CN104561223B - A kind of blueberry anthocyanin efficiently synthesizes extracting method - Google Patents
A kind of blueberry anthocyanin efficiently synthesizes extracting method Download PDFInfo
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- CN104561223B CN104561223B CN201510037796.2A CN201510037796A CN104561223B CN 104561223 B CN104561223 B CN 104561223B CN 201510037796 A CN201510037796 A CN 201510037796A CN 104561223 B CN104561223 B CN 104561223B
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Abstract
The invention discloses a kind of methods efficiently synthesizing anthocyanidin in extraction blueberry, specially blueberry anthocyanin synthesis extraction technology of preparing.Microbial fermentation culture blueberry cell, ultrafiltration chromatography is assisted to extract anthocyanidin in blueberry using high hydrostatic pressure.It assists microbial metabolic products to Blueberry cell wall damage using high hydrostatic pressure, anthocyanidin is made fully to discharge；The producing enzymes such as L-Phe and rhodotorula glutinis are added simultaneously to promote to be not converted into anthocyanidin precursor synthesis anthocyanidin in blueberry.Zymotic fluid obtains the anthocyanidin that purity is 99.31% through processing such as centrifugation, ultra-filtration and separation, chromatographic purifying, vacuum freeze dryings, increases 15% the 30% of anthocyanidin extracted amount.The present invention is easy to operate, generating process cleaning is harmless, at low cost, anthocyanidin yield is big, purity is high, does not need human body intestinal canal microbial degradation, and value medical health care is high, is suitble to large-scale production application, has great market prospects.
Description
Technical field
The present invention relates to a kind of blueberry anthocyanins to efficiently synthesize extracting method, belongs to technical field of bioengineering.
Background technology
Numerous studies show that diet is adjusted and nutritional supplementation can improve health and prevent disease.Rational nutrition
Therapy can improve the therapeutic effect of conventional medicament, drug usage amount be reduced, to reduce the side effect of drug therapy.In recent years
The study found that there is oxidation resistance rich in a large amount of natural anthocyanidin in blueberry, bodily fuctions can be improved and prevent certain
Ophthalmology disease.Therefore, how the anthocyanidin in efficient extraction and application blueberry has become the research topic in 21 century emerging forward position.
The anthocyanidin that the separation and extraction technology used at present obtains：Content is low, impurity is more, value medical health care is low.Due to
Anthocyanidin is more sensitive to pH value, temperature and light, therefore the separating and purifying technologies such as existing ultrasonic technique, solvent extraction method extract
Anthocyanidin content it is few, activity it is low, cause the effect to body-care, prevented and cured diseases reduction.
Blueberry is by the World Health Organization（World Health Organization, WHO）It is strongest to be described as antioxidant activity
One of fruits and vegetables.This is because rich in polyphenols such as flavonoids, phenolic acid in blueberry.Anthocyanidin in blueberry is considered as blueberry
Primary bioactivity.Therefore, the blueberry anthocyanin of high bioactivity and content is extracted, it is protection people rich health, pre-
Anti- disease is of great significance.
Invention content
Purpose of the present invention is to：It is concentrated mainly on that blueberry is intracellular, and lot of anthocyanin precursor is not filled for blueberry anthocyanin
Division leads to big target component dissolution hardly possible, active ingredient loss, energy consumption height, defect of high cost, proposes a kind of culture at utilization
Blueberry cell synthesizes anthocyanidin, establishes recovery rate height, strong operability, blueberry anthocyanin at low cost, mild condition synthesis extraction
Method.
Specifically operation carries out according to the following steps：
(1) pretreatment of raw material：It is raw material to take Blueberry fresh, that nothing is rotten, cleaned, mechanical to smash to pieces to pulpous state standby
With；
(2) blueberry cell culture：Blueberry in step (1) is homogenized through 0.7 mmolL-1Triton X-100 processing
After the high hydrostatic pressure (HHP) of 10 ~ 20 min, 50 ~ 60Mpa handle 5 min, inoculation：Aspergillus niger 5-8%, monascus 5-8%,
Clothing bacillus 1-3%, rhodotorula glutinis 4-7%, add 20 ~ 40 mg/L of L-phenylalanine, 3 ~ 8 mg/L of tyrosine, and culture medium is matched
Side：Sucrose 0.3-0.5%, peptone 1.0-2.0%, glucose 0.3-0.5%, corn steep liquor 1.0-3.0%, beancake powder 1.0-3.0%,
NaCl 0.3-0.8%, K2HPO40.1-0.3%, pH value 2.1-4.5,28 ~ 30 °C are cultivated 5 ~ 8 days, and culture solution is collected；
(3) separation and concentration of extraction mixture：By the zymotic fluid of step (2) to 8000-10000r/min room temperature conditions
Lower centrifugation 10-20min abandons precipitation, collects supernatant；Supernatant is subjected to ultrafiltration, pressure 0.1-0.6MPa, 25-40 ° of temperature
C collects concentrate, obtains anthocyanidin crude extract；
(4) it detaches, purify：The anthocyanidin crude extract of step (3) is placed in AB-8 absorption resin chromatography columns into Mobile state
Absorption washs pillar with the deionized water of 4-6 times of column volume, removes water-solubility impurity, then dense with 30-50% ethanol eluates etc.
