CN107418995A - Ellagic acid prepared by a kind of granatanine liquid state fermentation and preparation method thereof - Google Patents
Ellagic acid prepared by a kind of granatanine liquid state fermentation and preparation method thereof Download PDFInfo
- Publication number
- CN107418995A CN107418995A CN201710617149.8A CN201710617149A CN107418995A CN 107418995 A CN107418995 A CN 107418995A CN 201710617149 A CN201710617149 A CN 201710617149A CN 107418995 A CN107418995 A CN 107418995A
- Authority
- CN
- China
- Prior art keywords
- ellagic acid
- granatanine
- fermentation
- aqueous solution
- extraction
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
- C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
-
- 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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
-
- 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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
- C12N11/12—Cellulose or derivatives thereof
-
- 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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
-
- 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
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/181—Heterocyclic compounds containing oxygen atoms as the only ring heteroatoms in the condensed system, e.g. Salinomycin, Septamycin
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Inorganic Chemistry (AREA)
- Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Ellagic acid prepared by a kind of granatanine liquid state fermentation and preparation method thereof, category cosmetics, medicine, health product technology field.The present invention directly extracts concentrated aqueous solution as fermenting substrate tank liquid state fermentation using granatanine；Using black-koji mould (Aspergillus niger), Penicillium notatum（PencicillinG）And Escherichia coli（Escherichia. Coli）3 kinds of bacterium mix according to a certain percentage, and prepare immobilization micelle using sodium carboxymethylcellulose, sodium alginate and diatomite, can be used for multiple times, improve the utilization ratio of strain；Complementary effect can be formed using 3 kinds of bacterium mixed fermentations, makes ellagic acid transformation efficiency higher；The technique fermentation condition is gentle, greatly reduces the consumption of chemical substance, and environmental pollution is small, is more suitable for producing on a large scale.
Description
Technical field
To prepare ellagic acid and preparation method thereof the present invention relates to a kind of granatanine liquid state fermentation, category cosmetics, medicine,
The technical fields such as health products.
Background technology
Ellagic acid (ellagic acid), molecular formula C14H6O8, it is the dimerization derivative of gallic acid, is that one kind is more
Phenol dilactone.Ellagic acid sterling is a kind of yellow needle-like crystals, is slightly soluble in water, alcohol, is dissolved in hot methanol, alkali and pyridine.Its molecule
Structure is as follows：
Ellagic acid is the natural polyphenol class compound being widely present in various nuts, fruit, especially with rich content in granatum.
But free state is less, existed with condensed forms (such as Ellagitannins, glycosides).China's pomegranate cultivated area
Greatly, granatum aboundresources, it is most of all to go out of use in addition to small part is used for Chinese medicine, cause very big waste.With
Granatum is that raw material produces ellagic acid, and production cost is low, and added value of product is high, moreover it is possible to turns waste into wealth.
The research of nearly 20 years finds that ellagic acid has stronger anti peroxidation of lipid, catches the ability of free radical；It can press down
The metabolic activity of carcinogen processed, release carcinogenic toxicity；It is inhibited to HIV；Also there are stomach and intestine
Dynamics and urogenital kinetics function, and with blood coagulation, decompression, sedation.Therefore, paid close attention to by people.
Ellagic acid can be obtained by extraction, chemical synthesis and natural products edman degradation Edman, and specific preparation method mainly has
It is several below：
The first is that ellagic acid is extracted from plant.Contain in the vegetable materials such as chestnut spiny involucre, raspberry, pomegranate and sorghum kind shell
There is ellagic acid (about 1%) free on a small quantity, the ellagic acid of high-purity can be prepared with this method.But because cost of material is higher and tan
Flower acid content is low, does not have practical significance in production.
It is for second the oxidation polymerization by gallic acid (or its ester).Synthesizing precursor substance --- gallic acid (ester) needs
Oxidation polymerization ellagic acid is produced in the presence of enzyme.The yield of this method ellagic acid is 20%~30%.But gallic acid (ester)
Preparation process very complicated, enzyme is also required to polishing purification, and unknown quinones substance in synthetic product be present, and itself and tan are spent
The ratio of acid is 1: 2.4, and certain difficulty is brought to isolating and purifying for product, less at present to carry out ellagic acid with this method
Production.
