Note: Descriptions are shown in the official language in which they were submitted.
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
1
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. ,
6
. , ' ..~
CA 02006957 1999-02-16
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
3
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 '
11
