004] In recent years, with the rapid development of biotechnology, some emerging detection and sorting technologies have gradually been applied to actual production. Among them, FlowRACS flow Raman sorting technology has attracted widespread attention for its high efficiency, rapidity and accuracy. By combining flow cytometry and Raman spectroscopy, this technology can achieve rapid identification and sorting of fermented grains microorganisms (such as yeast).

[0005] However, due to the complex microbial environment in the mash, FlowRACS flow Raman sorting technology still faces many challenges when applied to the detection and sorting of single cells in the mash. First, the number of microorganisms in the mash is large and the types are diverse. How to quickly and accurately screen out the target bacteria from the numerous microorganisms is a difficult problem. Secondly, the strains in the mash often form a symbiotic relationship and influence each other. How to avoid the interference of non-target microorganisms and improve the accuracy of sorting is also a problem that needs to be solved. Summary of the invention

[0006] The present invention aims to provide a method and application for improving the efficiency of Raman sorting of single cells of oily microorganisms in wine mash, by fermenting and culturing with a culture medium mixed with IM culture medium and JP culture medium, the expression of oil performance of wine mash microorganisms is increased, the single cell population of wine mash microorganisms tends to show oil production characteristics, and the sorting efficiency of single cells of oily microorganisms with oil Raman signals is increased by 48.5 times, so that target bacteria can be quickly and accurately screened out from a large number of microorganisms with various types in wine mash, the interference of non-target microorganisms in wine mash is reduced, the accuracy of sorting is improved, and the rapid and accurate detection and sorting of single cells of oily microorganisms in wine mash are realized.

[0007] In a first aspect, the present invention provides a method for improving the efficiency of single-cell Raman sorting of oil microorganisms in fermented grains, comprising the following steps:

[0008] Step 1, extracting microorganisms from fermented grains: taking fermented grains to prepare plate colonies;

[0009] Step 2, culture medium fermentation culture: collecting the plate colonies in step 1 and fermenting them in a culture medium, wherein the culture medium is a mixture of IM culture medium and JP culture medium, and the volume ratio of the IM culture medium to the JP culture medium is 1:3-5;

[0010] Step 3: Raman spectroscopy detection.

[0011] In some embodiments, in step 1, the specific method for extracting the fermented grains microorganisms is:

[0012] Put the fermented grains into sterile water, shake on a shaker, dilute the supernatant in a gradient manner, spread the dilution on a nutrient agar medium, invert and culture to obtain plate colonies;

[0013] Preferably, the mass and volume ratio of the fermented grains to the sterile water is 1 g: 5-10 mL;

[0014] Preferably, the inverted culture temperature is 25-40°C and the time is 1-3 days.

[0015] In some embodiments, in step 2, the IM medium is prepared by: mixing yeast nitrogen base and amino acids, adding pure water to mix, sterilizing, adding filtered sterilized raffinose and filtered sterilized galactose, and adjusting the pH;

[0016] Preferably, the mass ratio of the yeast nitrogen base to the amino acid is 1:1-1.5;

[0017] Preferably, the volume ratio of the filtered sterilized raffinose to the IM culture medium is 1:8-12, and the final concentration of the filtered sterilized raffinose in the IM culture medium is 1.5-2.5% w / V;

[0018] Preferably, the volume ratio of the filtered sterilized galactose to the IM culture medium is 1:8-12, and the final concentration of the filtered sterilized galactose in the IM culture medium is 0.5-1.5% w / V.

[0019] In some embodiments, the sterilization method is high temperature sterilization, the specific sterilization temperature is 110-120°C, and the sterilization time is 20-40 minutes;

[0020] Preferably, the pH range is 5.5-6.0.

[0021] In some embodiments, in step 2, the JP medium is prepared by:

[0022] Mix the fermented grains and pure water, filter, centrifuge and filter again;

[0023] Preferably, the mixed volume ratio of the fermented grains and pure water is 1:1-5;

[0024] Preferably, the fermented grains are at least one of sauce-flavor fermented grains, light-flavor fermented grains, and strong-flavor fermented grains.

[0025] In some embodiments, in step 3, the Raman spectroscopy detection method is:

[0026] The bacterial solution to be tested was sterilely washed and resuspended to adjust its concentration to about 3×10 5 / mL, and add loading buffer to mix, inject the sample, observe the cells and record the spectrum.

