WO2000024921A1

WO2000024921A1 - Pectins and the preparation thereof

Info

Publication number: WO2000024921A1
Application number: PCT/GB1999/003429
Authority: WO
Inventors: Nigel Eric Banister; John Thomas Sime; Peter Samuel James Cheetham
Original assignee: Zylepsis Limited
Priority date: 1998-10-24
Filing date: 1999-10-22
Publication date: 2000-05-04
Also published as: GB9823245D0; AU6351199A; GB2342921A

Abstract

The invention relates to methods and reagents for preparing pectins, especially high molecular weight pectins having a high methoxy content which can form a gel or a viscous solution in the presence of polyvalent metal ions such as calcium ions.

Description

PECTINS AND THE PREPARATION THEREOF

The present invention relates to methods and reagents for preparing pectins, especially high molecular weight pectins having a high methoxy content formed by the block-wise demethoxylation of high molecular weight pectins having a high methoxy content. Such pectins can form a gel or a very viscous solution in the presence of metal ions with a valency of two or more (hereinafter referred to as polyvalent metal ions ie a metal ion with a valency of two or more, such as calcium).

A growing market need exists for calcium sensitive pectins of high methoxy content and high molecular weight. This need can only be met currently by using a very special source of extract pectin that is limited in supply and so cannot meet the rapidly increasing demands.

The high methoxy pectins extracted from plant sources such as citrus and apple do not gel with polyvalent metal ions eg calcium. Chemical demethoxylation into low methoxy pectins or chemical amidation allows gel formation. However, extensive demethoxylation or amidation is required due to the random pattern of these chemical reactions. Also, the harsh conditions required for chemical demethoxylation or amidation reactions causes damage to pectins, especially reductions in molecular weight which reduces the gelling and/or viscosifying performance of the pectin.

High molecular weight pectins having a high methoxy content formed by the block-wise demethoxylation of high molecular weight pectins having a high methoxy content offer exceptional polyvalent metal ion eg calcium sensitive performance compared with low methoxypectins and such pectins are greatly valued for a variety of applications especially in dairy products. Such pectins can be made using plant PMEs (ie plant pectin methylesterases) that have a block-wise mechanism of action. Unfortunately, plant extracts also contain other enzymes such as pectin and pectate lyases and polygalacturonidases which depolymerise the pectin thereby reducing the molecular weight of the pectin and its ability to form viscous solutions and gels. For example, pectin lyase acts on methoxy lated pectin i.e. before PME acts, and polygalacturonidase acts on demethoxylated pectin, i.e. after PME action, and β-elimination occurs adjacent to galacturonic acid residues, i.e. on partially demethoxylated pectins. This deleterious enzymic activity reduces the usefulness of the pectin. In addition, non-enzymic depolymerisation occurs by β- elimination, especially at alkaline pH, with some depolymerisation at neutral and even moderately acid pHs. Thus, the value of the pectin is greatly reduced and a practical way of making block demethoxylated pectins of high molecular weight has hitherto not yet been available.

PMEs having activity not inhibited by tyrosine selective inhibitors are disclosed in EP-A-0580252. The PMEs are used at neutral to alkaline conditions and calcium-sensitive low methoxy pectins are produced.

In an attempt to solve the above problem of adverse enzymic activities in plant extracts containing PMEs, the use of a recombinant PME derived from the fungus Aspergillus aculateus has been proposed.

The advantage of the recombinant PME is that it can easily be provided in a form which is free from the undesirable enzymes which are always present in plant extracts which contain PME. Similarly, WO97/03574 (Danisco) relates to a recombinant PME derived from oranges which can be produced free from adverse plant enzymes in microbial host cells.

Another factor that diminishes the functional value of pectins is the structural damage caused by processing, such as drying and milling as well as pH adjustments. Processing needs to be minimised to maximise pectin performance and financial value and keep energy and other operating costs involved in concentrating, precipitating and redissolving of pectins etc. to a minimum. However, whereas freshly extracted pectin solutions have an acidic pH, PMEs are normally active at neutral pHs, so that to achieve reasonable activity some pH adjustment would be required before PMEs could be used.

Hydrocolloids that interact with calcium to form gels or to greatly increase viscosity are widely used in the food and beverage industries as gelling and viscosifying agents. These include alginates and pectins (E440a).

Two forms of polyvalent metal ion eg calcium sensitive pectins are commercially available from a wide range of suppliers. These are low methoxy pectins in which the high methoxypectins extracted from citrus and apple sources are substantially demethoxylated by treatment with strong NaOH to produce pectins with a substantially different chemical structure. Secondly, there are amidated pectins in which high methoxypectins are treated with ammomum hydroxide to allow reaction with the ester groups of the pectin, producing an amidated pectin structure. Low methoxypectins occur naturally, but amidated pectins are artificial (E440b). Low methoxypectins are thought to gel in the presence of divalent ions by the "egg-box model". The "egg-box model" does not involve true electrostatic attractions, but involves intermolecular chelation instead thereby forming zones of aggregation of the polysaccharide between non- esterified galacturonic acid residues. This aggregation occurs more readily when the blocks of galacturonic acid residues on different polysaccharaide chains are adjacent to one another.

