CN112048456A - High-throughput method for screening microorganisms producing aldehyde or ketone flavor substances - Google Patents
High-throughput method for screening microorganisms producing aldehyde or ketone flavor substances Download PDFInfo
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- 239000000126 substance Substances 0.000 title claims abstract description 47
- 238000000034 method Methods 0.000 title claims abstract description 38
- 150000001299 aldehydes Chemical class 0.000 title claims abstract description 34
- 150000002576 ketones Chemical class 0.000 title claims abstract description 34
- 238000012216 screening Methods 0.000 title claims abstract description 33
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- 235000019634 flavors Nutrition 0.000 title claims abstract description 24
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- MIDXCONKKJTLDX-UHFFFAOYSA-N 3,5-dimethylcyclopentane-1,2-dione Chemical compound CC1CC(C)C(=O)C1=O MIDXCONKKJTLDX-UHFFFAOYSA-N 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention discloses a high-throughput method for screening microorganisms capable of producing aldehyde or ketone flavor substances, which comprises the following steps: collecting, dyeing and sorting microorganisms on unrolled cigar leaves in fermentation, and then verifying the screening effect by determining the concentration of aldehyde/ketone substances in microbial products and identifying the types of the aldehyde/ketone substances, wherein the yield of the aldehyde/ketone substances of the screened strains is 50-300 mu g/mL after the culture, the main metabolites are benzaldehyde, isophorone, 2, 4-dimethylbenzaldehyde, hydroxyacetone, phenylacetaldehyde, vanillin and isovaleraldehyde, the method can complete one-time separation culture within 3 days, and the yield of the aldehyde/ketone substances of 49 strains is 50-100 mu g/mL, and the yield of the aldehyde/ketone substances of 57 strains is 300 mu g/mL, so that the step of screening the microorganisms with practical application values is accelerated, the industrial application time is shortened, and the cost is saved.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a high-throughput method for screening microorganisms capable of producing aldehyde or ketone flavor substances.
Background
Flavoring substances are widely used in the food, cosmetic, chemical and pharmaceutical industries to supplement, enhance or modify the original taste/aroma of the product, and are of great importance in the consumer market. These compounds have extremely high volatility and chemical diversity, including lactones, hydrocarbons, alcohols, ketones, vanillin, terpenes, aldehydes and esters. Aldehyde ketone flavor substances (such as vanillin, benzaldehyde, phenylacetaldehyde and the like) are flavors which are widely utilized in the market at present; the aldehyde has pungent and pungent odor and has a function of harmonizing the fragrance of the soy sauce; ketones are not flavor substances per se, but small amounts of short-chain carbonyl compounds have a fruity, sweet-like caramel odor; phenylacetaldehyde has fragrance similar to hyacinth and lilac, and benzaldehyde has fragrance similar to dried almond. Currently, the main production method of these aroma compounds is chemical, because of the low concentration of the compounds extracted directly from nature, the high amount of impurities and often the geographical and seasonal limitations.
With the increasing awareness of health and nutrition, consumers are increasingly liking flavour compounds which can be labelled as "natural", which has prompted the emergence of new production methods, for example the biotechnological production of flavour substances. The bioconversion is performed by the action of microorganisms and enzymes, and the newly isolated microorganisms have more advantages than engineered microorganisms, having a wide variety of metabolic characteristics. However, it is not easy to isolate microorganisms that produce specific natural flavor compounds from complex ecosystems, especially if these organisms do not have a phenotype that is easily selectable.
Cigar fermentation is a traditional mixed bacterial fermentation, and can improve the quality of cigars through the metabolism of microorganisms. The natural microflora plays a crucial role in the formation of flavour compounds and the degradation of harmful substances. Aldehydes and ketones (e.g., benzaldehyde, phenylacetaldehyde, ionone and megastigmatrienone) are essential flavor substances in cigar leaves, most of which are produced by microbial metabolism during tobacco fermentation, and thus the present application screens flavor-producing microorganisms using fermented tobacco leaves as a screening sample. The high-throughput method is adopted to screen the strains, so that the working efficiency can be improved, the cost is reduced, the screening speed can be greatly improved, and the method has important significance for improving the product quality.
Disclosure of Invention
The invention aims to solve the problems and provide a high-throughput method for screening microorganisms producing aldehyde or ketone flavor substances.
The fluorescent dye cy5-hydrazide can be specifically combined with carbonyl bonds, a fluorescent substance can specifically mark strains of aldehyde ketone substances generated in the cigar fermentation process after entering cells, in addition, 2, 4-nitrophenylhydrazine can react with aldehyde or ketone to generate yellow, orange or red 2, 4-nitrophenylhydrazone precipitate, the yellow, orange or red 2, 4-nitrophenylhydrazone precipitate can be used for detecting the concentration of the aldehyde ketone substances, and the combination of the two detection modes can effectively improve the screening efficiency.
