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1. WO1999001564 - PROCEDE DE MODIFICATION DE COMPOSES TOXIQUES ET/OU AYANT MAUVAIS GOUT

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[ EN ]

Process for the modification
of toxic and/or off-flavoured compounds

Field of the invention

The present invention relates to enzymatic processes for the modification of undesirable compounds, especially in plant-derived material.

Background of the invention

A wide variety of plants contain so called anti-nutritional factors (ANFs) which are claimed to provide a natural protection against attacks of moulds, bacteria, insects and birds. Well known ANFs include trypsin- and chymotrypsin- inhibitors, lectins, tannins, saponins and (glyco) alkaloids.
In commercially important crops like soy, alfalfa and potato, the presence of non-proteinaceous ANFs like for example saponins and glycoalkaloids represent a considerable problem, because these compounds are toxic and bitter tasting. Unfortunately, these compounds are also very heat stable and, due to their hydrophobicity, difficult to extract.
For instance, the family of Solanaceae, which comprise potato and tomato, are known to contain several types of glycoalkaloids. Solanine is a general term used to describe the glycoalkaloids present in potato. The major compounds which form part of solanine are α-solanine and α-chaconine. Both substances are present in almost equimolar quantities and have a similar steroidal alkaloid part, the so-called, aglycone or, more specifically, solanidine.
The structures of both α-solanine and α-chaconine and their toxicity to man have been described in several publications (e.g. Angew. Chem. 66, nr. 20, 1951, pp.639- 640; Var Föda supplement 1, 1990, pp. 5-15; Trends in Food Science & Technology 7, 1996, pp. 126-128; Food Technology in Australia, 36(3) 1984, pp. 118-124).
Both potato glycoalkaloids consist of a nonpolar lipophilic steroid nucleus, called solanidine linked to a watersoluble trisaccharide. The carbohydrate components of α-solanine and α-chaconine differ. In the case of α-solanine, the aglycone carries a galactose (attached β-1,3 to the aglycone), a glucose (attached β-1,3 to the galactose) and a rhamnose (attached α-1,2 to the galactose). In the case of α-chaconine, the aglycone carries a glucose (attached β-1,3 to the aglycone) and two rhamnoses (attached α-1,2 and α-1,4 to the glucose) (Figure 1).
In addition, small amounts of partial glycosides occur e.g. (S-solanine (lacking either rhamnose or glucose), γ-solanine (lacking both rhamnose and glucose) (Figure 2), β-chaconine (lacking one rhamnose) and γ-chaconine (lacking two rhamnose molecules) (Figure 3). These partially glycosilated compounds are considered less toxic than either α-solanine or α-chaconine. (J. Agric. Food Chem. 1994, 42, 1511-1515). The aglycone solanidine is considerably less toxic than the various glycosides (Nishie et al . (1971), Toxicology and Applied Pharmacology 1981-1992).
On the basis of published data it is clear that a complete removal of the carbohydrate moiety of both solanine and chaconine will have a dramatic impact on not only the toxicity of these glycoalkaloids but also on their negative taste aspects.
Detoxification or debittering of glycosilated compounds by incubation with enzymes is not new and has been described before.
Bushway et al . , AM. Potato J. 65(11): 621-631, 1988, describe the stepwise hydrolysis of α-chaconine using a rhamnosidase from potato peels.
Stankovic et al . , Potato Res. 37(3): 271-278, 1994, describe the in si tu hydrolysis of potato glycoalkaloids leading to the formation of solanidine.

Swain et al . , Phytochemistry 171(4): 800-801, 1978 describe the enzymatic hydrolysis of α-chaconine and α-solanine by sprout and dormant tuber enzymes.
In all of these cases, it involves the hydrolysis of α-chaconine and/or α-solanine by unidentified and non-characterised enzymes from plant material. However, the level at which these desired enzymes are present in these plant materials is rather low so that it would be of great economic value to have the availability of microbiological sources producing the required enzymes or, alternatively, of plant materials in which the specific enzymes are present at high levels.

Brief description of the figures

Figure 1
The glycoalkaloids α-solanine and α-chaconine and their considerably less toxic aglycon solanidine.

