KR20110063081A - Process for purification and powdering of aronia berry extract - Google Patents

Abstract

PURPOSE: A purification and powdering method of an aronia berry extract is provided to improve the content of antocyanine of the aronia berry extract using the column chromatography. CONSTITUTION: A purification and powdering method of an aronia berry extract comprises the following steps: absorbing the aronia berry extract to a column filled with a diaion HP20 polymeric adsorbent; washing glycoside impurities from the column using a 5~20% ethanol solution acidified by sulfur acid or citric acid; eluting the purified aronia berry extract in the column using a 30~70% ethanol solution acidified by the sulfur acid or the citric acid; vacuum-concentrating the aronia berry extract at 20~60deg C, and diluting with purified water acidified with the sulfur acid or the citric acid; and freezing the diluted extract for 1~2hours, and freeze-drying the extract for 20~30hours.

Description

Process for purification and powdering of Aronia berry extract}

The present invention relates to a method for purifying and powdering an Aronia fruit extract, and more particularly, performing column chromatography on an extract of the Aronia fruit, using Diaion HP20 resin as an adsorption resin, and then to sulfuric acid or citric acid. By using ethanol acidified as a washing solvent, the present invention relates to a method for purifying an aronia fruit extract having a high content of anthocyanin as an indicator and an inexpensive treatment cost, and a method for powdering the purified product obtained by such a method.

Aronia is a shrub native to North America, belongs to the family Rosaceae, and opens small, sour berries, which ripen and fall immediately in late summer. Leaves are soft, glossy and pale green. This aronia is widely used in the food industry, especially aronia fruit is used as a variety of food, such as juice, beverages, concentrates, wine, jam, jelly, dried fruit, fruit tea.

Aronia berry (Aronia berry) is usually divided into two types according to the color, the red color of the fruit is called red berry (Aronia arbutifolia), and the black is called black berry (Aronia melanocarpa).

Aronia fruit contains a large amount of sugar (20%), anthocyanins (Antocyanins), polyphenols (Polyphenol), vitamins B1, B2, pantothenic acid, flavonoids (Flavonoid) and the like. Anthocyanins and polyphenols, among others, help blood circulation in the human body, help permeability of microvessels, elasticity of blood vessels, and normalize blood pressure, and in particular, provide a positive effect on eye microvessels to enhance vision. Is reported.

As the components of the Aronia fruit are known to have a useful effect on the human body, various clinical trials have been conducted on the dried powder of the Aronia fruit. That is, when 300 mg / day of dry powder of Aronia fruit was administered to the human body for 8 weeks, it showed an antioxidant effect to normalize the level of HbA1 (glycosylated hemoglobin) and decrease the level of intermediate product of reactive oxygen (ROI). (P. Pawlowicz, J. Wilczynski, T. Stetkiewicz.Influence of natural anthocyanins derived from chokeberry (Aronia melanocarpa) extract on glycosylated hemoglobin level in pregnant women with insulin-dependent diabetes mellitus.Polish Journal of Gynaecological Investigations. 3) .123-125).

In another study, consuming 30 mg of dry powder of Aronia fruit for 30 days reduced TBARS (oxidation product) and superoxide dismutase (oxidizing redox enzymes when oxides were produced) and Glutatione peroxidase (antioxidant enzyme). And Catalase (antioxidant enzyme) have been reported to show a significant increase (Kowalczyk E, Fijalkowski P, Kura M, Krzesinski P, Blaszczyk J, Kowalski J, Smigielski J, Rutkowski M, Kopff M. The influence of anthocyanins from Aronia melanocarpa on selected parameters of oxiidative stress and microelements contents in men with hipercholesterolaemia.Pol. Merk. Lek., 2005, XIX, 113, 651-653).

Such clinical effect is due to the efficacy of Anthocyanin, which is a major component of Aronia fruit. The anthocyanins are pigment glycosides of anthocyanidins contained in flowers or fruits, and are classified into one or two monosaccharides and aglycones by hydrolysis. The main indicator is Cyanidin 3-O-galactoside. to be. However, the content of anthocyanin in the fruit of Aronia is only about 3.0 mg / g (about 0.3% by weight). Therefore, in order to more effectively ingest the anthocyanin contained in the Aronia fruit, the anthocyanin needs to be concentrated to powder the Aronia fruit extract.

