CN112210512B - Erianthus capable of producing 3-methylindole and 4-methylphenol simultaneously and promoting ethyl hexanoate to produce hexanoic acid and application thereof - Google Patents
Erianthus capable of producing 3-methylindole and 4-methylphenol simultaneously and promoting ethyl hexanoate to produce hexanoic acid and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of microorganisms, and particularly relates to a novel erjohnsonia capable of simultaneously producing 3-methylindole and 4-methylphenol and promoting ethyl hexanoate to produce hexanoic acid and application thereof. Aiming at the problems that the research on the anabolism of 3-methylindole, 4-methylphenol and ethyl caproate is still in a blank stage in the conventional liquor brewing and the basis for liquor production is difficult to provide, the invention provides an acidianella abortus with the preservation number of CCTCC No. M2020358. The bacterium can produce 3-methylindole and 4-methylphenol and promote ethyl caproate to produce caproic acid, provides a theoretical basis for producing 3-methylindole and 4-methylphenol in the white spirit production, explains the reason for reducing the content of ethyl caproate in the white spirit, and provides the application of the bacterium in reducing the content of 3-methylindole and 4-methylphenol and improving the content of ethyl caproate in the white spirit brewing, thereby having good practical value.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a novel erjohnsonia capable of simultaneously producing 3-methylindole and 4-methylphenol and promoting ethyl hexanoate to produce hexanoic acid and application thereof.
Background
3-methylindole (3-methylindole) named as skatole can be used for preparing perfume at very low concentration, and reaches 0.00056ppm (0.0030 mg/m) at slightly high concentration3) A unique foul smell of the feces can be emitted. The existing research finds that the white spirit contains 3-methylindole, which can cause the white spirit to generate the odor of pit mud and influence the flavor of the white spirit. However, no report on how to reduce the content of 3-methylindole in the white spirit exists at present.
The production of 3-methylindole involves two steps, first deaminating the precursor L-Tryptophan (TRP) to indoleacetic acid (IAA), followed by the formation of 3-methylindole under the action of indoleacetic acid decarboxylase. At present, microorganisms have been found which convert TRP to 3-methylindole more under acidic conditions and to indole more under basic conditions. There have also been many studies finding that a variety of bacteria can achieve TRP to IAA conversion, but the catalytic process of IAA to 3-methylindole is currently only achieved by four bacteria (Clostridium scoragenes, Clostridium drakei, elsenella scoragenes, Lactobacillus sp.).
At present, the research on the source, precursor substances and metabolic pathways of 3-methylindole in white spirit brewing is still blank.
4-methylphenol (p-cresol, PC), an aromatic compound with pit mud odor, is produced by microorganisms anaerobically metabolizing Tyrosine (Tyr) under natural conditions. In the industrial and medical fields, PC is widely used as an anti-aging agent, a disinfectant, a plasticizer, and a dye. However, in the field of food hygiene, it affects the taste and quality of food as an off-flavor substance, and its sensory profile is described as pit mud odor, leather odor, burnt skin odor, animal odor. At present, the main synthetic method of PC is a chemical synthesis method, however, the chemical synthesis method has the problems of low safety, insufficient energy conservation, environmental protection and the like. Biologically, PC has been studied mainly as a microbial metabolite or as a by-product in other metabolic processes. In the seventies of the last century, cedony r. elsded et al have detected that PC is a metabolite of Clostridium difficile and Clostridium scoriogens. After a Xuyan team detects volatile components in pit mud, modern separation and flavor research technologies are applied to confirm that a compound generating pit mud odor is PC, and a Clostridium butyricum with high PC yield is found.
The ethyl caproate is a characteristic aroma component in Chinese strong aromatic Chinese spirits, has aroma of a koji flavor and a pineapple flavor, is used for preparing edible and tobacco essence and blending the flavor of the koji flavor, and the quality of the strong aromatic Chinese spirits is directly determined by the content of the ethyl caproate. The microorganisms for producing ethyl caproate can be divided into two types, one type is the microorganisms secreted to the outside by producing ethyl caproate through intracellular esterification, and the other type comprises yeast and caproic acid bacteria according to the existing report, and the other type is the microorganisms secreted to the outside by producing esterase by the microorganisms to esterify caproic acid and ethanol to form ethyl caproate, wherein the microorganisms comprise yeast (ester-producing yeast, aroma-producing yeast), mould (monascus and rhizopus) and bacteria (bacillus and burkholderia). However, no report about the bacterial source and hydrolysis mechanism of ethyl caproate hydrolysis in white spirit brewing exists at present.
