CN106176564B - The method for preparing ginseng PORIA ALBA fermentation liquid using ginseng endogenetic fungus - Google Patents
The method for preparing ginseng PORIA ALBA fermentation liquid using ginseng endogenetic fungus Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
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- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
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- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
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- 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
- A61Q19/08—Anti-ageing preparations
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/85—Products or compounds obtained by fermentation, e.g. yoghurt, beer, wine
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Abstract
The present invention provides a kind of methods for preparing ginseng PORIA ALBA fermentation liquid using ginseng endogenetic fungus comprising following steps: S1: preparing fungi in ginseng inside raw bacterium induced medium；S2: ginseng is prepared with the fungi that ginseng and step S1 are obtained respectively and extracts medicinal extract and fungus metabolite medicinal extract, and filters out aimed strain using ginseng extraction medicinal extract and fungus metabolite medicinal extract；S3: aimed strain seed liquor is prepared using the aimed strain；S4: by aimed strain seed liquor access by being cultivated at 28~32 DEG C, obtaining the ginseng PORIA ALBA fermentation liquid in ginseng and the fermentation medium of PORIA ALBA preparation.Compared with prior art, the present invention ferments to ginseng and PORIA ALBA, and the fermentation liquid of acquisition contains ginseng and PORIA ALBA active constituent, and whitening, antioxidant activity are more preferable than single fermented ginseng liquid.
Description
Technical field
The present invention relates to a kind of methods for preparing ginseng PORIA ALBA fermentation liquid using ginseng endogenetic fungus, belong to daily-use chemical industry
Technical field.
Background technique
Ginseng is described as the superfine product of " King of Herbs " " life is mended in enriching yin, is strengthened the body resistance to consolidate the constitution " from ancient times, containing there are many ginsenoside and people
Gracilis polysaccharide and vitamin and amino acids.Ginseng, which has, to be improved immunity of organisms, reducing blood lipid, anti-aging, removes free radical
Etc. a variety of effects.There is expansion capillary of skin for cosmetics, promote skin blood circulation, increase skin-nourishing, adjust
Skin water and oil balance, prevents skin dehydration, hardening, corrugation, and ginseng active substance inhibits the reducing property of melanin, keeps skin clean
White light is sliding, can enhance skin elasticity, obtains cell newborn.
PORIA ALBA is the white portion that Poria cocos cuts poria cocos thickness, usually the prepared slices of Chinese crude drugs.PORIA ALBA has for cosmetics
There is the effect of tender skin, whitening is moist.
The current existing patent about fermented ginseng liquid is the non-plants such as ginseng wine fermentation or utilization lactic acid bacteria mostly
Endogenetic fungus fermentation, if patent CN102366373 B is to ferment to obtain whitening effect more to red ginseng using Bulgarian lactic acid bacterium
Good fermented ginseng liquid；If patent CN10239183 B is to be fermented using lactic acid bacteria to ginseng and soapberry fermented mixture
Liquid.The present invention utilizes ginseng endogenetic fungus, and the fermentation liquid to be fermented to ginseng and PORIA ALBA compound is used for cosmetics
Application.
Existing patent, which is mainly studied, ferments to ginseng, makes full use of the active constituent of ginseng, fails to play fungi
Cometabolism function.Technical problem solved by the invention be filter out can generate it is the same or similar with ginseng active constituent
Endogenetic fungus；Ginseng and PORIA ALBA are compounded according to a certain percentage, and fermentation medium is used as after sterilizing；Optimize endogenetic fungus
Fermentation condition obtains fermentation liquid.
Summary of the invention
For the defects in the prior art, using ginseng endogenetic fungus, to prepare ginseng white the object of the present invention is to provide a kind of
The method of fermentation liquid of Poria cocos.
The present invention is achieved by the following technical solutions:
The present invention provides a kind of method for preparing ginseng PORIA ALBA fermentation liquid using ginseng endogenetic fungus comprising following step
It is rapid:
S1: fungi is prepared in ginseng inside raw bacterium induced medium；
S2: preparing ginseng with the fungi that ginseng and step S1 are obtained respectively and extract medicinal extract and fungus metabolite medicinal extract, and
Medicinal extract is extracted using the ginseng and fungus metabolite medicinal extract isolates aimed strain, which is monospore Mortierella,
It can be found in " ginseng endogenetic fungus and its secondary metabolite research ", The 2nd Army Medical College, Master's thesis, in June, 2009.
S3: aimed strain seed liquor is prepared using the aimed strain；
S4: the aimed strain seed liquor is accessed in the fermentation medium prepared by ginseng and PORIA ALBA, 28~32
It is cultivated at DEG C, obtains the ginseng PORIA ALBA fermentation liquid.
