CA2006957C

CA2006957C - Recovery of plant extractives

Info

Publication number: CA2006957C
Application number: CA002006957A
Authority: CA
Inventors: Frank William Collins; David Paton
Original assignee: Agriculture and Agri-Food Canada
Current assignee: Agriculture and Agri-Food Canada
Priority date: 1989-12-29
Filing date: 1989-12-29
Publication date: 2000-02-22
Anticipated expiration: 2009-12-29
Also published as: CA2006957A1

Abstract

A process for extracting minor components of economic value from food processing wastes such as fruit and vegetable wastes is described. An ion-exchange process for recovering pigments and other minor constituents from an aqueous ethanol effluent of beet waste processing shows that neutral and cationic fractions and neutral and anionic fractions such as phenolics, glucose, and uronic acids are recovered.

Description

200695' Field of Invention This invention relates to.a process for the extraction of minor components from plant and other extractives. More specificall-y the process relates to the continuous extraction and recovery of such minor components as coloured pigments, organic acids, phenolics and ,phenols from plant materials which have already been at least partially processed. .
Backsiround of Invention Plant materials such as cereals, pulses, fruits and vegetables contain minor components-such as pigments, organic acids, phenolics such as condensed tannins and lower molecular weight phenols (as esters or ethers). Such substances may, under certain processing conditions, cause deleterious effects during processing to food products or additives, such as development of coloured extracts under acid or alkaline treatment, binding of phenolics to proteins, or darkening of colour of solid and liquid streams upon application of heat. In wine making or fruit juice production there may be excessive pigmentation and/or astringency. Certain plant residues such as fruit press pulps or beet pulps contain pigments which are of interest to the food industry as natural colouring agents. Various processing techniques for either removing or recovering the minor components are, of course, known. Such techniques generally either destructively remove them as in 2oos9s~
flocculation.processes or utilize extractive procedures with strong acids or alkalis which require subsequent neutralization resulting in high salt levels in the final recovered product. Absorption on charcoal is frequently employed but many components bind very strongly to charcoal so that drastic conditions are necessary to recover extractives therefrom.
Gel filtration may also be employed, as in U.S. Patent 3,9.68,097 issued 6 July 1986 and assigned to Produits Nestle. A, wherein a soluble protein product is recovered from soya milk passed through a gel filtration step.
Extracts produced at alkaline pH levels above pH 6.5 have a marked odour and taste and exhibit a tendency to form gels.
Extracts produced a~t said pH levels below pH3 have a pleasant taste and smell and precipitate irreversibly on neutralization. Here again the high salt levels in the product require further processing steps to remove.
Ob.iect of Invention An object of the present invention is to provide a relatively inexpensive ion-exchange process for the continuous removal of minor components from plant and other extractions including fermented liquids. The minor components can then be eluted from the ion-exchange gel with a dissimilar solvent and the column regenerated under mild conditions.

2 2oos9s~
Brief Statement of Invention Thus, by one aspect of this invention there is provided a process for recovering values from food processing wastes comprising:
(a) extracting said wastes with an aqueous ethanol solution and separating a~ liquid effluent therefrom;
(b) acidifying said liquid effluent;
(c) passing said acidified effluent through an ion exchange column;
(d) eluting said column with an aqueous ethanol solvent; and (e) recovering said values from said solvent.
Brief Description of Drawings Fig. 1 is a schematic flow chart illustrating the invention, applied to an anionic sample.
Detailed Description of Preferred Embodiments Value-added food products and pigments can be recovered from several types of fruit and vegetable processing wastes.
For simplicity the invention will be discussed herein with reference to beet processing wastes, using ion exchange chromatography in aqueous organic sohvents. The irigredients/products consist of an extracted, decolourized pectin-cellulose-starch-protein powder, and a sugar-syrup fraction and one or more relatively pure magenta, and/or