Although 3-methylindole and PC are determined as main sources of pit mud odor flavor in the Luzhou-flavor liquor, a microorganism system for brewing the Luzhou-flavor liquor is complex, and pit mud anaerobic microorganisms need severer separation and culture conditions. Therefore, the research on the microbial sources and metabolic pathways of 3-methylindole and PC in the white spirit fermentation is still blank. In addition, people usually pay attention to the synthesis of ethyl caproate in white spirit brewing, and the hydrolysis of the ethyl caproate is not deeply studied.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: in the existing white spirit brewing, the research on the anabolism of 3-methylindole, 4-methylphenol and ethyl caproate is still in a blank stage, and the basis is difficult to provide for the production of white spirit.
The technical scheme for solving the technical problems comprises the following steps: provides the Lawsonia inermis which can produce 3-methylindole and 4-methylphenol simultaneously and promote ethyl caproate to produce caproic acid. The Lawsonia oracescens provided by the invention has the following preservation numbers: CCTCC No. M2020358. The preservation date is as follows: year 2020, 7, 27; the preservation center is China center for type culture Collection CCTCC with the address of Wuhan university preservation center of eight paths 299 in Wuchang area of Wuhan city, Hubei province.
Wherein, the 16S rDNA sequence of the Lawsonia inermis which can produce 3-methylindole and 4-methylphenol and promote ethyl caproate to produce caproic acid is shown as SEQ ID NO. 1.
16S rDNA sequence of Erianthus who can produce 3-methylindole and 4-methylphenol and promote ethyl caproate to produce caproic acid simultaneously with SEQ ID NO. 1
tgatgaacgctggcggcgcgcctaacacatgcaagtcgaacgattaaggcaccttcgggtgcgtatagagtggcgaacggctgagtaa cacgtgggcaacctgcccccctctccgggacagcctcgggaaaccgtggttaataccggatactccgcgacccccgcatggggggcgcggg aaagcccagacggagggggatgggcccgcggcctgttagctcgttggtggggcaacggcccaccaaggcaacgatgggtagctgggttga gagaccgaccagccagattgggactgagacacggcccagactcctacgggaggcagcagtggggaatattgcgcaatgggcgaaagcctg acgcagcgacgccgcgtgcgggacgaaggccttcgggtcgtaaaccgctttcagcagggacgaggccgcgaggtgacggtacctgcagaa gaagccccggctaactacgtgccagcagccgcggtaatacgtagggggcgagcgttatccggattcattgggcgtaaagcgctcgtaggcgg tccgtctggtcgggtgtcaaatccgggggctcaacccccgttcgcatccgataccggcggacttgagtttggtaggggaaggcggaattccaa gtgtagcggtggaatgcgcagatatttggaagaacaccggtggcgaaggcggccttctgggccacaactgacgctgaggagcgaaagctag gggagcgaacaggattagataccctggtagtcctagccgtaaacgatggacactaggtgtggggggatctgccctccgtgccgcagctaacg cattaagtgtcccgcctggggagtacggccgcaaggctaaaactcaaaggaattgacgggggcccgcacaagcagcggagcatgtggctta attcgaagcaacgcgaagaaccttaccagggcttgacatctaggtgaagcggcggaaacgccgtggccgagaggagcctagacaggtggtg catggctgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaaccctcgtcgcatgttgccagcggttcggccgggcac ccatgcgagaccgccggcgtcaagccggaggaaggtggggacgacgtcaagtcatcatgccccttatgtcctgggctgcacacgtgctacaa tggccggcacaatgggctgccaggcggcgacgccgagcgaatccccaaagccggccccagttcggatcggaggctgcaacccgcctccgt gaagtcggagttgctagtaatcgcggatcagcacgccgcggtgaatgcgttcccgggccttgtacacaccgcccgtcacaccacccgagtcga ttgcacccgaagtcgtcggcctaactctcacgagagggaggcgccgaaggtgtggttggtaaggggggtg。
Wherein the amount of 3-methylindole produced by the Lawsonia inermis which can simultaneously produce 3-methylindole and 4-methylphenol and promote ethyl hexanoate to produce hexanoic acid is 0.2 mg.L at most-1。
Wherein the amount of the 4-methylphenol produced by the Lawsonia inermis which can simultaneously produce 3-methylindole and 4-methylphenol and promote the ethyl caproate to produce caproic acid is 0.07 mg.L at most-1。
Wherein, the Erianthus who can produce 3-methylindole and 4-methylphenol and promote ethyl caproate to produce caproic acid has the following biological characteristics: the special anaerobic bacteria have circular colony, ellipsoidal shape, chain shape, paired or independent existence, no flagellum, optimal temperature of 37 deg.c, optimal production pH of 6.0; sucrose and trehalose can be utilized, lactose, mannitol, arabinose, rhamnose and cellobiose cannot be utilized.
