AU2021104833A4 - Simple and rapid commercial method for the production of anthocyanin pigment from the black carrot (daucus carota) juice by resin column chromatography - Google Patents

Abstract

The present invention relates to extraction of anthocyanin which is a flavonoid based colouring agent. The invention reports the use of black carrot (Daucus carota). The problem lies with purifying anthocyanin from natural sources which is a challenging task, further its isolation and purification methods for the anthocyanin are expensive, tedious and produce low yield with average purity. A chromatographic approach was adopted using a special industrial grade, non-ionic hydrophobic polymer resin to obtain anthocyanin in higher purity.

Description

SIMPLE AND RAPID COMMERCIAL METHOD FOR THE PRODUCTION OF ANTHOCYANIN PIGMENT FROM THE BLACK CARROT (DAUCUS CAROTA) JUICE BY RESIN COLUMN CHROMATOGRAPHY FIELD

[0001] The present invention relates to a method for generation of anthocyanin extracts of high purity.

BACKGROUND

[0002] Anthocyanin is a flavonoid which is known to have many health benefits which are attributed to its anti-oxidant and radical scavenging properties. Black carrot is a rich source of anthocyanin and has been investigated by a number of researchers on the health benefits of its inclusion in daily diet. The major anthocyanins have been identified as cyanidin- 3 - (2-xylosylgalactoside), cyanidin 3-xylosylglucosylgalactoside and cyanidin 3-ferulylxyloglucosyl galactoside. Many studies also detected acylated anthocyanins in the black carrot extracts, chemical structures of these compounds are presented below.

OH C" HO O OH

o

OH

[0003] Cyanidin 3-xylosylglucosylgalactoside (1)

OH 0H

0

H H HO

[0004] Cyanidin 3-xylosylgalactoside (2)

0CHa OH

0 HOHOH HO o

[0005] Peonidin 3-xylosylglucosylgalactoside (3)

[0006]Chemical structures of the non-acylated anthocyanins detected in the black carrot extracts: (1) Cyanidin 3-xylosylglucosylgalactoside, (2) Cyanidin 3-xylosylgalactoside, and (3) Peonidin 3-xylosylglucosylgalactoside.

OH

HO 0 OH - o

R HH

HO No. HO HO H 0

[0007]R 1=R 2=OCH 3 : Sinapic acid derivative of cyanidin 3 xylosylglucosylgalactoside (4)

[0008]Ri=H; R2=OCH 3 : Ferulic acid derivative of cyanidin 3 xylosylglucosylgalactoside (5)

[0009]Ri=R 2=H: Coumaric acid derivative of cyanidin 3-xylosylglucosylgalactoside (6)

OH OOH NON

Ferulic acid derivative of pelargonidin (7)

OCH

HO 0 I. Ho H H H Oft

0 OH

NO

Ferulic acid derivative of peonidin 3-xylosylglucosylgalactoside 3-xylosylglucosyl galactoside (8)

[00010] Anthocyanin also finds its use in food and drug industry as a natural food colour with low toxicity and relatively good stability. However, purifying anthocyanin from natural sources is a challenging task. Currently used isolation and purification methods for the anthocyanin are expensive, tedious and give low yield with average purity. A number of filtrations, aqueous two phases extraction and other chromatographic methods have been worked upon previously by other workers. Though many of these published methods find good industrial use and executable feasibility but these are either complex methods or expensive. Therefore, to get a good level of purity of anthocyanin using simple and non-reactive means, we thought to develop a method which could be used to purify anthocyanin from the juice of black carrot in good yield and high purity. One of the main problems in purification of anthocyanin is the presence of sugars in the extract that greatly hinders any attempts to purify the anthocyanin. Hence, a chromatographic approach was adopted using a special industrial grade, non-ionic hydrophobic polymer resin which would adsorb anthocyanin from the juice, allowing for the removal of sugars through subsequent washes of de-mineralized water. The anthocyanin was then eluted using acidified methanol.

[00011] The following prior art are being reported:

[00012] CN110101045 The invention discloses a preparation method of whole black carrot powder rich in anthocyanidin and application of the prepared whole black carrot powder in food. In the preparation process, the method comprises the following steps of cleaning, freezing and thawing, crushing, color protecting, ball milling, homogenization, enzymolysis, sterilization, spray drying and the like, and finally the whole black carrot powder can be obtained. The preparation process is simple, the production cost is low, the consumed time is short, the prepared whole black carrot powder retains the nutritional ingredients and flavor to a great extent, the content of anthocyanin is increased to the greatest extent, soluble solid matter is increased, and finally the homogeneity of the product is guaranteed; the powder is used for in baked food or frozen food or instant drinks, the taste can be enriched, the color and luster are added, and the powder has very high nutritional value.

[00013] CN111690267 The invention discloses a novel black carrot pigment refining method which comprises the steps: crushing black carrots, squeezing, filtering, adding an inorganic salt solution and hydrophilic low-molecular-weight organic matters into black carrot residues, uniformly stirring, standing for phase separation, extracting and separating anthocyanin to an upper phase, and carrying out vacuum concentration on the upper phase of the standing liquid to obtain a black carrot extracting solution; and adsorbing the obtained black carrot extracting solution by using macroporous adsorption resin, then carrying out vacuum concentration to obtain a black carrot concentrated solution, and carrying out spray drying to obtain black carrot pigment powder with the color value of 130-150. Compared with the prior art, the method provided by the invention has the advantages of less required organic solvent, quick separation and no need of heating, the technology of combining aqueous two-water phase extractionwith macroporous adsorption resin is adopted, the time can be shortened to a certain extent, the purity of the product is improved, meanwhile, the regeneration technology of the macroporous adsorption resin is suitable for mass production, the cost can be reduced to a great extent, and the content of effective components is improved.

[00014] W02018055108 A method for obtaining an anthocyanin pigment color composition from carrot plants (preferably black carrot plants) comprising pre-harvest foliar spraying of an ethylene-releasing compound and the use of the obtained anthocyanin pigment color composition for coloring of an edible product or a pharmaceutical product.

[00015] IN3130/DEL/2012 The present invention relates to heat stable anthocyanin rich composition with high nutraceutical value. More particularly, the invention relates to a process of preparation of anthocyanin and phenolic enriched products from biological resources such as black carrot, plum, grapes and alike through a novel enzyme mediated process. The enzyme mediated. A processing employs cell wall degrading enzymes to increase extraction efficiency. The process is green and solvent free, for effective extraction of the aqueous extract or juice rich in nutraceutical content. The process is governed by variables such as upstream and downstream process steps with optimized variables of enzyme concentration, extraction time and temperature. The concentrate can be used as value added ingredients for developing functional products such as beverages, functionalized juices and bakery products.

[00016] However, none of the prior arts mention the process to obtain concentrated anthocyanin as described in the instant invention.

SUMMARY

[00017] The following presents a simplified summary of the invention in order to provide a basic understanding of some aspects of the invention. This summary is not an extensive overview of the present invention. It is not intended to identify the key/critical elements of the invention or to delineate the scope of the invention. Its sole purpose is to present some concept of the invention in a simplified form as a prelude to a more detailed description of the invention presented later.

[00018] The chromatographic process in combination with a prior concentration step allows for the generation of anthocyanin extracts of good purity (EV ~ 100) and low cost. The method is relatively easy to execute, and the hardware required are easy to procure and simple to operate. The industrial application of this process thus seems promising.

DESCRIPTION OF EMBODIMENTS

[00019] The following description is of exemplary embodiments only and is not intended to limit the scope, applicability or configuration of the invention in any way. Rather, the following description provides a convenient illustration for implementing exemplary embodiments of the invention. Various changes to the described embodiments may be made in the function and arrangement of the elements described without departing from the scope of the invention.

[00020] EXAMPLES

[00021] Plant material and chemicals

[00022] Black carrots were purchased from the local market. They were stored at 4°C and were used as per requirement. Indion PA 800 (Industrial Grade Polymeric Adsorbent) resin was purchased from Ion Exchange and Purified de-ionized water was obtained from a Milli-Q (Millipore, MA, USA) water-purification system. Methanol, trifluoroacetic acid, citric acid, potassium chloride and sodium acetate of analytical grades were purchased from Fisher Scientific.

[00023] Apparatus and general experimental procedure

[00024] Thin layer chromatography was performed with precoated plates of silica gel 60F 254 (Merck, Darmstadt, Germany) in butanol: acetic acid: water (4:1:5) as mobile phase. Ten microliters of sample were spotted at 1 mg/mL concentration in methanol. Anisaldehyde-sulphuric acid was used as the spray reagent. UV spectra were recorded on UV-1800 UV- VIS Spectrophotometer from Shimadzu.