Generally, natural extracts are powdered using a spray dryer or a freeze dryer, but methods of spray drying or lyophilizing the Aaronia fruit extract have not been developed yet. Because Aronia fruit extract contains sugars, sugar alcohols, organic acids, amino acids, and proteins, in addition to anthocyanins, sugars are mainly composed of glucose ( Glucose, Fructose, and Sorbitol are present as impurities because these impurities inactivate anthocyanins, or prevent spray drying or lyophilization of Aronia fruit extracts.

Therefore, J Sci Food Agric 1998,78,435-440 has proposed a method of purifying extracts of aronia using column chromatography, but using Amberlite XAD-7 or silica gel RP-C18 as adsorptive resin. This completely eliminates glycoside impurities such as glucose, fructose, and sorbitol, but the silica gel RP-C18 is a resin that cannot be used for industrial purposes, and column chromatography using Amberlite XAD-7 has a glycoside in a low concentration of aqueous alcohol. There is also an advantage such as eluted in, but as a high-cost adsorption resin is also poor recovery, there is a problem that the processing cost is too high.

Therefore, the problem to be solved by the present invention is to purify the Aaronia fruit extract using column chromatography, the content of anthocyanin, which is an indicator, high glycoside impurities to inactivate the anthocyanin or hinder the powdering of the Aronia fruit extract It is to provide a new purification method with a low content, and further a low processing cost, and a method for effectively powdering the Aaronia fruit extract purified in this way.

Purifying method of the Aaronia fruit extract according to the present invention comprises the steps of adsorbing the Aaronia fruit extract stock in a column filled with Diaion HP20 adsorption resin; Washing glycoside impurities in the column using a 5% to 20% ethanol solvent acidified with sulfuric acid or citric acid; Eluting the purified Aronia fruit extract in the column using 30-70% ethanol solvent acidified with sulfuric acid or citric acid; Characterized in that comprises a.

On the other hand, in the powdering method of the Aronia fruit extract according to the present invention, the Aronia fruit extract purified in the same manner as described above concentrated under reduced pressure at 20 ~ 60 ℃, purified water adjusted to pH 2 ~ 6 by adding sulfuric acid or citric acid Dilution with to adjust the sugar content to 12-14 brix; Preliminarily freezing the diluted extract for 1 to 2 hours at -50 to 60 ° C, and lyophilizing for 20 to 30 hours at -30 to 40 ° C; Characterized in that comprises a.

In addition, the method of powdering the Aaronia fruit extract according to the present invention, the concentrated Aaronia fruit extract purified by the above method at 20 ~ 60 ℃ concentrated under reduced pressure, purified water adjusted to pH 2 ~ 6 by adding sulfuric acid or citric acid Diluting with 30 to adjust the sugar content to 30-40 brix; Adding a dextrin or starch 60-70% by weight to the Aaronia fruit extract, followed by mixing, spray drying at a temperature of 180-220 ° C .; Characterized in that comprises a.

Purification method of Aronia fruit extract according to the present invention is about 40 times the recovery rate compared to the conventional purification method using the adsorption resin such as Amberlite XAD-7 or silica gel RP-C18 (recovery rate increased from 0.4% to 16%) It is high and the regeneration efficiency is not only easy to work, the productivity is increased, there is an effect that can greatly reduce the purification cost.

In addition, the concentrated powder of the Aronia fruit extract prepared according to the powdering process of the present invention is an anthocyanin content of 162 ~ 243 mg / g (16 ~ 25% by weight) because it is formulated into easy to take tablets or capsules Is possible, even a small dose is enough to take an anthocyanin intake.

Hereinafter, the present invention will be described in detail.

Purification method of the Aronia fruit extract according to the present invention, first compressed by aging Aronia berry (Aronia berry) and filtered to obtain a primary filtrate, the residue of the filtered 20 to 70 acidified by sulfuric acid or citric acid After extracting with% ethanol solvent (pH 2 ~ 6) for 2 to 5 hours to obtain a secondary filtrate by filtration, the primary filtrate and the secondary filtrate is mixed to prepare an extract of aronia fruit.

