CN112481326B - Method for producing 6-pentyl-2H-pyran-2-one by virtue of loss fermentation - Google Patents
Method for producing 6-pentyl-2H-pyran-2-one by virtue of loss fermentation Download PDFInfo
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Abstract
The invention relates to a method for producing 6-pentyl-2H-pyran-2-one by virtue of fermented grains, belonging to the technical field of biological and chemical engineering. The invention provides a fermentation medium for producing 6-pentyl-2H-pyran-2-one, which contains lost grains and grains, wherein the grains are selected from at least one of sorghum, wheat, rice, corn and glutinous rice. According to the invention, the distiller grains are used as the fermentation medium components, and trichoderma can be metabolized to produce 6-pentyl-2H-pyran-2-one in a large quantity, so that the yield is high. In addition, the spent grains are recycled to produce the high-value 6-pentyl-2H-pyran-2-one, so that great economic benefits and environmental protection benefits are achieved.
Description
Technical Field
The invention relates to a method for producing 6-pentyl-2H-pyran-2-one by virtue of fermented grains, belonging to the technical field of biological and chemical engineering.
Background
6-pentyl-2H-pyran-2-one (6-pentylpyran-2-one) with molecular formula C 10 H 14 O 2 The structural formula is as follows, the boiling point is 85 ℃ (333 Pa), the grease is soluble, and the grease is insoluble in water.
The 6-pentyl-2H-pyran-2-one has the fragrance of mushroom, blue cheese, coconut or dairy, has the characteristics of both flavor compounds and biological active substances, is an extremely valuable food additive, but has limited supply. The 6-amyl-2H-pyran-2-ketone naturally exists in partial fruits and Chinese herbal medicines, and can also be obtained by chemical synthesis, microbial fermentation and other modes. Microorganisms reported to be capable of metabolizing to produce 6-pentyl-2H-pyran-2-one are commonly Trichoderma. The 6-amyl-2H-pyran-2-ketone is produced by utilizing the metabolism of microorganisms, and is extracted and applied, so that the effective way for overcoming the supply defect of the compound is provided.
The distilled grains are distilled grains discarded after the distilled grains are distilled in the traditional solid-state brewed white spirit, and are byproducts of brewing. At present, the annual white spirit yield of China is more than 600 million tons, and the annual distilled grain yield of China is up to 1800 million tons. Although some nutrients remain in the lost grains, 40 to 50% of the fermented filler rice hulls are contained, and the rice hulls mainly comprise crude fibers, so that the lost grains are limited to be recycled. In addition, the lost grains contain about 60 percent of water, are difficult to store and easy to mildew, and can only be discarded as wastes, thereby not only polluting the environment, but also causing great resource waste. Therefore, more utilization ways of the distilled grains are provided, the application value of the distilled grains is improved, and the distilled grains have great environmental protection benefits and economic benefits.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. To this end, the object of the present invention is to provide a fermentation medium for the production of 6-pentyl-2H-pyran-2-one. A second object of the present invention is to provide a fermentation feedstock for the production of 6-pentyl-2H-pyran-2-one. The third object of the present invention is to provide a process for producing 6-pentyl-2H-pyran-2-one. A fourth object of the invention is to provide a new use of a spent grain.
The invention provides a fermentation medium for producing 6-pentyl-2H-pyran-2-one, which comprises distilled grains and at least one grain selected from sorghum, wheat, rice, corn and glutinous rice.
Further, the spent grain: the weight ratio of the grains is 10: (1-10).
Preferably, the spent grain: the weight ratio of the grains is 10: (1-3).
Further, the lost grains are grains produced by brewing the Luzhou-flavor liquor.
Preferably, the raw materials for brewing the Luzhou-flavor liquor mainly comprise sorghum, wheat, rice, glutinous rice and corn.
The invention provides a fermentation raw material for producing 6-amyl-2H-pyran-2-ketone, which is obtained by inoculating Trichoderma strain which can metabolize to produce 6-amyl-2H-pyran-2-ketone into a fermentation culture medium.
Further, the fermentation raw material is obtained by the following method: taking the distiller grains and the grains, adding water, uniformly mixing, sterilizing, cooling, and inoculating the seed culture medium inoculated with the trichoderma to obtain the trichoderma strain.
Preferably, the sterilization is carried out by autoclaving at 120 ℃ for 30min.
