CN112961049B - Preparation method of optical dextro-3-hydroxybutyric acid - Google Patents
Preparation method of optical dextro-3-hydroxybutyric acid Download PDFInfo
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- CN112961049B CN112961049B CN202110140407.4A CN202110140407A CN112961049B CN 112961049 B CN112961049 B CN 112961049B CN 202110140407 A CN202110140407 A CN 202110140407A CN 112961049 B CN112961049 B CN 112961049B
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- 230000003287 optical effect Effects 0.000 title claims abstract 8
- 238000002360 preparation method Methods 0.000 title claims abstract 7
- WHBMMWSBFZVSSR-UHFFFAOYSA-N 3-hydroxybutyric acid Chemical compound CC(O)CC(O)=O WHBMMWSBFZVSSR-UHFFFAOYSA-N 0.000 claims abstract 17
- 238000000855 fermentation Methods 0.000 claims abstract 10
- 230000004151 fermentation Effects 0.000 claims abstract 10
- 241000894006 Bacteria Species 0.000 claims abstract 8
- 239000002253 acid Substances 0.000 claims abstract 6
- 239000000706 filtrate Substances 0.000 claims abstract 6
- 238000010438 heat treatment Methods 0.000 claims abstract 6
- 239000000126 substance Substances 0.000 claims abstract 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract 5
- 150000001720 carbohydrates Chemical class 0.000 claims abstract 3
- 238000004321 preservation Methods 0.000 claims abstract 3
- QRDZSRWEULKVNW-UHFFFAOYSA-N 6-hydroxy-2-oxo-1h-quinoline-4-carboxylic acid Chemical class C1=C(O)C=C2C(C(=O)O)=CC(=O)NC2=C1 QRDZSRWEULKVNW-UHFFFAOYSA-N 0.000 claims abstract 2
- 230000002378 acidificating effect Effects 0.000 claims abstract 2
- 238000001816 cooling Methods 0.000 claims abstract 2
- 238000001914 filtration Methods 0.000 claims abstract 2
- 239000001963 growth medium Substances 0.000 claims abstract 2
- 238000002156 mixing Methods 0.000 claims abstract 2
- 239000000203 mixture Substances 0.000 claims abstract 2
- 230000007935 neutral effect Effects 0.000 claims abstract 2
- 238000000926 separation method Methods 0.000 claims abstract 2
- 238000003756 stirring Methods 0.000 claims abstract 2
- 239000000758 substrate Substances 0.000 claims abstract 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims 4
- 230000007062 hydrolysis Effects 0.000 claims 3
- 238000006460 hydrolysis reaction Methods 0.000 claims 3
- 238000000034 method Methods 0.000 claims 3
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 claims 1
- 229930091371 Fructose Natural products 0.000 claims 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims 1
- 239000005715 Fructose Substances 0.000 claims 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 claims 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims 1
- 238000006243 chemical reaction Methods 0.000 claims 1
- 229930182830 galactose Natural products 0.000 claims 1
- 239000008103 glucose Substances 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 230000035484 reaction time Effects 0.000 claims 1
- 150000001875 compounds Chemical class 0.000 abstract 1
- 230000003301 hydrolyzing effect Effects 0.000 abstract 1
- 239000000047 product Substances 0.000 abstract 1
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- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
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- C07B2200/07—Optical isomers
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Abstract
The invention belongs to the technical field of compound preparation, and discloses a preparation method of optical dextro-3-hydroxybutyric acid. The preparation method comprises the following steps: (1) Adding a culture medium and halophilic bacteria into a saccharide serving as a fermentation substrate to form a fermentation mixture, and fermenting to obtain fermentation liquor; (2) Carrying out centrifugal separation on the fermentation liquor, breaking the walls of halophilic bacteria, and purifying to obtain a substance containing poly-beta-hydroxybutyric acid; (3) Mixing a substance containing poly-beta-hydroxybutyric acid, acid and alcohol, heating, stirring, reacting, then cooling, adjusting pH to be neutral or acidic, filtering, and taking filtrate; (4) Concentrating the filtrate to remove alcohol, adding acid into the concentrated filtrate, heating and hydrolyzing to obtain optical dextrorotation 3-hydroxybutyric acid; the biological preservation information of halophilic bacteria is CGCCNo.16437 2018.09.06. The optical dextro-3-hydroxybutyric acid product prepared by the preparation method does not contain optical levo-3-hydroxybutyric acid.
Description
Technical Field
The invention belongs to the technical field of compound preparation, and particularly relates to a preparation method of optical dextro-3-hydroxybutyric acid.
