ITPD20070065A1 - USE OF A PROTEIN INHIBITOR OF PECTINMETHYLESTERAS FOR THE REDUCTION OF THE FORMATION OF METHANOL IN GRAPE MUST AND WINE AND PROCESS FOR THE SAME - Google Patents

Description

Description of the industrial invention entitled:

"Use of a protein inhibitor of pectinmethylesterase for the reduction of methanol formation in grape musts and marc and process for the same"

FIELD OF THE INVENTION

The invention relates to the use of a protein or a portion thereof having pectinmethylesterase inhibitory activity to reduce the formation of methanol in grape musts and pomace and their derivative products for fermentation and distillation and to the process for the same. .

STATE OF THE TECHNIQUE

Pectin methylesterases (hereinafter also referred to as PME) are endogenous enzymes present ubiquitously in plants, where they have the important function of hydrolyzing the pectins secreted in the cell wall in their highly methyl-esterified form. The hydrolytic activity of these enzymes is aimed at the de-methylation of pectins with the formation of polygalacturonic acid domains capable of complexing calcium and thus forming gelatinous macromolecular structures. This deesterification also affects other physiological parameters and constitutes the prerequisite for the action of other degradative enzymes such as endopoligalacturonases which degrade polygalacturonic acids into pectic fragments.

However, this hydrolytic process is undesirable in the food canning industry for the production of juices, such as fruit juices, and concentrates, such as tomato concentrates, as this demethylation of the pectins results in a destabilization of the product with a loss of consistency. and flocculation of the vegetable preparation.

It is known that physical processes such as treatments of plant products at high temperatures and pressure can be used to inactivate endogenous plant pectinmethylesterases. However, these processes are complex and expensive in terms of energy consumption and negatively affect the quality of the final product. Furthermore, these processes are not completely efficient since there are extremely thermostable PME isoforms that can remain active even after severe heat treatments.

In the US patent 5,053,232 of 1991 and in the Italian patent RM2003A000346 of 2003 a protein inhibitor of PME (hereinafter also referred to as PMEI) is described which allows to overcome the limits deriving from these physical processes for the inactivation of pectinesterases. This protein inhibitor derived from kiwi (Actinidia chinensis) or obtained by recombinant route is capable of inhibiting vegetable PME in a way.

In fact, it has been shown that the PMEI of kiwifruit is able to inhibit the activity of PME present in tomatoes, apples, bananas and potatoes with high efficiency (Balestrieri C., Castaldo D., Giovane A., Quagliuolo L, Servillo L ( 1990) "A glycoprotein inhibitor of pectin methylesterase in kiwi fruii (Actinidia chinensis)" Eur.J.Biochem., 193, 183-187).

Protein inhibitors of PME were subsequently identified also in Arabidopsis thaliana (Raiola A., Camardella L, Giovane A., Mattei, B., De Lorenzo G., Cervone F. and Bellincampi D. (2004) "Two Arabidopsis thaliana genes encode functional pectin methylesterase inhibitors ”FEBS Leti., 557, 199-203).

The natural kiwi inhibitor has been extensively characterized biochemically. The inhibitor has a molecular mass of about 16 kDa and its complete amino acid sequence has been determined which presents some microheterogeneities (together with the most represented residues such as Alanine 56, Threonine 63, Tyrosine 78, Serine 117, Asparagine 123 and Vaiina 142 have been detected in smaller quantities Serine, Alanine, Phenylalanine, Asparagine, Aspartic acid and Isoleucine in the corresponding positions) attributable to the presence of multiple isoforms (Camardella L., Carratore V., Ciardiello M.A., Servillo L., Balestrieri C. and Giovane A (2000) "Kiwi protein inhibitor of pectin methylesterase amino-acid sequence and structural importance of two disulfide bridges" Eur. J. Biochem., 267, 4561-4565; Xiao-Hong Mei, Yan-Ling Hao, Hong-Liang Zhu, Hong-Yan Gao and Yun-Bo Luo (2006) “Cloning of pectin methylesterase inhibitor from kiwi fruii and its high expression in Pichia pastoris” · Article in print and published online in: Enzyme and Microbial Technology). The prevailing isoform, expressed at high efficiency in Pichia pastoris, has the same chemical characteristics of the natural inhibitor and its 3D structure has recently been resolved (Di Matteo A., Giovane A., Raiola A., Camardella L, Bonivento D ., De Lorenzo G., Cervone F., Bell incampi D., Tsernoglou D. (2005) “Structural Basis for the Interaction between Pectin Methylesterase and a Specific Inhibitor Protein” Plant Cell 17, 849-858).