Next, Diaion HP20 adsorption resin was regenerated using NaOH and HCl, and the adsorption resin was charged to a column for chromatography, and then the Aronia fruit extract stock was loaded on the upper layer of the column. In this way, the extract stock of Aaronia fruit is adsorbed to the Diaion HP20 adsorbent resin.

The Diaion HP20 resin used as the adsorption resin in the present invention is a highly porous synthetic adsorbent prepared by copolymerizing styrene and divinyl benzene. strong. However, when the pores are contaminated by impurities such as organic substances, the adsorption capacity is greatly reduced, and the adsorbents can be easily eluted with general organic solvents such as acids and bases. It is widely used for the purification of small-molecular weight proteins, antibiotics, other foods and pharmaceuticals, desalination, and discoloration. Currently, Mitsubishi Chemical Co., Ltd. Japan products can be easily purchased.

Next, the column is washed using 5% to 20% ethanol solvent (pH 2 to 6) acidified with sulfuric acid or citric acid as a washing solvent. At this time, it is preferable that the amount of the washing solvent used is about twice the bed volume of the column. This removes all impurities contained in the extract of the Aronia fruit, that is, glycoside impurities such as glucose, fructose and sorbitol.

Finally, the Aronia fruit extract purified from the adsorption resin is eluted by injecting 30-70% ethanol solvent (pH 2-6) acidified by sulfuric acid or citric acid to the column, and the Aronia fruit extract purified according to the present invention. Is obtained.

In the present invention, a solvent acidified to pH 2 to 6 in almost all processes is used to prevent oxidation of the aronia fruit extract, and especially to stabilize such anthocyanin because anthocyanin is unstable in neutrality and alkali. In addition, various experiments using hydrochloric acid, sulfuric acid, citric acid, acetic acid, etc. in the acidification process to prevent the oxidation of anthocyanin showed the best results to prevent the oxidation of anthocyanin and maintain a stable pH. .

When the Aronia fruit extract is injected into the Diaion HP20 adsorptive resin according to the present invention, the anthocyanin is slightly polarized and thus develops slowly in the Diaion HP20 adsorptive resin. However, since glycoside impurities such as glucose and fructose sorbitol are polar, they are first removed through the column during development. In this process, impurities mixed with polarity and nonpolarity are removed by washing with 5% to 20% ethanol, and anthocyanin, an active ingredient, is eluted last with 30 to 70% ethanol solvent.

On the other hand, the powdering method of the Aaronia fruit extract according to the present invention is a process of powdering the Aaronia fruit extract purified by the above method, it is divided into a freeze drying method and a spray drying method.

First, lyophilization method, the purified Aronia fruit extract was concentrated under reduced pressure at 20 ~ 60 ℃ to remove the ethanol solvent, diluted with purified water adjusted to pH 2 ~ 6 by adding sulfuric acid or citric acid, the sugar content of 12 ~ 14 Adjust to Briggs. At this time, if the sugar content is not adjusted to 12 to 14 brix, the concentration is too high, so lyophilization is not well gelled, it is preferable to adjust the sugar content of the concentrate to 12 to 14 brix before lyophilization.

Subsequently, the Aaronia fruit extract was pre-frozen at -50 to 60 ° C. for 1 to 2 hours, and then lyophilized at -30 to 40 ° C. for 20 to 30 hours, and then filled with a mesh to freeze the Aaronia fruit extract. Obtain dry powder.

The freeze-dried powder obtained according to the present invention was found to have an anthocyanin content of 16 to 25% by weight.

Next, the spray drying method, concentrated condensation of the Aronia fruit extract purified in the same manner as described above at 20 ~ 60 ℃, diluted with purified water adjusted to pH 2 ~ 6 by adding sulfuric acid or citric acid 30 ~ 40 Adjust with Brix. At this time, the sugar is preferably 30 ~ 40 Brix unlike the case of the lyophilization method.

Subsequently, 60 to 70% by weight of dextrin or starch is added to the Aaronia fruit extract, followed by spray drying at a temperature of 180 to 220 ° C. to obtain a spray-dried powder of the Aaronia fruit extract. The dextrin or starch is an excipient for facilitating spray drying. In the present invention, as shown in the following examples, the yield of the spray drying method is increased by about 60% compared to the freeze drying method, which is due to the addition of the dextrin or starch.