3 zoos9s~
yellow-orange and magenta colourant fractions. The colourants consist of two types of chromophore trivially ~Cnown as betacyanins (1) and betaxanthins (2) which are present in the tubers. Both pigments (collectively called batalains) are zwitterionic at physiological .pH's and contain both quaternary amine and carboxyl functions.
RO
r HO
H
Q~ C00' I
OOC . N ~ C00~ OOC ~ COO-N
(1) Betacyanins (magenta) (2) Betaxanthins (yellow-orange) , ...,.
._ R = f3-D-glucose, H, R' - glutamic acid, aspartic 13-D-glucose-6-sulfate, acid, glutamine, glucuroric acid, sophorose asparagine, proline ..

4 200695' In water, both types of pigment show iso-electric points (i.e. no net charge) around pH=2.0 while in mixed aqueous ethanol solvents this pI increases with increasing ethanol content. Batch ion-exchange experiments with Sephadex anion and cation exchange resins and 50% ethanol solvents at different pH's over the range pH 2 to 6.5 showed the following probable speciations.for example with betacyanins: ' - pI 50% ethanol - 4.5 betacyanins no net charge: not exchanged - at pH 3.0 betacyanin net charge +ve; strongly retained on SP-Sephadex C-25 - at pH 6.5 betacyanin net charge -ve; strongly retained on QAE-Sephadex A-25.
In aqueous solutions these pigments are relatively unstable especially in the presence of OZ and especially at pH values below 2 and above 9. However in aqueous ethanol they appear to be much more stable even in the presence of 02: Furthermore, since the iso-electric point is higher in aqueous ethanol than in water, less drastic pH conditions are required for retention and elution protocols resulting in higher recovery efficiency during isolation (extraction, ion-exchange, evaporation of solvents). The yields should therefore be higher when processing in aqueous ethanol than in water. A further advantage from the higher pI values in aqueous ethanol result from the fact that in these solvent s, 200695"
the pigments are, relatively stable both above and below their iso.-electric points enabling both anionic and cationic exchange processes to be utilized whereas in water alone, only anion exchange procedures can be used since the pigments are cationic only at pH's below 2 and undergo considerable degradation under these conditions.
Processing beet waste consists of blending, macerating, and/or chopping beet root ( Beta vul~xaris L ) pulp and waste material such as would be available from an industrial beet processing plant which washes, slices and cans/preserves whole beets. The beet waste tissues are mixed and blended at~room temperature in the presence of water and ethanol so as to give a ratio of solids to liquids ~of approximately 1 to 5. The proportions of water to ethanol can be varied from 30% to 80% to accommodate the initial water content of tFte beet waste without affecting the process. In the following examples 50% ethanol v/v was used. The beet puree is.filtered/pressed to produce a clear, deep red-purple liquid extract. Additional aqueous-ethanol can be added to the pressed cake and the extraction process repeated to recover additional material. The combined '50% ethanol extracts are considered in this process as the "waste effluent" the solids consisting primarily of cellulose, hemicellulose pectins, starchy polysaccharides and protein can be readily dried from ethanol: water to give an off-white (pale grey) powder useful as a filler/food ingredient rich in dietary fibre with high water regain capacity. , . , ' ..~

Example 1 Preparation of beet wastes Red beets were washed free of adhering dirt, the tops removed and the tubers manually dried to approximately 2 cm.
cubes. The beet tissue (100 gm fresh weight) was placed in a Waring blender along with 500 mls ethanol: water 150:50 v/v) and blended at high speed for approximately 2 minutes.
The mixture was then filtered by gravity through a coarse-porosity scintered glass filter, and the filtercake pressed to remove entrapped liquid. The cake was then re-suspended and re-extracted with a further 400 mls 50% ethanol and filtered as before to give a combined clear deep red filtrate (900 mls) and a grey filter cake. Th.e water regain capacity of the dried cake was 1 gm dry weight swells to 25.5 mls (2 hrs. in distilled H20). The betacyanin content of the filtrate (i.e. waste effluent) was determined proximately using spectrophotometry (~ax = 538nm) - waste effluent stream: 9.47 - 10 2 mg betanidin/ml effluent - Total present in 900 ml 85.2 mg betanidin equiv.
- pN waste effluent = 7Ø
To stabilize the pigments in the waste effluent, it was made acidic by addition of 4.5 mls glacial acetic acid (final acetic acid concentration 0.5% pH 5.5) and stored at -20°C until used.

