CN112625963B - Clostridium sarmentosum capable of producing 4-methylphenol and application thereof - Google Patents
Clostridium sarmentosum capable of producing 4-methylphenol and application thereof Download PDFInfo
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Abstract
The invention discloses a Clostridium plebei for producing 4-methylphenol and application thereof, wherein the preservation name is Clostridium sartagoforme bzxyb10007, the Clostridium sartagoforme bzxyb is preserved in China center for type culture collection, and the preservation number is CCTCC NO: m2020623. The method determines the production strain of the defect flavor substance 4-methylphenol of the strong aromatic white spirit, provides theoretical and technical support for analyzing the pit mud odor formation mechanism and constructing a mud odor elimination strategy, and can construct a base wine mud odor elimination strategy starting from pit mud culture and maintenance and prevention and control of mud odor generation microorganisms, thereby fundamentally solving the problem of pit mud odor in the strong aromatic white spirit base wine.
Description
Technical Field
The invention relates to the technical field of wine making and microorganisms, in particular to a strain of producing strain of 4-methylphenol which is a defective flavor substance of strong aromatic white spirit.
Background
The liquor industry is the traditional industry with thousands of calendar histories in China, and the unique brewing process of the liquor industry ensures that the Chinese liquor has unique flavor and is rich in various aroma-generating and flavor-developing compounds and functional active ingredients beneficial to health. The strong aromatic white spirit is not only a component of the economic industry of China, but also a component of the long-standing historical culture of China, and at present, how to apply biotechnology to the traditional process at the present of the rapid development of modern high technology is the problem concerned by workers in all wine circles on the basis of keeping the original cultural characteristics to realize technical breakthrough innovation.
In recent years, with the rapid development of society and science, the white spirit industry enters a new development period, people have higher and higher requirements on the flavor and taste of the white spirit, and the elimination of foreign flavor substances in the white spirit is urgent. The quality of the strong aromatic white spirit is in important relation with pit mud, pit mud odor is generated by the pit mud which is not well cultured in the fermentation process of fermented grains, the odor inevitably enters the raw wine, the odor cannot be weakened in the storage process, but is possibly amplified in the storage process, and the flavor of the strong aromatic white spirit is greatly influenced; it also affects the mood of the consumer. However, the formation mechanism of the mud odor in the white spirit is not clear in the current industry, and the further improvement of the product quality is hindered to a certain extent.
Xuyan et al at Jiangnan university confirm that a compound generating pit mud odor is 4-methylphenol [ Xuyan, Van Wen Lai, Wu qun, Wang Hai Yan's progress of flavor technology oriented white spirit brewing fundamental research, brewing science, No. 1 (No. 211) in 2012), the threshold value of the compound is determined to be 166.97 mug/L (46% vol alcohol solution), the most strong-flavor white spirit is used in all flavor white spirits, the content of the special strong-flavor white spirit is extremely high and reaches 1200 mug/L [ Duhai, Van Wen Lai, Xuyan's headspace solid phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS) are combined to quantify two kinds of foreign substances in white spirit, and the food industry, 2010,32(1) ]. However, no research on the cause of 4-methylphenol has been reported.
In addition, 4-methylphenol is an important chemical raw material, can be used as a raw material for preparing antioxidant 2, 6-di-tert-butyl-p-cresol and rubber antioxidant, and can also be used as an important raw material for producing medical TMP and dye British sulfonic acid. The synthesis method of 4-methylphenol mainly comprises a chemical synthesis method and a microbial fermentation method, wherein the chemical synthesis method has the problems of low safety and insufficient energy conservation and environmental protection, and the microbial fermentation method has the problem of low yield of 4-methylphenol, for example, the yield of 4-methylphenol of clostridium butyricum disclosed in patent CN106190924A is only 0.13 mg/L.
Therefore, the screening of the strain capable of highly producing the 4-methylphenol has great significance for eliminating the pit mud odor of the Luzhou-flavor liquor and improving the quality of the Luzhou-flavor liquor on one hand, and provides possibility for biologically and cleanly producing the 4-methylphenol on the other hand.
