WO2017113263A1 - Nouvelle utilisation de bouillon de fermentation de gluconacetobacter xylinus comme composition cosmétique - Google Patents

Nouvelle utilisation de bouillon de fermentation de gluconacetobacter xylinus comme composition cosmétique Download PDF

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Publication number
WO2017113263A1
WO2017113263A1 PCT/CN2015/100043 CN2015100043W WO2017113263A1 WO 2017113263 A1 WO2017113263 A1 WO 2017113263A1 CN 2015100043 W CN2015100043 W CN 2015100043W WO 2017113263 A1 WO2017113263 A1 WO 2017113263A1
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fermentation broth
cosmetic composition
xylobacteria
culture
stage
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PCT/CN2015/100043
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English (en)
Chinese (zh)
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林宜全
林圣凯
李亚洁
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奈菲儿生医股份有限公司
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Priority to PCT/CN2015/100043 priority Critical patent/WO2017113263A1/fr
Publication of WO2017113263A1 publication Critical patent/WO2017113263A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q1/00—Make-up preparations; Body powders; Preparations for removing make-up
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/02—Acetobacter

Definitions

  • the present invention relates to the use of a microbial fermentation broth as a cosmetic composition, in particular, to the use of a xyloacetic acid fermentation broth as a cosmetic composition.
  • the microbial fermentation broth is known to contain vitamins, minerals, peptides, amino acids or natural moisturizing factors, and has the effects of promoting metabolism, strengthening moisturizing, anti-oxidation, soothing inflammation, regulating sebum and the like on the skin.
  • SKII Procter &Gamble;P&G
  • Pirate TM a fermentation broth produced by yeast (Saccharomycopsis), as an active ingredient in the winemaking process to achieve skin care effects such as moisturizing
  • SKII Official Website www.sk-ii.com
  • Tsai et al. have also published that the fermentation broth of Saccharomycopsis has anti-inflammatory and cell repair effects (Journal of Dermatological Science, 2006, 42, pp. 249-257).
  • the present invention provides a cosmetic composition
  • a cosmetic composition comprising a fermentation broth of Gluconacetobacter xylinus as an active ingredient.
  • the present invention further provides a use of a fermentation broth of Gluconacetobacter xylinus as a cosmetic composition.
  • the above xyloacetic acid fermentation broth is produced by the following method:
  • the inoculum amount of the xylobacteria is preferably from 10 2 to 10 5 cells/mL.
  • the medium containing the xylobacteria is preferably cultured at 25 to 32 ° C, more preferably in two steps.
  • Stage culture which comprises culturing at a temperature of 25 to 28 ° C in the first stage and 29 to 32 ° C in the second stage after the first stage.
  • the method for producing the xylosonic acid fermentation broth may further include a purification step comprising at least one of adsorbent addition, ultrafiltration, or filtration.
  • the adsorbent may include a food adsorbent, activated carbon or a product having an adsorption effect.
  • the above method for producing the xylosonic acid fermentation broth may further comprise the step of drying the culture liquid collected in the step (iv).
  • the concentration of the xylosidum fermentation broth is 5% by weight or less based on the weight of the cosmetic composition.
  • Figure 1 is a graph showing the cytotoxicity of the fermentation broth of the xylobacteria in human skin keratinocytes (HaCaT cells) in an embodiment of the present invention.
  • Fig. 2 is a view showing the effect of the fermentation broth of the xylosylobacter oxysporum on the cell cycle distribution of human skin keratinocytes (HaCaT cells) in an embodiment of the present invention.
  • Fig. 3 is a view showing the ability of the acetic acid bacteria fermentation broth to remove DPPH (di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium) free radicals in an embodiment of the present invention.
  • DPPH di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium
  • Fig. 4 is a view showing the ability of the xyloacetic acid fermentation broth to remove ABTS (2, 2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) radicals in an embodiment of the present invention.
  • Fig. 5 is a view showing the ability of the fermentation broth of the xylobacteria bacterium to inhibit the production of intracellular reactive oxygen species (ROS) in an embodiment of the present invention.
  • ROS reactive oxygen species
  • Fig. 6 is a view showing the ability of the fermentation broth of the xylobacteria bacterium to inhibit the formation of lipoxygenase in an embodiment of the present invention.
  • Fig. 7 is a view showing the ability of the fermentation broth of the xylobacteria bacterium to inhibit the production of nitric oxide (NO) in an embodiment of the present invention.
