CZ374399A3 - Preparation containing pectin-methylesterase and two substrates - Google Patents

Abstract

A gel-forming composition suitable for use as a gel food or suitable for improving the stability of food, it contains pectin-methyl ester ester PME; first substrate for PME; and a second substrate for PME, wherein the first and second substrates for PMEs are not derived from each other. Advantageously Substrate for PME pectin or a derivative thereof.

Description

A composition comprising pectin methyl esterase and two substrates

Technical field

The present invention relates to a composition. More specifically, the present invention relates to a composition useful as a foodstuff or useful in the preparation of foodstuffs. More specifically, the present invention relates to a composition useful as a foodstuff or useful in the preparation of foodstuffs comprising or made from pectin or a pectin derivative.

BACKGROUND OF THE INVENTION

Pectin is currently an important industrial commodity. For example, it is used in the food industry as a thickening or gelling agent, for example in the preparation of jams.

Pectin is a structural polysaccharide that is commonly found in the form of protopectin in the wall of plant cells. The pectin skeleton contains α-1-4 linked galacturonic acid residues that are interrupted by a small number of 1,2-linked α-L-rhamnose units. In addition, pectin contains highly branched regions formed by an almost alternating rhamno-galacturonan chain. These highly branched regions also contain other sugar units (such as D-galactose, L-arabinose and xylose) linked by glycosidic linkages to the C3 or C4 atoms of the rhamnose units, or to the C2 or C3 atoms of the galacturonic acid units. Long chains of α-1-4 bound galacturonic acid residues are commonly referred to as smooth regions, while highly branched regions are commonly referred to as hairy regions.

Some carboxyl groups of galacturon residues are esterified (for example, carboxyl groups are methylated).

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Usually, esterification of the carboxyl groups takes place after the polymerization of the galacturonic acid residues. However, all carboxyl groups (for example methylated) are extremely esterified. Typically, the degree of esterification is in the range of 0 to 90%. When more than 50% of the carboxyl groups are esterified, the resulting pectin is referred to as highly esterified pectin (HE pectin) or highly methoxylated pectin. If less than 50% of the carboxyl groups are esterified, the resulting pectin is referred to as a low esterified pectin (LE pectin) or a low methoxy pectin. When 50% of the carboxyl groups are esterified, the resulting pectin is referred to as moderately esterified pectin (ME pectin) or moderately methoxylated pectin. If the pectin contains no - or only a few - esterified groups, it is usually referred to as pectinic acid.

The structure of pectin, particularly the degree of esterification (e.g. methylation) determines many of the resulting physical and / or chemical properties of pectin. For example, gelling of pectin depends on the chemical nature of the pectin, particularly the degree of esterification. However, pectin gel formation also depends on the solids content, the pH and the calcium ion content. It is believed that calcium ions form complexes with free carboxyl groups, especially groups on the LE pectin.

Pectin-acting enzymes are classified according to the way they act on the galacturonan portion of the pectin molecule. A review of some pectin-cleaving enzymes is given in Pilník and Voragen (Food Enzymology, Ed., P. F. Fox, Elsevier; (1991), pp. 303-337). Specifically, pectin methyl esterases (EC 3.1.1.11), otherwise referred to as PME, deesterify HE pectins into LE pectins or pectic acids. On the other hand, and for example, pectin depolymerases cleave glycosidic bonds between galacturonosyl methyl ester residues.

In particular, PME activity produces free carboxyl groups and free methanol. The increase in free carboxyl groups can be easily monitored by automatic titration. Previous studies have shown that some PMEs deesterify pectin in a random manner in the sense that they deesterify any esterified (for example, methylated) galacturonic acid residue on one or more than one pectin chain. Examples of PMEs that randomly de-esterify pectins can be obtained from fungi such as Aspergillus aculeatus (see WO 94/25575) and Aspergillus japonicus (Ishii et al., 1980, J. Food Sci. 44, 611-614). Baron et al. (1980, Lebensm. Wiss. M-Technol. 13, pp. 330-333) isolated PME from Aspergillus niger. This fungal PME has, as described, a molecular weight of 39,000 Da, an isoelectric point of 3.9, an optimum pH of 4.5, and K _m (mg / ml) 3.

Conversely, it is known that some PMEs deesterify pectin in a block fashion, i.e., act on pectins either at the non-reducing ends or after free carboxyl groups, and then proceed along single chain pectin molecules, creating blocks of unesterified galacturonic acid units that may be sensitive for calcium. Examples of such PMEs that block de-esterify pectin are plant PMEs. It has been reported that there are more than 12 PME isoforms in citrus (Pilník and Voragen A., G., J., (Food Enzymology,

Ed., P.F. Fox, Elsevier; (1991), pp. 303-337). These isoforms have different properties.

The random or block distribution of free carboxyl groups can be distinguished by high-resolution ion-exchange chromatography (Schols et al., Food Hydrocolloids, 1989, 9 9 9). 9 9 9 9 9 9

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6, pp. 115-121). These tests are often used to test unwanted, residual PME activity in citrus juices after pasteurization, as residual PME can cause a phenomenon called haze loss in orange juice, in addition to increasing the methanol content of the juice.

PME substrates, such as pectins from natural plant sources, are typically in the form of a highly esterified pectin having a DE of about 70%. To the extracts containing these highly esterified PME substrates, sugar must be added to achieve a sufficiently high dissolved solids content for gel formation. Usually a minimum of 55% dissolved solids is required. Syneresis occurs more frequently when the dissolved solids content is less than 55%. If syneresis occurs, expensive additives must be added to induce gel formation.

Versteeg et al., (J. Food Sci. 45 (1980) pp. 969-971) isolated PME from an orange tree. This plant PME has isoforms of various properties as described. Isoform I has a molecular weight of 36000 Da, an isoelectric point of 10.0, an optimum pH of 7.6 and a K _m (mg / ml) of 0.083. Isoform II has a molecular weight of 36200 Da, an isoelectric point of 11.0, an optimum pH of 8.8 and a K _m (mg / ml) of 0.0046. Isoform III (HMW-PE) has a molecular weight of 54,000 Da, an isoelectric point of 10.2, an optimum pH of 8.0 and a K _m (mg / ml) of 0.041. However, very little sequence data of such PMEs is available so far.

According to Nail File and Vooragen (above), PMEs can be found in many other higher plants, such as apple, apricot, banana, berry, lemon tree, grapefruit, tangerine, cherry, currant, vine, mango, papae, passion fruit, peach, pear , plums, beans, carrots,

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9 cauliflowers, cucumbers, leeks, onions, potatoes, radishes and tomatoes. However, very little sequence data of such PMEs is available so far.

Plant PME has been described in WO-A-97/03574 (the contents of which are incorporated herein by reference). This PME has the following characteristics: molecular weight from about 36 kDa to about 64 kDa; pH optimum 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 10 ° C to at least 40 ° C; K _m 0.07%; maximum activity at about 0.25 M NaCl; maximum activity at about 0.2 M Na ₂ SO ₄ ; and peak activity at about 0.3 M NaNO ₃ .

Another PME has been described in WO 97/31102 (the contents of which are incorporated herein by reference).

PMEs have significant applications in industry. For example, they can be used as sequestering agents for calcium ions. According to Pilník and Voragen (supra), cattle feed can be prepared by adding slurry of calcium hydroxide to citrus peel after juice extraction. Upon addition, high pH and calcium ions activate any natural PME in the skin, causing rapid de-esterification of pectin and coagulation of calcium pectate. The bound liquid phase is released and easily extruded, so that only a fraction of the original aqueous content has to be removed by expensive thermal drying. The extruded liquid can then be used as animal feed.

As mentioned above, PME was obtained from Aspergillus aculeatus (WO 94/25575). This PME can be used to improve the strength of pectin-containing materials, or to demethylate pectin, or to increase the viscosity of pectin-containing materials.

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It is also common to use pectin to prepare foods prepared from pectin-containing fruits or vegetables - such as jams or preservatives. For example, WO-A-94/25575 further describes the preparation of orange marmalade and tomato paste using PME obtained from Aspergillus aculeatus.

JP-A-63/209553 discloses pectin methyl esterase gels, in the presence of a polyvalent metal ion, on a pectin polysaccharide containing as a main component a highly methoxylated poly-α, 4-D-galacturonide chain and further describes a process for preparing such gels .

Nail file and Voragen (above) list the use of endogenous PMEs that include adding them to fruit juices to reduce the viscosity of the juice, if it contains too much fruit pectin, adding pectinase solution to gas bubbles in the citrus fruit albed that have been heated to internal temperature. 20 ° C to 40 ° C to remove skin and other membranes from intact segments (US-A-4284651) and their use in protecting and improving the structure and strength of processed vegetables and fruits such as apples (Wiley and Lee, 1970, Food Technol 24: 1168-70), canned tomatoes (Hsu et al., 1965, J. Food Sci. 30, pp. 583-588) and potatoes (Bartolome and Hoff, 1972, J. Agric. Food Chem. 20, p. 262-266).

Glahn and Rolin (1994, Food Ingredients Europe, Conf. Proceedings pp. 252-256) describe the hypothetical use of industrial GENU pectin type YM-100 for interaction with beverages prepared from sour milk. No details are given regarding the preparation of GENU pectin type YM-100.

EP-A-0664300 discloses a chemical fractionation method for preparing calcium sensitive pectin. It is stated that this φ

Calcium sensitive pectin is advantageous for the food industry.