Figure 2
Less toxic partial glycosides of α-solanine.

Figure 3
Less toxic partial glycosides of α-chaconine.

Detailed description of the invention

The present invention relates to a microorganism, to enzymes obtainable from this microorganism and to the use of these enzymes. In particular to the use of these enzymes in converting potato glycoalkaloids into less toxic compounds. In one aspect the invention provides a microorganism which was deposited as CBS 773.97 with the Centraal Bureau of Schimmelcultures, Baarn, the Netherlands, on 13 May 1997. It is a Gram positive irregular rod, which is strictly aerobic. It produces no acid or gas if grown on glucose. 16S rDNA sequence analysis of the most variable region showed 99.5% similarity to Brevibacterium helvolum (ATCC 13715). Similarity values to other members of the family Microbacteriaceae are below 95%.
The invention also encompasses derivatives of the deposited microorganism which produce the enzymes of the invention. These derivatives may be obtained by methods known in the art, such as for example spontaneous or man-induced mutation and genetic engineering.
Using a suspension of this microorganism, α-solanine and α-chaconine may be converted into their less toxic β-and γ-intermediates or the far less toxic solanidine.
In another aspect, the present invention provides novel microbial enzymes. In particular, it provides a β-glucosidase which is able to convert α-solanine into β2-solanine and which is obtainable from the above-mentioned microorganism. We have surprisingly found that the combination of this particular β-glucosidase with a suitable enzyme preparation, such as the AR2000® aroma enhancer (Gist-brocades, Seclin, France), is able to completely deglycosilate solanine.
This particular β-glucosidase may be isolated and purified from the microorganism by conventional biochemical techniques known in the art. A convenient method is the following. Cells of an 24-96 hr culture of the microorganism are collected by centrifugation. The cells are then destructed by means of sonification. The enzyme is solubilised by using ionic or non-ionic detergents and subsequently purified by a combination of ammonium sulphate precipitation, gel filtration, ion exchange and hydrophobic interaction chromotography.
An N-terminal or internal amino acid sequence of this particular β-glucosidase may be obtained by sequencing the substantially purified enzyme. The amino acid sequence may be used to obtain one or more DNA probes, which in turn may be used to recover the gene encoding the specific β-glucosidase and to identify the complete DNA sequence of the enzyme.

Once the DNA sequence of the enzyme is available, the enzyme of the invention may be produced by expressing it in a host cell, such as an animal cell, a plant cell, a fungal cell, a yeast cell or a bacterial cell, transformed with DNA encoding the enzyme, or a precursor thereof. The enzyme according to the invention may then be recovered in larger quantities from the lysed cells, or from the culture medium if secreted by the host cell.
A DNA probe of the invention may be used to screen for other enzymes which also display this particular β-glucosidase activity.
The isolated enzyme may be used on its own for the partial degradation of α-solanine or in combination with other enzymes, preferably with one or more glycosidases, for further degradation of α-solanine. Suitable combinations can be made with, for example, a rhamnosidase, a galactosidase or a mixture of enzymes. In one preferred embodiment, an extract or suspension of the microorganism deposited as CBS 773.97 is combined with AR2000® aroma enhancer (Gist-Brocades, Seclin, France) which is a fungal enzyme preparation which contains several enzyme activities. This extremely advantageous combination allows for the complete conversion of α-solanine into solanidine.
The person skilled in the art will understand that for the above described conversions, either substantially purified enzyme, partially purified enzyme or even a suspension of a microorganism which comprises the enzyme, may be used, whichever is convenient. The AR2000® fraction which is responsible for the conversion of β2-solanine into solanidine, i.e. the active fraction, may be isolated and purified from AR2000® using conventional biochemical techniques known in the art such as ion exchange chromatography, hydrophobic interaction chromatography and gel filtration chromatography.
It is known from WO 97/04107 that AR2000® also comprises a chaconinase, viz. an enzyme which cleaves the bond between the trisaccharide part and the aglycon part of chaconine. Therefore, yet another aspect of the present invention is that it provides a method for the complete deglycosilation of a mixture of α-solanine and α-chaconine into solanidine. Since the aglycon solanidine is far less toxic than the α-solanine, α-chaconine and less toxic than their β- or γ-intermediates, the present invention also provides a method for the detoxification of a mixture of α-solanine and α-chaconine.
The enzymes of the invention or an enzyme preparation which is enriched with the enzymes of the invention may be used to prepare human or animal food by upgrading potato-derived material. The potato-derived material, such as for example potato fruit juice derived material or potato peels, may be upgraded by incubating it in a vessel or reactor with the enzymes or enzyme preparation of the invention prior to consumption. However, especially in the case of animal food, the enzyme or enzyme preparation of the invention may also be administered at the same time as the potato-derived material. In this case, the upgrading will take place in the

