阅读说明：本技术 一种从蓝锭果中提取花青素的方法 (A method of extracting anthocyanidin from Lonicera caerulea ) 是由 何安乐 李伟 黄华学 于 2019-08-14 设计创作，主要内容包括：一种从蓝靛果中提取花青素的方法,包括以下步骤：(1)浸提：以水为溶剂浸提经破碎的蓝靛果,固液分离,液体为粗提液；(2)超滤：粗提液超滤,收集透过液为上柱清液；(3)树脂吸附洗脱；将上柱清液上阳离子交换树脂柱,采用盐溶液线性洗脱,收集含花青素的洗脱液,浓缩得浓缩液；(4)析晶：向浓缩液中加入低级醇进行析晶,析晶完成后,离心,洗涤,干燥得产品。利用本发明方法从蓝靛果中提取的花青素产品,花青素含量高；且整个提取工艺条件温和,具有收率高,环境友好,适宜于产业化生产等特点。(A method of it extracting anthocyanidin from indigo fruit, comprising the following steps: (1) extract: taking water as a solvent extraction through broken indigo fruit, be separated by solid-liquid separation, liquid is crude extract；(2) ultrafiltration: crude extract ultrafiltration, collection permeate are upper column clear liquid；(3) adsorption and desorption by resin；Cation exchange resin column in upper column clear liquid is collected the eluent containing anthocyanidin, be concentrated to give concentrate using salting liquid linear elution；(4) crystallization: being added lower alcohol into concentrate and carry out crystallization, after the completion of crystallization, is centrifuged, washing, dry product.The anthocyanidin product extracted from indigo fruit using the method for the present invention, anthocyanidin content are high；And entire extracting factor is mild, has high income, environmental-friendly, the features such as being suitable for industrialization production.)

1. a kind of method for extracting anthocyanidin from indigo fruit, which comprises the following steps:

(1) it extracts: taking water as a solvent extraction through broken indigo fruit, be separated by solid-liquid separation, obtain crude extract；

(2) ultrafiltration: by the resulting crude extract ultrafiltration of step (1), permeate is collected；

(3) adsorption and desorption by resin；Cation exchange resin column in the permeate that step (2) are collected, is linearly washed using salting liquid It is de-, the eluent containing anthocyanidin is collected, concentration obtains concentrate；

(4) crystallization: being added lower alcohol into step (3) resulting concentrate and carry out crystallization, after the completion of crystallization, is centrifuged, washs, does It is dry to obtain anthocyanidin product.

2. method according to claim 1, which is characterized in that the temperature of the extraction is 0 ~ 40 DEG C, it is preferred that the extraction Temperature be 4 ~ 20 DEG C；Preferably, the material water quality ratio of the extraction is 1:20 ~ 40, more preferably, the material water quality ratio For 1:25 ~ 35；Preferably, the extraction is extracted many times, and number is 2 ~ 5 times, and more preferably, the number of the extraction is 3 ~ 4 times；Preferably, the time of the extraction is 3 ~ 8h.

3. method according to claim 1 or claim 2, which is characterized in that in step (1), the mode of the separation of solid and liquid is successively Using strainer filtering and centrifuge separation, the mesh number of the strainer is 60 ~ 200 mesh, it is preferred that the mesh number of the strainer is 100 ~ 120 mesh；Preferably, the centrifugation uses disc centrifuge, Horizontal helical type centrifuge or link-suspended basket centrifuge.

4. any one the method according to claim 1 ~ 3, which is characterized in that in step (2), the molecular cut off of the ultrafiltration For 1 ~ 20KDa, it is preferred that the molecular cut off of the ultrafiltration is 4 ~ 10KDa.

5. any one the method according to claim 1 ~ 4, which is characterized in that in step (3), the cation exchange resin Type is selected from one of Dowex-50, D113, D131 and Amberlitc IRC-84, it is preferred that the cation exchange tree The type of rouge is Dowex-50；Preferably, the ratio of height to diameter of the cation exchange resin column is 3 ~ 10:1, it is preferred that the sun The ratio of height to diameter of ion exchange resin column is 5 ~ 7:1；Preferably, the upper column flow rate of the permeate is 0.3 ~ 4BV/h, it is preferred that The upper column flow rate of the permeate is 0.5 ~ 1.5 BV/h.

6. any one the method according to claim 1 ~ 5, which is characterized in that in step (3), the salting liquid is selected from chlorination One of sodium, potassium chloride, sodium sulphate and potassium sulfate solution are a variety of, and more preferably, the salting liquid is sodium chloride water Solution.