Therefore, pectins and de-esterified pectins, in addition to PME, are of industrial importance.

SUMMARY OF THE INVENTION

We have now found that the benefits associated with the use of PME in the preparation of, for example, foodstuffs depend to some degree on the quality, quantity and type of PME used and the quality, quantity and type of PME substrates used, particularly pectin. . For example, if the substrate is a fruit or vegetable, the amount and / or structure of the natural pectin in that substrate will vary for different types of fruit or vegetable. This corresponds to the data presented in WO-A-94/25575, in particular Fig. 7, where it is clearly seen that the PME system is not ideal.

We have now found that the addition of additional PME substrates allows for greater benefits associated with the use of PME in the preparation of, for example, foods.

The addition of another PME substrate will overcome any problems associated with the different amounts and qualities of PME substrates that may be present in the materials used in food preparation.

In a first aspect, the present invention comprises a composition comprising pectin methyl esterase (PME); a first substrate for PME; and a second substrate for PME; wherein the first and second PME substrates are not derived from each other.

· Φ Φ φ · · · φ φ φ · · · · · · · · · ·

In a second aspect, the present invention comprises a method of preparing a composition comprising making a PME mixture; a first substrate for PME; and a second substrate for PME; wherein the first and second PME substrates are not derived from each other.

In a third aspect, the present invention comprises a method comprising adding PME and a second PME substrate to a first PME substrate; wherein the first and second PME substrates are not derived from each other.

In a fourth aspect, the present invention comprises a foodstuff comprising a composition of the present invention or prepared by the methods of the present invention.

In a fifth aspect, the present invention also comprises a composition prepared from pectin methyl esterase (PME); a first substrate for PME; and a second substrate for PME; wherein the first and second PME substrates are not derived from each other.

In a sixth aspect, the present invention comprises a method for preparing a stable reaction medium comprising a first substrate for PME, said method comprising adding at least PME and a second substrate for PME; wherein the first and second PME substrates are not derived from each other.

More generally, the present invention comprises a composition comprising PME; a first substrate for PME; and a second substrate for PME; wherein the first and second PME substrates are not derived from each other.

In the present invention, the first and second PME substrates are not derived from each other. The term first and second PME substrates are not derived from each other meaning that the first PME substrate is not derived from the second PME substrate and / or the second · · φφφφ

ΦΦ · φ φ φ · · · · · φ φ φ φ φ φ φ φ φ φ φ • • • • • • • • • φ φ φ φ · · PME substrate is not derived from the first PME substrate. Therefore, in the present invention, the first PME substrate is not derived from the second PME substrate and vice versa. Therefore, for example, the composition of the present invention does not contain a quantity of PME substrate, wherein a portion of this PME substrate has been partially modified by the PME enzyme. Conversely, a second substrate for PME must also be present, and this second substrate for PME is not derived from the first substrate for PME.

Other, different substrates for PME may be present in the composition of the present invention.

Substrates for PME in the compositions of the present invention or compositions of the present invention may be obtained from different sources and / or may have different chemical compositions.

Similarly, other different PME enzymes may be present in the compositions of the present invention.

If more than one PME is present, these PME enzymes may be obtained from different sources and / or may have different compositions and / or may have different reactivity profiles (e.g., other optimum pH and / or different temperature optimum).

In the present invention, the PME enzyme may de-esterify substrates for PME at random or block. When more than one PME is used, each PME is independently selected from PMEs that can de-esterify substrates for PME randomly or from PMEs that can de-esterify substrates for PME block.

In one preferred embodiment, it de-esterifies (at least one)

PME enzyme substrates for PME block.

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If more than one PME is present, these PMEs may be selected from sodium ion sensitive (Na-sensitive) PME enzyme or sodium ion sensitive (Na-insensitive) PME enzyme. In a preferred embodiment, the (at least one) PME enzyme is Na-sensitive.

PME may be obtained from natural sources or may be synthesized chemically.

For example, PME may be obtained from fungi, as is the case with fungal-derived PMEs (i.e., PMEs which are produced from fungi).

Alternatively, PME may be obtained from bacteria, such as PME derived from bacteria (i.e., PMEs that are bacteria-derived).

Alternatively, PME may be obtained from plants, such as from plant-derived PME (i.e., plant-derived PME).

In one preferred embodiment, the PME! prepared by recombinant DNA techniques.

For example, the PME may be a recombinant PME as described in WO-A-97/03574 or a PME described in WO-A-94/25575 or WO-A-97/311102 as well as a variant, derivative or homolog of the sequences described in these patent applications.

In one preferred embodiment, the PME is a recombinant PME of WO-A-97/03574 (the contents of which are incorporated herein by reference) - such as a PME of SEQ ID NO: 1 or SEQ ID NO: 2 (which are encoded by the nucleotide sequences shown as SEQ ID NO. NO: 3 or SEQ ID NO: 4, respectively, or a variant, derivative thereof, or ·· ····

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99 99 99 99 99 homologue; and / or PME under WO-A-94/25575 (the contents of which are incorporated herein by reference) or a variant, derivative or homologue thereof.

The term variant, homolog, or fragment in connection with the recombinant enzyme of the present invention includes any substitutions, changes, modifications, substitutions, deletions or additions of one or more amino acids of the sequence provided that the resulting amino acid sequence has PME activity, preferably at least the same activity as recombinant an enzyme comprising one or more of the sequences shown as SEQ ID NO: 1 and SEQ ID NO: 2. The term homologue refers to homology with respect to structure and / or function, provided that the resulting recombinant enzyme has PME activity. In terms of sequence homology (i.e., similarities), a homology of at least 75%, preferably at least 85%, and even more preferably at least 90% with the sequence shown in the attached sequence listing is preferred. More preferably, the homology is at least 95%, most preferably at least 98% with the sequence in the appended sequence listing.

The term variant, homolog, or fragment in connection with a nucleotide sequence encoding a recombinant enzyme of the present invention includes any substitutions, changes, modifications, substitutions, deletions or additions of one or more nucleic acids of the sequence provided that the resulting nucleotide sequence encodes a recombinant enzyme having PME activity, preferably at least the same activity as a recombinant enzyme comprising one or more of the sequences shown as SEQ ID NO: 1 and SEQ ID NO: 2. The term homologue refers to homology with respect to structure and / or function, provided that the resulting nucleotide sequence encodes a recombinant an enzyme having PME activity.

In terms of sequence homology (i.e., similarity), a homology of at least 75%, more preferably at least 85% and even more preferably at least 90% is preferred. More preferably, the homology is at least 95%, most preferably at least 98%.

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The above terms are synonyms with the term allelic sequence variation.

In a preferred embodiment, the at least one PME substrate is pectin or a substrate obtained or obtainable from a pectin (e.g., a pectin derivative).

The term derived from pectin refers to derivatized pectin, degraded (e.g., partially degraded) pectin, and modified pectin. An example of a modified pectin is pectin that has been pretreated with an enzyme such as PME. An example of a pectin derivative is a pectin that has been chemically treated, for example, amidated.

Preferably, the first PME substrate and the second PME substrate are independently selected from pectin, a substrate obtainable from pectin or a substrate obtained from pectin.

In one preferred embodiment, the first substrate for PME and the second substrate for PME are pectin.

In another preferred embodiment, the first PME substrate or the second PME substrate is a modified pectin - in particular an enzymatically modified pectin, preferably a pectin treated with PME.

In a preferred embodiment, the second PME substrate is such a modified pectin.

In another preferred embodiment, the first PME substrate and the second PME substrate are such modified pectin.

Preferably, the first substrate for PME is present in (i.e. in situ) the plant or plant material. The plant may be a transgenic plant, for example, a plant that has been genetically modified to produce varying amounts and / or types of pectin. Similarly, plant material can be obtained from a transgenic plant, for example a plant that has been genetically modified to produce different amounts and / or types of pectin.

The plant or plant material may be obtained from a vegetable, fruit or other type of plant containing or producing pectin.

Preferably, the plant material is a material derived from vegetables and / or fruits.

Preferably, the material obtained from vegetables and / or fruit is a slurry.

The first PME substrate and / or the second PME substrate may be selected from low esterified pectin, intermediate esterified pectin or highly esterified pectin.

Preferably, the second substrate for PME is a low esterified pectin, a medium esterified pectin, or a highly esterified pectin. A protocol for determining the degree of substrate esterification for PME is given on page 58 of WO-A-97/03574 (the contents of which are incorporated herein by reference). This protocol is exemplified in the Examples (hereinafter).

In one preferred embodiment, the second substrate for PME is highly esterified pectin.

For the present invention, the first PME substrate and the second PME substrate are selected from a PME substrate that is calcium ion sensitive (Ca-sensitive) or which is not calcium ion sensitive (Ca-non-sensitive). Protocol for determination

3 4 · «4

Λ Λ Λ Λ calcium sensitivity is given on page 57 of WO-A-97/03574 (the contents of which are incorporated herein by reference). This protocol is also exemplified in the Examples.

In one preferred embodiment, the second PME substrate is Ca-sensitive.

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Preferably, the second PME substrate is added to the first PME substrate. The term added refers to the physical addition of a second PME substrate to a first PME substrate and vice versa.

The PME may be added at the same time as the second PME substrate, or before or after the second PME substrate is added.