Disclosure of Invention
Based on the problems in the prior art, the invention provides clostridium pleioides for producing 4-methylphenol and application thereof, so that the formation mechanism of pit mud odor can be deeply known, a mud odor eliminating strategy can be constructed, and the quality of strong aromatic white spirit can be improved.
In order to realize the purpose of the invention, the following technical scheme is adopted:
the invention separates the microorganism in the pit mud by simulating the micro-ecological environment of the pit microorganism, and can comprehensively separate and purify the pit microorganism by utilizing simulated culture conditions and simulated culture medium, which are more comprehensive close to the nutrient components and influencing factors of the fermented grains in the pit than the traditional synthetic culture medium or other culture mediums. Screening microbes which are easy to generate odor from the separated microbes in pit mud for fermentation experiments, extracting pit mud components by adopting a headspace-solid phase microextraction method, analyzing and detecting the fermented components by using a gas chromatography-mass spectrometer (GC-MS), searching strains with high 4-methylphenol yield, and analyzing and identifying the strains by using a BIOLoG microbe identification system and a 16SrDNA sequence analysis method.
The production bacteria of the 4-methylphenol screened by the invention are two strains:
the first strain is Clostridium sarmentosum, which has a preservation name of Clostridium sartagoforme bzxyb10007 and is preserved in China center for type culture collection with the preservation number of CCTCC NO: m2020623, the preservation date is 10 months and 22 days in 2020, and the preservation address is Wuhan, Wuhan university in China.
The second strain is Clostridium difficile, the preservation name of the third strain is Clostridium difficile bzxyb10008, the third strain is preserved in China center for type culture Collection, and the preservation number is CCTCC NO: m2020624, the preservation date is 10 months and 22 days in 2020, and the preservation address is Wuhan, Wuhan university in China.
The invention has the beneficial effects that:
1. the method determines the production strain of the defect flavor substance 4-methylphenol of the strong aromatic white spirit, provides theoretical and technical support for analyzing the pit mud odor formation mechanism and constructing a mud odor elimination strategy, and can construct a base wine mud odor elimination strategy starting from pit mud culture and maintenance and prevention and control of mud odor generation microorganisms, thereby fundamentally solving the problem of pit mud odor in the strong aromatic white spirit base wine.
2. The yield of the Clostridium sartagoformmebzxyb 10007, 4-methylphenol screened by the invention is up to 7.4mg/L, and the Clostridium sartagenii can be used as a production strain for producing 4-methylphenol by fermentation.
Detailed Description
The method for screening and identifying the 4-methylphenol producing strain comprises the following steps:
1 materials and methods
1.1 materials and reagents
Selecting different pit mud: no. 1 is pit mud without pit mud odor (from the cultivation and demonstration center for wine brewing of Bozhou institute), and No. 2 is pit mud with pit mud odor (from the cultivation and demonstration center for wine brewing of the Bozhou institute).
1.2 Main instruments and devices
WatersI-Class/XevoTQD gas chromatography-mass spectrometer (GC-MS); an AgilentPC420 solid phase micro-extraction instrument; CAR/PDMS75 μm headspace solid phase extraction head; BIOLOG microorganism identification instrument, anaerobic workstation.
1.3 Experimental methods
1.3.1 separation and purification of microorganisms in pit mud Using sugar as carbon Source
1.3.1.1 preparation of culture Medium
Liquid enrichment culture medium: preparing 500mL of liquid enrichment medium according to the mixture ratio of 100mL of fermented grain extract (obtained by adding fermented grain into water according to the mass ratio of 1:3, soaking for 1h at 80 ℃ and filtering, and the balance being water) of peptone 1%, beef extract 0.5%, glucose 3%, sodium chloride 0.05%, ammonium sulfate 0.09%, ferric sulfate 0.01%, magnesium sulfate 0.03%, calcium carbonate 2% and fermented grain extract, and sterilizing at 121 ℃ for 20min at the pH of 7.0.
