CN114806969B - Acinetobacter petite, esterification liquid, method for treating yellow serofluid and application - Google Patents
Acinetobacter petite, esterification liquid, method for treating yellow serofluid and application Download PDFInfo
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Abstract
The invention relates to the technical field of brewing, in particular to a method for treating yellow serofluid by using acinetobacter pith, esterified liquid and application of the method. The Acinetobacter of the invention is named Acinetobacter pipittii; the preservation number is CGMCC No.24023. The acinetobacter petite has the capability of efficiently catalyzing and synthesizing ethyl caproate, has strong specificity, hardly produces other esters, and can be applied to treating yellow serofluid.
Description
Technical Field
The invention relates to the technical field of brewing, in particular to a method for treating yellow serofluid by using acinetobacter pith, esterified liquid and application of the method.
Background
The sales of the strong aromatic Chinese spirits are about 70%. The wine has rich fragrance, sweet taste, coordinated fragrance and clean tail, and is deeply favored by consumers. Studies have shown that about 2% of the aroma-producing substances in white spirit are important substances for determining the quality of the wine. In the flavor substances, the ester components are important components, the synthesis of the ester substances is mainly generated by esterification reaction, the actual reaction is acid and alcohol, the dehydration is carried out to generate ester, the reaction is mainly carried out in a mild environment in a microorganism participating in white spirit brewing, and the process is completed by the participation of esterifying enzyme. The main aroma substance of the strong aromatic Chinese spirits is ethyl caproate, the production period is longer, the ethyl caproate yield is low, and the cost is high in the fermentation process in the cellar, so that the strong aromatic Chinese spirits become a technical problem in the production of the strong aromatic Chinese spirits.
The strains for producing the esterifying enzyme in the brewing process of the strong aromatic white spirit comprise fungi, bacteria and saccharomycetes, wherein the mold mainly comprises monascus, rhizopus, mucor and the like, the saccharomycetes mainly comprises aroma-producing yeasts, and the bacteria have fewer reports (mainly bacillus). Therefore, screening bacterial strains for efficient synthesis of ethyl caproate has important application potential.
Yellow serofluid contains abundant residual starch, reducing sugar and a large amount of organic acids, wherein the contents of acetic acid, butyric acid and lactic acid are the most abundant, and if the yellow serofluid is directly discharged without treatment, the yellow serofluid not only pollutes the environment, but also causes resource waste. At present, yellow serofluid can be prepared into esterified liquid through Daqu, and then is mixed in wine body by using a series steaming process. However, the characteristics of low ethyl caproate content and ethyl lactate content of the esterified liquid are often present at present.
Therefore, it is necessary to develop a strain for efficiently catalyzing and synthesizing ethyl caproate in yellow serofluid environment and a method for treating yellow serofluid.
Disclosure of Invention
The invention provides a high-yield ethyl caproate strain, namely the Acinetobacter pituitosum, which is obtained through a screening and purifying process.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a strain of Acinetobacter petite, the Latin name of which is Acinetobacter pittii; the preservation number is CGMCC No.24023, and the strain code is TGHSD ZWHG08.
The invention also provides application of the acinetobacter pituitosum in ethyl caproate production.
The invention further provides an esterification liquid which comprises the fermentation supernatant of the acinetobacter baumannii, mixed acid, ethanol and water.
Preferably, the mixed acid is caproic acid, acetic acid, butyric acid and lactic acid; the volume ratio of the caproic acid to the acetic acid to the butyric acid to the lactic acid is 1-3: 1 to 3:1 to 3:1 to 3;
the volume ratio of the fermentation supernatant to the mixed acid to the ethanol to the water of the Acinetobacter petite is 20-30: 4-8: 20-30: 70-80.
Preferably, the preparation method of the fermentation supernatant of the acinetobacter baumannii comprises the following steps: inoculating the acinetobacter baumannii into a liquid culture medium for culture to obtain fermentation liquor, centrifuging the fermentation liquor, and collecting supernatant to obtain the acinetobacter baumannii fermentation supernatant.