Therefore, the present invention includes at least the following options: adding PME to the first PME substrate at the same time as the second PME substrate is added; adding PME to the second PME substrate at the same time as adding the first PME substrate; adding a second PME substrate to the PME and at the same time adding a first PME substrate; adding PME and a first PME substrate and a second PME substrate to the reaction vessel at the same time; incubating the first PME substrate with PME before adding the second PME substrate; incubating the second PME substrate with PME before adding the first PME substrate; incubating the first PME substrate with PME before adding the second PME substrate and then adding another PME (which may be the same or different from the first PME); incubating the second PME substrate with PME before adding the first PME substrate and then adding another PME (which may be the same or different from the first PME); incubating the first PME substrate with PME before adding the second PME substrate and then adding additional PME substrates (which may be the same or different from the other substrates for PME).

PME); incubating the second PME substrate with PME before adding the first PME substrate and then adding additional PME substrates (which may be the same or different than the other PME substrates); adding a first PME substrate while incubating PME to a second PME substrate incubated with PME (which may be the same or different from the first PME); adding the first PME substrate after incubation with PME (optionally, the reaction may be terminated - for example by heat treatment) to a second PME substrate that has been incubated with PME, which may be the same or different from the first PME) (optionally, the reaction may be terminated) - for example by means of heat); as well as other combinations.

Preferably, the invention comprises any of the following embodiments: adding PME to the first PME substrate and at the same time adding a second PME substrate; adding PME to the second PME substrate and at the same time adding the first PME substrate; adding a second PME substrate to the PME and at the same time adding a first PME substrate; adding PME, a first PME substrate and a second PME substrate to the reaction vessel at the same time; incubating the first PME substrate with PME before adding the second PME substrate; incubating the second PME substrate with PME before adding the first PME substrate; incubating the first PME substrate with PME before adding the second PME substrate and then adding another PME (which may be the same or different from the first PME); incubating the second PME substrate with PME before adding the first PME substrate and then adding another PME (which may be the same or different from the first PME); incubating the first PME substrate with PME before adding to the second PME substrate and then adding another PME substrate (which may be the same or different than the other PME substrates); incubating the second PME substrate with PME before adding it to the first PME substrate and then adding another PME substrate (which may be the same or different from the other substrates for PME).

PME); adding a first PME substrate after incubation with PME to a second PME substrate (which may be the same or different from the first PME).

Thus, in one embodiment, a highly esterified second PME-treated PME substrate may be prepared, which may then be added to the first PME-substrate. Various PMEs (such as recombinant plant, fungal and bacterial PMEs) may be used in this procedure to modify the second substrate for PMEs, if appropriate for the use of PME substrates with different functions in the combined system.

That is, this embodiment of the present invention comprises a composition comprising PME; a first substrate for PME; and a second substrate for PME; wherein the first and second PME substrates are not derived from each other; and wherein at least the second substrate for PME has been treated with PME.

In another embodiment, it is possible to add a PME substrate to a highly esterified PME-treated first PME substrate.

Various PMEs (such as recombinant plant, fungal and bacterial PMEs) may be used in this procedure to modify the second substrate for PMEs, if appropriate for the use of PME substrates with different functions in the combined system.

In another embodiment, it is possible to prepare a highly esterified second substrate for PME treated PME, which may then be added to the highly esterified first substrate for PME treated PME. Various PMEs (such as recombinant plant, fungal, and bacterial PMEs) may be used in this procedure to modify the second substrate for PMEs when appropriate for the use of PME substrates with different PMEs.

functions in the combined system.

The composition may contain other ingredients, such as food ingredients. Typical food ingredients include acids such as citric acid, or sugars such as sucrose, glucose or invert sugar, or fruits, or other enzymes, preservatives, colorants, and other suitable ingredients.

The composition of the present invention may be used in the preparation of foods. For example, it may be a starting agent or intermediate in the preparation of foods.

Alternatively, the composition of the present invention may itself be a foodstuff.

The term food refers to food for a human and / or animal. Common foods include jams, marmalades, jellies, dairy products (such as milk or cheese), meat products, poultry products, fish products and pastries. The food may also be a beverage. The beverage may be a drinking yogurt, a fruit juice or a whey protein containing beverage.

Any PME can be used with other types of enzymes.

Examples of other types of enzymes are pectinases, pectin depolymerases, polygalacturonases, pectat lyases, pectin lyases, rhamno-galacturonases, galactanases, cellulases, hemicellulases, endo-E-glucanases, arabinases, acetylesterases, or pectin releasing enzymes, or combinations thereof.

These other types of enzymes may be added at the same time as PME, or may be added before or after PME addition.

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In one preferred embodiment, PME is used together with one or more polygalacturonasams, such as endo-polygalacturonases (for example according to WO-A-89/12648, the contents of which are incorporated herein by reference) and / or exo-polygalacturonases. (e.g., WO-A-94/14966, the contents of which are incorporated herein by reference). This embodiment is advantageous for the production of jams and marmalades because PME-treated substrates have a better controllable calcium sensitivity. As mentioned above, WO-A-97/03574 contains some useful knowledge for preparing suitable PMEs for use in the present invention regarding the use of recombinant DNA techniques. Some of these descriptions are provided below.

The recombinant organism for PME expression may be a prokaryotic or eukaryotic organism. Suitable prokaryotic organisms include E. coli and Bacillus subtilis. The transformation of prokaryotic hosts is well known in the art, see, for example, Sambrook et al., (Molecular Cloning: A Laboratory Manual, 2nd Edition, 1989, Cold Spring Harbor Laboratory Press). When using a prokaryotic organism, the gene must be suitably modified before transformation - for example, introns must be removed.

In one embodiment, the organism may be an organism of the genus Aspergillus, for example Aspergillus niger. Transgenic Aspergillus can be prepared by the technique described by Rambosek, J., and Leach, J., 1987, (Recombinant DNA in filamentous fungi:

Progress and Prospects. CRC Crit. Roar. Biotechnol. 6: 357-393), Davis RW, 1994 (Heterologous gene expression and protein secretion in Aspergillus, in Martinelli, SD, Kinghorn, JR (publishers) Aspergillus: 50 years on. Progress in industrial microbiology, vol. 29, Elsevier Amsterdam 1994, pp. 525-560), Ballance, DJ, 1991 (Transformation systems for Filamentous Fungi and an OverView of Fungal Gene structure. in Leong, SA, 99 99

Berka, R.M. (ed.), Molecular Industrial Mycology. Systems and

Applications for Filamentous Fungi, Marcel Dekker Inc., New York 1991, pp. 1-29) and Turner G., 1994 (Vectors for genetic manipulation, in Martinelli, SD, Kinghorn, JR (publishers)) Aspergillus: 50 years on. in industrial microbiology, Vol. 29, Elsevier Amsterdam 1994, pp. 641-666). However, the following description summarizes the knowledge necessary to prepare transgenic Aspergillus.

For nearly a hundred years, filamentous fungi have been used in many types of industrial production for the production of various organic compounds and enzymes. For example, traditional Japanese fermentations of breastfeeding and soybean utilized Aspergillus sp. In this century, Aspergillus niger was also used for the production of organic acids, especially citric acid, and for the production of various enzymes for industrial use.

There are two main reasons for using filamentous fungi in industrial production. The first is that the filamentous fungi can produce considerable amounts of extracellular products, for example enzymes and organic compounds such as antibiotics and organic acids. The second is that the filamentous fungi can grow on inexpensive substrates such as grains, bran, beet pulp, etc. The same reasons make filamentous fungi attractive for use in heterologous expression of recombinant PME.

For the preparation of transgenic organisms of the genus Aspergillus, expression constructs are prepared by insertion of the desired nucleotide sequences into a construct intended for expression in a filamentous fungus.

Several constructs for heterologous expression have been developed. These constructs may contain a promoter that is active in • φ φφφφ

Hou φφ · · · · φ · · · • • • • • φ φ φ φ φ φ φ φ φ φ φ φ φ φ Examples of promoters are fungal promoters for the intense expression of extracellular enzymes, such as the glucoamylase promoter or the α-amylase promoter. The nucleotide sequence may be fused to a signal sequence that causes secretion of the protein encoded by the nucleotide sequence. Usually a fungal signal sequence is used. The mushroom-active terminator is located at the end of the expression system.

Another type of expression system for fungal expression is one in which a nucleotide sequence can be fused to a minor or major portion of a fungal gene that encodes a stable protein. This protein may stabilize the protein encoded by the nucleotide sequence. In such a system, a cleavage site recognized by a specific protease may be inserted between the fungal protein itself and the protein encoded by the nucleotide sequence, so that the produced fusion protein can be cleaved at that position by the specific protease, releasing the protein encoded by the nucleotide sequence. For example, a peptidase-like site recognized by KEX-2 that is found in at least some Aspergillus organisms may be inserted. Such a fusion results in in vivo cleavage which results in the protection of the expressed product and in the absence of a larger fusion protein.

Heterologous expression in organisms of the genus Aspergillus has been described for several genes encoding bacterial, fungal, animal and plant proteins. Proteins may be deposited intracellularly if the coding sequence is not fused to the signal sequence. Such proteins accumulate in the cytoplasm and are usually not glycosylated, which may be beneficial for some bacterial proteins. When the nucleotide sequence is fused to a signal sequence, the protein accumulates extracellularly.

In terms of product stability and host modifications, • 4 · · ·· * ·

some heterologous proteins too stable in secretion into fungal culture medium. Most fungi produce several extracellular proteases that degrade heterologous proteins. To overcome this problem, special fungal strains with reduced protease production have been used to produce heterologous proteins.