Separating a culture medium: preparing 500mL of separation culture medium according to the mixture ratio of 1% of peptone, 0.5% of beef extract, 3% of glucose, 0.05% of sodium chloride, 0.09% of ammonium sulfate, 0.01% of ferric sulfate, 0.03% of magnesium sulfate, 20.5% of CaCl, 2% of agar, 50mL of fermented grain leaching liquor and the balance of water, sterilizing at the temperature of 121 ℃ for 20min, and then carrying out sterilization on the culture medium at the pH of 7.0.
Separating culture medium of fermented grain leaching liquor: preparing 500mL of fermented grain leaching liquor separation culture medium according to the mixture ratio of 200mL of fermented grain leaching liquor, 20.5% of CaCl, 2.0% of agar, 1.0% of glucose and the balance of water, and sterilizing at 121 ℃ for 15min at a pH of 6.5-7.0.
1.3.1.2 enrichment culture
Taking 10g of old cellar mud (No. 1 cellar mud and No. 2 cellar mud are respectively treated), heating at 85 ℃ for 10min, cooling, adding a liquid enrichment culture medium, and culturing at 35 ℃ for 5-7 d; and adding 10g of the aged cellar mud which is not subjected to heating treatment into a liquid enrichment medium for culturing for 5-7d at 35 ℃.
1.3.1.3 Dilute coated plates
Respectively selecting enrichment culture solution with good aerocyst, diluting by 10 times, 20 times and 30 times, coating on different separation culture media, and respectively performing aerobic culture and anaerobic culture under the conditions of different carbon dioxide concentrations (5 percent and 10 percent) and different temperatures (30 ℃ and 35 ℃).
After 12-24 hours of culture, 20-fold and 30-fold diluted and coated plate colonies are well separated to form single colonies, and the colonies grow well under the culture conditions that the concentration of carbon dioxide is 5% and the temperature is 35 ℃.
1.3.1.4 separating and purifying strains
Selecting single bacterial colony in the plate, inoculating and streaking on a separation culture medium, simulating a pit mud microenvironment to perform classified culture on the separated bacteria, and performing culture under the conditions of different carbon dioxide concentrations (0%, 5% and 10%) and different temperatures (30 ℃ and 35 ℃) for 24-36 hours.
1.3.1.5 results
Purifying and culturing to obtain more than 60 pure strains.
1.3.2 separation and purification of microorganisms in pit mud Using non-starch sugar as carbon Source
1.3.2.1 preparation of the culture Medium
Enrichment culture medium: 1000mL of enrichment medium is prepared according to the proportion of 0.5% of sodium acetate, 0.15% of yeast extract, 0.02% of magnesium sulfate heptahydrate, 0.04% of dipotassium hydrogen phosphate, 0.05% of ammonium sulfate, 1% of calcium carbonate, 200mL of fermented grain leaching liquor, 2.0% of ethanol (added before use after sterilization) and the balance of water, and the enrichment medium is sterilized by high-pressure steam at the temperature of 121 ℃ under the natural pH value of 0.12MPa for 20 min.
Separating a culture medium: according to the weight percentage of NaAc 0.5%, yeast extract 0.15%, MgSO4·7H2O 0.02％、K2HPO40.04％、(NH4)2SO40.05％、CaCl20.5 percent of agar, 2.0 percent of agar, 100mL of fermented grain leaching liquor, 2.0 percent of ethanol (added before use after sterilization) and the balance of water, preparing 500mL of separation culture medium, and sterilizing for 20min by high-pressure steam with natural pH and 0.12 MPa.
Separating culture medium of fermented grain leaching liquor: 200mL of wine mash leaching liquor and CaCl20.5 percent of agar, 2.0 percent of ethanol (added before use after sterilization) and the balance of water, and 500mL of fermented grain leaching liquor separation culture medium is prepared, and the pH value is 6.5-7.0.