Preferably, the liquid fermentation medium uses water as a solvent, and comprises the following components in concentration: 15-30 g/L of beef extract, 1-2 g/L of calcium chloride, 35-70 g/L of sucrose and 1-3 g/L of magnesium sulfate heptahydrate; the pH value of the liquid fermentation culture medium is 6.5-7.5;
the rotational speed of the centrifugation is 2500-3500 r/min; the centrifugation time is 13-17 min;
the inoculation proportion of the Acinetobacter petite is 1-5 multiplied by 10 6 individual/mL;
the culture time is 2-4 d; the culture temperature is 34-38 ℃.
The invention further provides a preparation method of the esterified liquid, which comprises the steps of mixing mixed acid with ethanol to obtain a mixture 1, mixing the mixture 1 with water to obtain a mixture 2, and mixing and esterifying the mixture 2 with the Acinetobacter peter fermentation supernatant.
Preferably, the esterification temperature is 30-36 ℃; the esterification time is 5-9 d.
The invention further provides a method for treating yellow serofluid, which comprises the following steps: mixing yellow serofluid, wine tails, pit mud culture solution, fermented grains and the esterified solution, and culturing at constant temperature.
Preferably, the mass ratio of the yellow serofluid to the wine tail to the pit mud culture solution to the fermented grains to the esterified liquid is 20-30: 65-75: 1-2: 1-2: 1.5 to 2.5;
the constant temperature culture time is 25-35 d; the constant temperature culture temperature is 30-36 ℃.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention screens out the strain of high-yield ethyl caproate, i.e. the Acinetobacter pituitosum, and reports that the strain has the capability of synthesizing ethyl caproate for the first time;
2. the esterase produced in the Acinetobacter fermentation broth has high specificity, almost no other esters are produced, and only a small amount of ethyl caproate is produced;
2. the acinetobacter petite can be applied to treating yellow serofluid, and the esterification liquid is obtained after the yellow serofluid is esterified, so that the ethyl caproate content of the esterification liquid is increased, and the method has the potential of improving the quality of wine. Therefore, the method not only can effectively treat the pollution of yellow serofluid to the environment, but also achieves the aim of high-value utilization of resources.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a plate photograph of the Acinetobacter baumannii strain;
FIG. 2 is a diagram showing the peak of the 27F-terminal sequencing part of the 16SrDNA sequence of the Acinetobacter picolina strain (200-300 pb range);
FIG. 3 shows the peak diagram (200-300 pb range) of the 1492R-end sequencing part of the 16SrDNA sequence of the Acinetobacter vinelandii;
FIG. 4 shows NCBI alignment of the 16SrDNA sequence of the Acinetobacter picolina species.
Description of biological preservation
Acinetobacter petite, latin name Acinetobacter pittii;
the strain is preserved in China general microbiological culture Collection center: the addresses are: no. 3 of North Chen Xili No. 1, the preservation date is 2021, 12, 02, and the preservation number is CGMCC No.24023.
Detailed Description
The invention provides a strain of Acinetobacter petite, the Latin name of which is Acinetobacter pittii; the preservation number is CGMCC No.24023. The bacterial colony of the strain is white bulge, and the surface is smooth and moist.
The invention also provides application of the acinetobacter pituitosum in ethyl caproate production.
The invention further provides an esterification liquid which comprises the fermentation supernatant of the acinetobacter baumannii, mixed acid, ethanol and water.
