CN116083288B - Leuconostoc mesenteroides producing 2,3-dihydro-2,2,6-trimethylbenzaldehyde and its application - Google Patents
Leuconostoc mesenteroides producing 2,3-dihydro-2,2,6-trimethylbenzaldehyde and its application Download PDFInfo
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- 241000192130 Leuconostoc mesenteroides Species 0.000 title claims abstract description 47
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- 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
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Abstract
The invention belongs to the technical field of biological fermentation, and particularly relates to leuconostoc mesenteroides for producing 2, 3-dihydro-2, 6-trimethylbenzaldehyde and application thereof. Aiming at the technical problem that few 2, 3-dihydro-2, 6-trimethylbenzaldehyde-producing microorganisms are reported, the invention provides a novel leuconostoc mesenteroides (Leuconostoc mesenteroides) strain for producing 2, 3-dihydro-2, 6-trimethylbenzaldehyde. The preservation number of the strain is CGMCC NO.25153. The strain has the content of the produced 2, 3-dihydro-2, 6-trimethyl benzaldehyde of 2.876mg/L in a fermentation medium, has short fermentation period and low cost, and has potential application in producing the 2, 3-dihydro-2, 6-trimethyl benzaldehyde.
Description
Technical Field
The invention belongs to the technical field of biological fermentation, and particularly relates to leuconostoc mesenteroides for producing 2, 3-dihydro-2, 6-trimethylbenzaldehyde and application thereof.
Background
The 2, 3-dihydro-2, 6-trimethylbenzaldehyde is also called crocin, saffron aldehyde, safflower aldehyde and saffron aldehyde, and mainly exists in crocus plants and semi-human equine plants, and can be used as a colorant and a condiment of food preparations, medicines and also can be used for preparing perfumes in crocus, siberia cornflower, chinese green tea and other plants. Researches show that the 2, 3-dihydro-2, 6-trimethyl benzaldehyde has important pharmacological effects in a plurality of systems and tissues, such as cardiovascular protection, anticonvulsant of a central nervous system, relieving clinical symptoms of AD and PD, easing pain, resisting cancer and the like, and the 2, 3-dihydro-2, 6-trimethyl benzaldehyde has obvious inhibition effect and anti-ultraviolet activity on matrix metalloproteinase, shows stronger sun protection, skin aging resistance and other advantages, is focused by students in recent years, and has wide application prospect in medicine, food and cosmetic industries.
Saffron is harsh to growth environment, and is planted in extreme weather, dry and calcized land, and at present Iran is the region with the largest planting area, but has short flowering period and extremely low yield, and under general conditions, only 0.5-0.9kg of saffron is harvested in one mu of land, the annual output in the whole world is only about 250 tons, and the investment cost is high. The 2, 3-dihydro-2, 6-trimethyl benzaldehyde is difficult to synthesize, the content of the existing saffron plant extract is low, the popularization and application of the 2, 3-dihydro-2, 6-trimethyl benzaldehyde are severely limited, and more fresh reports are provided for microorganisms producing 2, 3-dihydro-2, 6-trimethyl benzaldehyde, so that the search of strains capable of producing 2, 3-dihydro-2, 6-trimethyl benzaldehyde is of great significance in improving the yield of 2, 3-dihydro-2, 6-trimethyl benzaldehyde and researching the microbial preparation process.
CN103764818a, although disclosing methods and materials for recombinant production of saffron compounds, as well as nucleotides and polypeptides useful in establishing recombinant pathways for production of compounds such as crocin, crocin aldehyde, crocin acid or crocin ester, recombinant hosts including yeast, escherichia coli (e.coli), plant cells, mammalian cells and insect cells. However, the method only discloses the feasibility of microbial sources of 2, 3-dihydro-2, 6-trimethylbenzaldehyde, and does not consider genetic stability of recombinant microorganisms, expression stability of target genes and stability of continuous production of target products.
Disclosure of Invention
Aiming at the technical problem that few 2, 3-dihydro-2, 6-trimethylbenzaldehyde-producing microorganisms are reported, the invention provides a novel leuconostoc mesenteroides (Leuconostoc mesenteroides) strain for producing 2, 3-dihydro-2, 6-trimethylbenzaldehyde. The preservation number of the strain is CGMCC NO.25153. The preservation time is 2022 and 06 months 21, the preservation center is China general microbiological culture Collection center CGMCC, the address is China national academy of sciences of China, including the Xiyu No. 1 and the Xiyu No. 3 in the Korean area North Star of Beijing, and the postal code is 100101.