The invention also provides application of the aciduria opathia which can simultaneously produce 3-methylindole and 4-methylphenol and promote ethyl hexanoate to produce hexanoic acid in brewing white spirit.
Specifically, the application comprises the application of reducing the content of 3-methylindole and 4-methylphenol in the white spirit and increasing the content of ethyl caproate.
Further, in the above application, the increase of the ethyl caproate content is realized by increasing the amount of ethanol in the process of brewing the white spirit.
Further, the increasing of the ethanol amount means that the ethanol concentration is adjusted to 20 mL.L-1。
The invention has the beneficial effects that:
the invention separates the LaoSenensis which can produce 3-methylindole and PC and produce caproic acid by metabolizing ethyl caproate from wine brewing cellar mud of Luzhou Laojiao, and is named as LZLJ-2. The strain can achieve the highest 3-methylindole yield of 0.2 mg.L-1The yield of PC is 0.07 mg.L-1The method can better explain the source of microorganisms producing pit mud odor in the fermentation process of the Luzhou-flavor liquor, provides a certain theoretical basis for reducing 3-methylindole and PC in fermented food (such as liquor), and provides a certain possibility for cleanly producing raw materials of spice substances such as 3-methylindole, PC and the like. Meanwhile, the bacterium can hydrolyze ethyl caproate to produce caproic acid, and the addition of ethanol can inhibit hydrolysis reaction, so that a certain theoretical basis is provided for explaining possible reasons for reducing the content of ethyl caproate in the strong aromatic Chinese spirits and improving the yield of ethyl caproate which is a main aromatic substance in the strong aromatic Chinese spirits.
The Lawsonia inermis which can produce 3-methylindole and PC and metabolize ethyl hexanoate to produce hexanoic acid is preserved in China Center for Type Culture Collection (CCTCC) in 7 months and 27 days of 2020, and the preservation numbers are as follows: CCTCC No. M2020358. The preservation address is Wuhan university preservation center No. 299 eight roads in Wuhan district, Wuhan city, Hubei province. The classification is named as Olsenella immobilis LZLJ-2.
Drawings
FIG. 1 shows the colony morphology of LZLJ-2 of Lawsonia Oldhamii according to the invention.
FIG. 2 shows a transmission electron micrograph of LZLJ-2 of the Lawsonia Oldhamii of the present invention.
FIG. 3 shows phylogenetic evolution tree of Lawsonia olderia LZLJ-2 of the present invention.
FIG. 4 shows the production of 3-methylindole by LZLJ-2 of the Lawsonia inermis and the influencing factors thereof.
FIG. 5 shows PC production by LZLJ-2 of Lawsonia inermis and its influencing factors.
FIG. 6 shows the L.continentalis LZLJ-2 metabolizing ethyl hexanoate to produce hexanoic acid and its influencing factors.
Detailed Description
The invention provides an Erianthus capable of producing 3-methylindole and 4-methylphenol simultaneously and promoting ethyl caproate to produce caproic acid, which has a preservation number as follows: CCTCC number M2020358. The preservation date is as follows: year 2020, 7, 27; the preservation center is China center for type culture Collection CCTCC with the address of Wuhan university preservation center of eight paths 299 in Wuchang area of Wuhan city, Hubei province.
Wherein, the 16S rDNA sequence of the Lawsonia inermis which can produce 3-methylindole and 4-methylphenol and promote ethyl caproate to produce caproic acid is shown as SEQ ID NO. 1.
The Laurencia obtusifolia is obtained by separating and screening wine brewing cellar mud of Luzhou Laojiao Luzhou-flavor liquor, and is found to be capable of producing 3-methylindole and 4-methylphenol simultaneously and also capable of metabolizing ethyl hexanoate to produce hexanoic acid.
The research of the invention finds that the amount of 3-methylindole produced by the Lawsonia oralis LZLJ-2 is influenced by the contents of 3-indoleacetic acid and ferrous sulfate, and when 1 mg.L-1 of 3-indoleacetic acid is added, the yield of 3-methylindole is improved by 10 times, and the concentration reaches 0.20 mg.L-1. When 2 g.L-1 of ferrous sulfate is added, the yield of the 3-methylindole can be improved by about 3 times. And the L-tryptophan has no obvious influence on the yield of the 3-methylindole. Therefore, the invention provides the application of the Lawsonia inermis in reducing the content of 3-methylindole in the white spirit during the brewing of the white spirit, and provides a basis for reducing the content of 3-methylindole in the white spirit.