7. any one the method according to claim 1 ~ 6, which is characterized in that in step (3), the linear elution refers to, salt Solution concentration is eluted by 0M, until the concentration of salting liquid increases linearly to 2.0M and stops elution, the volume of the salting liquid For 5 ~ 10BV, in the linear elution, the flow velocity of eluent is 0.5 ~ 4BV/h；Preferably, the linear elution is that salting liquid is dense Degree is eluted by 0M, until the concentration of salting liquid increases linearly to 1.0M and stops elution, the volume of the salting liquid is 6 ~ 8BV, in the linear elution, the flow velocity of eluent is 0.8 ~ 2BV/h.

8. any one of according to claim 1 ~ 7 the method, which is characterized in that the concentration be concentrated into using reverse osmosis membrane it is dense The Brix value of contracting liquid is 20~60%, thickening temperature≤60 DEG C；Preferably, the concentration is to be concentrated into concentration using reverse osmosis membrane The Brix value of liquid is 40~50%, thickening temperature≤30 DEG C.

9. any one the method according to claim 1 ~ 8, which is characterized in that the lower alcohol is methanol or ethyl alcohol, it is preferred that The lower alcohol is ethyl alcohol；Preferably, the volume ratio of the lower alcohol and the concentrate is 1 ~ 20:1；More preferably, institute The volume ratio for stating lower alcohol and the concentrate is 8 ~ 15:1；Preferably, the temperature of the crystallization be -10 ~ 40 DEG C, crystallization when Between for 4 ~ for 24 hours, it is preferred that the temperature of crystallization is -4 ~ 10 DEG C, and the time of crystallization is 6 ~ 12h.

10. any one the method according to claim 1 ~ 9, which is characterized in that the washing refers to: being drenched using the lower alcohol It washes, the temperature of the washing lotion is -10 ~ 40 DEG C；Preferably, the drying is vacuum freeze drying, and temperature is -10~-70 DEG C, very Reciprocal of duty cycle is 5~15Pa.

Technical field

The present invention relates to a kind of methods for extracting anthocyanidin, and in particular to a kind of side that anthocyanidin is extracted from indigo fruit Method.

Background technique

Indigo fruit also known as black bear fruit are mainly distributed on the ground such as Chinese Heilungkiang, Jilin, Liaoning, fruit sweet and sour.It can It is edible, wine brewing and it is medicinal, be the important wild resource in Northeast Area of China.Indigo fruit is same rich in nutriment abundant When, also containing a large amount of active anthocyanidin, state to the report for extracting anthocyanidin in Lonicera caerulea and is studied relatively at present It is few, mainly also rest on fresh fruit and be processed into fruit wine, fruit juice, jam, preserved fruit and jelly, so, from its extract anthocyanidin it is non-often with There is development prospect.

CN105693680 discloses a kind of extracting method of indigo fruit anthocyanidin, and raw material indigo fruit is cleaned and removed by this method Miscellaneous, expression separation, macroporous resin adsorption, ethyl alcohol desorption, is concentrated and dried up to anthocyanidin finished product at acidic ethanol extraction.The technique Simply, and using universal D101 applied widely resin is adsorbed, but in obtained product, anthocyanidin content is relatively low.

CN105884737 discloses a kind of purifying technique of indigo fruit anthocyanidin, and this method is impregnated by ethanol water Broken, homogeneous, separation of solid and liquid, macroporous resin adsorption, ethanol water gradient elution, vacuum concentration, freeze-drying are to get production Product.In this method upper prop is simply just only concentrated to leaching liquor after being extracted after centrifugation, contained in upper prop liquid a large amount of Non- anthocyanidin substance, these non-anthocyanidin substances are easily attracted on resin and cause the anthocyanidin content of product low, and its General AB-8 resin, adsorption range is wider, and the anthocyanidin content for also resulting in product is low.

CN103130762 discloses a kind of method that anthocyanidin is extracted from indigo fruit, and this method passes through acidic aqueous solution Impregnate extraction, adjust pH value, resin adsorption, washing, the elution of acid alcohol, be concentrated and dried after, thus obtain anthocyanidin product.The work Skill is simple, and adsorbs resin using universal D101 applied widely, so that obtained product content is relatively low.

