U.S. patent application number 10/759294 was filed with the patent office on 2004-07-29 for production of polygalacturonides and their use in food additives.
This patent application is currently assigned to Technische Universitat Berlin. Invention is credited to Dornenburg, Heike, Lang, Christine.
Application Number | 20040146635 10/759294 |
Document ID | / |
Family ID | 7639102 |
Filed Date | 2004-07-29 |
United States Patent
Application |
20040146635 |
Kind Code |
A1 |
Lang, Christine ; et
al. |
July 29, 2004 |
Production of polygalacturonides and their use in food
additives
Abstract
The invention relates to the use of polygalacturonides as food
additives, said polygalacturonides being obtainable via the
following process steps: a) a pectinous plant material is subjected
to a pectin extraction in aqueous solution; b) the solids are
removed from the suspension obtained in step a), consisting of
liquid phase including dissolved pectin and solids from the plant
material; c) the pectin is precipitated from the liquid phase
obtained in step b); d) the pectin obtained in step c) is dissolved
in an aqueous solution and cleaved with purified
endo-polygalacturonase; e) the polygalacturonides obtained in step
d) are processed into a polygalacturonide preparation without using
an additional separation step and without hydrolyzing ester groups
that are present.
Inventors: |
Lang, Christine; (Berlin,
DE) ; Dornenburg, Heike; (Berlin, DE) |
Correspondence
Address: |
ROTHWELL, FIGG, ERNST & MANBECK, P.C.
1425 K STREET, N.W.
SUITE 800
WASHINGTON
DC
20005
US
|
Assignee: |
Technische Universitat
Berlin
Berlin
DE
10623
|
Family ID: |
7639102 |
Appl. No.: |
10/759294 |
Filed: |
January 20, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10759294 |
Jan 20, 2004 |
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10009055 |
Feb 25, 2002 |
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6696554 |
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10009055 |
Feb 25, 2002 |
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PCT/EP01/03998 |
Apr 6, 2001 |
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Current U.S.
Class: |
426/658 ;
536/2 |
Current CPC
Class: |
A23L 33/21 20160801;
A23L 29/231 20160801; C12Y 302/01015 20130101; A61K 31/70 20130101;
C08B 37/0045 20130101 |
Class at
Publication: |
426/658 ;
536/002 |
International
Class: |
C08B 037/06; A23G
003/00 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 6, 2000 |
DE |
100 19 076.6 |
Claims
1. Use of polygalacturonides as food additives, said
polygalacturonides being obtainable via the following process
steps: a) a pectinous plant material is subjected to a pectin
extraction in aqueous solution; b) the solids are removed from the
suspension obtained in step a), consisting of liquid phase
including dissolved pectin and solids from the plant material; c)
the pectin is precipitated from the liquid phase obtained in step
b); d) the pectin obtained in step c) is dissolved in an aqueous
solution and cleaved with purified endo-polygalacturonase; e) the
polygalacturonides obtained in step d) are processed into a
polygalacturonide preparation without using an additional
separation step and without hydrolyzing ester groups that are
present.
2. The use according to claim 1, wherein the pectin is precipitated
by adding C.sub.1-C.sub.10 alkylalcohols and/or inorganic
salts.
3. The use according to claim 1 or 2, wherein a pH of from 1.4 to
8.2, preferably from 3.5 to 5.0 is adjusted in step d).
4. The use according to any of claims 1 to. 3, wherein the
endo-polygalacturonase is purified using the gel filtration
technology.
5. The use according to any of claims 1 to 4, wherein from 10 to
1000, preferably from 20 to 400 U/g pectin of polygalacturonase is
used in step d).
6. The use according to any of claims 1 to 5, wherein the
polygalacturonase is immobilized.
7. The use according to any of claims 1 to 6, wherein the reaction
with polygalacturonase in step d) is performed at 4 to 80.degree.
C., preferably 30 to 70.degree. C., for 2 to 300 min, preferably 45
to 150 min.