Background
A large number of scientific data indicate that D-3-hydroxybutyric acid (D3 HB, CAS: 625-72-9) has a variety of uses. D-3-hydroxybutyrate can be used for weight control, exercise supplements, and various functional foods/beverages, and can also be used for promoting the growth of various cells and inhibiting inflammatory responses, and for the treatment of certain diseases. Generally, when the energy supply in the human body is reduced, fatty acids in the body are decomposed, and ketone bodies are naturally generated. When people are hungry or exercise, the exogenous dextro-3-hydroxybutyric acid is used for supplementing ketone bodies in vivo, so that extra energy can be provided, physical strength cannot be penetrated, and hunger tolerance is increased. Muscle composition may also be maintained or improved, improving cognitive function and motor performance. Dextro-3-hydroxybutyrate can regulate the fluctuation of lactic acid in human body during exercise, relieve exercise fatigue and promote the generation of muscle protein, and is used for various high-strength exercises and exercises in Europe and America at present. The dextro-3-hydroxybutyric acid also has the function of resisting osteoporosis, can inhibit the abnormal activation of osteoclast, reduce bone loss, promote the inflow of calcium ions, and can effectively promote the proliferation, differentiation and mineralization of embryo osteogenesis precursor cells. The D-3-hydroxybutyrate also has the effects of inhibiting inflammation and reducing tumorigenesis, and the D-3-hydroxybutyrate also has the effect of interacting with inflammatory bodies in immune cells and reducing the generation of inflammatory cytokines, thereby reducing inflammation. The D-3-hydroxybutyrate also has the function of promoting cell growth, and can promote the growth of various cells, including epithelium, endothelium, osteogenesis, neutrophile, fibroblast and the like. The D-3-hydroxybutyrate also has the effect of preventing and treating chronic syndromes, is used for preventing or treating chronic syndromes such as muscle injury, fatigue, muscle fatigue and myalgic encephalopathy, and can improve sub-health problems such as neurodegeneration, free radical toxicity, hypoxia or hyperglycemia (the effect of effectively regulating blood sugar fluctuation by D3 HB) and the like. D-3-hydroxybutyrate also has the effect of improving cognitive function, increasing brain phosphorylation potential and the Δ G' (Gibbs free energy) of ATP (adenosine triphosphate) hydrolysis to improve brain metabolic efficiency (28% increase in oxygen use efficiency), and increasing brain-derived neurotrophic factor (BDNF) level and amphetamine-responsive transcript (CART) level. The D-3-hydroxybutyrate is proved to be capable of obviously improving the Alzheimer disease and the Parkinson disease.
In the prior art, the production of optical L3-hydroxybutyrate (L3 HB) is often accompanied in the preparation process of optical D3-hydroxybutyrate, and the introduction of optical L3-hydroxybutyrate as an impurity can reduce the efficacy of optical D3-hydroxybutyrate.
Therefore, it is necessary to provide a method for preparing high-purity optical D-3-hydroxybutyric acid.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides a preparation method of optical dextrorotation 3-hydroxybutyric acid, the optical dextrorotation 3-hydroxybutyric acid prepared by the preparation method does not contain optical levorotation 3-hydroxybutyric acid, namely the optical dextrorotation 3-hydroxybutyric acid with the purity of 100% is prepared, and the preparation method has a positive propulsion effect on the application of the optical dextrorotation 3-hydroxybutyric acid in food or biological medicine.
The invention provides a preparation method of optical dextro 3-hydroxybutyric acid.
Specifically, the preparation method of the optical dextrorotation 3-hydroxybutyric acid comprises the following steps:
(1) Adding a culture medium and halophilic bacteria into a saccharide serving as a fermentation substrate to form a fermentation mixture, and fermenting to obtain fermentation liquor;
(2) Performing centrifugal separation on the fermentation liquor prepared in the step (1), breaking the walls of halophilic bacteria, and purifying to obtain a substance containing poly-beta-hydroxybutyrate (PHB);
(3) Mixing the substance containing poly-beta-hydroxybutyrate (PHB) prepared in the step (2), acid and alcohol, heating, stirring, reacting, then cooling, adjusting the pH value to be neutral or acidic, filtering, and taking filtrate;
(4) Concentrating the filtrate prepared in the step (3), removing alcohol, adding acid into the concentrated filtrate, and heating and hydrolyzing to obtain the optical dextro-3-hydroxybutyric acid;
the biological preservation information of the halophilic bacteria is CGCCNo.16437 2018.09.06 (see patent 2018114685727).