One problem with the above pectin preparation processes is that because of the harsh conditions that have to be used, some depolymerisation of the pectin always occurs which can often reduce the molecular weight of the pectin considerably and have a significant deleterious effect on the gel strength and/or viscosity that can be achieved. Thus, for instance, higher concentrations of low methoxy or amidated pectin have to be used in order to achieve the desired effect compared with the concentration that would have to be used in the absence of any depolymerisation. This, of course, also increases the pectin ingredient cost to the food/beverage manufacturer.

The present inventors have not chosen to follow the recent trend of providing PMEs using difficult genetic engineering techniques in order to eliminate the depolymerase activities of certain enzymes, such as pectin and pectate lysases and polygalacturonidases. Instead, by using extraction methods that select for the required PME and especially reaction conditions that allow the PME to operate, it is possible to prevent effectively 'depolymerase' activities by enzymes, such as pectin and pectate lyases and polygalacturonidase. This process can also allow the use of the full range of isoforms of the PME expressed in the plant material, whereas a genetically engineered PME is confined to only one isoform which is therefore more removed from the catalytic activity of a natural PME. Furthermore, the process can be performed at acidic pHs thereby substantially eliminating or reducing undesirable non-enzymatic depolymerisation by β-elimination.

Whereas, the existing methods are non-selective chemical methods, the biological process of the present invention creates opportunities in two critically important respects. Firstly, it is capable of eliminating depolymerisation of the pectin by carrying out the demethoxylation reaction with pectin methylesterases (PMEs) that do not exhibit any depolymerising activity due to pectin or pectate lyase or polygalacturonidase and by carrying out the reaction at a fairly low pH so that non-enzymic depolymerisation by β-elimination, which tends to occur under even mildly acidic conditions, can be avoided. Secondly, it can achieve "block-wise" demethoxylation of pectin rather than the random demethoxylated pectins achieved using chemical methods. This is advantageous because block demethoxylated pectins are more sensitive to calcium, presumably because of the presence of adjacent blocks of galacturonic acid ester and demethoxylated galacturonic acid residues. This improved polyvalent metal ion eg calcium sensitivity has several advantages such as for food, beverage, cosmetics and pharmaceutical systems that naturally only contain relatively low calcium content and for which gelling or viscosity ing is required. Also calcium has an unpleasant taste and so the addition of calcium in high enough concentrations to cause gellation or viscosifying effects in certain products could create a serious off-taste.

Although microbial forms of PME are known to function at low pHs, ie approximately pH4, these types of enzymes are not suitable for producing a polyvalent metal ion eg calcium sensitive high methoxy content high molecular weight pectin because they exhibit a random mode of demethoxylation rather than a blockwise demethylation mode of action.

According to the invention there is provided a method of preparing a demethoxylated pectin comprising reacting a high methoxy pectin with a plant extract containing a pectin methylesterase (PME) at a pH in the range of from approximately 1.0 to 5.5.

Preferably, the pectin is obtained from one or more sources selected from sugar beet, apple, sunflower and various citrus sources, such as lime and orange.

Conveniently, the method comprises a further step of recovering the pectin in a form suitable for storage/transportation etc prior to use (eg drying).

The reaction of pectin and PME is carried out at a pH in the range of from approximately 1.0 to 5.5, more preferably in the range of from approximately pH1.5 to 4.5, most preferably about pH4.0 to 4.4 and especially about pH4.2. It will be appreciated that the reaction may be carried out at pHs below the pka of pectin (ie below pH3.1-3.5).

It will be appreciated by those skilled in the art, that pectins are most stable at approximately pH4, with the lower limit for β-oxidation reported to be approximately pH5. The pka of polygalacturonic acid is believed to be in the range of pH3.1 to 3.5, consequently when the reaction is performed at a pH above the pH range of 3.1 to 3.5 then the PME will be reacting with pectin that is in a predominately unprotonated form, whereas when the reaction is performed at a pH below the pH range of 3.1 to 3.5 then the PME will be reacting with the pectin that is in a substantially protonated form. In contrast to prior art methods, it appears therefore that PMEs produced in accordance with the present invention are capable of reacting with both the unprotonated and protonated forms of pectins.