Description
[0001] The invention relates to polygalacturonides and to the use
thereof. Polygalacturonides are oligosaccharides obtained e.g. by
enzymatic decomposition of pectins. Pectin has galacturonic acid
monomers or galacturonic acid (methyl)ester monomers as major
components. Typically, both monomers are present at the same time,
the esterification level of the galacturonic acid groups ranging
from 0.5% to 70%. The galacturonic acid monomers or ester monomers
are .alpha.-1,4-linked to each other. In particular regions of the
pectin molecule, however, rhamnose monomers are inserted in the
chain, resulting in a zigzag structure of the polymer. The rhamnose
monomers may have side chains attached thereto which may be formed
of arabans (.alpha.-1,5 linkage) and arabinogalactans (linkage:
.beta.-1,3-1,6-D). The side chains may also include other sugar
monomers. As a supplement, reference is made to the citation
"Flussiges Obst" 6/97, pp. 301. This citation also describes that
the use of pectinases in juice production for clarification
purposes gives rise to undesirable colloids which interfere with
further processing and must be removed or prevented. In natural
unfiltrated juices, on the other hand, it is only the pectins
rather than the fragments thereof which have an advantageous
influence on the viscosity. It is clear from these considerations
that in juice production, for example, the presence of pectin
fragments is undesirable for technical reasons.
[0002] In addition to technical aspects, pectins also involve
physiological aspects. The citation Cerda, J. J., Trans. Am. Clin.
Climatol. Assoc. 99, 203-213 (1987), describes that pectin from
grapefruits plays an important role in promoting health in
consumers. The citation Matsumoto, T. et al, Int. J.
Immunopharmacol. 15, 683-693 (1993), describes that particular
fragments of Bupleuran 2IIc, a pectin-like polysaccharide from the
roots of Bupleurum falcatum, might be highly important in
pharmaceutical terms. The fragments are obtained by reaction with
endo-polygalacturonase. Ultimately, the citation Voragen, A. G. J.,
Trends Food Sci. Technol. 9, 328-335 (1998), provides information
that non-digestible polysaccharides or oligosaccharides may have a
number of health-promoting effects on persons consuming same.
[0003] Producing oligosaccharides for pharmaceutical purposes from
pectins using pectinases is known from the citation U.S. Pat. No.
5,683,991. The pectinases used therein are mixtures of various
enzymes which possibly also include endo-polygalacturonase, so that
cleavage also is effected in the side chain regions. Moreover, the
ester groups are hydrolyzed prior to reacting with the pectinases
so that, as a result, comparatively-small oligosaccharides (2-4
monomers) are obtained.
[0004] Additives in the food industry are to fulfill a variety of
functions. Essential functions are in the sector of preparing
processed foodstuffs, e.g. with respect to consistency, durability
and color appearance.
[0005] The invention is based on the technical problem of providing
an additive for foods which would improve the foods in a
health-promoting respect, with respect to taste, and optionally
with respect to consistency and/or other consumer-related
properties.
[0006] To solve said technical problem, the invention teaches the
use of polygalacturonides as additives in foods, said
polygalacturonides being obtainable via the following process
steps:
[0007] a) a pectinous plant material is subjected to a pectin
extraction in aqueous solution;
[0008] b) the solids are removed from the suspension obtained in
step a), consisting of liquid phase including dissolved pectin and
solids from the plant material;
[0009] c) the pectin is precipitated from the liquid phase obtained
in step b);
[0010] d) the pectin obtained in step c) is dissolved in an aqueous
solution and cleaved with purified endo-polygalacturonase;
[0011] e) the polygalacturonides obtained in step d) are processed
into a polygalacturonide preparation without using an additional
separation step and without hydrolyzing ester groups that are
present.
[0012] Within the range of the invention, it is essential that no
hydrolysis of ester groups takes place and that, owing to the use
of purified endo-polygalacturonase, only bonds in (naturally)
non-esterified galacturonic acid monomer units undergo cleavage. As
a result, a higher amount of comparatively large oligosaccharides,
e.g. with 5-20 monomer units (main and optionally side chains), is
obtained. Here, virtually all of the polygalacturonides are
saturated polygalacturonides, they are less reactive compared to
the unsaturated polygalacturonides formed by pectin lyase or
pectate lyase activity and therefore do not contribute to
non-enzymatic browning and formation of hydroxymethylfurfural (HMF)
from fructose or glucose. The mixture of various oligosaccharide
structures thus obtained has distinct advantages.
