CONVERSION AND REMOVAL OF STEROID GLYCOALKALOIDS

Abstract: The invention relates to microorganisms and cultures thereof, enzyme systems and enzymes capable of converting steroid glycoalkaloids (SGA) and removing same from Solanaceae species such as the potato, and relates to the use of such microorganisms, enzyme systems and enzymes in, for example, the potato processing industry. The invention also provides new methods for generating SGA metabolites and derivatives which, for example, can be used for steroid hormone synthesis. The invention also provides new crop potatoes that are resistant to diseases and plagues as a result of, or in conjunction with, a high content of SGA and the invention provides for the processing of such potatoes, whereby the SGA content is reduced to make the potato product useful for human or animal consumption. Such potatoes are eminently suitable as a source for SGA metabolites and derivatives, which can be used in steroid hormone synthesis.

Description: Title: Conversion and removal of steroid glycoal aloids . This invention lies in the field of agriculture and the processing of products from agriculture, and more particularly relates to the conversion of steroid glycoalkaloids from Solanaceae species . Steroid glycoalkaloids (SGA, Table 11) from Solanaceae species, such as the potato and the tomato, are bitter tasting and potentially toxic components that occur naturally in all parts of these plants. The highest SGA contents occur in parts having a high metabolic activity, such as flowers, leaves and offshoots (sprouts) [Jadhav, S.J.; Sharma, R.P.: Salunkehe, D.K. (1981) CRC Crit . Rev. Toxicol . 9, pp. 21-104] . It appears, for instance in potato tubers, that a variety of factors during growth, storage and processing [Burton, W.G. (1989) The Potato (ISBN 0-582- 46229-01)] can lead to an incidental and undesirable increase of the SGA content. Also, berries of, for instance, the potato, and seeds of, for instance, Solanaceae species Datura Stramonium are known for their toxicity [Friedman, M. (1992) Composition and safety evaluation of potato berries, potato and tomato seeds, potatoes, and potato alkaloids. In: Food Safety Assessment, Eds. J.W. Finley, S.F. Robinson, D.J. Armstrong, American Chemical Society, Washington D.C.] . SGA is considered responsible for an off-flavor which is described as bitter and sharp ("burning") [Maga, J.A. (1994) Potato Flavor. Food Reviews International, 10(1), pp. 1-48] . SGA contents in excess of 200 mg/kg of fresh weight can be sensed by most people as a bitter off-flavor, which discourages further consumption. [Maga. ibid.]. Zitnak & Filadelfi [Zitnak, A.; Filadelfi, M.A. (1985) Estimation of taste threshold of three potato glycoalkaloids. Can. Inst . Food Sci. Technol . J. 18(4) pp. 337-339] evaluated organoleptic properties of some potato SGA's. Chaconine is bitterer and causes a stronger burning sensation than solanine, while the bitterness of solanine is equal to that of solanidine. The aglycon solanidine in - 2 - the highest dose did not cause a burning sensation. High contents of SGA adversely affect the taste of, for instance, the tomato or potato and, for instance, products derived from these Solanum species, and are potentially toxic. The lethal dose of SGA for humans is estimated to be 3-6 mg/kg of body weight. There exists a considerable variation in the response among humans to high contents of glycoalkaloids [Morris, S.C.; Lee, T.H. (1984) The toxicity and teratogenicity of Solanaceae glycoalkaloids, particularly those of the potato ( Solanum tuberosum) ; a review. Food Techn. in Australia, vol 36(3), pp. 118-124] . In a study of the WHO [WHO (1993) Toxicological evaluation of certain food additives and naturally occurring toxicants, WHO Food Additives Series 30, p. 372], it is concluded that normal SGA contents (20-100 mg/kg of fresh weight) do not constitute a health risk. The oral LD 50 -value (dose for 50% mortality) for laboratory animals is highly species-dependent and is much higher than for humans. For rats, an LD S0 value of 590 mg/kg of body weight was found [Gull, D.D.; Isenberg, F.M.; Bryan, H.H. (1970) Hortic Sci. 5, p. 316], while in mice, at a dose of 1000 mg/kg of body weight, no toxic effects were observed [Nishie, K. ; Gubmann, M.R.; Keyl , A.C. (1971) Pharmacology of Solanine. Toxicol . and Appl . Pharmacol. 19, pp. 81-92] . Solanine was particularly toxic after parenteral administration in mice, rats and rabbits; the aglycon solanidine was significantly less toxic than the glycoside [Nishie, ibid] . However, the teratogenic effects experimentally demonstrated in laboratory animals, in particular hamsters [Renwick, J.H.; Claringbold, W.D.HB.; Earthy, M.E.; Few, J.D.; McLean, A.C.S., (1984) Neural tube defects produced in Syrian hamsters by potato glycoalkaloids, Teratology, 30, pp. 371-381], could not be confirmed in limited human studies [WHO, ibid.]. Glycoalkaloids also exhibit an insecticidal action [Tingey; American Potato Journal 61:157-167, 1984] inter alia against the Colorado beetle and grasshoppers. Also the growth of fungi, such as Phytophtora infestans, is suppressed, the EC 50 -value (effective concentration at which 50% inhibition occurs) for Trichoderma viride being 1153 μM solanine [Patil et al ; Food Cosmet . Toxicol . 10:395-398, 1972] . For Al ternaria brassicicola, 40% growth inhibition at 100 μM chaconine has been described; given the same concentration of solanine, inhibition was only 10% [Fewel et al; Phytochem. 33:323-328, 1993]. The toxicity of SGA, in particular after parenteral administration, is attributed to at least two mechanisms. They are the inhibition of acetylcholinesterase as neurotoxin and the disruption of membranes. For bovine and human acetylcholinesterase, the inhibitory effect of solanine is greater than that of chaconine; with enzyme from eel, for both glycoalkaloids 25% inhibition is found at a concentration of 40 μM, and for the aglycon solanidine only 15% inhibition at 85 μM [Busway et al ; American Potato Journal 64:409-413, 1987]. Disintegration of the membrane structure, and hence loss of functionality, depends on the type of (glyco) alkaloid and the membrane composition [Keukens, ijbid.] . In, for instance, potato and tomato breeding, traditionally, selection has been based on low contents of SGA. From the point of view of consumer safety, a content of 60-70 mg/kg of fresh weight is used [van Gelder, ibid . ] . In breeding, increasingly, use is made of wild Solanum species to introduce important properties such as resistance to diseases and plagues into a cultivated potato. However, an important barrier to introducing such wild genes resides in the fact that such genes might increase the contents of SGA in the crosses and possibly introduce other SGA's than solanine and chaconine. The guideline of average contents of 60-70 mg SGA per kg of fresh potato is therefore used in potato breeding to prevent toxicity problems of new potatoes. Admission to variety lists is denied to varieties having an average SGA content higher than 200 mg/kg. This limits the choice of possible crossing parents. Wild Solanum types having high or deviant SGA contents do not qualify to serve as ancestor, even though they may possess highly desirable (SGA-related or SGA-independent ) resistance characteristics which could improve the properties of a crop plant such as potato or tomato. By removal and/or modification of steroid glycoalkaloids (SGA) from Solanum products such as potato pulp, potato liquor, potato fibre, protamylasse, potato tops, potato protein, tomato pulp or tomato tops, a reduction of the toxicity of, for instance, potato and tomato tops or a taste improvement of, for instance, potato protein could be accomplished, with favorable consequences for utility or marketing possibilities. With regard to the available physico-chemical methods, in this connection supercritical extraction with C0 2 , extraction with organic solvents, extraction with acetic acid or citric acid, washing with inorganic acids and complex formation with cyclodextrin can be mentioned. However, such methods have various disadvantages, in that organic solvents are toxic and environmentally unsound, acids may not be discharged without neutralization, which in itself, due to the high salt quantities involved, has an adverse effect on the environment, and extraction with C0 2 or complex formation with cyclodextrin is expensive and little profitable. In addition to chemical methods, biotechnological treatments can be employed. Enzymes endogenously present in potato juice are capable of desugaring solanine and chaconine to form the aglycon solanidine [Swain et al ; Phytochem. 17:800-801, 1978]. Solanidine possesses a reduced acute toxicity compared with solanine and chaconine. However, these endogenous enzymes do not make a contribution to the removal or modification of SGA during the processing of potatoes. WO 97/04107 describes an enzyme preparation (chaconinase) which can deglycolyze (desugar) α-chaconine, but not β- or γ-chaconine, nor solanine, to solanidine . SGA-conversion is also possible with micro-organisms. Conversion of SGA and comparable glycoalkaloids and saponins has been described for various micro-organisms and enzymes therefrom. Deglycosylation of solanine or chaconine has been described for mixed populations of unidentified rumen micro-organisms [King et al ; J. Agric . Food Chem. 29:1101-1103, 1981] . Also, deglycosylations of tomatine, likewise a steroid glycoalkaloid, have been described for the fungi Septoria lycopersici , Botrytus cinerea and Fusarium oxysporum . For Gymnoascus reesii , both the conversion of tomatine to tomatidine and the further degradation of tomatidine have been reported [Gaberc- Porekar et al ; J. Ster. Biochem. 19:1509-1511, 1983]. Bacterial transformations can take place with Rhodococcus equi {Nocardia restrictus) , whereby tomatidine is converted to the 3 -oxo-1 , 4-di-ene derivative, and whereby, starting from tomatine, conjugation with lactate occurs without removal of the sugar units [Belie et al ; J. Ster. Biochem. 23:323-326, 1985]. Art rohacter simplex, after induction with cholesterol, is able to cleave the spirosolane side chain of tomatidine, thereby forming 1,4- androstadiene-3 , 17-dione, which is a starting product for the synthesis of female sex hormones [Belie et al; J. Ster. Biochem. 6:1211-1212, 1975]. Such a reaction, starting from solasodine and after cholesterol induction, proved to be possible also with Mycobacterium species NRRL B-3805 [Shukla et al ; Lett. Appl . Microbiol . 15:86-88, 1992], whereby l-androstene-3 , 17-dione was formed, which can likewise serve as starting material for sex hormone synthesis. From the above, it appears that a fair amount of knowledge is present with regard to the microbial conversions of tomatine and steroids derived therefrom. As for the possibilities of microbial conversion of - 6 - specifically solanine and chaconine and the toxicity of the conversion products, however, rather little is known. The invention provides isolated and/or recombinant micro-organisms and cultures thereof, and enzyme systems and enzymes, capable of converting substrates such as (toxic) steroid glycoalkaloids (SGA) and SGA derivatives. 'Isolated' in this specification means not only that the micro-organism in question, characterized in that it is capable of converting SGA and/or SGA derivatives, is provided by the invention in pure culture. 'Isolated' also means that the previously isolated micro-organism has been transferred to another environment. The invention also provides microbial cultures in which, through enrichment techniques or by administration from pure cultures, the organism in question is present in a substantial amount or in excess, in addition to the (obligatory or contaminatory) presence of other, possibly not SGA and/or SGA derivative- converting micro-organisms. The invention also provides isolated micro-organisms, and microbial cultures thereof, which relate to at least two micro-organisms, characterized in that a first one exhibits one particular aspect of SGA and/or SGA-derivative conversion, another exhibits one (possibly overlapping) aspect, and a next one again exhibits one (possibly overlapping) aspect. The invention further provides mixed cultures of isolated microorganisms, while (sequentially and/or simultaneously) use is made of the different enzyme systems and/or combinations thereof which enable the different micro-organisms to exhibit SGA and/or SGA-derivative conversion. The invention also provides recombinant micro-organisms which exhibit SGA and/or SGA-derivative conversion. Such recombinant micro-organisms can exhibit at least one aspect of the isolated micro-organisms previously provided by the invention. In particular, an enzyme and/or enzyme system, and/or nucleic acid coding therefore, which specifically exhibits SGA and/or SGA-derivative conversion, can be purified from an isolated micro-organism provided by the invention through molecular-biological techniques. With recombinant techniques which, per se, are within reach of those skilled in the art, the nucleic acid coding for such an enzyme can be incorporated into a recombinant micro- organism which, as a result, exhibits SGA and/or SGA- derivative conversion, resulting from activity of the desired enzyme. The invention also provides such an enzyme, either produced by an isolated micro-organism as provided by the invention or as commercially available as enzyme, or produced by a recombinant micro-organism provided by the invention, which is also by itself capable of exhibiting SGA and/or SGA-derivative conversion. Also combinations of such enzymes, or (specific) enzyme systems, are provided by the invention. By combinations of enzymes and/or by enzyme systems are meant those combinations of enzymes which, when used together for the conversion of SGA and/or SGA derivatives, exhibit a broader substrate spectrum and/or faster conversion than if only one enzyme is used. Combinations of enzymes are also useful to cause a specific reaction to proceed better. Combinations of enzymes and/or enzyme systems may also consist of enzymes which speed up conversion reactions which proceed sequentially, with an earlier conversion being followed systematically by a further conversion. The invention provides (pure cultures of) a micro-organism capable of deglycosylating SGA's such as the (toxic) solanine, chaconine and tomatine and (starting from solanine, chaconine and/or tomatine) converting same to, for instance, the (less toxic) aglycone . The invention provides an isolated micro-organism capable of deglycosylating steroid glycoalkaloids (SGA) , preferably solanine, and converting same to solanidine. The invention further provides a micro-organism capable of deglycosylating also chaconine and/or tomatine and converting same to aglycone. The invention provides a micro-organism capable of