[0027] In some embodiments, in step 3, the sorting and collection parameters of the Raman spectrum detection are shown in the following table:

[0028]

[0029] In some embodiments, the characteristic peak signal of the oil microorganism is 2877cm -1 ; The threshold limit of the oily microorganisms is the total lipid content X=0.006.

[0030] In a second aspect, the present invention provides an application of any of the above methods in the detection of oily microorganisms in fermented mash, wherein the method can improve the efficiency of single-cell Raman sorting of oily microorganisms in fermented mash.

[0031] In some embodiments, the fermented grains are at least one of sauce-flavor fermented grains, light-flavor fermented grains, and strong-flavor fermented grains.

[0032] In summary, the present application includes at least one of the following beneficial technical effects:

[0033] By extracting the microorganisms from the mash and fermenting them with a medium mixed with IM medium and JP medium, the expression of the oil performance of the microorganisms in the mash was increased, and the single-cell population of microorganisms in the mash tended to show oil-producing characteristics. The efficiency of sorting single cells of oily microorganisms with oil Raman signals was increased by 48.5 times, which enabled the target bacteria to be quickly and accurately screened out from the large number and variety of microorganisms in the mash, reduced the interference of non-target microorganisms in the mash, improved the accuracy of sorting, and achieved rapid and accurate detection and sorting of single cells of oily microorganisms in the mash. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the clustering diagram of oil-producing and non-oil-producing single cells of the positive control strain;

[0035] Figure 2 TAG is the positive control strain + Raman characteristic peak diagram of oil-producing single cells, where X≤0.006 is quadrant A; X≥0.006 is quadrant B; A is the cells in the second and third quadrants, i.e., low total lipids; B is the first and fourth quadrants, i.e., high total lipids;

[0036] Figure 3 FlowRACS Raman single-cell microscopic images of fermented grains microorganisms in Example 2 and Comparative Examples 1-2;

[0037] Figure 4 The single cell cluster analysis result diagram of the characteristic peaks of oil in the fermented grains samples in Example 2 and Comparative Example 2 is shown, wherein the x-axis is the total ester content, and the y-axis is the proportion of unsaturated fatty acids in the total fat, i.e., the degree of unsaturation;

[0038] Figure 5 This is a single cell population signal analysis diagram of the characteristic peak of the oil in the fermented grains sample in Example 2, where A represents: Samples 1 and 2, based on Figure 4 , cell signal peak diagram with X<0.006, all signal peaks are merged (merged when signal peaks are counted); B represents: samples 1 and 2, based on Figure 4 , cell signal peak graph with X>0.006, all signal peaks are merged. (Signal peaks are merged when statistics are performed);

[0039] Figure 6 This is the single-cell Raman spectrum of the oil-producing mash sample in Example 2. DETAILED DESCRIPTION

[0040] The technical solution of the present invention is further described below by specific embodiments, which do not limit the protection scope of the present invention. Some non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the protection scope of the present invention.

[0041] Due to the complex microbial environment in the mash, it is a difficult problem to quickly and accurately screen out the target bacteria from a large number of microorganisms when FlowRACS flow Raman sorting technology is applied to the detection and sorting of single cells in the mash. Secondly, the strains in the mash often form a symbiotic relationship and influence each other, and there is interference from non-target microorganisms, which leads to low sorting accuracy.

[0042] In order to solve the above problems, the present invention is committed to reducing the influence of the fermented mash microbial environment on the Raman sorting of single-cell oil microorganisms in the fermented mash. The present invention tried a variety of different culture media for fermentation and culture, and finally found that the fermentation culture of IM culture medium and JP culture medium mixed in a volume ratio of 2:8 increased the expression of the oil performance of the fermented mash microorganisms, and the single-cell population of the fermented mash microorganisms tended to show oil production characteristics. Other culture media cannot achieve the same technical effect. At the same time, the mixing ratio of IM culture medium and JP culture medium also affects the expression of the oil performance of the fermented mash microorganisms. The effect of IM culture medium that accounts for too low or too high a proportion gradually shows a decreasing trend. Therefore, the present invention finally uses IM culture medium and JP culture medium mixed in a volume ratio of 2:8 for fermentation and culture.

[0043] The materials and reagents of the present invention are from the following sources:

[0044] Fermented grains samples: provided by Kweichow Moutai Distillery. DO Supplement, raffinose, glucose, and 95% ethanol were purchased from Sinopharm Group, and LB broth medium and Yeast Nitrogen Base medium were purchased from Qingdao Haibo Biological Co., Ltd. The two strains of oleaginous yeast (TAG+) and non-oleaginous yeast (TAG-) were glycerol tubes from strains deposited in Qingdao Xingsai Biotechnology Co., Ltd.