Furthermore, the research of the invention finds that the Lawsonia Oldhamii LZLJ-2 can improve the yield of PC by 70 percent, the yield of PC is influenced by ferrous sulfate, and after 2 g.L-1 ferrous sulfate is added, the yield of PC can be improved by 193 percent to reach 0.07 mg.L-1; while the PC yield did not change significantly with the addition of 20 mL.L-1 of ethanol. Therefore, the invention provides the application of the Lawsonia inermis in reducing the content of PC in the white spirit during the white spirit brewing, and provides a basis for reducing the content of PC in the white spirit.
Furthermore, the research of the invention finds that the Lawsonia Ovalifolia LZLJ-2 can hydrolyze ethyl hexanoate to generate hexanoic acid, so that the yield of the ethyl hexanoate can be reduced by 83%, the content of the hexanoic acid can be increased by 308%, and the content of the hexanoic acid is obviously higher than that of the ethyl hexanoate; when 20mL of ethanol of the concentration of 1 is added, the hydrolysis of ethyl caproate can be inhibited, and the content of the ethyl caproate is recovered and is higher than that of the caproic acid; the ferrous sulfate can obviously promote the conversion of ethyl caproate to caproic acid, and compared with the condition that the ferrous sulfate is not added, the addition of 2 g.L-1 ferrous sulfate can improve the yield of the caproic acid by 2.64 times. Therefore, the invention provides the application of the eusenna in improving the content of the ethyl caproate in the white spirit during the white spirit brewing, and also provides a basis for reducing the conversion from the ethyl caproate to the caproic acid in the white spirit.
Specifically, the specific method for producing 3-methylindole by fermenting the Lawsonia Oldhamii LZLJ-2 comprises the following steps: inoculating the Lawsonia Oldhamii LZLJ-2 into a culture medium containing 3-indoleacetic acid or ferrous sulfate, and performing anaerobic culture. The concentration of the 3-indoleacetic acid is 1 mg.L-1, the concentration of the ferrous sulfate is 2 g.L-1, and the culture medium is a PYG liquid culture medium. Comprising (g, L-1): peptone 20.0; 5.0 of glucose; 10.0 parts of yeast extract powder; 0.08 parts of sodium chloride; cysteine hydrochloride 0.5; 0.008 parts of calcium chloride; 0.008 of magnesium sulfate; dipotassium phosphate 0.04; potassium dihydrogen phosphate 0.04; 0.4 parts of sodium bicarbonate; dd H2O 1000 mL, pH 7.2. Autoclaving at 121 deg.C for 15min, adding 1mL of sterile filtered vitamin K1 solution and 5mL of hemin solution (5 mg/mL), and mixing.
Further, the preparation method of the vitamin K1 solution comprises the following steps: 0.1 mL vitamin K1 was dissolved in 20mL 95% ethanol and filter sterilized. Stored in a brown bottle at 4 ℃ in a refrigerator.
Further, the preparation method of the hemin solution comprises the following steps: weighing 50 mg of hemin, dissolving in 1mL of 1mol/L sodium hydroxide solution, adding distilled water to 100mL, autoclaving at 121 deg.C for 15min-20min, and storing in refrigerator at 4 deg.C.
Specifically, the specific method for producing PC by fermenting the Lawsonia Oldhamii LZLJ-2 comprises the following steps: inoculating the Lawsonia Oldhamii LZLJ-2 into a culture medium containing ferrous sulfate for anaerobic culture; the concentration of the ferrous sulfate is 2 g.L-1, and the culture medium is the PYG liquid culture medium.
Specifically, the specific method for producing the caproic acid by hydrolyzing ethyl caproate with the Lawsonia Oldhamica LZLJ-2 comprises the following steps: inoculating the Lawsonia Oldhamii LZLJ-2 into a culture medium containing ferrous sulfate for anaerobic culture; the concentration of the ferrous sulfate is 2 g.L-1, and the culture medium is the PYG liquid culture medium.
The following examples are intended to illustrate specific embodiments of the present invention without limiting the scope of the invention to the examples.