Preferably, in the step (1), the saccharide is at least one selected from glucose, mannose, fructose or galactose; further preferably, the saccharide is glucose.
Preferably, in the step (1), the concentration of the sugar substances in the fermentation mixture is 3-50g/L; further preferably, the concentration of the saccharide is 5 to 25g/L.
Preferably, in step (1), the concentration of said halophilic bacteria in the fermentation mixture is 0.01g/L to 150g/L. By OD 600 (optical Density) value measures the concentration of halophilic bacteria in the fermentation mixture, OD 600 The value is 0.2-700.
Preferably, in step (1), the culture medium is a conventional commercially available culture medium, and the culture medium contains organic nitrogen and trace elements; further preferably, the organic nitrogen is yeast extract.
Preferably, in step (1), the composition of the culture medium is: 5-22g/L of corn steep liquor dry powder, 5-22g/L of yeast powder and 5-22g/L, mgSO of peptone 4 0.01-1g/L of urea and 0.5-8g/L, KH 2 PO 4 0.5-15g/L、NaCl 0-50g/L。
Preferably, in the step (1), the fermentation time is more than 24 hours; further preferably, the fermentation time is 36-60 hours; more preferably, during the fermentation, when the concentration of halophilic bacteria is maintained constant for 2 to 3 hours continuously, the fermentation is stopped.
Preferably, in the step (1), the temperature of the fermentation is 15-39 ℃; further preferably, the temperature of the fermentation is 33-38 ℃.
Preferably, in the step (1), during the fermentation, the sugar substance is added into the fermentation mixture, and the concentration of the sugar substance in the fermentation mixture is maintained to be 5-30g/L.
Preferably, the concentration of the carbohydrate substance added to the fermentation mixture is 500-900g/L; further preferably, the concentration of the saccharide added to the fermentation mixture is 600 to 800g/L.
Preferably, in the step (2), after the fermentation liquid is centrifuged, a washing process is further included.
Preferably, in step (2), the purification is to isolate and extract PHB in the cells of halophilic bacteria.
Preferably, in the step (2), after the purification, a concentration and drying process is further included.
Preferably, in the step (2), the poly- β -hydroxybutyrate (PHB) -containing material is in the form of powder.
Preferably, in the step (2), the mass concentration of the poly-beta-hydroxybutyric acid in the poly-beta-hydroxybutyric acid (PHB) -containing substance is greater than 90%; further preferably, the mass concentration of poly-beta-hydroxybutyrate is greater than 95%.
In the step (2), the substance containing poly-beta-hydroxybutyrate (PHB) does not contain optical levorotatory 3-hydroxybutyrate monomer. Poly-beta-hydroxybutyrate (PHB) is a high molecular polymer of optical dextro-3-hydroxybutyrate (D3 HB).
Preferably, in the step (3), the mass ratio of the poly-beta-hydroxybutyrate (PHB) -containing substance to the acid to the alcohol is 1: (0.1-1.2): (2-8); more preferably, the mass ratio of the poly-beta-hydroxybutyrate (PHB) -containing substance, the acid and the alcohol is 1: (0.4-0.6): (3-6).
Preferably, in the step (3), the acid is hydrochloric acid; further preferably, the mass concentration of the hydrochloric acid is 20-45%; more preferably, the mass concentration of the hydrochloric acid is 30-40%.
Preferably, in step (3), the alcohol is ethanol.
Preferably, in the step (3), the temperature of the reaction is 75-85 ℃; further preferably, the temperature of the reaction is 78-82 ℃.
Preferably, in the step (3), the reaction time is 36-180 hours; further preferably, the reaction time is 48 to 72 hours.
Preferably, in the step (3), the reaction is carried out in a reaction kettle containing a condensed reflux device.
Preferably, in step (3), the temperature is reduced to room temperature, for example to 5-35 ℃.
Preferably, in the step (3), the pH is adjusted to 6.0-7.0; further preferably, the pH is adjusted to 6.5-7.0.
Preferably, in the step (3), at least one of calcium bicarbonate, sodium bicarbonate or potassium bicarbonate is used in the process of adjusting the pH.
Preferably, in the step (4), the filtrate obtained in the step (3) is concentrated, and the alcohol is removed by distillation at 38-45 ℃ under a vacuum degree of-0.06 MPa to-0.10 MPa. The alcohol obtained by distillation can be recycled.
Preferably, in the step (4), acid is added to adjust the pH to 0.5-3; further preferably, an acid is added to adjust the pH to 1-2.