(animal) body. Either way, the invention will have great economic advantages for the potato processing industry.
The enzyme preparation may be prepared in accordance with methods known in the art. It may be formulated in any convenient way, including as a paste, liquid, emulsion, powder, flakes, extrudate, granulate or pellet. It may be stabilised in accordance with methods known in the art.
The present invention will be ilustrated by the following examples.

EXAMPLES
Example 1 - Enzymatic hydrolysis of α-chaconine and α-solanine
In this experiment we test the ability of selected enzymes to hydrolyse the various sugar linkages occurring in α-chaconine and α-solanine.

Experimental
To test degradation of the glycoalkaloids, either α-chaconine or α-solanine (Sigma Chemicals) was dissolved at a concentration of 1 mg/ml in D2O, buffered with 100 mmol/l sodium acetate at pH 5. Per ml glycoalkaloid solution 15 μl of the liquid enzyme preparations and 2 mg of the powdered enzyme preparations were added. To this solution the various enzyme preparations were added. Incubation took place at 37ºC for 8 hours.

Results
The effect of the various enzyme preparations on the sugar moiety of the glycoalkaloid was established by means of 600 Mhz 1H NMR. Of the many enzyme preparations tested, only AR2000® and Cytolase® were able to affect the sugar moiety of α-chaconine. The latter enzymes showed no effects towards α-solanine. Quite surprisingly none of the other enzymes, specifically selected for their galactosidase, glucosidase or rhamnosidase hydrolytic activity, was able to cleave any of the sugar linkages present in α-chaconine or α-solanine. This demonstrates that only very selected enzymes will be able to detoxify α-solanine.

Example 2 Screening for a microorganism which is capable of hydrolysing α-chaconine and α-solanine.

Micro-organisms were obtained from a potato fruit juice solution (Potato washing unit at Emsland Starke GmbH, Germany). This solution was diluted 2×104 fold with a physiological salt solution and 100 μl was plated on BHI-agar or mout-agar. The plates were incubated for a few days in the dark at 30°C, followed by a week in daylight at room temperature. Of the isolated colonies, approx. twenty colonies exhibiting different morphology were selected and plated out to single colony isolates. These cultures were inoculated and maintained on a slant containing BHI-agar.
The isolated microorganisms were screened by an adaptation of the method described by D.J. Wijbenga , D. Binnema and A. Veen, (in: Bacterial modification of steroidal glycoalkaloids from Solanum species, 7th European Congress on Biotechnology, Nice, 19-23 February 1995) on agar plates on defined medium with glycoalkaloids as the sole C-source.
Preparation of agar plates:
Solution A: 22.56 g Na2HPO4.2H2O, 9.0 g KH2PO4 and 750 ml
H2O.
Solution B: 0.6 g MgSO4.7 H2O, 1.5 g NaCl, 3.0 g NH4Cl and
750 ml H2O.
Solution C: 10 mg α-solanine and 10 mg α-chaconine and 10 ml H2O. 10 ml of solution C was first mixed with 5 ml of solution B (sterile) and then 5 ml of solution A (sterile) was slowly added. The pH of the total solution was kept at 6.0.

