Leuconostoc mesenteroides producing 2,3-dihydro-2,2,6-trimethylbenzaldehyde and its application

Abstract

The present invention belongs to the field of biological fermentation technology, and in particular to Leuconostoc mesenteroides and its application for producing 2,3-dihydro-2,2,6-trimethylbenzaldehyde. In view of the technical problem that there are few reports of microorganisms producing 2,3-dihydro-2,2,6-trimethylbenzaldehyde, the present invention provides a new strain of Leuconostoc mesenteroides producing 2,3-dihydro-2,2,6-trimethylbenzaldehyde. The strain is deposited as CGMCC NO.25153. In the fermentation medium, the strain produces 2,3-dihydro-2,2,6-trimethylbenzaldehyde in a content of 2.876 mg / L, and the fermentation cycle is short and the cost is low, and it has potential application in producing 2,3-dihydro-2,2,6-trimethylbenzaldehyde.

Description

Technical Field

[0001] The invention belongs to the technical field of biological fermentation, and particularly relates to Leuconostoc mesenteroides producing 2,3-dihydro-2,2,6-trimethylbenzaldehyde and application thereof. Background Art

[0002] 2,3-Dihydro-2,2,6-trimethylbenzaldehyde, also known as crocinaldehyde, crocinaldehyde, crocinaldehyde, and crocinaldehyde, is mainly found in plants of the genus Crocus and Centaurus. It can be used as a colorant and seasoning for food preparations, as a medicine, and can also be used in the preparation of perfumes. Studies have shown that 2,3-dihydro-2,2,6-trimethylbenzaldehyde has important pharmacological effects in multiple systems and tissues due to its antioxidant, oxidative stress inhibition, anti-inflammatory, and anti-apoptotic effects, such as cardiovascular protection, central nervous system anticonvulsant, relief of clinical symptoms of AD and PD, analgesia, and anti-cancer. In addition, 2,3-dihydro-2,2,6-trimethylbenzaldehyde has a significant inhibitory effect on matrix metalloproteinases and anti-ultraviolet activity, showing strong advantages in sun protection and anti-skin aging. It has attracted much attention from scholars in recent years and has broad application prospects in the pharmaceutical, food, and cosmetics industries.

[0003] Saffron has a demanding growth environment. Its cultivation requires extreme weather and dry and calcified land. Currently, Iran is the largest region with the largest planting area, but its flowering period is short and its yield is extremely low. Generally, only 0.5-0.9 kg of saffron can be harvested from one acre of land. The annual output in the world is only about 250 tons, and the investment cost is high. In addition, the synthesis of 2,3-dihydro-2,2,6-trimethylbenzaldehyde is difficult. The existing saffron plant extract has a low content, which seriously limits the promotion and application of 2,3-dihydro-2,2,6-trimethylbenzaldehyde. There are few reports on microorganisms that produce 2,3-dihydro-2,2,6-trimethylbenzaldehyde. Therefore, finding strains that can produce 2,3-dihydro-2,2,6-trimethylbenzaldehyde is of great significance to the research on improving the yield of 2,3-dihydro-2,2,6-trimethylbenzaldehyde and the microbial preparation process.

[0004] Although CN103764818A discloses methods and materials for recombinant production of saffron compounds, as well as nucleotides and polypeptides that can be used to establish a recombinant pathway for producing compounds such as crocin, crocinaldehyde, crocin, crocetic acid or crocetate, and recombinant hosts, including yeast, Escherichia coli (E. coli), plant cells, mammalian cells and insect cells, this patent method only discloses the feasibility of the microbial source of 2,3-dihydro-2,2,6-trimethylbenzaldehyde, and does not consider the genetic stability of the recombinant microorganism, the stability of the expression of the target gene, and the stability of the continuous production of the target product. Summary of the invention

[0005] In view of the technical problem that there are few reports on microorganisms producing 2,3-dihydro-2,2,6-trimethylbenzaldehyde, the present invention provides a new strain of Leuconostoc mesenteroides that produces 2,3-dihydro-2,2,6-trimethylbenzaldehyde. The strain is deposited with a number of CGMCC NO.25153. The deposit time is June 21, 2022, and the deposit center is the General Microbiology Center of the China Microbiological Culture Collection Administration CGMCC, with an address of Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a zip code of 100101.