CN105294632 discloses a kind of industrial method that indigo fruit anthocyanidin is prepared from indigo fruit, and this method is logical The alkaline aqueous solution extraction containing natural is crossed, acid is adjusted, flocculation, cationic exchange resin adsorption, acid solution elution, adjusts Up to product after being concentrated under reduced pressure, being spray-dried after pH.The technique is related to the adjusting of multiple pH, easily causes to degrade to anthocyanidin And process is tedious by tune pH, subsequent to be eluted with the hydrochloric acid of 2-5% to cation exchange resin, on the one hand this concentration hydrochloric acid is higher It easily causes product to degrade, and a large amount of chloride ion can be remained in desorbed solution, influence the anthocyanidin content and stabilization of product.

CN105153739 discloses a kind of preparation method of Lonicera caerulea dyestuff that pigment is stable, and this method in water by stirring It mixes and smashes, filters, dehydrated alcohol extracted many times, concentration, adjusting after acid, ethyl alcohol dissolution up to product.This method simple process, raw material It is its product after simple process, products obtained therefrom anthocyanidin content is extremely low, and the handicraft product is dyestuff, is only applicable to close and spin The dyeing of fabric.

CN102659870 discloses a kind of extracting method of Lonicera edulis anthocyanin, and this method is squeezed the juice by ethanol water Up to product after homogenate, homogeneous, centrifuge separation, film concentration, vacuum freeze-drying.This method simple process is concentrated dry after extraction It is dry, it is indigo fruit crude extract, the anthocyanidin content of product is low.

CN107325139 discloses a kind of method that anthocyanin is rapidly and efficiently extracted from indigo fruit, and this method passes through equal Matter is crushed enzymatic hydrolysis, alcohol steep, ethyl alcohol low temperature flocculating setting, reversed phase column chromatography, methanol elution, is concentrated and dried up to product.It should Technology utilization prepares chromatography and carries out chromatography, so separating capacity is limited, treatment effeciency is low, and measures small.It is subsequent to be washed with methanol It is de-, it may cause methanol and remain exceeded, the unsuitable industrialized production of the technique.

CN105885469 discloses a kind of ultrasonic-microwave collaboration auxiliary acidization ethyl alcohol extraction Lonicera edulis anthocyanin technique, This method is to extract acidic ethanol under the conditions of Lonicera caerulea freeze-drying, crushing, ultrasound-microwave cooperating, be centrifugated up to flower Color glycosides extracting solution.The simple process also stays at crude extract, product content is low although ultrasound-microwave cooperating extraction is utilized.

CN106117285 discloses a kind of high-pressure pulse electric auxiliary acidization ethyl alcohol extraction Lonicera caerulea anthocyanin technique, should Technique after the crushing of freezing Lonicera caerulea, sieving, acidic ethanol extraction, high-pressure pulse electric processing, centrifuge separation by extracting to obtain the final product Liquid.The simple process, products obtained therefrom are only crude extract, and anthocyanidin content is low.

CN101463056 discloses the preparation process of purifying Lonicera edulis anthocyanin by using macroporous resin, which passes through Lonicera caerulea Drying, crushing, acidic ethanol extraction, rotary evaporation, macroporous resin adsorption, ethyl alcohol are disposably desorbed, are concentrated, are dried in vacuo, and are obtained To product.The technique is related to drying, and high temperature may cause target product degradation, single adsorption resin X-5 and disposable desorption, The anthocyanidin content of resulting product is lower.

CN102532219 discloses a kind of method of anthocyanin in enriching and purifying Lonicera edulis, and this method passes through raw material Crush, ethyl alcohol be heated to reflux, pressurize recycling ethyl alcohol, adjust acid, washing, macroporous resin adsorption, gradient elution, silicagel column, acetone and Methanol solution elution, recrystallizes up to product active carbon decoloring.This method primary extract target substance is water-soluble, water after tune acid It is relatively difficult to achieve to be washed till neutral technique；Silicagel column elution has used acetone and methanol, residual may be brought in subsequent product, and most After used active carbon decoloring and recrystallization, will necessarily be that product yield is relatively low.

CN101434972 discloses a kind of technique of preparing Lonicera edulis anthocyanin by solid state biological reaction, and this method passes through Liquid nitrogen condition pomace crushes, the wooden enzyme solid state fermentation, ultrasonic wave added alcohol steep, is centrifuged to obtain clear liquid, X-5 resin adsorption, scraper-type Concentration, vacuum freeze drying, to obtain the final product product.The raw material that this method uses is indigo fruit pomace, itself anthocyanin substance is few, And it is subsequent using the wooden enzyme solid state fermentation, harmful bacteria synchronous fermentation is easily led to be metabolized out poisonous and harmful substance.Entire opposite fruit For slag refuse reclamation, the process is more complicated, and production cost is relatively too high, is not suitable for large-scale production.