It will be appreciated that if the pectin and PME solution is initially at pH4.2, without maintaining the pH of the solution, as the reaction proceeds the pH of the solution may drop to a more acidic pH which causes subsequent enzyme inactivation and pectin precipitation without the need for making additional pH adjustments. In other words, the overall process requires fewer steps for processing the pectin/PME solution (minimal processing) compared with known approaches with the advantage of causing less damage to the pectin such as, and especially, a reduction in molecular weight of the pectin.

Carrying out the reaction of pectin and PME within the pH range of the invention permits PME activity but prevents activity of undesirable enzymes eg. pectin/pectate lyase and polygalacturonidase whose action leads to depolymerisation. Also, reaction in the acid pH range of the invention prevents non-enzymic depolymerisation by β-elimination which occurs predominantly at alkaline pH with some depolymerisation at neutral and even mildly acidic pHs.

Preferably, the reaction of pectin and PME is carried out in the presence of NaCl at a concentration of at least 0.05 Molar (M), preferably in the range of 0.05 to 0.2 M, most preferably 0.15 M.

PMEs are normally active at neutral pHs. Hence, the acidic pH range of the method of the invention reduces the level of PME activity. However, the presence of NaCl at a concentration of at least 0.1M increases PME activity significantly, thereby compensating for the reduced level of PME activity at the acidic pH range, so that surprisingly large amounts of demethoxylated pectins can be produced. Demethoxylation of the pectin can be continued to produce demethoxylated pectins with degrees of demethoxylation consistent with lower methoxy pectins, thereby demonstrating that the PMEs exhibit substantial resistance to the by-products of the demethoxylation reactions, such as methanol.

Pectins as obtained by extraction from the plant source or as redissolved from commercial supplies give fairly acidic solutions. Hence if the PME reaction was to be done at pH 7 then the pH of the pectin solution would have to be adjusted up to pH 7 and then readjusted back down to an acid pH after reaction to enable recovery of the solid pectin. This involves additional processing costs, NaOH and HCl costs, damage to the pectins molecular weight (M.Wt.) by the two big pH adjustments and extra processing time etc. By contrast, when the reaction is performed at pH 4.2 according to a preferred embodiment of the present invention, only a small further reduction in pH is necessary to both achieve inactivation of the PME and allow recovery of the pectin. Hence, the invention provides a processing approach which involves fewer steps than known approaches. The process is particularly advantageous as it allows easier, superior and more consistent product standardisation and product quality, as compared with prior art methods, which is an important criteria for the effective and reproducible use of hydrocolloids.

Preferably, the PME containing plant extract is from acceptable plant material, especially food acceptable material of one or more of the various strains and cultivars of the following plants: tomato, banana, orange. Preferably the PME containing plant extract is from plant material of tomatoes. The present invention provides a more cost effective means for extracting larger amounts of PME, using simple techniques, such as by homogenising the plant extract, and with the resulting enzyme preparations being generally regarded as safe.

Preferably, the plant material source of PME is extracted by homogenising the plant material (which typically has an acidic pH) in the presence of sufficient NaCl to produce an extract having an NaCl concentration of at least 0.1 molar (M), preferably in the range of about 0.1 to 0.2M, more preferably about 0.15M.

The inventors have observed that the above NaCl concentration is advantageous because it increases the amount of PME which is solubilised in the extract and also activates the PME. Furthermore, the presence of NaCl at such concentrations has no adverse effect on the preparation of pectin when used according to the method of the invention.

The low pH of the PME reaction also facilitates easy inactivation of the PME once the desired degree of reaction has been achieved, eg by heat treatment under acidic conditions for a short period.

The process of the present invention can be operated in a continuous fashion if desired.

The polyvalent metal ion sensitive pectins of the invention may be used in combination with other biopolymers which interact with polyvalent metal ions such as calcium. Such other biopolymers include caseins and alginates.

In a second aspect the invention provides a plant extract containing PME wherein the extract contains NaCl, preferably at a concentration of at least 0.1M, preferably in the range of 0.1 to 0.2M, more preferably 0.15M. The inventors have observed that at such a NaCl concentration the PME is stabilised and can be stored for long periods even under ambient conditions of temperature and pressure.

In a further aspect the invention provides use of a PME for extracting pectins from raw materials, such as citrus fruits, apples, sugar beet and other agricultural products. This may allow higher molecular weight pectins and/or demethoxylated pectins to be extracted from such raw materials.

In a further aspect the invention provides use of a PME for treating pectins present in process streams, such as fruit juices or dairy products (eg yoghurts and milk based drinks) where it is advantageous to either increase the viscosity of or gell the product.

Surprisingly, the inventors have discovered a PME containing plant extract which has activity at an acidic pH, even at a pH of 4.0 or less and still more unexpectedly at a pH of 3.5 or less.

Accordingly, the invention also provides a PME containing plant extract having a significant activity at an acidic pH, preferably pH 4.0 or less, most preferably pH 3.5 or less.