Wherein the 16S rDNA sequence of the leuconostoc mesenteroides is shown as SEQ ID NO. 1.
The leuconostoc mesenteroides is characterized in that colony forms are round or bean-shaped, the diameter of the colony is smaller than 1.0mm, the surface is smooth and milky, no pigment is generated, the cell forms are spherical, bean-shaped or short-stalk-shaped, some are arranged in pairs or short chains, do not move and have no spores, and gram staining is positive.
The leuconostoc mesenteroides has the growth conditions of microaerobic nature, good anaerobic culture growth, a growth temperature range of 2-53 ℃, strong acid resistance and a growth optimum pH of 5.5-6.5.
Wherein, the optimal growth temperature of the leuconostoc mesenteroides is 30-40 ℃.
Wherein, the leuconostoc mesenteroides can produce 2, 3-dihydro-2, 6-trimethylbenzaldehyde in a fermentation medium with the yield of 2.876mg/L.
The fermentation medium comprises 16g of glucose, 10g of tryptone, 2.5g of yeast powder, 5g of beef extract, 3.85g of beef brain, 4.9g of beef heart, 0.5g of tween 80,1g of ammonium citrate, 2.5g of sodium chloride, 2.5g of anhydrous sodium acetate, 0.05g of magnesium sulfate, 0.025g of manganese sulfate, 1.25g of disodium hydrogen phosphate and 1g of dipotassium hydrogen phosphate.
The invention also provides application of the leuconostoc mesenteroides in producing 2, 3-dihydro-2, 6-trimethylbenzaldehyde.
The invention has the beneficial effects that the invention separates a new leuconostoc mesenteroides strain with high yield of 2, 3-dihydro-2, 6-trimethylbenzaldehyde from the fermented grains sample of the Lao jiao of Luzhou for the first time. The content of 2, 3-dihydro-2, 6-trimethylbenzaldehyde produced by the strain in a fermentation medium is 2.876mg/L. The method has the advantages of short fermentation period and low cost, provides possibility of preparing the 2, 3-dihydro-2, 6-trimethyl benzaldehyde by a microbiological method, can be used as a research object of recombinant organisms, and has important significance for promoting the industrialized production of the 2, 3-dihydro-2, 6-trimethyl benzaldehyde.
The invention provides a novel leuconostoc mesenteroides strain for producing 2, 3-dihydro-2, 6-trimethylbenzaldehyde, which has a preservation number of CGMCC No.25153. The preservation time is 2022 and 06 months 21, the preservation center is China general microbiological culture Collection center CGMCC, the address is China national academy of sciences of China, including the Xiyu No. 1 and the Xiyu No. 3 in the Korean area North Star of Beijing, and the postal code is 100101. The classification was named Leuconostoc mesenteroides Leuconostoc mesenteroides.
Drawings
FIG. 1 is a mass spectrum of GC-MS molecular fragments of 2, 3-dihydro-2, 6-trimethylbenzaldehyde produced by the strain of the present invention;
FIG. 2 is a mass spectrum of GC-MS molecular fragments of 2, 3-dihydro-2, 6-trimethylbenzaldehyde standard.
Detailed Description
The invention provides a novel leuconostoc mesenteroides (Leuconostoc mesenteroides) strain for producing 2, 3-dihydro-2, 6-trimethylbenzaldehyde. The preservation number of the strain is CGMCC NO.25153. The preservation time is 2022 and 06 months 21, the preservation center is China general microbiological culture Collection center CGMCC, the address is China national academy of sciences of China, including the Xiyu No. 1 and the Xiyu No. 3 in the Korean area North Star of Beijing, and the postal code is 100101.
Wherein the 16S rDNA sequence of the leuconostoc mesenteroides is shown as SEQ ID NO. 1.
