USE OF A PROTEIN INHIBITOR OF PECTIN METHYLESTERASE FOR REDUCING METHANOL FORMATION IN GRAPE MUST AND MARC, AND PROCESS THEREFOR

Field of the invention

The invention relates to the use of a protein or a part thereof having pectin methylesterase inhibitor activity, for reducing methanol formation in grape must and marc and products thereof derived from fermentation and distillation, and to a process therefor. State of the art

Pectin methylesterases (hereinafter also referred to as PME) are endogenous enzymes widely present in plants, where they have the important function of hydrolysing pectins, secreted in cell walls, in their highly methyl-esterified form. The hydrolytic activity of these enzymes is aimed at the de-methylation of pectins, with formation of polygalacturonic acid domains able to complex calcium, thus forming gelatinous macromolecular structures. Said de-esterification also influences other physiological parameters and represents the basis for the action of other degradative enzymes such as endo-polygalacturonases that degrade polygalacturonic acids into pectic fragments. However, this hydrolytic process is undesirable in the preserved food industry with regard to the production of juice, such as fruit juice, and concentrates, such as tomato paste, in that as a result of this pectin demethylation a destabilization of the product arises with loss of consistency and flocculation of the vegetable or fruit product. It is known that to inactivate endogenous plant pectin methylesterases, physical processes such as high temperature and pressure treatments of vegetable and fruit products can be utilized. These processes, however, are complex and costly in terms of energy consumption and impact negatively on final product quality. Moreover, these processes are not completely efficient as there exist extremely heat stable PME isoforms which can remain active even after severe heat treatments. A protein inhibitor of PME (hereinafter also known as PMEI), which enables the

limitations resulting from these physical processes for pectinesterase inactivation to be overcome, is described in US 5,053,232 by Balestrieri C. et al. dated 1991 and Italian patent RM2003A000346 by Bellincampi D. et al. dated 2003. This protein inhibitor derived from kiwi fruit (Actinidia chinensis) or obtained by recombinant technology is capable of inhibiting plant PMEs.

It has been demonstrated that kiwi PMEI is highly efficient in inhibiting the activity of PME present in tomatoes, apples, bananas and potatoes (Balestrieri C, Castaldo D., Giovane A., Quagliuolo L, Servillo L. (1990) "A glycoprotein inhibitor of pectin methylesterase in kiwi fruit (Actinidia chinensis)" Eur.J.Biochem., 193, 183-187).

Proteinaceous inhibitors of PME were subsequently also identified 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 Lett., 557, 199-203). The natural inhibitor from kiwi has been amply characterized biochemically. The inhibitor has a molecular mass of about 16 kDa and its entire amino acid sequence has been determined, presenting some microheterogeneities (in addition to the more represented residues such as alanine 56, threonine 63, tyrosine 78, serine 1 17, asparagine 123 and valine 142, the following have been found in smaller amounts: 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 ami no-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 (2007) "Cloning of pectin methylesterase inhibitor from kiwi fruit and its high expression in Pichia pastoris", Enzyme and Microbial Technology, 40, 1001-1005). The predominant isoform, expressed with high efficiency in Pichia pastoris, presents the same chemical characteristics as the natural inhibitor and its 3-D structure has recently been resolved (Di Matteo A., Giovane A., Raiola A., Camardella L., Bonivento D., De Lorenzo G., Cervone F., Bellincampi D., Tsernoglou D. (2005) "Structural Basis for the Interaction between

Pectin Methylesterase and a Specific Inhibitor Protein" Plant Cell 17, 849-858). As well as the previously mentioned destabilization effect in juices and concentrates due to PME, it should be noted that in the PME catalysed reaction where highly methylated pectin is converted into low methoxy pectin and pectic acids, methanol is released, this being a known volatile compound very toxic to man. This aspect is particularly significant in fruit juice or concentrates from vegetable sources in which the pectins are present in a highly methylated form and/or the pectin esterases are particularly active. Methanol taken with the diet can also arise from the degradation of artificial sweeteners (e.g. 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 observation that methanol can also be produced in several ways in the human body therefore means that its intake from the diet has to be lowered in order to avoid a cumulative effect of such a noxious alcohol. For this reason the European Union has fixed limits for methanol concentration in foods which must be complied particularly for fermented and distilled products from wine industry byproducts such as grappa (EC1576/89), obtained from the distillation of grape marc. In this alcoholic beverage in particular, the presence of methanol can easily exceed the threshold of 1 mg per 100 ml of anhydrous alcohol. In the specific case of distillates from grape must fermentation by-products, to prevent methanol exceeding legal limits, as is often the case when preserved grape marc is distilled, no attempt is made to inactivate the enzyme responsible for methanol release, but instead a second distillation is carried out with a demethylating column. This measure however has the disadvantage of resulting in a loss in the aromatic fraction of the distillate. As a result, the distillate is qualitatively down-graded since it has lost its typical aroma, this being a fundamental characteristic for differentiating between different distillates. It is therefore the purpose of the present invention to identify methods capable of effectively reducing methanol production in grape must and marc and their fermented and distilled products. Summary Following thorough research the inventors have now demonstrated that the pectin

methylesterase inhibitor obtained from kiwi or obtained by recombinant technology is also active against pectin methylesterase from grape and marc and is able to effectively inhibit methanol formation in grape must and marc.