The third is the technique (patent No. that acid-hydrolysis method prepares ellagic acid：200510002081.X), this method has higher
Yield and purity, but high concentration strong acid need to be consumed, and completed under the conditions of long-time heating, to the requirement of equipment resistance to corrosion
Very high, environmental pollution is heavier.
4th kind is presently disclosed using granatum as the non-chemical method for preparing ellagic acid of raw material.One is enzyme catalysis method
(the patent No.：200810056007.X), its main process is divided into two steps：Liquid state fermentation producing enzyme stage and enzymatic transformation stage, this
Method process is cumbersome, and the requirement of enzymatic conversion condition is strict, is difficult to control.Disclosed another method is aspergillus niger solid state fermentation
（The patent No.：201010178430.4）, its process is that Pomegranate Rind is placed in fermentation tank, and granatum is with accounting for gross weight 47%
~60% initial wetting aqueous solution soaks, added with 0.1% NaCl, 0.1% MgSO in fountain solution used4With 0.5%
NH4NO3, wherein NH4NO3As N sources, 5~7,115 DEG C of 30 min of sterilizing of pH, accessed after cooling aspergillus niger (Aspergillus niger3.316) usage ratio of spore suspension, granatum and aspergillus niger be 5 g granatums add 1 mL concentration be 1 ×
1010Individual/mL aspergillus niger spore suspension.Simultaneously quiescent culture is mixed, 30 DEG C ferment 5~7 days, obtain the fermentation containing ellagic acid
Product.This method uses fermentation tank solid state fermentation, and charging is cumbersome with discharging, and top and the bottom fermentation is uneven；It is this again
Method is fermented using only single culture, and efficiency has much room for improvement, and these aspects have certain limitation to large-scale industrial production.
The content of the invention
It is an object of the invention to provide a kind of the more efficient liquid state fermentation side of ellagic acid is prepared by substrate of granatanine
Method.By using granatanine extract solution as fermentation substrate, through black-koji mould(Aspergillus niger), Penicillium notatum
（PencicillinG）With Escherichia coli（Escherichia. Coli）Co-immobilization liquid fermentation method produces ellagic acid, through first
High-purity ellagic acid is obtained by extraction in alcohol.This method is simple to operate, and the purity and yield of ellagic acid are higher.
The present invention uses sodium carboxymethylcellulose, marine alga using black-koji mould, Penicillium notatum and Escherichia coli as fermented bacterium
Sour sodium is fixed altogether, using granatanine extract solution as fermentation substrate, carries out liquid state fermentation.In fermentation process, aspergillus niger, mould and
The equal syntannin enzyme of Escherichia coli is simultaneously secreted into extracellular, the Ellagitannins degraded generation tan flower in tannin enzymatic granatum
Acid, tunning obtain high-purity ellagic acid through extracting and washing purifying, comprised the following steps：
（1）It is prepared by granatanine extract solution
Dry granatum is crushed to 40 mesh, uses 60-80 DEG C of refluxing extraction of ethanol solution 3 of 61%~70% volume fraction
Secondary, the 1st addition ethanol solution is 10~15 times of Pomegranate Rind quality, extracts 2 h, centrifuges filtrate and filter residue；2nd
It is secondary that 8~10 times of ethanol solution is added into filter residue, 1.5 h are extracted, are centrifuged；The 3rd addition ethanol solution 6 into filter residue~
8 times, 1 h is extracted, is centrifuged；Merge 3 filtrates, through being distilled to recover ethanol, obtain granatanine extraction concentrated aqueous solution, can
It is directly used in fermenting and producing.Tannin content 1%~1.5% in concentrated aqueous solution is extracted after measured.
（2）The culture of fermented bacterium is fixed together
By black-koji mould, Penicillium notatum and Escherichia coli respectively in liquid complete medium in 33 DEG C of h (80r/ of shaken cultivation 12
Min after), 3 kinds of nutrient solutions is centrifuged into 12~10 min respectively at 5200~6000 r/min, obtain spore and thalline suspension, with
Sterilized water dilutes bacteria suspension, makes spore or thalline number up to 106~107Individual/mL, then by 3 kinds of bacteria suspensions with 3: 2: 2 volume ratio
After example is well mixed, then mixed with the colloid aqueous solution of sodium carboxymethylcellulose and sodium alginate in 1: 1 ratio, colloid is water-soluble
The mass percent of sodium carboxymethylcellulose is 1% in liquid, and the mass percent of sodium alginate is 4%, then by 1%~2% quality
Than the diatomite for adding 60 mesh, stir, what then instillation was stirred continuously contains 4%CaCl2And 2%MgSO4Mixed aqueous solution
In, 2 h are soaked, the mm of diameter 2~3 spheric granules is made, it is standby after being washed twice with sterilized water.