  • Figure 8 is a graph showing the ability of the fermentation broth of the xylobacteria bacterium to inhibit the activity of mushroom tyrosinase in an embodiment of the present invention.
  • the object of the present invention is to provide a novel use of a fermentation broth of Gluconacetobacter xylinus as a cosmetic composition.
  • Traditional acetic acid bacteria are often used in the production of vinegar, such as cereal vinegar Or fruit vinegar and so on.
  • acetic acid bacteria are also known to be metabolized to produce biocellulose, which is formed into a film shape, and can be applied to foods, industrial materials, biomedical materials, cosmetic compositions and the like.
  • biomedical materials it is known as, for example, a burn dressing, artificial skin, or the like.
  • a microbial fiber membrane is also commercially available as a mask or the like.
  • prior to the present invention there has not been a technique in which a fermentation broth of xyloacetic acid bacteria is used as a cosmetic composition.
  • the fermentation broth of Gluconacetobacter xylinus described in the present invention is a fermentation broth left by removing the microbial cellulose membrane produced by fermentation of xylobacteria.
  • the fermentation broth described in the present case may contain partially residual cellulose, based on the aerobic nature of the xyloacetic acid bacterium, the microbial cellulose is formed at a liquid surface in contact with the liquid medium to form a film, and thus the removal is performed. After the membrane, there is almost no microbial cellulose in the fermentation broth.
  • the xylobacteria fermentation broth of the present invention is produced by the following methods:
  • the medium prepared in the above step (i) contains a carbon source, a nitrogen source, a phosphorus source, an inorganic salt, and an organic acid.
  • the carbon source may be derived from a five-carbon sugar, a six-carbon sugar, a hydrolyzed sugar liquid, a sugar-containing wastewater, etc.
  • the nitrogen source may be derived from a compound such as yeast extract or peptone.
  • the phosphorus source can be derived from Na 2 HPO 4 , NaH 2 PO 4 , K 2 HPO 4 , and the like.
  • the inorganic salts may be derived from minerals, vitamins such as NaCl, CaCO 3 , C 2 H 3 O 2 Na, and the like.
  • the organic acid can be derived from citric acid, acetic acid or the like.
  • glucose having a concentration of 10 to 30 g/L is used as a carbon source, and 2 to 10 g/L of Na 2 HPO 4 is used as a phosphorus source.
  • the composition of the medium formulated in this case can be appropriately adjusted according to the conditions of culture.
  • the bacterial inoculation amount of Gluconacetobacter xylinus may be 10 2 to 10 5 cells/mL, but is not limited thereto, and the culture conditions and desired fermentation broth may be used. Adjust the concentration appropriately.
  • the step (iii) of the culture medium containing the xylosylic acid bacterium may be carried out at 25 to 32 °C.
  • the above static culture is carried out at room temperature.
  • the static culture is a two-stage culture comprising culturing at a temperature of 25 to 28 ° C in the first stage and 29 to 32 ° C in the second stage after the first stage.
  • a purification step may be further included, including the addition of an adsorbent, ultrafiltration, or Filter at least one of them.
  • the adsorbent may include a food adsorbent, activated carbon or a product having an adsorption effect.
  • the adsorbent may be activated carbon, which adsorbs odor and decolorizes by the porous structure of activated carbon, and then removes activated carbon by filtration or the like.
  • Ultrafiltration uses a nanoporous ultrafiltration membrane to filter out impurities in the fermentation broth.
  • the step of drying the culture liquid collected in the step (iv) may be further included.
  • the dried lactic acid bacteria fermentation liquid can be stored for a long period of time, and only needs to be added with water to dissolve, and the activity can be reproduced.
  • the xyloglucanase fermentation liquid preferably contains a concentration of 5% by weight or less, more preferably 0.1% by weight to 5% by weight, even more preferably 2% by weight to 5%, based on the total weight of the cosmetic composition to be prepared. weight%.
  • the cosmetic composition containing the lactic acid bacteria fermentation liquid having a concentration of 5 wt% or less has an effective DPPH (di(phenyl)-(2,4,6-trinitrophenyl) iminoazanium) free radical scavenging effect, ABTS (2, 2'-azino- Bis (3-ethylbenzthiazoline-6-sulfonic acid) free radical scavenging effect, inhibitory effect of reactive oxygen species (ROS) production, inhibition of nitric oxide (NO) production, inhibition of lipoxygenase activity, and
  • ROS reactive oxygen species
  • NO nitric oxide
  • the inhibitory effect of mushroom tyrosinase activity can be seen that the cosmetic composition described in the present invention has anti-oxidation, anti-aging, moisturizing, whitening and the like, and in the cytotoxicity test, the concentration is 5 weight.
  • the cell viability of the xylobacteria fermentation broth of less than % is more than 50%, and the biosafety of the