The present invention therefore relates to the use of a pectin methylesterase inhibitor or a part thereof having pectin methylesterase inhibitor activity for reducing methanol formation in grape must and marc and in products derived therefrom by fermentation and distillation.

Preferably the pectin methylesterase inhibitor is from Actinidia chinensis and is obtained by kiwi purification or by recombinant technology. The invention also relates to a process for reducing methanol formation in grape must and marc and in products derived therefrom based on the use of the PME inhibitor from Actinidia chinensis or a part thereof having pectin methylesterase inhibitor activity, obtained by kiwi purification or by recombinant DNA methods.

Brief description of the figures Figure 1 : The figure shows the results obtained with 5 μl of Muscat grape extract and 5 μl of various possible inhibitor solutions, including that containing PMEI, loaded into the various wells, so maintaining the final volume constant at 10 μl.

The petri plates were incubated at 30 °C for 21 hours, this being a sufficient time for the loaded sample to fully exert its enzymatic activity. The wells contained (1 ) 0.1 M disodium EDTA, (2) 0.1 M CaCI ₂ , (3) 0.1 M (NH ₄ )SO ₄ , (4) 0.1 M saccharose,

(5) 0.1 M (NH ₄ )CI, (6) 0.1 M D-(+)-galacturonic acid, (7) 0.6 mg/ml kiwi PMEI, (8) blank, (9) control (Muscat grape extract only).

Figure 2: The figure shows the results obtained on the inhibitory capacity of PMEI using scaling protein quantities corresponding to different enzyme units present in Muscat grape extract. The right plate was loaded only with the enzymatic extract, while the left plate also contained 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 for methanol production inhibition in grape must and marc by this kiwi-derived inhibitor are given by way of non-limiting example.

The PME inhibitory capacity of the Actinidia chinensis natural protein and of the

same protein expressed in Pichia pastoris, its biochemical characteristics and other industrial applications of this protein inhibitor are, as already previously stated, described in patents US 5,053,232 and RM2003A000346 and in works published by Camardella L. et al. and Di Matteo A. et al. (Camardella L, et al. (2000) ret cit; Di Matteo A, et al. (2005) ret cit).

This notwithstanding, the ability of this protein to significantly inhibit methanol formation resulting from demethylation of pectin in general and from grapes in particular has never been studied. The present invention is therefore based on the use of these PME inhibitors for reducing methanol concentration in grape must or marc and in products obtained from fermentation or distillation therefrom. For this purpose the use of kiwi PMEI which, depending on what is more industrially convenient, can be obtained either naturally from kiwi fruit or expressed in Pichia pastoris, is found to be preferable. Research and consumer attention has only recently been focussed on the close link between food and health. This has also drawn the attention of public opinion to the toxic compounds present in foods and systems for removing them. In the preserved food sector the presence of the PME enzyme has always been associated with the production of juices of greater or lesser consistency and/or cloudiness. The problem of methanol in juices has always been ignored, with consideration only given to PME inhibition to avoid enzymatic activity of other polygalacturonase enzymes which act sequentially (and are much more thermostable), but which are unable to catalyse the reaction unless the PME has acted first. In the case of grappa, where methanol concentration is fixed by law, achieving these legal limits (which are almost always exceeded in distillates obtained without using a demethylating column) leads to an impairment in the quality since a demethylating column has to be used which eliminates the typical aroma of the distillate itself. In conclusion, being able to inhibit the PME plant enzyme, whose activity is responsible for methanol release, can have a two-fold advantage: lowering methanol concentration in juices (hence resulting in its lower dietary intake) and, in the case of distillates, simultaneously providing a superior quality in terms of the typical aroma. For the purposes of the present invention, the usable

PME inhibitor is a 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 can be a polypeptide obtained by recombinant technology, and comprises the following amino acid sequence or parts thereof: Seq. ID No 1

ENHLISEICPKTRNPSLCLQALESDPRSASKDLKGLGQFSIDIAQASAKQTSKIIAS

LTNQATDPKLKGRYETCSENYADAIDSLGQAKQFLTSGDYNSLNIYASAAFDGAG

TCEDSFEGPPNIPTQLHQADLKLEDLCDIVLVISNLLPGS.