Degree elution pillar, flow velocity 2-4ml/min obtain the higher anthocyanidin eluent of purity；
(5) dry：Rotary evaporation recycles ethyl alcohol, and vacuum freeze drying obtains the anthocyanidin powder product that purity is 96.34%.
In step (2), strain can select aspergillus niger 6-7%, monascus 6-7%, lactic acid bacteria 0.5-0.8%, lichens gemma bar
The combination of one or more of the microorganisms such as bacterium 1.5-2.5%, rhodotorula glutinis 5-6%, brewer's yeast 1.0-1.5% carries out biology
Degradation and synthesis.
In step (2), culture medium prescription can select：Sucrose 0.4-0.5%, peptone 1.2-1.8%, glucose and corn
The mass ratio of the addition of slurry is 1:2.
In step (2), the addition mass ratio of L-phenylalanine and l-tyrosine is 1:1.
In step (4), ultrafiltration uses the ultrafiltration apparatus of molecular cut off 500Da.
The advantage of the invention is that：
1, compared with prior art, the present invention is super using high hydrostatic pressure-microbial fermentation-using Blueberry as raw material
The new and high technologies such as filter, column chromatography extract already contg anthocyanidin in fruit.
2, plant cell wall is destroyed using aspergillus niger and monascus and high hydrostatic pressure, keeps anthocyanidin release complete.
3, phenylalaninase is generated using rhodotorula glutinis, lactic acid bacteria, adds anthocyanidin precursor substance L-phenylalanine and L-
Tyrosine continues to promote to accelerate more natural anthocyanidin to synthesize using original ingredient in blueberry.
4, the technology of the present invention low temperature, low ph value, be protected from light, it is ensured that anthocyanidin chemical property stability, organic solvent is using few
Easily recycling, the production cycle is only 5-8 days.
5, the anthocyanidin finished product purity obtained greatly improves, and can reach 99.31%.
6, the present invention overcomes temperature in traditional mode of production is higher, dissolution rate is relatively low, and organic reagent dosage is more, of high cost, effect
The low disadvantage of rate.Therefore the achievable scale of the present invention, industrialization production, increase the economic value of blueberry, extend blueberry industry
Chain.
Specific implementation mode
Embodiment 1
(1) pretreatment of raw material：It is raw material to take Blueberry fresh, that nothing is rotten, cleaned, mechanical to smash to pieces to pulpous state standby
With；
(2) blueberry cell culture：Blueberry in step (1) is homogenized through 0.7 mmolL-1Triton X-100 processing
After high hydrostatic pressure (HHP) the processing 3-4 min of 15 ~ 20 min, 55 ~ 58Mpa, inoculation：Aspergillus niger 6%, monascus 6%, lichens
Bacillus 1%, rhodotorula glutinis 5% add L-phenylalanine 30mg/L, l-tyrosine 5mg/L, add L-phenylalanine and L-
The mass ratio of the addition of tyrosine is 1:1, culture medium prescription：Glucose 0.4%, corn steep liquor 1.5%, NaCl 0.4%,
K2HPO40.2%, 2.5,28 °C of pH value is cultivated 7 days, and culture solution is collected；
(3) separation and concentration of extraction mixture：The zymotic fluid of step (2) is centrifuged under room temperature to 80000r/min
10-20min abandons precipitation, collects supernatant；Supernatant is subjected to ultrafiltration, pressure 0.1MPa, 25 °C of temperature collects concentrate,
Obtain anthocyanidin crude extract；
(4) it detaches, purify：The anthocyanidin crude extract of step (3) is placed in AB-8 absorption resin chromatography columns into Mobile state
Absorption washs pillar with the deionized water of 5-6 times of column volume, removes water-solubility impurity, then dense with 30-50% ethanol eluates etc.