  • the type of the cosmetic composition described in the present invention may be selected from the group consisting of a lotion, a gel, a frozen film, a mud film, an emulsion, a cream, a lipstick, a foundation, a powder, a powder, and a makeup cosmetic. , but not limited to, cleansing oil, cleansing milk, facial cleanser, shower gel, shampoo, hair lotion, sunscreen lotion, hand cream, nail polish and perfume.
  • the cosmetic compositions of the present invention may optionally contain cosmetically acceptable ingredients such as emulsifiers, penetration enhancers, softeners, solvents, excipients, antioxidants, or combinations of these.
  • the cosmetic composition described in the present invention may further comprise a second active ingredient such as hyaluronic acid, salicylic acid, ursolic acid, kojic acid, a polypeptide, an oligopeptide, a growth factor, an enzyme, or the like, or a combination thereof.
  • a second active ingredient such as hyaluronic acid, salicylic acid, ursolic acid, kojic acid, a polypeptide, an oligopeptide, a growth factor, an enzyme, or the like, or a combination thereof.
  • a culture solution of 10 to 30 g/L glucose, 5 to 10 g/L yeast extract powder, 2 to 10 g/L Na 2 HPO 4 , and 1 to 5 g/L citric acid (sterilization at 121 ° C for 30 minutes) is prepared.
  • the previously cultured inoculum was added to control the initial concentration of xyloacetic acid bacteria at 10 2 to 10 5 cells/mL, and the culture was carried out in an A4-sized culture pot.
  • the initial culture temperature was 25-28 ° C. After 2-4 days, it is raised to 29 to 32 ° C, and further cultured for 3 to 10 days. Thereafter, the crude fermentation broth was collected, and the fermentation broth was brown in color and was a flowable liquid.
  • the obtained filtrate was allowed to stand for 10 hours by adding 10 g of activated carbon to decolorize and deodorize. Then, suction filtration was performed using Whatman filter paper, and the collected filtrate was 90 mL of the purified fermentation liquid.
  • the purified fermentation broth was dried under vacuum to obtain 2.25 g of a dried fermented product, which was stored at 25 °C. The dried fermented product was added with water and dissolved back before use to obtain a 100%-concentrated fermentation broth of xylobacter aceti to carry out the following tests.
  • the previously cultured inoculum was added to control the initial concentration of xyloacetic acid bacteria at 10 2 to 10 5 cells/mL, and the culture was carried out in an A4-sized culture pot.
  • the initial culture temperature was 25-28 ° C. After 2-4 days, it is raised to 29 to 32 ° C, and further cultured for 3 to 10 days. Thereafter, the crude fermentation broth was collected, and the fermentation broth was brown in color and was a flowable liquid.
  • the collected fermentation broth will further add 1-10% by weight of adsorbent for pre-decolorization and deodorization treatment, the treatment condition is stirring at normal temperature for 10 minutes, and then the adsorbent is removed by suction filtration, and the obtained product is obtained. It can be used directly after sterilization without any processing.
  • Human skin keratinocytes (HaCaT cells obtained from the Center for Bioresource Conservation and Research - Food Industry Development Institute, BCRC 60038) were cultured in a 96-well plate (NEST 701001) at 1 ⁇ 10 5 cells/mL. Incubate for 24 hours at 37 ° C in a 5% CO 2 incubator.
  • the above-mentioned xylosonic acid fermentation broth was diluted with water to prepare a fermentation broth of xylobacter xylinum at concentrations of 1 wt%, 2 wt%, 5 wt% and 10 wt%, respectively.
  • the human skin keratinocyte culture medium was composed of Dube's improved Ig's culture medium, and 10% (v/v) fetal bovine serum and 1 mM sodium pyruvate were additionally added.
  • Fig. 1 The cell viability of the control group to which no fermentation broth was added (100%), human skin keratinocytes (HaCaT cells) and the concentrations of 1 wt%, 2 wt%, 5 wt%, and 10 wt% above. After 24 hours of co-cultivation of the xylobacter acetate fermentation broth, the cell viability was 97.5%, 95.4%, 70.0% and 50.5%, respectively. This result indicates that the xylobacteria fermentation broth having a concentration of 5% or less is not cytotoxic.
  • HaCaT cells human skin keratinocytes
  • the above microcentrifuge tube was centrifuged at 1200 rpm for 5 minutes at 4 ° C, and the supernatant was removed. Then, 445 ⁇ L of PBS, 5 ⁇ L of RNase (10 mg/mL), and 50 ⁇ L of 10% Triton X-100 were sequentially added to disrupt the RNA of the cells, and then reacted at 37 ° C for 30 minutes, centrifuged at 1200 rpm for 5 minutes, and the supernatant was removed. .
  • the content of DPPH radical detected by the positive control group to which 1000 ⁇ g/mL of vitamin C was added was used as a reference (100%), and as shown in Fig. 3, the concentrations were 1 wt%, 2 wt%, 5 wt%, and 10 wt% as described above.
  • the DPPH clearance rates of the xylobacteria fermentation broth were 37.8%, 54%, 77.2% and 84.0%, respectively, and the IC 50 of the DPPH free radical scavenging was ⁇ 10%. The higher the value of DPPH free radical scavenging, the stronger the antioxidant capacity of the fermentation broth.