[0013] On the one hand, fragments with largely retained side chains
have a supporting effect on the immune system in physiological
terms. In addition, the comparatively long oligosaccharides of the
invention assume a roughage effect, as is the case with other
long-chain polymers as well. Roughage is important in the
prophylaxis and therapy of a number of diseases such as
constipation, diverticulosis, colon carcinoma, diabetes mellitus,
and lipid metabolic diseases. However, the drawbacks of
conventional roughage, namely, binding of essential nutrients, will
be reduced when using oligosaccharides of the invention ranging
from 5 to 20 monomer units. Moreover, by virtue of the intestinal
bacterial flora as found in organisms, the polygalacturonides
according to the invention are converted relatively readily to
short-chain fatty acids such as acetate, butyrate and propionate
which in turn have a positive effect on the intestinal flora and on
the intestinal pH value. These fatty acids can be utilized
energetically by the organism (energy content of the basic
oligosaccharides: about 2 kcal/g), serving particularly mucosa
cells as energy supplier, with consequent modulation of
proliferation. The colon motility is activated and thus, the mucosa
blood circulation as well. In addition, the polygalacturonides used
according to the invention have an antibacterial and
emulsion-stabilizing effect, which is particularly advantageous in
food-technological terms. This also enables the use as fat
substitutes, e.g. in mayonnaises and the like.
[0014] The use according to the invention can be effected with a
wide variety of processed foods, such as ready meals, baby food,
canned food (including food filled in glassware), such as canned
vegetables and canned fruits, beverages candy and pastry (including
chips and the like). In addition to plants themselves, vegetable
extraction residues, e.g. from juice production, as well as
vegetable cell cultures are possible as pectinous plant material.
Plants suitable for use as pectin source can be exemplified as
follows:
[0015] Any type of fruits, particularly apples and citrus fruits,
vegetables, particularly sugar beets, carrots and tomatoes. The
pectin extraction can be effected in the neutral (pH 6.0-8.0) or
acidic range (pH 2.0-3.0; acid: e.g. sulfuric acid, hydrochloric
acid, phosphoric acid, citric acid, lactic acid, and/or tartaric
acid). Typical process conditions are as follows:
[0016] 40-120.degree. C., preferably 90-100.degree. C., 1-20 hours,
preferably 6-10 hours, optionally multiple repetitions, e.g.
twice.
[0017] The solids can be removed by (e.g. hydraulic) pressing
and/or centrifugation. Prior to further processing, the resulting
solution can be concentrated using vacuum evaporation and/or
ultrafiltration.
[0018] The pectin precipitation can be effected in a variety of
ways. On the one hand, water-miscible organic (non-ionic) solvents
can be used, removing the hydrate cage from the pectin, so as to
cause precipitation. For example, acetone and C.sub.1-C.sub.10
alkylalcohols are possible, with ethanol being preferred from a
food-technological view. Alternatively or additionally, inorganic
salts such as sulfates and/or phosphates of aluminum, copper and/or
calcium can be employed.