Further, the seed culture medium is prepared by the following method: according to the lost grain: sorghum: and (2) uniformly mixing water in a mass ratio of 10.
The loss grains used for preparing the seed culture medium are preferably the loss grains produced by brewing Luzhou-flavor liquor.
Preferably, the raw materials for brewing the Luzhou-flavor liquor mainly comprise sorghum, wheat, rice, glutinous rice and corn.
Preferably, the sterilization is carried out by autoclaving at 120 ℃ for 30min.
Preferably, the Trichoderma strain is inoculated and cultured at a constant temperature of 26 ℃ for 5 days.
Furthermore, the inoculation amount of the seed culture medium accounts for 10-20% of the weight of the fermentation raw materials.
Further, grain: the weight ratio of water is 10: (6-10).
Further, the Trichoderma is Trichoderma viride Trichoderma virride.
Preferably, the Trichoderma is Trichoderma viride WLY-L-M-01CGMCC No.20254.
The invention provides a production method of 6-amyl-2H-pyran-2-ketone, which comprises the following steps: taking the fermentation raw material, performing fermentation culture, extracting the 6-amyl-2H-pyran-2-ketone generated by fermentation, and purifying the crude extract to obtain the product.
Further, the fermentation culture meets at least one of:
the culture temperature is 26-30 ℃;
the culture period is 15-20 days.
Further, the extracting satisfies at least one of:
the extraction solvent is methanol and/or ethanol;
the extraction times are 2-3 times;
volume of extraction solvent for each extraction: the weight of raw materials to be extracted is = (3-5): 1,ml/g;
for each extraction, the extraction time is 20-40 min;
the extraction mode is oscillation extraction or ultrasonic extraction;
the extraction temperature is 10-30 ℃.
Further, the purification comprises the following steps:
a. carrying out liquid-liquid extraction on the crude extract by using petroleum ether and/or ethyl acetate, and concentrating the extract to obtain a secondary extract;
b. carrying out normal phase chromatography on the secondary extract, collecting eluent containing 6-amyl-2H-pyran-2-ketone, and concentrating to obtain a crude product;
c. and (3) performing reversed-phase chromatographic separation on the crude product, collecting eluent containing 6-pentyl-2H-pyran-2-one, and concentrating to obtain 6-pentyl-2H-pyran-2-one.
Further, the normal phase chromatography satisfies at least one of the following conditions:
the stationary phase adopts normal phase silica gel;
preferably, the normal phase silica gel is 100-300 meshes;
petroleum ether is used as an elution reagent.
Further, the reverse phase chromatographic separation satisfies at least one of the following conditions:
adopting preparative chromatography;
the stationary phase is a C18 column;
the mobile phase is a mixed solvent of methanol and/or ethanol and water;
gradient elution is adopted, and the mobile phases are as follows in sequence: 0-1 min:20% v/v methanol and/or ethanol; 1.1-5 min: 20-100% v/v methanol and/or ethanol; 5.1-15 min:100% v/v methanol and/or ethanol; 15.1-17 min:20% v/v methanol and/or ethanol; stopping elution;
the chromatographic flow rate is 10-20 mL/min;
preferably, the chromatography flow rate is 10mL/min.
Further, the concentration in the step a, the step b and the step c is carried out at 40-80 ℃.
Wherein, the methanol and/or the ethanol are/is used as an extraction solvent, the extraction efficiency is high, and meanwhile, components such as saccharides, proteins and the like in the raw materials are not easy to enter an organic solvent system, so that preliminary impurity removal can be realized.
Wherein, petroleum ether and/or ethyl acetate are/is used as a liquid-liquid extraction solvent, and the target 6-amyl-2H-pyran-2-one can effectively enter an organic solvent, so that the extraction efficiency is high. Meanwhile, water and water-soluble components in the ethanol and/or methanol crude extract can be effectively separated from the target substance, so that impurity removal can be further realized.
Wherein the eluent collection mode is ultraviolet absorption peak collection, and the ultraviolet absorption wavelength is 290-310 nm.
Wherein, the concentration is carried out at 40-80 ℃, which not only can reduce the loss of the target 6-amyl-2H-pyran-2-ketone in the concentration process, but also can achieve the purpose of concentrating the target.
The invention provides application of a distiller grain in preparation of a culture medium for fermentation production of 6-pentyl-2H-pyran-2-one.