[0045] The apparatus and equipment of the present invention are as follows:

[0046] FlowRACS flow Raman sorting instrument (Qingdao Xingsai Biotechnology Co., Ltd.);

[0047] High-speed refrigerated centrifuge (Eppendorf);

[0048] Ultrasonic cleaning machine (VCX 500, SONICS, Newtown, CT, USA);

[0049] Vertical pressure steam sterilizer (Boxun).

[0050] Example 1

[0051] The construction of a positive reference method for oil-producing microorganisms includes the following steps:

[0052] (1) Activation and fermentation of oleaginous / non-oleaginous yeast

[0053] In 1mL of IM medium, inoculate 100uL of oleaginous / non-oleaginous yeast glycerol tube, shake well, and culture at 30℃ for 2 days to complete the first expansion culture. In a 50mL centrifuge tube, add 10mL of IM medium, inoculate 1mL of oleaginous / non-oleaginous yeast activated bacterial solution, culture at 30℃ for 2 days to complete the fermentation culture, and obtain the IM fermentation broth of oleaginous yeast and non-oleaginous yeast.

[0054] In this embodiment, the two strains of glycerol tubes of oleaginous yeast (TAG+) and non-oleaginous yeast (TAG-) are from strains deposited in Qingdao Xingsai Biotechnology Co., Ltd., and the strain information can be found in the reference (Positive dielectrophoresis-based Raman-activated droplet sorting for culture-free and label-free screening of enzyme function in vivo, published in the journal: SCIENCE ADVANCES. DOI: 10.1126 / sciadv.abb3521).

[0055] The IM medium was prepared by adding 10.7 g / L Yeast Nitrogen Base (w / V), 1.74 g / L DO Supplement (-ura) (CloneTech) (amino acid mixture) (w / V) and H 2 O to 800mL / L, sterilize at 115℃ for 30min, add 100mL of filtered sterilized raffinose (2% final concentration, w / V), add 100mL of filtered sterilized galactose (1% final concentration, w / V), and adjust the pH to 5.8 with 5% NaOH.

[0056] (2) Construction of positive oil production method

[0057] Take 1 mL of IM fermentation broth of oleaginous yeast and non-oleaginous yeast respectively, wash the samples three times, and keep 10 μL of bacterial solution for mixing; then take 1 μL of bacterial solution for spotting on calcium fluoride slide. After spotting, wash the remaining bacterial solution with 1 mL of ddH 2 O was resuspended, and the oleaginous yeast (TAG+) and non-oleaginous yeast (TAG-) were mixed in equal volume ratio (1:1). After mixing, the supernatant was discarded by centrifugation, and 10 μL of bacterial liquid was retained. 1 μL was also taken for spotting on calcium fluoride slides. After the slides were air-dried, FR Raman equipment was used for detection. At least 200 spectra were detected for each sample. If the cells were severely accumulated during the sample detection, appropriate dilution was performed.

[0058] In this example, the positive method was constructed using oleaginous yeast and non-oleaginous yeast with known oleaginous properties in order to clarify the reference limit of the target characteristic cell sorting threshold and provide a reference standard for subsequent sorting. Figure 1 As shown in the figure: After fermentation and cultivation, the single cells were clearly divided into groups, and grouped cells were produced in the second, third, and fourth quadrants. Subsequently, Raman spectroscopy was performed, and the results were as follows: Figure 2 As shown: In the Raman spectrum of single cells in quadrant B with X ≥ 0.006, there is an obvious oil production characteristic peak signal at 2877 cm -1 .

[0059] Therefore, combined with the Raman spectrum detection results of the positive control strain, it can be seen that in terms of the construction of the positive method platform, the characteristic peak signal value and threshold of oil production are clearly defined, which are the characteristic peak position signal of oil substances at 2877cm -1 ; The threshold limits are total fat content X=0.006 and lipid unsaturation Y=0.12.