In addition to the destabilizing effect of juices and concentrates due to PME mentioned above, it should also be taken into account that in the reaction catalyzed by PME, in which the highly methylated pectin is converted into pectin with low methoxyl and pectic acids, methanol is released, which is known as a volatile compound, very toxic to humans. This aspect is particularly significant in fruit juices or concentrates from vegetable sources in which the pectins are present in a highly methylated form and / or the pectin esterases are particularly active. Dietary methanol can also result from the degradation of synthetic sweeteners (such as aspartame) (Davoli E., et al. (1986). "Serum Methanol Concentrations in Rats and in Men After a Single Dose of Aspartame" Food and Chemical Toxicology, 24, 187-189).

The finding that methanol can also be produced in different ways in the human body, therefore makes it necessary to lower its intake with the diet to avoid an accumulation effect of this harmful alcohol. For this reason, the European Union has set limits on the concentration of methanol in foods, limits that must be respected especially for products deriving from fermented and distilled by-products of the winemaking process such as grappa (EC1576 / 89), obtained from distillation of the marc. In this particular alcoholic beverage, the presence of methanol can easily exceed the threshold of 1 mg per 100ml of anhydrous alcohol.

In the specific case of distillates from by-products of the fermentation of grape musts to avoid the presence of methanol beyond the legal limits, a common situation when distilling preserved pomace, for example, we do not intervene by trying to deactivate the enzyme responsible for the release of methanol, but a second distillation with de-methylating column is used. However, this intervention has the disadvantage of leading to a loss of the aromatic fraction of the distillate. The distillate is therefore qualitatively downgraded, having lost its typical aroma, a fundamental characteristic for differentiating the different distillates from each other.

The object of the present invention is therefore to identify means capable of effectively reducing the production of methanol in grape musts and marc and their fermented and distilled products.

SUMMARY

Following extensive research, the inventors have now shown that the inhibitor of pectin methylesterase obtained from kiwifruit or obtained recombinantly is also active against pectin methylesterase in grapes and marc and is capable of effectively inhibiting the formation of methanol in musts. grapes and in marc.

Therefore, the object of the present invention is the use of a pectin methylesterase inhibitor or a portion thereof having inhibitory activity of pectin methylesterase for the reduction of methanol formation in grape musts and marc and products derived from these by fermentation and distillation.

Preferably, the pectinmethylesterase inhibitor is from Actinidia chinensis and is obtained by purification from kiwi or recombinantly. The object of the invention is also the process for reducing the formation of methanol in grape musts and marc and products derived therefrom based on the use of the PME inhibitor from Actinidia chinensis or of a portion thereof having inhibitory activity of pectin methylesterase. obtained by purification from kiwi or by recombinant method. BRIEF DESCRIPTION OF THE FIGURES

Figure 1: the figure shows the results obtained on 5 µl of Muscat grape extract and 5 μΙ of different solutions of possible inhibitors, including the one containing PMEI, were loaded into the different wells, thus keeping the final volume constant equal to 10 μl. The Petri dishes were incubated at 30 ° C for 21 h, time necessary for the complete performance of the enzymatic activity of the loaded sample. The wells contained (1) 0.1 M disodium EDTA, (2) 0.1 M CaCI2, (3) 0.1 M (NH4) SO4, (4) 0.1 M sucrose, (5) 0.1 M (NH4) CI, (6) 0.1 M D - (+) - galacturonic acid, (7) 0.6 mg / ml PMEI kiwi, (8) blank, (9) control (Muscat grape extract only).