Further, the lost grains are grains produced by brewing the Luzhou-flavor liquor.
Preferably, the raw materials for brewing the Luzhou-flavor liquor mainly comprise sorghum, wheat, rice, sticky rice and corn.
Further, the culture medium is a culture medium of Trichoderma capable of metabolizing to produce 6-pentyl-2H-pyran-2-one.
Further, the Trichoderma is Trichoderma viride Trichoderma virride.
Preferably, the Trichoderma is Trichoderma viride WLY-L-M-01CGMCC No.20254.
The production method of 6-amyl-2H-pyran-2-ketone provided by the invention mainly has the following advantages:
1. the invention innovatively adopts the brewing byproduct lost grains as the components of the fermentation medium. With the lost grains as the substrate, trichoderma can be metabolized in a large amount to produce 6-pentyl-2H-pyran-2-one with high yield. In addition, the distiller grains are recycled to produce high-value 6-amyl-2H-pyran-2-one, so that the distiller grains have great economic benefit and environmental protection benefit.
2. The trichoderma used for fermentation is easily obtained in a wine brewing production environment, can adapt to the wine brewing production environment, can be metabolized in a large amount to generate 6-pentyl-2H-pyran-2-one by utilizing the wine brewing by-product lost grains, and is high in yield.
3. The extraction process adopted in the production method of 6-pentyl-2H-pyran-2-one is simple and efficient, and the purification process combines forward chromatography and reverse phase chromatography, so that the 6-pentyl-2H-pyran-2-one and impurities in the crude product can be effectively separated.
Drawings
FIG. 1 is a total ion flow diagram and an extracted ion flow diagram of 6-pentyl-2H-pyran-2-one at a standard solution concentration of 1000. Mu.g/L in example 1;
FIG. 2 is a total ion flow diagram and an extracted ion flow diagram of sample No. 3 in example 1;
FIG. 3 is a total ion flow graph and an extracted ion flow graph of sample # 1 in example 1;
FIG. 4 is a total ion flowsheet and extracted ion flowsheet for sample # 2 from example 1;
FIG. 5 is a graph showing the effect of the mixed medium composition on productivity in comparative example 1 and comparative example 2;
FIG. 6 is a graph comparing the effect of fermentation period on yield in comparative example 3.
Detailed Description
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
The spent grains used in the examples and comparative examples were spent grains from brewing multi-grain Luzhou-flavor liquor, and the brewing materials were mainly sorghum, wheat, rice, glutinous rice and corn.
Trichoderma viride virus WLY-L-M-01 used in the examples and comparative examples was collected by the inventors from the air of the brewing production environment of the laboratory of the wuliangye technology center, yibin, sichuan, with the preservation number of CGMCC No.20254, the preservation time of 2020, 9, month and 2 days, and the preservation center of CGMCC: the general microbiological center of China Committee for culture Collection of microorganisms, address No. 3 West Lu No. 1 of the morning-Yangtze district of Beijing, institute of microbiology, china academy of sciences, postal code: 100101.
1. screening method of Trichoderma viride strain
Collecting a culture medium: lactic acid 2%, glucose 1%, peptone 0.5%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.05%, chloramphenicol 0.01%, agar 2%, and distilled water, with natural pH, and autoclaving at 121 deg.C for 20min.
PDA agar medium: 1% of glucose, 0.5% of peptone, 0.1% of potassium dihydrogen phosphate, 0.05% of magnesium sulfate, 0.01% of chloramphenicol, 2% of agar and distilled water, wherein the pH is natural, and the sterilization is carried out at 121 ℃ for 20min.
100mL of the prepared collection culture medium is placed in a laboratory of the technical center of wuliangye in Yibin, sichuan for 6 hours in an open manner, sterile gauze is sealed and then cultured for 5 days at a constant temperature of 28 ℃, trichoderma filamentous thalli grow out in the culture medium, the thalli are picked by an inoculating loop and placed in a PDA agar culture medium, numbering and streaking are carried out for purification, and purification is repeated for 2-3 times until bacterial colonies on a flat plate are all in the same shape. Selecting a single bacterial colony to prepare a water immersion piece, observing the cell morphology of the thallus under a microscope, completing the separation and purification of the environmental trichoderma, and storing the slant test tube in a refrigerator at 4 ℃.