The usable PME inhibitor for reducing methanol production in grape must and marc or in products obtained from fermentation or distillation therefrom, can be obtained by known methods such as extraction and separation from fruit as previously described (Balestrieri C. et al. ( 1990) ref. cit.) or, on the basis of the aforementioned sequence, obtained by the molecular biology technique of recombinant DNA and biochemical purification as already described by Di Matteo A. et al. (D/ Matteo A. et al. (2005) ref. cit.)

For the purposes of the present invention, methanol formation in grape must and marc is reduced by incubating the PME inhibitor obtained from kiwi or obtained by recombination DNA methods and grape must or marc in a ratio inhibitor : grape must or marc of at least 1 part of inhibitor per grape marc or must parts from 2,000 to 20,000 w/w and in particular under the following conditions:

- incubation of grape must with inhibitor at a ratio of 20,000 parts must to at least 1 part inhibitor, respectively, at a temperature between 25-35 °C for a period of at least 7 days, with daily top-ups.

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

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

Experimental part With the aim of evaluating whether the PME inhibitor from kiwi can result in PME

inhibition in grape must and marc as well as a significant reduction in methanol production in grape must and marc, the inventors conducted the experiments described below.

Example 1 : preparation of the PME inhibitor A-Purification of the natural inhibitor

Ripe kiwi fruit were homogenized with water in a 1 :1 ratio (mass:volume) and centrifuged at 15,00Og for 20 minutes at 4°C. The supernatant was brought to pH 7.5 with NaOH and directly precipitated with 70% saturated ammonium sulphate. The precipitate was resuspended in 10 mM Tris-HCI at pH 7.5, 50 mM NaCI and dialyzed overnight against the same buffer. The dialysate was loaded onto a Q- Sepharose column equilibrated with 10 mM Tris-HCI, pH 7.5 and 100 mM NaCI. After washing the column was eluted with a linear gradient from 100 to 400 mM NaCI. The fractions containing inhibitory activity were collected, concentrated by ultrafiltration and loaded onto a preparative Superose 12 column equilibrated with 10 mM Tris-HCI, pH 7.5 and 100 mM NaCI. The inhibitor purified in this manner was analysed by 12.5% SDS-gel electrophoresis. B-Purification of PMEI expressed in Pichia pastoris

Culture broth of Pichia pastoris expressing pectin methylesterase inhibitor as described by Xiao-Hong Mei et al. {ref. cit.) was filtered and precipitated with 80% saturated ammonium sulphate. The precipitate obtained in this manner was collected by centrifugation at 20,00Og for 20 minutes, resuspended in a 10 mM TrisCI buffer, pH 8.5 and dialyzed exhaustively against the same buffer. After dialysis, the solution was centrifuged at 20,00Og for 20 minutes and loaded onto a MonoQ ^® column (HR 10/10, Pharmacia) equilibrated with the same buffer. The PMEI was then eluted with a linear gradient from 0 to 0.5 M NaCI over 30 minutes at a flow rate of 1.5 ml/minute. The fractions with inhibitory activity were combined, concentrated by ultrafiltration on Centricon 3 filters (Amicon) and loaded onto a Sephadex ^® G75 column (1.5 x 90 cm) equilibrated with 10 mM TrisCI, pH 7.5, 250 mM NaCI at a flow rate of 5 ml/hour. The fractions with inhibitory activity tested pure when analysed by 12.5% SDS-gel electrophoresis and stained with a solution of Coomassie ^® brilliant blue G250. The calculated molecular weight of 16 kDa corresponded to that deduced from the amino acid

sequence (16,753 Da), in which respect glycosylation sites were not present in the protein. The protein presented a single peak when analysed by reverse phase column HPLC. The N-terminal sequence (EAEFENHLISEI-PK) corresponded to the cloned and expressed gene sequence, and showed the presence of 4 additional amino acids (in bold) corresponding to the signal peptide residue of Pichia.

Example 2: inhibition of pectin methylesterase activity in grape must with added methylated pectin The inventors then proceeded to establish a sensitive diffusion method on petri plates (Downie B., Dirk LM. , 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 enzymatic activity of grape pectin methylesterase. The plant extracts obtained from Muscat grapes gathered at the same stage of ripening were incubated on plates in the presence of highly methylated pectin (94%) at 25-35 °C for 21 hours. Enzyme activity was expressed according to a standard curve (ranging from 0.72 U to 0.04 U) obtained from a logarithmic conversion of the activity obtained from using different concentrations of a commercial pectin methylesterase derived from oranges against the diameter of the stained halo produced therefrom.