[0060] Example 2

[0061] A method for improving the efficiency of single-cell Raman sorting of oil microorganisms in fermented grains comprises the following steps:

[0062] Step 1: Extraction of fermented grains microorganisms

[0063] Take 10g of fermented grains and put them into a 250mL conical flask with 90mL of sterile water. Shake on a shaker for 30min. Dilute the supernatant in a gradient manner. -1 , 10 -2 , 10 -3 0.1 mL of the dilutions were spread on nutrient agar medium, and 3 plates were spread for each dilution concentration. After spreading on the plates, they were inverted and cultured at 30°C for 2 days to obtain plate colonies. Another group of identical parallel samples were set up, and after spreading on the plates, they were inverted and cultured at 37°C for 2 days to obtain plate colonies.

[0064] In other embodiments, the mass and volume ratio of the mash to the sterile water is 1g:5-10mL, such as 1g:5mL, 1g:6mL, 1g:7mL, 1g:8mL, 1g:8.5mL, 1g:10mL, etc.

[0065] In other embodiments, the inverted culture temperature is 25-40°C, and the time is 1-3 days, such as 25°C for 3 days, 29°C for 2 days, 35°C for 1 day, etc.

[0066] In this example, two different types of fermented grain samples were selected for oil-producing microbial screening tests. In order to maintain the cells in a good state, the two fermented grain samples were sampled for production and then tested. The specific sample information is shown in the following table:

[0067] Fermented Grains Sample Information

[0068]

[0069] Step 2: Fermentation culture of culture medium

[0070] Collect single colonies from the fermented mash microbial plate and mix them in 5 mL of water. Take 1 mL of the bacterial solution and ferment it in 10 mL of IM+JP medium. Culture it at 30°C for 1 day. Take 1 mL and centrifuge it to discard the supernatant. Add 500 uL of sterile water to wash it, centrifuge it to discard the supernatant, take 50 uL of sterile water to dilute it, take 2 uL and add it to 2 mL of loading buffer to obtain a test bacterial solution with more yeast cells and fewer slender rods.

[0071] The IM+JP medium is a mixture of IM medium and JP medium in a volume ratio of 2:8, wherein the IM medium is prepared as follows: 10.7 g / L Yeast Nitrogen Base (w / V), 1.74 g / L DO Supplement (-ura) (CloneTech) (amino acid mixture) (w / V) and H 2 O to 800mL / L, sterilize at 115℃ for 30min, add 100mL of filtered sterilized raffinose (2% final concentration, w / V), add 100mL of filtered sterilized galactose (1% final concentration, w / V), and adjust the pH to 5.8 with 5% NaOH.

[0072] The preparation method of JP culture medium is as follows: add sauce-flavored mash and pure water in a volume ratio of 1:3, shake at 400 rpm for 10 min, ultrasonicate for 20 min, filter with gauze, centrifuge at 1400 rpm for 10 min, and filter with 0.22 μm filter membrane for sterilization.

[0073] In other embodiments, the volume ratio of the IM medium and the JP medium is 1:3-5, such as the volume ratio of the IM medium and the JP medium is 1:3, the volume ratio of the IM medium and the JP medium is 1:3.5, or the volume ratio of the IM medium and the JP medium is 1:5, etc.

[0074] In other embodiments, the mass ratio of the yeast nitrogen base to the amino acids is 1:1-1.5, such as the mass ratio of the yeast nitrogen base to the amino acids is 1:1, 1:1.2, 1:1.3, 1:1.5, etc.

[0075] In other embodiments, the volume ratio of the filtered sterilized raffinose to the IM culture medium is 1:8-12, and the final concentration of the filtered sterilized raffinose in the IM culture medium is 1.5-2.5% w / V; for example, the volume ratio of the filtered sterilized raffinose to the IM culture medium is 1:8, the volume ratio of the filtered sterilized raffinose to the IM culture medium is 1:9, the volume ratio of the filtered sterilized raffinose to the IM culture medium is 1:12, etc., the final concentration of the filtered sterilized raffinose in the IM culture medium is 1.5% w / V, the final concentration of the filtered sterilized raffinose in the IM culture medium is 2.5% w / V, etc.

[0076] In other embodiments, the volume ratio of the filtered sterilized galactose to the IM culture medium is 1:8-12, and the final concentration of the filtered sterilized galactose in the IM culture medium is 0.5-1.5% w / V; such as the volume ratio of the filtered sterilized galactose to the IM culture medium is 1:8, and the final concentration of the filtered sterilized galactose in the IM culture medium is 0.5% w / V; the volume ratio of the filtered sterilized galactose to the IM culture medium is 1:11, and the final concentration of the filtered sterilized galactose in the IM culture medium is 0.9% w / V; the volume ratio of the filtered sterilized galactose to the IM culture medium is 1:12, and the final concentration of the filtered sterilized galactose in the IM culture medium is 1.5% w / V, etc.