Preferably, in the step (4), the temperature of the heating hydrolysis is 95-105 ℃; further preferably, the temperature of the heated hydrolysis is 98-100 ℃.
Preferably, in the step (4), the time for heating hydrolysis is 24-96 hours; further preferably, the time for the heating hydrolysis is 40 to 60 hours.
Preferably, in the step (4), after the hydrolysis by heating, the pH is adjusted to 2 to 4 by adding an alkali, and more preferably, the pH is adjusted to 2.5 to 3.5 by adding an alkali.
The dextro-3-hydroxybutyrate obtained in the step (4) is in a solution state, and the solution does not contain optical levo-3-hydroxybutyrate.
Preferably, in the step (4), after heating and hydrolyzing, concentrating to obtain a solution containing the optical dextrorotation 3-hydroxybutyric acid; the mass concentration of the optical dextrorotation 3-hydroxybutyric acid in the solution containing the optical dextrorotation 3-hydroxybutyric acid is 40-90%.
An optical dextro 3-hydroxybutyric acid prepared by the above-mentioned preparation method, and the optical 3-hydroxybutyric acid does not contain optical levorotatory 3-hydroxybutyric acid.
The application of the optical dextro-3-hydroxybutyric acid in preparing food or biomedicine.
The halophilic bacteria are applied to the preparation of optical dextro-3-hydroxybutyric acid; the biological preservation information of the halophilic bacteria is CGCCNo.16437 2018.09.06.
Compared with the prior art, the invention has the following beneficial effects:
(1) The preparation method provided by the invention adopts halophilic bacteria for fermentation, and the obtained substance containing poly-beta-hydroxybutyrate (PHB) does not contain optical levorotatory 3-hydroxybutyrate monomer or optical levorotatory 3-hydroxybutyrate derivative, which is very important for preparing pure optical dextrorotatory 3-hydroxybutyrate.
(2) And (4) adding acid twice in the step (3) and the step (4), and the reaction process is favorable for preparing pure optical dextro-3-hydroxybutyric acid.
(3) The optical dextro-3-hydroxybutyric acid product prepared by the preparation method does not contain optical levo-3-hydroxybutyric acid.
(4) The optical dextro-3-hydroxybutyric acid prepared by the preparation method disclosed by the invention has good biocompatibility, can be completely digested, absorbed and degraded by a human body, and can be widely applied to the fields of food or biomedicine.
Detailed Description
In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples are given for illustration. It should be noted that the following examples are not intended to limit the scope of the claimed invention.
The starting materials, reagents or apparatuses used in the following examples are conventionally commercially available or can be obtained by conventionally known methods, unless otherwise specified.
The following media were used: 5-22g/L of corn steep liquor dry powder, 5-22g/L of yeast powder and 5-22g/L, mgSO of peptone 4 0.01-1g/L of urea and 0.5-8g/L, KH 2 PO 4 0.5-15g/L、NaCl 0-50g/L。
Example 1: preparation of optical dextro 3-hydroxybutyric acid
A preparation method of optical dextro-3-hydroxybutyric acid comprises the following steps:
(1) Adding culture medium and halophilic bacteria to glucose as fermentation substrate to form fermentation mixture (OD of halophilic bacteria in fermentation mixture) 600 Value of 0.2), glucose concentration of 10g/L, fermentation at 20 deg.C for 2 hr, and stopping fermentation (at which time the OD of halophilic bacteria in the fermentation mixture is maintained) 600 Value of about 700) to obtain a fermentation broth; the biological preservation information of halophilic bacteria is CGCCNo.16437 2018.09.06 (see patent 2018114685727);
(2) Performing centrifugal separation, washing, wall breaking by halophilic bacteria, purifying, concentrating and drying on the fermentation liquor prepared in the step (1) to obtain a substance containing poly-beta-hydroxybutyrate (PHB); in the substance containing poly-beta-hydroxybutyrate (PHB), the mass concentration of the poly-beta-hydroxybutyrate is more than 90%, and the substance containing the poly-beta-hydroxybutyrate (PHB) does not contain an optical levorotatory 3-hydroxybutyrate monomer;
(3) Mixing 100 kg of the substance containing poly-beta-hydroxybutyrate (PHB) prepared in the step (2), 140 kg of hydrochloric acid (with the mass concentration of 37%) and 400 kg of absolute ethyl alcohol in a reaction kettle with a condensing reflux device, heating to 80 ℃, stirring, reacting at the temperature of 80 ℃ for 40 hours, cooling to 20 ℃, adjusting the pH value to 6.8 by using calcium bicarbonate, filtering, and taking filtrate;
(4) Concentrating and distilling the filtrate obtained in the step (3) in a concentration tank with the vacuum degree of-0.08 MPa at 40 ℃, recycling alcohol obtained by distillation, adding hydrochloric acid (the mass concentration is 37%) into the concentrated filtrate until the pH value is 1, heating for hydrolysis at the temperature of 100 ℃ for 30 hours, then adding sodium bicarbonate to adjust the pH value to 3.0, and concentrating to obtain a solution containing optical dextrorotatory 3-hydroxybutyric acid; the mass concentration of the optical dextrorotation 3-hydroxybutyric acid in the solution is 50%, and the solution containing the optical dextrorotation 3-hydroxybutyric acid does not contain the optical levorotation 3-hydroxybutyric acid.