2. Morphological characteristics and molecular identification of the Trichoderma viride strains
1. Observation of colony and cell morphology
Inoculating the activated new Trichoderma strain on a PDA culture medium by using an inoculating loop streak, culturing for 3d at 28 ℃, and observing the morphological characteristics of a bacterial colony; the mycelia are picked, stained with laccolic acid cotton blue staining solution, and subjected to flaking, and the characteristics of the somatic cells are observed under a microscope.
The morphological characteristics of the bacterial colony are as follows: the colony is circular and is in a white hypha divergence shape, and green spores appear in the center.
And (3) thallus cell characteristics: conidiophores with septa, vertically or acutely symmetrical branching, conidiophores, which are single or clustered, round or oval, are visible.
2. Molecular identification
Extracting strain genome DNA, selecting an ITS universal primer to a forward primer ITS1: the genomic DNA was amplified with tccgtggaacctgcgg (SEQ ID NO: 2) and reverse primer ITS4: tcctccgcttatgatatgc (SEQ ID NO: 3) under the following reaction conditions: after 5min of pre-denaturation at 95 ℃, the following cycle was entered: denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 30s, extension at 72 ℃ for 90s, and 30 cycles; extension for 10min at 72 ℃. The result is good after 2% agarose gel electrophoresis. Sending the PCR amplification product to a company of bioengineering (Shanghai) GmbH for sequencing, carrying out BLAST sequence comparison on the sequencing result on an NCBI database to determine that the product is Trichoderma viride, and naming the Trichoderma viride as WLY-L-M-01. The strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms (CGMCC) in 2020, 9 months and 2 days, the preservation number is CGMCC No.20254, and the preservation address is the microorganism research institute of China academy of sciences No. 3, xilu No. 1 Hospital, beijing, chaoyang, beijing, chaozhong, etc. The ITS sequence of the strain is shown as SEQ ID NO. 1: <xnotran> tttcctccggctattgatatgcttaagttcagcgggtattcctacctgatccgaggtcaacatttcagaagttgggtgttttacggacgtggacgcgccgcgctcccggtgcgagttgtgcaaactactgcgcaggagaggctgcggcgagaccgccactgtatttcggggccgggatcccgtcttaggggctcccgaggtccccaacgccgaccccccggaggggttcgagggttgaaatgacgctcggacaggcatgcccgccagaatactggcgggcgcaatgtgcgttcaaagattcgatgattcactgaattctgcaattcacattacttatcgcatttcgctgcgttcttcatcgatgccagaaccaagagatccgttgttgaaagttttgattcattttgaatttttgctcagagctgtaagaaataacgtccgcgaggggactacagaaaagagtttggttggtccctccggcgggcgcctggttccggggctgcgacgcacccggggcgtgaccccgccgaggcaacagtttggtatggttcacattgggtttgggagttgtaaactcggtaatgatccctccgcaggttcaccctacggaaggat. </xnotran>
EXAMPLE 1 production of 6-pentyl-2H-pyran-2-one
Weighing 750g of distilled grains, putting the distilled grains into a stainless steel barrel of 3L, adding 200g of sorghum powder and 200g of water, uniformly mixing, sealing with gauze, and sterilizing at 120 ℃ for 30 minutes. After the sterilization is finished, taking out, pouring into a sterilized shallow tray, cooling to below 30 ℃, inoculating 230g of seed culture medium under the aseptic condition, mixing uniformly, placing at the constant temperature of 28 ℃, intermittently spraying sterile water and stirring. The preparation method of the seed culture medium comprises the following steps: the loss is as follows: sorghum: and (2) uniformly mixing the components according to the mass ratio of 10 to 10, carrying out autoclaving at 120 ℃ for 30min, cooling, then, selecting trichoderma viride plate hyphae by using an inoculating loop, inoculating the trichoderma viride plate hyphae to a seed culture medium, and carrying out constant temperature culture at 26 ℃ for 5 days, wherein the inoculation amount is 10%.
Taking out 1000g of fermentation medium cultured for 15 days, placing in a 5L stainless steel barrel, adding 3L ethanol, ultrasonically extracting at room temperature for 40min, filtering, repeatedly extracting and filtering for three times, and rotary evaporating and concentrating the filtrate at 65 deg.C to obtain 600g crude extract.
And extracting the crude extract by using 1L of petroleum ether, repeating the extraction twice, and performing rotary evaporation and concentration on the extract at the temperature of 30 ℃ to obtain 12.5g of secondary extract.