In the present invention, the mixed acid is caproic acid, acetic acid, butyric acid and lactic acid; the volume ratio of the caproic acid to the acetic acid to the butyric acid to the lactic acid is 1-3: 1 to 3:1 to 3:1 to 3; preferably 1 to 2: 1-2: 1-2: 1 to 2; further preferably 2:2:2:1 to 2; more preferably 1:1:1:1.
in the invention, the volume ratio of the fermentation supernatant of the Acinetobacter petite, the mixed acid, the ethanol and the water is 20-30: 4-8: 20-30: 70-80 parts; preferably 22 to 28:5 to 7: 22-28: 72-78; more preferably 24 to 26:6: 24-26: 74-76; more preferably 25:6:25:75.
in the invention, the preparation method of the fermentation supernatant of the acinetobacter baumannii comprises the following steps: inoculating the acinetobacter baumannii into a liquid culture medium for culture to obtain fermentation liquor, centrifuging the fermentation liquor, and collecting supernatant to obtain the acinetobacter baumannii fermentation supernatant.
In the invention, the liquid fermentation medium takes water as a solvent and comprises the following components in concentration: 15-30 g/L of beef extract, 1-2 g/L of calcium chloride, 35-70 g/L of sucrose and 1-3 g/L of magnesium sulfate heptahydrate; preferably 20-25 g/L of beef extract, 1.5g/L of calcium chloride, 50-60 g/L of sucrose and 1.5-2.5 g/L of magnesium sulfate heptahydrate; further preferably, the beef extract is 22-23 g/L, calcium chloride is 1.5g/L, sucrose is 54-56 g/L, and magnesium sulfate heptahydrate is 2g/L; more preferably, the beef extract is 22.5g/L, calcium chloride is 1.5g/L, sucrose is 55g/L, and magnesium sulfate heptahydrate is 2g/L.
In the invention, the pH of the liquid fermentation medium is 6.5-7.5; preferably 6.7 to 7.3; further preferably 6.9 to 7.1; more preferably 7.
In the invention, the rotational speed of the centrifugation is 2500-3500 r/min; preferably 2700 to 3300r/min; further preferably 2900 to 3100r/min; more preferably 3000r/min.
In the invention, the centrifugation time is 13-17 min; preferably 14 to 16 minutes; further preferably 15min.
In the present invention, the ratio of the inoculation of the Acinetobacter petite is 1 to 5X 10 6 individual/mL; preferably 2 to 4X 10 6 individual/mL; further preferably 3X 10 6 And each mL.
In the invention, the culture time is 2-4 d; preferably 3d.
In the invention, the culture temperature is 34-38 ℃; preferably 35 to 37 ℃; further preferably 36 ℃.
The invention further provides a preparation method of the esterified liquid, which comprises the steps of mixing mixed acid with ethanol to obtain a mixture 1, mixing the mixture 1 with water to obtain a mixture 2, and mixing and esterifying the mixture 2 with the Acinetobacter peter fermentation supernatant.
In the invention, the esterification temperature is 30-36 ℃; preferably 31-35 ℃; further preferably 32 to 34 ℃; more preferably 33 ℃.
In the invention, the esterification time is 5-9 d; preferably 6 to 8 days; further preferably 7d.
The invention further provides a method for treating yellow serofluid, which comprises the following steps: mixing yellow serofluid, wine tails, pit mud culture solution, fermented grains and the esterified solution, and culturing at constant temperature.
In the invention, the mass ratio of the yellow serofluid, the wine tails, the pit mud culture solution, the fermented grains and the esterified liquid is 20-30: 65-75: 1-2: 1-2: 1.5 to 2.5; preferably 22 to 28: 67-73: 1.5:1.5:1.7 to 2.3; more preferably 24 to 26:69 to 71:1.5:1.5:1.9 to 2.1; more preferably 25:70:1.5:1.5:2.
in the invention, the constant temperature culture time is 25-35 d; preferably 27 to 33d; further preferably 29 to 31d; more preferably 30d.