SEQ ID NO. 1 16S rDNA sequence of Leuconostoc mesenteroides
TCCTGGCTCACATCAGCTGCGATAGGTAGGGTGCCCCGTTTAGTGTGAGTGGCGAACGGGTGAGTAACACGTGGACAACCTGCCTCAAGGCTGGGGATAACATTTGGAAACAGATGCTAATACCGAATAAAACTTAGTGTCGCATGACACAAAGTTAAAAGGCGCTTCGGCGTCACCTAGAGATGGATCCGCGGTGCATTAGTTAGTTGGTGGGGTAAAGGCCTACCAAGACAATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCTGCAGTAGGGAATCTTCCACAATGGGCGAAAGCCTGATGGAGCAACGCCGCGTGTGTGATGAAGGCTTTCGGGTCGTAAAGCACTGTTGTATGGGAAGAACAGCTAGAATAGGAAATGATTTTAGTTTGACGGTACCATACCAGAAAGGGACGGCTAAATACGTGCCAGCAGCCGCGGTAATACGTATGTCCCGAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGACGGTTTATTAAGTCTGATGTGAAAGCCCGGAGCTCAACTCCGGAATGGCATTGGAAACTGGTTAACTTGAGTGCAGTAGAGGTAAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTTACTGGACTGCAACTGACGTTGAGGCTCGAAAGTGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACACCGTAAACGATGAACACTAGGTGTTAGGAGGTTTCCGCCTCTTAGTGCCGAAGCTAACGCATTAAGTGTTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTTTGAAGCTTTTAGAGATAGAAGTGTTCTCTTCGGAGACAAAGTGACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTGTTAGTTGCCAGCATTCAGATGGGCACTCTAGCGAGACTGCCGGTGACAAACCGGAGGAAGGCGGGGACGACGTCAGATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGCGTATACAACGAGTTGCCAACCCGCGAGGGTGAGCTAATCTCTTAAAGTACGTCTCAGTTCGGATTGTAGTCTGCAACTCGACTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGTTGATGTACACCCCGCCTCTCACACCATAGGCAAGGTAACCGTAGAGTTTGATCCTGGCTCAGTAAGTCGTAACAAGGTAGCCGTAGAG.
The leuconostoc mesenteroides has the biological characteristics that colony forms are round or bean-shaped, the diameter of the colony is smaller than 1.0mm, the surface is smooth and milky, no pigment is produced, the cell forms are spherical, bean-shaped or short-stalk-shaped, some are arranged in pairs or short chains, no movement and no spore are produced, and gram staining is positive.
The leuconostoc mesenteroides has good growth conditions of microaerobic anaerobic culture, the growth temperature ranges from 2 ℃ to 53 ℃, the optimal growth temperature ranges from 30 ℃ to 40 ℃, the acid resistance is strong, and the optimal growth pH is 5.5 to 6.5.
The invention also provides a screening and identifying method of the leuconostoc mesenteroides, which comprises the following steps of scattering, separating, marking, purifying and preserving strain of a luzhou Laojiao fermented grain sample, and obtaining the leuconostoc mesenteroides producing 2, 3-dihydro-2, 6-trimethylbenzaldehyde by taking the capability of producing 2, 3-dihydro-2, 6-trimethylbenzaldehyde as a screening standard.
The fermented grain sample is diluted by taking suspension after being scattered by shaking, and a plurality of gradient dilutions are coated on a solid culture medium flat plate so as to perform anaerobic culture. The micro-aerobic bacteria can grow well in an anaerobic state, and anaerobic culture is selected during screening.
Wherein, single colonies with different colony morphologies are picked on a plate on which colonies are grown, and all single colonies are purified by secondary streaking.
Wherein each strain after purification is subjected to stationary culture in MRS liquid medium in the form of single colony.
The method comprises the steps of respectively fermenting different strains to obtain fermentation bacteria liquid, extracting and enriching a target compound by using a headspace solid-phase microextraction method, and then performing chromatographic detection analysis by using GC-MS (gas chromatography-mass spectrometry) to screen and obtain the leuconostoc mesenteroides with high yield of 2, 3-dihydro-2, 6-trimethyl benzaldehyde by comparing the content of volatile substances.
The invention also provides application of the leuconostoc mesenteroides in producing 2, 3-dihydro-2, 6-trimethylbenzaldehyde.
Specifically, the application is that leuconostoc mesenteroides is subjected to static culture in a fermentation medium at 37 ℃ for 2 days, the pH value is 6.0, and the inoculation amount of the leuconostoc mesenteroides is 2% (. Times.10 7 cfu/mL).
More specifically, the fermentation medium comprises 16g of glucose, 10g of tryptone, 2.5g of yeast powder, 5g of beef extract, 3.85g of beef brain, 4.9g of beef heart, 0.5g of tween 80,1g of ammonium citrate, 2.5g of sodium chloride, 2.5g of anhydrous sodium acetate, 0.05g of magnesium sulfate, 0.025g of manganese sulfate, 1.25g of disodium hydrogen phosphate and 1g of dipotassium hydrogen phosphate per liter of medium.
By adopting the fermentation process, the content of 2, 3-dihydro-2, 6-trimethylbenzaldehyde produced by the strain is 2.876mg/L.