Example 1 preparation of a novel bacterium of the genus Lawsonia LZLJ-2
10g of pit mud is dug at the bottom of a one hundred year pit in a Luzhou Laojiao Luohan brewing base, is quickly filled into a sterile No. five self-sealing bag and is taken back to a laboratory for further study. The sample was then added to a conical flask containing sterile 90mL of physiological saline, sterilized glass beads were added, and the mixture was shaken thoroughly on a vortex shaker for 10min to allow the sample to disperse thoroughly. Diluting the supernatant with 10 times gradient to obtain 10 dilution-3、10-4、10-5、10-6、10-70.05mL of sample solution is sucked by a pipette gun, the sample solution is uniformly coated on a PYG screening culture medium by adopting a plate coating method, and the prepared plate is inverted and placed in an anaerobic culture tank for culture at 37 ℃ for 14 days. Separating out bacterial strain by plate streaking, selecting bacterial strain capable of growing well on culture medium, separating out single bacterial colony by multiple streaking separation culture, inoculating it into PYG liquid culture medium, culturing, and storing glycerine tube at-20 deg.C for use.
Example 2 identification of LZLJ-2 of the genus Lawsonia
(1) Morphological identification of Lawsonia olderia LZLJ-2
The separated strain was observed for colony, shape, gram stain and other structures using a PYG plate and a microscope. As shown in FIG. 1, when the strain was cultured on PYG medium at 37 ℃ for 7 days, the colony was round, smooth and opaque on the surface, pale white, gram-positive, and exhibited the presence of a chain, paired or individual, and no flagella by transmission electron microscopy. The selected strain was initially considered to be Larresia terrestris (Olsenlansp.) with reference to the handbook of bacteriological identification.
(2) Physiological and biochemical characteristics of Lawsonia olderia LZLJ-2
The physiological and biochemical characteristics of the strain Lawsonia Oldhamii LZLJ-2 are shown in Table 1
TABLE 1 physiological and biochemical Properties of Strain LZLJ-2
| Experimental project | Olsenella sp.LZLJ-2 |
| Aerobic conditions | Strict anaerobism |
| Colony morphology | Circular shape |
| Size of cell | 1.0-1.8μm×0.6-0.8μm |
| Cell morphology | Ellipsoidal, usually existing in a chain shape, or in pairs or singly |
| Flagellum | Is free of |
| Movement property | Is free of |
| Optimum growth temperature (. degree. C.) | 37 |
| Growth pH Range (optimum) | 6-7(6) |
| NaCl tolerance range (%) | 0-2 |
| Saccharides and their use as anti-inflammatory agents | |
| Lactose | - |
| Mannitol | - |
| Sucrose | + |
| Arabinose | - |
| Rhamnose | - |
| Trehalose | + |
| Cellobiose | - |
Note: +: indicates that the microorganism is capable of utilizing the carbon source; -: indicating that the microorganism cannot utilize the carbon source.
(3) Molecular biological identification of Lawsonia olderia plantarii new bacterium LZLJ-2
The genome of the novel bacterium Lawsonia Oldhamii LZLJ-2 was extracted using a bacterial genome extraction kit (purchased from Shanghai, Order NO. B518255), and PCR amplification was performed using bacterial 16S rRNA gene universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO:2) and 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO: 3).
The PCR reaction system is as follows: 1. mu.l of the template DNA obtained in step 2, 12.5. mu.l of PCR Premix, 1. mu.l of forwarder (20 pmol/. mu.l), 1. mu.l of Reverse primer (20 pmol/. mu.l), ddH2O make up to a total volume of 25. mu.l.
PCR amplification procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 1min, annealing at 52 ℃ for 30s, extension at 72 ℃ for 1min, and 30 cycles; extending for 5min at 72 ℃, cooling to 4 ℃, and taking out the product.
The sequence determination work is carried out by the amplified PCR product sample and sequence determination, the sequence determination is completed by Shanghai Biotechnology limited, and the 16S rDNA nucleotide sequence is shown as SEQ ID NO: 1.
According to the morphological characteristics (figure 1 and figure 2), physiological and biochemical indexes and the blast comparison result of the 16SrDNA gene sequence in NCBI, the similarity between the gene sequence and Olsenella profusa is 94.92%, a phylogenetic evolution tree (shown in figure 3) is further constructed, and the Lagerella abortus LZLJ-2 and the Olsenella strain are gathered together, so that the strain can be identified as the suspected new bacterium of the Lagerella abortus (Olsenella), and is named as the Lagerella abortus LJ-2(Olsenella sp.