Several transformation protocols have been developed to transform filamentous fungi (Ballance, 1991, supra). Many of these protocols are based on preparation of protoplasts and introduction of DNA into protoplasts using PEG and Ca ²⁺ ions. * The transformed protoplasts then regenerate and the transformed fungi are selected using various selective markers. Markers used for transformation include auxotrophic markers such as argB, trpC, niaD, and pyrG, antibiotic resistance markers such as benomyl resistance, hygromycin resistance and phleomycin resistance. A commonly used transformation marker is the amdS gene of A. nidulans, which, when present in many copies, allows the fungus to grow on acrylamide as the sole nitrogen source.

In another embodiment, the transgenic organism may be yeast. Yeasts are also used as organisms for the expression of heterologous genes. The Saccharomyces cerevisiae strain has a long history of industrial use, including for the expression of heterologous genes. Expression of heterologous genes in Saccharomyces cerevisiae is reported in Goodey et al. (1987, Biotechnology, D. R. Berry et al., Eds., Pp. 401-429, Allen and Unwin, London), and in King et al. (1989, Molecular and Cell Biology of Yeast, E. F. Walton and G. T. Yarronton, eds., Pp. 107-133, Blackie,

Glasgow).

Saccharomyces cerevisiae is suitable for expression of heterologous genes for several reasons. The first is that it is non-pathogenic to humans and is unable to produce certain endotoxins. The second is that it has a long history of safe industrial use for various purposes. This leads to being widely accepted. The third is that extensive commercial use and research on the organism has led to a good understanding of genetics and physiology, as well as to the determination of the characteristics of industrial fermentation of Saccharomyces cerevisiae.

An overview of the principles of expression of heterologous genes in Saccharomyces cerevisiae and secretion of gene products is given in E. Hinchcliffe, E. Kenny (1993, Yeast as a Vehicle for Expression of Heterologous Genes, Yeasts, Volume 5, Anthony H. Rose and J. Stuart Harrison , 2nd edition, Academie Press, Ltd.).

There are several types of yeast vectors, including integration vectors that require recombination with the host genome for maintenance, and auto-replicating plasmid vectors.

For the preparation of transgenic organisms of the genus Saccharomyces, expression constructs are prepared by inserting a nucleotide sequence into a construct intended for expression in yeast. Several types of constructs for heterologous expression have been developed. The constructs contain a yeast-active promoter fused to the nucleotide sequence, usually a yeast promoter, for example the GAL1 promoter, is used. Typically, a yeast signal sequence, such as a sequence encoding a SUC2 signal peptide, is used. The yeast-active terminator is located at the end of the expression system.

Several transformation protocols have been developed for yeast transformation. For example, transgenic organisms of the genus Saccharomyces can be prepared according to Hinnen et al. (1978, Proceedings of the National Academy of Sciences of the USA, 75:

• · · · · ·

1929); Beggs, J.D. (1978, Nature, London, 275: 104); and Ito, H. et al., (1983, J. Bacteriology 153: 163-168).

Transformed yeast is selected using a variety of selective markers. Markers used in transformation include auxotrophic markers such as LEU2, HIS4 and TRP1, dominant antibiotic resistance markers such as aminoglycoside resistance markers, for example G418.

Plants are another host organism. There are not many references in the art regarding the preparation of transgenic plants. There are two documents listing techniques that can be used to prepare transgenic plants, EP-B-0470145 and CA-A-2006454 - some of these techniques are listed below.

The basic principle of the construction of genetically modified plants is the insertion of genetic information into the plant genome so that the inserted genetic material is stably maintained.

There are several techniques for inserting genetic material where the main principle is either the direct introduction of genetic information or the indirect introduction of genetic information using a vector system. An overview of the techniques is given in Potrykus (Annu. Rev. Pian. Physiol. Plant Mol. Biol. (1991), 42: 205-225) and Christou (Agro-Food-Industry Hi-Tech (March / April 1994, 17-27). ).

A suitable plant transformation system may contain one vector, but may also contain two vectors. In the case of two vectors, the vector system is commonly referred to as a binary vector system. Binary vector systems are described in more detail in Gynheung An. et al. (1980), Binary Vectors, Plant Molecular Biology Manual A3, 1-19.

* Φ φ * * * * * * * * * * * * φ · ·

One system used to transform plant cells with said promoter or nucleotide sequence is a construct based on the use of an Agrobacterium tumefaciens Ti plasmid or an Agrobacterium rhizogenes Ri plasmid as described in An et al., (1986), Plant Physiol. 81: 301-305 and Butcher, D.N. et al. (1980) Tissue Culture Methods for Plant Pathologist, Ed .: D.S. Ingrams and J.P. Helgeson, 203-208.

Several different Ti and Ri plasmids have been constructed which are suitable for the construction of plants or plant cell constructs described above. An example of such a plasmid is the Ti plasmid pGV3850.

The nucleotide sequence or construct should be inserted into the Ti-plasmid between the T-DNA terminal sequences or adjacent T-DNA sequences so as not to disrupt the sequences immediately adjacent to the T-DNA, at least those sequences that are likely to be essential for inserting the modified T-DNA. -DNA into the plant genome.

From the above description, it is clear that when the organism is a plant, the vector system is preferably one that contains the sequences necessary for infection of the plant (e.g., a vir region) and at least one terminal portion of the T-DNA sequence that is located on the same vector. as a genetic construct.

Preferably, the vector system is the Ti-plasmid Agrobacterium tumefaciens or the Ri-plasmid Agrobacterium rhizogenes or a derivative thereof, since these plasmids are well known and widely used in the construction of transgenic plants and there are many vector systems based on these plasmids or derivatives thereof.

In the construction of a transgenic plant, it may be nucleotide

4444

4 4 4 4 ·

the sequence is first constructed in a microorganism in which the vector can replicate and where vector modifications can be readily made before insertion into the plant. An example of such an organism is E. coli, but other microorganisms having suitable properties may also be used. If the vector of the vector system as defined above has been prepared in E. coli, it is transferred, if necessary, to a suitable Agrobacterium strain, for example Agrobacterium tumefaciens. Thus, the plasmid bearing the nucleotide sequence or construct is transferred to a suitable Agrobacterium organism, for example Agrobacterium tumefaciens, so that an Agrobacterium cell carrying the nucleotide sequence is obtained, which is then transferred to a plant cell to be modified.

As disclosed in CA-A-2006454, there are a number of cloning vectors that contain an E. coli replication system and a marker that allows the selection of transformed cells. Vectors include, for example, pBR322, pUC series, M13 mp series, PACYC184, etc.

In this procedure, the nucleotide sequence may be inserted into a suitable restriction site in the vector. The plasmid is then used to transform E. coli. E. coli cells are cultured in a suitable nutrient medium and then harvested and lysed. A plasmid is thus obtained. Sequence analysis, restriction analysis, electrophoresis and other biochemical - molecular biological techniques are usually used as the analysis. After each manipulation, the DNA sequence may be cleaved and linked to another DNA sequence. Each sequence may be cloned in the same or in a different plasmid.

In any method for the insertion of the desired promoter or construct or nucleotide sequence into a plant, the presence and / or insertion of additional DNA sequences may be necessary. If φ · · 4 · · 4 4 4 4 4 4 4 4 4 4 4

For example, a Ti- or Ri-plasmid can be used to transform at least the right border and often the right and left borders of the Tia Ri-plasmid DNA as regions surrounding the inserted genes. The use of T-DNA for the transformation of plant cells has been extensively studied and is described, for example, in EP-A-120516; Hoekema, in: The Binary Plant Vector System Offset-drukkerij, Kanters, B.B., Alblasserdam, 1985 ,. Chapter V; Fraley et al., Crit. Roar.

Plant. Sci., 4: 1-46; and An et al., EMBO J. (1985), 4: 277-284.

Direct infection of plant tissue with Agrobacteria is a simple technique that is commonly used and described in Butcher D.N. et al., (1980) Tissue Culture Methods for Plant

Pathologist, Ed .: D.S. Ingrams and J.P. Helgeson, 203-208. For further description of these techniques, see Potrykus (Annu. Rev. Pian. Physiol. Plant Mol. Biol. (1991), 42: 205-225) and Christou (Agro-Food-Industry Hi-Tech (March / April 1994, 17- Using this technique, infection of a plant can be performed on some parts or tissues of the plant, ie on a part of a leaf, root, stem or other part of the plant.

Typically, in direct infection of plant tissues bearing the promoter and / or GOI of Agrobacterium, the infected plant is injured, for example, by incision with a knife, or by needle or scratching. The wound is then inoculated with Agrobacteria. The inoculated plant or plant part is then cultured on a suitable culture medium and developed into a mature plant.

In the construction of plant cells, these cells can be cultured and maintained using conventional culture methods, such as cell culture methods in a suitable culture medium supplemented with the necessary growth factors such as amino acids, plant hormones, vitamins, etc. The regeneration of transformed cells into genetically modified plants can be performed. using known techniques for regenerating plants from cells or tissue cultures, for example by selecting transformed shoots using antibiotics or by subculturing the shoots on a medium containing suitable nutrients, plant hormones, etc.

Further details regarding plant transformation can be found in EP-A-0449375.

In conclusion, a composition suitable for use as a foodstuff or suitable for use in the preparation of foodstuffs is described. The composition comprises PME; a first substrate for PME; and a second substrate for PME; wherein the first and second PME substrates are not derived from each other.