deglycosylating α-solanine, β-solanine and/or γ-solanine and a micro-organism capable of deglycosylating also α-chaconine, β-chaconine and/or γ-chaconine. The invention further provides a micro-organism characterized in that it can detoxify solanine and/or chaconine. The invention also provides a micro-organism capable of converting solanidine to a less toxic component, such as 4-solanidine-3-one . In the experimental section of the description of the invention, by way of example, a bacterial micro-organism is discussed. A micro-organism according to the invention relates to, for instance, an Arthrobacter species or a micro-organism with related functional properties with regard to SGA, specifically those relating to deglycosylation of solanine and/or solanine and chaconine and the conversion to (less toxic) solanidine, and/or with regard to the conversion of solanidine to (less toxic) 4-solanidine-3 -one . An example of such a micro-organism is A . histidinolovorans/nicotinovorans . A representative of such a micro-organism with such functional properties with regard to SGA is isolate 6B, deposited under CBS 100108 on Monday, September 29, 1997 with the Centraalbureau voor Schimmelcultures , Oosterstraat 1, 3742 SK, Baarn, the Netherlands. Also a representative of such a micro-organism with such functional properties with regard to SGA is Arthrobacter isolate 13A. Another example of such a microorganism according to the invention relates to an Aureobacterium or a micro-organism with related functional properties with regard to SGA. A representative of such a micro-organism with such functional properties with regard to SGA is isolate 2B1, deposited under CBS 100107 on October 7, 1997 with the Centraalbureau voor Schimmelcultures, Oosterstraat 1, 3742 SK, Baarn, the Netherlands. Another example of such a micro-organism according to the invention relates to an isolate 10B or a micro-organism having functional properties regarding SGA - 9 - that are related to isolate 10B. Another example of such a micro-organism according to the invention relates to a Rhodococcus or a micro-organism with related functional properties with regard to SGA. An example of such a micro- organism described in the experimental section is R . erythropolis . A representative of such a micro-organism with such functional properties with regard to SGA is Rhodococcus erythropolis (for example, AVB-sol 6) , deposited under CBS 100109 on Monday, September 29, 1997 with the Centraalbureau voor Schimmelcultures, Oosterstraat 1, 3742 SK, Baarn, the Netherlands. The invention further provides a micro-organism capable of converting 4-solanidine-3 -one to a (less toxic) derivative or a microorganism capable of converting solanidine to other derivatives than 4-solanidine-3-one, for example via an intermediary formation of 4-solanidine-3 -one . The invention further provides a micro-organism capable of mineralizing 4-solanidine-3-one to C0 2 Such a micro-organism according to the invention relates to a Rhodococcus or a micro- organism with related functional properties with regard to SGA, which properties relate to the conversion of 4- solanidine-3 -one to a (less toxic) derivative. A representative of such a micro-organism with such functional properties with regard to SGA is Rhodococcus erythropolis (for example, AVB-sol 6) , deposited under CBS 100109. Other representatives of such a micro-organism with such functional properties concern, for example, Paenibacillus pabuli (for example, AVB-sol 1, 2, 5) and/or Enterococcus flavescens (for example, AVB-sol 3) . The invention provides a micro-organism, enzyme system and/or enzyme, capable of (enzymatically) converting steroid glycoalkaloids (SGA) and SGA metabolites or derivatives (such as solanidine and 4-solanidine-3 -one) to (culture) mixtures which, in addition to SGA, also contain (substantial amounts of) (potato, aubergine or tomato) protein and/or (potato, aubergine or tomato) carbohydrates. - 10 - The invention provides a micro-organism which exhibits SGA and/or SGA-derivative conversion in a culture medium which, in addition to SGA, also contains simple and/or complex carbohydrates and/or proteins. The invention provides a micro-organism which exhibits preferentially SGA and/or SGA-derivative conversion, also in complex and/or nutrient rich media such as (potato, aubergine or tomato) protein suspensions and protamylasse, which is a concentrate of deproteinized potato liquor. The invention further provides a method for isolating micro-organisms which exhibit (preferentially) SGA and/or SGA-derivative conversion. Such a method provided by the invention is concerned with the enrichment of microorganisms in a medium containing at least one SGA followed by isolating and identifying such micro-organisms growing on culture plates such as agar plates which are wholly or partly, for example in a top layer, provided with at least one SGA. Micro-organisms can be introduced into the medium from inoculum, such as, for example, soil and/or potato pulp, but also other sources of micro-organisms can be used. In one embodiment of this method, the base medium is a relatively poor carbon and energy source medium, in which only the SGA itself is a complex nutrient, whose concentration can vary. In another embodiment of a method according to the invention, the base medium is rich in substrates with complex nutrients, for example (potato, aubergine of tomato) protein and/or (potato, aubergine or tomato) carbohydrates or a slurry deriving from substrates pretreated with chemical or physical methods. Another embodiment of a method according to the invention relates to the enrichment of micro-organisms in a base medium which contains at least one SGA derivative (in different possible concentrations) , followed by streaking such enriched microorganisms on culture plates such as agar plates which are wholly or partly, for example in a top layer, provided with at least one SGA derivative. A further embodiment of a method according to the invention relates to the repetitive - 11 - performance of the method, while the SGA and/or SGA- derivative that is present in the base medium or in the agar in the second performance is preferably different than in the first performance, while a choice can be made, for instance, for an SGA or SGA-derivative (such as 4- solanidine-3-one) which has been formed by a previously invented micro-organism. By proceeding, during a second or further performance of a method according to the invention, to limit the base medium or the concentration of components therein (for example nitrogen (N) in (in) organically bound form) or otherwise modifying it, a micro-organism is selected which can obtain the necessary nutrients through SGA and/or SGA-derivative conversion, which yields a successive SGA-derivative and associated micro-organism. The invention also provides the (further) isolation and identification of an enzyme (or enzyme system or enzyme combination) isolated from a micro-organism according to the invention, which exhibits SGA and/or SGA-derivative conversion. Such an isolation and identification of a micro-organism according to the invention are per se within reach of those skilled in the art and can be performed according to any one of the many purification methods and/or activity tests which are common in microbiology. The invention provides an enzyme combination and/or an enzyme which is capable of converting (α-, β-, and/or γ-) solanine and/or (α- , β-, and/or γ-) chaconine to solanidine. The invention further provides an enzyme capable of converting solanidine to, for example, 4-solanidine-3 -one, keto-4- solanidine-3 -one , 4-solanidine-l , 3 -dione, 4-solanidine-2 , 3 - dione or 4-solanidine-3 , 6-dione or solanidiene-3 -one and also an enzyme capable of converting 4-solanidine-3-one , keto-4-solanidine-3-one, 4 -solanidine-2 , 3-dione, 4- solanidine-3 , 6-dione or keto-4-solanidine-3 -one or solanidiene-3-one . Also, with recombinant DNA and/or RNA techniques known per se, the gene and/or the corresponding nucleic acid molecule that codes for an enzyme according to the invention can now be identified and isolated, for - 12 - example after selection of appropriate primers and/or probes and use of appropriate hybridization and/or polymerization techniques. Also, such a gene can be modified, so that mutants are obtained. The invention also provides a vector, such as a plasmid or a viral vector, equipped with such a gene or genes. The invention also provides a (host) cell or micro-organism or expression system provided with such a gene or vector, and the invention also provides an enzyme produced by a (recombinant) cell according to the invention. An example of such a cell provided by the invention is a recombinant or transgenic plant cell or a naturally SGA-producing plant, which plant now exhibits, during growth or during processing, enzymatic SGA-conversion and/or detoxification through the enzyme produced by the gene introduced. The invention further provides a test method in which the content of SGA and/or SGA derivatives in a sample is determined by means of an enzymatic reaction in which at least one enzyme according to the invention is used. An example of such a test method provided by the invention is a test method in which the content of solanidine and/or tomatidine in a sample, such as taken, for example, from a potato, potato protein, tomato, tomato pulp and the like, is determined on the basis of the enzymatic conversion to, for example, 4-solanidine-3 -one or keto-4-solanidine-3 -one, such as for example catalyzed by the enzymes solanidine oxidase or solanidine dehydrogenase which are provided by the invention. In another example of a test method provided by the invention, the content of solanine or tomatine is determined through selective conversion to solanidine or tomatidine, catalyzed by an enzyme provided by the invention, whereafter the content of solanidine or tomatidine is determined as described. In an embodiment of such a test method, the hydrogen peroxide released in the conversion can be measured and serve as a measure for the converted aglycone. The invention further provides a test kit comprising requisite means, for example an enzyme and - 13 - (control) substrates for carrying out a test method according to the invention. The invention further provides the use, and a method for the use, of an isolated microorganism or (recombinant) micro-organism according to the invention for the conversion and/or removal of SGA and SGA derivatives, as applicable, for instance, in the potato and/or tomato processing industry. The invention also provides a method in which such a micro-organism is used for the production of raw material for (sex) hormone synthesis and for further synthesis of bioactive steroids. Especially steroid hormones such as testosterone, aldosterone, progesterone, dexamethasone, corticosterone and cortisol can now be produced from SGA from Solanaceae species such as the potato or tomato. The invention also relates to the industrial application of such micro-organisms, enzyme systems and enzymes and methods in, for example, the potato and/or tomato processing industry and in combination with new agricultural methods which are sustainable and affect the environment less, if at all. The invention provides the selection of a crop plant such as a crop potato which has a high or strongly elevated average SGA content (for example >200-250, or >250-500 or >500mg SGA/kg of fresh weight) . Such a potato can be selected on the basis of a test method provided by the invention for determining the SGA or SGA- derivative content. The invention provides the use of potatoes with a high SGA content in the potato processing industry. The invention provides a method for the industrial processing of such potatoes, in which the SGA and SGA-derivative content is reduced to detoxify the potato product and make it suitable for human or animal consumption. This is now possible by virtue of the microorganisms and methods (capable of converting SGA and SGA derivatives) which are provided by the invention. The invention provides the selection of crop plants, such as tomatoes and/or crop potatoes that are resistant to - 14 - diseases and plagues as a result of a high or greatly elevated content of SGA or as a result of crossing in specific resistance characteristics, whereby at the same time a high SGA content has been introduced into the new potato. Solanum types with a high or deviating SGA content now do qualify to serve as ancestor, because they possibly possess highly desirable (SGA-related or SGA-independent ) resistance characteristics which could improve the properties of a crop plant. By crossing in (breeding) such characteristics, the (toxic) SGA content of, for example, the crossing potato will rise, which results in potatoes having an increased resistance to diseases and plagues, such as fungi (e.g. Phytophthora) and nematodes, which are sensitive to SGA. However, non SGA-related resistance can now be readily crossed in, too, since the selection of such crossing products no longer needs to be hindered in that they exhibit a high SGA content. The invention also provides new potatoes with a high productivity. This high productivity can now be easily crossed in, since the selection of such crossing products no longer needs to be hindered because of a high SGA content . The invention further provides potatoes and/or tomatoes (having a high or strongly elevated SGA content) which are pre-eminently suitable as a source for SGA-metabolites and derivatives which can be used in steroid hormone synthesis. This SGA can be economically recovered and used in steroid synthesis. Residual SGA (for example, in the residue protein) can subsequently be converted according to the methods mentioned hereinabove; residue problems are thus prevented or controlled. The crops which are provided by the invention can also contain other SGA's than solanine and/or chaconine. While the conventional crop potato (S . tujberosum) principally contains the above-mentioned two SGA's, other Solanum species that are eligible as crossing parent (such as S.vernei) also have other types of SGA. The - 15 - invention provides an integrated (sustainable) agricultural method whereby crops such as potatoes having a high SGA content and a high productivity and/or a high resistance to diseases and plagues (and hence having a low need for crop protection chemicals) are processed by the potato processing industry in a manner hardly, if at all, affecting the environment. A crop potato with a high SGA content as provided by the invention can also be used as seed potato to breed new potatoes and new potato varieties. Such a potato variety with a high SGA content has, for example, a high resistance to one or more diseases and plagues and/or a high productivity. As a result, fewer diseases and plagues need to be controlled, resulting in a lower use of