Degree elution pillar, flow velocity 2-4ml/min obtain the higher anthocyanidin eluent of purity；
(5) dry：Rotary evaporation recycles ethyl alcohol, and vacuum freeze drying obtains the anthocyanidin powder product that purity is 98.27%.
Embodiment 2
(1) pretreatment of raw material：It is raw material to take Blueberry fresh, that nothing is rotten, cleaned, mechanical to smash to pieces to pulpous state standby
With；
(2) blueberry cell culture：Blueberry in step (1) is homogenized through 0.7 mmolL-1Triton X-100 processing
After high hydrostatic pressure (HHP) the processing 3-4 min of 15 ~ 20 min, 55 ~ 58Mpa, inoculation：Monascus 6%, glues at lactic acid bacteria 0.6%
Rhodotorula 5%, brewer's yeast 1.2%, add 30 mg/L of L-phenylalanine, 6 mg/L of l-tyrosine, addition L-phenylalanine with
The mass ratio of the addition of l-tyrosine is 1:1, culture medium prescription：Sucrose 0.5%, corn steep liquor 2.0%, NaCl 0.5%,
K2HPO40.3%, 3.5,28 °C of pH value is cultivated 7 days, and culture solution is collected；
(3) separation and concentration of extraction mixture：The zymotic fluid of step (2) is centrifuged under room temperature to 9000r/min
15min abandons precipitation, collects supernatant；Supernatant is subjected to ultrafiltration, pressure 0.3MPa, 30 °C of temperature is collected concentrate, obtained
To anthocyanidin crude extract；
(4) it detaches, purify：The anthocyanidin crude extract of step (3) is placed in AB-8 absorption resin chromatography columns into Mobile state
Absorption washs pillar with the deionized water of 5-6 times of column volume, removes water-solubility impurity, then dense with 30-50% ethanol eluates etc.
Degree elution pillar, flow velocity 3-4ml/min obtain the higher anthocyanidin eluent of purity；
(5) dry：Rotary evaporation recycles ethyl alcohol, and vacuum freeze drying obtains the anthocyanidin powder product that purity is 99.18%.
Embodiment 3
(1) pretreatment of raw material：It is raw material to take Blueberry fresh, that nothing is rotten, cleaned, mechanical to smash to pieces to pulpous state standby
With；
(2) blueberry cell culture：Blueberry in step (1) is homogenized through 0.7 mmolL-1Triton X-100 processing
After high hydrostatic pressure (HHP) the processing 3-4 min of 10 ~ 15 min, 55 ~ 58Mpa, inoculation：Monascus 7%, glues at lactic acid bacteria 0.8%
Rhodotorula 6%, brewer's yeast .5%, the mass ratio for adding the addition of L-phenylalanine and l-tyrosine are 1:1, culture medium prescription：
The mass ratio of sucrose 0.5%, peptone 11.8%, the addition of glucose and corn steep liquor is 1:2, NaCl 0.6%, K2HPO40.3%, pH
3.0,28 ~ 30 °C of value is cultivated 7 days, and culture solution is collected；
(3) separation and concentration of extraction mixture：The zymotic fluid of step (2) is centrifuged under room temperature to 10000r/min
15 min abandon precipitation, collect supernatant；Supernatant is subjected to ultrafiltration, pressure 0.5MPa, 40 °C of temperature is collected concentrate, obtained
To anthocyanidin crude extract；
(4) it detaches, purify：The anthocyanidin crude extract of step (3) is placed in AB-8 absorption resin chromatography columns into Mobile state
Absorption washs pillar with the deionized water of 4-6 times of column volume, removes water-solubility impurity, then dense with 45-50% ethanol eluates etc.
Degree elution pillar, flow velocity 3.5-4ml/min obtain the higher anthocyanidin eluent of purity；
(5) dry：Rotary evaporation recycles ethyl alcohol, and vacuum freeze drying obtains the anthocyanidin powder product that purity is 99.31%.