  • ABTS analysis solution The ABTS+ stock solution prepared above was diluted 40 times with secondary ddH 2 O, and the absorbance was measured at a light absorption value of 734 nm at about 0.7. Thereafter, 2.45 mM K 2 S 2 O 8 and 7 mM ABTS solution were mixed, and after standing for 16-18 hours in the dark, the reaction generated a radical. Thereafter, 1.5 ⁇ L of a concentration of 1 wt%, 2 wt%, 5 wt%, and 10 wt% of the above-mentioned fermentation liquid of xylobacteria and 1000 ⁇ g/ml of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) were respectively taken.
  • a positive control group 3.5 ⁇ L of ddH 2 O and 145 mL of a 7 mM ABTS solution were mixed and allowed to stand for 20 minutes, and the absorbance at 734 nm was measured by a spectrophotometer. The lower the absorbance value, the better the ability of the sample to scavenge ABTS free radicals.
  • the control group was a solution in which the above fermentation broth or Trolox was not added.
  • the ABTS free radical is a cationic free radical.
  • This test can evaluate whether the above-mentioned xyloacetic acid fermentation broth has the effect of scavenging cationic radicals.
  • the above-mentioned xylobacteria were added at a concentration of 1 wt%, 2 wt%, 5 wt%, and 10 wt% based on the concentration of ABTS radical detected by adding a 1000 ⁇ g/ml Trolox positive control group.
  • the ABTS free radical scavenging rates of the fermentation broth were 33.0%, 76.6%, 77.2% and 84.2%, respectively, and the IC 50 of the ABTS free radicals was eliminated by ⁇ 10%.
  • the number of cells of 1 ⁇ 10 5 cells/mL was cultured in a 96-well plate, and grown in a 37° C., 5% CO 2 incubator for 24 hours or more, and then added at a concentration of 0 wt%, 1 wt%, 2 wt%, and 5 wt%, respectively. % and 10% by weight of the above-mentioned xylosonic acid fermentation broth were co-cultured. After the reaction time, the supernatant was removed, washed with PBS, and freshly cultured with 0.1 mM H 2 O 2 was added thereto for further 1 hour.
  • the culture solution was removed, and a fresh culture solution containing 10 ⁇ m of DCFH 2 DA (2',7'-dichlorofluorescin diacetate) was added, and after 1 hour from the light, the culture solution was removed.
  • the excitation light 502nm / 524nm scattered light measured at the absorbance value (BioTek, Synergy TM 2, USA ).
  • the control group was a cell to which no fermentation broth or H 2 O 2 was added.
  • DCFH 2 DA is a fluorescent agent that emits fluorescence when combined with reactive oxygen species, and uses this property to detect intracellular reactive oxygen species.
  • the ROS concentration generated by the cells to which the fermentation broth or H 2 O 2 was not added was used as a reference (100%), and the cells in which the fermentation broth was not added were subjected to H 2 O 2 treatment, and the intracellular activity was observed.
  • the amount of oxygen production (121%) increased by 21%.
  • ROS production has an IC 50 ⁇ 10%.
  • the LOX-1 enzyme is one of the important enzymes in the inflammatory reaction. This test can be used to evaluate whether the above-mentioned xyloacetate fermentation broth has anti-inflammatory efficacy. Since this test has a wavelength limitation, since the above-mentioned xyloacetic acid fermentation broth is dark brown at a high concentration, only the inhibition rate of the lipoxygenase in the low concentration of the xylobacteria fermentation broth can be measured. The results are shown in Fig. 6. Based on the LOX-1 concentration measured by the positive control group to which 1000 ⁇ g/ml of caffeic acid was added (100%), the concentration of 0.1% and 0.2% of the above-mentioned lignin fermentation broth was inhibited in LOX. The ability of -1 activity was 58.7% and 65.2%, respectively, and its IC 50 of inhibition of LOX-1 activity was ⁇ 10%.
  • concentrations of 1% by weight, 2% by weight, 5% by weight and 10% by weight of the above-mentioned xylosidum fermentation broth to remove NO radicals were about 34.0%, 38.9%, 47.1% and 51.2%, respectively, and the ability of the rutin to remove NO radicals was about 84%, which inhibits 50 ⁇ 10% of the enzyme tyrosinase IC.
  • the test group was 2 ⁇ L of the concentration of 1 wt%, 2 wt%, 5 wt%, and 10 wt% of the above-mentioned xyloacetic acid fermentation broth mixed with 18 ⁇ L of DMSO, and added to a 96-well plate, followed by 25 ⁇ L.
  • a 100 unit solution of mushroom tyrosinase was reacted at room temperature for 10 minutes. Thereafter, the background value of the mushroom tyrosinase was measured at a wavelength of 475 nm.
  • the above-mentioned xyloacetate fermentation broths having a concentration of 1% by weight, 2% by weight, 5% by weight, and 10% by weight, respectively, based on the concentration of the mushroom tyrosinase measured by adding 1000 ⁇ g/mL of vitamin C, respectively. It has a tyrosinase inhibition rate of 30.4%, 55.8%, 93.8% and 94.3%, indicating that the above-mentioned xyloacetic acid fermentation broth has a good whitening effect, and its inhibitory tyrosinase has an IC 50 ⁇ 10%.