By "significant activity" we mean that PME is capable of a pectin demethoxylation activity which is measurable using the methods described herein eg by monitoring the rate of addition of a base to maintain a solution of the pectin and the PME at a constant pH under the conditions mentioned in example 2.2 and/or testing the polyvalent metal ion sensitivity of the pectin after treatment with the PME. The methods described in WO97/03574 may also be used and are hereby incorporated by reference.

By "plant extract" we mean plant material in a different state from that in which it exists in nature. Preferably, the extract is in the form of an aqueous solution obtained by mechanical processing of plant material, optionally with the addition of water and/or sodium chloride, to form a homogenate which may be filtered.

Preferably, the PME activity is blockwise demethoxylation of pectin rather than random demethoxylation.

Preferably, the PME containing extract is derived from tomatoes.

The invention also relates to the use of the above purified PME in the demethoxylation of a pectin.

Skilled persons will appreciate that the PME contained in the extracts of the invention can be used to obtain amino acid and nucleic acid sequences which permit the enzyme to be produced using recombinant DNA technology. Suitable methodologies for producing a recombinant PME are described in WO97/03574, the disclosure of which is incorporated herein by reference. DEFINITIONS

1. The phrase pectin having a "high" methoxy content is intended to mean pectin having a degree of methoxylation of more than 50% . Typically, a pectin having a "high" methoxy content is not calcium sensitive. However, when a pectin having a "high" methoxy content is formed by "block-wise demethoxylation" it may be polyvalent metal ion eg calcium sensitive.

2. Similarly the phrase low methoxy content refers to pectins having a degree of methoxylation of 50% or less. Typically, these pectins are polyvalent metal ion eg calcium sensitive.

3. The term "high average molecular weight" pectin includes pectins having an average molecular weight of greater than about 200,000, preferably greater than 400,000 and less than 1000 kDa. It will be appreciated that the molecular weight of a pectin and the range of molecular weights of a pectin in a given sample depend on a variety of factors, such as, the source of the pectin (ie type of plant source), the growth conditions employed, the maturity of the plant source and the degree of reprocessing required. Preferably, the difference in molecular weight between the pectin formed by the block-wise demethoxylation of the high molecular weight pectin having a high methoxy content (substate pectin) and the substrate pectin itself is not more than 15% as a result of demethoxylation, preferably it is not more than 12% , most preferably no more than 10% , especially no more than 5%.

4. "Polyvalent metal ion eg calcium sensitive" is intended to mean that the pectin is capable of forming a gel or a solution having a significantly greater viscosity in the presence of polyvalent metal ions, such as calcium ions. The term includes pectins which have a viscosity in the range of approximately 50 to 100,000 centipoise (cP) in the presence of calcium ions at a concentration of 0.1M or less.

5. "Block-wise demethoxylation" is intended to mean a mechanism of demethoxylation which produces a pectin comprising consecutive galacturonic acid residues surrounded by regions of consecutive galacturonic acid methyl ester residues.

6. "PME activity" can be measured by monitoring the rate of addition of a base, such as 0.1M aqueous NaOH, to maintain a solution of the pectin and the plant extract at a constant pH. The rate of addition of the base is a measure of PME actvity. In a typical procedure, an aqueous solution of pectin (60 ml of a 1 % solution (w/v) of pectin (Sigma P95611) at pH 7 containing a plant extract (ie. tomato extract 3.0 ml) is automatically titrated with a solution of aqueous NaOH (0.1M) with stirring at 30 °C using an autotitrator and the rate of addition of base required to maintain the solution at a constant pH is monitored for a certain time period, typically between 30 minutes and 2 hours.

7. The neutral sugar content of a pectin can be determined by hydrolysis and monosaccharide analysis using HPLC.

The invention will now be described with reference to the following non- limiting examples and figure which embody certain aspects of the invention.

Figure 1 represents the pH activity profile of PME from tomato extract.

la. Effect of NaCl

In a typical experiment the rates of reaction were as follows using PME extracted (purified or fractionated) using water or NaCl solution, and assayed in the absence and presence of NaCl.

Extraction with NaCl (0.15M) increases the amount of PME obtained 53.8 fold when assayed in the absence of NaCl. Also the presence of NaCl (0.15M) activates the activity of the PME 14.8 fold compared with enzyme extracted without NaCl.

2. Production of tomato fraction containing PME activity

A tomato fraction was initially produced by simple homogenisation of tomatoes, centrifugation and filtration. However, a greater yield of PME activity was produced when sodium chloride was added to the tomatoes prior to fractionation to give a concentration of approximately 150mM in the final fraction and this was adopted as standard methodology. The fraction may be produced without pH adjustment giving a fraction having a pH of approximately pH4.

