SE525503C2 - Process for purification of anthocyanins / anthocyanidins using hydrophobic zeolite - Google Patents

Abstract

A method and a kit for the extraction and purification of anthocyans from plant materials, comprising the steps of providing plant material(s) comprising anthocyans, providing one or more hydrophobic dealuminised zeolite(s), adding the plant material(s) to one or more hydrophobic dealuminised zeolite(s), allowing adsorption of the anthocyans to one or more hydrophobic zeolite(s), eluting the anthocyans from the hydrophobic zeolite(s), and obtaining the anthocyans which have been extracted and purified form the plant material(s). A method for separating the compounds anthocyanidines and anthocyanins. The use of extracted product(s) preferably for direct or indirect use for human consumption, in food or pharmaceutical production.

Description

Hydrophobic zeolites have previously been used to adsorb specific organic compounds in water. For example, U.S. Patent No. 5,108,617 (7) discloses adsorption of detergents I to hydrophobic zeolites, SB Patent Specification 9503731-3 (8, 9) adsorption of phenol and m-cresol, SE patent application 9802140-5 adsorption of pesticides (10) and SE PCT application SE 99/02281 adsorption of phthalates (1 l). The pores and cavities of zeolites have previously been used to adsorb organic compounds.

A limiting factor for the use of zeolites is considered to be that the zeolites cannot adsorb molecules that are too large to be able to pass into the cavities and channel systems of the zeolites. Anthoeyarins / anthocyariidines are clearly too large molecules to be able in this context to enter and be adsorbed in the cavities of the zeolites. Nevertheless, dealurninated zeolite Y exhibits a high adsorption capacity of anthocyanins / anthocyanidins from various plant materials (see patent examples) and * zeolites can be effectively used as aids to purify these antioxidants while maintaining antioxidant activity.

Description of the invention The invention relates to a new use of hydrophobic zeolites, namely purification of anthocyanins / anthocyanidins. In this case, it is usually the fi- idea of purifying anthocyanins / anthliocyanidines that is to be used as antioxidants is human. -_ consumption, food production or in the pharmaceutical industry. The use itself can in this case take place according to per se known principles, which generally means that the solution in question is brought into contact with the zeolite, using such an amount as the desired degree of adsorption of anthocyanins / anthocyanidins. to the zeolite is obtained.

The use of the invention can generally be carried out both batchwise and continuously or semi-continuously. An alternative here means that the hydrophobic zeolite is added directly to the solution, while another alternative is represented by the case where the hydrophobic zeolite is filled in a column, filter or the like, through which the solution to be purified is passed. Various specific applications may, of course, be considered, but these should not need to be described in more detail here, as they can be taken from the technology known per se.

However, the zeolites cannot adsorb unlimited amounts of anthocyanins / anthocyanidins, which means that the zeolite must eventually be eluted and! regenerated.

Treatment with organic solvents is a way of breaking the bond between zeolite in and adsorbed molecules and thus obtaining an elution of adsorbed material from a zeolite column. Adsorbed anthocyanins / anthocyanidins can be eluted from dealuminated zeolites by eluting the zeolite preferably with organic solvents such as ethanol or more preferably with acidified ethanol.

The zeolite used is generally of the type corresponding to the composition [(A1, o,), (sio,),] aa: x and y heat and y / x> 1s, preferably> 1oo, rather> zoo and often According to the invention, hydrophobic (dealuminated) zeolites of the type indicated are preferably selected from the group consisting of silicalite, mordenite and zeolite Y.

Overall, however, zeolite Y is most often particularly preferred. _ In other respects, principles known per se apply to zeolites. To the basic zeolite structure, other atoms such as B, P, Fe, Ga, Ge can to some extent replace Al 'and Si to provide the basic zeolitic structure and can thus also be used in the process according to the invention.