crop protection chemicals. The tops and other components of SGA-containing crop plants can now be better made suitable by the detoxification and/or taste improvement through enzymatic conversion as provided by the invention. (Animal) food with such treated material is eminently suitable, now that the toxic and/or taste- affecting components have been removed. The invention is further elucidated in the experimental section, which does not imply any limitation of the invention. EXPERIMENTAL SECTION MATERIAL AND METHODS 1. Enrichment In enrichment, use was made inter alia of a base medium having the following composition: NaN0 3 1 g; MgS0 4 .7aq 0.2 g; KCl 0.5 g; FeCl 3 .6aq 0.02 g; tryptone 1 g; glucose 1 g; aquadest 960 ml. To portions of 24 ml of this medium, 5 grams of inoculum were added. As inoculum, soil was used in which potatoes have been grown, potato pulp of non-washed potatoes and a mixture (l/l, v/v) of soil and - 16 - potato pulp. Next, 1 ml of a solution of 25 mg/ml SGA in 7.9% H 3 P0 4 was administered. For neutralization of the medium, 0.6 ml 2N NaOH was added. The final SGA content in the medium was 1 g/1, as was the P-content. SGA was obtained from the Department of Crop Research of NIKO-TNO. The preparation was isolated from potato pulp. Based on dry matter, the preparation contained 84% glycoalkaloids with an excess of solanine and chaconine. The enrichment cultures were incubated for three days at 30°C in a shaking incubator at 200 rpm. The enrichment was continued in Bioscreen cuvettes, (Labsystems Oy, Finland) . With regard to the medium composition, the following variants were used on the basis of the above-described enrichment medium: C-sources: In the presence or absence of 1 g/1 glucose, 1 or 2 g/1 SGA was added to the medium. N-sources: 1 g/1 (NH 4 ) 2 S0 4 or 1 g/1 NaN0 3 or 0.5 g/1 (NH 4 ) 2 S0 4 and 0.5 g/1 NaN0 3 or 0.5 g/1 NaN0 3 and 0.5 g/1 tryptone. Vitamins: Variation consisted in adding, or not adding, 10 ml/1 of a stock solution. The same procedure was followed with regard to the administration of spore elements. The total volume in the Bioscreen cuvettes was 350 μl . To 260 μl base medium, the necessary amount of SGA was added as a solution in 3.7% H 3 P0 4 in a volume of 30 μl . Next, for neutralization, 30 μl 0.51N NaOH was added. As inoculum, 30 μl of the above-described enrichment cultures from soil and/or potato pulp was used. The inoculum was added as a 10-fold, 10 3 -fold and 10 5 -fold dilution. In enrichment, further, use was made of slurry, obtained after treatment of the acid washing water, which is released upon the treatment of potato protein for the chemical removal of SGA with a method according to WO 97/03571. The acid washing water was treated with lye to pH 10. The precipitate was in turn treated with acid to extract SGA and the acid fraction was again precipitated with lye (pH 10) . This material was stored for a few months, whereby the pH started to decrease to, finally, - 17 - neutral. It concerns material with a high SGA content (5% based on dry matter) . 2. Isolation From different Bioscreen cuvettes, 0.1 ml culture (in tenfold, 10 3 -fold and 10 4 -fold dilution) was spread onto agar substrates with 1.8% agar and the above-mentioned composition of the base medium excluding the C- and N- sources glucose, tryptone and NaN0 3 . Variation with regard to the N-sources resulted in the following media: A: The above-described medium with 1 g/1 tryptone, B: As A, but with a lowered tryptone content to 25 mg/l, C : As A with 1 g/1 NaN0 3 instead of tryptone. In all cases, further, 10 ml/1 of spore elements and vitamins were added, as well as 40 ml/1 7.9% H 3 P0 4 , whereafter the pH was adjusted to 6.9 with 4 N NaOH. After inoculation, the basic agar was overlayed with 3 to 3.5 ml top agar. The top agar contained 0.75% agar and 2.5 g/1 SGA. The preparation thereof occurred as follows: After dissolving 150 mg SGA in 5 ml 3.7% H 3 P0 4 , 5 ml 0.51N NaOH was added thereto. The thus formed suspension of 15 mg/ml SGA was added in an amount of 1 ml to 5 ml 0.9% agar. Thus the inoculated agar substrates were provided with an even, slightly turbid covering layer. The procedure described was used because direct addition of SGA (without first dissolving in acid followed by precipitation with NaOH) to an agar suspension does not yield a uniform turbidity of the top layer. Instead, flocculation is observed, a problem which may also arise upon direct addition of SGA to the basic agar followed by neutralization and sterilization. The cultures were incubated at 30°C for five days and selected isolated colonies were replica plated onto the media A and C. Upon further selection, a number of colonies were maintained on medium A and also streaked onto agar substrates identical in composition to medium A. Instead of - 18 - H 3 P0 4 , however, phosphate buffer was added to the basic agar, in the form of 2.5 g/1 K 2 HP0 4 and 2.5 g/1 KH 2 P0 4 . Also, no top agar with SGA was administered, but in the bottom agar 2 g/1 glucose (medium D) or 2 g/1 soluble starch (medium E) were present as carbohydrate source. From medium D the isolates were again cultured onto medium D and subsequently preserved in liquid nitrogen in a Nutrient Broth medium to which 20% (m/v) glycerol had been added. In addition, the cultures were maintained on agar slants (Nutrient Agar) . From a 20% slurry suspension in physiological salt, decimal dilutions were spread onto mineral agar medium with 1 g/1 tryptone, spores and vitamins and in addition onto Tryptone Soy Agar. Plates were inoculated and covered with a covered top layer with 0.75% agar and 2.5 g/1 SGA. Isolated colonies were transferred to Tryptone Soy Agar (TSA) with covering layer. After 72 hours at 30°C, colonies with a clearing zone were found. These were spread onto TSA and Nutrient Agar (NA) with and without covering layer of SGA. Isolated colonies were plated onto TSA and NA and maintained as pure cultures of Nutrient Agar slants. 3. Identification of micro-organisms Different isolates were further identified by DSM-Z (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany) . Prior thereto, microscopic evaluation of the isolates occurred, as well as investigation of growth at different temperatures on Nutrient Agar (Difco) . Anaerobic growth was examined by streaking from a suspension onto Plate Count Agar (Difco) having added thereto 0.5 g/1 cysteine-HCl . The plates were incubated at 30°C for 7 days in anaerobic jars in the presence of Anaerogen™-AN35 (Oxoid) . In addition, Gram- stains were performed, as well as spore stains. The motility of the bacteria was investigated by means of streaking in API-M (otility) medium (API-Biomerieux, Den - 19 - Bosch) . In case of turbidity of the medium after 24-48 hours of growth at 30°C, the bacteria are considered as motile; in the case of exclusive growth adjacent to the inoculation pitch, as non-motile. The oxidative/fermentative conversion of glucose was examined with API-OF-medium (API-Biomerieux, Den Bosch) . For assaying into catalase activity, three colonies were smeared out onto an object glass. Next, per colony, 1 drop of 3% H 2 0 2 was added. In the case of gas development the reaction is positive. The presence of oxidase was investigated with oxidase reagent (1% tetramethyl-p- phenylene-diamide in isoamyl alcohol; API-Biomerieux, Den Bosch) . Further (biochemical and physiological) characterization (enzyme reactions, conversion of substrates, acid formation and growth on different substrates) occurred according to instructions with the identification systems (Biomerieux, Den Bosch) API-20E [Enterobacteriaceae) , API-20NE (determination of non- Enterojbacteriaceae) and API-Coryne (determination of coryneform bacteria) . 4. SGA-degradation in shaking cultures Of a number of isolates, the SGA-degradation was monitored in shaking cultures. Growth occurred in a mineral medium with SGA as carbon and carbohydrate source . The composition of the medium was, for example, as follows: NaN0 3 1 g/1; KCl 0.5 g/1; KH 2 P0 4 1 g/1; K 2 HP0 4 1 g/1; MgS0 4 .7aq 0.2 g/1; FeCl 3 .6aq 0.02 g/1; MnCl 2 .4aq 0.004 g/1; spore solution 10 ml/1; vitamin solution 10 ml/1; SGA- suspension 100 ml/1. The SGA-suspension was prepared by dissolving 1 g SGA in 25 ml 3.7% H 3 P0 4 followed by neutralization to pH 7 with 25 ml 0.51 N NaOH. The final concentration of SGA in the medium was 2 g/1. After inoculation with a colony of medium D, 25 ml medium was - 20 - incubated up to 7 days at 30°C in a shaking incubator at approx. 150 to 180 rpm. 5. Conversion of SGA in protein and protamylasse Investigation of the conversion of SGA in protein occurred amongst others with the isolates 6B and 13A. In a first orientation, these organisms were cultured overnight in Tryptic Soy Broth (TSB) at 30°C and 200 rpm (shaking incubator) . Then, 25 ml culture was added to 25 ml protein suspension (potato protein AVEBE-PF-PCR) with 10 g protein in 40 mM phosphate buffer pH 7.0. As a control, 25 ml sterile TSB was added. In the next experiments with regard to the conversion in potato protein, in each case 12.5 ml overnight culture was centrifuged and resuspended in 12.5 ml physiological salt solution (0.85% NaCl with 1 g/1 peptone) . This suspension was added to 12.5 ml protein suspension (2.5 g protein) in 12.5 ml 40 mM phosphate buffer pH 7.0) . As a control, sterile physiological salt solution was used. In the first experiment, a protein concentration of 20% was used; in the other experiments, it was 10%. All protein incubations occurred at 30°C and 200 rpm in a shaking incubator. For growth and conversion of SGA in protamylasse, amongst others isolate 6B was used. Protamylasse (AVEBE-December 1996) with 60% dry matter was diluted with aquadest to 0.5-2.0% dry matter. To these media, in several cases glucose was added in a concentration of 0.1-0.5%. Glucose was separately sterilized and the diluted protamylasse was, prior to heat-sterilization, aerated for 0.5 hours to prevent a possibly inhibitory effect of sulfite. The different media were inoculated (1 ml/50 ml) with an overnight cultured pre-culture in TSB. Incubation of pre-culture and main cultures occurred at 30°C in a shaking incubator (200 rpm) . The growth was monitored by plating 0.1 ml of suitable dilutions on - 21 - Tryptic Soy Agar or Plate Count Agar (Difco) . Counting of the number of colonies occurred after overnight incubation at 30 °C. In another set up, growth of the micro-organisms in diluted protamylasse was combined in a 1 -unit -operation with SGA removal from potato protein or potato protein filtercake. In a typical experiment Arthrobacter 6B was cultured in 0.5% (dry matter) protamylasse at 30 °C . After 10 hours of cultivation, potato protein filtercake or dried potato protein was added to a final concentration of 10% (dry matter) to establish the removal of SGA. 6. Identification of modified aglycons and other analyses. Preparation of the modified aglycon was carried out as described hereinbelow in 7.1 and 8.3. NMR-spectroscopy (1H and 13 C) was done with a Varian VXR 300 (300 Mhz) spectrometer. The components to be examined are dissolved in CDC1 3 , with which also internal calibration took place. In addition, analysis of the aglycon conversion was carried out with the aid of direct MS performed with a Finnigan mass spectrometer using APcI (Atmospheric Pressure Chemical Ionization) in "positive mode" in the range of 100-1000 Da. SGA-analysis occurred according to an HPLC method and the aglycon was determined with a GC method, both as described hereinbelow. In addition, the microbial consumption of SGA and aglycons was monitored in certain cases through a total -carbohydrate analysis (Anthron) or TOC analysis with a Dohrmann DC- 190 TOC-analyzer . Prior to the TOC and Anthron determinations, the samples were acidified to pH 2.3 with 0.1 ml 3.7% H 3 P0 4 per ml sample, so that SGA, and components derived therefrom, were completely in solution. The activity of protease was measured with 2.5% azocasein as substrate in 10 mM phosphate buffer pH 7.0. The reaction occurred through incubation of 0.25 ml substrate with 0.25 ml sample for 2 hours at 30°C. As control, 0.25 ml - 22 - substrate was incubated with 0.25 ml phosphate buffer. After addition of 2 ml 5% trichloroacetic acid, centrifugation occurred (8000 rpm; 15 min) followed by administration of 1.25 ml supernatant to 1.25 ml 0.5 N NaOH. As a measure for the protease activity, E 366 was determined, after correction for the control. A protease unit corresponds to an increase of E 366 of 1/hour. Determination of glycoalkaloids occurred using an HPLC method. An amount of 4 ml sample was added to 25 ml Na-heptanesulfonate/acetic acid, followed by centrifugation and filtration. Next, 20 ml liquid was purified over a SepPak-column. An amount of 3 ml liquid was eluted from the column, of which 20 μl was injected for HPLC-analysis . The conditions were as follows : mobile phase: 50% acetonitrile buffer (0.3 g/1 (NH 4 ) 2 HP0 4 ) ; isocratic 1 ml/min; column: Zorbax RX-C18 with Zorbax RX-C18 precolumn; detection: UV-detector at 202 nm. For the determination of aglycons, a gas chromatographic method was used. An amount of 2 ml sample is extracted in freeze-dried form with 10 ml chloroform/methanol (1/2 (v/v) ) by incubating overnight at room temperature. Next, the extract is divided into two portions of 4 ml (total aglycon and free aglycon) , whereafter the extraction agent was removed by passing through nitrogen gas. For determining the total aglycon, 0.1 ml aquadest and 0.9 ml 3 N HCI are added to the evaporated extract in a screw cap tube. Thereafter, 2 ml internal standard in trichloroethane (150 mg/l heptadecane and 150 mg/l cholesterol) were added, followed by 2 hours' incubation at 75°C under vigorous mixing of the two phases. Upon cooling to room temperature, 1 ml 4N NaOH is added, followed by mixing for 30 minutes to extract the released aglycon to the organic phase. After centrifugation, 0.5 μl of the trichloroethane phase is injected. For determining the free aglycon, no hydrolysis occurs (no administration of HCI, no incubation at 75°C) . To the evaporated extract, - 23 - directly 2 ml internal standard in trichloroethane was added, followed by administration of 4N NaOH. The follow-up procedure is identical to that for the total aglycon. The GC-conditions for determining the aglycons were as follows: CP Sil 5 CB column (50 m * 0.32 mm (internal diameter) film thickness 0.12 μm) with a deactivated precolumn of ca . 