2.1 Preferred method for preparing tomato PME

Tomatoes (200g) are roughly chopped and sodium chloride (1.124g) added (equivalent to 150mM in 128 ml of fraction produced). The tomatoes are then pureed using a kitchen blender until homogeneous and shaken at 200 rpm for 15 minutes at room temperature. The puree is centrifuged (25 min @ 3362xg) and the supernatant filtered under vacuum through a double thickness of Whatman No. 1 filter paper. The resulting fraction is typically between 120 ml and 130 ml and pH4.5 and pH4.6. The fraction is routinely divided into smaller aliquots and stored at >0°C.

The tomato extract can then be concentrated if required by standard methods, such as ultrafiltration, to prepare a more concentrated enzyme preparation.

2.2 Comparison of PME activity at pH7.0, pH5.5, pH4.5 and pH3.5

PME activity was deteπnined over a range of pHs by adding pectin (0.8g) and sodium chloride (0.35 g) to water (36.79 ml). The solution was adjusted to the appropriate pH by the addition of sodium hydroxide (2.0 M or 0.1 M). Tomato fraction (3.21 ml) was added to initiate the reaction and an autotritrator was used to hold the pH at the desired value by the addition of sodium hydroxide (0.1 M). The rate of this addition was used as a measure of the PME activity.

2.3 Effect of temperature on PME activity

Tomato fraction was used at 30 °C for all of the experiments described. We have found that exposing the enzyme to 60 °C for 10 minutes removes all activity. Thus, we would suggest that the enzyme could be utilised at any temperature up to 60° C.

2.4 Treatment of pectin with tomato pectin methyl esterase

Pectin (6 g) is dissolved in distilled water (300 ml) containing sodium chloride (2.63 g, 150 mM) to give a 2% (w/v) solution. The pectin was mixed using a Silverson homogeniser and the pH of the solution adjusted to pH 4.2 using sodium hydroxide (2 M and 0.1 M). Tomato fraction (350μl) was added to the pectin solution and the solution incubated at 30°C and shaken at 240 rpm. The pH of the pectin solution was held at pH4.2 throughout the incubation by the use of a pH stat that added sodium hydroxide solution (0.1 M) as necessary. The reaction was stopped after 16000 seconds by adjusting the pH of the pectin solution to pH 3.0 by the addition of hydrochloric acid (2.0 M) whilst still shaking at 240 rpm the pectin solution. The pectin was precipitated from solution by pouring it in an isopropanol/water solution at 30 to 35°C (450 ml of specific gravity 0.82, 162.5 ml water plus 837.5 ml isopropanol). The solution was stirred for 20 seconds and left to stand for 30 minutes. The precipitated pectin was recovered on a 500 μm sieve and pressed dry by hand. The precipitated pectin was resuspended in a isopropanol/water (300 ml of specific gravity 0.82) stirred for 20 seconds and left to stand for 30 minutes. The precipitated pectin was recovered on a 500 μm sieve and pressed dry by hand. The precipitated pectin was pressed between sheets of paper kitchen towel and dried overnight 50 °C followed by 40 minutes at lOO°C. Yield 4.86 g (81 %).

Analysis of a pectin subjected to the whole process but without PME treatment showed that the loss in molecular weight was due to the physical processing and not to the enzyme action.

2.5 Treatment of liquid pectin stream with tomato pectin methylesterase. Recovery of pectin by precipitation.

Sodium chloride (2.63 g, 150 mM) was added to a liquid pectin stream (300 ml) containing ca 2% pectin. The pectin was mixed using a Silverson homogeniser and the pH of the solution adjusted to pH4.2 by addition of sodium hydroxide (2 M and 0.1 M). Tomato fraction (350 μ\) was added to the pectin solution and the solution incubated at 30°C and shaken at 240 rpm. The pH of the pectin solution was held at pH4.2 throughout the incubation by the use of a pH stat that added sodium hydroxide solution (0.1 M) as necessary. The reaction was stopped after 4 hours by adjusting the pH of the pectin solution to pH 3.0 by the addition of hydrochloric acid (2.0 M) whilst still shaking at 240 rpm the pectin solution. The pectin was precipitated from solution by pouring it into an isopropanol/water solution at 30 to 35 °C (450 ml of specific gravity 0.82, 162.5 ml water plus 837.5 ml isopropanol). The solution was stirred for 20 seconds and left to stand for 30 minutes. The precipitated pectin was recovered on a 500 μm sieve and pressed dry by hand. The precipitated pectin was resuspended in a isopropanol/water (300 ml of specific gravity 0.82) stirred for 20 seconds and left to stand for 30 minutes. The precipitated pectin was recovered on a 500 μm sieve and pressed dry by hand. The precipitated pectin was pressed between sheets of paper kitchen towel and dried overnight 50 °C followed by 40 minutes at 100°C.