[0006] The 16S rDNA sequence of the above-mentioned Leuconostoc mesenteroides is shown in SEQ ID NO:1.

[0007] Among them, the biological characteristics of the above-mentioned Enteric Leuconostoc are: the colony morphology is round or bean-shaped, the colony diameter is less than 1.0 mm, the surface is smooth, milky white, and no pigment is produced; the cell morphology is spherical, bean-shaped or short-stalked, some are arranged in pairs or in short chains, non-motile, and without spores; Gram staining is positive.

[0008] Among them, the growth conditions of the above-mentioned Enteric Leuconostoc mesenteroides are: microaerobic, good growth in anaerobic culture; growth temperature range of 2°C to 53°C; strong acid resistance, and the optimal pH for growth is 5.5 to 6.5.

[0009] Among them, the optimal growth temperature of the above-mentioned Enterococcus mesenteroides is 30℃~40℃.

[0010] Among them, the above-mentioned Leuconostoc mesenteroides has a high yield of 2,3-dihydro-2,2,6-trimethylbenzaldehyde in the fermentation medium, and its yield is 2.876 mg / L.

[0011] The fermentation medium comprises 16 g glucose, 10 g tryptone, 2.5 g yeast powder, 5 g beef extract, 3.85 g bovine brain, 4.9 g bovine heart, 0.5 g Tween 80, 1 g ammonium citrate, 2.5 g sodium chloride, 2.5 g anhydrous sodium acetate, 0.05 g magnesium sulfate, 0.025 g manganese sulfate, 1.25 g disodium hydrogen phosphate and 1 g dipotassium hydrogen phosphate per liter of medium.

[0012] The present invention also provides application of the above-mentioned Leuconostoc mesenteroides in producing 2,3-dihydro-2,2,6-trimethylbenzaldehyde.

[0013] Beneficial effects: The present invention is the first to isolate a new strain of Leuconostoc mesenteroides that produces high 2,3-dihydro-2,2,6-trimethylbenzaldehyde from a sample of Luzhou Laojiao fermented grains. The strain produces 2.876 mg / L of 2,3-dihydro-2,2,6-trimethylbenzaldehyde in the fermentation medium. The fermentation cycle is short and the cost is low, which provides the possibility of preparing 2,3-dihydro-2,2,6-trimethylbenzaldehyde by microbial method. It can also be used as a research object for recombinant organisms, which is of great significance for promoting the industrial production of 2,3-dihydro-2,2,6-trimethylbenzaldehyde.

[0014] The present invention provides a new strain of Leuconostoc mesenteroides producing 2,3-dihydro-2,2,6-trimethylbenzaldehyde, with a deposit number of CGMCC NO.25153. The deposit time is June 21, 2022, and the deposit center is the General Microbiological Center of China Microbiological Culture Collection Administration CGMCC, the address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, zip code 100101. Classification name: Leuconostoc mesenteroides. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the GC-MS molecular fragment mass spectrum of 2,3-dihydro-2,2,6-trimethylbenzaldehyde produced by the strain of the present invention;

[0016] Figure 2 This is the GC-MS molecular fragmentation mass spectrum of 2,3-dihydro-2,2,6-trimethylbenzaldehyde standard. DETAILED DESCRIPTION

[0017] The present invention provides a new strain of Leuconostoc mesenteroides that produces 2,3-dihydro-2,2,6-trimethylbenzaldehyde. The strain is deposited with a number of CGMCC NO.25153. The deposit date is June 21, 2022, and the deposit center is the General Microbiological Center of the China Microbiological Culture Collection Administration CGMCC, with an address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, zip code 100101.

[0018] The 16S rDNA sequence of the above-mentioned Leuconostoc mesenteroides is shown in SEQ ID NO:1.

[0019] SEQ ID NO:1 16S rDNA sequence of Leuconostoc mesenteroides

[0020]

[0021] The biological characteristics of the enteric Leuconostoc mesenteroides are as follows: the colony morphology is round or bean-shaped, the colony diameter is less than 1.0 mm, the surface is smooth, milky white, and no pigment is produced; the cell morphology is spherical, bean-shaped or short stalk-shaped, some are arranged in pairs or in short chains, are non-motile, and have no spores; Gram staining is positive.

[0022] The growth conditions of the enteric Leuconostoc mesenteroides are as follows: microaerobic, good growth in anaerobic culture; growth temperature range is 2°C to 53°C, the optimum growth temperature is 30°C to 40°C; strong acid resistance, the optimum growth pH is 5.5 to 6.5.