As mentioned above, PME substrates such as pectins obtained from plant sources are generally in the form of highly esterified pectin with a DE of greater than 70%. Sugar must be added to extracts containing these highly esterified PME substrates to achieve a dissolved solids content that induces gel formation. Usually a content of 55% dissolved solids is required. Syneresis occurs more frequently when the dissolved solids content is less than 55%. If syneresis occurs, expensive additives must be added to induce gel formation.

In the present invention we have surprisingly found that it is possible to induce gel formation in an extract containing a highly esterified PME substrate by adding a second highly esterified substrate for PME. The higher gel-forming ability of these combined highly esterified PME substrates at less than 55% dissolved solids content is completely unexpected. Previously, it has always been reported in the art that highly esterified pectins usually require a minimum of 55% content of ekt φ φ φ φ φ · · · · · · · · · · · · · · φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ dissolved solids to allow gel formation.

Therefore, in a more general aspect, the present invention comprises an aqueous system in a solidified gel state having a soluble solids content of less than 50% w / w; wherein gel formation is induced using a highly esterified PME substrate. In this aspect, the state of the solidified gel can be determined as described in the Examples (hereinafter).

The present invention differs from WO-A-94/25575 because this patent application does not disclose or suggest a composition comprising PME; a first substrate for PME and a second substrate for PME; wherein the first and second PME substrates are not derived from each other. This also applies to JP-A-63/209533.

It should also be noted that the description given on page 12 (lines 6-14) of WO-A-94/25575 differs from the present invention. The term vegetable or fruit products as used in lines 7 and 8 of WO-A-94/25575 does not explicitly describe a substrate for PME. In addition, the following sentence Alternatively (in our belief), natural pectin can be demethylated by the enzyme ... on lines 12 to 14 of WO-A-94/25575 clearly shows that the reaction medium proposed by WO-A-94/25575 contains only one PME substrate, not two PME substrates, as in the present invention.

Description of the figures in the attached drawings

Giant. 1 is a bar graph showing the effect of PPME modification, pectin addition and +/- calcium.

Giant. 2 is a bar graph showing the effect of fruit and pectin processing (P66) on PPME.

* · • • * * * * * * * * * * * * *

9

DETAILED DESCRIPTION OF THE INVENTION

Protocol 1: Calcium Sensitivity Index (CF)

Calcium sensitivity is measured as the viscosity of pectin dissolved in solution with 57.6 mg calcium per g pectin divided by the viscosity of exactly the same amount of pectin in solution, but in the absence of calcium. Calcium insensitive pectin has CF = 1.

Dissolve 4.2 g of pectin in 550 ml of hot water with sufficient mixing. The solution is cooled to approximately 20 ° C and the pH is adjusted to 1.5 with 1 N HCl. Adjust the volume of the pectin solution to 700 ml with water and mix. 145 g of this solution is dispensed into 4 viscosity tubes. To two tubes, add 10 mL of water (double measurement) and, while mixing, to the other two tubes, add 10 mL of 250 mM CaCl ₂ solution.

ml of acetate buffer (0.5 M, pH about 4.6) is added to all four tubes for measuring viscosity under magnetic stirring, causing the pH of the pectin solution to rise to more than 4.0. The magnets were then removed and the tubes were allowed to stand overnight at 20 ° C. Viscosity is measured the next day using a Brookfield viscometer. The calcium sensitivity index is calculated as follows:

viscosity of solution with 57.6 mg Ca ²⁺ / g pectin CF = —viscosity of solution with 0.0 mg Ca ² * / g pectin

Protocol 2: Degree of esterification (% DE)

2.5 g is added to 50 ml of 60% isopropanol and 5% HCl solution

30 / φ φ • φ φ ♦ • • • • φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ • • • • • The pectin solution is filtered through a glass filter and washed 6 times with 15 ml of a 60% isopropanol / 5% HCl solution and then washed with 60% isopropanol until the chloride is removed from the filtrate. The filtrate was dried overnight at 80 ° C.

ml of 0.5 N NaOH and 20.0 ml of HCl are mixed in a conical tube and 2 drops of phenolphthalein are added. Titrate this solution with 0.1 N NaOH until a permanent color change is obtained. The 0.5 N HCl solution should be slightly stronger than 0.5 N NaOH. The added volume of 0.1 N NaOH is indicated by V.

O

0.5 g of dried pectin (filtrate) is added to a conical tube and moistened with 96% ethanol. 100 ml of boiled and cooled distilled water are added and the resulting solution is mixed until all the pectin is completely dissolved. Then 5 drops of phenolphthalein are added and the solution is titrated with 0.1 N NaOH (until the color of the solution changes and the pH is 8.5). The amount of 0.1 N NaOH used is referred to herein as ν _χ . 20.0 ml of 0.5 N NaOH is added and the tube is shaken vigorously and then left to stand for 15 minutes. Add 20.0 mL of 0.5 N HCl and shake the tube until the pink color disappears. 3 drops of phenolphthalein are added and the solution is titrated with 0.1 N NaOH. The amount of 0.1 N NaOH used is referred to herein as V.

The degree of esterification (% DE:% carboxyl groups) is calculated as follows:

V - V

O% DE = ⁺ (V ₂ - in _o ) · ft ft ·· · ft · ft · 9 · ft · 9 · ft · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

Food preparations

Introduction

The food preparations of the present invention may contain other ingredients such as food ingredients. Typical food ingredients include acids such as citric acid, or sugars such as sucrose, glucose or invert sugar, or fruits, or other enzymes, preservatives, colorants, and other suitable ingredients.

For example, fruit causes not only its taste, color and texture in the gel, but also contributes to pectin and small amounts of solids. Depending on the quality and quantity of the flavor and coloring in the fruit, fruit usually constitutes from 25% to 60% of the jam. The solids content of ordinary fruit is about 10% brix, but a fruit concentrate containing about 65-70% brix may also be used. The pH of the fruit varies depending on the type of fruit, but most fruits have a pH between 3.0 and

3.5.

The pectin content also varies according to the type of fruit. For example, redcurrants, blackcurrants and oranges have a high pectin content and satisfactory gels can be obtained from this fruit by adding only small amounts of additional pectin. The choice of GRINDSTED ™ pectin depends on the type of jam. For example, GRINDSTED ^- ™ Pectin SS 200 is used in jams that do not contain pieces of fruit or contain very few pieces of fruit. The separation of fruits in such jams is not a problem and therefore slower setting pectin and lower filling temperature can be used. GRINDSTED ™ Pectin RS 400 is used in jams containing large pieces of fruit or whole fruits such as cherries or strawberries. In jams containing whole fruits, separation of fruits can be a problem and therefore fast-setting pectin such as GRINDSTED ™ Pectin RS 400 should be used.

»0000

0000

0 1 0 0 0 0 0 0 0

0 0 0

The choice of pectin also depends on the type of container used. If standard preserving jars are used, the filling temperature is less important with respect to the stability of the pectin, since the jars are cooled relatively quickly after filling and the pectin is not degraded. However, when jams are filled into large containers, for example 500 or 1000 kg, the cooling time is very long. At the center of such a large container, pectin will degrade and the gel will be less dense at the center than at the edges. Therefore, slower pectin is usually used for larger containers, which allows filling at lower temperatures and therefore protection against pectin degradation.

Sugar is added to jams for various reasons, such as:

1. for the delivery of soluble solids - HE pectins may require the presence of a minimum solids content of 55% in order to form a gel;

2. for sweetening;

3. to increase physical, chemical and microbiological stability;

4. to improve mouthfeel;

5. to improve color and gloss.

Sucrose is usually used, but other sugars may also be used, depending on taste requirements, sweetening efficiency, crystallization desired and structure. Sugar selection can also affect price.

Invert sugar has the same sweetening effect as sucrose, while glucose syrup, glucose and sorbitol have lower sweetening activity. High fructose corn syrup and fructose have a higher sweetening effect than sucrose.

The structure and stiffness of the gel, as well as the temperature required for gel formation, are to some extent influenced by changes in sugar composition.

* «♦ *..... 9

9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 99 99

99

9 9 9

9 9 9

9 9 9

99

The acid is added for two reasons: (1) partially reduces the pH to 3.0-3.2, allowing a satisfactory gel to be obtained from pectin; and (2) partially enhances the taste of the fruit. The optimum pH for gel formation using HE pectin depends on the pectin type and the solids content.

When GRINDSTED ™ Pectin SS 200 is used in a 65-68% Brix jam, a pH of 3.0-3.2 is optimal.

If the solids content is higher, the optimum pH is 3.1-3.3.

Conversely, if the solids content is lower, the optimum pH is 2.8-3.0.

When GRINDSTED ™ Pectin RS 400 is used, the optimum pH is approximately 0.2 units higher than the pH for GRINDSTED ™ Pectin SS 200.

The most commonly used acid is citric acid, monohydrate, in a 50% (w / v) solution.

Other acids (such as malic, tartaric or phosphoric acid) may be used but must already be in solution.

The choice of acid depends on the regulations, price and sweetness of the final product.

Citric acid causes a relatively strong acidity of the final product, while malic acid produces a weaker but longer lasting taste.

Tartaric acid can cause a slightly bitter taste and acid

Phosphorous causes sweeter flavor ..

Enzymatically processed pectin can prevent the syneresis that often occurs in the production of marmalades and jams with a low content of soluble solids.