2.6 Treatment of liquid pectin stream with tomato pectin methylesterase. Termination of reaction by heat inactivation of the enzyme.

Sodium chloride (11.01 g, 150 mM) was added to a liquid pectin stream

(1400 ml). The pectin was mixed using a Silverson homogeniser and the pH of the solution adjusted from pHl.8 to pH4.2 using sodium hydroxide (2 M and 0.1 M). A sample of pectin was separated (300 ml) and the pH adjusted to pH1.4. This sample was a control for the affect of the pH adjustment steps. Tomato fraction (1.2 ml) was added to the remaining 1100 ml of pectin solution and the solution incubated at 30°C and shaken at 240 rpm. The pH of the pectin solution was held at pH4.2 throughout the incubation by the use of a pH stat that added sodium hydroxide solution (0.1 M) as necessary. The reaction was stopped after 5.5 hours by adjusting the pH of the pectin solution to pH 1.4 by the addition of hydrochloric acid (2.0 M) whilst mixing using the Silverson homogeniser. The pectin solution was then split so that 800 ml was treated at 60 °C for 10 minutes by pumping through silicon tubing suspended in a heated water bath. The heat treatment removes all PME activity.

The remaining 300 ml of pectin solution was treated to recover the pectin by precipitation. The pectin was precipitated from solution by pouring it into an isopropanol/water solution at 30 to 35 °C (450 ml of specific gavity 0.82, 162.5 ml water plus 837.5 ml isopropanol). The solution is stirred for 20 seconds and left to stand for 30 minutes. The precipitated pectin was recovered on a 500 μm sieve and pressed dry by hand. The precipitated pectin was resuspended in a isopropanol/water (300 ml of specific gravity 0.82) stirred for 20 seconds and left to stand for 30 minutes. The precipitated pectin was recovered on a 500 μm sieve and pressed by hand. The precipitated pectin was pressed between sheets of kitchen towel and dried overnight 50°C followed by 40 minutes at 100°C. Yield 5.8 g.

This demonstrates that the heat inactivation step is successful and has no deleterious effect on the pectin produced and that enzyme treatment produces a very great increase in calcium sensitivity as measured by the viscosity of a pectin solution after addition of calcium.

2.7 Assessment of enzyme stability

The tomato fraction was stored for a period of 3 months under the following conditions:

i. Freeze dried, 4°C & Room Temperature (RT) ii. Plus NaCl 2.5 % , 4°C & RT iii. Plus NaCl 9.25 % , 4°C & RT iv. Plus NaCl 16%, 4°C & RT

The PME activity of these samples was assessed periodically and after 3 months all of the sodium chloride containing samples exhibited PME activity equal to or greater than the freeze dried samples. The PME activity of the tomato fraction has also been shown to be stable over a one- month period in the presence of sodium metabisulphite (0.4 mg ml^"1).

2.8 Importance ofpH of pectin for recovery by precipitation

It is critical for the pH of the pectin solution to be adjusted to pH3 or below if the molecular weight of the recovered pectin is not to be adversely effected. When a 2% solution of a pectin (molecular weight > 500 kDa) adjusted to pH4.5 was immediately recovered (without any enzyme treatment) the molecular weight of the recovered pectin had dropped to 266 kDa. However, if the pH of the pectin is adjusted to pH3.0 prior to precipitation of the pectin then no loss in molecular weight is observed. For example, a pectin that has been incubated for 5.5 hours at pH4.2 and then recovered by precipitation after adjustment to pH3 exhibited a molecular weight of 472 kDa compared to a molecular weight of 449 kDa for pectin that was not treated with enzyme but recovered without pH adjustment to pH3 (these results are within the error for the assay).

2.9 Continuous demethoxylation process

A continuous demethoxylation process could consist of continually pumping the pectin solution formed as part of the normal manufacturing process through a heated tube to maintain a temperature of 30° C. The pH of the pectin solution would have to be adjusted to pH4.2 by the addition of base. The tomato fraction containing PME activity could be introduced into the pectin solution at the start of the tube and the pH monitored along the reactor length. Acid or base could be added to the pectin as appropriate along the reactor length to ensure the correct pH (ie pH4.2) was maintained for the PME activity and to prevent any reduction in molecular weight of the pectin. The demethoxylation reaction catalysed by PME could be teirriinated by re-adjusting the pH of the pectin solution to its original pH (eg between pHl and pH3) and by raising the temperature of the pectin solution to 60 °C for 10 minutes. The resulting pectin solution could then continue through the normal manufacturing process. The degree of methoxylation of the treated pectin could be controlled to yield a specific level by altering the amount of tomato fraction added and/or by adjusting the flow rate (and thus the residence time) of the pectin solution through the reaction tube.