[0077] In other embodiments, the specific sterilization temperature is 110-120°C, and the sterilization time is 20-40min; such as the sterilization temperature is 110°C, and the sterilization time is 40min; the sterilization temperature is 118°C, and the sterilization time is 32min; the sterilization temperature is 120°C, and the sterilization time is 20min, etc.

[0078] In other embodiments, the pH range is 5.5-6.0, such as pH 5.5, pH 5.7, pH 5.9, pH 6.0, etc.

[0079] In other embodiments, when JP culture medium is prepared, the mixing volume ratio of the mash and pure water is 1:1-5, for example, the mixing volume ratio of the mash and pure water is 1:1, the mixing volume ratio of the mash and pure water is 1:2, the mixing volume ratio of the mash and pure water is 1:4, the mixing volume ratio of the mash and pure water is 1:5, etc.

[0080] In other embodiments, when the JP culture medium is prepared, the fermented grains are at least one of sauce-flavor fermented grains, light-flavor fermented grains, and strong-flavor fermented grains, such as light-flavor fermented grains or strong-flavor fermented grains, or a mixture of light-flavor fermented grains and strong-flavor fermented grains.

[0081] Step 3: Raman spectroscopy detection

[0082] The test bacterial liquid was tested on the FlowRACS high-throughput flow Raman sorter. The total ester content and ester unsaturation of the cell population changed, the single-cell population was classified, and tended to have oil-producing characteristics.

[0083] In this embodiment, the FlowRACS instrument operation process refers to the FlowRACS instrument startup calibration, chip assembly, gas and liquid connection instrument user manual provided by Qingdao Xingsai Biotechnology Co., Ltd. Specifically, the test bacterial solution is sterile cleaned and resuspended, and the sterile cleaning and resuspending operations are as follows:

[0084] 1. Centrifuge the bacterial solution to be tested to remove the culture medium at 4°C, 12,000 rpm, and 2 min.

[0085] 2. Take 1 mL of sterile water to resuspend and wash the centrifuged cells. Centrifuge again and remove the supernatant. The centrifugation conditions are 4°C, 12000 rpm, 2 min. Resuspend and wash with sterile water three times.

[0086] 3. After washing, add sterile water to dilute the bacterial solution to 3×10 5 Pieces / mL.

[0087] Adjust the concentration to about 3 × 10 5 After 100 μl / mL, add loading buffer and mix well for later use.

[0088] Before sorting, start the air pump, observe the cells entering the chip smoothly through a microscope, and accurately adjust the laser point to the detection point. After that, set the Raman acquisition and sorting parameters, observe the cells passing through the detection point in an orderly manner and being accurately recorded in the spectrum. After the test, use 75% ethanol to thoroughly clean the chip to ensure that the equipment is clean and closed.

[0089] Among them, the specific detection and sorting parameters of single cell detection and sorting in this embodiment are shown in the following table.

[0090] Parameters for collecting and sorting fermented mash microbial cells

[0091]

[0092] Comparative Example 1

[0093] A method for improving the efficiency of single-cell Raman sorting of oil microorganisms in fermented grains, which differs from Example 2 in that: after the fermented grains microorganisms are extracted in step 1, a plate colony detection is directly performed, and then a FlowRACS on-machine detection is performed. The specific method of the plate colony detection is as follows:

[0094] Collect the single colonies on the plate after the extraction of the fermented grains microorganisms in step 1 of Example 2, add them to 5 mL of water, mix them well, obtain the original mixed bacterial solution, take 2 uL for microscopic examination, and observe the microbial morphology. Take 1 mL of the above original mixed bacterial solution, centrifuge and discard the supernatant, add 500 uL of sterile water to wash once, centrifuge and discard the supernatant, take 50 uL of sterile water to dilute the bacteria, and obtain a diluted bacterial solution. Take 2 uL of the above diluted bacterial solution and add it to 2 mL of loading buffer, without medium fermentation culture, and directly perform the FlowRACS machine detection step.