1 m * 0.53 mm (internal diameter); injector: cold-on- column with H.O.T. -option (sec. cooling from 2 min before injection to 10 min after injection); carrier gas He with 1 bar gauge pressure; FID-detector at 300 °C; temperature programming: 23 min at 270 °C, followed by an increase of 5 °C/min to 320°C and cooling off to 270°C. 7. Toxicity of SGA and SGA metabolites and derivatives 7.1 Material For the toxicity tests, an SGA mixture was ;used, which has been isolated from potato pulp. This material contains 56% solanine and 44% chaconine based on dry matter. In addition, use was made of commercially available solanine, chaconine and the aglycon solanidine (Sigma) . On the basis of the SGA mixture, also 4-solanidine-3 -one was prepared. To that end, Arthrobacter 13A was cultured in a mineral medium with spore elements and vitamins in the presence of 2 g/1 SGA mixture as carbon and energy source. After 48 hours' incubation and complete deglycosylation of SGA, the pH of the culture was adjusted to 9.0, followed by 15 minutes' centrifugation at 10,000 rpm. After washing with slightly alkaline water (pH 8.5 to 9.0), the precipitate was resuspended in water, followed by addition of 0.1 part of H 3 P0 4 . After 15 to 30 minutes' mixing, whereby the aglycons are dissolved, centrifugation occurred at 10,000 rpm for 15 minutes. The supernatant was subsequently decanted and, with mixing, neutralized to pH 7.3 with 0.51N - 24 - NaOH. After centrifugation and washing (slightly alkaline demi water pH 8.5), the precipitate was dried in vacuum at 50°C. From GC-analysis, it can be concluded that 99.9% of the total aglycon is present as modified aglycon (4-solanidine-3-one) with only 0.1% solanidine. 7.2 Growth inhibition as a measure for the toxicity for micro-organisms It was determined whether the toxicity of glycoalkaloids and derivatives thereof can be established by means of turbidimetric analysis in a Bioscreen C incubator, (Labsystems Oy, Finland) . To that end, the bacteria Escherichia coli (ATCC 25922) , Pseudomonas putida (RIVM MT2) and Bacillus subtilis (ATCC 15841) , as well as yeast ( Saccharomyces cerevisiae) were cultured at 30°C and pH 6.5 in buffered peptone water (BPW, E. coli and P. putida) , Tryptic Soy Broth (TSB, Difco, B . subtilis) or Potato Dextrose Broth (PDB, Difco) . E. coli and P. putida, after overnight incubation (OD 540 = 1.4), were added in an amount of 100 μl to 30 ml 2.8 -times concentrated BPW. B . subtilis and baking yeast (OD 540 = 1.7) were added in an amount of 50 μl to, respectively, 2.8-times concentrated TSB and PDB. In all cases, the volume was supplemented to 60 ml with sterile demi water. For the incubations in the Bioscreen, 250 μl of the above-mentioned bacteria and yeast suspensions were added to 100 μl solutions of glycoalkaloids and aglycons of different concentration. The fungi Aspergillus niger (DSM 821) , A . oryzae (ATCC 7252) and Penicillium rubrum (AVEBE) were cultured for 1 to 2 weeks on PDA (Potato Dextrose Agar, Difco, 40 ml in Erlenmeyer of 250 ml, pH 6.5) at 25 °C . After addition of 25 ml sterile demi water with 0.1% Tween 80, after mixing and suspending of the mycelium, the suspensions were transferred to sterile bottles. The amount of spores - 25 - (usually 10 6 to 10 7 per ml) were counted microscopically in a Thoma counting chamber. Of a suitable dilution, a certain amount was subsequently added to 30 ml 2.8-times concentrated PDB (pH 6.5). Thereafter, this was supplemented with sterile demi water to 60 ml and of the spore suspension 250 μl was added to the wells in the Bioscreen plates, which were supplemented to 350 μl with solutions of glycoalkaloids and aglycons. The initial density of the fungal spores in the wells was thus set at 400 spores per ml. The preparation of (stock) solutions of SGA and aglycons occurred by dissolving 35 mg (dry matter) in 1.5 ml 0.25% H 3 P0 4 followed by supplementation to 2 ml with 0.51N NaOH (pH 6.5) . Dilutions thereof were prepared in 20 mM phosphate buffer of pH 6.5. For determining the toxicity for bacteria and baking yeast, incubation occurred in the Bioscreen at 30°C for 24 hours. Once every 10 minutes, an OD measurement (wideband filter) took place, as did mixing (big- ast) . For fungi the same conditions were used; the total incubation time, however, was 40 hours, an OD measurement taking place once every 20 minutes. After analysis of the growth curves at different concentrations of SGA and aglycons, the percent inhibition was determined as : % inhibition = ( (OD control -OD toxi J /OD control ) ) *100 ( l ! wherein for the OD the final absorption value was taken as a starting point. The percentage of inhibition is plotted against the toxicant concentration. Through graphic interpolation, the EC 50 -value (effective concentration) was determined as the concentration at which 50% inhibition occurs . - 26 - 7.3. Inhibition of the enzyme synthesis: Toxi-Chromotest The Toxi-Chromotest [Kwan; Toxicology and Water Quality 8: 223-230, 1993] is supplied as a complete kit (version 3.0, AquaSense, Amsterdam) . It was used according to the instructions of the supplier. 7.4 Genotoxicity: SOS-Chromotest For determining genotoxic effects [Quillardet et al ; PNAS 79: 5971-5975, 1982] use was made of the SOS-Chromo testkit version 6.0 (AquaSense, Amsterdam) . All operations occurred in accordance with the instructions supplied along with the kit. 7.5 Microbiotests for toxicity in higher organisms To indicate the toxicity in higher organisms, use was made of the Rotoxkit F and the Thamnotoxkit F (AquaSense, Amsterdam) and the instructions supplied along with them. The sensitivity of these tests is comparable to that of a standard toxicity test with Daphnia [Maas et al ; Rapport RIZA 93.171, 1996] . 8. Screening enzyme preparations Many commercially available enzyme preparations were investigated. SGA had been isolated from potato protein (June 1994) and contained, based on initial sample weight, (DM 95%) 42% α-solanine, 36% α-chaconine and 4% β-chaconine. Pure α-chaconine and α-solanine is from Sigma and ICN, pure solanidine from Sigma and Rohm. 4-solanidine- 3 -one had been obtained by incubation of Arthrobacter species 13A on SGA. Potato juice (AVW) was obtained from potatoes, which involved standard addition of 0.1% sodium pyrosulfite (Na 2 S 2 0 5 ) during grinding. Potato protein - 27 - (initial weight 3500 mg SGA/kg, 46% α-chaconine, 45% α-chaconine, 9% β-chaconine and 91.7% DM) came from the potato starch plant Oostermoer (Avebe, PF-PCR AV 9410.001). Incubations in buffer were carried out in Eppendorf vessels of 2 ml with 0.2 ml enzyme and 0.2% SGA in 0.05 molar maleate buffer with 0.02% sodium azide, and optionally 25 ppm ampicillin, in a final volume of 2.0 ml. pH and temperature of the main activity of the enzyme preparation in question were used. An enzyme dosage of 2% (w/v) or 10% (v/v) was used. For incubations with powdered material, first 200 mg of the preparation was dissolved in 1 ml 0.05 molar sodium maleate buffer with 0.02% sodium azide. If interfering components, such as sugars, were present in the enzyme preparation, enzyme preparations were dialyzed before incubation. The incubation period for samples that were analyzed with thin-layer chromatography as described below, was minimally 18 hours. Thin-layer chromatography (TLC) was carried out essentially as described in M. Friedman et al J.Agric. Food Chem. 41:1397-1406, 1993. To Silica gel 60 plates of a thickness of 0.20 mm (Merck art.105553), 5.0 μl sample and 5.0 ml of standard solutions of 0.1% α-chaconine, α-solanine, solanidine and 4-solanidine-3 -one were applied. Solanidine and 4 -solanidine-3 -one were priorly dissolved, with heating to 50°C, in 0.05 molar maleate buffer pH 2.2 and 0.02% sodium azide and then adjusted with NaOH to pH 5.8. The TLC plates, after being dried, were placed in the incubator which was saturated with chloroform-methanol- 2% ammonia (70:30:5) . The components were visualized with saturated iodine vapor or by spraying with anisaldehyde reagent (0.5 ml anisaldehyde, 10 ml methanol, 0.5 ml sulfuric acid and 0.1 ml acetic acid) and heating for 5 minutes at 120°C. - 28 - 8.1 Activity in potato juice and potato protein 0.2 ml enzyme solution was incubated at 38°C in a final volume of 2.0 ml with potato juice (AVW) to which 0.2 % SGA, 0.02% sodium azide and 25 ppm ampicillin had been added. For determining the deglycosylating activity in potato protein, a suspension of 10% potato protein in demi water with 0.02% sodium azide was incubated with 0.02% (w/v) enzyme at pH 5.8 and 40°C. 8.2 Removal of alkaloid by fractionation AVW of the variety Elkana (pH 4.8), to which 0.2 % SGA had been added, was treated with chaconine deglycosylating enzyme. After conversion of chaconine, a part of the AVW was adjusted to pH 6.5 and pH 7.5. This material was subjected to three different treatments: centrifugation (5 min. 3,000 rpm, Eppendorf 5415C) , filtration (0.2 μm) and a combination of heating for 0.5 hours at 48 °C and centrifugation. 8.3 Solanidine modification Incubations were carried out as described under 8. For identification of AG-X1, in different batches, with a total of 2.65 g SGA, incubation took place up to 23 days at a temperature of 38 °C. Of that, 0.1 g SGA in 20 ml was incubated with 5 ml enzyme and 2 ml Helix pomatia HP2 (Sigma) . After incubation, all batches were pooled (total volume 255 ml) . SGA with α-solanine, α-chaconine and β- chaconine in the ratio of 1:1.07:0.26 proved, after incubation, to be converted into a product in which, as appears from HPLC analysis of glycoalkaloid content, only α-solanine (0.75 g) was still present. Ca . 65% of the glycoalkaloid has been deglycosylated. Analysis of the aglycon composition through GC gave - 29 - chromatograms in which, in addition to the peaks of the internal standards heptadecane (6.0 min.) and cholesterol (18.8 min.), there were found peaks of solanthrene (15.7 min. (samples of total aglycon)), solanidine (19.6 min.), 4-solanidine-3-one (21.6 min.), AG-X1 (24.1 min.) and another two components present in lower concentrations, with retention times of 22.0 (unknown 1) and 24.5 minutes (unknown 2) . GC assays showed that about 47% of the total aglycon was present in free form. The difference between the GC and the HPLC outcome can be attributed to conversion of the aglycon to products which do not occur in the GC-chromatograms . This supposition is supported by the given that the calculated amount of total aglycon (about 0.78 g) was well over 20 percent lower than the total initial weight of aglycon (a good 1 g) . It is striking that aglycon unknown 2 was found in the GC assay of the free aglycon but hardly in the determined total aglycon. This may mean that this aglycon is not acid-stable. The other aglycons, except for solanidine (which is converted to solanthrene), are acid-stable. 250 ml of the pooled product was, after pH- increase with sodium hydroxide solution to pH 10.1, extracted with about 250 ml 1 , 1 , 1-trichloroethane . After centrifugation, a light-brown opaque trichloroethane layer was formed. When, after removal of the water layer, the opaque and rather viscous trichloroethane layer was diluted 1 on 1 with trichloroethane, it was found the insoluble parts coagulate to a brown pellet and a bright extract was obtained (extract 1: 485 ml) . From the analysis of the extracts, it appears that in this extract the greater part (71%) of the Ag-Xl is present. Solanidine (24%) extracts significantly more poorly than the other aglycons . Isolation of the modified aglycon AG-X1 from extract 1 (13% AG-X1) was done through column chromatography over silica (Silica gel 60, 230-400 mesh ASTM, Merck), using chloroform :methanol 95:5 (v:v) as mobile phase. The crude - 30 - AG-X1 (with 66% AG-X1 and further consisting mainly of 4 -solanidine- 3 -one and unknown 1) which had been obtained in this way, was purified further (to 89% AG-X1) by column chromatography over silica (Silica gel 60, 230-400 mesh ASTM, Merck) , using chloroform with a gradient MeOH as mobile phase. The thus obtained product with ca. 5 mg AG-X1 was completely evaporated for identification. Eluting in front of AG-X1 a fraction could be collected with less than a mg of aglycone unknown 1, free of the other aglycones . Conversion of 4-solandine-3-one was carried out in a mixture with 0.5 ml 0.1 g/ml Hemicellulase F35000 in 0.05 molar maleate buffer pH 5.8 and 0.02% sodium azide added to 1 ml 4-solanidine-3-one which, under heating at 50°C, had been dissolved to 3 g/1 in 0.05 molar maleate buffer pH 2.2, 0.02% sodium azide and then adjusted with NaOH to pH 5.8. RESULTS 1 Enrichment In the enrichment from soil and/or potato pulp, microbial growth was found to occur in all cases. It was striking that exclusively bacterial growth was involved when SGA was present in the medium. In the absence of SGA, also fungi and protozoans were found. The above indicates the toxicity of SGA with regard to the disturbance of steroid structures in eukaryotic cell membranes. Upon continuation of the enrichment in Bioscreen cuvettes utilizing more selective media, growth was found to occur in each combination of C- and N-sources. This is indicative of the presence of micro-organisms which are capable of growth in a mineral medium to which SGA has been added as carbon and energy source. The degradation of SGA in the different media was confirmed by carbohydrate assays according to Anthron. Some results are reported in Table 1. The SGA content is derived from the carbohydrate content, - 3 1 - 1 gram SGA corresponding to 560 mg carbohydrate. The table shows that, independently of the enrichment source, the N-source in the Bioscreen medium and the presence of vitamins and spore elements, a concentration of 2 g/1 SGA has almost completely been converted. With 1 g/1 and other concentrations, this was also the case (not shown) . When in addition to SGA, glucose had been added as carbohydrate source, also a extensive SGA degradation occurred. 