1.3.2.2 enrichment culture
Taking 10g of old cellar mud (No. 1 cellar mud and No. 2 cellar mud are respectively treated), heating at 85 ℃ for 10min, cooling, adding an enrichment medium, and culturing at 35 ℃ for 5-7 d; and adding 10g of the aged cellar mud which is not subjected to heating treatment into an enrichment medium to culture at 35 ℃ for 5-7 d.
After 48 hours of culture, the culture solution starts to produce gas, and after 168 hours of culture, the enrichment culture condition is good, the liquid is turbid, the gas is produced, the pit mud sample No. 1 is pit aroma, and the pit mud sample No. 2 is pit mud odor.
1.3.2.3 Dilute coated plates
Respectively selecting enrichment culture solution with good aerocyst, diluting by 10 times, 100 times and 1000 times, coating on different separation culture media, and respectively performing aerobic culture and anaerobic culture under the conditions of different carbon dioxide concentrations (5 percent and 10 percent) and different temperatures (30 ℃ and 35 ℃).
After 12-24 hours of culture, the colonies of the plate which is diluted and coated by 10 times and 100 times are better separated to form single colonies, and the colonies grow well under the culture conditions that the concentration of carbon dioxide is 5 percent and the temperature is 35 ℃.
1.3.2.4 isolation and purification of the strains
On 16 plates with various fungi to be separated, selecting single colonies with good growth in the plates, inoculating and streaking on a separation culture medium, simulating a pit mud microenvironment to culture the separated fungi in a classified manner, and respectively culturing under the conditions of different carbon dioxide concentrations (0%, 5%, 10%) and different temperatures (30 ℃ and 35 ℃).
After 24-36 hours of culture, the single colony which grows well is placed into a refrigerator for identification, and the un-grown single colony is continuously placed into an incubator for culture.
1.3.2.5 results
Purifying and culturing to obtain more than 60 pure strains.
1.3.3 fermentation experiment of odor-producing microorganism separated from pit mud
1.3.3.1 fermentation experiment of microbial strains in pit mud with sugar as carbon source
1.3.3.1.1 preparation of culture Medium
Strain activation medium: peptone 1%, beef extract 0.5%, glucose 3%, sodium chloride 0.05%, ammonium sulfate 0.09%, ferric sulfate 0.01%, magnesium sulfate 0.03%, CaCl20.5%, fermented grain leaching liquor 20%, agar 2%, and water in balance, pH7.0, and sterilizing at 121 deg.C for 20 min.
Fermentation medium: saccharifying liquid (corn, sorghum, and rice) 9%, peptone 1%, beef extract 0.5%, ammonium sulfate 0.09%, magnesium sulfate 0.03%, and water in balance, pH7.0, and sterilizing at 121 deg.C for 20 min.
1.3.3.1.2 fermentation test of bacterial species
9 strains of microorganisms which generate odor on a solid culture medium are selected from the separated microorganism strains in the pit mud which takes sugar as a carbon source for fermentation, wherein the 9 strains of microorganisms are respectively numbered from 1 to 9.
Activating strains: respectively selecting strain activating culture medium according to strain characteristics, inoculating, and activating under aerobic condition at 35 deg.C for 12-48 h.
Seed culture: taking two-ring strains from a fresh inclined plane, inoculating the two-ring strains into a test tube (25mL of shake flask liquid containing amount is 10-15mL), and performing shaking table (35 ℃, 140r/min) or static culture for 24h to prepare a seed solution.
Fermentation culture: inoculating the aerobic strain seed liquid into a fermentation medium (250mL of triangular flask, liquid loading capacity 150mL), and performing shake culture at 35 ℃ for 72h at 140 r/min.
1.3.3.1.3 results of the experiment
After 12-24 hours of culture, bottles No. 1, 2, 3, 5, 7, 8 and 9 start to produce gas in the fermentation, bottles No. 4 and 6 do not produce gas, little gas is produced in the fermentation for 72 hours, and the fermentation liquid has odor except for the bottles No. 4 and 6, and the fermentation liquid waits for gas chromatography.