[0023] The present invention also provides a screening and identification method for the above-mentioned Leuconostoc mesenteroides, comprising the following steps: breaking up and separating, streaking and purifying, and preserving the strain of Luzhou Laojiao fermented grains sample, and then using the ability to produce 2,3-dihydro-2,2,6-trimethylbenzaldehyde as a screening standard to obtain Leuconostoc mesenteroides that produces 2,3-dihydro-2,2,6-trimethylbenzaldehyde.

[0024] The fermented grains sample is shaken and then the suspension is diluted, and multiple gradient dilutions are applied on a solid culture medium plate for anaerobic culture. Microaerobic bacteria can also grow well in an anaerobic state, and the present invention selects anaerobic culture during screening.

[0025] Among them, single colonies with different colony morphologies were picked from the plates where colonies grew, and all single colonies were purified by secondary streaking.

[0026] Among them, each purified strain was statically cultured in MRS liquid medium in the form of a single colony.

[0027] Among them, different strains were fermented to obtain fermentation broth, and the target compounds were extracted and enriched by headspace solid phase microextraction, and then GC-MS was used for chromatographic detection and analysis, so as to screen out Enterococcus mesenteroides with high production of 2,3-dihydro-2,2,6-trimethylbenzaldehyde by comparing the content of volatile substances.

[0028] The present invention also provides application of the above-mentioned Leuconostoc mesenteroides in producing 2,3-dihydro-2,2,6-trimethylbenzaldehyde.

[0029] Specifically, the application is to culture Leuconostoc mesenteroides in a fermentation medium at 37°C for 2 days, with a pH value of 6.0, and the inoculation amount of Leuconostoc mesenteroides is 2% (×10 7 cfu / mL).

[0030] More specifically, the composition of the fermentation medium is 16g of glucose, 10g of tryptone, 2.5g of yeast powder, 5g of beef extract, 3.85g of bovine brain, 4.9g of bovine 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.

[0031] Using the above fermentation process, the content of 2,3-dihydro-2,2,6-trimethylbenzaldehyde produced by the strain is 2.876 mg / L.

[0032] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0033] The culture medium formula involved in the embodiment is:

[0034] MRS liquid culture medium: glucose 20g / L, peptone 10g / L, yeast powder 4g / L, beef extract powder 5g / L, Tween 801g / L, triammonium citrate 2g / L, sodium acetate 5g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.05g / L, dipotassium hydrogen phosphate 1g / L, pH 6.0, high pressure steam sterilization at 115℃ for 20 minutes.

[0035] MRS solid medium: Add 15 g / L agar to MRS liquid medium and sterilize with high pressure steam at 115°C for 20 minutes.

[0036] Fermentation medium: glucose 16 g / L, tryptone 10 g / L, yeast powder 2.5 g / L, beef extract 5 g / L, bovine brain 3.85 g / L, bovine heart 4.9 g / L, Tween 80 0.5 g / L, ammonium citrate 1 g / L, sodium chloride 2.5 g / L, anhydrous sodium acetate 2.5 g / L, magnesium sulfate 0.05 g / L, manganese sulfate 0.025 g / L, disodium hydrogen phosphate 1.25 g / L, dipotassium hydrogen phosphate 1 g / L, pH 6.0, high-pressure steam sterilization at 115°C for 20 min.

[0037] Example 1: Isolation and purification of strains

[0038] (1) Separation method: Take 20g of Luzhou Laojiao fermented grains sample and put it into a conical flask filled with 180mL of sterile distilled water. Oscillate on a constant temperature shaker for 10min to fully disperse and mix the sample. Take 1mL of the sample suspension and dilute it to 10% by multiple dilution method. -2~10 -7 , take 100 μL from each gradient dilution and spread it evenly on the MRS solid culture medium plate, prepare two plates in parallel, invert them, and culture them at 37℃ under anaerobic conditions for 36-48h, and observe them in time.

[0039] (2) Streak purification: Take out the plate with colonies, pick out single colonies with different colony morphologies, and perform streaking a second time until all single colonies are purified.

[0040] (3) Preservation of bacterial strains: After purification, a single colony of each strain was picked into 5 mL of MRS liquid culture medium and cultured at 37°C under anaerobic conditions for 20-24 h. 1 mL of bacterial solution was transferred to a bacterial preservation tube, and 0.5 mL of 60% sterile glycerol solution was added, resuspended, and stored at -80°C.