Uniformly stirring the secondary extract and 100-200 meshes of normal phase silica gel, loading the normal phase silica gel into a column with 200-300 meshes of normal phase, and carrying out normal phase column chromatography. Petroleum ether is used as an elution reagent, and the elution reagent is separately collected every 50 mL. And (3) performing liquid phase mass spectrum detection on the eluent, combining the eluents of which the 6-amyl-2H-pyran-2-one is determined, concentrating under reduced pressure at 40 ℃, and drying to obtain a crude product S1-17.
Diluting the crude product S-17 with methanol, introducing sample to prepare a chromatogram, wherein the sample amount is 5mL, and performing gradient elution by using a C18 column as a stationary phase and a mixed solvent of methanol and water as a mobile phase. The mobile phase composition in elution is as follows in sequence: 0-1 min:20% v/v methanol; 1.1-5 min: 20-100% v/v methanol (gradient change is automatically effected by the instrument); 5.1-15 min:100% v/v methanol; 15.1-17 min:20% v/v methanol; the elution was stopped. The chromatography flow rate was 10mL/min. The section having an absorption peak at an ultraviolet absorption wavelength of 300nm was collected. Performing liquid phase mass spectrum detection on the eluate, combining the eluates determined to be 6-pentyl-2H-pyran-2-one, concentrating at 55 ℃ under reduced pressure, and drying to obtain a component Z1-9.
The obtained component Z1-9 is dissolved in methanol, 6-amyl-2H-pyran-2-ketone standard substance is used as reference, the mass spectrum quantification is carried out, and the product purity is 98%. The total amount of the obtained pure product is 1.12g, the fermentation yield is 1276mg/L, and the extraction and purification efficiency is 87.77%.
In addition, a sample comparison, a liquid quality test, sample # 1: ethanol crude extract; sample # 2: 1-17 of a crude product S; sample # 3: pure component Z1-9. The total ion flow chart and the extracted ion flow chart of the 6-amyl-2H-pyran-2-ketone when the concentration of the standard solution is 1000 mu g/L are shown in figure 1, the total ion flow chart and the extracted ion flow chart of a sample 3# are shown in figure 2, the total ion flow chart and the extracted ion flow chart of the sample 1# are shown in figure 3, and the total ion flow chart and the extracted ion flow chart of the sample 2# are shown in figure 4. In FIGS. 1 to 4, the abscissa represents time/min, and the ordinate represents ion intensity. As can be seen from the figure, more strongly polar and weakly polar impurities are present in sample # 1; separating by a normal phase silica gel column to completely remove the strong polar impurities and most of the weak polar impurities in the sample No. 2; and separating by a reversed-phase preparative column, wherein the spectrogram of the sample No. 3 has no other impurity peaks except for 6-amyl-2H-pyran-2-ketone.
EXAMPLE 2 production of 6-pentyl-2H-pyran-2-one
Weighing 1000g of distiller grains, putting the distiller grains in a 3L stainless steel barrel, adding 100g of mixed grain powder (sorghum, rice, glutinous rice, corn and wheat) and 60g of water, mixing, sealing with gauze, and sterilizing at 120 deg.C for 30min. Taking out after sterilization, pouring into a sterilized shallow tray, cooling to below 30 ℃, inoculating 220g of seed culture medium under aseptic condition, mixing uniformly, placing at the constant temperature of 26 ℃, intermittently spraying sterile water and stirring. The preparation method of the seed culture medium comprises the following steps: the loss is as follows: sorghum: and (2) uniformly mixing the components according to the mass ratio of 10 to 10, carrying out autoclaving at 120 ℃ for 30min, cooling, then, selecting trichoderma viride plate hyphae by using an inoculating loop, inoculating the trichoderma viride plate hyphae to a seed culture medium, and carrying out constant temperature culture at 26 ℃ for 5 days, wherein the inoculation amount is 10%.
Taking out 1000g of fermentation medium cultured for 20 days, placing in a 5L stainless steel barrel, adding 5L methanol, ultrasonically extracting at room temperature for 30min, filtering, repeatedly extracting and filtering twice, and rotary evaporating the filtrate at 55 deg.C for concentrating to obtain 580g of crude extract.