Figure 2: the figure shows the results obtained on the inhibition capacity of PMEI with scalar doses of protein corresponding to different enzymatic units present in the Muscat grape extract. In the right plate only the enzymatic extract was loaded, while in the left one there was also PMEI.

DETAILED DESCRIPTION OF THE INVENTION

The characteristics and advantages of the present invention will be better understood from the following detailed description, in which the preparation of the inhibitor and the data of inhibition of the production of methanol with this inhibitor of kiwi in grape musts and pomace.

The ability to inhibit the PMEs of the natural protein of Actinidia chinensis and of the same protein expressed in Pichia pastoris, the biochemical characteristics and other industrial applications of this protein inhibitor are, as already anticipated, widely described in US patents 5,053,232 and RM2003A000346 and in the works published by Camardella L. et al. and by Di Matteo et al. (Camardella L, et al. (2000) ref. Cit.; Di Matteo A, et al. (2005) ref. Cit.).

Nevertheless, the ability of this protein to significantly inhibit the formation of methanol resulting from the demethylation of pectins in general and in particular in grapes has never been studied. The present invention is therefore based on the use of these PME inhibitors to obtain a reduction in the concentrations of methanol in grape musts or marc and in products obtained from fermentation or distillation from these. For this purpose, the use of kiwi PMEI which, according to the greater industrial convenience, can be obtained both naturally from kiwi fruit and expressed in Pichia pastoris, is preferential.

In fact, only in recent times has the attention of both research and consumers been focused on the close link between nutrition and health. Hence the attention of public opinion to the toxic compounds present in food and systems to eliminate them. In the canned sector, the presence of the PME enzyme has always been related to the production of juices with greater or lesser consistency and / or turbidity. The problem of the presence of methanol in juices has always been ignored, considering as important only the possibility of inhibiting PME to avoid the enzymatic activity of the other polygalacturonase enzymes which act in sequence (and are much more thermostable), but are not able to catalyze the reaction if the PME has not acted before.

In the case of grappa, where the concentration of methanol is fixed by law, the achievement of these legal limits (almost always exceeded in the distillate obtained without the use of the demethylating column) involves a damage in terms of quality because it obliges to use the demethylating column. which cancels the aromatic typicality of the distillate itself. In conclusion, the possibility of inhibiting the vegetable PME enzyme, whose activity is responsible for the release of methanol, can have a double advantage: lowering the concentration of methanol in the juices (thus determining a lower ingestion of it with the diet) and at the same time offering, in the in the case of distillates, a superior quality in terms of aromatic typicality. For the purposes of the present invention, the PME inhibitor that can be used is the natural single-chain polypeptide having a molecular mass of about 16 kDa and an isoelectric point of about 4.3, it is preferably extracted from kiwi fruit, or it can be a polypeptide obtained by recombinant route and comprising the following amino acid sequence or portions thereof:

Seq.IDNI ENHLISEICPKTRNPSLCLQALESDPRSASKDLKGLGQFSIDIAQASAKQ TSKIIASLTNQATDPKLKGRYETCSENYADAIDSLGQAKQFLTSGDYNS LNIYASAAFDGAGTCEDSFEGPPNIPTQLHQADLKVISLK.

The PME inhibitor that can be used to reduce the production of methanol in grape musts and marc or in products obtained by fermentation or distillation from these, can be obtained by known means such as for example extraction and separation from fruits as described above (Balestrieri C. et al. {1990) ref. cit.) or on the basis of the sequence indicated above obtained by molecular biology techniques of recombinant DNA and biochemical purification as already described by Di Matteo A. et al. {Di Matteo A. et al. (2005) ref. cit.)

For the purposes of the present invention, the reduction of methanol formation in grape musts and marc is obtained by using the PME inhibitor from kiwifruit or obtained recombinantly under the following conditions:

- incubation of grape must with the inhibitor in the ratio of 20,000 parts of must and at least 1 part of inhibitor respectively, at a temperature between 25-35 * C for at least 7 days, with daily pumping over.