[0041] Example 2: Detection of the ability to produce 2,3-dihydro-2,2,6-trimethylbenzaldehyde

[0042] (1) Prepare the bacterial solution to be tested:

[0043] After the glycerol preservation tubes of the screened strains were dissolved, they were inoculated into MRS liquid culture medium and cultured at 37°C under anaerobic conditions for 24 hours. After activation for three generations, they were inoculated into 50 mL MRS liquid culture medium at a 2% (volume ratio) inoculation rate and cultured for 24 hours to obtain the bacterial solution to be tested.

[0044] (2) HS-SPME / GC-MS detection method for 2,3-dihydro-2,2,6-trimethylbenzaldehyde:

[0045] Using headspace solid phase microextraction / gas chromatography-mass spectrometry technology: take the supernatant, add it to the headspace bottle, add saturated NaCl solution, use 0.822mg / ml 2-octanol as the internal standard, keep the prepared sample at 60℃ for 5min, extract it with 50 / 30μmDVB / CAR / PDMS extraction head at 60℃ for 50min, and desorb it at 250℃ at the GC injection port for 5min after extraction. The compound search results are matched with the NIST standard spectral library, and the similarity reaches more than 80% to confirm the target compound. The culture broth without bacterial fermentation is used as the blank control group to calculate the content of each volatile substance.

[0046] GC-MS detection chromatographic conditions:

[0047] Gas chromatography conditions: HP-INNOWAX column (60m×0.25mm×0.25μm); temperature program: starting temperature 40°C, hold for 5min, increase to 100°C at 4°C / min, then increase to 230°C at 6°C / min, hold for 10min, carrier gas is high-purity helium (1.0mL / min); injection port temperature 250°C, no split.

[0048] Mass spectrometry conditions: electron ionization source, electron energy 70 eV; electron multiplier voltage 350 V; ion source temperature 230 °C; transfer line temperature 250 °C; mass range 40-450 m / z.

[0049] Through the above detection method, a lactic acid bacteria with a strong ability to produce 2,3-dihydro-2,2,6-trimethylbenzaldehyde was screened out from the isolated strains, and this strain was selected for further study.

[0050] Example 3: Molecular identification of strains

[0051] After the target strain was expanded, fresh bacterial liquid in the logarithmic growth phase was taken, centrifuged to collect the bacteria, and genomic DNA was extracted using a bacterial genome extraction kit. The universal primers 27F / 1541R for lactic acid bacteria were used to amplify the full-length sequence of 16S rDNA, as follows:

[0052] SEQ ID NO: 2: 27F (5′-AGAGTTTGATCCTGGCTCAG-3′)

[0053] SEQ ID NO: 3: 1541R (5′-AAGGAGGTGATCCAGCC-3′)

[0054] ①Reaction system (50 μl)

[0055]

[0056] ②Reaction procedure

[0057]

[0058] According to the above process, 35 cycles of "denaturation-annealing-extension" PCR reaction were carried out, and the PCR products were separated and tested by 1.0% agarose gel electrophoresis with a voltage of about 11 V / cm and an electrophoresis time of 20 min.

[0059] The purification of PCR products was carried out according to the instructions of the mini-gel recovery PCR product purification kit of Shanghai Sangon Biotechnology Co., Ltd., and the sequencing was completed by Shanghai Sangon Biotechnology Co., Ltd.

[0060] The gene sequence of the 16S rDNA fragment obtained by sequencing was compared with NCBI's BLAST to determine the species information of the strain, and it was identified as Leuconostoc mesenteroides. It was deposited in the collection center: China General Microbiological Culture Collection Center CGMCC on June 21, 2022, with the collection number CGMCC NO.25153. It was named Leuconostoc mesenteroides zqw121.

[0061] Example 4 Physiological and biochemical characteristics of Leuconostoc mesenteroides zqw121

[0062] Leuconostoc mesenteroides zqw121 was inoculated into MRS solid culture medium and cultured at 37°C for 2 days, and then the colonies and cell morphology of the strain were observed.