EXAMPLE 3 measurement of 3-methylindole production by LZLJ-2 of Lawsonia Oldhamii of the present invention
(1) Selectively separating Lawsonia oldhamii LZLJ-2 to study 3-indoleacetic acid, L-tryptophan and Fe2+The effect on the production of 3-methylindole.
(2) The control group adopts PYG liquid culture medium, and the culture medium used in the experimental group is PYG liquid culture medium added with 1 mg.L-13-indoleacetic acid or 1 mg. L-1Or 2 g.L of-1Ferrous sulfate. Inoculating according to 1% of inoculum sizeAfter the Lawsonia oldhamii LZLJ-2, the strain is cultured in an anaerobic jar at 37 ℃ for 7 days, and the concentration of 3-methylindole in the fermentation liquor is detected.
(3) The detection method of the 3-methylindole in the fermentation liquor comprises the following steps: taking 5mL of fermentation liquor obtained in the step (2), adding 4g of sodium chloride until saturation, and taking 2-octanol as an internal standard. The 3-methylindole concentration is determined by adopting a headspace solid phase microextraction-gas chromatography-mass spectrum combined (HS-SPME-GC-MS) technology.
GC-MS conditions:
extraction conditions are as follows: the DVB/CAR/PBDS extraction head is pre-extracted for 15min and 45min at the extraction temperature of 60 ℃.
GC conditions were as follows: the sample inlet temperature is 230 ℃, the carrier gas He, the flow rate is 3 mL/min-1The sample is injected without splitting, and the chromatographic column is DB-Wax (30m × 0.32m mi.d.. times.0.25 μm, J)&W Scientific). The temperature rise procedure during detection is as follows: keeping the temperature at 36 deg.C for 8min, and keeping the temperature at 2.5 deg.C/min-1Heating to 40 deg.C, maintaining for 0min at 5 deg.C/min-1Heating to 100 deg.C, maintaining for 0min at 10 deg.C/min-1Heating to 200 deg.C, maintaining for 0min at 20 deg.C/min-1The temperature was raised to 220 ℃ and maintained for 10 min.
MS conditions: the EI ionization source has electron energy of 70eV, the ion source temperature of 230 ℃ and the scanning range of 35.00-350 amu. The mass spectrometry database was derived from NIST14s (Shimadzu).
As shown in FIG. 4, the concentration of 3-methylindole after inoculation with L.continentalis LZLJ-2 was increased by 10.3 times to 0.02 mg. multidot.L in the blank medium-1(ii) a 3-Indolylacetic acid has very significant influence on the generation of 3-methylindole, and 1 mg.L is added-1The concentration of 3-indoleacetic acid and 3-methylindole can be increased by 10 times to 0.20 mg.L-1(ii) a The L-tryptophan has no obvious influence on the yield of the 3-methylindole; the ferrous sulfate can increase the yield of 3-methylindole, and 2 g.L is added-1The yield of the ferrous sulfate and the 3-methylindole can be improved by about 3 times.
EXAMPLE 4 measurement of PC production by LZLJ-2 of Lawsonia Oldhamii of the present invention
(1) Research on Fe by selecting and separating Lawsonia olderia LZLJ-22+And the effect of ethanol on PC production.
(2) The control group adopts PYG liquid culture medium, and the culture medium used in the experimental group is 2 g.L-1Ferrous sulfate or 20 mL. L-1Ethanol (c) in the presence of a base. Inoculating 1% of the strain into Lawsonia Oldhamii LZLJ-2, culturing in an anaerobic jar at 37 deg.C for 7 days, and detecting the concentration of PC in the fermentation liquid.
(3) The method for detecting PC in fermentation liquor comprises the following steps: taking 5mL of fermentation liquor obtained in the step (2), adding 4g of sodium chloride until saturation, and taking 2-octanol as an internal standard. The PC concentration in the sample is determined by adopting a headspace solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) technology.
GC-MS conditions:
extraction conditions are as follows: the DVB/CAR/PBDS extraction head is pre-extracted for 15min and 45min at the extraction temperature of 60 ℃.
GC conditions were as follows: the sample inlet temperature is 250 ℃, the carrier gas He, the flow rate is 2 mL/min-1The sample is injected without splitting, and the chromatographic column is DB-Wax (30m × 0.32m mi.d.. times.0.25 μm, J)&W Scientific). The temperature rise procedure during detection is as follows: keeping the temperature at 36 deg.C for 8min, and keeping the temperature at 2.5 deg.C/min-1Heating to 40 deg.C, maintaining for 0min at 5 deg.C/min-1Heating to 100 deg.C, maintaining for 0min at 10 deg.C/min-1Heating to 200 deg.C, maintaining for 0min at 20 deg.C/min-1The temperature was raised to 220 ℃ and maintained for 10 min.