- incubation of the marc with the inhibitor in the ratio of 2,000 parts of marc and at least 1 part of inhibitor respectively, at a temperature between 25-35 * C for a period of between 14 and 28 days.

At the end of the process, the PME inhibitor can be eliminated from the must by known means already used in the sector, such as protein stabilization processes. In the case of marc, the protein inhibitor is denatured during the distillation process.

Experimental part

In order to verify if the kiwifruit PME inhibitor is able to determine an inhibition of PME in grape musts and pomace and also to determine a significant reduction in the production of methanol in grape musts and pomace, the inventors conducted the experiment described below.

Example 1: preparation of the PME inhibitor

A-Purification of the natural inhibitor

Ripe kiwi fruits are homogenized in water in the ratio 1: 1 (mass: volume) and centrifuged at 15000 x g for 20 minutes at 4 * C. The supernatant is brought to pH 7.5 with NaOH and directly precipitated with ammonium sulphate 70% saturation, The precipitate is resuspended in 10 mM tris / HCI pH 7.5, 50 mM NaCl and dialyzed overnight against the same buffer, the dialysate is loaded on Q-Sepharose column equilibrated in 10 mM tris / HCI pH 7.5 and 100mM NaCl. After washing, the column is eluted in a linear gradient from 100 to 400 mM NaCl. The fractions containing inhibitory activity are collected, concentrated by ultrafiltration and loaded onto a preparative Superose 12 column equilibrated in 10 mM Tris / HCl, pH 7.5 and 100mM NaCl. The inhibitor thus purified is analyzed SDS-gel electrophoresis at 12.5%.

B-Purification of PMEI expressed in Pichia pastoris

The culture broth of Pichia pastoris is filtered and precipitated with ammonium sulphate at 80% saturation. The precipitate thus obtained is collected by centrifugation at 20000xg for 20 min, resuspended in a 10 mM TrisCl buffer, pH 8.5 and exhaustively dialyzed against the same buffer. After dialysis, the solution is centrifuged 20000xg for 20 min and loaded onto a MonoQ <®> column (HR 10/10, Pharmacia) equilibrated with the same buffer. The PMEI is then eluted with a linear gradient from 0 to 0.5 M NaCl in 30 min at a flow of 1.5 ml / min. The fractions containing inhibitory activity are pooled, concentrated by ultrafiltration on Centricon 3 filters (Amicon) and loaded on a Sephadex <®> G75 column (1.5 x 90 cm) equilibrated in 10 mM TrisCI, pH 7.5, 250 mM NaCI at one flow of 5 ml / h. The fractions containing inhibitory activity are pure when analyzed by SDS-gel electrophoresis at 12.5% and stained with a solution of Coomassie® brillant blue G250. The calculated molecular weight of 16 KDa corresponds to that deduced from the amino acid sequence (16,753 Da), and in fact there are no glycosylation sites in the protein. The protein shows only one peak when analyzed by HPLC on a reversed phase column. The N-terminal sequence (EAEFENHLISEI-PK) corresponds to the sequence of the cloned and expressed gene, and shows the presence of 4 additional amino acids (in bold) corresponding to the residue of the Pichia signal peptide.

Example 2: inhibition of pectinmethylesterase activity in grape musts with the addition of methylated pectin

The inventors then proceeded to develop a sensitive method of diffusion on a petri dish (Downie B., Dirk L.M., Hadfield K.A., Wilkins T.A., Bennett A.B., Bradford K.J. (1998) "A gel diffusion assay for quantification of pectin methylesterase activity "Anal.Biochem.

264, 149-157) to determine the pectinmethylesterase enzymatic activity of the grape.

The plant extracts obtained from Moscato grapes harvested at the same stage of ripeness were incubated on a plate, in the presence of highly methylated pectin (94%), at 25-35'C for 21 hours. The activity of the enzyme was expressed on the basis of the standard curve (range from 0.72U up to 0.04U) obtained from the logarithmic transformation of the activity obtained using different concentrations of a commercial pectin methylesterase from orange against the diameter of the colored zone. product.