[0063] Determination of suitable growth temperature: Take Leuconostoc mesenteroides zqw121 in the logarithmic growth phase and inoculate it into MRS liquid culture medium at an inoculum rate of 2%. After culturing for 18 hours at 2, 10, 15, 20, 30, 40, 45, and 53°C, measure the OD of the bacterial solution. 600 value;

[0064] pH tolerance determination: 2M hydrochloric acid and 1M sodium hydroxide were used to adjust the pH of MRS liquid culture medium to 3.0, 5.0, 5.5, 6.0, 6.5 and 7.0 respectively. Leuconostoc mesenteroides zqw121 in the logarithmic growth period was inoculated with 2% inoculation volume, mixed thoroughly, and the OD of the bacterial solution was determined after culturing at 37°C for 18 hours. 600 value;

[0065] The deep agar method is used to determine the effect of oxygen on bacterial growth: Inoculate enteroconstricta mesenteroides zqw121 into an MRS solid culture medium tube and culture it at 37°C for 2 days. The degree of oxygen requirement of various bacteria is determined based on the location where the strain grows in the test tube culture medium.

[0066] The experimental results show that the biological characteristics of Enterobacter mesenteroides zqw121 are as follows: the colony morphology is round or bean-shaped, the colony diameter is less than 1.0 mm, the surface is smooth, milky white, and no pigment is produced; the cell morphology is spherical, bean-shaped or short-stalked, some are arranged in pairs or in short chains, non-motile, and without spores; Gram staining is positive.

[0067] The growth conditions of Enterobacter mesenteroides zqw121 are: microaerobic, and anaerobic culture grows well; the growth temperature range is 2℃~53℃, the optimum growth temperature is 30℃~40℃; the optimum pH for growth is 5.5~6.5.

[0068] Example 5: Fermentation culture of Leuconostoc mesenteroides zqw121

[0069] After dissolving the obtained Leuconostoc mesenteroides zqw121 glycerol storage tube, inoculate it into 10 mL of MRS liquid culture medium at a 5% inoculation amount, incubate it at 37°C for 24 hours, inoculate it into 400 mL of fermentation medium at a 2% inoculation amount, and incubate it at 37°C for 2 days. The detection method is the same as the volatile component determination method in Example 2. After continuous cultivation of Leuconostoc mesenteroides for 2 days, the production of 2,3-dihydro-2,2,6-trimethylbenzaldehyde can reach 2.876 mg / L.

Claims

1. Leuconostoc mesenteroides producing 2,3-dihydro-2,2,6-trimethylbenzaldehyde ( Leuconostoc mesenteroides ) strain, characterized in that: The strain preservation number is CGMCC NO. 25153.

2. The strain according to claim 1, characterized in that: Its 16S rDNA sequence is shown in SEQ ID NO:

1.

3. The strain according to claim 1, characterized in that: The biological characteristics of the strain are: the colony morphology is round or bean-shaped, the colony diameter is less than 1.0 mm, the surface is smooth, milky white, and no pigment is produced; the cell morphology is spherical, bean-shaped or short-stalked, some are arranged in pairs or in short chains, are non-motile, and have no spores; Gram staining is positive.

4. The strain according to claim 1, characterized in that: The growth conditions of the strain are: microaerobic, good growth in anaerobic culture; growth temperature range of 2°C to 53°C; strong acid resistance, and the optimal growth pH is 5.5 to 6.

5.

5. The strain according to claim 4, characterized in that: The optimum growth temperature is 30℃ ~ 40℃.

6. The strain according to claim 1, characterized in that: The strain can produce a high yield of 2,3-dihydro-2,2,6-trimethylbenzaldehyde in a fermentation medium, with the yield being 2.876 mg / L.

7. The strain according to claim 6, characterized in that: The fermentation medium comprises 16 g glucose, 10 g tryptone, 2.5 g yeast powder, 5 g beef extract, 3.85 g bovine brain, 4.9 g bovine heart, 0.5 g Tween 80, 1 g ammonium citrate, 2.5 g sodium chloride, 2.5 g anhydrous sodium acetate, 0.05 g magnesium sulfate, 0.025 g manganese sulfate, 1.25 g disodium hydrogen phosphate and 1 g dipotassium hydrogen phosphate per liter of medium.

8. Use of the 2,3-dihydro-2,2,6-trimethylbenzaldehyde-producing Leuconostoc mesenteroides according to any one of claims 1 to 7 in producing 2,3-dihydro-2,2,6-trimethylbenzaldehyde.

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