（3）Inoculation and fermentation
（1）Obtained granatanine extraction concentrated aqueous solution loads fermentation tank, adds (the NH of mass ratio 0.2%4)2HPO4,
Immobilized bacteria micelle is accessed fermentation tank again, inoculum concentration is the 10%~15% of zymotic fluid mass ratio, and fermentation temperature is controlled 33
~35 DEG C, filtrated air is pumped into from fermenter base, air mass flow for 0.4~0.6 times of fermentating liquid volume per hour, fermentation 4
~5 days.Zymotic fluid and immobilized bacteria micelle are separated by filtration, strain micelle can be used continuously more than 20 times, and zymotic fluid is used to carry
Take ellagic acid.
（4）Extraction
Zymotic fluid containing ellagic acid obtains the precipitation containing ellagic acid by centrifuging, and adds the anhydrous of 4~6 times of volumes for the 1st time
70 DEG C of h of reflux extraction 4 of methanol, filtering；2nd h of absolute methanol reflux extraction 3 that 2~3 times of volumes are added into filter residue, mistake
Filter；Merge time filtrate, revolving recovery methanol to saturation, the concentrate of saturation is stood still for crystals at room temperature, filtering gained solid knot
Crystalline substance is freeze-dried after deionized water is washed produces ellagic acid product.
The essence of the present invention is black-koji mould, Penicillium notatum and Escherichia coli hair using granatanine as liquid state fermentation substrate
Ferment produces tannase, and its specificity is high, the ester bond in the degraded Ellagitannins of selectivity, makes the degraded generation of granatum Ellagitannins
Ellagic acid, the ellagic acid of high-purity is obtained after abstraction purification.
It is an advantage of the invention that：The present invention can form complementary effect using 3 kinds of bacterium mixed fermentations, make ellagic acid transformation efficiency
It is higher；3 kinds of bacterium mix according to a certain percentage, and prepare immobilization glue using sodium carboxymethylcellulose, sodium alginate and diatomite
Grain, add the utilization ratio for utilizing number, improving strain of immobilization micelle.The technique fermentation condition is gentle, subtracts significantly
The consumption of few chemical substance, environmental pollution is small, is more suitable for producing on a large scale.
Embodiment
Embodiment 1：
（1）It is prepared by granatanine extract solution
Dry granatum is crushed to 40 mesh, using 72 DEG C of the ethanol solution refluxing extraction 3 times of 62% volume fraction, the 1st time plus
Enter 12 times that ethanol solution is Pomegranate Rind quality, extract 2 h, centrifuge filtrate and filter residue；Added into filter residue for 2nd time
10 times of ethanol solution, 1.5 h are extracted, centrifuged；3rd 8 times of the ethanol solution of addition into filter residue, extracts 1 h, centrifugation point
From；Merge 3 filtrates, through being distilled to recover ethanol, obtain granatanine extraction concentrated aqueous solution, can be directly used for fermenting and producing.
Tannin content 1%~1.5% in concentrated aqueous solution is extracted after measured.
（2）The culture of fermented bacterium is fixed together
By black-koji mould, Penicillium notatum and Escherichia coli respectively in liquid complete medium in 33 DEG C of h (80r/ of shaken cultivation 12
Min after), 3 kinds of nutrient solutions is centrifuged into 12 min respectively at 5200 r/min, spore and thalline suspension is obtained, is diluted with sterilized water
Bacteria suspension, make spore or thalline number up to 106~107Individual/mL, then 3 kinds of bacteria suspensions are well mixed with 3: 2: 2 volume ratio
Afterwards, then in 1: 1 ratio mixed with the colloid aqueous solution of sodium carboxymethylcellulose and sodium alginate, carboxymethyl in colloid aqueous solution
The mass percent of sodium cellulosate is 1%, and the mass percent of sodium alginate is 4%, then the silicon of mass ratio 60 mesh of addition by 1%%
Diatomaceous earth, stir, what then instillation was stirred continuously contains 4%CaCl2And 2%MgSO4Mixed aqueous solution in, soak 2 h, be made
The mm of diameter 2~3 spheric granules, it is standby after being washed twice with sterilized water.