"~".
Example 2 Recovery of Betalain Pigments using Agueous Ethanol ANION Exchange Protocols A portion 1450 ml) of the acidified waste effluent from Example 1) was passed through a column of QAE-Sephadex A-25 anion exchanger in the formate form, pre-equilibrated in 50%
ethanol (50 mls bed volume of gel). The waste effluent was fed onto 'the column by gravity flow at a flow rate of approximately 1 to 2 ml/min.
After all the waste effluent had been passed through the column, the column was washed with a further 2 bed volumes of 50% ethanol to displace the interstitial fluid.
The combined eluent and washings (approx. 500 mls, grey-brown in colour) hereafter referred to as tkie NEUTRAL AND
CATIONIC FRACTION was analyzed for betalain pigments spectrophotometrically and evaporated to a brown syru p by rotary evaporation at reduced pressure. The residue was then taken up in 50% isopropanol and examined by qualitative ttain-layer chromatographic procedures. The column was then eluted with the solvent ethanol: water: formic acid (70:20:10 v/v/v) to remove sequentially the betaxanthins and betacyanins. the first 5 bed volumes (250 mls) of eluate contained a mixture of betaxanthins (Structure 2). The next 4 bed volumes (200 mls) contained primarily the pigment betanidin-5-O-13-D-gl_ucoside ( Structure 1 ; R=glt.~cose ) . A
further 4 bed volumes remove the aglycone bet~~nidin (Structure 1 R---Ii). Finally, the last pigment, identified as prebetanin (Structure 1, R=glucose-6-sulfate) was recovered A a 200695' using 2 additional bed volumes of the solvent (100 mls).
The recovery/separation scheme is summarized in Figure 1.
Total recovered betacyanin by this process from all fractions amounted to '72.6% of the total betanidin equivalents originally present in the waste effluent. Using this elution profile the individual types of pigments can be isolated in relatively pure form in good yield on a continuous basis since no further recycling of the column is required before re-use.
Example 3 Recoverv of Betalain Pistments Using Aaueous Ethanol CATION Exchanste Protocols A portion (450 mls) of the acidified. waste effluent from Example 1 was adjusted to pH 3.5 by addition of formic acid (= 2 mls 98% foYrmic acid). The solution was passed through a column of SP Sephadex C-25 cation exchanger in the hydrogen ion form, pre-equilibrated in 50% ethanol (50 ml bed volume of gel). The solution was fed onto the column by gravity flow at a rate of approximately 1 to 2 ml/min.
After all the re-acidified waste effluent had been passed thxough the column, the column was washed with a further 2 bed volumes of the solvent ethanol: water: glacial acetic acid (50:45:5 v/v/v) to displace the remaining intersitial waste effluent in the column. The eluate and washings hereafter ,.
referred to as the NEUTRAL AND ANIONIC FRACTION was concentrated to a thick brownish yellow syrup resuspended and washed with 50 mls of 50% ethanol and re-evaporated by ' 9 2oos9s~
rotary evaporation, to give a syrup free of the last traces of volatile acids (i.e. formic and acetic). the syrup was taken up in 50% isopropanol and stored at -20°C until further analyzed. The betalain pigments were then collectively recovered from the column by elution with 2 bed volumes of the solvent ethano1:0.2 molar aqueous ammonium acetate (50:50 v/v). The betalain pigment fraction thus recovered in 100 mls was found to contain 82.5% of the total pigment originally present in the waste effluent. If desired the excess ammonium acetate can be removed by vacuum spray drying (ammonium acetate readily decomposes in vacuo to ammonia and acetic acid, both of which are volatile).
The cation exchanger, now in the NH4+ form is then recycled to the H+ form using ..a dilute formic acid solution in 50%
ethanol (e. g. 5%) and is ready for re-use.
a) Analysis of the NEUTRAL AND CATIONIC FRACTION- from Example 2 Qualitative TLC analyses revealed the presence of substantial quantities of sucrose lesser quantities of glucose and fructose. Amino acids included Glvcine, Histidine, Proline, Phenvlalanine, Tyrosine and a number of peptides. Phenolics included several flavonoid glycosides based on quercetin and kaempferol possibly chlorogenic acids (chlorogenic, iso-chlorogenic, neochlorogenic) and/or several coumarins.
-~ 10 200695' b) Analysis of the NEUTRAL AND ANIONIC FRACTION from Example Qualitative TLC analyses revealed the same sugar profile as above along with several uron.ic acids (Glucuronic, Galacturonic acids Mannuronic acid'~).~ Amino acids included Glycine, Glutamic, Aspartic acids, Proline, Tyrosine, Phenylalanine and several peptides. Phenolics detected included Caffeic acid, Ferulic acid, P-coumaric acid, possibly chlorogenic acids and/or coumarins along with several flavonoid glycosides as seen above.
S '