And (3) extracting the crude extract by using ethyl acetate 1.5L for three times, and performing rotary evaporation and concentration on the extract at 40 ℃ to obtain 18.8g of secondary extract.
Uniformly stirring the secondary extract and 100-200 meshes of normal phase silica gel, loading the normal phase silica gel into a column with 200-300 meshes of normal phase, and carrying out normal phase column chromatography. Petroleum ether is used as an elution reagent, and the elution reagent is separately collected every 50 mL. And performing liquid phase mass spectrum detection on the eluent, combining the eluents determining the 6-pentyl-2H-pyran-2-one, concentrating under reduced pressure at 50 ℃, and drying to obtain a crude product S2-19.
Diluting the crude product S2-19 with methanol, and carrying out sample introduction preparation chromatography with the sample introduction amount of 5mL, and carrying out gradient elution by using a C18 column as a stationary phase and a mixed solvent of ethanol and water as a mobile phase. The mobile phase composition in elution is as follows in sequence: 0-1 min:20% v/v ethanol; 1.1-5 min: 20-100% v/v ethanol (gradient change is automatically realized by the instrument); 5.1-15 min:100% v/v ethanol; 15.1-17 min:20% v/v ethanol; the elution was stopped. The chromatography flow rate was 10mL/min. The section having an absorption peak at an ultraviolet absorption wavelength of 290nm was collected. Performing liquid phase mass spectrum detection on the eluent, combining the eluents determined to be 6-amyl-2H-pyran-2-ketone, decompressing and concentrating at 60 ℃, and drying to obtain a component Z2-8.
The obtained component Z2-8 was dissolved in methanol, and the purity of the compound was 97% by mass spectrometry using 6-pentyl-2H-pyran-2-one as a reference. The total amount of the obtained pure product is 0.90g, the fermentation yield is 1016mg/L, and the extraction and purification efficiency is 88.58%.
Comparative example 1 removal of grain from Mixed culture Medium
6-pentyl-2H-pyran-2-one was produced as in example 1, except that 200g of sorghum flour initially charged was replaced with a lost grain of equal mass, and no further water was added as it was not required to be mixed with the grain. The fermentation yield of 6-pentyl-2H-pyran-2-one was determined to be 125mg/L, see FIG. 5 condition (1).
Comparative example 2 with no loss of grain in the mixed culture medium
6-pentyl-2H-pyran-2-one was produced as in example 1, except that 750g of the initially charged spent grain was replaced with an equivalent mass of sorghum flour. The fermentation yield of 6-pentyl-2H-pyran-2-one was found to be 645mg/L, as shown in FIG. 5, condition (2).
As can be seen by comparison, in comparative example 2, since no spent grains were added, the culture medium was mainly made of sorghum, and as a result, the yield of 6-pentyl-2H-pyran-2-one was greatly reduced from that of example 1. Comparative example 1 had mostly spent grain made medium, but the yield of 6-pentyl-2H-pyran-2-one was also very low due to the absence of added food raw material. As can be seen, in example 1, the mixture of the distilled grains and the grain raw materials is used as the fermentation medium, the growth and metabolism of the microorganisms are the best, and thus, higher fermentation yield is obtained.
Comparative example 3 optimization of fermentation conditions
A fermentation medium was prepared in the same manner as in example 1 except that the culture period of the fermentation culture was varied, and the fermentation yield of 6-pentyl-2H-pyran-2-one was measured for 7 days, 9 days, 12 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, and 21 days, respectively. The results are plotted on the ordinate of the 6-pentyl-2H-pyran-2-one yield and on the abscissa of the number of days in culture, and are shown in FIG. 6.
As can be seen from the figure, the fermentation yield of 6-amyl-2H-pyran-2-ketone is higher when the fermentation culture lasts for 15-20 days, and is superior to the effect obtained by adopting other culture periods.
It should be appreciated that the particular features, structures, materials, or characteristics described in this specification may be combined in any suitable manner in any one or more embodiments. Furthermore, the various embodiments and features of the various embodiments described in this specification can be combined and combined by one skilled in the art without contradiction.