[0019] Preferably, step d) is performed using a pH of from 1.4 to
8.2, more preferably from 3.5 to 5.0. The endo-polygalacturonase
which is used can be recovered from plants or from microorganisms
occurring therein, e.g. cotton/Aspergillus flavus/Aspergillus
parasiticus, rye/Claviceps purpurea, maize/Cochliobolus
carbonum/Fusarium moniliforme, American walnut/Cryphonectria
parasitica, tomato/Fusarium oxysporum/Ralstonia (Pseudomonas)
solanacearum, rice/Rhizoctonia solani, grass/Sclerotinia borealis,
sunflower/Sclerotinia sclerotiorum, and apple/Stereum purpureum,
carrot/Erwinia carotorova or Burkholderia (Pseudomonas) cepacia, or
from microorganisms genetically engineered so as to produce a
well-defined endo-polygalacturonase. The latter is preferred for
its comparatively easy purification. In addition to well-known
genes of the above-exemplified microorganisms, well-known cDNAs or
cDNAs from plants themselves which encode well-known enzyme
structures are possible for recombinant operations. Examples of
well-known plant cDNAs are those from Arabidopsis thaliana, Persea
americanus and Prunus persica. Examples of well-known
endo-polygalacturonases of plants are those from Lycopersicon
esculentum, Musa acuminata, Gossypium barbadense, Gossypium
hirsutum, Cucumus sativus, Phaseolus vulgaris, Citrus limon,
Mangifers indica, Cucumis melo, Passiflora edulis, Prunus persica,
Pyrus communis, Rubus idaeus, and Fragaria ananassa. It is
preferred to use one or more endo-polygalacturonases selected from
the endo-polygalacturonases which can be obtained from organisms of
the group consisting of Aspergillus carbonarius, Aspergillus niger,
Aspergillus oryzae, Aspergillus tubingensis, Aspergillus ustus,
Kluyveromyces marxianus, Neurospora crassa, Penicillium
frequentans, and Saccharomyces cerevisiae SCPP, Lactobacillus
casei, Lactobacillus plantarum, Lactococcus lactis, Bacteroides
thetaiotaomicron, Piromonas communis, Neocalimastix patriciarum, or
from other microorganisms modified with DNA sequences of the above
organisms which encode the endo-polygalacturonases. The
purification can be effected using e.g. the gel filtration
technology which is well-known to those skilled in the art. The
amount of polygalacturonase employed in step d) advantageously
ranges from 10 to 1000, preferably from 20 to 400 U/g pectin.
Determination of the activity is effected using the following
method:
[0020] 10 mg/l polygalacturonic acid (e.g. Sigma P 7276, according
to the product specifications valid on Mar. 1, 2000) is dissolved
in substrate buffer (2 mM citric acid solution, 1 mM CaCl.sub.2)
and added with a defined quantity of enzyme solution. Incubation is
effected for 15 min at 25.degree. C. The reaction then is stopped
by adding an equal volume of 4.4 mM 2-hydroxy-3,5-dinitrobenzoic
acid solution, boiling for 5 min, and cooling to 0.degree. C.
Eventually, the absorption at 540 nm is detected, and the
conversion is determined from these values against a standard curve
in the usual manner. One unit is the conversion of 1 .mu.M of
galacturonic acid per minute.
[0021] In a particularly preferred embodiment of the invention, the
polygalacturonase is immobilized by inclusion in enzyme membrane
reactors, e.g. flat membrane or hollow fiber membrane reactors, or
binding to a conventionally prepared support material can be
effected in an adsorptive, ionic, chelating, covalent way, or by
crosslinking.
[0022] The immobilization allows to make sure that the final
product is virtually free of polygalacturonase and, in fact,
without an extra separation. In this way, interfering reactions due
to otherwise possible entraining of the employed polygalacturonase
with pectins possibly included in the food is virtually excluded.
In particular, this is advantageous because undesirable
decomposition of pectins included in the foods might have an
unfavorable effect on the consistency of the foods, e.g. the
viscosity thereof. Moreover, the polygalacturonase consumption is
comparatively low.
[0023] The reaction with polygalacturonase in step d) preferably is
effected at 4 to 80.degree. C., more preferably 30 to 70.degree.
C., for 2 to 300 min, preferably 45 to 150 min. The operations can
be performed in a continuous or discontinuous (batchwise) fashion.
As to the continuous process, the above-mentioned time period
relates to the average residence time in a reaction volume
containing polygalacturonase.
[0024] The polygalacturonides according to the invention are
employed at concentrations of from 0.01 to 1.0 g/kg food,
preferably from 0.1 to 0.5 g/kg food. The concentration should be
adjusted with regard to normal human food consumption so as to
maintain doses of from 0.5 to 50 mg, preferably from 1 to 25 mg per
kg body weight per day.
[0025] With reference to the examples which merely represent
embodiments, the invention will be illustrated in more detail
below.
EXAMPLE 1
[0026] Isolation and Purification of a Polygalacturonase from
Tomatoes.
[0027] Tomatoes, 1 kg, are homogenized in 1 liter (1) of water, and
the suspension obtained is adjusted to pH 3.0.