MS conditions: the EI ionization source has electron energy of 70eV, the ion source temperature of 230 ℃ and the scanning range of 35.00-350 amu. The mass spectrometry database was derived from NIST14s (Shimadzu).
As shown in FIG. 5, the addition of the strain LZLJ-2 increased the yield of PC by 70% compared to the blank medium; the yield of PC is affected by ferrous sulfate, and 2 g.L is added-1After ferrous sulfate, the yield of PC can be increased by 193% to 0.07 mg.L-1(ii) a Ethanol had almost no effect on the yield of PC, and 20 mL. L was added-1The yield of PC in the ethanol is not obviously changed.
EXAMPLE 5 measurement of caproic acid and ethyl caproate production by LZLJ-2 of Lawsonia inermis of the present invention
(1) Selectively separating Lawsonia oldhamii LZLJ-2 to research ethanol and Fe2+Influence on caproic acid and ethyl caproate.
(2) The control group adopts PYG liquid culture medium, and the culture medium used in the experimental group is PYG liquid culture medium added with 20mL L -12 g.L of ethanol or-1Ferrous sulfate. Inoculating 1% of Lawsonia Oldhamii LZLJ-2, culturing in anaerobic jar at 37 deg.C for 7 days, and detecting the concentration of caproic acid and ethyl caproate in the fermentation liquid.
(3) The detection method of caproic acid and ethyl caproate in the fermentation broth was the same as that of 3-methylindole in example 3.
As shown in FIG. 6, the ethyl hexanoate content in the fermentation broth after inoculation of the strain LZLJ-2 was significantly reduced and the hexanoate content was significantly increased, compared to the blank medium. The strain LZLJ-2 can hydrolyze ethyl caproate to produce caproic acid, and the addition of the strain LZLJ-2 can reduce the yield of the ethyl caproate by 83 percent, increase the content of the caproic acid by 308 percent and obviously increase the content of the caproic acid to be more than that of the ethyl caproate; adding 20mL of L-1The ethanol can inhibit the hydrolysis of the ethyl caproate, and the content of the ethyl caproate is recovered and is higher than that of the caproic acid; the ferrous sulfate can obviously promote the conversion of ethyl caproate to caproic acid, and compared with the method without the ferrous sulfate, 2 g.L is added-1The ferrous sulfate can increase the yield of the caproic acid by 2.64 times.
From the results of the above examples, it can be seen that: the screening method obtains the Lawsonia Oersonii which can produce 3-methylindole and 4-methylphenol simultaneously and promote ethyl caproate to produce caproic acid, provides a basis for reducing the content of 3-methylindole and 4-methylphenol and reducing the hydrolysis of ethyl caproate in the process of brewing white spirit, and has good application value.
Sequence listing
<110> Luzhou Production technologies, Inc
LUZHOULAOJIAO BREWING Co.,Ltd.
<120> Lawsonia oracescens capable of producing 3-methylindole and 4-methylphenol simultaneously and promoting ethyl hexanoate to produce hexanoic acid and application thereof
<130> A200875K (preface)
<141> 2020-10-15
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1438
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tgatgaacgc tggcggcgcg cctaacacat gcaagtcgaa cgattaaggc accttcgggt 60
gcgtatagag tggcgaacgg ctgagtaaca cgtgggcaac ctgcccccct ctccgggaca 120
gcctcgggaa accgtggtta ataccggata ctccgcgacc cccgcatggg gggcgcggga 180
aagcccagac ggagggggat gggcccgcgg cctgttagct cgttggtggg gcaacggccc 240
accaaggcaa cgatgggtag ctgggttgag agaccgacca gccagattgg gactgagaca 300
cggcccagac tcctacggga ggcagcagtg gggaatattg cgcaatgggc gaaagcctga 360
cgcagcgacg ccgcgtgcgg gacgaaggcc ttcgggtcgt aaaccgcttt cagcagggac 420
gaggccgcga ggtgacggta cctgcagaag aagccccggc taactacgtg ccagcagccg 480