（3）Inoculation and fermentation
（1）Obtained granatanine extraction concentrated aqueous solution loads fermentation tank, adds (the NH of mass ratio 0.2%4)2HPO4,
Immobilized bacteria micelle is accessed fermentation tank again, inoculum concentration is the 10% of zymotic fluid mass ratio, and fermentation temperature is controlled at 33 DEG C, from
Fermenter base is pumped into filtrated air, and air mass flow per hour, is fermented 5 days for 0.4 times of fermentating liquid volume.It is separated by filtration fermentation
Liquid and immobilized bacteria micelle, strain micelle can be used continuously more than 20 times, and zymotic fluid is used to extract ellagic acid.
（4）Extraction
Zymotic fluid containing ellagic acid obtains the precipitation containing ellagic acid by centrifuging, add for the 1st time 4 times of volumes without water beetle
70 DEG C of h of reflux extraction 4 of alcohol, filtering；2nd h of absolute methanol reflux extraction 3 that 3 times of volumes are added into filter residue, filtering；Close
And secondary filtrate, revolving recovery methanol to saturation, the concentrate of saturation are stood still for crystals at room temperature, filtering gained solid crystal is through going
It is freeze-dried after ionized water washing and produces ellagic acid product.
Embodiment 2：
（1）It is prepared by granatanine extract solution
Dry granatum is crushed to 40 mesh, used:73 DEG C of the ethanol solution refluxing extraction 3 times of 65% volume fraction, the 1st time
14 times that ethanol solution is Pomegranate Rind quality are added, 2 h is extracted, centrifuges filtrate and filter residue；2nd time into filter residue plus
Enter 9 times of ethanol solution, extract 1.5 h, centrifuge；3rd 7 times of the ethanol solution of addition into filter residue, extracts 1 h, centrifugation point
From；Merge 3 filtrates, through being distilled to recover ethanol, obtain granatanine extraction concentrated aqueous solution, can be directly used for fermenting and producing.
Tannin content 1%~1.5% in concentrated aqueous solution is extracted after measured.
（2）The culture of fermented bacterium is fixed together
By black-koji mould, Penicillium notatum and Escherichia coli respectively in liquid complete medium in 33 DEG C of h (80r/ of shaken cultivation 12
Min after), 3 kinds of nutrient solutions is centrifuged into 11 min respectively at 5500 r/min, spore and thalline suspension is obtained, is diluted with sterilized water
Bacteria suspension, make spore or thalline number up to 106~107Individual/mL, then 3 kinds of bacteria suspensions are well mixed with 3: 2: 2 volume ratio
Afterwards, then in 1: 1 ratio mixed with the colloid aqueous solution of sodium carboxymethylcellulose and sodium alginate, carboxymethyl in colloid aqueous solution
The mass percent of sodium cellulosate is 1%, and the mass percent of sodium alginate is 4%, then adds 60 purposes by 1.5% mass ratio
Diatomite, stir, what then instillation was stirred continuously contains 4%CaCl2And 2%MgSO4Mixed aqueous solution in, soak 2 h, system
The mm of diameter 2~3 spheric granules is obtained, it is standby after being washed twice with sterilized water.
（3）Inoculation and fermentation
（1）Obtained granatanine extraction concentrated aqueous solution loads fermentation tank, adds (the NH of mass ratio 0.2%4)2HPO4,
Immobilized bacteria micelle is accessed fermentation tank again, inoculum concentration is the 12% of zymotic fluid mass ratio, and fermentation temperature is controlled at 34 DEG C, from
Fermenter base is pumped into filtrated air, and air mass flow per hour, is fermented 4.5 days for 0.5 times of fermentating liquid volume.It is separated by filtration hair
Zymotic fluid and immobilized bacteria micelle, strain micelle can be used continuously more than 20 times, and zymotic fluid is used to extract ellagic acid.