2.10 pH profile of PME activity of tomato extract

PME activity was assayed using an autotitrator by monitoring the rate of base added (NaOH 0.1M) to hold a solution of pectin (60 ml of 1 % solution also containing 150 mM NaCl (w/v; Sigma P9561)) containing tomato extract (3.0 ml) at the desired pH whilst stirring and mamtaining the temperature at 30°C. The reaction was monitored for 30 min and the rate of base addition was used as a measure of the rate of deesterification of the pectin by PME in the tomato extract.

The tomato extract exhibited a large reduction in activity as the pH of the reaction was reduced from pH7.0 to pH3.0 although the rate of base consumption remained near linear for each individual pH tested. At pHs below pH3.0 the enzyme activity was not constant, ie the results obtained for pH2.8, pH2.5 and pH2.0 consisted of an initial phase of activity (and thus base comsumption) which rapidly declined during the 30 minutes of incubation. The rate data shown in Table 1 and Figure 1 represents the rate of base consumption calculated from the total volume of base consumed in the 30 minutes of the incubation.

Table 1. Variation in PME activity of tomato extract between pH2.0 and pH7.0 pH Rate (ml min^"1) Activity (% of pH7 activity)

7.0 0.148 100.0

4.2 0.057 38.4

3.5 0.039 28.0

3.0 0.018 12.5

2.8 0.017 11.1

2.5 0.018 11.8

2.0 0.023 15.3

Control 0.008 4.1

(pH2.5 + Inactivated enzyme)

2.11 Gel testing of pectins

Samples of pectin were routinely removed from PME treatment and control incubations to assess their ability to form a gel in the presence of calcium ions. High methoxy pectin does not form a gel in the presence of calcium, whilst low methoxy pectin produced by the action of PME does form calcium sensitive gels.

Solutions of pectin were assessed for their ability to form a calcium sensitive gel by taking a 1 ml aliquot of the pectin solution and adding 100 μl of an aqueous calcium chloride solution (0.1M). The samples were mixed and left to stand at room temperature. The formation of a gel was visually assessed periodically and compared to samples of the pectin solution without the addition of calcium chloride and to samples taken at the initiation of the deesterification reaction.

Claims

1. A method of preparing a demethoxylated pectin comprising: reacting a high methoxy pectin with a plant extract containing a pectin methylesterase (PME) at a pH in the range of from approximately 1.0 to 5.5.

2. A method as claimed in Claim 1 wherein the pH is in the range of from approximately 1.5 to 4.5.

3. A method as claimed in Claim 2 wherein the pH is in the range of from approximately 4.0 to 4.4.

4. A method as claimed in Claim 2 wherein the pH is in the range of from approximately 2.0 to 3.0

5. A method as claimed in any one of the preceding claims wherein the reaction is carried out in the presence of NaCl.

6. A method as claimed in Claim 5 wherein the NaCl concentration is at least 0.05M.

7. A method as claimed in Claim 6 wherein the NaCl concentration is in the range of approximately 0.05 to 0.2M, preferably about 0.15M.

8. A method as claimed in any one of the preceding claims wherein the pectin produced comprises a polyvalent metal ion sensitive, preferably calcium sensitive, high methoxy pectin.

9. A method as claimed in Claim 8 wherein the pectin produced has a degree of methoxylation of more than 50% .

10. A method as claimed in any one of Claims 1 to 8 wherein the pectin produced has a degree of methoxylation of less than 50 % .

11. A method as claimed in any one of the preceding claims wherein the pectin produced has a high average molecular weight.

12. A method as claimed in any one of the preceding claims wherein the pectin produced has a neutral sugar content not significantly different from that of the starting pectin as determined by high performance liquid chromatography.

13. A method as claimed in any one of the preceding claims wherein the demethoxylation is block-wise.

14. A method as claimed in any one of the preceding claims wherein the plant extract containing PME is obtained directly from one or more available plant sources, such as tomato, banana and/or orange.

15. A method as claimed in Claim 14 wherein the extract is from tomato.

16. A method as claimed in any one of the preceding claims wherein the plant extract is extracted with NaCl.

17. A method as claimed in Claim 16 wherein the NaCl is at a concentration of at least 0.05M.

18. A method as claimed in Claim 17 wherein the NaCl concentration is in the range of approximately 0.05 to 0.2M, preferably about 0.15M.

19. A method as claimed in any one of Claims 1 to 18 wherein the reaction is terminated by heating and/or reducing the pH to inactivate the PME.

20. A method as claimed in Claim 19 wherein the pH is reduced to approximately 1.4 to 3.0 prior to precipitation of the pectin.

21. A method as claimed in any one of Claims 1 to 20 wherein the demethoxylated pectin produced is capable of forming a gel in a solution containing polyvalent metal ions, preferably calcium ions.

22. A method as claimed in any one of Claims 1 to 21 wherein the demethoxylated pectin produced is capable of forming a solution having an increased viscosity.

23. A method as claimed in any one of the preceding claims wherein the molecular weight of the pectin is not reduced by more than 15% as a result of demethoxylation.

24. A method as claimed in Claim 23 wherein the molecular weight is not reduced by more than 5 % as a result of demethoxylation.

25. A method as claimed in any one of the preceding claims wherein the content of neutral sugars in the pectin is not reduced by more than 10% as a result of demethoxylation.

26. A method as claimed in any one of the preceding preceding claims wherein the solution of pectin to be treated contains a source of S0₂, preferably sodium metabisulphate.

27. A method as claimed in any one of the preceding claims wherein the pectin solution is produced by dissolving pectin in solid form.

28. A method as claimed in any one of the preceding claims wherein the pectin solution is provided as an untreated liquid plant extract.

29. A method as claimed in any one of the preceding claims wherein the method is performed as a continuous process.

30. A method as claimed in any one of the preceding claims further including an additional PME.

31. Use of the pectin obtained by a method as claimed in any preceding claim for direct addition to a food, beverage, personal care, pharmaceutical, product or ingredient.

32. A polyvalent metal ion sensitive, preferably calcium sensitive, high molecular weight pectin obtainable by a method as claimed in any one of the preceding claims.

33. A polyvalent metal ion sensitive, preferably calcium sensitive high molecular weight pectin as claimed in Claim 32 wherein the pectin has a degree of methoxylation of more than 50% .

34. Use of a pectin as claimed in Claim 32 or 33 in a food, beverage, personal-care, or pharmaceutical, product or ingredient.

35. Use of a pectin as claimed in Claim 34 wherein the product is a fruit product, confectionary product, beverage product, dairy product, a skin care product, a dental product, or a hair care product.

36. Use of a pectin as claimed in Claim 32 or 33 for stabilizing protein components of a food, beverage, pharmaceutical, personal-care, product or ingredient.

37. Use of a pectin as claimed in Claim 36 for stabilizing dairy proteins preferably caseins.

38. Use of the PME obtained by a method as claimed in any one of Claims 1 to 31 for stabilizing a protein component of a food, beverage, pharmaceutical or personal-care product.

39. The use as claimed in Claim 38 wherein the PME is added to the product during preparation of the product and/or to the final product.

40. A plant extract containing a PME wherein the extract is in the form of an aqueous solution having a NaCl concentration effective to stabilise the PME.

41. An extract as claimed in Claim 40 wherein the NaCl concentration is at least 0.05M.

42. An extract as claimed in Claim 41 wherein the NaCl concentration is in the range of approximately 0.05 to 0.2M, preferably 0.15M.

43. A PME containing extract obtainable by a method comprising providing a plant material and treating the material with NaCl.

44. A PME containing extract as claimed in Claim 43 wherein the method involves homogenising the plant material in the presence of NaCl.

45. A PME containing extract as claimed in Claim 43 or 44 wherein the NaCl is provided as a aqueous colution.

46. An extract as claimed in any one of Claims 43 to 45 wherein the NaCl is at a concentration of at least 0.05M.

47. An extract as claimed in Claim 46 wherein the NaCl concentration is in the range of approximately 0.05 to 0.2M, preferably about 0.15M.

48. Use of a PME containing extract as claimed in any one of Claims 32, 33 and 40 to 47 for producing a calcium sensitive high molecular weight pectin having a degree of methoxylation of more than 50 % .

49. Use of a PME containing extract as claimed in any one of Claims 32, 33 and 40 to 47 for extracting pectins from citrus fruits, apples, sugar beet and other agricultural products.

50. A PME containing plant extract having a significant pectin demethoxylation activity at an acidic pH.

51. An extract as claimed in Claim 50 having a significant PME activity at a pH of 4.0 or less.

52. An extract as defined in Claim 51 having a significant PME activity at a pH of 3.5 or less.

53. An extract as claimed in any one of Claims 50 to 52 wherein the extract comprises or consists of an extract from a tomato.

54. Use of a PME containing plant extract as claimed in any one of Claims 50 to 53 in the demethoxylation of a pectin.

55. A method of preparing a demethoxylated pectin substantially as described herein, preferably with reference to one or more of the examples.

56. Use of a PME in a food, beverage, personal care or pharmaceutical product or ingredient.

57. Use of a polyvalent metal ion sensitive high molecular weight pectin having a degree of methoxylation of more than 50% obtained by a method substantially as described herein, preferably with reference to one or more of the examples, in a food, beverage, personal care, or pharmaceutical product or ingredient.

58. A PME containing plant extract having significant demethoxylation activity at an acidic pH, preferably pH 4.0 or less, substantially as described herein, preferably with reference to one or more of the examples.