Preferably, the endophyte induced medium the preparation method comprises the following steps: by PDA solid medium melt after fall
Enter in culture dish, forms endophyte induced medium.
Preferably, the preparation method of the fungi includes following operation:
Ginseng is inoculated in endophyte induced medium after pretreatment as cultured tissue, is cultivated at 28 DEG C
7~10 days, Pick Wire purifying is carried out while culture, obtains the fungi.
Preferably, the pretreated method are as follows:
After taking fresh ginseng to be rinsed well with tap water, it is washed with deionized water 3 times；
The ginseng cleaned is cut into after the root segment of 1cm long and carries out surface sterilization according to the following steps: the leaching of 75wt% ethyl alcohol
It steeps 30s, 5wt% sodium hypochlorite and impregnates the immersion of 30s, 75wt% ethyl alcohol 10s, aseptic water washing 3 times；
Ginseng root segment is cut into 5mm sheet with sterilized scalpel, as cultured tissue by filter paper wipe dry.
Preferably, the preparation method of the fungal metabolite medicinal extract includes following operation:
Fungi is accessed in PDA liquid medium, shaker fermentation 15 days, obtains fermentation liquid, be extracted with ethyl acetate 3 times,
It is concentrated under reduced pressure to give fungus metabolite medicinal extract.
Preferably, it includes following operation that the ginseng, which extracts the preparation method of medicinal extract:
Ginseng methanol is impregnated 3 days, ginseng is concentrated under reduced pressure to give and extracts medicinal extract.
Preferably, the separation method of the aimed strain includes following operation:
It takes fungus metabolite medicinal extract and ginseng to extract medicinal extract respectively to be dissolved with methanol, thin-layer chromatography detection, which is compared, obtains two
The similar fungi of person's ingredient is as aimed strain.
Thin-layered chromatography detection method are as follows:
Silica gel plate: G type silica gel plate；
Solvent: chloroform: methanol: water=8:2:0.2；
Color developing agent: the mixed liquor of sulfuric acid and ethyl alcohol；
Ginseng extracts medicinal extract as standard specimen, and each fungus metabolite medicinal extract is sample to be tested, after being dissolved respectively with methanol, uses
Capillary point sample on silica gel plate, drying after being put into expansion cylinder expansion, impregnate color developing agent, the ratio of more each ingredient of baking colour developing
Shifting value RfSize filters out RfIdentical sample.Rf=solute moving distance/solution moving distance.
Preferably, the preparation method of the fermentation medium includes following operation:
Ginseng and PORIA ALBA are sliced respectively, the crushed after being dried at 40~50 DEG C obtains ginseng pulverate and poria alba powder；
After the ginseng pulverate and poria alba powder are mixed, adds water dispersion, sterilize at 120 DEG C, after being cooled to room temperature
Obtain fermentation medium.
Preferably, the mass ratio of the ginseng pulverate and poria alba powder is 50:1~100:1.
Preferably, the dosage of the water is 5~10 times of ginseng pulverate and poria alba powder total weight.
Compared with prior art, the present invention have it is following the utility model has the advantages that
1, the present invention is using ginseng endogenetic fungus as fermented fungal, ginsenoside Rg2 in fermentation liquid, Rg3, Rh1, Rh2,
Rb2 has different degrees of raising；
2, the present invention ferments to ginseng and PORIA ALBA, and the fermentation liquid of acquisition contains ginseng and PORIA ALBA active constituent, whitening,
Antioxidant activity is more preferable than single fermented ginseng liquid.
Specific embodiment
The present invention is described in detail combined with specific embodiments below.Following embodiment will be helpful to the technology of this field
Personnel further understand the present invention, but the invention is not limited in any way.It should be pointed out that the ordinary skill of this field
For personnel, without departing from the inventive concept of the premise, various modifications and improvements can be made.These belong to the present invention
Protection scope.