[0028] The solids (cell residues) are removed by centrifugation
(10,000 g, 20 min) and washed in water. The pellets are taken up in
50 mM sodium acetate/1.25 mM NaCl (pH 6.0) at 4.degree. C. and
stirred at 4.degree. C. for 1 hour. Proteins are precipitated by
means of 70% ammonium sulfate saturation and removed by
centrifuging (10,000 g, 20 min). The protein pellet is dissolved in
0.125 M sodium acetate (pH 6.0) and dialyzed against said buffer.
The proteins then are separated on a CM Sepharose column in a
2-stage gradient (0.45 M sodium acetate, pH 6.0, and 1.0 M sodium
acetate, pH 6.0). The endo-polygalacturonase elutes with the first
stage.
EXAMPLE 2
[0029] Preparation and Purification of a Polygalacturonase from
Genetically Engineered Microorganisms.
[0030] Yeast (Saccharomyces cerevisiae) is transformed using an
expression plasmid including cDNA of the Aspergillus niger
endo-polygalacturonase gene under yeast ADH1 promoter control.
[0031] The plasmid still includes the yeast replication origin and
yeast selection marker (e.g. LEU2 gene). The yeast strain obtained
is pre-grown in nutrient medium (minimal medium) and cultured in a
fermentation process wherein the endo-polygalacturonase is
discharged into the nutrient medium.
[0032] The yeast cells are harvested from 500 ml of medium
(centrifuging: 6000 g, 10 min).
[0033] The clear medium supernatant is transferred on a
carboxymethylcellulose cation exchange column and equilibrated with
10 mM sodium acetate (pH 4.0). The proteins are eluted using a
linear gradient of from 1 to 1.5 M NaCl in 10 mM sodium acetate (pH
4.0). Fractions including pure endo-polygalacturonase elute between
0.6 and 0.75 M NaCl.
EXAMPLE 3
[0034] Isolation and Purification of a Polygalacturonase from a
Commercially Available Pectinase
[0035] 50 g of a commercially available enzyme mixture is dissolved
in 200 ml of 0.02 M sodium acetate (pH 3.6) with stirring for 3
hours. Solid components are removed by centrifuging (25,000 g, 0.10
min). The solution then is desalted using a Sephadex g 50 column.
The proteins are transferred on an alginate column (matrix by
crosslinking alginate with epichlorohydrin) and equilibrated with
0.02 M sodium acetate (pH 3.6). Elution is effected using 0.1 M
sodium acetate, pH 4.2, then pH 5.6, and subsequently using a
linear NaCl gradient of from 0 to 1 M in acetate buffer, pH
5.6.
[0036] The endo-polygalacturonase is eluted in the salt
gradient.
EXAMPLE 4
[0037] Immobilization of a Polygalacturonase
[0038] 3 g of a particulate carrier, e.g. silicate or glass carrier
derivatized with surface-bound NH2 groups (Solvay Enzymes,
Hanover), is suspended in 20 ml of 0.01 M S.o slashed.rensen
phosphate buffer (pH 7.0) and degassed. 0.2 g, i.e. 1000 U of
endo-polygalacturonase from Example 3 is dissolved in 5 ml buffer,
pH 7.0, and added to the carrier suspension. With stirring, the
decline of the extinction at 280 nm is monitored for about 30 to 60
min. The solution is removed, followed by washing 5 times with
water, and coupling is effected by adding 10 ml of glutaric
dialdehyde solution (5%) and stirring for 30 min. Thereafter, this
is washed 5 times in water, 1 hour, followed by washing 3 times
with buffer (pH 5.0). Finally, this is suspended in 50 ml of
buffer, pH 5.0, to obtain covalently immobilized
endo-polygalacturonase.
EXAMPLE 5
[0039] Preparation of Oligogalacturonides from Bupleurum
falcatum
[0040] Washed roots of Bupleurum falcatum, 1 kg, are chopped
mechanically and subjected to an extraction process step using 30
kg of deionized water. The extraction is performed twice for 8
hours at 98.degree. C. and atmospheric pressure. Following cooling
below 60.degree. C., the suspension obtained is centrifuged (4000 g
for 10 min.) to remove solids. The clear supernatant obtained is
concentrated to 1/10 of the original volume using vacuum
evaporation.
[0041] The pectin is precipitated by adding 4 parts by weight of
ethanol (96%) per part by weight of solution and separated from the
liquid phase by centrifuging (10,000 g for 10 min) at 4.degree. C.,
optionally with subsequent dialysis. The pectin obtained then is
dissolved in a 0.1 M sodium acetate buffer (pH 4.2) and reacted
with 300 U/g pectin employed of endo-polygalacturonase (from
Aspergillus japonicus, available from the Sigma Company under the
product designation P 3304, according to the product specifications
valid on Mar. 1, 2000) for 4 hours at 370.degree. C., said reaction
being done batchwise. The polygalacturonides obtained are recovered
in solid form by evaporating the water.
Example 6
[0042] Preparation of Galacturonides from Beet Pressed Chips
[0043] Beet pressed chips, 1 kg, from the beet-processing industry
are extracted in 15 kg of an acidic aqueous solution (phosphoric
acid, pH 1.5) for 1 hour at 90.degree. C. Following cooling to
2.degree. C., the solid phase is removed by hydraulic pressing and
optional filtration. Using vacuum evaporation, this is concentrated
to {fraction (1/10)} the volume of the liquid phase. The pectin is
precipitated by adding 2 parts by weight of isopropanol per part by
weight of concentrate. Following centrifugation (10,000 g, 10 min)
and drying (70.degree. C., 1 hour), the enzymatic hydrolysis is
accomplished by adding an aqueous solution of 5 g/kg water
(adjusted to pH 4.0) of pectin extract with 20 U/g pectin of
endo-polygalacturonase from Example 1 and performing the reaction
for 60 min at 60.degree. C. The polygalacturonides obtained are
recovered in solid form by evaporating the water.
EXAMPLE 7
[0044] Production of Baby Food Containing Polygalacturonides
[0045] A ready-to-eat baby food processed as a mash is mixed
homogeneously with 0.1 g/kg mash of polygalacturonides from Example
6, and the mash then is packaged in a ready-for-sale fashion. The
mash proves to be emulsion-stabilized.
EXAMPLE 8
[0046] Production of a Soft Drink Containing Polygalacturonides
[0047] A tea is prepared from green tea leaves in the usual manner.
Following cooling, 1 g/kg beverage of polygalacturonides from
Example 5 are added to the tea to form a solution.
[0048] The ready-to-bottle product thus obtained has an agreeably
sour taste.
EXAMPLE 9
[0049] Immobilization by Crosslinking
[0050] 500 mg of endo-polygalacturonase is dissolved in 15 ml of
distilled water. With stirring in an ice bath, 30 ml of ice-cold
acetone is slowly added, followed by addition of 2 ml of a 25%
glutaric dialdehyde solution. Thereafter, this is agitated for 60
min at 30.degree. C. and subsequently centrifuged. The supernatant
is discarded, and the residue is stirred up with 40 ml of distilled
water and homogenized using an Ultra Turrax. Following
centrifugation and discarding of the supernatant, the residue is
washed once more with 40 ml of distilled water. The crosslinked
preparation obtained is suspended to make 100 ml.
EXAMPLE 10
[0051] Fractionation of Polygalacturonides
[0052] 1.5 g of the polygalacturonides from Example 6 are dissolved
in 15 ml of distilled water. The polygalacturonides are separated
using chromatography. The solution is transferred on an anion
exchange column (2.5/40 cm, BioRad AGMP 1) 150 ml in volume,
equilibrated with eluent (0.2 M sodium formate buffer, pH 4.7). The
polygalacturonides are eluted using a linear gradient between 0.2
and 0.7 M sodium formate buffer (pH 4.7).
[0053] The composition of the individual fractions is subsequently
analyzed using thin layer chromatography (TLC). The fractions are
transferred on the stationary phase, Silicagel 60 (Merck); a
mixture of one part ethanol and one part acetate (25 mM) is used as
mobile phase. The development is performed at 35.degree. C. The
individual polygalacturonides are made visible by spraying with a
reagent (200 mg of naphthalene-1,3-diol in 50 ml of methanol and 50
ml of 20% (g/g) sulfuric acid). Fractions of equal composition are
pooled, and the polygalacturonides contained in these fractions are
precipitated with double the volume of acetone and separated from
the acetone after centrifugation (6000 g, 10 min) to obtain
polygalacturonides of well-defined size.
* * * * *