Sequence listing
<110> Yibin wuliangye Co., ltd
<120> method for producing 6-pentyl-2H-pyran-2-one by virtue of fermented grains
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tttcctccgg ctattgatat gcttaagttc agcgggtatt cctacctgat ccgaggtcaa 60
catttcagaa gttgggtgtt ttacggacgt ggacgcgccg cgctcccggt gcgagttgtg 120
caaactactg cgcaggagag gctgcggcga gaccgccact gtatttcggg gccgggatcc 180
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cgatgattca ctgaattctg caattcacat tacttatcgc atttcgctgc gttcttcatc 360
gatgccagaa ccaagagatc cgttgttgaa agttttgatt cattttgaat ttttgctcag 420
agctgtaaga aataacgtcc gcgaggggac tacagaaaag agtttggttg gtccctccgg 480
cgggcgcctg gttccggggc tgcgacgcac ccggggcgtg accccgccga ggcaacagtt 540
tggtatggtt cacattgggt ttgggagttg taaactcggt aatgatccct ccgcaggttc 600
accctacgga aggat 615
<210> 2
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tccgtaggtg aacctgcgg 19
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Claims (19)
1. The fermentation raw material for producing 6-amyl-2H-pyran-2-ketone is characterized by comprising the following steps: it will be able to metabolize to produce 6-pentylTrichoderma sp of (E) -2H-pyran-2-oneTrichodermaInoculating into fermentation medium for producing 6-pentyl-2H-pyran-2-one; the Trichoderma is Trichoderma viride WLY-L-M-01CGMCC No.20254; the fermentation medium contains lost grains and grains generated by brewing the Luzhou-flavor liquor, wherein the grains are selected from at least one of sorghum, wheat, rice, corn and glutinous rice.
2. The fermentation feedstock of claim 1, wherein: the loss is as follows: the weight ratio of the grains is 10: (1 to 10).
3. The fermentation feedstock of claim 2, wherein: the loss is as follows: the weight ratio of the grains is 10: (1 to 3).
4. The fermentation feedstock of claim 1, wherein: the raw materials for brewing the Luzhou-flavor liquor mainly comprise sorghum, wheat, rice, glutinous rice and corn.
5. The fermentation feedstock of claim 1, wherein: obtained by the following method: and (3) taking the distiller grains and the grains, adding water, uniformly mixing, sterilizing, cooling, and inoculating the seed culture medium inoculated with the trichoderma bacteria to obtain the trichoderma strain liquid.
6. The fermentation feedstock of claim 5, wherein: the seed culture medium is prepared by the following method: according to the lost grain: sorghum: and (2) uniformly mixing the water mass ratio of 10 to 6, sterilizing, cooling, and inoculating the trichoderma hyphae with the inoculation amount of 10% to obtain the trichoderma hyphae strain.
7. The fermentation feedstock of claim 6, wherein: inoculating the Trichoderma strain, and culturing at 26 deg.C for 5 days.
8. The fermentation feedstock of claim 5, wherein: the inoculation amount of the seed culture medium accounts for 10-20% of the weight of the fermentation raw materials.
9. The fermentation feedstock of claim 5, wherein: grain: the weight ratio of water is 10: (6 to 10).
A method for producing 6-pentyl-2H-pyran-2-one, characterized in that: the method comprises the following steps: taking the fermentation raw material of any one of claims 1 to 9, carrying out fermentation culture, extracting 6-pentyl-2H-pyran-2-one generated by fermentation, and purifying a crude extract to obtain the compound.
11. The method of claim 10, wherein: the fermentation culture meets at least one of the following conditions:
the culture temperature is 26 to 30 ℃;
the culture period is 15 to 20 days.
12. The method of claim 10, wherein: the extracting satisfies at least one of:
the extraction solvent is methanol and/or ethanol;
the extraction times are 2 to 3;
volume of extraction solvent for each extraction: and (3) extracting the raw materials by weight (3 to 5): 1,ml/g;
for each extraction, the extraction time is 20 to 40min;
the extraction mode is oscillation extraction or ultrasonic extraction;
the extraction temperature is 10 to 30 ℃.
13. The method of claim 10, wherein: the purification comprises the following steps:
carrying out liquid-liquid extraction on the crude extract by using petroleum ether and/or ethyl acetate, and concentrating the extract to obtain a secondary extract;
b. carrying out normal phase chromatography on the secondary extract, collecting eluent containing 6-amyl-2H-pyran-2-ketone, and concentrating to obtain a crude product;
c. and (3) carrying out reverse phase chromatographic separation on the crude product, collecting eluent containing 6-pentyl-2H-pyran-2-one, and concentrating to obtain the 6-pentyl-2H-pyran-2-one.