（4）Extraction
Zymotic fluid containing ellagic acid obtains the precipitation containing ellagic acid by centrifuging, add for the 1st time 5 times of volumes without water beetle
70 DEG C of h of reflux extraction 4 of alcohol, filtering；2nd h of absolute methanol reflux extraction 3 that 3 times of volumes are added into filter residue, filtering；Close
And secondary filtrate, revolving recovery methanol to saturation, the concentrate of saturation are stood still for crystals at room temperature, filtering gained solid crystal is through going
It is freeze-dried after ionized water washing and produces ellagic acid product.
Embodiment 3：
（1）It is prepared by granatanine extract solution
Dry granatum is crushed to 40 mesh, using 75 DEG C of the ethanol solution refluxing extraction 3 times of 70% volume fraction, the 1st time
15 times that ethanol solution is Pomegranate Rind quality are added, 2 h is extracted, centrifuges filtrate and filter residue；2nd time into filter residue plus
Enter 8 times of ethanol solution, extract 1.5 h, centrifuge；3rd 6 times of the ethanol solution of addition into filter residue, extracts 1 h, centrifugation point
From；Merge 3 filtrates, through being distilled to recover ethanol, obtain granatanine extraction concentrated aqueous solution, can be directly used for fermenting and producing.
Tannin content 1%~1.5% in concentrated aqueous solution is extracted after measured.
（2）The culture of fermented bacterium is fixed together
By black-koji mould, Penicillium notatum and Escherichia coli respectively in liquid complete medium in 33 DEG C of h (80r/ of shaken cultivation 12
Min after), 3 kinds of nutrient solutions is centrifuged into 10 min respectively at 6000 r/min, spore and thalline suspension is obtained, is diluted with sterilized water
Bacteria suspension, make spore or thalline number up to 106~107Individual/mL, then 3 kinds of bacteria suspensions are well mixed with 3: 2: 2 volume ratio
Afterwards, then in 1: 1 ratio mixed with the colloid aqueous solution of sodium carboxymethylcellulose and sodium alginate, carboxymethyl in colloid aqueous solution
The mass percent of sodium cellulosate is 1%, and the mass percent of sodium alginate is 4%, then the silicon of mass ratio 60 mesh of addition by 2%
Diatomaceous earth, stir, what then instillation was stirred continuously contains 4%CaCl2And 2%MgSO4Mixed aqueous solution in, soak 2 h, be made
The mm of diameter 2~3 spheric granules, it is standby after being washed twice with sterilized water.
（3）Inoculation and fermentation
（1）Obtained granatanine extraction concentrated aqueous solution loads fermentation tank, adds (the NH of mass ratio 0.2%4)2HPO4,
Immobilized bacteria micelle is accessed fermentation tank again, inoculum concentration is the 15% of zymotic fluid mass ratio, and fermentation temperature is controlled at 35 DEG C, from
Fermenter base is pumped into filtrated air, and air mass flow per hour, is fermented 4 days for 0.6 times of fermentating liquid volume.It is separated by filtration fermentation
Liquid and immobilized bacteria micelle, strain micelle can be used continuously more than 20 times, and zymotic fluid is used to extract ellagic acid.
（4）Extraction
Zymotic fluid containing ellagic acid obtains the precipitation containing ellagic acid by centrifuging, add for the 1st time 6 times of volumes without water beetle
70 DEG C of h of reflux extraction 4 of alcohol, filtering；2nd h of absolute methanol reflux extraction 3 that 2 times of volumes are added into filter residue, filtering；Close
And secondary filtrate, revolving recovery methanol to saturation, the concentrate of saturation are stood still for crystals at room temperature, filtering gained solid crystal is through going
It is freeze-dried after ionized water washing and produces ellagic acid product.