The invention realizes the purpose through the following technical scheme:
a high throughput method of screening for an aldehyde or ketone flavor producing microorganism, the method comprising: collecting, dyeing and sorting microorganisms on unrolled cigar leaves in fermentation, and then verifying the screening effect by determining the concentration of aldehyde/ketone substances in microbial products and identifying the types of the aldehyde/ketone substances, wherein the yield of the aldehyde/ketone substances of the screened strains is 50-300 mu g/mL after the screened strains are cultured, and the main metabolites are benzaldehyde, isophorone, 2, 4-dimethylbenzaldehyde, hydroxyacetone, phenylacetaldehyde, vanillin and isovaleraldehyde, namely, the method can be effectively used for separating and screening the strains producing the aldehyde-ketone flavor substances.
The method comprises the following specific steps:
(1) weighing tobacco leaves, adding sterilized normal saline or neutral phosphate buffer solution, oscillating to separate the tobacco leaves and microorganisms, and centrifugally collecting the microorganisms;
(2) re-dissolving the collected microbes, adding a fluorescent dye cy5-hydrazide, and dyeing in a dark place;
(3) differential centrifugation is carried out to separate microorganisms and unbound dye, the staining condition of the strain is detected by a fluorescence microscope, and then the strain marked by fluorescence is sorted into a 96 deep-well plate filled with YH culture medium for culture;
(4) determining the content of aldehyde ketone compounds in the culture medium by using 2, 4-nitrophenylhydrazine, and identifying the type of the product by using GC-MS;
(5) the species of the selected microorganism was then identified based on 16s rRNA.
In another preferred example, the collection of the microorganisms on the tobacco leaves in the step (1) is to weigh 1-5g of tobacco leaves, add sterilized normal saline or neutral phosphate buffer solution in a ratio of 1: 40-50(g/v), oscillate for 1-2h at a rotation speed of 200-.
In another preferred example, the microorganism is stained in step (2) by adding 1-2mL of buffer solution to resuspend the microorganism, adding the fluorescent dye cy5-hydrazide into the system, wherein the dye concentration is 1-2mg/mL, and staining for 1-2h in the dark.
In another preferred example, in the step (3), the strain with higher fluorescence intensity is screened by a flow cytometer.
In another preferred example, the YH medium of step (3) comprises the following components: 20g/L glucose, 10g/L yeast powder, 20g/L peptone, 10g/L soybean peptone and MgSO40.5g/L，KH2PO41g/L，CaCl20.2g/L。
In another preferred example, the culturing in the step (3) is performed under conditions of 35-45 ℃ and 600-800rpm for 36-48 h.
In another preferred example, the concentration of the aldone compound in step (4) is determined by adding 50. mu.L of 2, 4-nitrophenylhydrazine to 50. mu.L of culture supernatant, standing for 30min, adding 500. mu.L of KOH (1mol/L) and 400. mu.L of deionized water, and determining the absorbance value at 500 nm.
In another preferred example, the identification method of the aldehyde ketone compound in the step (4) is that 500. mu.L of culture supernatant is frozen in liquid nitrogen, and then freeze-dried in a vacuum freezer, and then 500. mu.L of absolute ethanol is added for redissolution, and then 500. mu.L of acetone is added for protein removal, and then the compound is identified by GC-MS.
The second purpose of the invention is to provide the application of the method in microorganism screening, aldehyde ketone substance development and production and natural flavor substance production.
The invention has the beneficial effects that:
(1) the method has the characteristics of small screening system, large flux, simple and convenient operation, easy repetition, high efficiency and the like, and can effectively solve the problems of less screening methods of strains producing natural flavor substances, less strains producing natural flavor substances, difficult production of natural flavor substances and the like in the current market.
(2) By applying the method of the invention, one-time separation culture is completed within 3 days, the yield of aldehyde ketone substances of 49 strains of bacteria is 50-100 mu g/mL, and the yield of aldehyde ketone substances of 57 strains of bacteria is 300 mu g/mL, thereby quickening the step of screening the microorganisms with practical application value, shortening the industrial application time and saving the cost.
(3) The metabolites of the strains screened by the method are identified and analyzed, the main metabolites are benzaldehyde, isophorone, 2, 4-dimethylbenzaldehyde, hydroxyacetone, phenylacetaldehyde, vanillin and isovaleraldehyde, which are the most commonly used flavor additives in the market at present, but the methods for producing the substances through biotechnology are few, and the method disclosed by the invention is applied to microbial screening, aldehyde ketone substance development and production and natural flavor substance production.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the embodiments or the drawings needed to be practical in the prior art description, and obviously, the drawings in the following description are only some embodiments of the embodiments, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1: the invention has the screening process;
FIG. 2: performing fluorescence imaging and sorting on the strains;
FIG. 3: standard curve of aldehyde ketone compound concentration and aldehyde ketone substance yield of partial strain.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.