CN109053834 discloses a kind of method that high-purity Cy-3-G is extracted in blue fruit honeysuckle, should Method by the way that freezable fruit is ground, methanol extraction, be centrifuged supernatant, low-temperature rotary concentration, SPE purifying, ethyl acetate washes De-, acidic methanol elution, rotary evaporation and filter membrane, HPLC detect up to product.This method methanol is extracted and is eluted, and can be led Product is caused to have methanol residual, and this method is adsorbed using C18 column, yield amount is few and column packing is expensive, production cost original text, it is difficult to advise Modelling production.

Summary of the invention

The technical problems to be solved by the present invention are: overcoming the above deficiency, one kind is provided and extracts anthocyanidin from indigo fruit Method.This method extracts the high income of anthocyanidin product from indigo fruit, and the purity of obtained anthocyanidin product is also high, production Cost.

The technical solution adopted by the present invention to solve the technical problems is:

A method of extracting anthocyanidin from indigo fruit, comprising the following steps:

(1) it extracts: taking water as a solvent extraction indigo fruit, be separated by solid-liquid separation, obtain crude extract；

(2) ultrafiltration: by the resulting crude extract ultrafiltration of step (1), permeate is collected；

(3) adsorption and desorption by resin；Cation exchange resin column in the permeate that step (2) are collected, is linearly washed using salting liquid It is de-, the eluent containing anthocyanidin is collected, concentrate is concentrated to give；

(4) crystallization: being added lower alcohol into concentrate and carry out crystallization, after the completion of crystallization, is centrifuged, washing, dry that anthocyanidin produces Product.

The anthocyanidin product that the present invention is extracted from Lonicera caerulea, product content is high, and entire extracting factor is mild, has High income, environmental-friendly, the features such as being suitable for industrialization production.

Preferably, in step (1), the temperature of the extraction is 0 ~ 40 DEG C, more preferably, the temperature of the extraction is 4 ~ 20℃。

Preferably, in step (1), the material water quality ratio of the extraction is 1:20 ~ 40, more preferably, the material water quality Amount is than being 1:25 ~ 35.

Preferably, in step (1), the extraction is extracted many times, and number is 2 ~ 5 times, more preferably, the extraction Number be 3 ~ 4 times.

Preferably, in step (1), the time of the extraction is 3 ~ 8h.

Preferably, in step (1), the mode of the separation of solid and liquid is successively to use strainer filtering and centrifuge separation, described The mesh number of strainer is 60 ~ 200 mesh, it is preferred that the mesh number of the strainer is 100 ~ 120 mesh.

Preferably, in step (1), the centrifugation uses disc centrifuge, Horizontal helical type centrifuge or link-suspended basket centrifuge.

Preferably, in step (2), the molecular cut off of the ultrafiltration is 1 ~ 20KDa, it is preferred that the retention of the ultrafiltration Molecular weight is 4 ~ 10KDa.

The ultrafiltration can effectively remove a large amount of macromolecular substances, while cross column convenient for subsequent, and pillar is avoided to be blocked.

Preferably, in step (3), the type of the cation exchange resin be selected from Dowex-50, D113, D131 or One of Amberlitc IRC-84, more preferably, the type of the cation exchange resin are Dowex-50.

Preferably, in step (3), the ratio of height to diameter of the cation exchange resin column is 3 ~ 10:1, more preferably, described The ratio of height to diameter of cation exchange resin column is 5 ~ 7:1.

Preferably, in step (3), the upper column flow rate of the permeate is 0.3 ~ 4BV/h, more preferably, the permeate Upper column flow rate is 0.5 ~ 1.5BV/h.

There are high molecular conjugated systems in anthocyanidin molecule can carry out containing acidic-group with cation exchange resin Ion exchange and be adsorbed, ratio of height to diameter appropriate and loading flow velocity can shorten loading while ion exchange sufficiently occurs Time, column efficiency in raising.

Preferably, in step (3), the salting liquid is in sodium chloride, potassium chloride, sodium sulphate and potassium sulfate solution One or more, more preferably, the salting liquid is sodium-chloride water solution.