In addition, the spray drying process may be a loss of the active ingredient because it proceeds at a high temperature of 180 ~ 220 ℃, freeze-drying process has the advantage that there is little loss of the active ingredient. However, the concentrated powder obtained by the spray drying method has an advantage of easy storage compared to the concentrated powder obtained by the freeze drying method.

The present invention is a fruit that contains glycosides containing a lot of antioxidants in addition to the Aronia fruit extract, such as blueberries, bilberries, acai berry, mulberry, bokbunja, strawberry (strawberry), blackberry (blackberry), cranberry ( It is also applicable to purifying or powdering extracts such as bilberry, gooseberry (western currant) and currant (currant fruit).

Hereinafter, examples of the present invention will be described.

A) Aaronia  Preparation of Fruit Extract

500 kg of frozen aronia berries are screened and thawed until the temperature reaches 1-5 ° C., and the aronia berries are compressed and extracted in a compression extractor to obtain 280 liters of the primary filtrate, and the filtration residue is transferred to the extractor. do.

The primary filtrate is transferred to a storage tank and adjusted to have a pH of 2 to 6 using sulfuric acid or citric acid. Then, 1360 liters of 20-70% alcohol ethanol solvent acidified with sulfuric acid or citric acid was added to the filtrate residue, followed by extraction for 2 to 5 hours, followed by filtration to obtain a secondary filtrate. The secondary filtrate is concentrated with a reduced pressure concentrator to obtain 680 liters of ethanol-free concentrate. The concentrate is diluted with about 2 times purified water, and then sulfuric acid or citric acid is added to adjust the pH to 2-6. .

The primary filtrate and the secondary filtrate were combined to obtain 1500 liters of Aaronia extract extract.

B) Purification by Chromatography

First, regenerate Diaion HP20 adsorption resin (manufactured by Mitsubishi Chemical Co., Ltd.) using 1N NaOH and 1N HCL. Adsorb the Aaronia Fruit Extract. Then, using 5% to 20% ethanol acidified with sulfuric acid or citric acid as a washing solvent, the column was washed twice with bed volumes of glucose or fructose contained in the extract of Aronia fruit, Glycoside impurities such as sorbitol are removed.

Then, about 200 liters of purified Aronia fruit extract was obtained by adding 20-70% alcohol ethanol solvent acidified with sulfuric acid or citric acid by twice the amount of the bed volume.

C) Preparation of Lyophilized Powder

Concentrated under reduced pressure at 40 ℃ in order to volatilize the ethanol present in the Aaronia fruit extract, dilute the Aaronia fruit extract with purified water adjusted to pH 2-6 by adding citric acid to reduce the sugar content of 12-14 brix Adjust with

Subsequently, the Aaronia fruit extract is preliminarily frozen in a preliminary freeze dryer at a temperature of -60 ° C for 1 hour, and then lyophilized at -40 ° C for 24 hours in a main freeze dryer. Finally, the lyophilisate was filled with a mesh to obtain 80 kg (yield: 16%) of lyophilized powder of the Aronia fruit extract.

D) Spray drying  Manufacture of powder

Concentrate the Aronia fruit extract purified according to the process B) at 40 ° C. under reduced pressure, dilute the Aronia fruit extract with purified water adjusted to pH 2-6 by adding sulfuric acid to adjust the sugar content to 30-40 brix. .

Subsequently, 60% by weight of dextrin was added to 40% by weight of the Aaronia fruit extract, spray dried at a temperature of 200 ° C., and the resulting dried material was filled with a mesh to obtain 130 kg of spray dried powder of Aronia fruit extract (yield: 26%). Got it.

[Activity test]

A) HPCL  Measure

HPLC was performed on the glycoside standard as an impurity, Cyanidin 3-O-galactoside as an indicator of anthocyanin, a conventional Aronia fruit extract, and the lyophilized powder prepared according to the above example under the following conditions. And the results are shown in FIGS. 1 to 5, respectively. However, the lyophilized powder was repeatedly tested twice, and the results are shown in FIGS. 4 and 5, respectively.

As can be seen in Figures 1 to 5, the lyophilized powder prepared according to the present invention (Figs. 4 and 5) of the glycoside which is an impurity as compared to the Aronia fruit extract stock (Fig. 3) not subjected to the purification process The content was significantly reduced, but the content of anthocyanin, an active ingredient, was found to be almost unchanged.