1.3.3.2 fermentation experiment of microbial strains in pit mud with non-starch sugar as carbon source
1.3.3.2.1 preparation of culture Medium
Strain activation medium: NaAc0.5%, yeast extract 0.15%, MgSO4·7H2O 0.02％、K2HPO40.04％、(NH4)2SO40.05％、CaCl20.5%, fermented grain leaching solution 20%, ethanol 2.0% (added after sterilization), and water in balance, natural pH, and 0.12MPa high pressure steam sterilizing for 20 min. The solid medium was added with 2.0% agar.
Fermentation medium: 0.5% of NaAc, 0.15% of yeast extract and MgSO4·7H2O 0.02％、K2HPO40.04％、(NH4)2SO40.05％、CaCO31%, ethanol 2.0% (added after sterilization) and water in balance, and sterilizing with 0.12MPa high pressure steam for 20min under natural pH.
1.3.3.2.2 fermentation test of bacterial species
Selecting 12 strains of microorganism strains which generate odor on a solid culture medium from microorganism strains in separated pit mud which takes non-starch sugar as a carbon source, fermenting, wherein the 12 strains of microorganism strains are obtained in a micro-aerobic state and are respectively numbered from No. 1 to No. 12.
Activating strains: respectively selecting strain activating culture medium according to strain characteristics, inoculating, and activating under the conditions of micro-oxygen and 35 deg.C for 12-48 h.
Seed culture: taking two-ring strains from a fresh inclined plane, inoculating the two-ring strains into a test tube (25mL of shake flask liquid containing amount is 10-15mL), and statically culturing for 24h to prepare a seed solution.
Fermentation culture: inoculating anaerobic and facultative strain seed liquid into fermentation medium (200mL triangular flask, liquid loading amount 180mL), and culturing at 35 deg.C under micro-aerobic condition for 72-120 h.
1.3.3.2.3 results of the experiment
After 12-24 hours of culture, the bottles No. 1, 3, 5, 7, 8, 9, 10, 11 and 12 start to produce gas in the fermentation, the bottles No. 2, 4 and 6 do not produce gas, the gas production is little after 96 hours of fermentation, the bottles except No. 2, 4 and 6 have odor, and the fermentation is basically finished.
1.3.4 analysis of microbial fermentation products susceptible to odor development
1.3.4.1 sample pretreatment
And (3) a pipette sucks 8mL of fermentation liquor and adds the fermentation liquor into a headspace bottle, 3.0g of sodium chloride is weighed and added into the headspace bottle, a stirring magneton is added, the temperature of a magnetic stirrer is set to be 50 ℃, the rotating speed is 400r/min, and the headspace of a PDMS extraction head is extracted for 30 min.
The extraction head is selected: the head was extracted with 50/30 μm DVB/CAR/PDMS three phase.
1.3.4.2 Experimental gas chromatography
Analyzing and detecting the components of the strong aromatic white spirit by using a gas chromatography-mass spectrometer (GC-MS), and smelling by using a fragrance smelling instrument.
Analysis conditions of the gas chromatograph-mass spectrometer are as follows: the flow rate of the non-flow distribution is 0.9 mL/min; sample inlet temperature: 230 ℃; temperature rising procedure: keeping the temperature at 35 ℃ for 4min, heating to 60 ℃ at 2 ℃/min without keeping, and heating to 180 ℃ at 6 ℃/min for 15 min.
The analysis results of the fermentation liquid of the microbial strains in 9 strains of pit mud with sugar as a carbon source are shown in table 1; the results of analysis of fermentation liquid of microbial species in 12-strain pit mud using non-starch sugar as a carbon source are shown in Table 2.
1.3.4.3 results of the analysis
From the analysis results of the fermentation liquid of 9 strains of pit mud microbial strains using sugar as a carbon source, it can be seen that: the 9 pit mud microorganisms produce little 4-methylphenol, the threshold value of 4-methylphenol is 166 mu g/L, and 9 strains are not enough to cause wine to have pit mud odor.