The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The medium formulation referred to in the examples:
MRS liquid culture medium comprises 20g/L glucose, 10g/L peptone, 4g/L yeast powder, 5g/L beef extract powder, 1g/L Tween 80, 2g/L triammonium citrate, 5g/L sodium acetate, 0.2g/L magnesium sulfate, 0.05g/L manganese sulfate, 1g/L dipotassium hydrogen phosphate, pH6.0 and high-pressure steam sterilization at 115 ℃ for 20 minutes.
MRS solid Medium 15g/L agar was added on the basis of MRS liquid Medium and autoclaved at 115℃for 20 minutes.
The fermentation medium comprises 16g/L of glucose, 10g/L of tryptone, 2.5g/L of yeast powder, 5g/L of beef extract, 3.85g/L of beef brain, 4.9g/L of beef heart, 0.5g/L of tween 80, 1g/L of ammonium citrate, 2.5g/L of sodium chloride, 2.5g/L of anhydrous sodium acetate, 0.05g/L of magnesium sulfate, 0.025g/L of manganese sulfate, 1.25g/L of disodium hydrogen phosphate, 1g/L of dipotassium hydrogen phosphate and sterilizing by high-pressure steam at the pH of 6.0,115 ℃ for 20 minutes.
EXAMPLE 1 isolation and purification of strains
(1) The separation method comprises taking 20g of fermented grains of Luzhou Laojiao, placing into a conical flask containing 180mL sterile distilled water, shaking with a constant temperature shaking table for 10min, and thoroughly dispersing and mixing. Taking 1mL of sample suspension, diluting to 10 -2～10-7 by adopting a double-ratio dilution method, sucking 100 mu L of the diluted solution of each gradient, uniformly coating the diluted solution on an MRS solid culture medium plate, preparing two plates in parallel, inverting, culturing for 36-48h under anaerobic condition at 37 ℃ and observing in time.
(2) And (3) streaking and purifying, namely taking out a plate with the colonies, picking single colonies with different colony morphologies, and streaking for the second time until all the single colonies are purified.
(3) And (3) preserving the strain, namely picking single colonies of each strain after purification into a 5mLMRS liquid culture medium, standing and culturing for 20-24 hours at 37 ℃ under anaerobic condition, sucking 1mL of bacterial liquid into a bacteria-preserving tube, adding 0.5mL of 60% sterile glycerol solution, re-suspending, and preserving at-80 ℃.
Example 2 detection of 2, 3-dihydro-2, 6-trimethylbenzaldehyde producing ability
(1) Preparing a bacterial liquid to be tested:
after the glycerol preservation tube of the strain obtained by screening is dissolved, the glycerol preservation tube is respectively inoculated into an MRS liquid culture medium, and is subjected to static culture for 24 hours at 37 ℃, and after three generations of activation, the glycerol preservation tube is inoculated into a 50mLMRS liquid culture medium according to the inoculum size of 2% (volume ratio) for culture for 24 hours, so that the bacterial liquid to be detected is obtained.
(2) HS-SPME/GC-MS method for detecting 2, 3-dihydro-2, 6-trimethylbenzaldehyde:
Taking supernatant, adding the supernatant into a headspace bottle, adding saturated NaCl solution, taking 0.822mg/ml of 2-octanol as an internal standard substance, carrying out heat preservation and balance on the prepared sample at 60 ℃ for 5min, extracting the sample at 60 ℃ for 50min by using a 50/30 mu mDVB/CAR/PDMS extraction head, and desorbing the sample at 250 ℃ for 5min at a GC sample inlet after the extraction is finished. And (5) matching the compound search result with an NIST standard spectrum library, and confirming that the similarity reaches more than 80% as a target compound. The culture solution without bacteria is used as a blank control group to calculate the content of each volatile substance.
GC-MS detection chromatographic conditions:
The gas chromatography conditions are HP-INNOWAX chromatographic column (60 m×0.25mm×0.25 μm), heating program, the initial temperature is 40 ℃, the temperature is kept for 5min, the temperature is raised to 100 ℃ at 4 ℃ per min, the temperature is raised to 230 ℃ at 6 ℃ per min, the temperature is kept for 10min, the carrier gas is high-purity helium (1.0 mL/min), and the temperature of the sample inlet is 250 ℃ without split flow.
The mass spectrum conditions comprise an electron ionization source, electron energy of 70eV, electron multiplier voltage of 350V, ion source temperature of 230 ℃, transmission line temperature of 250 ℃ and mass range of 40-450 m/z.