[0095] Comparative Example 2

[0096] A method for improving the efficiency of single-cell Raman sorting of oil microorganisms in fermented grains, which is different from Example 2 in that: the medium used for medium fermentation culture in step 2 is IM medium. Specifically, the preparation method of IM medium is as follows:

[0097] 10.7 g / L Yeast Nitrogen Base (w / V) and 1.74 g / L DO Supplement (-ura) (CloneTech) (w / V) were added to the H 2 O to 800mL / L, sterilize at 115℃ for 30min, add 100mL of filtered sterilized raffinose (2% final concentration, w / V), add 100mL of filtered sterilized galactose (1% final concentration, w / V), and adjust the pH to 5.8 with 5% NaOH.

[0098] Result detection and analysis:

[0099] 1. FlowRACS Raman single cell microscopy images of fermented grains microorganisms in Example 2 and Comparative Examples 1-2 are as follows: Figure 3 As shown, in Comparative Example 1, bacteria account for a large proportion of the colonies directly cultured with LB and have strong adhesion, and the microorganisms are in the form of slender rods, which causes these microorganisms to stick to the Raman chip and cannot be effectively detected by separating cells in the dielectric capture field. Therefore, the method of directly using colonies cultured with LB for general screening is not suitable for current equipment, and a strategy for sorting different types of bacterial colonies after culture is considered.

[0100] By comparing the plate colonies cultured in LB (Comparative Example 1), the bacterial liquid after IM fermentation (Comparative Example 2), and the bacterial liquid after IM+JP fermentation (Example 2), when detecting single cells on the FlowRACS device, it was found that the single-cell flow rate of the bacterial liquid after IM+JP fermentation in the FlowRACS Raman dielectric capture field was significantly increased. At the same time, the aggregation amount at the electrode extreme was large and the cell aggregation degree was high, and the efficiency of single-cell capture and sorting was significantly increased, which ultimately resulted in more effective data for single-cell detection, which was more conducive to the application of FlowRACS flow Raman sorting of single cells in fermented mash. Therefore, the present invention finally selected IM+JP medium for fermentation and cultivation of fermented mash microorganisms for FlowRACS detection.

[0101] 2. Further single cell cluster analysis of oil characteristic peaks was performed on the fermented grains samples of Example 2 and Comparative Example 2. The results are as follows: Figure 4 As shown: It was found that after fermentation of mash microorganisms in different culture media, the total ester content and ester unsaturation of cell groups in the mash samples changed, and single cell groups were classified. Specifically, after IM+JP culture, the number of cells of mash microorganisms in the high-fat partition with X>0.06 increased, indicating that the sorted cells tended to have oil-producing characteristics, that is, under the conditions of IM+JP fermentation liquid, the cells tended to cluster in the high-fat area with X>0.06. Therefore, single cells in the high-fat area with X>0.06 were detected by Raman spectroscopy.

[0102] 3. The single cell population signal analysis diagram of the characteristic peak of oil in the fermented grains sample in Example 2 is as follows Figure 5 As shown, from Figure 5 The results show that all single cells in the high-fat area of ​​the samples have obvious oil-producing characteristic signal peaks. Subsequently, the single cells with oil-producing signals detected in the high-fat area were sorted out using the FR flow Raman device, and the sorted oil-producing single cells were re-tested and verified. The Raman spectrum of the oil-producing single cells of the fermented grains sample in Example 2 is as follows: Figure 6 As shown, from Figure 6 The results showed that the sorted oil-producing target cells of all samples were verified to be at Wavenumber = 2877cm -1 All of them show characteristic signal peaks of oil production.

[0103] 4. The results of oil-producing single cell sorting of the middle-fermented mash samples in Example 2 and Comparative Example 2 are shown in the following table:

[0104] Number of sorted cells

[0105]

[0106] According to the results of the sorted cell numbers in the statistical experiment, compared with the IM medium, the number of cells with the oil-producing characteristic signal peak after fermentation in the IM+JP medium increased significantly, and the target single cell sorting efficiency increased by 48.5 times. Therefore, compared with the traditional medium, the improved medium IM+JP not only significantly increased the single-cell throughput of fermented mash microorganisms in the dielectric capture field of flow Raman cell sorting, but also increased the cell aggregation at the extreme of the capture electrode by 71.42%. In addition, the improved medium also increased the expression of oil performance of fermented mash microorganisms, and the single-cell population of fermented mash microorganisms tended to show oil-producing characteristics, and the target single-cell sorting efficiency with oil Raman signals increased by 48.5 times. It shows that the present invention provides an effective pre-treatment method for the subsequent sorting of functional oil-producing microorganisms in fermented mash samples.