Claims (2)
1. ellagic acid prepared by a kind of granatanine liquid state fermentation, it is characterised in that comprise the following steps：
（1）It is prepared by granatanine extract solution
Dry granatum is crushed to 40 mesh, uses 60-80 DEG C of refluxing extraction of ethanol solution 3 of 61%~70% volume fraction
Secondary, the 1st addition ethanol solution is 10~15 times of Pomegranate Rind quality, extracts 2 h, centrifuges filtrate and filter residue；2nd
It is secondary that 8~10 times of ethanol solution is added into filter residue, 1.5 h are extracted, are centrifuged；The 3rd addition ethanol solution 6 into filter residue~
8 times, 1 h is extracted, is centrifuged；Merge 3 filtrates, through being distilled to recover ethanol, obtain granatanine extraction concentrated aqueous solution, can
Fermenting and producing is directly used in, extracts tannin content 1%~1.5% in concentrated aqueous solution after measured；
（2）The culture of fermented bacterium is fixed together
By black-koji mould, Penicillium notatum and Escherichia coli respectively in liquid complete medium in 33 DEG C of h (80r/ of shaken cultivation 12
Min after), 3 kinds of nutrient solutions is centrifuged into 12~10 min respectively at 5200~6000 r/min, obtain spore and thalline suspension, with
Sterilized water dilutes bacteria suspension, makes spore or thalline number up to 106~107Individual/mL, then by 3 kinds of bacteria suspensions with 3: 2: 2 volume ratio
After example is well mixed, then mixed with the colloid aqueous solution of sodium carboxymethylcellulose and sodium alginate in 1: 1 ratio, colloid is water-soluble
The mass percent of sodium carboxymethylcellulose is 1% in liquid, and the mass percent of sodium alginate is 4%, then by 1%~2% quality
Than the diatomite for adding 60 mesh, stir, what then instillation was stirred continuously contains 4%CaCl2And 2%MgSO4Mixed aqueous solution
In, 2 h are soaked, the mm of diameter 2~3 spheric granules is made, it is standby after being washed twice with sterilized water；
（3）Inoculation and fermentation
（1）Obtained granatanine extraction concentrated aqueous solution loads fermentation tank, adds (the NH of mass ratio 0.2%4)2HPO4,
Immobilized bacteria micelle is accessed fermentation tank again, inoculum concentration is the 10%~15% of zymotic fluid mass ratio, and fermentation temperature is controlled 33
~35 DEG C, filtrated air is pumped into from fermenter base, air mass flow for 0.4~0.6 times of fermentating liquid volume per hour, fermentation 4
~5 days.Zymotic fluid and immobilized bacteria micelle are separated by filtration, strain micelle can be used continuously more than 20 times, and zymotic fluid is used to carry
Take ellagic acid；
（4）Extraction
Zymotic fluid containing ellagic acid obtains the precipitation containing ellagic acid by centrifuging, and adds the anhydrous of 4~6 times of volumes for the 1st time
70 DEG C of h of reflux extraction 4 of methanol, filtering；2nd h of absolute methanol reflux extraction 3 that 2~3 times of volumes are added into filter residue, mistake
Filter；Merge time filtrate, revolving recovery methanol to saturation, the concentrate of saturation is stood still for crystals at room temperature, filtering gained solid knot
Crystalline substance is freeze-dried after deionized water is washed produces ellagic acid product.
2. a kind of method that granatanine liquid state fermentation as described in power 1 prepares ellagic acid, it is characterised in that including following step
Suddenly：
（1）It is prepared by granatanine extract solution
Dry granatum is crushed to 40 mesh, uses 60-80 DEG C of refluxing extraction of ethanol solution 3 of 61%~70% volume fraction
Secondary, the 1st addition ethanol solution is 10~15 times of Pomegranate Rind quality, extracts 2 h, centrifuges filtrate and filter residue；2nd
It is secondary that 8~10 times of ethanol solution is added into filter residue, 1.5 h are extracted, are centrifuged；The 3rd addition ethanol solution 6 into filter residue~
8 times, 1 h is extracted, is centrifuged；Merge 3 filtrates, through being distilled to recover ethanol, obtain granatanine extraction concentrated aqueous solution, can
Fermenting and producing is directly used in, extracts tannin content 1%~1.5% in concentrated aqueous solution after measured；
（2）The culture of fermented bacterium is fixed together
By black-koji mould, Penicillium notatum and Escherichia coli respectively in liquid complete medium in 33 DEG C of h (80r/ of shaken cultivation 12