Zeolites with a high proportion of silicon have highly hydrophobic properties and are stable in aqueous systems, within a wide pH range and are also insensitive to oxidizing and reducing agents. Furthermore, they withstand high pressures and high temperatures without changing. For the zeolite it can be used as such or in the form of sintered. zeolite crystals or in the form of crystals contained or suspended in non-zeolitic materials. It may also be deposited on or otherwise combined with one or more, preferably permeable, non-zeolitic materials. Of course, in the zeolite can not adsorb unlimited amounts of anthocyanins / anthocyanidins. The adsorption capacity of the zeolite can be restored by washing the zeolite alternately at low and high pH, preferably between pH 1 and pH 8.5. After washing, the zeolite is equilibrated at the desired pH to obtain adsorption of anthocyanins / anthocyanidins. The zeolites can withstand heating and if the adsorption capacity of the zeolite is lost due to clogging with organic material or microbial growth, the zeolites can be regenerated by heating. A preferred embodiment is that the zeolite is heated to a temperature exceeding 700 ° C, more preferably exceeding 850 ° C, and most preferably in the range 900-1100 ° C. If you also want to eliminate any risks of chemical contamination and microbial infections before starting a purification process, you can also carry out a corresponding heating, such as a pre-treatment of the zeolite in question. The high binding strength and the binding rate of anthocyanins / anthocyanidins to ultrastable zeolites means that in the contact between water and zeolite in a column or filter, a high water fl fate, ie short contact time; 4 which is preferably in the range of a few tens of seconds and up to 20-30 minutes or even less, such as 10 seconds I - 15 minutes or more preferably 20 seconds - 10 minutes.

The term anthocyanidins is used in connection with the invention as names of molecules with the following general structure and anthocyanins are used when a glycosylation has taken place in one of or in both positions 3 and 5. n, n, Anmocymiam HH Pelargonidine in Ol H - Cyanidine OIH Peonidine M ' 01 'or Del fi nidin O OI Petunidin M »OO Malvidin Mn - Mn Figure 1. Chemical structure for some different anthocyanidins where R, and R, are hydrogen g (H) and for anthocyanins where R, and R ,, are hydrogen ( H) or Carbohydrates (II 1 »D (S1 (II LTD (_, J Example 5) The present invention will now be further illustrated by the following concrete examples, which are intended solely to illustrate the invention and therefore may not be construed as limiting the same in other than that set out in the appended claims.

Example 1 Batch incubation of blueberry Va (Vaccinium myrtillus) and dealunxinated and aluminum-containing zeolite, respectively.

P * o 10 "" Dnlurinated zeolite Y 2_5 "" 'U_ Almniniuninixel lulluide zeolite Y 2.0 1.5 angd anthocyanins left in solution (mg / ml) 1.0 0.5 M 0.0 I' | I I l I 0 l 2 3 4 5 6 Number of batch incubations Figure 2. Blueberry mg was incubated on a tilting table with 20 mg / ml of dealuminem zeolite Y (O) or aluminum-containing zeolite Y (I) and after 15 minutes the samples were centrifuged. A new portion of the zeolites was added to the samples which were re-incubated 15 minutes before centrifugation. A total of 5 additions of zeolite were made.

In Example 2. Purification of anthocyanins / anthocyanidins from blueberries (Vaccinium myrtillus) by adsorption to dealuminated zeolite Y particles packed in a column.

ElutionBQH + 0J Ml-lCl 0.20 'ä ä' 0.15- g ö TI a f; == -5. ä 2 9.2 å- g 0.10 g EQ + ï s * å “'5' 5 4 å g å '“ å oos š š å i: m oooi,' 1 za 4 so 1 as 1o 11 Fraction Figure 3. A column packed with dealuminated zeolite Y particles that had been deaerated and washed with H 2 O. Juice predicted 1: 5 in H 2 O was applied to the column which was then washed first with H 2 O (fraction 2-3) and then with 50 mM NaCl (fraction 4-5). To elute adsorbed anthocyanins / anthocyanidins, 96% ethanol (fraction 6-7), ethanol with 0.1 M HCl (fraction 8-9) and finally ethanol with 0.4 M HCl (fraction 10-1 1) were pumped through the column. 10 ml fractions were pooled.

HPLC analysis of blueberry fi (Vaccinium myrtillus) and anthocyariins / anthliocyanidines eluted from a zeolite column. The analyzes were performed with an ODS Hypersil column (50 x 3.00 mm, 3 μm) with a gradient elution 0 - 2 minutes 100% mobile phase A and there for 2 - 11 minutes a gradient from 0 to 30% of mobile phase B. Mobile phase A : 1% phosphoric acid, 10% acetic acid in water. Mobile phase B: 100% acetonitrile. The flow rate * was 0.5 ml / minute and anthocyanins / anthliocyanidines were detected by absobance at 503 nm. Deglycosylation of anthocyanins occurs if these are incubated in 4 M HCl at 100 ° C. Anthocyanins eluting from the zeolite column with ethanol and 0.1 M HCl (Fig. 4G) are retarded on the HPLC column when deglycosylated (Fig. 4D) and the same deglycosylated HPLC profile is obtained from material eluted from the zeolite column with pure ethanol (Fig. 4B ). This shows that an fi action is eluted, i.e. separation of anthocyanidins and anthocyanins, can be obtained by eluting the zeolite column first with pure ethanol, which elutes the anthocyanidins, and then with acidified ethanol, which elutes the anthocyanins.