In the invention, the constant temperature culture temperature is 30-36 ℃; preferably 31-35 ℃; further preferably 32 to 34 ℃; more preferably 33 ℃.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
The titration methods used in the examples below were:
(1) 30% absolute ethanol: 30mL of absolute ethyl alcohol is put into a volumetric flask with 100mL, the volume is fixed to 100mL, and the absolute ethyl alcohol and the volumetric flask are uniformly mixed;
(2) 0.1mol/LNaOH solution: weighing 4g of sodium hydroxide, adding a small amount of distilled water for dissolution, fixing the volume to 1000mL, and uniformly mixing;
(3)0.1mol/L H 2 SO 4 solution: measuring 5.43mL of concentrated sulfuric acid, slowly adding into proper amount of water under continuous stirring, cooling, diluting to 1000mL, and mixingUniform;
(4) The method for measuring the esterifying enzyme comprises the following steps: 1.5mL of hexanoic acid was added, 25mL of absolute ethanol was added, and after sufficient shaking, 75mL of pure water was added, and shaking was again sufficient. 25ml of enzyme solution is weighed and added into a conical flask mixed with the liquid, shaking is carried out, the conical flask is tightly tied by a transparent sealing film, and meanwhile, a blank test is carried out according to the same sequence, but the enzyme solution is replaced by the enzyme solution after the enzyme solution is sterilized by high-pressure steam at 115 ℃ for 20min, and other reagents and operation steps are kept unchanged.
Finally, the esterifying enzyme sample conical flasks of the experimental group and the control group are sequentially placed into a constant temperature incubator for thermal insulation and esterification for 7 days, and meanwhile, marks are made. The distillation device is assembled in advance, the bacteria liquid after 7 days of esterification is washed, the bacteria liquid is poured into a 250mL distillation flask, a small amount of absolute ethyl alcohol with the concentration of 30% is used for washing an conical flask of an esterifying enzyme sample for many times, the washed liquid is poured into the flask together to ensure the accuracy of an experiment, a 50mL volumetric flask is adopted to collect the distilled liquid at a collecting device, the volumetric flask is placed in the beaker, a proper amount of ice water is placed in the beaker, then distillation is started, when the liquid of the small volumetric flask is close to a 50mL score line, the volumetric flask is placed in a constant temperature box for heat preservation, the temperature is kept at 20 ℃, and the liquid in the volumetric flask is fully and uniformly mixed for later use.
After the liquid distilled in the last step is kept at 20 ℃, the liquid is poured into a 250mL beaker with a plug, a few drops of phenolphthalein are added, the neutralization titration is carried out by using a freshly prepared NaOH solution (0.1 mol/L), the consumption of the solution is recorded, then the amount of the solution is measured and added with 25mL of sodium hydroxide solution, the solution is poured into the flask with the plug, the solution is fully vibrated and shaken uniformly, the solution is put into boiling water and boiled for 30 minutes, after heating, the liquid with the plug beaker is cooled to the room temperature, the titration is carried out by using a prepared sulfuric acid solution (0.1 mol/L), and when the red color disappears, the consumption H is recorded 2 SO 4 Is a combination of the amounts of (a) and (b).