2 Isolation Further isolation occurred on agar media with a top layer of SGA as carbohydrate source. Isolated colonies were found in the case of inoculation with a 10 -dilution from the Bioscreen cuvettes. On media with 1 g/1 tryptone (medium A) or NaN0 3 (medium C) as N-source, after 2 days of incubation, around a large number of colonies a clearing zone (pointing to the uptake of SGA or SGA derivatives) was present in the turbid covering layer. After 5 days of incubation, in turn, often an additional turbid zone is found (pointing to a possible secretion of aglycon and/or SGA derivatives) . On medium B with only 25 mg/l tryptone as N- source, growth was considerably slower and after 5 days of incubation, still many colonies had a clearing halo. In the further selection of micro-organisms, the capacity for a rapid clearing has been used as an important criterion. A number of isolated colonies of the media A, B and C were replica plated to media A and C. Of these, depending on the extent of clearing and (different) colony morphology, colonies were maintained on medium A and, further, streaked onto media D and E (with glucose and starch, respectively, as a carbohydrate source) . Some properties of such cultures are reported in Table 2. The growth of isolate 10B proceeds slower than that of a number of other isolates. In 10B, after three days of incubation, still clearing zones around the colonies are present. In other isolates, clearing was obvious after 1 to 2 days of incubation and after - 32 - subsequent growth, as already discussed, in a number of cases additional turbidity around the colonies was shown. The pigmentation on the glucose medium was more pronounced than on the other media. The isolate 10B exhibited slime formation on the glucose medium, which was not the case after growth on the starch medium E. Isolates as AVB-Sol 1- 6 were further isolated in a corresponding manner, as previously described in Chapter 2 (Isolation) of Material and Methods . Identification of micro-organisms The pure cultures present, 2B1, 6B and 13A, display a microscopic image of irregularly shaped, Gram-positive rods. Sometimes, Gram-positive granular structures are present in Gram-negative cells. Also, rapid decolorization to Gram-negative can take place, so that in the same culture both Gram-a substrate, acid formation occurs neither under fermentative conditions nor under oxidative conditions. Isolate 2B1 forms a yellow pigment and is motile. The other two isolates are non-motile and form cream-colored colonies. Spore formation and anaerobic growth have not been demonstrated in any of the cases examined. For cultures 13A and 6B, a clear coc-rod cycle was observed. The peptidoglycan of culture 6B has been characterized as A3α. An A3α-type means that the third (di) amino acid is L-lysine [Schleifer et al ; Bact . Rev. 36:407-477, 1972]. For strain 6B, the tripeptide L-alanine- L-threonine-L-alanine is present as interpeptide bridge between L-lysine and D-alanine. The combination of rod- coccus cycle, lysine as (third) (di) amino acid and other characteristics lead to the conclusion that culture 6B falls under the irregular, non-sporulating Gram-positive rods, within the genus Arthrobacter {Bergey's Manual of systematic Bacteriology 2 (Ed: J.G. Holt, Williams and Wilkins, Baltimore, USA, 1986). The peptidoglycan described - 33 - has been demonstrated in A . histidinilovorans , A . aurescens and A . ureafaciens . From the sequence determination of the most variable regions of the 16S rDNA, it has been derived that 99.8% percent homology is present between Arthrobacter 6B and A . histidinolovorans/A . nicotinovorans . Further discrimination between these two very closely related species is virtually impossible. The result of the identification of Arthrobacter 6B is thus Arthrobacter histidinolovorans /nicotinovorans . The peptidoglycan of strain 13A has been characterized as A4α . The combination of peptidoglycan composition (type A, L-lysine as di-amino acid) , the observed rod-coccus cycle and other characteristics again leads to the genus Arthrobacter [Bergey's, ibid] . The highest degree of 16S rDNA-homology with different Arthrobacter species was found with Arthrobacter nicotianae and is 96.9%. However, this is not sufficient to characterize the organism at species level. The identification result can be described for Arthrobacter 13A as a new Arthrobacter species within the Arthrobacter nicotianae group. Due to the morphological characteristics described, it is plausible that strain 2B1 also belongs to the irregular, Gram-positive non-sporulating rods. The peptidoglycan of this culture has been identified as B2β having as interpeptide bridge the dipeptide glycine-D- ornithine. B-type peptidoglycan is found in the coryneform bacteria, especially in the genera Agromyces, Aureobacteriu , Curtobacterium, Microbacterium and Corynebacterium . The presence of D-ornithine in combination with the B2β-type peptidoglycan leads to the genera Curtobacterium and Aureobacterium . In Curtobacterium, however, only one amino acid (D-ornithine) is present as bond, while the dipeptide found is characteristic of Aureobacterium. On the basis of 16S rDNA-homology, 97.5% agreement with Aureobacterium luteolum and A . liquefaciens - 34 - was found. This value, however, is too low (<98%) for an identification at species level. The organism has thus been identified as a new Aureobacterium species. The microorganisms isolated from slurry have been identified by DSM as: AVB-sol 1, 2 and 5: Paenibacillus pabuli ; AVB-sol 3: Enterococcus flavescens; AVB-sol 6: Rhodococcus erythropol i s . 4 SGA-degradation in shaking cultures Shaking cultures were inoculated with colonies from glucose agar plates. SGA degradation was monitored in a mineral medium with SGA as carbon and energy source. In all cases, with the cultures, a an extensive degradation of both solanine and chaconine was established after 24 to 48 hours of incubation. In Fig. 1, by way of example, the rapid degradation with culture 13A is represented, as well as the somewhat slower degradation with culture 10B. With 13A, after 24 hours of incubation, both solanine and chaconine have largely disappeared (according to HPLC analysis) . This is confirmed by the strong decrease of the carbohydrate content. Also the TOC-content decreases to a virtually constant level, with about 60% of the initially present carbon remaining present . This value shows proper agreement with the amount of carbon that is present as aglycon in SGA. With culture 10B, the SGA degradation proceeds somewhat slower. After 48 hours, here too a major degradation of solanine and chaconine has occurred. The carbohydrate content and the TOC-content have decreased less than may be expected on the ground of the solanine and chaconine removal . The previously mentioned slime formation (polysaccharides) may have been the cause of this. Also, intermediately, the presence of β- and γ-glycoalkaloids may have been involved. The peaks in Fig. 2 corresponding with retention times of 11.23, 11.27, 14.42 and 14.43 minutes might derive from these components. For that matter, after - 35 - 168 hours of incubation, these components had disappeared. It is also clear that a microbial effect is involved. As Fig. 2 shows, in the absence of bacteria, even after 168 hours of incubation, no conversion of SGA has occurred. From microscopic and macroscopic observations, it can be derived that during the cultivation of, for example, 13A, initially (after six hours of incubation) , conglomerates of SGA are involved, while free microorganisms are hardly in evidence. In culture 13A, subsequently, after 24 hours, free micro-organisms (short, non-motile rods) were visible. Also, the size of the conglomerates had decreased strongly. The turbidity of the culture had increased strongly. In culture 10B, after 48 hours of cultivation, micro-organisms were visible (very short rods) , while the turbidity of the medium had likewise increased strongly. It is plausible to suppose that the turbidity increase is a consequence of the SGA- conglomerates falling apart after degradation of the carbohydrate and the formation of the less soluble aglycon, as well as the liberation and growth of free bactereria in the cultures. Modification of the aglycon Using gas chromatography, it was determined to what extent modification of the aglycon had arisen in the shaking cultures. In each case, the concentration of total aglycon and the concentration of free aglycon were determined. In the determination of total aglycon, cleavage of carbohydrate, if any is still present, takes place through hydrolysis in 1 N HCI at 75°C. The solanidine formed is thereby in part converted further. It is plausible that after this conversion the more volatile solanthrene is present. In a standard mixture in which, in addition to solanidine .HCI , solanthrene is present, a peak is found for solanidine at a retention time of 17.7 min. and for solanthrene t r is 14.6 minutes (Fig. 3) . In the - 36 - investigated cultures 2B-1, 6B, 12B, 13A and 16A, however, after virtually complete conversion of SGA, neither solanidine nor solanthrene is demonstrated (Fig. 3) . In all cases, a component with a retention time of 20.1 min. (T20.1) is involved, both for the free aglycon and for the total aglycon. This means that in these cultures, conversion of the aglycon has taken place to a component which, in view of the chromatographic properties, is more apolar. When a partial degradation of SGA is involved as determined with the HPLC method, then, with gas chromatography, in the determination of the total aglycon, in addition to T20.1, also solanthrene and, in a minor amount, solanidine are found. In determining the free aglycon, again virtually exclusively T20.1 is involved. The proportion of free aglycon and the proportion of T20.1 within the total aglycon correlates very well with the degredation of SGA, as determined by HPLC (Fig. 4; slope and intercept do not significantly deviate from 1 and 0 according to regression analysis (P=0,05) ; r is 0.99 for % free aglycon and 1.00 for (T20.1). This means that the above-mentioned modification of the aglycon to T20.1 apparently follows instantaneously after the removal of the sugars of solanine and chaconine. A different picture is observed after growth of the culture 10B (Fig. 5) , where after 168 hours of incubation, the free aglycon is mainly present as solanidine and to a slight extent as T20.1. In determining total aglycon, after hydrolysis, solanthrene in excess and to a slight extent T20.1 are detected. Also, despite the complete degradation of solanine and chaconine (according to HPLC analysis, see Figs. 1 and 2), the amount of total aglycon is greater than the amount of free aglycon. In relation to this, it has been established before that after complete degradation of solanine and chaconine, more carbohydrate and TOC are present than may be expected from the degradation of solanine and chaconine. The presence of bound aglycon and carbohydrate in the absence of solanine and chaconine may signify that the - 37 - micro-organism 10B, in addition to forming solanidine from SGA, is capable of forming other products from solanine and/or chaconine. A possibility is (in relation to the observed slime formation) the formation of a polysaccharide based on SGA. Identifica tion of the modified aglycon For the identification of the modified aglycon as demonstrated with the aid of gas chromatography, use was made of 1 H-NMR and 13 C-NMR. Striking in the proton spectrum (not shown) , regarding solanidine, is the CH-OH-group at (δ=3.5 ppm) and the CH (δ=5.34 ppm) of the unsaturated bond. In the modified aglycon, the CH-OH-resonance is no longer present and the CH has shifted from the unsaturated bond to lower field (δ=5.70 ppm) . In the C-spectrum (Fig. 17) of solanidine, resonances are present, amongst others, at 60 ppm, 68.9 ppm, 71, 6 ppm, 74.5 ppm, 121.5 ppm and 140.6 ppm. These can be attributed to, respectively, N-CH 2 , N-CH, CHOH, N-CH, the tertiary C-atom of the double bond between C-5 and C-6 and the quaternary C-atom of this bond. After modification, the resonance at 71.6 ppm (CHOH) has disappeared and a resonance is present at 199.5 ppm, which is characteristic of a ketone group on C-3. The peak of the quaternary C-atom of the double bond (C-5) has shifted to 171.4 ppm and that of the tertiary C-atom to 123.9 ppm. This indicates that the double bond will now be present between C-4 and C-5. The resonances at 60 ppm, 68.9 ppm and 74.5 ppm are also present in the modified aglycon, so that, apparently, no modification with regard to bonds to the N-atom has arisen. It is concluded that solanidine has been converted to 4-solanidine-3-one . - 38 - 5 Conversion of SGA in protein and protamylasse A well known problem in microbiology is the phenomenon that (cultures of) micro-organisms which during their growth in a particular (poor) medium exhibit a particular enzymatic activity and/or affinity for a particular substrate, do not (always) exhibit this activity or affinity, or actually lose it, when apart from this specific substrate, other substrates are also available in the medium, which are preferentially converted by the organism. However, the micro-organisms of this invention have been found to maintain their specific activity and/or affinity and functional properties regarding SGA and SGA derivatives in rich media as well. Tested as rich media were potato protein-containing suspensions and protamylasse, both also containing SGA. In a first experiment, the conversion of SGA by Arthrobacter culture 6B in a suspension of about 20% potato protein was determined. After 24 hours of incubation of an overnight culture in TSB, diluted twice in the protein suspension, no SGA was detectable anymore. In the control, where sterile TSB had been added to the non-sterilized protein, bacterial growth took place, but after 24 and 48 hours of incubation, the SGA content was equal to that at the start of the incubation (675 mg/l) . In a next series of experiments 10% protein suspensions were applied, while the overnight cultures were first resuspended in physiological salt solution before being added (diluted twice) to the protein suspensions. As a control, sterile physiological salt solution was added. In Tables 3, 4 and 5, the conversions are represented for the cultures 6B and 13A, as well as for the control . Both with Arthrobacter 6B and 13A, within 8 hours, complete deglycosylation of SGA in a suspension of previously dried protein can take place. This is accompanied by the formation of free aglycon, which is chiefly present as 4-solanidine-3 -one . Over the first 8 - 3 9 - hours of incubation, no decrease, or only a slight decrease, of the dry matter contentis observed. In all cases, high amounts of bacteria were involved (3*10 9 /ml) . In addition, some protease activity is present (at least after 24 hours) , which, in the presence of strain 13A, is somewhat higher than after addition of Arthrobacter 6B . As a follow-up experiment, the conversions within a period of 8 hours were further analyzed. The results are represented in Figs. 6 and 7. With Arthrobacter 6B, the decrease of SGA, both solanine and chaconine, already starts after 2 hours of incubation. After 6 hours of incubation, SGA is substantially converted. The aglycon is already present for a large part in a free form as 4-solanidine-3 -one . After 8 hours of incubation, virtually all aglycon is present as 4-solanidine-3-one . With culture 13A, the conversion starts somewhat later, but also in this case, after 8 hours, virtually complete conversion of SGA to 4-solanidine-3-one is established. In the absence of (inoculated) Arthrobacter, over the investigated incubation time, no conversion of SGA occurs. In addition to the conversion of SGA, it is of interest to determine whether the microbial activity leads to a decrease of the dry matter content. Theresults, as well as the pH change upon incubations with Arthrobacter, is represented in Fig. 7. The control incubation involves some fluctuation in the dry matter content (partly as a result of sample-taking) . This varies from 11.8% at the beginning to 8.8% after 4 hours of incubation and eventually 9.6% after 8 hours of incubation. The pH changes hardly, from 5.7 at the