Min after), 3 kinds of nutrient solutions is centrifuged into 12~10 min respectively at 5200~6000 r/min, obtain spore and thalline suspension, with
Sterilized water dilutes bacteria suspension, makes spore or thalline number up to 106~107Individual/mL, then by 3 kinds of bacteria suspensions with 3: 2: 2 volume ratio
After example is well mixed, then mixed with the colloid aqueous solution of sodium carboxymethylcellulose and sodium alginate in 1: 1 ratio, colloid is water-soluble
The mass percent of sodium carboxymethylcellulose is 1% in liquid, and the mass percent of sodium alginate is 4%, then by 1%~2% quality
Than the diatomite for adding 60 mesh, stir, what then instillation was stirred continuously contains 4%CaCl2And 2%MgSO4Mixed aqueous solution
In, 2 h are soaked, the mm of diameter 2~3 spheric granules is made, it is standby after being washed twice with sterilized water；
（3）Inoculation and fermentation
（1）Obtained granatanine extraction concentrated aqueous solution loads fermentation tank, adds (the NH of mass ratio 0.2%4)2HPO4,
Immobilized bacteria micelle is accessed fermentation tank again, inoculum concentration is the 10%~15% of zymotic fluid mass ratio, and fermentation temperature is controlled 33
~35 DEG C, filtrated air is pumped into from fermenter base, air mass flow for 0.4~0.6 times of fermentating liquid volume per hour, fermentation 4
~5 days.Zymotic fluid and immobilized bacteria micelle are separated by filtration, strain micelle can be used continuously more than 20 times, and zymotic fluid is used to carry
Take ellagic acid；
（4）Extraction
Zymotic fluid containing ellagic acid obtains the precipitation containing ellagic acid by centrifuging, and adds the anhydrous of 4~6 times of volumes for the 1st time
70 DEG C of h of reflux extraction 4 of methanol, filtering；2nd h of absolute methanol reflux extraction 3 that 2~3 times of volumes are added into filter residue, mistake
Filter；Merge time filtrate, revolving recovery methanol to saturation, the concentrate of saturation is stood still for crystals at room temperature, filtering gained solid knot
Crystalline substance is freeze-dried after deionized water is washed produces ellagic acid product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710617149.8A CN107418995B (en) | 2017-07-26 | 2017-07-26 | A kind of ellagic acid and preparation method thereof of granatanine liquid state fermentation preparation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710617149.8A CN107418995B (en) | 2017-07-26 | 2017-07-26 | A kind of ellagic acid and preparation method thereof of granatanine liquid state fermentation preparation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107418995A true CN107418995A (en) | 2017-12-01 |
| CN107418995B CN107418995B (en) | 2019-12-03 |
Family
ID=60431323
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710617149.8A Active CN107418995B (en) | 2017-07-26 | 2017-07-26 | A kind of ellagic acid and preparation method thereof of granatanine liquid state fermentation preparation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107418995B (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110357900A (en) * | 2019-08-02 | 2019-10-22 | 中国科学院新疆理化技术研究所 | A kind of intermediate processing obtaining ellagic acid from pomegranate rind extract |
| CN110668453A (en) * | 2019-11-12 | 2020-01-10 | 东海县博汇新材料科技有限公司 | Method for purifying superfine silica powder by using microorganism mixed strain |
| CN111544355A (en) * | 2020-06-04 | 2020-08-18 | 爱西美(珠海)生物技术有限公司 | Ellagic acid enzyme and preparation method thereof |
| CN112772789A (en) * | 2020-12-31 | 2021-05-11 | 内江师范学院 | Functional additive for pomegranate peel feed, preparation method and application |
| US11122828B2 (en) * | 2018-11-05 | 2021-09-21 | MarvelBiome, Inc. | Microbial compositions comprising ellagitannin and methods of use |
| CN114107161A (en) * | 2021-11-15 | 2022-03-01 | 泸州老窖股份有限公司 | Method for producing ellagic acid by degrading pomegranate rind with mixed domesticated strain |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101481714A (en) * | 2008-01-11 | 2009-07-15 | 北京化工大学 | Method for preparing ellagic acid from pomegranate bark by enzyme process |
| CN102864184A (en) * | 2012-08-29 | 2013-01-09 | 太仓市茂通化建有限公司 | Method for producing citric acid by utilizing immobilized aspergillus niger |
| CN104341431A (en) * | 2014-09-30 | 2015-02-11 | 桂林三宝生物科技有限公司 | Method for extracting ellagic acid from pomegranate peel |
-
2017