In order to establish whether it is possible to inhibit grape pectin methylesterase, tests were then carried out on plates in the presence of the following inhibitors: Na ₂ EDTA, calcium chloride, ammonium sulphate, saccharose, galacturonic acid and PME inhibitor (PMEI) extracted from kiwi. The plate assay of pectin methylesterase activity in the presence of possible inhibitors was conducted in the following manner:

5 μl of Muscat grape extract and 5 μl of several possible inhibitor solutions including that containing the PME inhibitor, were loaded into the different wells, so maintaining the final volume constant at 10 μl. The petri plates were incubated at 25-35°C for 21 hours, this being a sufficient time for the loaded sample to fully exert its enzyme activity. The wells contained (1 ) 0.1 M disodium EDTA, (2) 0.1 M CaCI ₂ , (3) 0.1 M (NH ₄ )SO ₄ , (4) 0.1 M saccharose, (5) 0.1 M (NH ₄ )CI, (6) 0.1 M D-

(+)-galacturonic acid, (7) 0.6 mg/ml kiwi PMEI, (8) blank, (9) control (Muscat grape extract only).

The results are shown in table 1 below and in figure 1.

Table 1

Of the 7 possible inhibitors of Muscat grape PME tested on the plate, only that derived from kiwi (PMEI) showed a positive response (total inhibition of pectin methylesterase activity) observable by the complete absence of a red inhibition halo on the plate (fig. 1 ). The inhibitory activity of PMEI was subsequently calculated: a) on the basis of enzyme concentration; b) on the basis of inhibitor concentration. In the first case (a) the inhibitory capacity of PMEI was tested with scaling quantities of protein corresponding to varying enzyme units present in the Muscat grape extract. The results are shown in table 2 below.

Table 2

In the second case (b) in order to determine the minimum inhibitory quantity able to inhibit pectin methylesterase activity of a fixed amount of enzyme, different aliquots of PMEI were added to the same quantity of protein, thus including also PME, present in the Muscat grape extract. The results obtained have enabled the establishment of 0.12 micrograms of inhibitor as being the minimum amount necessary to provide complete inhibition of 0.135 enzyme units, corresponding to 4.60 micrograms of protein, present in the extract. The results are shown in table 3 below.

Table 3

It was also demonstrated that the PMEI inhibits Muscat grape PME even at pH 3.5 (0 = 4.0 mm if loading 4.6 μg Muscat protein) (equal to 8 μl) 0 = 0 mm if loading 4.6 μg Muscat protein and 1.2 μg PMEI (equal to 2 μl). Example 3: inhibition of methanol formation in grape must with added methylated pectin

It was then determined whether, in addition to the inhibitory capacity of kiwi PMEI towards PME, there was also a corresponding significant inhibition of methanol release following pectin demethylation, this being measured by HS-SPME-GC (table 4). Under the same temperature, pH and incubation time conditions, 74.43 ppm of methanol were produced by the control consisting of pectin methylesterase enzyme extract from grape and a 0.1 % solution of 94% methylated pectin, while the same extract in the presence of 6 micrograms of inhibitor released only 18.86 ppm of methanol.

Table 4

Sample Protein of Volume of PMEI (μg) Methanol (ppm) loaded Muscat loaded PMEI

(μg) inhibitor (μl)

Muscat grape 155 0 0 74.43 extract without PMEI

Muscat grape 155 10 6 18.86 extract with PMEI

Accordingly, use of the inhibitor enables the concentration of released methanol to be reduced by 75%.

Example 4: inhibition of methanol formation in grape must without added methylated pectin

The inhibitor was also tested on Muscat grape must and marc under similar environmental conditions to those normally found in wine cellars and distilleries. In the test with must, 50 μl of inhibitor equal to 75 μg of PMEI were added to 1.0 ml of unfiltered grape juice. Instead, 50 μl of distilled water were added to the blank. The samples were incubated in hermetic vials at 25-35 °C for 7 days and maintained under constant agitation. At the end of the incubation, HS-SPME-GC analysis showed that, if the inhibitor was present, an 8.5% inhibition in methanol formation was found. In the case of the grape marc, two different amounts of inhibitor equal to 75 and 150 μg of PMEI were added to 100 mg of marc, and the samples were incubated at 25-35 °C for 14 and 28 days. In the samples analysed after 14 days a 16.6% reduction in methanol was found where inhibitor was present; after 28 days' incubation the inhibition was found to be between 19.5% and 33.3%. The results obtained which were confirmed by using both the natural and the recombinant inhibitors, showed that the kiwi inhibitor can find advantageous industrial use for reducing methanol in grape must and marc, particularly in products obtained from fermentation and/or distillation thereof, such as wines and distillates.