B) HPLC  Condition

1) Column; Aminopac PA 20 analytical, 4.6ⅹ250 mm

Aminopac PA 20 guard, 4.6ⅹ50 mm

2) Mobile phase; 80 mM NaOH

3) Sample Injection; 10 μL

4) Flow rate; 1 mL / min

5) Pressure; 12.4 MPa

6) Detection; DIONEX ICS-3000 Pulsed Amperometric Detector (USA)

C) Determination of the content of active ingredients

The test method for anthocyanin as an active ingredient in the lyophilized powder of Aronia fruit extract was revised as the standard of Korean medicines (Korea Food and Drug Administration Notification No. 2007-25) Part 2 of the herbal medicine “Bakinium Mytileus X” The test was modified with some modifications.

4 batches of lyophilized powder of the Aronia fruit extract were prepared in the same manner as in the above example, and a sample of a predetermined amount was taken from each batch, and the anthocyanin content was measured as described above. It was confirmed that it was 243 mg / g (16.2-24.3%).

D) Recommended daily intake

Recommended daily intake of the Aaronia fruit extract lyophilized powder prepared according to the present invention is 240 ~ 300 mg / day, which corresponds to 38.8 ~ 72.9 mg / day based on the content of anthocyanin as an active ingredient. The recommended daily intake is based on the results of the following two clinical trials.

1) P. Pawlowicz, J. Wilczynski, T. Stetkiewicz. Influence of natural anthocyanins derived from chokeberry (Aronia melanocarpa) extract on glycosylated hemoglobin level in pregnant women with insulin-dependent diabetes mellitus. Polish Journal of Gynaecological Investigations. 2001.3 (3). 123-125.

2) Kowalczyk E, Fijalkowski P, Kura M, Krzesinski P, Blaszczyk J, Kowalski J, Smigielski J, Rutkowski M, Kopff M. The influence of anthocyanins from Aronia melanocarpa on selected parameters of oxiidative stress and microelements contents in men with hipercholesterolaemia. Pol. Merk. Lek., 2005, XIX, 113, 651-653.

1 is an HPLC graph of a glycoside standard as an impurity,

2 is an HPLC graph of Cyanidin 3-O-galactoside, an indicator of anthocyanin,

3 is a HPLC graph of a typical Aronia fruit extract,

4 and 5 are HPLC graphs of the lyophilized powder of the Aronia fruit extract according to the present invention, respectively.

Claims (4)

Adsorbing the Aaronia fruit extract stock to a column filled with a Diaion HP20 adsorbent resin; Washing glycoside impurities in the column using a 5% to 20% ethanol solvent acidified with sulfuric acid or citric acid; Eluting the purified Aronia fruit extract in the column using 30-70% ethanol solvent acidified with sulfuric acid or citric acid; Purifying method of aronia fruit extract, characterized in that comprises a. According to claim 1, Aronia (Aronia) fruit extract is obtained by compressing the Aronia fruit after squeezing to obtain a primary filtrate, the residue of the filtrate in 20 ~ 70% ethanol solvent acidified by sulfuric acid or citric acid 2 After extracting for ˜5 hours to obtain a secondary filtrate by filtration, the method of purifying Aaronia fruit extract, characterized in that it is prepared by mixing the primary filtrate and the secondary filtrate. Aronia fruit extract purified according to claim 1 or 2 is concentrated under reduced pressure at 20 ~ 60 ℃, diluted with purified water adjusted to pH 2 ~ 6 by the addition of sulfuric acid or citric acid and the sugar content of 12 to 14 brix ( Brix); Pre-freezing the diluted extract for 1 to 2 hours at -50 to 60 ° C, and lyophilizing for 20 to 30 hours at -30 to 40 ° C; Powdered method of aronia fruit extract, characterized in that comprises a. Aronia fruit extract purified according to claim 1 or 2 is concentrated under reduced pressure at 20 ~ 60 ℃, diluted with purified water adjusted to pH 2 ~ 6 by adding sulfuric acid or citric acid to reduce the sugar content of 30 ~ 40 brix ( Brix); Adding a dextrin or starch 60-70% by weight to the Aaronia fruit extract, followed by mixing, spray drying at a temperature of 180-220 ° C .; Powdered method of aronia fruit extract that comprises a.

Images