[00025] Blanching and juicing

[00026] Blanching of the black carrot was done by using a modified procedure. The black carrots were immersed in a thermostatic water bath at (98±1)°C for previously determined time of 3 and 6 min. The ratio of sample mass to water volume was 1:2. During the isothermal heat treatment, the temperature was controlled with a thermometer. After blanching, the samples were drained to remove the excess water. 500 g of blanched carrot was juiced using a juicer and then filtered through 1p filter cloth.

[00027] Pretreatment of Resin

[00028] Pretreatment INDION 800 polymeric adsorbent is shipped as a water wet product imbibed with sodium chloride and sodium carbonate salts to retard bacterial growth. These salts must be washed from the adsorbent prior to use and this be achieved by washing with water at a linear flow rate of 5-10 m/h until the required level is achieved.

[00029] Isolation of Anthocyanins

[00030] A chromatography glass column with a radius of 1.5 cm and height of 40 cm was packed with 100 g of pretreated Indion PA 800 (Industrial Grade Polymeric Adsorbent) resin suspended in de-mineralized water. The filtered black carrot juice was either loaded directly onto the column or was concentrated under vacuum until its anthocyanin content was raised to 2.0%w/w and then loaded onto the column. The flow rate was set to 5 ml/min during the period of adsorption post which, 2 L of de-mineralized water was passed through the column at a flow rate of 10 ml/min. The column was then eluted using 400 ml of acidified methanol 0.1% TFA (trifluro acetic acid) at a flow rate of ml / min. The acidified methanol eluate was evaporated under vacuum at 40°C until a solid, purified mass was obtained. Sugar detection was done using Molisch's reagent to test the last column volume of the de-mineralized water which was passed through the column.

[00031] All experiments were performed in quadruplicates.

[00032] Colorimetric Assay

[00033] The total anthocyanins content was determined by the differential pH method based on the property of anthocyanin pigments to change the color with pH. Two dilutions of the same sample were prepared, the first one in potassium chloride buffer (0.025 M, pH 1.0) and the second one in sodium acetate buffer (0.4 M, pH 4.5), pH being adjusted with HCl 0.2N. After equilibration at room temperature for 15 min, the absorbance of two dilutions was read at 510 nm and 700 nm. Total monomeric anthocyanins (mg cyanidin 3-glucoside equivalent/100 g Fresh Weight) were calculated as follows:

A V %of Anthocyan (w/w)= MWDF 100 -L W

A V ColourValue(EV)= MW DF 100x3 gL Wt

A = (Asien - A70a)pH=1 - (A510no - A7Toom)pH=4.5

[00034]

[00035] The semnifications of symbols used in these relations are:

[00036] %w/w - percentage weight/weight (grams of solute in100g of solution)

[00037] A - Absorbance

[00038] , - Molar extinction coefficient (26900 L/mol cm)

[00039] L - Path length

[00040] MW - Molecular weight (449.2 g/mol for cyanidin 3-glucoside)

[00041] DF - Dilution factor

[00042] V - Volume

[00043] Wt - sample weight

[00044] The Brown Index [BI= (A43 0nm / A5 30 nm)] and Violet Index [VI= (A5 8 0 A 520 m)] are widely used for assessing color quality, but also for estimating the shade or hue. Hence they reflect the quantity of brown (A 430 )nm or blue (A5 8 0 nm) in anthocyanin based red colorants.

[00045] The pH of the juice and elutes were left unadjusted. The pH observed in the juice was found to be around 6. The last column volume of all the experiments showed negative result for the Molisch's test, indicating the absence of sugars. A concise representation of the results is given in the tables 1 & 2.

[00046] The comparative study between the performances of direct chromatographic purification of the black carrot juice versus the addition of a concentration step prior to the chromatographic purification procedure yielded results in favour of the latter process. From the data obtained, it was evident that the effectiveness of the chromatographic purification process was dependent upon the prior concentration step. The use of a concentration step prior to the chromatographic purification procedure almost doubles the purity of the final eluate and the recovery of the anthocyanin from the juice is increased by about 10% Furthermore, the concentration step seems to improve the consistency of the process, as indicated by the variance values. This indicates the importance of increasing the anthocyanin concentration and the total dissolved solids in the sample prior to the chromatographic purification step.

[00047] A direct consequence of this process is the ability to generate anthocyanin extracts of good purity, which facilitate the lowering of effective dosages in final products. The increased purity of the extracts could contribute towards generating more consistent colour hues when used for food colouring applications. The ability to reuse the resin several times reduces the production cost of thefinal extract. The consequent requirement for lowered dosages would help contribute towards decreasing the cost of the final product of application. Furthermore, the chromatographic process, starting from sample loading to complete elution of the anthocyanin in methanol required no more than three working hours. This allows for quick production of the final product.