The fermentation screening of ginseng endogenetic fungus
Fresh ginseng is taken, tap water is rinsed well, is washed with deionized water 3 times.Ginseng is cut into the root segment of 1cm long
Press following procedure surface sterilization afterwards: 75wt% ethyl alcohol impregnates 30s, 5wt% sodium hypochlorite and impregnates the immersion of 30s, 75wt% ethyl alcohol
10s, aseptic water washing 3 times.Root segment is cut into 5mm thickness with sterilized scalpel, as culture group by filter paper wipe dry
It knits.In superclean bench, it is inoculated with 4 pieces of ginseng tissues in each PDA culture dish, puts into and cultivates 10 days in 28 DEG C of incubators, often
Fungi upgrowth situation in its observation culture dish, when having new mycelia to grow out, picking mycelia is accessed in new culture dish and cultivates,
Continue to purify, obtains fungi strain after purification.24 plants of endogenetic fungus are obtained altogether by counting.By this 24 plants of endogenetic fungus difference
Access shaker fermentation 15 days, obtains fermentation liquid and is extracted with ethyl acetate equipped in the 250mL conical flask of 100mLPDA fluid nutrient medium
It takes 3 times, is concentrated under reduced pressure to give fungus metabolite medicinal extract.Ginseng 95wt% ethyl alcohol is impregnated 3 days, people is concentrated under reduced pressure to give
Ginseng extracts medicinal extract, and a small amount of fungus metabolite medicinal extract and ginseng is taken to extract the dissolution of medicinal extract dehydrated alcohol, thin-layer chromatography detection respectively
Compare and obtains the similar fungi of the two ingredient as aimed strain.By the secondary metabolite of screening discovery bacterial strain RC18 bacterial strain
It is most like with the active constituent of ginseng, therefore using bacterial strain RC18 as aimed strain.
Embodiment 1
A kind of method for preparing ginseng PORIA ALBA fermentation liquid using ginseng endogenetic fungus that the present embodiment is related to, including it is as follows
Step:
Ginseng, PORIA ALBA are cut into medicine materical crude slice, and 45 DEG C of dryings for 24 hours, crush in an oven, the Ginseng Root Powder 100g after taking drying
PORIA ALBA is not added, the pure water for adding 6 times of amounts in mass ratio stirs evenly, 120 DEG C of high-temperature sterilization 30min.After sterilizing, put to
Room temperature accesses 6mL aimed strain RC18 seed liquor with liquid-transfering gun in superclean bench, then by fermentation flask in constant incubator
Middle fermentation 10 days.Filtered through gauze removes the mycelium in fermentation liquid, and bacterium solution is filtered with diatomite, is diluted with butanediol and pure water
It is settled to 1L.
Embodiment 2
A kind of method for preparing ginseng PORIA ALBA fermentation liquid using ginseng endogenetic fungus that the present embodiment is related to, including it is as follows
Step:
Ginseng, PORIA ALBA are cut into medicine materical crude slice, and 45 DEG C of dryings for 24 hours, crush in an oven, the Ginseng Root Powder 100g after taking drying
Ginseng in mass ratio: after mixing, the pure water for adding 6 times of amounts stirs evenly PORIA ALBA=50:1,120 DEG C of high-temperature sterilizations
30min.It after sterilizing, puts to room temperature, accesses 6mL aimed strain RC18 seed liquor with liquid-transfering gun in superclean bench, then will
Fermentation flask ferments 10 days in constant incubator.Filtered through gauze removes the mycelium in fermentation liquid, and bacterium solution is filtered with diatomite,
1L is settled to butanediol and pure water dilution.
Embodiment 3
A kind of method for preparing ginseng PORIA ALBA fermentation liquid using ginseng endogenetic fungus that the present embodiment is related to, including it is as follows
Step:
Ginseng, PORIA ALBA are cut into medicine materical crude slice 45 DEG C of dry 48h in an oven, crushed, the Ginseng Root Powder 100g after taking drying
Ginseng in mass ratio: after mixing, the pure water for adding 8 times of amounts stirs evenly PORIA ALBA=75:1,120 DEG C of high-temperature sterilizations
30min.It after sterilizing, puts to room temperature, accesses 8mL aimed strain RC18 seed liquor with liquid-transfering gun in superclean bench, then will
Fermentation flask ferments 12 days in constant incubator.Filtered through gauze removes the mycelium in fermentation liquid, and bacterium solution is filtered with diatomite,
1L is settled to butanediol and pure water dilution.
Embodiment 4
A kind of method for preparing ginseng PORIA ALBA fermentation liquid using ginseng endogenetic fungus that the present embodiment is related to, including it is as follows
Step:
Ginseng, PORIA ALBA are cut into medicine materical crude slice, and 50 DEG C of dryings for 24 hours, crush in an oven, the Ginseng Root Powder 100g after taking drying
Ginseng in mass ratio: after mixing, the pure water for adding 10 times of amounts stirs evenly PORIA ALBA=100:1,120 DEG C of high-temperature sterilizations
30min.It after sterilizing, puts to room temperature, accesses 10mL aimed strain RC18 seed liquor with liquid-transfering gun in superclean bench, then will
Fermentation flask ferments 14 days in constant incubator.Filtered through gauze removes the mycelium in fermentation liquid, and bacterium solution is filtered with diatomite,
1L is settled to butanediol and pure water dilution.