Flg. 4A. HPLC analysis of blueberry fi predicted 200 times.

Fig. 411. HPLC analysis of the ethanol eluate from a zeolite column Blueberry fi 'was applied to a zeolite column and after washing, the column is eluted with pure ethanol. The elute corresponds to fraction 6 in Example 2.

Fig. 4C. HPLC analysis of eluate with acidified ethanol from a zeolite column. Blueberry juice was applied to a zeolite column and after washing, the column is first eluted with pure ethanol and then with surgical ethanol. The elute corresponds to fraction 9 in Example 2. to Mt Flg. 4D. HPLC analysis of hydrolyzed material from Fig. 4C. Hydrolysis by incubating the surgical ethanol elution action for 60 minutes with 4 M I-ICL on a boiling water bath Example 4 - Table I. Purification of anthocyanins / anthocyanidins from blueberries (Vaccinium myrtillus) by adsorption to dealuminated zeolite Y particles packed in a column. - A column was packed with dealuninated zeolite Y particles that had been precipitated and washed with H 2 O. So diluted 1: 5 in H 2 O was applied to the column which was then washed first with H 2 O (fraction 2) and then with 50 mM NaCl (fraction 3). To elute adsorbed anthocyanins / anthocyanidins, 96% ethanol (fi-action 4-6), ethanol with 0.1 M HCl (fi-action 7-8) and finally ethanol with 0.4 M HCl (fraction 9-10) were pumped through the column. By visually following washing and elution, different large fraction volumes were collected. A total of 5.1 mg of anthocyanins with an antioxidant capacity corresponding to 584 nmol trolox is applied to the column. A total of 4.0 mg of anthocyanins with an antioxidant capacity corresponding to 473 nmol trolox is eluted from the column. Fraction Volume Total amount of anthocyanin Total antioxidant capacity (ml) (mg) (nmol Trolox) 1. Filtrate 8 0.026 6.7 2. Wash H 2 O 13. 0.273 _. 53 3. Wash NaCl 32 _ 0.486 x 631 4. Elution ethanol 1 0.128 9.1 ï s. Eme fi ng ethanol 10 0.190 _27 6. Elution ethanol 8.5 0.099 13 7. Emamg 7.5 0.309 ii “32 ethanol / 0, 1 M is HCl. Elution 15 in 2.916 387 ethanol / 0.1 M HCl 9. Elution 1.0 0.347 35 ethanol / 0.4 M HCl 10. Elution 4 0.026 VI 5.2 ethanol / 0.4 M HCl t TI 1 Q C- " I few <3 8 Example 5 a Purification of anthocyanins / anthocyanidins å- from blackberries, Rubus fi- utícosus, (Fig. 5A) and aronia, Aronia melanocarpa, (Fig. SB) by adsorption to dealuminated zeoht Y particles packed in a column . 0-12 _ Wash with water (å) f: 'få g. Ä 0.10- 5 2 + 5 E' å, ä 'ä m ._- æø q- Ü + HJ 2 U * _ 0.08 å., M - _, ma ~ ez 2 å 'f 2 -g _ 3. š “' .a: n å * ä '' ë s 9 w 2“ 'ä .É 004-' "a '“' å 'm ii "of” '* i.' I »ä eo: IE) g> _ _-.

J 'P 0.00' '1 2 3 4 5 6 1 a 9 10 11 Fraction Figure 5A. One column was packed with dealuminated zeolite Y particles which had been precipitated and washed with 50 mM phosphate buffered pH 6.4. Juice án- without corner bar, Rubus fi utzšcosus, 10 ml, was applied to the column which was then washed first with 5 mM phosphate buffer pH 6.4 (fraction 2-3) and there with -50 mM NaCl 5 mM phosphate buffer pH 6.4 (fraction 4) -5). To elute adsorbed anthocyanins / anthocyanidins, 96% ethanol (fractions 6-7), ethanol with 0.1 M HCl (fractions 8-9) and finally ethanol with 0.4 M were pumped.