Low sugar jam and jam with 25% SS

Means

Folders % % soluble solids Pre-prepared pectin solution Enzymatically processed pectin ¹ 0,6-0,8 0,6-0,8 (highly reactive with Ca) Sugar 3.6 3.6 Water 18.0 Fruit base PME ² 7-8 units Fruit with natural pectin * 45.0 4,5 Sugar 15.9 15.9 Water 21.1 Preservatives as required Citric acid** as required Total • * 104 Evaporator -4 Yield 100.0 Final solub content. solids 25 -25 Final pH 3.2

* Common fruits are strawberries, apples, cherries, oranges and blackcurrants.

** or any other food acid.

^The enzymatically modified pectin may be a pectin according to WO-A-97/03574.

² PME may be PME according to WO-A-97/03574.

Method

Preparation of pectin solution:

1. Dry blend of enzymatically modified pectin and sugar.

2. Dissolve the pectin-sugar mixture in hot water (80 ° C) with vigorous mixing.

Jam

1. Mix fruit, sugar and water.

2. Boil the fruit mixture briefly and cool to 40 ° C.

3. After cooling to 40 ° C, a solution of PME is added.

4. The reaction time for the fruit mixture is one hour.

5. Heat the fruit mixture at 85 ° C for several minutes and allow the jam to evaporate to the desired soluble solids content.

6. Add pectin solution.

7. Add preservatives and adjust pH.

8. Cool the jam to the filling temperature, fill and cool to ambient temperature.

This example may be modified by the addition or substitution of at least one food additive and / or the addition of another suitable enzyme (such as glucanase).

• · · · · · · · · · ·

Low sugar jam and jam with 50% SS

Means

Folders % % soluble solids Pre-prepared pectin solution Enzymatically processed pectin ¹ 0,4-0,7 0,4-0,7 (highly reactive with Ca) Sugar 2.2 2.2 Water 11.0 Fruit base PME ² 7-8 units Fruit with natural pectin * 45.0 4,5 Sugar 42.7 42.7 Water 3.1 Preservatives as required Citric acid** as required Total -105 Evaporator -5 Yield 100.0 Final solub content. solids 50 -50 Final pH 3.2

* Common fruits are strawberries, apples, cherries, oranges and blackcurrants.

** or any other food acid.

^The enzymatically modified pectin may be a pectin according to WO-A-97/03574.

² PME may be PME according to WO-A-97/03574.

• · · ·

Method

Preparation of pectin solution:

1. Dry-blended enzymatically modified pectin and sugar.

2. Dissolve the pectin-sugar mixture in hot water (80 ° C) with vigorous mixing.

Jam

1. Mix fruit, sugar and water.

2. Boil the fruit mixture briefly and cool to 40 ° C.

3. After cooling to 40 ° C, a solution of PME is added.

4. The reaction time for the fruit mixture is one hour.

5. Heat the fruit mixture at 85 ° C for several minutes.

6. Add the pectin solution and the remaining sugar and jam then allow to evaporate to the desired dissolved solids content.

7. The jam is cooled to the filling temperature, filled and cooled to ambient temperature.

This example may be modified by the addition or substitution of at least one food additive and / or the addition of another suitable enzyme (such as glucanase).

Modifications of the present invention will be apparent to those skilled in the art. For example, as mentioned in the examples above, both glucanase and PME can be used to obtain pectin with a low degree of esterification (e.g., slow-setting pectin).

Preparation of orange slurry processed with plant PME

Stage 1

The pieces of orange are homogenized in a kneading machine and boiled for 15 minutes to inactivate the endogenous enzymes. After freezing / thawing, 20% (w / w) sugar is added and the orange slurry is diluted 1: 1 with preheated (95-100 ° C) deionized water. The slurry is transferred to glass beakers and the pH is adjusted to 7.0 (with 10% NaOH) and the temperature is adjusted to 40 ° C.

The purified plant PME (300 μπιοΐ / min / ml) at a concentration of 113 μ1 / 200 g of orange slurry is incubated with the slurry at 40 ° C for 15 minutes and then the pH of the slurry is adjusted with citric acid (50% w / v). to 3.2 (± 0.6). To inactivate the added plant PME, the slurry is heated at 85 ° C for 3 minutes. While plant PME usually requires the addition of NaCl for its activity, this enzyme-modified orange slurry preparation does not require the addition of any exogenous NaCl, since sufficient endogenous NaCl (24 ppm) is present to provide PME activity.

Plant PME treated slurry (approximately 90 g) is stored in crystallization beakers (60 mm diameter, 35 mm depth) at 5 ° C. All pectin gelling measurements were performed over three days of processing and reproducible results were obtained.

Step 2: Selection, preparation and addition of pectin substrates

Selection: Three pectin substrates were selected for use. All three substrates, GRINDSTED ™ pectin SS 200, P66 and P60, had a high degree of esterification (% DE). These degrees of esterification were 65%, 66% and 60%, respectively. P60 and P66 were prepared by processing the GRINDSTED ™ Ultra Rapid Set (URS) pectin with PME, while GRINDSTED ™ pectin SS200 was unprocessed. Two of the three substrates, P60 and P66, were calcium sensitive, while GRINDSTED ™ pectin SS200 was calcium sensitive.

Only one substrate, GRINDSTED ™ pectin SS200, was commercially available.

Preparation: P60 and P66 were prepared by treating GRINDSTED ™ URS pectin with PME using the following procedure: GRINDSTED ™ URS pectin was solubilized in 0.15 M NaCl and treated with PME for several hours at pH 7.0 at 40 ° C. After adjusting the pH to 3.0, the solubilized pectin was heated at 100 ° C for 5-10 minutes to inactivate any PME present, and then was precipitated with isopropanol and dried before use. All three pectin substrates were prepared as 8% solutions and were completely dissolved in pre-heated (80 ° C) deionized water using a magnetic stirrer.

Addition: Addition of pectin substrates to the slurry of enzymatically modified PME was performed using a high speed magnetic stirrer to ensure solution homogeneity.

Step 3: Preparation and addition of calcium citrate

Pectin gelling can be induced by the addition of calcium, as a slurry or as a hydrate, at high temperature. Ca-citrate (C H Ca O 4 H O) was added to the mixture to a final concentration

IC 3 2 mm.

Step 4: Measurement of gel stiffness

The degree of gel formation / gel stiffness was determined by compression tests using a structure analyzer. The procedure is described in The Chemistry and Technology of Pectin, edited by Reginald H. Walker; Academie Press; (1991), p. 240 (the contents of which are incorporated herein by reference).

• · · · · · · · · · · · · · ·

The viscosity induced by compression was performed on a SMS TA-XT2 Texture Analyzer (Reciproter) using a cold storage probe (5 ° C) at a rate of 2.0 nun / s and a penetration of 30%. The sample temperature was 5 ° C. The peak force in the compression curve shows the gel strength in Newtons (N).

Step 5: Tests for syneresis and solidification

Syneresis is defined as the inability of pectin gel to form a solid. The gel showed syneresis unless it remained intact after the gel-containing reaction vessel was tipped over. The gel showed solidification if it remained intact after the gel-containing reaction vessel was tipped over. The intact gel had a very flat surface compared to the non-intact gel.

Results

Characterization of orange slurry processed by PME

Treatment of the orange slurry with plant PME resulted in a highly esterified pectin with an average degree of esterification (% DE) of 55.2% (% DE was determined according to protocol 2).

• ·

Table 1: Effect of calcium (0.096 / g Ca-citrate / 100 g orange slurry) and / or plant PME treatment on the orange slurry gel stiffness.

Treatment plant PME Ca-citrate (5 mM) Gel Stiffness (N) Solidification Gel (Yes / No) - - 0.175 No - + 0.234 No + - 0, 227 No + + 0.226 No

The gel stiffness of the extracted orange slurry was slightly affected by plant PME treatment or calcium addition (Table 1). However, sequencing the orange slurry with plant PME followed by the addition of calcium did not have a synergistic effect on gel stiffness. Visual examination of the gels showed that they were all in liquid form.

These results show that although the processing of the orange slurry with PME plant yields a more homogeneous product in terms of degree of esterification (% DE is 55.2%) and sensitivity to low endogenous calcium concentrations, the enzymatically modified slurry does not correspond to exogenous calcium calcium or in terms of higher gel stiffness.

These results also demonstrate that the reaction of PME treated orange slurry with endogenous or exogenous calcium ions is not sufficient to satisfactorily increase the gel stiffness.

The addition of a second pectin substrate, such as P60 or P66, to the plant PME treated slurry, in the presence or absence of calcium, induces a significant increase in gel stiffness compared to the treated control. orange mash (Table 2, Figure 1). Specifically, both P66 and P60 pectin substrates induced a 3- and 5-fold increase in gel stiffness, respectively, after the addition of each substrate to the plant PME treated orange slurry, in the presence of calcium. The increase in gel stiffness is slightly less pronounced for both pectin substrates in the absence of calcium in the reaction mixture.

Adding the GRINDSTED ™ pectin SS200 substrate to the plant PME treated orange slurry did not induce an increase in gel stiffness because the GRINDSTED ™ pectin SS200 substrate was not pre-treated with PME and therefore the combined substrates did not form a gel.