beginning to 5.6 after 4 to 8 hours of incubation. Also after addition of culture 6B, the dry matter content fluctuates. At the beginning, 11.2% dry matter was present. A maximum decrease over the first 8 hours of incubation is found after 5 hours of incubation, when 10.4% dry matter was still present. The decrease of the dry matter content will therefore be maximally in the order of 0.5-0.8% and is equal to the - 40 - fluctuations already found in the control . Over the first 8 hours, the pH rises slowly from 5.7 to 6.3. For culture 13A, the pH increases slightly more than after incubation with Arthrobacter 6B . After 8 hours of incubation, it has risen from 5.7 at the start to 6.7. The dry matter content again exhibits variation from minimally 9.1% after 7 hours of incubation to a peak of 13.6% after 5 hours of incubation. Calculated with a dry matter content of 10.0% at the start, the maximum decrease is 0.9% after 7 hours of incubation. It is important to establish here that despite the excess of protein over SGA, the bacteria do not initially consume a large proportion of the protein before proceeding to SGA-conversion. Accordingly, the loss of dry matter is limited to 0.5 to 1.0% in the incubation of Arthrobacter bacteria with protein. The conversion of SGA in protein was also investigated with AVB-sol 1, 3 and 6 (Tables 8, 9 and 10) . In AVB-sol 1, after 24 hours, the solanine is converted to free solanidine. Hardly any 4-solanidine-3-one has been formed and there is virtually no chaconine conversion. In AVB-sol 3: Enterococcus flavescens, after 7 hours, solanine is converted to free solanidine; hardly any 4 -solanidine-3 -one has been formed and there is no chaconine degradation. In AVB-sol 6: R . erythropolis, after 7 hours, the chaconine has largely disappeared, 24 ppm free aglycon is found with principally 4-solanidine-3-one and some free solanidine. In addition, intermediately, a component (X) to be further identified is present in a low concentration. After 16 hours, solanine has likewise disappeared and hardly any aglycon is measured anymore, 4-solanidine-3-one is further converted, and so is the known component (X) . Further, there were not found any other aglycons, such as demissidine, solanthrene, solanidine or comparable components. The further conversion of 4-solanidine-3-one was investigated by means of cultivation of R . erythropoli s in a mineral medium with 620 mg/l 4-solanidine-3-one and 800 mg/l yeast extract as - 41 - organic components. By way of control, incubation also occurred in a mineral medium supplemented with yeast extract. From the decrease of the TOC-content (total organic carbon) in the 4-solanidine-3-one culture, corrected for the culture in a mineral medium with yeast extract alone, it can be concluded that in the cell -free culture liquid the organic carbon of the originally present 4-solanidine-3-one has disappeared for 74%. To a considerable extent, therefore, mineralization to C0 2 (and formation of cell material) occurs. Using direct MS- analysis of these cultures, this picture was confirmed. No detectable concentrations of conversion products specific as to molecular weight are found. Growth and conversion of SGA in protamylasse The growth and conversion of SGA in protamylasse was determined inter alia with Arthrobacter culture 6B. Protamylasse is a suitable and inexpensive substrate for culturing (for example, Arthrobacter) cell material for the desired SGA conversions on a practical scale. The influence of the protamylasse concentration on the growth, as well as the presence of extra added glucose, was determined (Fig. 8) . Also, in the presence and absence of glucose, it was determined in how far conversion of the SGA likewise present in protamylasse can take place (Fig. 9) . At concentrations of 0.5-2.0% protamylasse (Fig. 8A) , the highest growth rate is found at 0.5-1% protamylasse; the cell yield after 28 hours of growth is in the order of 5-7*10 9 /ml. In the presence of glucose with 0.5% protamylasse (Fig. 8B) , comparable growth rates and yields are found with regard to incubations without glucose. At a higher protamylasse concentration (Fig. 8C) , a negative influence of glucose is present. The minimum doubling time increases from 1.2 hours (without glucose) to 1.9-2.6 hours - 42 - at the different glucose concentrations, and in all cases the eventual cell yield is about 4*10 9 /ml. For the growth of Arthrobacter 6B, a medium with 0.5-1% protamylasse without additional glucose is suitable. As represented in Fig. 9, in 2% protamylasse about 90 mg/l SGA is present. Both in the presence and absence of 5 g/1 glucose, a decrease of SGA occurs. At the start, 60% of the SGA consisted of solanine. The decrease of solanine and chaconine progressed at a comparable rate, 4-solanidine-3- one (T20.1) being the most important product, as in the protein incubations. During the different incubations of protamylasse and glucose, the pH was 6.9-7.3 at the start. After 8 hours of incubation, it had risen to 7.1-7,6 and at the end of growth, after 28 hours, the pH varied from 8.3 to 9.0. SGA removal from protamylasse is accompanied by growth of the micro-organism to a level of about 5*10 9 per ml. This means that protamylasse can be utilized as a (pre) culture medium for the micro-organisms. In an experiment with Arthrobacter 6B, cultivation was performed in 0.5% (dry matter) protamylasse for 10 hours at 30 °C, followed by the addition of dried potato protein or potato protein filtercake to a final level of 10% dry matter, corresponding to a concentration of about 300 mg/l SGA in the medium. After a period of 2-4 hours of further incubation, complete removal of SGA was established, concomitant with the formation of 4-solanidine-3 -one . It is concluded that with both protein substrates, dried potato protein as well as potato protein filtercake (about 40% dry matter) , a rapid removal of SGA is possible in the described 1 -unit -operation process, combining growth in protamylasse and SGA-removal from protein in one fermentation unit. - 43 - 7 Toxicity of SGA and SGA metabolites and derivatives The EC 50 -values for bacteria, yeast and fungi, as well as the MIC-values of the Toxi-Chromotest and the LC 50 -values for a rotifer (Brachionus calyciflorus) and a crustacean ( Thamnocephalus platyurus) are summarized in Table 6. The toxicity of the glycoalkaloids is greater for fungi than for bacteria or yeast. An explanation might be the disruption of sterols present in the cell membrane of fungi. The toxicity for the fungi examined, for chaconine, shows a fair agreement with that for Al ternaria brassicicola [Fewell et al ; Phytochem. 33;323-328, 1993]. For solanine, a lower LC S0 -value is found than described previously for Trichoderma viride [Patil et al ; Food Cosmet . Toxicol . 10: 395-398, 1972]. For fungi, the toxicity of chaconine is slightly greater than that of solanine. Also with bacterial cells, in view of the results of the Toxi-Chromotest, a toxic effect of glycoalkaloids can be demonstrated. The sensitivity of this bacterial system, is considerably greater than the growth inhibition found with three different bacteria. The highest sensitivity to glycoalkaloids is found in a rotifer and a crustacean and is in the order of a few μM. Thus, the microbiotests are more sensitive than most membrane- disturbance tests, the hemolysis of erythrocytes, the LDH- release of intestinal epithelium cells and the inhibition of acetylcholinesterase where the EC 50 -values are typically about 10 to tens of μM. In a number of cases, the glycoalkaloids are more toxic than the aglycon solanidine, which is in line with the literature, but in yeast and in two cases with fungi, the EC 50 -value of solanidine is lower than that of the glycoalkaloids. Deglycosylation of SGA to the aglycon solanidine is therefore not generally adequate for detoxification. In all cases examined, the detoxification manifests itself much more strongly after further conversion of the aglycon. For yeast, 4 -solanidine- - 44 - 3 -one is 20 times less toxic than solanidine; for fungi, the detoxification varies by a factor of 3 to 45. With the Toxi-Chromotest, the MIC-value has decreased by a factor of 1.3 to 5.3 compared with solanidine, although it should be noted here that in this test solanidine is already considerably less toxic than the glycoalkaloids. In the microbiotests, the toxicity of 4-solanidine-3 -one proves to be reduced by a factor of 100-133 with Brachionus calycifloris and by a factor of 6-15 with Thamnocephalus platyurus . For the investigated concentrations of the glycoalkaloids and aglycons, no direct genotoxicity has been demonstrated with the SOS-Chromotest; the investigated maximum concentration varied for the different components from 580 to 3788 μM. Absence of genotoxicity as for chaconine, solanine and solanidine corresponds with observations described in the literature [Friedman et al ,- Food Chem. Toxicol . 30:689-694, 1992], where these components were investigated with the aid of the Ames- Salmonella test and the foetal erythrocyte micronucleus assay. The modified aglycon (4-solanidine-3 - one) was not involved in the Friedman study, but from the investigations performed here with the SOS-Chromotest it may be concluded that at the investigated maximum concentration of 3788 μM no direct genotoxicity is present. From the experiments described here, it can be concluded that deglycosylation of glycoalkaloids to form solanidine will generally lead to (minor) detoxification. Really favorable effects are envisaged with regard to detoxification when further conversion of solanidine to 4 -solanidine- 3 -one and other derivatives can take place. 8 Screening enzyme preparations As described hereinabove, micro-organisms and enzymes can be isolated that can be used in a method to convert in materials SGA such as solanine and chaconine into - 45 - considerably less toxic aglycons. The use of commercially available enzyme preparations would be another possibility, if applicable preparations could be found. .About 190 commercially available enzyme preparations were examined. Enzyme preparations are already used on a large scale for improving the properties of foods. As described, for example, in Industrial Enzymology, second edition (ISBN 0- 333-594649; ed. T. Godfrey & S. West, Macmillan Press Ltd, London) (1996) pp. 255-260 and 373-382, in the food industry, enzymes, in particular also glycosidases, are used for improving the aroma (wine industry) , reducing the bitter taste in citrus juice (fruit juice industry) and for cell wall treatment. To investigate whether enzyme preparations can be used for conversion of steroid glycoalkaloids (SGA) , a large number of preparations from different producers were screened for SGA-deglycosylating activity, using the TLC method described. As described in PCT/EP96/03232 , in certain enzyme preparations produced by Aspergillus niger, an activity can be found which cleaves the chacotriose of α-chaconine. Found to be highly active in the screening carried out by applicant were the Aspergillus niger- produced preparations Rapidase C 80, Arabinase strain Hemi.23, Hemicellulases Reg 2 and pectinase preparation P 9179. Formation of partially deglycosylated intermediates was not established in these preparations, nor after incubation periods in which the substrate had not been completely converted. Preparations acting on α-chaconine were found to be capable not only of deglycosylating α-chaconine but additionally also of deglycosylating the disaccharide-containing β 2 -chaconine to the aglycon solanidine . From the investigation presented by applicant in this application, it appears that chaconine-deglycosylating activity is not limited to Aspergillus niger preparations. Also preparations of Aspergillus japonicus (Pectolyase, - 46 - ICN) , Penicillium decumbens (Naringinase, Sigma) , potato, oats, Helix aspersa and mollusc are found to have utility for deglycosylating chaconine. Instead of a method which involves cleavage of the chacotriose as described in PCT/EP96/03232 , deglycosylation could also be realized by selective cleavage of the different monosaccharides . From the investigation presented by applicant in this application, it appears that such a method in which enzyme preparations of different glycosidase activities are utilized, is possible: partial deglycosylation of α-chaconine to - - and γ-chaconine, in addition to aglycon formation, was established in incubations with a pectinase preparation from A . niger from Merck. In incubations with the preparations LPL-L, LPL-NL (A . niger) and a pectinase preparation of ICN, mainly β χ -chaconine, in addition to solanidine, was formed. Further, some preparations formed from α-chaconine one partially deglycosylated product. Thus, β x -chaconine formation was established with Rohament P, the β-glucuronidase L-2 from Patella vulgata and the β-glucuronidase from Abalone entrails . The preparation LPL-G from A . niger formed exclusively β 2 -chaconine and saccharase from Merck γ-chaconine. A number of preparations exhibited activity on β 2 -chaconine . This was converted into solanidine. This concerned the α-amylase preparations X-A and 4a and Fungamyl 800L from A . oryzae, amyloglucosidase (Merck) and Pectinet Ultra SP from A . niger. The above- described preparations, as well as those described in PCT/EP96/03232 , can be used to deglycosylate chaconine but do not possess, in contrast to the micro-organisms and associated enzymes described in the present application, the ability to deglycosylate (the different forms of) solanine. In view of the structure of the solatriose, it was investigated if α-solanine degradation was possible with endo- and exo-β-galactosidases with different - 47 - specificity, alone or in combination with β-glucosidase . For this purpose, the commercially available endo- galactosidase from Bacteroi ' des fragilis and exo- galactosidases from bovine testes, Diplococcus pneumoniae, E. coli and A . oryzae were used. Not in any of the enzymes or combinations thereof was any degradation of solanine established. Nor was any effect caused by combination with a chaconine-deglycosylating A . niger preparation or with a pectinase preparation from Merck which, as described above, forms β x - and γ-chaconine and solanidine from α-chaconine. Nor did available multi -enzyme preparations from Trichoderma harzianum and from Basidiomycetes exhibit any activity on the SGA preparation. A considerable activity on both solanine and chaconine was found to be present in preparations from Helix pomatia . Such preparations can be used in a method for conversion of chaconine- and solanine-containing materials. The above-mentioned enzyme preparations also exhibited SGA conversion in potato liquor and potato protein. The demonstrated presence of said activities in preparations of different origin can be used by one skilled in the art for him, after purification