Comparative example 1
The difference from example 1 is that comparative example 1 does not produce a solution containing optical dextro-3-hydroxybutyric acid without adding hydrochloric acid in step (3).
Comparative example 2
The difference from example 1 is that in comparative example 2, hydrochloric acid was not added in step (4), and the mass concentration of optical 3-hydroxybutyric acid in the finally obtained solution was less than 10%.
Product effectiveness testing
The purity, appearance, odor, taste, pH and impurity content of the solution containing optical 3-hydroxybutyric acid obtained in example 1 are shown in table 1.
TABLE 1
As can be seen from Table 1, the solution containing optical D-3-hydroxybutyrate prepared by the preparation method of the invention does not contain optical L-3-hydroxybutyrate, which is beneficial to the application of the solution containing optical D-3-hydroxybutyrate in the fields of food or biomedicine.
In addition, it should be noted that, in the technical scheme of the present invention, the solution containing the optical D-3-hydroxybutyrate obtained by changing the pH, the amount of each substance, the fermentation temperature and time, and the reaction temperature and time according to example 1 does not contain the optical L-3-hydroxybutyrate, and if the solid optical D-3-hydroxybutyrate is desired to be obtained, the solution is further dried and concentrated.
Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.
Claims (6)
1. A preparation method of optical levorotatory 3-hydroxybutyric acid is characterized by comprising the following steps:
(1) Adding a culture medium and halophilic bacteria into a saccharide serving as a fermentation substrate to form a fermentation mixture, and fermenting to obtain fermentation liquor;
(2) Performing centrifugal separation on the fermentation liquor, breaking the walls of halophilic bacteria, and purifying to obtain a substance containing poly-beta-hydroxybutyric acid;
(3) Mixing the substance containing poly-beta-hydroxybutyric acid, acid and alcohol, heating, stirring, reacting, then cooling, adjusting pH to be neutral or acidic, filtering, and taking filtrate;
(4) Concentrating the filtrate to remove alcohol, adding acid into the concentrated filtrate, adjusting the pH to 0.5-3, and heating for hydrolysis to obtain the optical levorotatory 3-hydroxybutyric acid;
the biological preservation information of the halophilic bacteria is CGCCNo.16437 2018.09.06;
in the step (3), the mass ratio of the poly-beta-hydroxybutyric acid-containing substance to the acid to the alcohol is 1: (0.1-1.2): (2-8);
in the step (3), the reaction temperature is 75-85 ℃; the reaction time is 36-180 hours;
in the step (4), the temperature of the heating hydrolysis is 95-105 ℃; the time for heating hydrolysis is 24-96 hours.
2. The method according to claim 1, wherein in the step (1), the saccharide is at least one selected from glucose, mannose, fructose and galactose.
3. The method according to claim 1, wherein in the step (1), the fermentation time is more than 24 hours; the temperature of the fermentation is 15-39 ℃.
4. The method according to claim 1, wherein in the step (2), the poly- β -hydroxybutyric acid-containing substance has a mass concentration of poly- β -hydroxybutyric acid of greater than 90%.
5. The production method according to claim 1, wherein in the step (3), the acid is hydrochloric acid; the mass concentration of the hydrochloric acid is 20-45%.
6. The halophilic bacteria are applied to the preparation of optical levorotatory 3-hydroxybutyric acid; the biological preservation information of the halophilic bacteria is CGCCNo.16437 2018.09.06.
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| CN100344280C (en) * | 2005-08-01 | 2007-10-24 | 清华大学 | 3-hydroxy butanoic acid and novel use of derivative thereof |
| CN109486710B (en) * | 2018-12-03 | 2022-10-21 | 北京微构工场生物技术有限公司 | Method for continuously fermenting and culturing microorganisms by recycling wastewater and bacteria with self-flocculation and self-sedimentation characteristics used by method |
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