Comparative example 1
A kind of preparation method for normal fermentation liquid that this comparative example is related to, includes the following steps:
Ginseng is cut into medicine materical crude slice, and 45 DEG C of dryings for 24 hours, crush in an oven, and the Ginseng Root Powder 100g after taking drying is not added
PORIA ALBA, the pure water for adding 6 times of amounts stir evenly, 120 DEG C of high-temperature sterilization 30min.It after sterilizing, puts to room temperature, in ultra-clean work
Make to access the sub- liquid of 6mL Bulgaria lactic acid strain with liquid-transfering gun in platform, then fermentation flask ferments 10 days in constant incubator.
Filtered through gauze removes the mycelium in fermentation liquid, and bacterium solution is filtered with diatomite, is settled to 1L with butanediol and pure water dilution.
Comparative example 2
The preparation method for another normal fermentation liquid that this comparative example is related to, includes the following steps:
PORIA ALBA is cut into medicine materical crude slice, and 45 DEG C of dryings for 24 hours, crush in an oven, the PORIA ALBA powder 100g after taking drying, no
Ginseng is added, the pure water for adding 6 times of amounts in mass ratio stirs evenly, 120 DEG C of high-temperature sterilization 30min.After sterilizing, put to room
Temperature accesses 6mL aimed strain RC18 seed liquor with liquid-transfering gun in superclean bench, then by fermentation flask in constant incubator
Fermentation 10 days.Filtered through gauze removes the mycelium in fermentation liquid, and bacterium solution is filtered with diatomite, and it is fixed to be diluted with butanediol and pure water
Hold to 1L.
The content of comparative analysis ginsenoside.
Using comparative example fermentation liquid as experiment contrast, embodiment 1 is test specimen, utilizes efficient liquid phase chromatographic analysis ratio
The content of ginsenoside Rg2, Rg3, Rh1, Rh2, Rb2 in the fermentation liquid obtained compared with two kinds of fungi fermentations.Its result such as 1 institute of table
Show.
HPLC analysis condition is established: column temperature: room temperature (25 DEG C), flow velocity 1.0mL/min, Detection wavelength 203nm.
1. content of ginsenoside of table
As shown in table 1, ginsenoside Rg2, Rg3, Rh1, Rh2, Rb2 increase than comparative example 1 in embodiment 1, say
Bright ginseng endogenetic fungus is as fermented fungal, and content of ginsenoside is higher in the fermentation liquid of acquisition.
Comparative analysis inhibits tyrosinase activity
Experimental material: the fermentation liquid that above-described embodiment prepares.
Tyrosinase inhibitory activity is detected using microplate reader.Accurately weigh the fermented ginseng liquid and embodiment 1 of different weight
Middle fermentation liquid is configured to different concentration with DMSO.Accurately draw the fermented ginseng liquid and the white Fu of ginseng of configured various concentration
Siberian cocklebur fermentation liquid is added in 96 orifice plates as sample to be tested, and the phosphate buffer mixing for sequentially adding l-tyrosine and pH6.8 is equal
Even, 30 DEG C of water-bath 10min are added tyrosinase and mix, be quickly transferred in cuvette, measure the absorbance at 475nm.L- junket
Propylhomoserin is test substrate, and sample to be tested is tester.3 repetitions of each concentration, 3 repetitions of each sample.With the phosphoric acid of pH6.8
Buffer is as blank control.The inhibiting rate calculation formula of tyrosinase is as follows:
Inhibiting rate (%)={ [(A-B)-(C-D)]/(A-B) } * 100%；
A: absorbance when having substrate without tester；B: the absorbance of blank control；C: there is the absorbance of substrate and tester；
D: there is absorbance of the tester without substrate.
Experimental result is as shown in table 2, it can be seen that embodiment 1 is compared with other embodiments to tyrosinase inhibition rate IC50
It is worth bigger than normal, but difference is not it is obvious that illustrating that the beauty of fermentation liquid can be enhanced to a certain extent by adding PORIA ALBA in fermentating formula
White activity；1 tyrosinase inhibition rate IC of embodiment50Value is obviously smaller than comparative example 1 and comparative example 2, illustrates the hair that the present invention obtains
Zymotic fluid is more preferable than normal fermentation liquid whitening active.