- 2017-07-26 CN CN201710617149.8A patent/CN107418995B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101481714A (en) * | 2008-01-11 | 2009-07-15 | 北京化工大学 | Method for preparing ellagic acid from pomegranate bark by enzyme process |
| CN102864184A (en) * | 2012-08-29 | 2013-01-09 | 太仓市茂通化建有限公司 | Method for producing citric acid by utilizing immobilized aspergillus niger |
| CN104341431A (en) * | 2014-09-30 | 2015-02-11 | 桂林三宝生物科技有限公司 | Method for extracting ellagic acid from pomegranate peel |
Non-Patent Citations (1)
| Title |
|---|
| 张立华: "鞣花酸和三萜类物质在石榴不同器官中的分布", 《食品科学》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11122828B2 (en) * | 2018-11-05 | 2021-09-21 | MarvelBiome, Inc. | Microbial compositions comprising ellagitannin and methods of use |
| AU2019377423B2 (en) * | 2018-11-05 | 2023-09-21 | MarvelBiome, Inc. | Microbial compositions comprising ellagitannin and methods of use |
| CN110357900A (en) * | 2019-08-02 | 2019-10-22 | 中国科学院新疆理化技术研究所 | A kind of intermediate processing obtaining ellagic acid from pomegranate rind extract |
| CN110668453A (en) * | 2019-11-12 | 2020-01-10 | 东海县博汇新材料科技有限公司 | Method for purifying superfine silica powder by using microorganism mixed strain |
| CN111544355A (en) * | 2020-06-04 | 2020-08-18 | 爱西美(珠海)生物技术有限公司 | Ellagic acid enzyme and preparation method thereof |
| CN112772789A (en) * | 2020-12-31 | 2021-05-11 | 内江师范学院 | Functional additive for pomegranate peel feed, preparation method and application |
| CN112772789B (en) * | 2020-12-31 | 2023-05-09 | 内江师范学院 | Functional additive for pomegranate rind feed, preparation method and application |
| CN114107161A (en) * | 2021-11-15 | 2022-03-01 | 泸州老窖股份有限公司 | Method for producing ellagic acid by degrading pomegranate rind with mixed domesticated strain |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107418995B (en) | 2019-12-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107418995B (en) | A kind of ellagic acid and preparation method thereof of granatanine liquid state fermentation preparation | |
| CN105838622B (en) | Aspergillus niger HC306 and conversion aurantiin prepare the application in naringenin | |
| CN101481714A (en) | Method for preparing ellagic acid from pomegranate bark by enzyme process | |
| CN102250981B (en) | Method for preparing ellagic acid by solid fermentation with granatum as raw material | |
| CN102827896B (en) | Method for extracting theaflavin and theanine from tea leaves | |
| CN102676614B (en) | Method for preparing galacto-oligosaccharide | |
| CN102690846A (en) | Method for catalytically synthesizing gamma-aminobutyric acid from glutamate biological solid-phase enzyme | |
| CN107083406A (en) | (R) production method of 3 hydroxybutyric acids | |
| CN110438015B (en) | Immature bitter orange endophytic fungus for producing hesperidinase and method for producing hesperidinase by fermenting immature bitter orange endophytic fungus | |
| WO2017016199A1 (en) | Use of streptomyces psammoticus and method for producing vanillin | |
| CN107828834A (en) | A kind of preparation method of galactooligosaccharide | |
| AU2020102334A4 (en) | Ellagic acid prepared from pomegranate rind by solid-state fermentation and its preparation method | |
| CN102864190A (en) | Producing method of gamma-aminobutyric acid | |
| CN102703405A (en) | Method for preparing lactase by taking bran as raw material | |
| CN104800110B (en) | A kind of method that microbial fermentation prepares total saponin of sapindusmukerossi | |
| CN104031109A (en) | Method for purifying tea saponin by microbial fermentation | |
| CN101225418A (en) | Method for preparing gamma-reanal by using rice grain as raw material | |
| CN108384814A (en) | A kind of preparation method of phloretin | |
| CN102533565B (en) | Aspergillus niger capable of producing glycosidase and application thereof in improving resveratrol content in Japanese knotweed | |
| CN104774794A (en) | Strain capable of producing D-mannose isomerase and method for producing D-mannose isomerase by using same | |
| CN101857886B (en) | Method for preparing xylitol and co-producing L-arabinose | |
| CN111518860B (en) | Preparation method of cowberry fruit extract | |
| CN104561157A (en) | Method for producing gamma-aminobutyric acid (GABA) by fermentation method | |
| CN104357527A (en) | Method for extracting tea saponin from tea seed meal with microbial fermentation method | |
| CN107904075A (en) | A kind of preparation method of Chinese data wine |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |