Process for enzymatically modifying pectin

Details Process for enzymatically modifying pectin Process for enzymatically modifying pectin

2001-07-16

2003-09-30

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Process of utilizing an enzyme or micro-organism to destroy...

Treating animal or plant material or micro-organism

C435S041000, C435S183000, C435S197000, C435S072000, C435S101000, C435S243000, C435S252100, C435S252300, C435S254100

Reexamination Certificate

active

06627429

ABSTRACT:

The present invention relates to a process.
In particular, the present invention relates to a process which comprises the use of an enzyme.
More in particular, the present invention relates to a process for enzymatically modifying pectin.
Pectin is a structural polysaccharide commonly found in the form of protopectin in plant cell walls. The backbone of pectin comprises linked (1→4)-&agr;-D-galacturonic acid residues which are interrupted with a small number of 1→2 linked &agr;-L-rhamnose units.
In addition, pectin comprises highly branched regions with an almost alternating rhatnno-galacturonan chain. These highly branched regions also contain other sugar units (such as D-galactose, L-arabinose and xylose) attached by glycosidic linkages to the C3 or C4 atoms of the rhamnose units or the C2 or C3 atoms of the galacturonic acid units. . The long chains of -(1→4)-&agr;linked galacturonic acid residues are commonly referred to as “smooth” regions, whereas the highly branched regions are commonly referred to as the “hairy regions”.
Some of the carboxyl groups of the galacturonic residues are esterified (e.g. the carboxyl groups are metbylated). By way of example some galacturonic acid residues are esterified with methanol. Typically esterification of the carboxyl groups occurs after polymerisation of the galacturonic acid residues. However, it is extremely rare for all of the carboxyl groups to be esterified (e.g. methylated).
Usually, the degree of esterification will vary from 0-90%. If 50% or more of the carboxyl groups are esterified then the resultant pectin is referred to as a “high ester pectin” (“HE pectin” for short) or a “high methoxyl pectin”. If less than 50% of the carboxyl groups are esterified then the resultant pectin is referred to as a “low ester pectin” (“LE pectin” for short) or a “low methoxyl pectin”. If 50% of the carboxyl groups are esterified then the resultant pectin is referred to as a “medium ester pectin” (“ME pectin” for short) or a “medium methoxyl pectin”. If the pectin does not contain any—or only a few—esterified groups it is usually referred to as pectic acid.
The structure of the pectin, in particular the degree of esterification (e.g. methylation), dictates many of the resultant physical and/or chemical properties of the pectin. For example, pectin gelation depends.on the chemical structure of the pectin, especially the degree of esterification. In addition, however, pectin gelation also depends on the soluble-solids content, the pH and calcium ion concentration. With respect to the latter, it is known that the calcium ions form complexes with free carboxyl groups, particularly those on a LE pectin.
Pectic enzymes are classified according to their mode of attack on the galacturonan part of the pectin molecule. A review of some pectic enzymes has been prepared by Pilnik and Voragen (Food Enzymology, Ed.: P. F. Fox; Elsevier; (1991); pp: 303-337). In particular, pectin methylesterases (EC 3.1.1.11), otherwise referred to as PMEs, de-esterify HE pectins to LE pectins or pectic acids. In contrast, and by way of example, pectin depolymerases split the glycosidic linkages between galacturonosyl methylester residues.
In more detail, PME activity produces free carboxyl groups and free methanol. The increase in free carboxyl groups can be easily monitored by automatic titration. In this regard, earlier studies have shown that some PMEs de-esterify pectins in a random manner, in the sense that they de-esterify any of the esterified (e.g. methylated) galacturonic acid residues on one or more than one of the pectin chains. Examples of PMEs that randomly de-esterify pectins may be obtained from fungal sources such as
Aspergillus aculeatus
(see WO 94/25575) and
Aspergillus japonicus
(Ishii et al 1980 J Food Sci 44 pp 611-14). Baron et al (1980 Lebensm. Wiss. &mgr;-Technol. 13pp 330-333) apparently have isolated a fungal PME from
Aspergillus niger
. This fungal PME is reported to have a molecular weight of 39000 D, an isoelectric point of 3.9, an optimum pH of 4.5 and a K
m
value (mg/ml) of 3.
In contrast, some PMEs are known to de-esterify pectins in a block-wise manner, in the sense that it is believed they attack pectins either at non-reducing ends or next to free carboxyl groups and then proceed along the pectin molecules by a single-chain mechanism, thereby creating blocks of un-esterified galacturonic acid units which can be calcium sensitive. Examples of such enzymes that block-wise enzymatically de-esterify pectin are plant PMEs. Up to 12 isoforms of PME have been suggested to exist in citrus (Pilnik W. and Voragen A. G. J. (Food Enzymology (Ed.: P. F. Fox); Elsevier; (1991); pp: 303-337). These isoforms have different properties.
Random or blockwise distribution of free carboxyl groups can be distinguished by high performance ion exchange chromatography (Schols et al Food Hydrocolloids 1989 6 pp 115-121). These tests are often used to check for undesirable, residual PME activity, in citrus juices after pasteurisation because residual PME can cause, what is called, “cloud loss” in orange juice in addition to a build up of methanol in the juice.
PME substrates, such as pectins obtained from natural plant sources, are generally in the form of a high ester pectin having a DE of about 70%. Sugar must be added to extracts containing these high ester PME substrates to provide sufficient soluble solids to induce gelling. Usually a minimum of 55% soluble solids is required.
Syneresis may occur. By way of example, syneresis in marmalades and jams with low soluble solid content (<55%) may occur when using HE-pectin. However, HE-pectin is not usually used in such applications. If pectins are to be used, then typically amidated pectins or LE-pectin are used, such as for jams with <55% SS.
When syneresis does occur, expensive additives must be used to induce gelline.
Versteeg et al (J Food Sci 45 (1980) pp 969-971) apparently have isolated a PME from orange. This plant PME is reported to occur in multiple isoforms of differing properties. Isoform I has a molecular weight of 36000 D, an isoelectric point of 10.0, an optimum pH of 7.6 and a K
m
value (mg/ml) of 0.083. Isoform II has a molecular weight of 36200 D. an isoelectric point of 11.0, an optimum pH of 8.8 and a K
m
value (mg/ml) of 0.0046. Isoform III (HMW-PE) has a molecular weight of 54000 D, an isoelectric point of 10.2, an optimum pH of 8 and a k
m
value (mg/ml) of 0.041. However, to date there has been very limited sequence data for such PMEs.
According to Pilnik and Voragen (ibid), PMEs may be found in a number of other higher plants, such as apple, apricot, avocado, banana, berries, lime, grapefruit, mandarin, cherries, currants, grapes, mango, papaya, passion fruit, peach, pear, plums, beans, carrots, cauliflower, cucumber, leek, onions, pea, potato, radish and tomato. However, likewise, so far there has been very limited sequence data for such PMEs.
A plant PME has been reported in WO-A-97/03574 (the contents of which are incorporated herein by reference). This PME has the following characteristics: a molecular weight of from about 36 kD to about 64 kD; a pH optimum of pH 7-8 when measured with 0.5% lime pectin in 0.15 M NaCl; a temperature optimum of at least 50° C.; a temperature stability in the range of from 10°—at least 40° C.; a k
m
value of 0.07%; an activity maximum at levels of about 0.25 M NaCl; an activity maximum at levels of about 0.2 M Na
2
SO
4
; and an activity maximum at levels of about 0.3 M NaNO
3
.
Another PME has been reported in WO 97/31102 (the contents of which are incorporated herein by reference).
PMEs have important uses in industry. For example, they can be used in or as sequestering agents for calcium ions. In this regard, and according to Pilnik and Voragen (ibid), cattle feed can be prepared by adding a slurry of calcium hydroxide to citrus peels after juice extraction. After the addition, the high pH and the calcium ions activate any native PME in the peel causing rapid de-esterification of the pectin and calcium pectate coagulation oc

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