[00048] Table 1 Anthocyanin content of Black carrot juice

Weigh Anthocyani Anthocyani Anthocyani t of Juice Anthocyani . n purity in n content n content purifie Recover obtaine n purity in purified in purified in juice d y (0) d (g) juice (%) extract extract (g) extract (%o) (g) (g)

Tria 314 0.64 2.01 11.45 15.04 1.72 85.57 11

Tria 316 0.65 2.05 10.86 16.82 1.82 88.78

12

)Tria 311 0.65 2.02 11.82 14.89 1.76 87.13 13

Tria 313 0.61 1.91 9.62 16.1 1.55 81.15 14

Avg313.5 0.637 1.997 10.54 15.71 1.712 85.66

Std. Dev 1.802 0.0164 0.0526 0.834 0.791 1.000 2.839

Var. 3.25 0.0002 0.0027 0.695 0.626 0.010 8.060

[00049] A trial wise detail of the parameters measured during the experiment where black carrot juice was directly subjected to the chromatographic purification step without a prior concentration step. Avg. indicates the average values. Std. Dev. indicates the standard deviation of the values obtained from the trials and Var. indicates the variance of the values. Recovery is the percentage measure of the anthocyanin present in the final extract against the anthocyanin originally present in the black carrot juice.

[00050] Table 2 Anthocyanin content of concentrated Black carrot juice after chromatography

Anthocyani Anthocyani Weigh Anthocyani Anthocyan Juice n content in t of n purity in in content Recover obtaine n purity in juice purifie purified in purified y(%) d(g) juice( (g) d extract extract extract (%) (g)

(g)

Trial 1 312 0.72 2.25 6.15 33.82 2.08 92.44

Trial 2 320 0.65 2.08 5.72 34.63 1.98 95.19

Trial 3 315 0.65 2.04 5.99 32.89 1.97 96.57

Trial 4 310 0.68 2.11 6.03 33.33 2.01 95.26

Avg. 314.25 0.675 2.12 5.97 33.67 2.01 94.86

Std. 3.7666 0.0287 0.0790 0.157 0.645 0.0430 1.504 Dev.

14.187 Var. 0.0008 0.0062 0.0247 0.4170 0.0018 2.262 5

[00051] A trial wise detail of the parameters measured during the experiment where black carrot juice was first subjected to a concentration step till the anthocyanin content reached 2.0% (EV 6) and then subjected to the chromatographic purification step. Avg. indicates the average values. Std. Dev. indicates the standard deviation of the values obtained from the trials and Var. indicates the variance of the values. Recovery is the percentage measure of the anthocyanin present in the final extract against the anthocyanin originally present in the black carrot juice

[00052] While considerable emphasis has been placed herein on the specific features of the preferred embodiment, it will be appreciated that many additional features can be added and that many changes can be made in the preferred embodiment without departing from the principles of the disclosure. These and other changes in the preferred embodiment of the disclosure will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation.

Claims (2)

1. 1. A method of extraction and concentration of anthocyanin from Daucus carota comprising of the steps:

i. blanching of the black carrot by immersing in a thermostatic water bath at 98±1°C for 3 and 6 min wherein, the ratio of black carrot mass to water volume is 1:2;

ii. draining to remove the excess water to obtain drained black carrot mass;

iii. juicing the drained black carrot mass and filtering to obtain black carrot juice;

iv. packing a chromatography glass column of radius of 1.5 cm and height of 40 cm with 100 g of pretreated Industrial Grade Polymeric Adsorbent Indion PA 800 resin suspended in de-mineralized water;

v. loading filtered black carrot juice onto the column after concentrating under vacuum until its anthocyanin content was raised to 2.0%w/w;

vi. setting the flow rate to 5 ml/min during the period of adsorption post which, 2 L of de-mineralized water was passed through the column at a flow rate of 10 ml/min.;

vii. eluting the column using 400 ml of acidified methanol 0.1% trifluro acetic acid at a flow rate of 20 ml/min.;

viii. evaporating the acidified methanol eluate under vacuum at 40°C until a solid, purified mass was obtained;

ix. determining the total anthocyanins content was determined by the differential pH method based.

2. The method of extraction and concentration of anthocyanin as claimed in claim 1, wherein the concentration of anthocyanin determined is 2%w/w.