Claims (3)
1. a kind of blueberry anthocyanin synthesizes extracting method, it is characterised in that operation carries out according to the following steps：(1) raw material is located in advance
Reason：Take it is fresh, be raw material without rotten Blueberry, it is cleaned, mechanical smash to pieces to pulpous state after it is spare；(2) blueberry cell is trained
It supports：By the blueberry pulp obtained in step (1) through 0.7 mmol L-1Triton X-100 handle 10 ~ 20 min, 50 ~ 60MPa's
After high hydrostatic pressure handles 5 min, inoculation：Aspergillus niger 6-7%, monascus 6-7%, lactic acid bacteria 0.5-0.8%, bacillus licheniformis
1.5-2.5%, rhodotorula glutinis 5-6%, brewer's yeast 1.0-1.5% add 20 ~ 40 mg/L of L-phenylalanine, l-tyrosine 3 ~ 8
Mg/L, culture medium prescription：Sucrose 0.3-0.5%, peptone 1.0-2.0%, glucose 0.3-0.5%, corn steep liquor 1.0-3.0%, beans
Cake powder 1.0-3.0%, NaCl 0.3-0.8%, K2HPO4 0.1-0.3%, pH value 2.1-4.5,28 ~ 30 °C are cultivated 5 ~ 8 days, and training is collected
Nutrient solution, above-mentioned percent concentration are mass percent concentration, and the addition mass ratio of L-phenylalanine and l-tyrosine is 1:1；
(3) separation and concentration of extraction mixture：The culture solution of step (2) is centrifuged under room temperature to 8000-10000r/min
10-20min abandons precipitation, collects supernatant；It is 0.1-0.6MPa in pressure, at 25-40 °C of temperature, supernatant is subjected to ultrafiltration,
Filtrate is collected, anthocyanidin crude extract is obtained；(4) it detaches, purify：The anthocyanidin crude extract that step (3) obtains is placed in AB-8
Dynamic Adsorption is carried out in absorption resin chromatography column, pillar is washed with the deionized water of 4-6 times of column volume, removes water-solubility impurity,
30-50% ethanol eluate Isocratic clution pillars, flow velocity 2-4ml/min is used to obtain the higher anthocyanidin elution of purity again
Liquid；(5) dry：Rotary evaporation recycles ethyl alcohol, and vacuum freeze drying obtains the anthocyanidin powder product that quality purity is 96.34%.
2. blueberry anthocyanin according to claim 1 synthesizes extracting method, it is characterised in that in step (2), culture medium is matched
Side：The mass ratio of sucrose 0.4-0.5%, peptone 1.2-1.8%, the addition of glucose and corn steep liquor are 1:2.
3. blueberry anthocyanin according to claim 1 synthesizes extracting method, which is characterized in that in step (3), ultrafiltration uses
The ultrafiltration apparatus of molecular cut off 500Da.
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Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106137936A (en) * | 2016-08-30 | 2016-11-23 | 杭州高峰蓝莓种植有限公司 | Blue berry elite skin care liquid and preparation method thereof |
| CN108094313A (en) * | 2017-12-15 | 2018-06-01 | 柳州旭至自动化科技有限公司 | A kind of method for breeding for protecting pig liver |
| CN108271979A (en) * | 2018-02-07 | 2018-07-13 | 淄博职业学院 | A kind of blueberry ginkgo composite fruit juice and preparation method thereof |
| CN108576817B (en) * | 2018-03-23 | 2021-07-02 | 河南科技大学 | Preparation method of slow-release type anthocyanin microcapsules |
| CN108576142A (en) * | 2018-05-21 | 2018-09-28 | 遵义盛林农业发展有限公司 | A kind of production method of blueberry biscuit |
| CN108719395A (en) * | 2018-05-21 | 2018-11-02 | 遵义盛林农业发展有限公司 | A kind of production method for blueberry biscuit |
| CN108925985A (en) * | 2018-07-22 | 2018-12-04 | 贵州蓝茅生物科技有限公司 | A kind of aroma blueberry oral preparation and preparation method thereof |
| CN113582959A (en) * | 2020-04-30 | 2021-11-02 | 湖南今汉药业有限公司 | Extraction process of mulberry anthocyanin |
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| CN102924423A (en) * | 2012-10-29 | 2013-02-13 | 杨公明 | Method for extracting cyanidin from banana peel |
| CN103013936A (en) * | 2012-12-25 | 2013-04-03 | 浙江惠松制药有限公司 | Method for extracting anthocyanidin by using compound enzyme, and compound enzyme preparation thereof |
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| CN102924423A (en) * | 2012-10-29 | 2013-02-13 | 杨公明 | Method for extracting cyanidin from banana peel |
| CN103013936A (en) * | 2012-12-25 | 2013-04-03 | 浙江惠松制药有限公司 | Method for extracting anthocyanidin by using compound enzyme, and compound enzyme preparation thereof |
Non-Patent Citations (2)
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| Blue light irradiation affects anthocyanin content and enzyme activities involved in postharvest strawberry fruit;Xu Feng et al;《Journal of Agricultural and Food Chemistry》;20140430;4778-4783 * |
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