The calculation formula is as follows:
(1) Total ester content of sample (in the case of ethyl caproate)
A＝Ai-A0
50—volume of experimental esterified sample, unit milliliter mL;
25.0-volume of sodium hydroxide solution (0.1 mol/L) added during saponification in mL;
0.142-mass of ethyl hexanoate in grams equivalent to 1.00mL sodium hydroxide solution;
a-the unit is gram per liter, and the content of the total ester of the experimental sample;
ai-the total ester content measured in grams per liter without subtraction of the blank;
a0-the total ester content measured in grams per liter in a blank test,
ci-concentration of sulfuric acid titration solution in moles per liter;
c-the concentration of sodium hydroxide solution in moles per liter;
v1-the unit is milliliter ml, consume H 2 SO 4 (0.1 mol/L);
(2) Esterification force of sample y=a×50×2
2-conversion coefficient of the units of the koji enzyme activity;
y-the esterification force of the sample in u;
50—sample volume in milliliters;
a-total esters of distillate in grams per liter;
example 1
A method for screening high-yield ethyl caproate acinetobacter sp comprises the following steps:
(1) Taking 1.0g of pit mud sample, adding the pit mud sample into 99mL of sterile water with glass beads under aseptic condition, shaking uniformly to prepare 10 -2 The diluted solution is respectively diluted to 10 -3 、10 -4 、10 -5 、10 -6 100 mu L of each gradient dilution is coated on a selective culture medium, and the culture is carried out for 3 days at the temperature of 30 ℃;
(2) Screening and picking 10 strains (with the numbers of TG-1 to TG-10) with larger hydrolysis circles from a selection medium, streaking and separating the strains on a plate to obtain pure seeds, numbering the strains obtained by screening and preserving the strains in a 20% glycerol tube, and preserving the strains in a refrigerator at a temperature of minus 20 ℃;
(3) Inoculating the pure strain screened by the streak into an inclined plane filled with LB solid culture medium, and culturing in a constant temperature incubator at 30 ℃ for 3d;
(4) Preparing the strain obtained by primary screening into seeds according to the concentration of 1×10 6 Inoculating the strain/mL into a triangular flask filled with a liquid fermentation culture medium, and placing the triangular flask into a constant-temperature shaking table at 36 ℃ to culture for 3d at 180 r/min;
(5) 20ml of fermentation broth is added into a 50ml centrifuge tube, and after centrifugation for 15min at 3000r/min, the strain esterification force is measured by titration of the supernatant, and the results are shown in Table 1. The four strains TG-3, TG-5, TG-6 and TG-9 obtained by measurement have higher esterification force.
Wherein the selection medium formulation is 1L: 90mL of emulsion, 3.5mL of olive oil, 2g/L of sodium nitrate, 1g/L of dipotassium hydrogen phosphate, 0.5g/L of magnesium sulfate, 0.5g/L of potassium chloride, 0.01g/L of ferrous sulfate, 12g/L of glucose, 16g/L of agar and the balance of water, and natural pH.
The formula of the screening culture medium is as follows: 10g/L of fish meal peptone, 10g/L of sodium chloride, 6.5g/L of yeast extract powder, 30g/L of agar and the balance of water, and pH7.0.
The formula of the liquid fermentation medium is as follows: 30g/L of beef extract, 2g/L of calcium chloride, 70g/L of sucrose, 3g/L of magnesium sulfate heptahydrate and the balance of water, wherein the pH value is 7.5.
Table 1 comparison of esterification force results for strains
Example 2
Inoculating the four strains TG-3, TG-5, TG-6 and TG-9 selected in the example 1 into an inclined plane filled with LB solid culture medium, and culturing in a constant temperature incubator at 30 ℃ for 3d;
(2) Inoculating four bacterial inclined plane strains into a conical flask filled with a liquid culture medium, putting into a constant temperature shaking table at 36 ℃, and culturing for 3d at 180 r/min;
(3) Adding 20ml of fermentation liquor into a 50ml centrifuge tube, centrifuging for 15min at 3000r/min in a centrifuge, and obtaining supernatant as enzyme solution;
(4) Taking 1.5ml of caproic acid, acetic acid, butyric acid, lactic acid and mixed acid of four acids (1:1:1), adding 25ml of absolute ethyl alcohol, shaking uniformly, adding 75ml of pure water, shaking uniformly, adding 25ml of supernatant of four strains of TG-3, TG-5, TG-6 and TG-9 respectively, shaking uniformly, fastening by using a sealing film, and placing into an incubator for esterification for 7d to prepare esterified solutions of different acids;
(5) The ability of the strain to produce ethyl caproate was detected by subjecting the esterified liquid to gas phase chromatography, and the data of the gas chromatography analysis result are shown in Table 2. The peter's immobilized lever (the amplification sequence is shown as SEQ NO: 3) of TG-3 is detected to have the strongest capacity for producing ethyl caproate and has stronger specificity in mixed acid esterification.