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Abstract

L'invention concerne une composition cosmétique, avec un bouillon de fermentation de Gluconacetobacter xylinus comme ingrédient actif, qui a des effets antivieillissement, blanchissants et de rétention de l'humidité, et présente une propriété de biostabilité.
PCT/CN2015/100043 2015-12-31 2015-12-31 Nouvelle utilisation de bouillon de fermentation de gluconacetobacter xylinus comme composition cosmétique WO2017113263A1 (fr)

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PCT/CN2015/100043 WO2017113263A1 (fr) 2015-12-31 2015-12-31 Nouvelle utilisation de bouillon de fermentation de gluconacetobacter xylinus comme composition cosmétique

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PCT/CN2015/100043 WO2017113263A1 (fr) 2015-12-31 2015-12-31 Nouvelle utilisation de bouillon de fermentation de gluconacetobacter xylinus comme composition cosmétique

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113046394A (zh) * 2021-03-17 2021-06-29 天津强微特生物科技有限公司 一种覆膜酵母发酵滤液的制备工艺

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1872022A (zh) * 2006-03-29 2006-12-06 钟春燕 细菌纤维素凝胶面膜
JP2006345796A (ja) * 2005-06-17 2006-12-28 Mitsukan Group Honsha:Kk セラミド高生産性酢酸菌
EP2028276A1 (fr) * 2006-05-22 2009-02-25 Mizkan Group Corporation Procédé de production de céramide de type bactérie acétique
CN105002254A (zh) * 2015-07-03 2015-10-28 北京工商大学 一种银耳发酵提取物的制备方法及其应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006345796A (ja) * 2005-06-17 2006-12-28 Mitsukan Group Honsha:Kk セラミド高生産性酢酸菌
CN1872022A (zh) * 2006-03-29 2006-12-06 钟春燕 细菌纤维素凝胶面膜
EP2028276A1 (fr) * 2006-05-22 2009-02-25 Mizkan Group Corporation Procédé de production de céramide de type bactérie acétique
CN105002254A (zh) * 2015-07-03 2015-10-28 北京工商大学 一种银耳发酵提取物的制备方法及其应用

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113046394A (zh) * 2021-03-17 2021-06-29 天津强微特生物科技有限公司 一种覆膜酵母发酵滤液的制备工艺
CN113046394B (zh) * 2021-03-17 2022-08-12 天津强微特生物科技有限公司 一种覆膜酵母发酵滤液的制备工艺

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