Subsequently, to determine if it is possible to inhibit grape pectinmethylesterase, plate tests were carried out in the presence of the inhibitors: Na2EDTA, calcium chloride, ammonium sulfate, sucrose, galacturonic acid and the PME inhibitor (PMEI) extracted from kiwifruit.

The plate assay of pectinmethylesterase activity in the presence of possible inhibitors was conducted as follows:

5 µl of Muscat grape extract and 5 μl of different solutions of possible inhibitors, including the one containing the PME inhibitor, were loaded into the different wells, thus keeping the final volume equal to 10 μΙ constant. The Petri dishes were incubated at 25-35 'C for 21 h, the time necessary for the complete performance of the enzymatic activity of the loaded sample. The wells contained (1) 0.1 M disodium EDTA, (2) 0.1 M CaCl2, (3) 0.1 M (NH4) S04, (4) 0.1 M sucrose, (5) 0.1 M (NH4) CI, (6) 0.1 M D - (+) - galacturonic acid, (7) 0.6 mg / ml PMEI of kiwifruit, (8) blank, (9) control (Muscat grape extract only).

The results are shown in Table 1 below and Figure 1.

Table 1

Of the 7 possible inhibitors of Muscat grape PME tested on the plate, only the one derived from kiwifruit (PMEI) showed a positive response (total inhibition of pectinmethylesterase activity) which can be viewed with the complete absence of a red halo on the plate (Fig. 1).

The inhibitory activity of PMEI was then calculated: a) based on the concentration of the enzyme; b) based on the concentration of the inhibitor.

In the first case (a) the inhibition capacity of PMEI was tested with scalar doses of protein corresponding to different enzymatic units present in the Muscat grape extract.

The results are shown below in Table 2 below.

Table 2

Protein Extract PMEI volume 0 Unit loaded with PMEI extract [μg] (mm) enzymatic (μl) (μg) loaded and (U) (μl)

4 3.112 0 0 12 0.1603 4 3.112 2 1.2 0 0 6 4.668 0 0 12.7 0.1966 6 4.668 2 1.2 0 0 8 6.224 0 0 13.7 0.2633 8 6.224 2 1.2 0 0 In the second case (b) to determine the minimum dose of inhibitor in able to inhibit the pectinmethylesterase activity of a fixed amount of enzyme, different aliquots of PMEI were added to the same amount of protein present in the Muscat grape extract. The results obtained made it possible to establish that 0.12 micrograms of inhibitor is the minimum dose necessary for there to be a total inhibition of 0.135 enzyme units, corresponding to 4.60 micrograms of protein, present in the extract.

The results are shown below in Table 3 below.

Table 3

No. of Protein Extract PMEI Volume 0 Well unit loaded with enzymatic PMEI extract [μg] (mm) (μl) (μg) loaded (U) (pi)

1 8 4.60 0 0 12.8 0.2024 2 8 4.60 2 0.12 0 0 3 8 4.60 2 0.012 12.8 0.2024 It has also been shown that the PMEI inhibitor inhibits the PME of Moscato grapes even at pH 3.5 (0 = 4.0 mm loading 4.6 pg protein Moscato (equal to 8 pi)

0 = 0 mm by loading 4.6 pg Muscat protein and 1.2 pg PMEI (equal to 2 μl).

Example 3: inhibition of methanol formation in grape musts with the addition of methylated pectin

It was therefore verified whether the inhibitory capacity towards PME of kiwifruit PMEI also corresponded to a significant inhibitory activity of the release of methanol following the de-methylation of pectin and measured via HS-SPME-GC (table 4). Under the same conditions of temperature, pH and incubation time 74.43 ppm of methanol are produced by the control consisting of the enzymatic extract of grape pectinmethylesterase and a 0.1% solution of 94% methylated pectin, while the same extract in the presence of 6 micrograms of inhibitor releases only 18.86 ppm of methanol.