Wherein, the formula of the liquid fermentation medium is as follows: 20g/L of beef extract, 2g/L of calcium chloride, 60g/L of sucrose, 1.5g/L of magnesium sulfate heptahydrate and the balance of water, and the pH value is 6.5.
TABLE 2 results of gas chromatography analyses (g/100 ml) of esterified solutions of four strains of bacteria
Example 3
After the extraction of the strain DNA, PCR amplification was performed: sequence amplification using universal primers:
the gene amplification adopts bacterial universal primer amplification.
The primer sequences are respectively upstream primers (27F: 5 '-AGAGTTTGATCCTGGCTCAG-3') (shown as SEQ NO: 1);
the downstream primer (1499R: 5 '-GGTTACCTTGTTACGACTT-3') (shown in SEQ NO: 2).
The 20. Mu.l PCR reaction system included: 2.0. Mu.l of 10 XEx Taq buffer, 0.2. Mu.l of 5U Ex Taq, 1.6. Mu.l of 2.5mM dNTP mix, 1. Mu.l of primer 1/primer 2 (sequence 1 and sequence 2), 0.5. Mu.l of DNA template, 13.7. Mu.l of ddH 2 O。
The amplification conditions were 95℃for 5min pre-denaturation, followed by cycling, 94℃for 30S denaturation, 56℃for 30S annealing, 72℃for 1.5min extension, 35 cycles, and most preferably 72℃for 1.5min extension. The obtained product is purified by a kit, cloned in, and positive colonies are identified for sequencing.
The amplified products were sequenced using a generation sequencing platform 3730, and each sample was sequenced to yield a peak image in abl format as shown in FIG. 2 (27F end sequencing portion peak plot) and FIG. 3 (1492R end sequencing portion peak plot). The quality of the sequences at the two ends of the general sequencing is poor, the sequences with low quality at the two ends of the general sequencing are removed through quality shearing, and the quality-controlled double-end sequencing result is assembled to obtain the 16S rRNA sequence or 26S rDNA.
NCBI database alignment confirms species: and (3) carrying out database comparison by using the assembly sequence of the sample, judging according to coverage, similarity and the like of the comparison result, and determining the species of the sample. Typically, the highest alignment score is chosen, as shown in FIG. 4.
Example 4
A method for treating yellow serofluid comprises the following steps: mixing yellow serofluid, wine tails, pit mud culture solution, fermented grains and esterified solution, and culturing at 30deg.C for 25 days;
the mass ratio of the yellow serofluid to the wine tails to the pit mud culture solution to the fermented grains to the esterified liquid is 20:65:1:1:1.5;
the preparation method of the esterification liquid comprises the steps of mixing mixed acid and ethanol to obtain a mixture 1, mixing the mixture 1 with water to obtain a mixture 2, and mixing the mixture 2 with the fermentation supernatant of the Acinetobacter peter at 30 ℃ for esterification for 5d;
the volume ratio of TG-3 fermentation supernatant to mixed acid, ethanol and water in the esterified liquid is 20:4:20:70; the mixed acid is caproic acid, acetic acid, butyric acid and lactic acid, and the volume ratio is 1:3:3:3, a step of;
preparation of the fermentation supernatant of Acinetobacter pitiThe method comprises the following steps: inoculating the Acinetobacter cutanea (inoculation ratio of 1×10) 6 Culturing in liquid culture medium at 34 deg.C for 2d to obtain fermentation broth, centrifuging 2500r/min for 13min, and collecting supernatant to obtain Acinetobacter pituitosus fermentation supernatant;
the liquid fermentation medium takes water as a solvent and comprises the following components in concentration: 15g/L of beef extract, 1g/L of calcium chloride, 35g/L of sucrose and 1g/L of magnesium sulfate heptahydrate; the pH of the liquid fermentation medium was 6.5.
Example 5
A method for treating yellow serofluid comprises the following steps: mixing yellow serofluid, wine tails, pit mud culture solution, fermented grains and esterified solution, and culturing at 36 ℃ for 35d;
the mass ratio of the yellow serofluid to the wine tails to the pit mud culture solution to the fermented grains to the esterified liquid is 30:75:2:2:2.5;
the preparation method of the esterification liquid comprises the steps of mixing mixed acid and ethanol to obtain a mixture 1, mixing the mixture 1 with water to obtain a mixture 2, and mixing the mixture 2 with the fermentation supernatant of the Acinetobacter peter for esterification at 36 ℃ for 9d;
the volume ratio of the fermentation supernatant of the Acinetobacter vintelco, the mixed acid, the ethanol and the water in the esterified liquid is 30:8:30:80; the mixed acid is caproic acid, acetic acid, butyric acid and lactic acid, and the volume ratio is 3:1:1:1, a step of;
the preparation method of the fermentation supernatant of the Acinetobacter piti comprises the following steps: inoculating the Acinetobacter cutanea (inoculation ratio is 5×10) 6 Culturing in liquid culture medium at 38deg.C for 4d to obtain fermentation broth, centrifuging the fermentation broth 3500r/min for 17min, and collecting supernatant to obtain Acinetobacter picoteatum fermentation supernatant;
the liquid fermentation medium takes water as a solvent and comprises the following components in concentration: 30g/L of beef extract, 2g/L of calcium chloride, 70g/L of sucrose and 3g/L of magnesium sulfate heptahydrate; the pH of the liquid fermentation medium was 7.5.
Example 6
A method for treating yellow serofluid comprises the following steps: mixing yellow serofluid, wine tails, pit mud culture solution, fermented grains and esterified solution, and culturing at a constant temperature of 33 ℃ for 30 days;
the mass ratio of the yellow serofluid to the wine tails to the pit mud culture solution to the fermented grains to the esterified liquid is 25:70:1.5:1.5:2;
the preparation method of the esterification liquid comprises the steps of mixing mixed acid and ethanol to obtain a mixture 1, mixing the mixture 1 with water to obtain a mixture 2, mixing the mixture 2 with the fermentation supernatant of the Acinetobacter peter for esterification at 33 ℃ for 7d;
the volume ratio of the fermentation supernatant of the Acinetobacter vintelco, the mixed acid, the ethanol and the water in the esterified liquid is 25:6:25:75; the mixed acid is caproic acid, acetic acid, butyric acid and lactic acid, and the volume ratio is 1:1:1:1, a step of;
the preparation method of the fermentation supernatant of the Acinetobacter piti comprises the following steps: inoculating the Acinetobacter cutanea (inoculation ratio is 3×10) 6 Culturing in liquid culture medium at 36deg.C for 3d to obtain fermentation broth, centrifuging the fermentation broth at 3000r/min for 15min, and collecting supernatant to obtain Acinetobacter picoteatum fermentation supernatant;
the liquid fermentation medium takes water as a solvent and comprises the following components in concentration: beef extract 22.5g/L, calcium chloride 1.5g/L, sucrose 55g/L, magnesium sulfate heptahydrate 2g/L; the pH of the liquid fermentation medium was 7.
Example 7
Comparison test: the esterifying ability of the Acinetobacter pituitum was compared with that of the conventional yeast powder.
Example 5 was used as the experimental group;
control group: the other methods were the same as in example 3, except that the esterified liquid was replaced with Daqu powder in the sulcus.
After the treatment of the experimental group and the control group respectively, the yellow serofluid treated by the experimental group and the control group is respectively subjected to content detection of ethyl acetate, ethyl butyrate, ethyl lactate and ethyl caproate, and the results are shown in table 3.
TABLE 3 detection results (g/100 mL)
Therefore, the yellow serofluid obtained by the treatment of the method has greatly improved ethyl caproate, and the esterification effect is obviously better than that of the traditional yeast powder.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Sequence listing
<110> Jiangsu Tanggou two-phase Hejiu industry Co., ltd
<120> a strain of Acinetobacter petite, esterified liquid, method for treating yellow serofluid and application thereof
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taccaccaat ggcaaggtcg agcggagaga ggtagcttgc tactgatctt agcggcggac 60
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aataccgcat acgtcctacg ggagaaagca ggggatcttc ggaccttgcg ctaatagatg 180
agcctaagtc ggattagcta gttggtgggg taaaggccta ccaaggcgac gatctgtagc 240
gggtctgaga ggatgatccg ccacactggg actgagacac ggcccagact cctacgggag 300
gcagcagtgg ggaatattgg acaatgggcg caagcctgat ccagccatgc cgcgtgtgtg 360
aagaaggcct tatggttgta aagcacttta agcgaggagg aggctacttt agttaatacc 420
tagagatagt ggacgttact cgcagaataa gcaccggcta actctgtgcc agcagccgcg 480
gtaatacaga gggtgcaagc gttaatcgga tttactgggc gtaaagcgcg cgtaggcggc 540
taattaagtc aaatgtgaaa tccccgagct taacttggga attgcattcg atactggtta 600
gctagagtgt gggagaggat ggtagaattc caggtgtagc ggtgaaatgc gtagagatct 660
ggaggaatac cgatggcgaa ggcagccatc tggcctaaca ctgacgctga ggtgcgaaag 720
catggggagc aaacaggatt agataccctg gtagtccatg ccgtaaacga tgtctactag 780
ccgttggggc ctttgaggct ttagtggcgc agctaacgcg ataagtagac cgcctgggga 840
gtacggtcgc aagactaaaa ctcaaatgaa ttgacggggg cccgcacaag cggtggagca 900
tgtggtttaa ttcgatgcaa cgcgaagaac cttacctggc cttgacatag taagaacttt 960
ccagagatgg attggtgcct tcgggaactt acatacaggt gctgcatggc tgtcgtcagc 1020
tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc aacccttttc cttatttgcc 1080
agcgagtaat gtcgggaact ttaaggatac tgccagtgac aaactggagg aaggcgggga 1140
cgacgtcaag tcatcatggc ccttacggcc agggctacac acgtgctaca atggtcggta 1200
caaagggttg ctacctagcg ataggatgct aatctcaaaa agccgatcgt agtccggatt 1260
ggagtctgca actcgactcc atgaagtcgg aatcgctagt aatcgcggat cagaatgccg 1320
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Claims (6)
1. Acinetobacter petite strainAcinetobacter pittii) The method comprises the steps of carrying out a first treatment on the surface of the The preservation number is CGMCC No.24023.
2. Use of acinetobacter petri according to claim 1 for the production of ethyl caproate.
3. A preparation method of an esterification liquid is characterized in that: mixing the mixed acid with ethanol to obtain a mixture 1, mixing the mixture 1 with water to obtain a mixture 2, and mixing and esterifying the mixture 2 with the fermentation supernatant of the acinetobacter pituitarium according to claim 1; the esterified liquid comprises fermentation supernatant of the acinetobacter bauhini, mixed acid, ethanol and water; the mixed acid is caproic acid, acetic acid, butyric acid and lactic acid.
4. The method for producing an esterified liquid according to claim 3, wherein the temperature of the esterification is 30 to 36 ℃; the esterification time is 5-9 d.
5. A method for treating yellow serofluid, comprising the steps of: mixing yellow serofluid, wine tails, pit mud culture solution, fermented grains and esterified liquid prepared by the method of claim 3, and culturing at constant temperature.
6. The method for treating yellow serofluid according to claim 5, wherein the mass ratio of yellow serofluid, wine tails, pit mud culture fluid, fermented grains and esterified liquid is 20-30: 65-75: 1-2: 1-2: 1.5 to 2.5;
the constant temperature culture time is 25-35 d; the temperature of the constant temperature culture is 30-36 ℃.
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