Preferably, in step (3), the linear elution is that concentration of salt solution is eluted by 0M, until salting liquid is dense Degree increases linearly to 2.0M and stops elution, and the volume of the salting liquid is 5 ~ 10BV, in the linear elution, the flow velocity of eluent For 0.5 ~ 4BV/h.

Preferably, in step (3), the linear elution is that concentration of salt solution is eluted by 0M, until salting liquid is dense Degree increases linearly to 1.0M and stops elution, and the volume of the salting liquid is 6 ~ 8BV, in the linear elution, the flow velocity of eluent For 0.8 ~ 2BV/h.

Sodium chloride is cheap and is suitable for food, and easily obtains, and is particularly suitable for eluant, eluent；Linear elution is more advantageous to The ion of different ionic strength is eluted from resin, realizes the separation of anthocyanidin and other impurities.

Preferably, in step (3), the concentration is that reverse osmosis membrane is used to be concentrated into the Brix value of concentrate as 20~60%, Thickening temperature is not higher than 60 DEG C.

Preferably, in step (3), the concentration is that reverse osmosis membrane is used to be concentrated into the Brix value of concentrate as 40~50%, Thickening temperature is not higher than 30 DEG C.

Preferably, in step (4), the lower alcohol is methanol or ethyl alcohol, it is preferred that the lower alcohol is ethyl alcohol.

Preferably, in step (4), the volume ratio of the lower alcohol and the concentrate be 1 ~ 20:1 more preferably, institute The volume ratio for stating lower alcohol and the concentrate is 8 ~ 15:1.

Preferably, in step (4), the temperature of the crystallization is -10 ~ 40 DEG C, the time of crystallization is 4 ~ for 24 hours, it is more highly preferred to , the temperature of crystallization is -4 ~ 10 DEG C, and the time of crystallization is 6 ~ 12h.

The alcoholic solution and cryogenic conditions of debita spissitudo are conducive to the growth of crystal, and can accelerate crystalline growth speed with stirring Degree.

Preferably, in step (4), the washing is eluted using the lower alcohol, and the temperature of the washing lotion is -10 ~ 40 DEG C.

The washing lotion elutes the impurity that can rinse out plane of crystal to crystallization.

Preferably, in step (4), the drying be vacuum freeze drying, temperature be -10~-70 DEG C, vacuum degree be 5~ 15Pa。

The temperature of vacuum freeze drying is low, and the influence to anthocyanidin is small.

Preferably, in step (4), the indigo fruit is in fresh indigo fruit, freezing indigo fruit and dehydration indigo fruit It is one or more.

Fresh indigo fruit best can keep anthocyanidin not to be degraded, and freeze and be dehydrated indigo fruit and will lead to by processing The degradation of part anthocyanidin.

The beneficial effects of the present invention are:

(1) cation exchange resin is eluted by the way of linear elution due to this method, more accurately by target Substance is eluted, while the last method using crystallization purifies again, so that anthocyanidin content further mentions in product Height can extract anthocyanidin product in the slave indigo fruit of high yield using extracting method of the present invention, and obtained anthocyanidin product contains It measures higher；

(2) entire extracting factor of the invention is mild, and simple process is environmental-friendly, and lower production costs are suitable for industry Metaplasia produces.

Specific embodiment

The present invention is further detailed with reference to embodiments.

Indigo fruit of the present invention through being crushed includes through the new vivid blue of such as conventional fragmentation procedure such as squeezing, grinding and crushing Indigo fruit, freezing indigo fruit and dehydration indigo fruit.

The detection method of anthocyanidin content is that plant extracts Bilberry fruit P.E SW/T-2013 is attached in the embodiment of the present invention The measuring method of record A.2.

Raw material or chemical reagent used in the embodiment of the present invention are obtained by routine business approach unless otherwise specified .

Cation exchange resin used in the embodiment of the present invention is first activated using preceding, specific method are as follows: will The alcohol solution dipping of fresh resin volume fraction 95% for 24 hours after, with distilled water flushing to no alcohol taste, then with 3BV, quality point The NaOH solution of number 3.5% impregnates 5.5h, with distilled water flushing to neutrality, then, with 3BV, the hydrochloric acid solution of mass fraction 2.5% 5.5h is impregnated, with distilled water flushing to neutrality.

The high performance liquid chromatography detection instrument that the embodiment of the present invention uses is Anjelen Sci. & Tech. Inc Agilent Technologies production, model: HPLC-1260.