Table 2: Effect of combined pectin substrates and calcium on gel stiffness achieved

Pectin % pectin % solub. of particles PH Processed. grew. PME Citrate vápe- natý (5 mM) Rigidity gel (N) Relat. increase stiffness gel Solidification gel Y / N Porridge 0 0.175 100 ALIGN! No SS200 0.8 20 May 3.1 + 0 0.195 111 No SS200 0.8 20 May 3.2 + + 0.223 127 No P66 0.8 20 May 3.1 + 0 0.316 180 Viscous P66 0.8 20 May 3.1 + + 0.457 312 Yes P60 0.8 20 May 3.1 + 0 0.530 302 Yes P60 0.8 20 May 3.1 + + 0,782 446 Yes

The solidifying gel was obtained after mixing the P66 pectin substrate with an orange slurry treated with plant PME in the presence of • 999

9999

but not in the absence of calcium. In contrast, the setting gel was obtained after mixing the P60 pectin substrate with an orange slurry treated with plant PME in the presence and absence of calcium.

The additive effect of PME processing and the presence of calcium on pectin gelling is illustrated in Figure 2. This figure shows the percentage of relative increase in gel stiffness (or hardness) observed after adding P66 pectin substrate to the untreated and PME treated orange slurry in the presence or absence of calcium. Absolute gel stiffness values (N) are shown next to each bar, and the percent increase in gel stiffness is shown above each bar. When reading from left to right, the first column shows that the unprocessed orange slurry has a low gel stiffness, which is in liquid form upon visual inspection. After the addition of a second pectin substrate, such as P66, to the untreated orange slurry in the absence of calcium, no effect on the gel stiffness is achieved (column 2). However, when P66 is added to the PME treated orange slurry, a higher gel viscosity is observed (column 3). A similar increase in gel viscosity is observed when P66 is mixed with untreated orange slurry in the presence of calcium (column 4). Finally, when P66 is added to the PME treated orange slurry, a solid gel is obtained (column 5). These results are obtained regardless of the order in which the mixing and processing of the PME was performed. Thus, a solid gel is obtained when the GRINDSTED ™ URS pectin substrate and the orange slurry are mixed and treated with PME in the presence of calcium, or when the GRINDSTED ™ URS pectin substrate and the orange slurry are treated separately with PME and then mixed in the presence of calcium .

The experiments described in Figures 1 and 2 were repeated and the results are φφ φφφφ

ΦΦ ΦΦΦΦ φ φ φ φ φ φ φ φ φ φ · · · · · · · · · · · · · · · • • The visual examination of the gels obtained in these experiments confirmed the earlier finding that:

(i) the orange slurry alone or after treatment with plant PME and calcium, alone or in combination, does not induce an increase in gel stiffness;

(ii) adding calcium alone to GRINDSTED ™ URS pectin does not alter pectin function. Similarly, unless GRINDSTED ™ URS pectin is treated with a PME plant, an increase in viscosity but not solidification is observed when mixed with PME treated orange slurry in the presence or absence of calcium;

(iii) treating the GRINDSTED ™ URS pectin with plant PME to give P66, which is more effective when mixed with PME treated orange slurry in the presence of calcium. This higher efficiency results in induction of gel solidification in the sample;

(iv) processing of two pectin substrates, such as orange slurry and GRINDSTED ™ URS pectin, plant PME, leads to more potent highly esterified pectins that are capable, in the presence of calcium, of inducing solidification of the low solids jam.

Table 3: Effect of combined pectin substrates and calcium on gel stiffness

Pectin Plant. % % solub. treatment Citrate State PME pectin solid PME porridge Calcium gel of particles (+/-) Porridge 0 liquid Porridge + + liquid/ viscous Porridge 0 + liquid

·· φφφφ

9999 • φ φ • * · · · φ

9 9 Φ • 9 φ φ · »

ΦΦ ΦΦ Φ · · · · • • • • • • • •

Φ Φ ΦΦ

Table 3: Effect of combined pectin substrates and calcium on gel stiffness - continued

Pectin Plant. % pectin % solub. solid of particles treatment PME porridge (+/-) Citrate Calcium State gel PME Porridge + + liquid/ viscous URS * 0 0.8 20 May - 0 liquid URS * 0 0.8 20 May - + liquid/ viscous URS * 0 0.8 20 May + 0 liquid/ viscous URS * 0 0.8 20 May + + liquid/ viscous P66 * - 0.8 20 May - 0 viscous P66 * - 0.8 20 May - + viscous P66 * - 0.8 20 May + 0 solid P66 * - 0.8 20 May + + solid

* is GRINDSTED ™ URS pectin

Discussion

When the low soluble solid jam is prepared from an orange slurry homogenate, gel formation is not observed either after treatment with plant PME or after addition of exogenous calcium. Similarly, when a second highly esterified pectin substrate, such as P66, is added to the unprocessed orange slurry, it does not affect the ability to gel even when the second pectin substrate is pretreated with a PME plant. However, the increase in gel viscosity is ·· ·· 4 ·

4 is observed when a second highly esterified pectin substrate, such as P66, is combined with untreated orange slurry in the presence of calcium. A similar effect, in terms of increasing the viscosity of the gel, is observed when the same highly esterified pectin substrate (P66) is combined with PME treated orange slurry in the absence of calcium.

These results indicate that an increase in gel viscosity can be induced both by treating the orange slurry with plant PME prior to mixing with a highly esterified pectin substrate such as P66, and mixing the untreated orange slurry with P66 in the presence of calcium. These results also show that mixing PME treated orange slurry with P66 in the presence of calcium will result in a solid gel. This effect is observed regardless of the order in which the PME is mixed and processed. Thus, gel solidification is observed both in the treatment of PME pectin substrates prior to mixing, and in the treatment of mixed PME substrates in the presence of calcium.

Conclusion

When low soluble solid jam is prepared from orange slurry, gel formation is not observed either after treatment of orange slurry with plant PME or addition of exogenous calcium.

When the second highly esterified pectin substrate is added to the unprocessed orange slurry, it does not affect the ability to gel even when the second substrate is pretreated with plant PME.

When mixing PME treated orange slurry with highly esterified pectin substrates in advance ·· · ♦ ··

Gels with significant PME treatment, in the presence or absence of calcium, give gels with significant levels of PME treatment in the presence or absence of calcium. higher rigidity and better function. Therefore, processing of PME pectin substrates, alone or in combination, improves their gel formation capacity by preparing pectin substrates that are more potent highly esterified substrates in the presence of calcium.

Sequence ssq.i.d. no. and

MIKNMTDTDM MIMRTSNNRK LIESTSTVDG WPAWL3TGDR RLLQSSSVTP 50 NVWAADGSG NFXTVAAAVA AAPQGGTKRY IIRIKAGVYR ENVEVTKKHK 100 NIMFIGDGRT RTIITGSRNV VDGSTTFKSA TVAWGEGFL ARDITFQNTA 15 0 GPSKHQAVAL RVGADLSAFY NCDMLAYQDT LYVHSNRQFF VNCLIAGTVD 200 FIFGNAAAVL QNCDIHARKP NSGQKNMVTA QGRADPNQNT GIVIQKSRIG 250

ATSDLKPVQG SFPTYLGRPW KEYSRTVIMQ LNTLFYGEHQ NAGAGAGTSG RVKWKGFRVI LGSTGFPFSL GL

SSQ.I.D. NO. 2

MTRIKEFFTK LSESSTNQNI SNIPKKKKKL VNSRKNSGDN GNEPHKAILK SSC3STRYPD DVIEMSLNIT TTAVEHNYFG IQKLLKRTNL ELHKAVEDLE EYPNKKSLSQ HADDLKTLMS HVRDALSDGQ VHVEKMCSNA LAMIKNMTDT DGWPAWLSTG DRRLLQSSSV TPNVWAADG RYIIRIKAGV YRENVEVTKK HKNIMFIGDG SATVAWGEG FLARDITFQN TAGPSKHQAV DTLYVHSNRQ FFVNCLIAGT VDFIFGNAAA TAQGRADPNQ NTGIVIQKSR IGATSDLKPV MQSSITDVIH PAGWHEWDGN FALNTLFYGE VITSATEAQA FTPGSFIAGS SWLGSTGFPF

SSITDVIHPA GWHEWDGNFA 300 TSATEAQAFT PGSFIAGSSW 350 362

FLALFATLLV VAAVIGIVAG 50 LCFSAIAAVP EASXKVTSQK 100 ALIGN! TKREKVALHD CLETIDETLD 150 AAMTNQGTCL DGFSHDDANK 200 DMMIMRTSNN RKLIEETSTV 250 SGNFKTVAAS VAAAPQGGTK 300 RTRTIITGSR NWDGSTTFK 350 ALRVGADLSA FYNCDMLAYQ 400 VLQNCDIHAR KPNSGQKNMV 450 QGSFPTYLGR PWKEYSRTVI 500 HQNAGAGAGT SGRVKWKGFR 550