From the analysis results of the fermentation liquid of the microbial strains in the 12 strains of pit mud which takes non-starch sugar as the carbon source, it can be seen that: the amount of 4-methylphenol produced by the bacterium No. 5 is 7432.50 mug/L, and the amount of 4-methylphenol produced by the bacterium No. 9 is 2846.49.50 mug/L; although the amount of 4-methylphenol product produced in No. 7 is greater than its threshold, the amount of 4-methylphenol product is still small.
The strains No. 5 and No. 9 are analyzed to be high in 4-methylphenol product production amount and enough to cause wine to have pit mud odor, and the strains No. 5 and No. 9 are considered to be the strains which are searched for and produce 4-methylphenol.
1.3.5 pit mud microorganism identification
The No. 5 and No. 9 strains are sent to the company Limited of biological engineering (Shanghai) for molecular biological identification, and the molecular 16SrDNA sequencing and comparison result shows that 2 strains are: the collection name of the bacterium No. 5 is Clostridium sartagoforme bzxyb10007, the collection name of the bacterium No. 9 is Clostridium terrium bzxyb10008, and the two strains are all Clostridium.
2 discussion and conclusions
The invention finds out the bacterial strain with high yield of 4-methylphenol by separating and purifying pit mud microorganisms, analyzing and detecting fermentation products thereof, wherein the preservation names of the bacterial strain with high yield of 4-methylphenol are Clostridium sartagoforme bzxyb10007 and Clostridium terrium bzxyb10008, and the two bacterial strains belong to the genus Clostridium. The single strain which can be cultured and has high yield is obtained, and theoretical and technical support is provided for analyzing the pit mud odor formation mechanism and constructing a mud odor elimination strategy, so that the pit mud odor elimination strategy can be constructed by starting from pit mud culture and maintenance and prevention and control of mud odor generation microorganisms, and the problem of pit mud odor in the strong aromatic white spirit base liquor is fundamentally solved. Meanwhile, the method provides possibility for producing 4-methylphenol in a biological cleaning way.
The present invention is not limited to the above exemplary embodiments, and any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (2)
1. Clostridium tetani (Clostridium tetani) for producing 4-methylphenolClostridium sartagoforme) The method is characterized in that: the Clostridium pleionis has a preservation name of Clostridium sartagoforme bzxyb10007, is preserved in the China center for type culture collection, and has a preservation number of CCTCC NO: m2020623, the preservation date is 10 months and 22 days in 2020, and the preservation address is Wuhan, Wuhan university in China.
2. Use of a strain of clostridium pleriiformis according to claim 1, wherein: is used for producing 4-methylphenol by fermentation as a production strain.
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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 |
| WO2017074566A1 (en) * | 2015-10-30 | 2017-05-04 | Dean Falb | Bacteria engineered to treat diseases that benefit from reduced gut inflammation and/or tightened gut mucosal barrier |
| CN109971681A (en) * | 2019-03-29 | 2019-07-05 | 泸州品创科技有限公司 | Old cellar clostridium and application thereof |
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- 2020-12-29 CN CN202011589183.7A patent/CN112625963B/en active Active
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017074566A1 (en) * | 2015-10-30 | 2017-05-04 | Dean Falb | Bacteria engineered to treat diseases that benefit from reduced gut inflammation and/or tightened gut mucosal barrier |
| 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 |
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| Complete genome sequence of a malodorant-producing acetogen, Clostridium scatologenes ATCC 25775(T);ZhengangZhu et al.;《Journal of Biotechnology》;20151031;第212卷(第20期);第19-20页 * |
| 浓香型白酒中窖泥异味物质4-甲基苯酚的产生机制研究;刘博;《中国优秀博硕士学位论文全文数据库(硕士) 工程科技Ⅰ辑》;20170215(第02期);第B024-1228页 * |
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