By the detection method, a lactobacillus with stronger capability of producing 2, 3-dihydro-2, 6-trimethylbenzaldehyde is screened from the separated strain, and the strain is selected for further research.
Example 3 molecular characterization of strains
And (3) amplifying and culturing the target strain, taking fresh bacterial liquid in the logarithmic growth phase, centrifugally collecting bacterial cells, and extracting genome DNA by using a bacterial genome extraction kit. The full-length sequence of the 16S rDNA is amplified by adopting a lactobacillus universal primer 27F/1541R, and the method is concretely as follows:
SEQ ID NO:2:27F(5′-AGAGTTTGATCCTGGCTCAG-3′)
SEQ ID NO:3:1541R(5′-AAGGAGGTGATCCAGCC-3′)
① Reaction system (50 μl)
② Reaction procedure
The PCR reaction of the "denaturation-annealing-extension" was performed 35 times in the above procedure, and the PCR product was checked by electrophoresis on a 1.0% agarose gel at a voltage of about 11V/cm for 20min.
The purification of PCR products was performed as described in the Shanghai Biotechnology Co small amount of gel recovery PCR product purification kit, and sequencing was performed by Shanghai Biotechnology Co.
The gene sequence of the 16S rDNA fragment obtained by sequencing is compared by BLAST of NCBI, the strain species information is determined, the strain species information is identified as leuconostoc mesenteroides (Leuconostoc mesenteroides), and the leuconostoc mesenteroides is preserved in the preservation center of China general microbiological culture collection center (CGMCC) in the year 2022 and the month 21, and the preservation number is CGMCC NO.25153. And is designated Leuconostoc mesenteroides zqw121,121.
Example 4 physiological and Biochemical characteristics of Leuconostoc mesenteroides zqw121,121
Leuconostoc mesenteroides zqw121,121 was inoculated to MRS solid medium, and after 2 days of stationary culture at 37 ℃, the colony and cell morphology of the strain were observed.
Measuring temperature suitable for growth, namely inoculating Leuconostoc mesenteroides zqw121 in logarithmic growth phase into MRS liquid culture medium according to 2% of inoculum size, and respectively culturing at 2, 10, 15, 20, 30, 40, 45 and 53 ℃ for 18 hours to measure the OD 600 value of the bacterial liquid;
the pH tolerance measurement comprises adjusting MRS liquid culture medium pH to 3.0, 5.0, 5.5, 6.0, 6.5 and 7.0 respectively with 2M hydrochloric acid and 1M sodium hydroxide, inoculating Leuconostoc mesenteroides zqw121 in logarithmic phase, mixing completely and uniformly, culturing at 37deg.C for 18 hr, and measuring bacterial liquid OD 600;
the effect of oxygen on bacterial growth was determined by submerged agar method by inoculating Leuconostoc mesenteroides zqw, 121 in MRS solid medium tube, culturing at 37deg.C for 2 days, and judging the oxygen demand of various bacteria according to the growth position of strain in tube medium.
Experimental results show that the leuconostoc mesenteroides zqw121,121 has the biological characteristics that colony forms are round or bean-shaped, the diameter of the colony is smaller than 1.0mm, the surface is smooth and milky, no pigment is produced, the cell forms are spherical, bean-shaped or short-stalk-shaped, some are paired or arranged in short chain, do not move and have no spores, and gram staining is positive.
The Leuconostoc mesenteroides zqw121 has the growth conditions of microaerobic nature, good anaerobic culture growth, the growth temperature range of 2-53 ℃, the optimal growth temperature of 30-40 ℃ and the optimal growth pH of 5.5-6.5.
EXAMPLE 5 fermentation culture of Leuconostoc mesenteroides zqw121
After the obtained leuconostoc mesenteroides zqw-121 glycerol storage tube was dissolved, the obtained leuconostoc mesenteroides was inoculated into 10mL of a liquid-filled MRS liquid medium according to an inoculum size of 5%, and after 24 hours of stationary culture at 37 ℃, the obtained leuconostoc mesenteroides was inoculated into 400mL of a fermentation medium according to an inoculum size of 2%, and then stationary culture was carried out for 2 days at 37 ℃. The detection method was the same as the volatile component measurement method in example 2. After continuous culture of Leuconostoc mesenteroides for 2 days, the yield of the produced 2, 3-dihydro-2, 6-trimethylbenzaldehyde can reach 2.876mg/L.
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