Table 4

PMEI Protein Sample Volume [μg] Methanol Muscat Inhibitor [ppm] Loaded (μg) PMEI

loaded (μl)

Grape extract 155 0 0 74.43 Moscato

without PMEI

Grape extract 155 10 6 18.86 Moscato con

PMEI

Consequently, the use of the inhibitor allows to reduce the concentration of methanol released by 75%.

Example 4: inhibition of methanol formation in grape musts without the addition of methylated pectin

The inhibitor was also tested on grape must and Moscato marc in environmental conditions similar to those that can normally be found in the cellar and distillery. In the test with the must, 50 µl of inhibitor equal to 75 μg of PMEI was added to 1.0 ml of unfiltered grape juice. In the white, however, there were 50 µl of distilled water. The samples were incubated in hermetic vials at 25-35 ° C for 7 days and kept under constant shaking. At the end of the incubation, after HS-SPME-GC analysis, where the inhibitor was present, an inhibition of the formation of methanol equal to 8.5% was found. In the case of pomace at 100 mg of the same, two different amounts of inhibitor equal to 75 and 150 pg of PMEI were added and the samples were incubated at 25-35 ° C for 14 and 28 days. In the samples analyzed after 14 days, where the inhibitor is present, a reduction of methanol of 16.6% occurred, while after 28 days of incubation the inhibition was between 19.5% and 33.3%.

The results obtained, confirmed using both the natural inhibitor and the recombinant, show that the kiwi inhibitor can find an advantageous industrial use in the reduction of methanol in grape musts and marc and in particular in the products obtained from fermentation and / or distillation. of the same, such as wines and spirits.

Claims (12)

CLAIMS 1. Use of a protein inhibitor of pectin methylesterase from Actinidia chinensis or of a portion thereof having inhibitory activity of pectin methylesterase for the reduction of methanol formation in grape musts and marc. 2. Use according to claim 1 in which the inhibitor is obtained by purification from Actinidia chinensis extracts. 3. Use according to claim 1 in which the inhibitor is obtained recombinantly. 4. Use according to claims 1-3 in which the inhibitor comprises the amino acid sequence or portions thereof ENHLISEICPKTRNPSLCLQALESDPRSASKDLKGLGQFSIDI AQASAKQTSKI IASLTNQATD PKLKG RYETCSE N YADAI DSLG QAKQFLTSGDYNSLNIYASAAFDGAGTCEDSFEGPPNIPTQL HQADLKLEDLCDIVLVISNLLPGS. 5. Process for reducing the formation of methanol in grape musts or marc comprising the use of a protein inhibitor of pectin methylesterase from Actinidia chinensis or a portion thereof having inhibitory activity of pectin methylesterase. 6. Process according to claim 5 in which the inhibitor is obtained by purification from Actinidia chinensis extracts. 7. Process according to claim 5 in which the inhibitor is obtained recombinantly. 8. Process according to claims 5-7 in which the inhibitor comprises the amino acid sequence or portions of it ENHLISEICPKTRNPSLCLQALESDPRSASKDLKGLGQFSIDI AQASAKQTSKI IASLTNQATD PKLKG RYETCSE N YADAI DSLG QAKQFLTSGDYNSLNIYASAAFDGAGTCEDSFEGPPNIPTQL HQADLKLEDLCDIVLVISNLLPGS. 9. Process according to one of claims 5 to 8 in which the reduction of methanol formation is obtained by incubating grape musts with at least 1 part of inhibitor in relation to 20,000 parts of must, at a temperature between 25-35 ° C for a period of at least 7 days, with daily pumping over. 10. Process according to one of claims 5 to 8 wherein the reduction of methanol formation is obtained by incubating the marc with at least 1 part of inhibitor in relation to 2,000 parts of marc, at a temperature between 25-35 ° C for a period between 14 and 28 days. 11. Process according to claim 9 in which the inhibitor is eliminated by protein stabilization. 12. Process according to claim 10 in which the inhibitor is eliminated by denaturation during the distillation process.