of the responsible proteins using the amino acid sequence, to isolate the DNA in question and, optionally after genetic modification, to use it for the development of enzyme preparations with an adapted activity and/or action. Removal of SGA by fractionation During the degradation of SGA in buffer solution as well as in potatojuice, it was found that generally a turbidity arose; invariably, solanidine formation was involved. The possibility was examined whether by partial fractionation of enzymatically treated potato juice a part of the SGA could be removed. After the enzymatic treatment, it appeared that chaconine had been completely converted into solanidine. This was first investigated with SGA in a - 48 - buffer. After deglycosylation of chaconine, it appears that through centrifugation a considerable amount of the alkaloid can be removed from the solution. From Fig. 13 it appears that at the investigated SGA concentration at pH .8 , approximately half of the solanidine is removed from the solution. At a slightly higher pH, this effect is found to be enhanced and also a considerable part of the solanine disappears. Removal of alkaloid from the liquid phase resulted in a rise of the mass of the pellet obtained after centrifugation. Next, it was attempted to remove alkaloid from liquor through centrifugation or filtration. Here, the influence of pH and temperature was investigated. Results are represented in Fig. 14. As in buffer, through centrifugation (Fig. 14A) alkaloid can be removed from potato liquor (AVW) . In contrast to the buffer system, the amount of material in the pellet obtained after centrifugation decreases upon increase of the pH . This can be explained by an increased solubility of the protein present. In Fig. 15A the effect is represented of the pH on the protein concentration in the supernatant/filtrate obtained upon the different treatments; at higher pH more protein is found in supernatant/filtrate. Pretreatment of the AVW by incubating it at 48°C for 30 minutes seems to have an additional positive effect on the amount of alkaloid that can be removed. In particular at pH 7.5, clearly less alkaloid is present in the supernatant (Fig. 14A and B) . Also, more pellet was obtained, but this was also established in a control AVW. Filtration using a 0.2 μm filter is also possible. Treatment of AVW in which the chaconine present is deglycosylated can lead to a considerable improvement in the ratio of alkaloid/recoverable protein. From Fig. 15B it appears that through centrifugation and filtration, in the investigated SGA-enriched juice, the SGA/protein ratio can be improved by a factor of three compared with the initial situation. From the measured values it can be calculated - 49 - that an SGA-enriched protein product is separated. In particular at pH 7.5, a product is obtained having a 10 times higher SGA/protein ratio. Solanidine modifica tion Upon incubation of Hemicellulase F35000 from Aspergillus niger, Viscozym L (Novo) from an Aspergillus species and Pectolyase (ICN and Sigma) from Aspergillus japonicus, two stains were established with R F -values (R F 0.94 and 0.95) corresponding with two products present in the solanidine standard (Rohm) . Analyses with GC showed that in addition to solanidine, even several forms of the aglycon occurred. From TLC analyses of products obtained with these aglycon-modifying enzymes, it appeared that Hemicellulase F35000, Pectolyase (ICN), Pectinase (Sigma) and the moderately active preparations Viscozym L Pectinet Ultra SP (both Novo) and Pectolyase (Sigma) were capable of converting α-chaconine to solanidine and to modified aglycon. From GC-analysis, it appeared that in the incubation of Hemicellulase F35000 on SGA, in addition to the solanidine, three other forms of the aglycon had been formed, of which two occurred to a considerable extent. By standard addition, it could be established that one aglycon form is identical to the modified 4-solanidine-3 -one as was obtained after an incubation of Arthrobacter species on SGA. Another aglycon form (AG-X1) , as demonstrated with the aid of gas chromatography, was characterized with GC/MS, 'H-NMR and 13 C-NMR. From the X H-NMR spectrum of this modified aglycon (not shown) , it appears that in comparison with the spectrum of 5 -solanidine, the CHOH resonance at δ 3.50 ppm has disappeared and the CH hydrogen atom has shifted from the unsaturated bond to lower field (δ 6.18 ppm) . Further striking is the shift of one of the methyl resonances to lower field (δ 1.18 ppm) . In the 13 C-NMR spectrum of the modified aglycon, 4 methyl, 9 methylene, 8 - 50 - methyne, 1 monosubstituted alkene (C=C(R)H, δ 125.38 ppm)) and 5 quaternary carbon atoms (2 quaternary carbon atoms, 1 disubstituted alkene (C=C (R. ) R 2 ) (δ 160.82), and 2 ketones (C=0) (δ 199.34 and δ 202.05 ppm) are observable. In comparison with the starting material 5-solanidine, the unsaturated bond has shifted and the hydroxyl group has been oxidized to a ketone (see structure clarification 4 -solanidine-3 -one) . Further, one of the methylene groups is oxidized to a ketone. The .NMR data correspond with an m/z peak of 409. With GC/MS also for AG-Xl a m/z peak was determined of 409, 12 higher than that for solanidine (m/z 397) . From the structure clarification using X H- and 13 C-NMR spectroscopy and GC/MS, it can be concluded that an oxidized 4-solanidine-3-one (keto-4-solanidine-3-one) has been formed. The ^-NMR data of the A- and B-ring of keto- 4 -solanidine- 3 -one correspond with the values found for cholest-4-ene-3 , 6-dione by Teng and Smith (Steroids 61, 627-633, 1996): 19-CH 3 , δ 1.18 ppm and 4-H, δ 6.18 ppm (cholest-4-ene-3, 6-dione: 19-CH 3 , δ 1.17 ppm and 4-H, δ 6.18 ppm) . Further in line with the reactions described in the above article is the finding that keto-4-solanidine-3- one was not formed upon incubation with 4-solanidine-3-one as substrate . From these data it can be concluded that AG-Xl is 4-solanidine-3 , 6-dione . Aglycone unknown 1 was analysed with the aid of direct MS performed with a Finnigan Navigator massaspectrometer with Apcl in "positive mode" in the range of 100-1000 Da. Under these conditions the molecular mass of solanthrene was determined to be 380, of 4-solanidine-3-one 396, of solanidine 398, of AG-Xl 410 and of aglycone unknown 1 394 Da. Based on these data aglycone unknown 1 is a solanidiene-3 -one . Aglycon modification Upon bacterial conversion of SGA by Arthrobacter 6B, the solanidine formed is rapidly converted to an oxidized - 51 - form, 4-solanidine-3-one . Such a conversion of the 3β-hydroxyl group of a steroid is catalyzed by oxidases such as cholesterol oxidase. From the literature, it is known that these enzymes display a fairly broad substrate specificity [A.G. Smith, C.J.W. Brooks: J. Steroid Biochemistry 7 (1976) 705-713; B. Wortberg: Z. Lebensm. Unters . -Forsch. 157 (1975) 333-338; R. Saunders, P.S. Cheetham, R. Hardman: Enzyme Microb. Technol. 8 (1986) 549] . Thus, the enzyme from Rhodococcus (Nocardia erythropolis can oxidize, in addition to cholesterol, a large number of other steroids, including an alkaloid such as diosgenin. This enzyme, however, hardly exhibits activity on solanidine. Now, from the investigation presented by applicant in this application, it appears that enzyme preparations produced by industrially used Aspergillus strains are capable of modifying the aglycon solanidine. The activities found, however, are very low. The aglycon is converted into several products. Upon continued incubation, in the GC-analyses performed, the recovery of total determined alkaloid decreased. Possibly, there occurs a further enzymatic conversion into forms which, under the GC-conditions used, are not detected anymore and/or are not extracted anymore in the work-up procedure. In NMR-analysis of purified AG-XI, the strength of the signal was found to decrease over time. This means that 'spontaneous' chemical conversion should also be taken into account. One main product, formed in the incubations with preparations such as Hemicellulase F35000, could be identified as 4-solanidine-3 -one . The influence of oxygen and sulfite on the conversion then points to conversion of the aglycon catalyzed by oxidase. This points to the presence of a 3β-hydroxysteroid oxidase (solanidine oxidase) . In addition to 4-solanidine-3 -one, however, preparations such as Hemicellulase F35000 form yet another - 52 - product, AG-XI. After purification, this product could be identified as solanidine-4-ene-3 , 6-dione . A similar component, cholest-4-ene-3 , 6-dione, is described in an article by J.I. Teng and L.L. Smith, [Steroids 61 (1996) 627-633] as a product formed upon the oxidation of cholesterol by Pseudomonas fluorescens cholesterol oxidase. In the route for cholesterol oxidases heretofore postulated, as intermediate in the oxidation of a steroid such as cholesterol (represented for solanidine in Fig. 14) a 5-ene-3-one is formed which via a 3,5-diene- 3-ol(2) with the aid of isomerase activity present in the enzyme is converted to a 4-ene-3-one (4) . By Teng and Smith it is rendered plausible that the cholesterol oxidase from P. fluorescens also works as a dioxigenase. According to the proposed reaction mechanism, cholesterol is also converted by this enzyme into the intermediary product cholesta-3 , 5-diene-3-ol (2) , whereby the cofactor FAD is reduced. The 3 , 5-diene-3-ol (2) formed is thereupon peroxidized by the resulting flavin peroxide (FAD.H 2 .0 2 ) to a 6β-hydroperoxide (3) . Reoxidation of FAD.H 2 thereby formed can lead to a repetition of this last reaction or to the release of H 2 0 2 . From the unstable 6β-hydroperoxide (3) , within a few hours, the 3,6-dione(5) is formed as well as the 3-one, 6-ol (6) . The formation of solanidine-4-ene-3 , 6- dione (keto-4-solanidine-3 -one) , 4-solanidine-3-one as well as the fact that solanidine-4-ene-3 , 6 -dione is not formed upon incubations with 4-solanidine-3-one as substrate can then be explained by the presence of a similar 3β-hydroxysteroid oxidase in the Aspergillus preparations examined. To applicant's knowledge, the presence of such an activity has not been described in the literature. That, in deviation of what Teng and Smith find for the oxidation of cholesterol by the Pseudomonas cholesterol oxidase, in the conversions with Aspergillus preparations investigated here also 4-solanidine-3 -one is found, can be - 53 - explained by very low enzyme activities which have been employed. In view of the instability of hydroperoxide 3, this will not have been present anymore in the reaction mixtures analyzed. In these reaction mixtures, as is clear with GC, other products are present; by analogy with reaction mechanisms found by Teng and Smith, this also concerns 6β-hydroxysolanide-4-ene-3 -one (6) . From the investigation presented in this application, it is clear that the toxicity of the modified aglycon 4-solanidine-3 -one is considerably lower than that of solanidine, chaconine and solanine. 4-solanidine-3 , 6-dione is a further modified product whose toxicity is further lowered. For an incubation of Hemicellulase F35000 on SGA, specific activities were calculated on the basis of the determined concentrates of the products formed: solanidine, 4-solanidine-3-one and the AG-Xl (Table 7). The invention further provides a test method, in which the content of SGA and/or SGA derivatives in a sample is determined by means of an enzymatic reaction in which at least one enzyme according to the invention is used. An example of such a test method provided by the invention is a test method in which the content of solanidine in a sample, such as, for example, taken from a potato, potato protein, and the like, is determined on the basis of the enzymatic conversion to 4-solanidine-3-one, such as, for example, catalyzed by solanidine oxidase or solanidine dehydrogenase provided by the invention. In another example of a test method provided by the invention, the content of solanine is determined by selective conversion to solanidine, catalyzed by an enzyme provided by the invention, whereafter the content of solanidine is determined as described. In one embodiment of such a test method, the hydrogen peroxide released in the conversion can be measured and serve as a measure for the converted solanidine. The hydrogen peroxide content can be - 54 - determined, for example, through the use of 4-aminoantipyrine and p-hydroxybenzene sulfonate, which, together with hydrogen peroxide and under the influence of a peroxidase, is converted to a quinone-imine color which can be measured. However, many other possible methods for measuring hydrogen peroxide are known. The invention also provides a test kit comprising requisite means, for example an enzyme and (control) substrates, such as 4-aminoantipyrine and p-hydroxybenzene sulfonate, for carrying out a test method. Use can also be made of the above-mentioned deglycosylating preparations such as those of Helix pomatia . Such a test method can also be applied to samples which have already been extracted. An important aspect in the SGA conversion by microorganisms, enzyme systems and/or enzymes represented by the micro-organisms with functional properties related to the SGA and/or SGA derivative-converting properties of, for example, Aureobacterium species such as 2B1, Arthrobacter species such as 6B and 13A, Paenibacillus species such as AVB-sol 1, 2, 5, Enterococcus species such as AVB-sol 3 and Rhodococcus species such as R . erythropolis (AVB-sol 6) , is the formation of solanidine or of a modified and further detoxified aglycon or the complete mineralization to C0 2 . Another important aspect is the fact that such microorganisms (preferentially) exhibit this conversion also in "rich" media, which, in addition to SGA, also contain other carbohydrate sources and/or protein sources. A first modified aglycon has been identified as 4-solanidine-3-one, whose toxicity was found to be reduced with respect to solanidine. For a rotifer, a ca . 100 times lesser toxicity was found and for a crustacean a reduction by a factor of 10. The reduced toxicity of 4-solanidine-3 -one with respect to solanidine has been confirmed with lower organisms such as yeast, fungi and bacteria. In view of the type of conversion, an activity comparable to cholesterol oxidase - 55 - must be involved. In comparison with the described cholesterol oxidase from Nocardia erythropolis [Smith et al; J. Ster. Biochem. 