Claims

We Claim:

1. A process for recovering values comprising added food products and pigments from food processing wastes comprising:
(a) extracting said wastes with an aqueous ethanol solution and separating a liquid effluent therefrom;
(b) acidifying said liquid effluent with an aqueous ethanol-volatile acid solution so as to provide an acidified effluent containing at least 50% by volume ethanol and having a pH in the range 3.0 to 6.5:
(c) passing said acidified effluent through an ion exchange column comprising a macroporous cross linked polysaccharide gel;
(d) eluting said column with an aqueous ethanol solvent comprising at least 50% by volume ethanol and a volatile acid or an ammonium salt thereof; and (e) recovering said values from said eluting solvent.

2. A process as claimed in claim 1 wherein said ion exchange column is an anionic exchange column of the dextran type.

3. A process as claimed in claim 2 wherein said anionic exchange column is a column of QAE-Sephadex R A-25 anionic exchange gel in the formate form.

4. A process as claimed in claim 3 wherein said values are selected from sucrose, fructose, glycine, histidine, proline, phenylalanine, tyrosine, peptides, coumarins, flavonoid glycosides and chlorogenic acids.

5. A process as claimed in claim 1 wherein said column is additionally eluted with an ethanol-water-formic acid solvent so as to recover additional values.

6. A process as claimed in claim 5 wherein said additional values are selected from betaxanthins and betacyanins.

7. A process as claimed in claim 6 wherein said betacyanins comprise betanidin-5-glucoside, betanidin and prebetanin.

8. A process as claimed in claim 1 wherein said ion exchange column is a cationic exchange column of the dextran type.

9. A process as claimed in claim 8 wherein said cationic exchange column comprises SP Sephadex ~ C-25 cationic exchange gel in the hydrogen ion form.

10. A process as claimed in claim 9 wherein said aqueous ethanol solvent additionally contains acetic acid.

11. A process as claimed in claim 10 wherein said values comprise sucrose, fructose, uronic acids, glycine, glutamic acid, aspartic acid, proline, tyrosine, phenylalanine, peptides, caffeic acid, ferulic acid, p-coumaric acid, chlorogenic acids, coumarins and flavonoid glycosides.

12. A process as claimed in claim 9 wherein said column is additionally eluted with an ethanol-aqueous ammonium acetate solution.

13. A process as claimed in claim 12 wherein a betalain pigment fraction is recovered from said ethanol-aqueous ammonium acetate solution.