[0107] In summary, after fermentation and cultivation with IM+JP medium, the single-cell flow rate of fermented mash microorganisms in the FlowRACS Raman dielectric capture field increased significantly. At the same time, the aggregation amount was large and the cell aggregation degree was high at the electrode extreme, which significantly increased the efficiency of single-cell capture and sorting, ultimately resulting in more effective data for single-cell detection, which is more conducive to the application of FlowRACS flow Raman sorting of single-cell fermented mash. Moreover, after fermentation with IM+JP medium, the oil production performance expression of the cells can be increased, which provides an effective pretreatment method for the subsequent sorting of oil-producing microorganisms in actual samples.

[0108] It is to be understood that the present invention is described by some embodiments, and it is known to those skilled in the art that various changes or equivalent substitutions may be made to these features and embodiments without departing from the spirit and scope of the present invention. In addition, under the teachings of the present invention, these features and embodiments may be modified to adapt to specific circumstances and materials without departing from the spirit and scope of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed herein, and all embodiments falling within the scope of the claims of this application are within the scope of protection of the present invention.

Claims

1. A method for improving the efficiency of single-cell Raman sorting of oil microorganisms in fermented grains, characterized in that: The following steps are involved: Step 1, extracting microorganisms from fermented grains: taking fermented grains to prepare plate colonies; Step 2, culture medium fermentation culture: collecting the plate colonies in step 1 and fermenting them in a culture medium, wherein the culture medium is a mixture of IM culture medium and JP culture medium, and the volume ratio of the IM culture medium to the JP culture medium is 1:3-5; Step 3: Raman spectroscopy detection.

2. The method according to claim 1, characterized in that In step 1, the specific method of extracting the fermented grains microorganisms is: Put the fermented grains into sterile water, shake on a shaker, dilute the supernatant in a gradient manner, spread the dilution on a nutrient agar medium, invert and culture to obtain plate colonies; Preferably, the mass and volume ratio of the fermented grains to the sterile water is 1 g: 5-10 mL; Preferably, the inverted culture temperature is 25-40°C and the time is 1-3 days.

3. The method according to claim 1, characterized in that In step 2, the IM medium is prepared by mixing yeast nitrogen base and amino acids, adding pure water, sterilizing, adding filtered sterilized raffinose and filtered sterilized galactose, and adjusting the pH; Preferably, the mass ratio of the yeast nitrogen base to the amino acid is 1:1-1.5; Preferably, the volume ratio of the filtered sterilized raffinose to the IM culture medium is 1:8-12, and the final concentration of the filtered sterilized raffinose in the IM culture medium is 1.5-2.5% w / V; Preferably, the volume ratio of the filtered sterilized galactose to the IM culture medium is 1:8-12, and the final concentration of the filtered sterilized galactose in the IM culture medium is 0.5-1.5% w / V.

4. The method according to claim 3, characterized in that The sterilization method is high temperature sterilization, the specific sterilization temperature is 110-120°C, and the sterilization time is 20-40min; Preferably, the pH range is 5.5-6.

0.

5. The method according to claim 1, characterized in that In step 2, the JP culture medium is prepared as follows: Mix the fermented grains and pure water, filter, centrifuge and filter again; Preferably, the mixed volume ratio of the fermented grains and pure water is 1:1-5; Preferably, the fermented grains are at least one of sauce-flavor fermented grains, light-flavor fermented grains, and strong-flavor fermented grains.

6. The method according to claim 1, characterized in that In step 3, the Raman spectroscopy detection method is: The bacterial solution to be tested was sterilely washed and resuspended to adjust its concentration to about 3×10 5 / mL, and add loading buffer to mix, inject the sample, observe the cells and record the spectrum.

7. The method according to claim 1, characterized in that In step 3, the sorting and collection parameters of the Raman spectrum detection are shown in the following table:

8. The method according to claim 1, characterized in that The characteristic peak signal of the oil microorganism is 2877 cm -1 ; The threshold limit of the oily microorganisms is the total lipid content X=0.

006.

9. An application of the method according to any one of claims 1 to 8 in the detection of oil microorganisms in fermented grains, characterized in that: The method is used to improve the efficiency of single-cell Raman sorting of oil microorganisms in fermented grains.

10. The use according to claim 9, characterized in that The fermented grains are at least one of sauce-flavor fermented grains, light-flavor fermented grains, and strong-flavor fermented grains.

Images