14. The method of claim 13, wherein: the normal phase chromatography satisfies at least one of the following conditions:
the stationary phase adopts normal phase silica gel;
the normal phase silica gel is 100 to 300 meshes;
petroleum ether is used as an elution reagent.
15. The method of claim 13, wherein: the reverse phase chromatographic separation satisfies at least one of the following conditions:
a preparative chromatograph is adopted;
the stationary phase is a C18 column;
the mobile phase is a mixed solvent of methanol and/or ethanol and water;
gradient elution is adopted, and the mobile phase comprises the following steps in sequence: 0-1 min:20% v/v methanol and/or ethanol; 1.1-5 min: 20-100% v/v methanol and/or ethanol; 5.1-15 min:100% v/v methanol and/or ethanol; 15.1-17 min:20% v/v methanol and/or ethanol; stopping elution;
the chromatographic flow rate is 10 to 20mL/min.
16. The method of claim 15, wherein: the chromatography flow rate was 10mL/min.
17. The method of claim 13, wherein: and (c) concentrating at 40-80 ℃.
18. The application of the loss grain in preparing a culture medium for fermentation production of 6-pentyl-2H-pyran-2-one; the lost grains are produced by brewing the Luzhou-flavor liquor; the culture medium is Trichoderma capable of metabolizing to produce 6-amyl-2H-pyran-2-ketoneTrichodermaThe culture medium of (1); the Trichoderma strain is Trichoderma viride WLY-L-M-01CGMCC No.20254.
19. Use according to claim 18, characterized in that: the raw materials for brewing the Luzhou-flavor liquor mainly comprise sorghum, wheat, rice, glutinous rice and corn.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0568531A (en) * | 1991-09-09 | 1993-03-23 | Mercian Corp | Method for solubilizing lees of rice wine (sake) |
| CN101565688A (en) * | 2008-04-25 | 2009-10-28 | 中国科学院沈阳应用生态研究所 | Trichodrema spp. microbial agent and preparation method thereof |
| CN102559766A (en) * | 2010-12-20 | 2012-07-11 | 北京林业大学 | Method for producing trichoderma biocontrol agent by using potato starch waste water and beer grains |
| CN109837220A (en) * | 2017-11-27 | 2019-06-04 | 刘大兴 | A kind of utilization method of spirit stillage |
-
2020
- 2020-12-01 CN CN202011399179.4A patent/CN112481326B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0568531A (en) * | 1991-09-09 | 1993-03-23 | Mercian Corp | Method for solubilizing lees of rice wine (sake) |
| CN101565688A (en) * | 2008-04-25 | 2009-10-28 | 中国科学院沈阳应用生态研究所 | Trichodrema spp. microbial agent and preparation method thereof |
| CN102559766A (en) * | 2010-12-20 | 2012-07-11 | 北京林业大学 | Method for producing trichoderma biocontrol agent by using potato starch waste water and beer grains |
| CN109837220A (en) * | 2017-11-27 | 2019-06-04 | 刘大兴 | A kind of utilization method of spirit stillage |
Non-Patent Citations (8)
| Title |
|---|
| Characterization of the Major Aroma Constituent of the Fungus Trichoderma viride;Collins等;《J. AGR. FOOD CHEM.》;19721231;全文 * |
| Recovery of 6-pentyl-alpha-pyrone from Trichoderma viride culture medium by perevaporation;Bengtson等;《Biotechnology Techniques》;19920131;全文 * |
| 一株绿色木霉的筛选、发酵条件优化及利用白酒丢糟发酵的初步研究;胡梦 等;《中国酿造》;20080530;全文 * |
| 多粮型白酒酒糟成分分析研究;胡伟 等;《食品工业科技》;20140728;全文 * |
| 微生物发酵白酒糟的研究;郭素环 等;《广东农业科学》;20120425;第115页"1.1试验材料" * |
| 渐绿木霉抑菌物质的分离纯化及其对植物病原菌的抑制作用;张量 等;《中国农业科学》;20150302;全文 * |
| 王淼 等.七、6-戊基-2-吡喃酮.《食品风味物质与生物技术》.中国轻工业出版社,2004,第104页. * |
| 白酒酿造环境绿色木霉新菌株及其在低度潮饮酒方面的应用;廖勤俭 等;《酿酒科技》;20210918;全文 * |
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