To the final solution (20 ml) 300 mg agar was added. The agar was dissolved and the solution was heated in a micro-wave oven. The solution was cooled down to 50°C and subsequently the agar plates were poured. One plate was divided into various areas.
In some experiments the mix of both α-solanine and α-chaconine (solution C) has been replaced by 15 mg of either α-solanine or α-chaconine in the same volume.
The isolated colonies were numbered, inoculated on the plates and incubated at 30°C in the dark for about a week. In a control plate solution C consisted only of water.
Finally two growing colonies were identified on the plates containing either α-solanine or α-chaconine or both the glycoalkaloids. On the reference plate only one of these colonies was present. The organism growing solely on the agar plates containing α-solanine or α-chaconine or both the glycoalkaloids was sent in for full characterisation and used in further experiments. It was deposited as CBS 773.97 with the Centraal Bureau of Schimmelcultures, Baarn, the Netherlands, on 13 May 1997.

Example 3 Reaction mechanism of the hydrolysis of α-solanine.

To obtain more information on the mechanism of glycoalkaloid hydrolysis by the strain isolated according to the procedure described in Example 2, a number of incubations with the various glycoalkaloid forms were carried out. In addition the possible synergistic effect with a commercial enzyme preparation AR2000® was studied.
The isolated microorganism from Example 2 was inoculated in 25 ml BHI-medium in a 100 ml flask. The microorganism was grown for 96 hours at 30°C and 280 rpm. The cells were collected by centrifugation for 20 minutes at 20°C and thawed prior to use. Hydrolysis for glycoalkaloids was assayed with an adaptation of the method described by Kudou et al . (1990, Agric. Biol. Chem. 54: 1341-1342).

A. Hydrolysis wi th the mi croorganism suspension .
40 μl of a α-solanine (Sigma) solution, (2 mg/ml in 100 mM sodiumacetate pH 5.0), was incubated at 37°C with 45 μl of the cell suspension and 10 μl 50 mM NaAc pH 5.5. At several time intervals samples were taken and analysed by thin layer chromatography analysis. To that end 15 μl of the incubation mixture was added to 35 μl methanol to stop the reaction and to precipitate protein. After centrifugation, 25 μl of the supernatant was applied to a TLC plate. TLC analysis was performed on a Silica 60F2S4 plate with a solvent system of chloroform-methanol-water (65:35:10, v/v, lower layer).

During this incubation, two α-solanine intermediates were formed, and, depending on the time of incubation, the most hydrophilic intermediate accumulated. No detectable solanidine was formed during this incubation.

B. Hydrolysi s wi th the microorganism suspension in combination wi th AR2000® .
40 μl of a α-solanine solution was incubated at 37°C with 45 μl of the cell suspension and 10 μl AR2000® (Gist-brocades, Seclin, France), (50 mg/ml in 50 mM NaAc pH 5.5).After 6-20 hrs a 15 μl sample of the incubation mixture was added to 35 μl methanol to stop the reaction and to precipitate protein. After centrifugation, 25 μl of the supernatant was applied to a TLC plate. TLC analysis was performed as described before under A.
During the incubation with the resuspended cells in combination with AR2000® the substrate α-solanine was hydrolysed into the aglycon solanidine. This shows that AR2000® in combination with the cell suspension is able to hydrolyse α-solanine directly into solanidine, whereas the experiments shown in Example 1 indicated that α-solanine is not a substrate for AR2000®. So obviously, incubation of α-solanine with the cell suspension renders the (modified) α-solanine susceptible to AR2000®.
To establish whether an enzyme is involved capable of cleaving off glucose or rhamnose, α-solanine was incubated with cells only. After 20 hours, the reaction was stopped with 234 μl methanol. The total amount of the supernatant was applied to a TLC plate in different lanes and chromatographed in the above mentioned solvent mixture. Subsequently, the accumulated intermediate was scratched off the plate and eluted for identification.
Identification of the collected intermediate took place by means of 1H NMR at 600 MHz. As solvent CD3OD was used. The isolated intermediate could be confirmed to consist of the aglycon solanidine with rhamnose and galactose as the sugar residues, i.e. β2-solanine. From these results three conclusions can be drawn:
(1) the microorganism isolated contains an enzyme able to deglucosilate α-solanine;
(2) whereas α-solanine is resistant to enzymatic hydrolysis, deglucosilated α-solanine provides a good substrate for AR2000®;
(3) a combination of the β-glucosidase present in the microorganism CBS 773.97 together with AR2000®, or the , active fraction thereof, yields an enzyme mixture able to detoxify mixtures of α-solanine and α-chaconine.