Example 1 isolation and staining of microorganisms on tobacco leaves
The method comprises the steps of weighing 5g of tobacco leaves in a fermentation process, adding 100mL of sterilized normal saline, oscillating and eluting for 2 hours in a shaking table at 10 ℃, separating tobacco leaf fragments and bacterial liquid, centrifugally collecting thalli, washing for 2-3 times by using the normal saline, oscillating and uniformly mixing the thalli, dyeing, adding a fluorescent dye cy5-hydrazide to the concentration of 1-2mg/L, placing for 1 hour in a dark place, carrying out differential centrifugal collection and separation on microorganisms and the fluorescent dye, washing for 1-2 times by using the normal saline, removing the dye which is not combined, collecting strains for further analysis, observing the dyeing condition of cells under a fluorescent microscope, and sorting one part of the strains in a flow cytometer.
Example 2 fluorescence imaging and sorting of strains
To verify whether the strain was labeled, staining of the strain was first examined by fluorescence microscopy and the staining of the cells was analyzed using a Nikon Eclipse Ti microscope equipped with a 60X/1.40 oil objective (Nikon) and a Nikon Eclipse Ti camera. After cell staining was determined, cell sorting was performed using a flow cytometer, and the sample was first vortexed to obtain single cells. Fluorescence detection and cell sorting were then performed by forward scattering (FSC-SSC) using BD-FACSAria III equipped with 70 μm nozzles and a 488nm laser. Gating parameters were selected for each experiment and sample based on FSC-SSC and fluorescence levels. Negative controls included unstained microbial populations (check for autofluorescence), and samples treated in parallel with the sample but without the microorganisms (check for fluorescent precipitates). The cells were sorted into 96 shallow well plates (200. mu.L per well), cultured at 37 ℃ at 220rpm for 12 hours, and then transferred to 96 deep well plates (1 mL per well) for fermentation.
Example 3 Standard Curve for aldehyde ketone Compound concentration and measurement of product content
After the strain is fermented for 36-48h, 50 mu L of fermentation supernatant is mixed with 2, 4-dinitrophenylhydrazine with the same volume, the mixture reacts for 30 minutes at room temperature, then 500 mu L of KOH (1mol/L) and 400 mu L of distilled water are added, and the absorbance of the reddish brown solution is measured by a microplate reader at 500 nm. In addition, acetone is used as a substrate, the absorbance of the acetone at different concentrations is measured, and a linear regression standard curve of carbonyl concentration and absorbance is drawn for calculating the concentration of the aldehyde ketone substance in the strain product (see fig. 3).
Example 4 product identification
After the content of aldehyde and ketone in the fermentation supernatant is measured, a gas chromatography-mass spectrometer is used for identifying a product with the content of more than 200mg/mL, 500 mu L of the fermentation supernatant is frozen in liquid nitrogen, dried by a vacuum freezer, then 500 mu L of ethanol is added for resuspension, 100 mu L of acetone is added for high-speed centrifugation to remove protein, and the supernatant is transferred to a GC-MS system after passing through a 0.22 mu m filter membrane. The fractions of the product were detected by means of a DB-5MS (60 m. times.id 0.25 mm. times.film thickness 0.25 μm) column. The detection conditions of the gas chromatography are as follows: helium C-60 flow rate was 1mL/min and the injector port was heated at 250 ℃. The oven temperature was first fixed at 40 ℃ for 2 minutes and then increased to 250 ℃ at a rate of 10 ℃/min for 5 minutes. The transport line temperature and the ion source temperature were 280 ℃ and 210 ℃ respectively. The ionization mode is Electron Impact (EI), and the EI voltage is 70 eV. In full scan mode, a mass scan range of 33-400m/z is used at an acquisition rate of 10 scans per second. Metabolite identification was accomplished by comparing mass spectra to national institute of standards and technology (NIST 14) database spectral libraries and comparing experimental and theoretical (literature) Kovats indices.
Example 5 Strain identification
After product identification, 16s rRNA sequence sequencing was used to identify the aroma-producing strains, genomic DNA was extracted from the sorted strains using a bacterial genome extraction kit, amplified and sequenced using primers 27F (5 '-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'), and sequence similarity analysis was performed using the blast program, with the results shown in Table 1;
by applying the method of the invention, one-time separation culture is completed within 3 days, the yield of aldehyde ketone substances of 49 strains of bacteria is 50-100 mu g/mL, and the yield of aldehyde ketone substances of 57 strains of bacteria is 300 mu g/mL, thereby quickening the step of screening the microorganisms with practical application value, shortening the industrial application time and saving the cost.