V HCl (II-action 10-11) through the column. 10 ml fractions were pooled. 0 8_ å alumn; Ezornonmucl 'š å ä É ë å g' e ä '0.6- å .f e .e 6 5 v å .e 5 ä m I a: g ä å' z m ä ä.

. , In g 0.4 't:' g ä ä Q + +> »E e» 2 - ä ä ä få 'B' 3 -ä m fl- l .e »= gs š f» .- å 0.2- I ee '-' É É I - Oy MV 1 2 s 4 se 1 ae 10 11 Fraction Figure SB. One column was packed with dealuminated zeolite Y particles which had been deaerated and washed with the 50 mM phosphate buffer pH 6.4. The aronia, Aronia melanocarpa, 10 ml, was applied to the column which was then washed first with 5 mM phosphate buffer pH 6.4 (fraction 2-3) and then with 50 mM NaCl in 5 mM phosphate buffer pH 6.4 (fraction 4). -5). To elute adsorbed anthocyanins / anthocyanidins, 96% ethanol (fraction 6-7), ethanol with 0.1 M HCl (fraction 8-9) and finally ethanol with 0.4 M HC] (fraction 10-11) were pumped through the column. 10 ml fractions were collected. "C11 i n.) Å," I (T '| CD xjä! References. E? 1. Ames, B., Shigena, MK and Hagen, TM (1993) Oxidants, antioxidants and the degenerative diseases of aging Proc. Natl. Acad.Sci. USA, 90: 7915-7922 2. Magistretti, MJ. and Conti, M. Anthocyanidins for the treatment of ophthalmic diseases EP 90308197.4 3. Seghizzi, R., Gabetta, B. And Morazzoni, P. Oral pharmaceutical compositions containing anthocyanosides US 5,320,841 4. Viberg, Uno. (2000) Blålaär - the functional berry. Food technology No. 11 5. Goiffon, JP. Et. Al. (1999) Anthocyariic pigment determination in red fi uit ' juices, concentrated juices and syrups using liquid chromatography. Analytical Chimica Acta, 382 6. 'Breck, DW (1974) Zeolite Molecular Sieves, Wiley, New York. 7. Eriksson, H. and Blum, Z. Method of using zeolites for adsorbing detergents.United States Patent, Patent number 5, l08,617 Date of Patent Apr, 28, 1992 8. Larsson, K, Eriksson, H and Andersson, S. Use of a hydro fob zeolites ßr removal of preservatives used for drug solutions in a drug context from a protein solution. Swedish patent SE 9503731-3 9. Hjertmanß, Eriksson, H and Andersson, SA system for microbial control of a fl uia, Swedish patent application sE 0102265-6 10. Eriksson, H and Larsson, K. Zeolites for adsorption Swedish patent application SE 9802140-5 'i' 1 l. Eriksson, H. Purification of water PCT application SE 99/02281 I

Claims (10)

C71 1 ..) year: r / ~ PATENTKRAV lo Process for the purification of anthocyanins / anthocyariidines and intended for direct or indirect use for human consumption, in food production or for drug production, characterized in that an extract containing anthocyanins / anthocyanidins is brought into contact with such an amount of a hydrophobic zeolite that desired degree of adsorption of anthocyanins / anthocyanidins is obtained. A compound according to mv 1, characterized in that a hyaphophobic zeolite uses one having the composition [(Al 2 O 2), (SiO 2),], where x and y are integers and y / x> 15, preferably> 100. Process according to Claim 2, characterized in that y / x> 200, in particular> 1000. Process according to one of Claims 1 to 3, characterized in that the zeolite is selected from the group consisting of silicalite, mordenite and zeolite Y. Process according to Claim 4, characterized in that the zeolite consists essentially of zeolite Y. Process according to one of the preceding claims, characterized in that the zeolite is used in the form of sintered zeolite crystals or zeolite crystals contained or coated with or suspended in one or more non-zeolitic materials. Process according to one of the preceding claims, characterized in that the zeolite is attached to a surface or the zeolite is packed in a column or an alter or the zeolite is formed into a column or an alter and the extract is passed through it. (this). Process according to one of Claims 1 to 6, characterized in that the zeolite is added directly to the extract containing anthocyanins / anthocyariidines. Process according to one of the preceding claims, characterized in that the anthocyanins / anthocyanidins which have been adsorbed to the zeolite are eluted with ethanol and / or acidified ethanol. g and fl Process according to any one of the preceding claims, characterized in that dealurninated zeolites are used to separate anthocyanidins and anthocyanins for analytical or prepaxative purposes.