SLGL

584 9 9 9 9 9 9 9 9

9 • 9

9 9

99

SEQ.I.D. NO. 3

GTAGCAATGG GCTTGCTATG ATCAAGAACA TGACTGACAC TGACATGATG 50 ATCATGAGGA CTTCAAACAA CAGGAAGCTG ATAGAGGAGA CCAGTACGGT 100 ALIGN! TGATGGGTGG CCGGCGTGGC TGTCCACCGG AGACAGGAGG CTGTTGCAGT 150 CCTCGTCGGT GACACCGAAC GTGGTGGTGG CAGCAGATGG CAGCGGAAAC 200 TTTAAGACGG TGGCGGCAGC GGTGGCGGCG GCTCCTCAGG GAGGCACTAA 250 GCGGTATATT ATTAGGATTA AAGCCGGTGT TTATCGGGAA AATGTTGAGG 300 TGACAAAGAA GCATAAAAAT ATAATGTTCA TCGGTGACGG GAGGACTAGA 350 ACTATCATCA CAGGAAGTAG AAATGTGGTT GATGGAAGCA CAACTTTCAA 400 GTCTGCTACA gttgctgttg TTGGTGAAGG ATTCTTGGCC CGAGACATTA 450 5 CATTCCAAAA CACAGCCGGC CCCTCAAAGC ACCAGGCGGT GGCACTACGA 500 GTGGGAGCTG ACCTTTCAGC ATTTTACAAT TGCGATATGT TAGCTTACCA 550 AGACACACTC TACGTCCACT CGAACCGCCA GTTCTTTGTG AACTGCTTAA SOO TTGCTGGCAC GGTTGATTTT ATTTTTGGTA ACGCTGCAGC CGTGTTACAA 650 AATTGTGACA TCCATGCACG AAAGCCCAAT TCCGGCCAAA AAAATATGGT 700 10 CACAGCCCAA GGCAGGGCTG ACCCTAACCA AAACACCGGC ATTGTCATTC 750 AAAAATCTAG GATTGGTGCC ACCTCCGATT TAAAACCGGT TCAGGGTAGT 800 TTCCCGACGT ACCTCGGCAG GCCCTGGAAG GAGTACTCGA GGACGGTGAT 850 CATGCAGTCA TCGATTACTG ACGTGATCCA CCCTGCCGGG TGGCACGAGT 900 GGGATGGTAA CTTCGCGTTG AACACATTGT TTTACGGAGA GCATCAGAAC 950 15 Dec GCCGGAGCCG GTGCCGGAAC TTCAGGGAGA GTGAAATGGA AGGGATTTAG 1000 GGTTATTACA AGTGCTACCG AGGCTCAAGC TTTTACTCCT GGAAGCTTCA 1050 TTGCTGGTAG TAGCTGGCTG GGCTCCACTG GTTTCCCATT CTCCCTTGGT 1100 TTGTAATATT CACTAGGAGT TTTAATTAAT ATGTTTTGTA TTAGTGGATC 1150 CATAGGTCTC TGGTCTTTCA ATTTGTAATA TTTGATTGAG CGTGTCTTAT 1200 20 May TCGTGGCTTC GATTTCACAA ATACTATTGT GTGATTAACA AGAAATAAAA 1250 TAGCATGGGA AGAATAATAA TTTCCGGCTT CTTTAAAAAA AAAAAAAAAA 1300 AAAAAAAAAA AAAAAAAAAA AAA 1323 SEQ.I.D. NO. 4 CTTTTGTTCT CTCTTATCGA GAAAAAAAAT GACCCGCATA AAAGAATTCT 50 TCACAAAACT TTCTGAATCT TCTACCAACC AAAACATTTC CAATATTCCC 100 ALIGN! AAGAAAAAAA AGAAACTATT CTTAGCTCTT TTTGCAACGC TACTCGTTGT 150 CGCTGCCGTA ATCGGCATTG TCGCCGGAGT GAACTCAAGA AAAAACTCCG 200 30 GCGACAACGG CAACGAGCCT CATCATGCTA TCCTCAAATC ATCATGTAGC 250 AGCACAAGGT ACCCGGACTT ATGCTTTTCG GCTATTGCTG CCGTTCCAGA 300 GGCCTCCAAA AAGGTGACAA GCCAAAAGGA CGTTATTGAG ATGTCCTTAA 350 ACATCACAAC AACAGCCGTG GAACACAACT ACTTCGGGAT TCAGAAGCTC 400 TTGAAGAGAA CGAATCTCAC CAAACGGGAA AAGGTTGCTC TCCATGACTG 450 35 TCTTGAGACG ATCGATGAGA CTCTTGATGA GTTACACAAA GCCGTCGAGG 500

44 ··

44

4 4 4

4 4 4

4 4 4 4 4

4 43 ·· * «

ATCTTGAGGA GTACCCGAAC AAGAAATCTT TATCACAGCA TGCGGATGAT 550 CTCAAAACCC TAATGAGTGC CGCGATGACC AATCAGGGGA CGTGTCTTGA 600 TGGGTTCTCT CATGATGATG CTAATAAGCA CGTGCGGGAT GCGTTGTCAG 650 ACGGCCAGGT TCATGTTGAG AAGATGTGTA GCAATGCGCT TGCTATGATC 700 AAGAACATGA CTGACACTGA CATGATGATC ATGAGGACTT CAAACAACAG 750 GAAGCTGATA GAGGAGACCA GTACGGTTGA TGGGTGGCCG GCGTGGCTGT 800 CCACCGGAGA CAGGAGGCTG TTGCAGTCCT CGTCGGTGAC ACCGAACGTG 850 GTGGTGGCAG CAGATGGCAG CGGAAACTTT AAGACGGTGG CGGCATCGGT 900 GGCGGCGGCT CCTCAGGGAG GCACTAAGCG GTATATTATT AGGATTAAAG 950 CCGGTGTTTA TCGGGAAAAT GTTGAGGTGA CAAAGAAGCA TAAAAATATA 1000 ATGTTCATCG GTGACGGGAG GACTAGAACT ATCATCACAG GGAGTAGAAA 1050 TGTGGTTGAT GGAAGCACAA CTTTCAAGTC TGCTACAGTT GCTGTTGTTG 1100 GTGAAGGATT CTTGGCCCGA GACATTACAT TCCAAAACAC AGCCGGCCCC 1150 TCAAAGCACC AGGCGGTGGC ACTACGAGTG GGAGCTGACC TTTCAGCATT 1200 TTACAATTGC GATATGTTAG CTTACCAA.GA CACACTCTAC GTCCACTCGA 1250 ACCGCCAGTT CTTTGTGAAC TGCTTAATTG CTGGCACGGT TGATTTTATT 1300 TTTGGTAACG CTGCAGCCGT GTTACAAAAT TGTGACATCC ATGCACGAAA 1350 GCCCAATTCC GGCCAAAAAA ATATGGTCAC AGCCCAAGGC AtíGGCTGACC 1400 CTAACCAAAA CACCGGCATT GTCATTCAAA AATCTAGGAT TGGTGCCACC 1450 TCCGATTTAA AACCGGTTCA GGGTAGTTTC CCGACGTACC TCGGCAGGCC 1500 CTGGAAGGAG TACTCGAGGA CGGTGATCAT GCAGTCATCG ATTACTGACG 1550 TGATCCACCC TGCCGGGTGG CACGAGTGGG ATGGTAACTT CGCGTTGAAC 1600 ACATTGTTTT ACGGAGAGCA TCAGAACGCC GGAGCCGGTG CCGGAACTTC 1650 AGGGAGAGTT AAATGGAAGG GATTTAGGGT TATTACAAGT GCTACCGAGG 1700 CTCAAGCTTT TACTCCTGGA AGCTTCATTG CTGGTAGTAG CTGGCTGGGC 1750 TCCACTGGTT TCCCATTCTC CCTTGGTTTG TAATATTCAC TAGGAGTTTT 1300 AATTAATATG TTTTGTATTA GTGGATCCAT AGGTCTCTGG TCTTTCAATT 1850 TGTAATATTT GATTGAGCGT GTCTTATTCG TGGCTTCGAT TTCACAAATA 1900 CTATTGTGTG ATTAACAAGA AATAAAATAG CATGGGAAGA ATAATAATTT 1950

1975

Claims (18)

A gel-forming composition comprising pectin methyl esterase (PME), a PME substrate and a pectin or PME-treated pectin derivative; wherein the first and second PME substrates are not derived from each other. The composition of claim 1, wherein the PME is recombinant. A composition according to claim 1 or 2, wherein the PME substrate is pectin or a derivative thereof. Composition according to any one of the preceding claims, characterized in that the PME substrate is present in the plant and / or plant material. A composition according to claim 4 wherein the plant or plant material is a vegetable, a vegetable derived material, a fruit or a fruit derived material. Composition according to claim 1, characterized in that the vegetable-derived material and / or the fruit-derived material is a slurry. A composition according to any one of the preceding claims wherein the pectin or PME-treated derivative thereof is highly esterified pectin. Composition according to any one of the preceding claims, characterized in that the PME substrate is a highly esterified substrate. • · • · β λ Composition according to any one of the preceding claims, characterized in that it is in a solid gel state and has a content of soluble solid material of less than 50% (w / w). Composition according to any one of the preceding claims, characterized in that it is a foodstuff or is used for preparing foodstuffs. 11. A method of preparing a composition comprising a PME substrate and a pectin or pectin derivative treated by PME, comprising treating the PME substrate and the pectin or pectin derivative PME before, during and / or after mixing said substrate and pectin or pectin derivative. in the composition; wherein the PME substrate and the pectin or pectin derivative are not derived from each other. The method of claim 11, wherein the PME is recombinant. The method according to claim 11 or 12, wherein the PME substrate is the same as defined in any one of claims 3 to 6. Process according to any one of claims 11 to 13, characterized in that it is intended for the preparation of foodstuffs. A method for improving the stability of a reaction medium comprising a substrate for PME, comprising adding at least PME and a pectin or pectin derivative; wherein the PME substrate and the pectin or pectin derivative are not derived from each other. • · Process according to claim 15, characterized in that the reaction medium is a foodstuff or is intended for the preparation of foodstuffs. A composition which is obtainable by a process according to any one of claims 11 to 13. The composition of claim 17, wherein the composition is in a solid gel state and has a soluble solids content of less than 50% (w / w).