cggtaatacg tagggggcga gcgttatccg gattcattgg gcgtaaagcg ctcgtaggcg 540
gtccgtctgg tcgggtgtca aatccggggg ctcaaccccc gttcgcatcc gataccggcg 600
gacttgagtt tggtagggga aggcggaatt ccaagtgtag cggtggaatg cgcagatatt 660
tggaagaaca ccggtggcga aggcggcctt ctgggccaca actgacgctg aggagcgaaa 720
gctaggggag cgaacaggat tagataccct ggtagtccta gccgtaaacg atggacacta 780
ggtgtggggg gatctgccct ccgtgccgca gctaacgcat taagtgtccc gcctggggag 840
tacggccgca aggctaaaac tcaaaggaat tgacgggggc ccgcacaagc agcggagcat 900
gtggcttaat tcgaagcaac gcgaagaacc ttaccagggc ttgacatcta ggtgaagcgg 960
cggaaacgcc gtggccgaga ggagcctaga caggtggtgc atggctgtcg tcagctcgtg 1020
tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc tcgtcgcatg ttgccagcgg 1080
ttcggccggg cacccatgcg agaccgccgg cgtcaagccg gaggaaggtg gggacgacgt 1140
caagtcatca tgccccttat gtcctgggct gcacacgtgc tacaatggcc ggcacaatgg 1200
gctgccaggc ggcgacgccg agcgaatccc caaagccggc cccagttcgg atcggaggct 1260
gcaacccgcc tccgtgaagt cggagttgct agtaatcgcg gatcagcacg ccgcggtgaa 1320
tgcgttcccg ggccttgtac acaccgcccg tcacaccacc cgagtcgatt gcacccgaag 1380
tcgtcggcct aactctcacg agagggaggc gccgaaggtg tggttggtaa ggggggtg 1438
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
agagtttgat cctggctcag 20
<210> 3
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ggttaccttg ttacgactt 19
Claims (1)
1. The aciduria opathia which can produce 3-methylindole and 4-methylphenol and promote ethyl caproate to produce caproic acid is characterized in that the preservation number is CCTCC No. M2020358.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106190924A (en) * | 2016-08-17 | 2016-12-07 | 江南大学 | One plant height produces the clostridium tyrobutyricum of 4 methylphenols |
| CN108884435A (en) * | 2015-12-18 | 2018-11-23 | 比奥森蒂亚有限公司 | Gene modification type bacterial cell factory for thiamine production |
| CN109971681A (en) * | 2019-03-29 | 2019-07-05 | 泸州品创科技有限公司 | Old cellar clostridium and application thereof |
-
2020
- 2020-10-15 CN CN202011102804.4A patent/CN112210512B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108884435A (en) * | 2015-12-18 | 2018-11-23 | 比奥森蒂亚有限公司 | Gene modification type bacterial cell factory for thiamine production |
| CN106190924A (en) * | 2016-08-17 | 2016-12-07 | 江南大学 | One plant height produces the clostridium tyrobutyricum of 4 methylphenols |
| CN109971681A (en) * | 2019-03-29 | 2019-07-05 | 泸州品创科技有限公司 | Old cellar clostridium and application thereof |
Non-Patent Citations (6)
| Title |
|---|
| Characterization of novel human oral isolates and cloned 16S rDNA sequences that fall in the family Coriobacteriaceae:description of Olsenella gen.nov, reclassification of Lactobacillus uli as Olsenella uli comb.nov and description of Olsenella profusa sp.;Floyd E. Dewhirst等;《International Journal of Systematic and Evolutionary Microbiology》;20010930;第51卷(第5期);参见第1797–1804页全文 * |
| Deep sequencing reveals high bacterial diversity and phylogenetic novelty in pit mud from Luzhou Laojiao cellars for Chinese strong-flavor Baijiu;Mao-Ke Liu等;《Food Research International》;20170928;第102卷;参见第68-76页全文 * |
| Draft genome sequence of Olsenella scatoligenes SK9K4T, a producer of 3-methylindole (skatole) and 4-methylphenol (p-cresol), isolated from pig feces;Li XiaoQiong等;《Genome Announcements》;20160229;第4卷(第1期);文献号e00042-16，参见第1-2页全文 * |
| Olsenella scatoligenes sp nov., a 3-methylindole- (skatole) and 4-methylphenol- (p-cresol) producing bacterium isolated from pig faeces;Li,XQ等;《INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY》;20140430;第65卷(第4期);参见第1227–1233页全文 * |
| Olsenella sp. strain LZLJ-2 16S ribosomal RNA gene, partial sequence;lu,l.F.;《GenBank》;20200906;ACCESSION NUMBER MT525318.1 * |
| 乳酸菌的系统分类概况;王阶平等;《生物资源》;20191231;第41卷(第6期);参见第471-485页全文 * |
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