Table 2: tyrosinase inhibition rate result
| Sample | IC50(ug/mL) |
| Embodiment 1 | 564.1 |
| Embodiment 2 | 509.8 |
| Embodiment 3 | 518.6 |
| Embodiment 4 | 534.5 |
| Comparative example 1 | 812.8 |
| Comparative example 2 | 695.4 |
Specific embodiments of the present invention are described above.It is to be appreciated that the invention is not limited to above-mentioned
Particular implementation, those skilled in the art can make various deformations or amendments within the scope of the claims, this not shadow
Ring substantive content of the invention.
Claims (8)
1. a kind of method for preparing ginseng PORIA ALBA fermentation liquid using ginseng endogenetic fungus, which comprises the steps of:
S1: fungi is prepared in ginseng inside raw bacterium induced medium；
S2: ginseng is prepared with the fungi that ginseng and step S1 are obtained respectively and extracts medicinal extract and fungus metabolite medicinal extract, and is utilized
The ginseng extracts medicinal extract and fungus metabolite medicinal extract filters out aimed strain；
S3: aimed strain seed liquor is prepared using the aimed strain；
S4: by aimed strain seed liquor access by being trained at 28 ~ 32 DEG C in ginseng and the fermentation medium of PORIA ALBA preparation
It supports, obtains the ginseng PORIA ALBA fermentation liquid；
The aimed strain be monospore Mortierella (mortierella monospora), strain name Yuan-27；
The preparation method of the fermentation medium includes following operation:
Ginseng and PORIA ALBA are sliced respectively, the crushed after being dried at 40 ~ 50 DEG C obtains ginseng pulverate and poria alba powder；
After the ginseng pulverate and poria alba powder are mixed, adds water dispersion, sterilize at 120 DEG C, obtained after being cooled to room temperature
Fermentation medium；
The mass ratio of the ginseng pulverate and poria alba powder is 50:1 ~ 100:1.
2. the method for preparing ginseng PORIA ALBA fermentation liquid using ginseng endogenetic fungus as described in claim 1, which is characterized in that
The endophyte induced medium the preparation method comprises the following steps: by PDA solid medium melt after pour into culture dish, formed endophyte
Induced medium.
3. the method for preparing ginseng PORIA ALBA fermentation liquid using ginseng endogenetic fungus as described in claim 1, which is characterized in that
The preparation method of the fungi includes following operation:
Ginseng is inoculated in endophyte induced medium after pretreatment as cultured tissue, 7 ~ 10 are cultivated at 28 DEG C
It, carries out Pick Wire purifying while culture, obtains the fungi.
4. the method for preparing ginseng PORIA ALBA fermentation liquid using ginseng endogenetic fungus as claimed in claim 3, which is characterized in that
The pretreated method are as follows:
After taking fresh ginseng to be rinsed well with tap water, it is washed with deionized water 3 times；
The ginseng cleaned is cut into after the root segment of 1cm long and carries out surface sterilization according to the following steps: 75wt% ethyl alcohol immersion 30s,
5wt% sodium hypochlorite impregnates 30s, 75wt% ethyl alcohol and impregnates 10s, aseptic water washing 3 times；
Ginseng root segment is cut into 5mm sheet with sterilized scalpel, as cultured tissue by filter paper wipe dry.
5. the method for preparing ginseng PORIA ALBA fermentation liquid using ginseng endogenetic fungus as described in claim 1, which is characterized in that
The preparation method of the fungus metabolite medicinal extract includes following operation:
Fungi is accessed in PDA liquid medium, shaker fermentation 15 days, obtain fermentation liquid, be extracted with ethyl acetate 3 times, depressurizes
It is concentrated to get fungus metabolite medicinal extract.
6. the method for preparing ginseng PORIA ALBA fermentation liquid using ginseng endogenetic fungus as described in claim 1, which is characterized in that
The preparation method that the ginseng extracts medicinal extract includes following operation:
Ginseng 95wt% ethyl alcohol is impregnated 3 days, ginseng is concentrated under reduced pressure to give and extracts medicinal extract.
7. the method for preparing ginseng PORIA ALBA fermentation liquid using ginseng endogenetic fungus as described in claim 1, which is characterized in that
The separation method of the aimed strain includes following operation:
It takes fungus metabolite medicinal extract and ginseng to extract medicinal extract respectively to be dissolved with dehydrated alcohol, thin-layer chromatography detection, which is compared, obtains two
The similar fungi of person's ingredient is as aimed strain.
8. the method for preparing ginseng PORIA ALBA fermentation liquid using ginseng endogenetic fungus as described in claim 1, which is characterized in that
The dosage of the water is 5 ~ 10 times of ginseng pulverate and poria alba powder total weight.
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