The metabolites of the strains screened by the method are identified and analyzed, the main metabolites are benzaldehyde, isophorone, 2, 4-dimethylbenzaldehyde, hydroxyacetone, phenylacetaldehyde, vanillin and isovaleraldehyde, which are the most commonly used flavor additives in the market at present, but the methods for producing the substances through biotechnology are few, and the method disclosed by the invention is applied to microbial screening, aldehyde ketone substance development and production and natural flavor substance production.
Table 1: the concentration and species of the metabolite of the strain and the strain species
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims. It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition. In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.
Claims (10)
1. A high throughput method for screening microorganisms for producing aldehydes or ketones, comprising:
collecting, dyeing and sorting microorganisms on unrolled cigar leaves in fermentation, and then verifying the screening effect by determining the concentration of aldehyde/ketone substances in microbial products and identifying the types of the aldehyde/ketone substances, wherein the yield of the aldehyde/ketone substances of the screened strains is 50-300 mu g/mL after the screened strains are cultured, and the main metabolites are benzaldehyde, isophorone, 2, 4-dimethylbenzaldehyde, hydroxyacetone, phenylacetaldehyde, vanillin and isovaleraldehyde, namely, the method can be effectively used for separating and screening the strains producing the aldehyde-ketone flavor substances.
2. A high-throughput method for screening microorganisms capable of producing aldehydes or ketones according to claim 1, wherein the method comprises the following steps:
(1) weighing tobacco leaves, adding sterilized normal saline or neutral phosphate buffer solution, oscillating to separate the tobacco leaves and microorganisms, and centrifugally collecting the microorganisms;
(2) re-dissolving the collected microbes, adding a fluorescent dye cy5-hydrazide, and dyeing in a dark place;
(3) differential centrifugation is carried out to separate microorganisms and unbound dye, the staining condition of the strain is detected by a fluorescence microscope, and then the strain marked by fluorescence is sorted into a 96 deep-well plate filled with YH culture medium by a flow cytometer for culture;
(4) determining the content of aldehyde ketone compounds in the culture medium by using 2, 4-nitrophenylhydrazine, and identifying the type of the product by using GC-MS;
(5) the species of the selected microorganism was then identified based on 16s rRNA.
3. The high-throughput method for screening microorganisms capable of producing aldehydes or ketones according to claim 2, wherein the collection of microorganisms on the tobacco leaves in step (1) comprises weighing 1-5g of tobacco leaves, adding sterilized normal saline or neutral phosphate buffer solution at a ratio of 1: 40-50(g/v), oscillating at a rotation speed of 200-250rmp for 1-2h, separating tobacco leaf fragments and microorganisms, and collecting microorganisms at a rotation speed of 4000-5000 rmp.
4. The high-throughput method for screening microorganisms capable of producing aldehydes or ketones according to claim 2, wherein the microorganisms are stained in step (2) by adding 1-2mL of buffer solution to resuspend the microorganisms, adding the fluorescent dye cy5-hydrazide to the system, wherein the concentration of the dye is 1-2mg/mL, and staining the system in the dark for 1-2 h.
5. The high-throughput method for screening microorganisms capable of producing aldehydes or ketones according to claim 2, wherein the strains having higher fluorescence intensity are screened by flow cytometry in the step (3).
6. The high-throughput method for screening microorganisms capable of producing aldehydes or ketones according to claim 2, wherein the YH medium of step (3) comprises the following components: 20g/L glucose, 10g/L yeast powder, 20g/L peptone, 10g/L soybean peptone and MgSO4 0.5g/L，KH2PO4 1g/L，CaCl2 0.2g/L。
7. The high-throughput method for screening microorganisms capable of producing aldehydes or ketones according to claim 2, wherein the culturing in step (3) is carried out under conditions of 35-45 ℃, 600-800rpm for 36-48 h.
8. The high-throughput method for screening microorganisms capable of producing aldehydes or ketones according to claim 2, wherein the concentration of aldehydes or ketones in step (4) is determined by adding 50. mu.L of 2, 4-nitrophenylhydrazine to 50. mu.L of culture supernatant, allowing the mixture to stand for 30min, adding 500. mu.L of KOH (1mol/L) and 400. mu.L of deionized water, and determining the absorbance at 500 nm.
9. The high-throughput method for screening microorganisms capable of producing aldehydes or ketones according to claim 2, wherein the identification of the aldehydes or ketones in step (4) is carried out by freezing 500. mu.L of culture supernatant in liquid nitrogen, freeze-drying in a vacuum freezer, adding 500. mu.L of absolute ethanol for reconstitution, adding 500. mu.L of acetone for deproteinization, and identifying the compounds by GC-MS.
10. Use of the method according to any one of claims 1 to 9 for screening microorganisms, developing and producing aldehydes and ketones, and producing natural flavors.
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