7:705-713, 1976], which effects oxidation of solanidine to a very low extent, the added value of conversion of solanidine with Arthrobacter , Aureobacterium or Rhodococcus (or solanidine oxidase therefrom) is based on the rate and completeness of the conversion, in other words, on the substrate specificity or preference for solanidine. With cholesterol as substrate, the situation is the obverse: a high degree of conversion with the Nocardia enzyme and hardly any conversion with Arthrobacter. In this application, it has further been demonstrated that other cholesterol oxidases, from Schizophillum commune and Pseudomonas fluorescens , in contrast to the enzyme from Nocardia, are capable, to a reasonable extent, of forming 4 -solanidine- 3 -one . The oxidation of C-3β-OH is the first step in the biodegradation route for steroids. In general, it is followed by dehydrogenation, resulting in double bonds to C-1 and C-4. When further degradation is possible, this mostly means cleavage of the side chain to C-17, resulting in the formation of the important intermediate 1,4- androstadiene-3 , 17-di-one, a precursor for bioactive steroids such as female sex hormones. By hydroxylation to C-9, also the B-ring is opened. Thereafter, further fragmentation arises, with (possibly) eventual mineralization to inter alia C0 2 and H 2 0. Particularly the effectuation of a double bond to C-1 is of industrial importance for the synthesis of prednisolone from cortisone with the aid of Arthrobacter. In addition to oxidation and dehydrogenation, hydroxylation plays an important role in steroid transformations to bioactive steroids. In the presence of solanidine and microsomes from Solanum chacoense, a hydroxy solanidine is formed. In addition, there occurs glycosylation to C-23 to form - 56 - 23β-hydroxysolanidine-glycoside . Glycosylation can also occur with an isolated bacterium, described as isolate 10B. After incubation with this bacterium, no SGA is present anymore, but the aglycon is not present in free form. Also, slime formation is signalled and carbohydrate remains present in the medium, this in contrast to the conversions with the Arthrobacter and Aureobacterium strains described here. The micro-organisms, enzyme systems and/or enzymes represented by, for example, isolate AVB-sol 6, which deglycosylate solanine and can further be specific for chaconine, can further transform the 4-solanidine-3-one or convert same to a (less toxic) SGA-derivative and can accomplish further steroid transformation. Conversions in vivo based on 4-solanidine-3-one here indicate that eventually mineralization to C0 2 occurs. Process technological and economic aspects The results presented here indicate that, starting from potato protein with high contents of SGA (such as about 3500 mg SGA/kg) , a rapid conversion of SGA to solanidine or 4-solanidine-3-one, keto-4-solanidine-3-one or further metabolites or derivatives to mineralization to C0 2 is possible. Conversion occurs, for instance, in a suspension of (for example, 20%) protein to which 1 volume part of a mature pre-culture of a suitable (recombinant) micro-organism is added. With such a pre-culture, for example cultured in 0.5% protamylasse, it is possible to treat several batches of protein. In a fed-batch process, protein in a 10% suspension, with about 1500-3500 mg SGA per kg, can be detoxified within 8 hours at a maximum, preferably within 3 hours. After the detoxification of a first batch, with conventional methods, such as sedimentation, centrifugation and filtration, a separation is effected between the detoxified protein present in undissolved form and a liquid phase characterized by a low - 57 - content of suspended material. The distribution of the added bacteria over the two phases is such that the liquid phase with little suspended material - for example in the form of a supernatant after sedimentation - can be used again as an inoculum of the desired micro-organisms for the detoxification of a next batch. In one embodiment of this process, it is possible to treat several batches, preferably 3-10 batches, with a pre-culture cultured a single time (for example in protamylasse) . The possibility of separation of (treated) protein and a liquid phase with little protein, but a high bacterial density, and the use of this liquid phase for re-inoculation, imply a major saving on the use of substrates with regard to the culture of the desired micro-organisms. With comparable techniques of separation by density and/or size, it is also possible to utilize (recombinant) micro-organisms and/or enzymes therefrom, which are present in immobilized form. Immobilization can be accomplished with known techniques, for example with the aid of alginate or carrageen. With the immobilized systems, rapid detoxification of SGA can take place in, for example, potato juice and protamylasse. In a preferred embodiment, use is made of potato juice, while using, in addition to the immobilized biocatalyst, the free and endogenous enzymes present in potato juice for detoxifying SGA. Detoxifications that are possible in such systems relate to conversions of SGA to solanidine, as with the class of (recombinant) micro-organisms (and (recombinant) enzymes therefrom) comparable or identical to Paenibacillus pabuli (AVB-sol 1, 2 and 5 ) , Enterococcus flavescens (AVB-sol 3) and the class of (recombinant) enzyme systems such as Helix aspersa , Aspergillus japonicus , Penicillium decumbens and/or Helix pomatia preparations . Another class of further-reaching detoxifications to be applied is characterized by the further conversion of SGA via solanidine to 4-solanidine-3-one, as described for the - 58 - group of (recombinant) micro-organisms (and (recombinant) enzymes therefrom) comparable or identical to Aureobacterium species 2B1, Arthrobacter histidinolovorans/nicotinovorans 6B and Arthrobacter species 13A, as well as the class of (recombinant) enzyme systems such as Hemicellulase F35000, Pectolyase (151804, ICN), Pectinase (art. P9179, Sigma), Pectinase (art. P2829, Sigma) Viscozym L and Pectinet Ultra SP (NOVO Nordisk) and Pectolyase (art. P3026, Sigma) and Pectolyase (art. P5936, Sigma) . With the class of (recombinant) micro-organisms (and (recombinant) enzymes therefrom) comparable or identical to Rhodococcus erythropolis (AVB-sol 6) , it is further possible to effect derivatization and detoxification beyond the level of 4-solanidine-3 -one . Also the conversion to keto-4-solanidine-3-one is possible now, with (recombinant) enzyme systems and/or enzymes as found for, for example, Hemicellulase F35000 preparations. A method in which detoxification is achieved through conversions of SGA by the use of the class of (recombinant) micro-organisms (and (recombinant) enzymes therefrom) comparable or identical to Paenibacillus pabuli (AVB-sol 1, 2 and 5), Enterococcus flavescens (AVB-sol 3) and the class of (recombinant) enzyme systems such as Helix aspersa , Aspergillus japonicus , Penicillium decumbens and/or Helix pomatia preparations, can be combined with techniques for removing detoxified alkaloid, preferably through separation by specific weight and/or size, as can be achieved by centrifugation and/or filtration. In one embodiment of this process, a method in which, with an enzyme preparation or micro-organism, only a partial detoxification is achieved, such as a deglycosylation which is limited to the chaconine present, is expanded with a method such as described in the application, in which alkaloid is removed by techniques such as centrifugation or filtration at neutral or slightly alkaline pH. In another embodiment of the method, after conversion of the SGA, a supplementary treatment of the material to be detoxified is carried out to enhance the possibility of removing the alkaloid present. In one embodiment of such a process, for a limited period of time, preferably less than half an hour, the temperature of the material to be detoxified is raised to values preferably below 50°C, whereby limited coagulation arises. A method for detoxification of treated material can also mean removal of detoxified alkaloid through sedimentation, for example during residence of potato liquor AVW in storing basins at temperatures below 15 °C. The above-mentioned methods can be carried out, as described in the application, under conditions where the native conformation of the potato protein is maintained. In this way, detoxified native protein can be obtained. With the conventionally used methods, in which, as a result of pH and/or temperature treatments, denaturation of the potato protein occurs, this is not possible. The above-described methods can also be carried out to obtain a fraction enriched with modified alkaloid, aimed at further processing and application of the alkaloid. Further to this, it is supposed, for practice, that SGA as found in, or recovered from, for instance, potato protein, liquor or protamylasse, under conditions as mentioned above, can be treated to yield a high degree of detoxification and/or further transformation to a bioactive steroid. In this connection, it is also important to mention that the potato product to be treated in practice will typically have a lower SGA content than the protein having the high content of 3500 mg/kg which is investigated here, thus enabling short processing times. Also when potatoes with a selected high average SGA content are going to be used in the production process, and the contents in, for example, concentrated protein become higher still, the SGA will be converted to a less toxic derivative by microorganisms, enzyme systems and/or enzymes as provided in this invention. Such a derivative can also be (further) - 60 - transformed to a bioactive steroid or be mineralized to C0 2 . Fishfeed In a comparative trial with rainbow trout ( Oncorchymus mykiss) , the effect of potato protein treated according to the invention, untreated potato protein and high quality fish meal, on acceptance, growth and feed conversion was tested. The test was carried out in triplicate in 9 tanks, with 12 fish per tank. Fish weight was approximately 110-120 g/pc. The tanks were randomly distributed to the feeds (3 tanks per feed) . At the start of the trial the biomass and all fish in each tank were individually weighed. The same procedure was carried out at the end of the trail, after three weeks' trial. The trial was carried out in cylindric conical acrylic glass tanks. The system was a flow-through system without filtration or water purification units. The tanks were equipped with a bottom grate so that uneaten feed and faeces could easily be collected. The oxygen concentration of the water was regulated to a minimum of 80% saturation. Measurements of temperature and oxygen were carried out daily. At least once a week the contents of ammonia/ammonium (NH 3 /NH 4 + ) , nitrite (N0 2 J and nitrate (N0 3 J were measured as well as the pH level. The feeds were formulated to be isonitrogenous and isocaloric and were pelleted into 4mm pellets. The composition of the feeds is listed in Table 12. 61 Table 1. Degradation of SGA after 72 hours of incubation of micro-organisms in different media. Enrichment Bioscreen media SGA sources C-sources 1 N-sources Spo /Vit J (mg/l) Soil 2 g/1 SGA 0.5 g/1 NaN0 3 1 g/i Glue. 0.5 g/1 Tryptone - 86 Soil 2 g/1 SGA 1.0 g/1 NaN0 3 1 g/1 Glue. - 85 Soil 2 g/1 SGA 0.5 g/1 NaN0 3 0.5 g/1 Tryptone _ 95 Pot.+ Soil 2 g/1 SGA 0.5 g/1 NaN0 3 0.5 g/1 Tryptone -. 194 Pot.+ Soil 2 g/1 SGA 1.0 g/1 NaN0 3 - 64 Pot . 2 g/1 SGA 1.0 g/1 (NH4) 2 S0 4 - 128 Pot.+ Soil 2 g/1 SGA 1.0 g/1 NaN0 3 + 92 1) If two C- or N-sources are present, these are listed under each other. 2) .Abbreviations : Spo/Vit. = Spore and vitamin solution; Glue. = Glucose; Pot. = Potato pulp. Table 2. Properties of a number of SGA-degrading cultures. Name N-source J Clearing Growth and Growth and Starch and growth on appearance appearance degradation med Lum A 2 on medium D on medium E on medium E 2B-1 tryptone + + yellow +/- yellow - 2B-2 tryptone + + yellow +/- yellow - 6B tryptone + ++ + - 10B tryptone +/- + slime + + 12B tryptone + +/- yellow +/- yellow - 13A NaN0 3 ++ ++ ++ + 13B-1 tryptone ++ ++ ++ + 13B-2 tryptone ++ ++ ++ + 16A NaN0 3 + ++ yellow + - 1) Nitrogen source on which the original colony was grown. 2) ++ = strong; + = clear; +/- = moderate; - = none. 3) According to iodine stain. Table 3. Conversion of SGA in protein with Arthrobacter 6B. Time Dry matter pH SGA Aglycon (hours) (%) (mg/l) (mg/l t/f/m) 11 0 11.4 6.6 411 154/6/0 8 10.1 6.0 13 178/158/107 16 10.2 7.0 0 160/160/155 24 9.7 7.5 0 174/171/169 1) respectively, total, free and modified aglycon. Table 4. Conversion of SGA in protein with Arthrobacter 13A. Time Dry matter pH SGA Aglycon (hours) (%) (mg/l) (mg/l t/f/m) 1 ' 0 10.0 6.6 451 169/5/0 8 11.9 6.7 42 167/150/120 16 9.5 7.4 0 160/160/155 24 9.1 8.0 0 159/152/150 1) respectively, total, free and modified aglycon. Table 5. Conversion of SGA in protein after addition of physiological salt solution. Time Dry matter pH SGA (mg/l) Aglycon (hours) (%) (mg/l t/f/m) 1 ' 0 12.1 6.5 364 179/7/0 8 9.6 5.6 478 173/7/0 16 12.4 5.6 377 152/9/1 24 9.7 5.8 454 173/7/0 1) respectively, total, free and modified aglycon. 63 Table 6. Summary of the results of the dif erent toxicity tests with SGA and derived components. Toxicant EC 50 (μM) MIC LC 50 (μM) (μM) Yeas Bact Fungus Enzyme Brachionus Thamno - terium produccalyciflorus cephalus tion platyurus SGA- 1394- 523 107- 122-144 1.2-1.7 2.9 mixture 2324 140 Solanine 519- 980 231- 144-288 1.7-2.3 4.0-4.6 750 461 Chaconine 1407- 469 82-176 147-293 2.3 2.3-2.9 >1758 Solanidine >1889- 101 25-504 945-1889 3.8 101-252 >2519 4-Sol-3- 2525- 2020 1136- 2525-5050 379-505 1515 one 3288 1515 Table 7. Specific activities of Hemicellulase F35000 products solanidine, 4-solanidine-3 -one and AG-Xl. Enzyme specific activity (U/g protein) Hemicellulase α-chaconine 4 -sol -3 -one AG-Xl F35000 degradation 69 21xl0 2 64xl0 ~2 - 64 - O 99/43837 PCT/NL99/00102 Table 8: Conversion of SGA in protein by Paenibacillus pabuli (AVB-sol 1) t=0 (h) 7 16 24 40 SGA 129 124 117 80 62 (mg/l) solanine 49 50 47 4 0 chaconine 80 74 70 76 62 total 59 67 63 64 59 aglycon (mg/l) free 0.9 0.3 2.3 13 18 aglycon (mg/l) free 0.8 0.15 2.2 12.7 17.3 solanidin e (mg/l) free 4- 0.1 0.15 0.1 0.3 0.7 sol-3-one (mg/l) pH 5.8 5.6 5.5 5.6 6.7 65 Table 9: Conversion of SGA in protein by Enterococcus flavescens (AVB-sol 3) t=0 (h) 7 16 24 40 SGA 130 74 86 66 77 (mg/l) solanine 50 2 2 0 3 chaconine 80 72 84 66 74 total 65 71 58 65 62 aglycon (mg/l) free 1.6 15 11 13 15 aglycon (mg/l) free 1.2 14.3 10.1 12.0 14.1 solanidin e (mg/l) 4-S01-3- 0.4 0.7 0.9 1.0 0.9 one (mg/l) pH 5.8 5.7 5.7 5.7 6.5 66 Table 10 : Conversion of SGA in protein by Rhodococcus erythropolis (AVB-sol 6 ) t=0 (h) 7 16 24 40 SGA 122 58 4 3 4 (mg/l) solanine 46 51 0 3 2 chaconine 76 7 4 0 2 total 58 50 5 3.5 3.2 aglycon (mg/l) free 0.8 24 1.4 0.3 0.3 aglycon (mg/l) free 0.6 5 0.7 0 0.3 solanidin e (mg/l) 4-S01-3- 0.2 17 0.7 0.3 0 one X (mg/l) 0 2 0 0 0 pH 5.9 6.2 6.4 6.7 7.1 - 67 - Table 11: Some glycoalkaloids, aglycons and sugar groups, Glycoalkaloid Aglycon Sugar group D-glu α-Solanine Solanidine -D-gal< L-rha β-Solanine Solanidine -D-gal-ϋ-giu γ-Solanine Solanidine -υ-gai L-rna α-Chaconine Solanidine -D-glu< L-rha (3-Chaconine solanidine -D-glu-L-rna γ-Chaconine solanidine -D-glu D-glu α-Solamarine Tomatidenol -D-gal< L-rha L-rna β-Solamarine Tomatidenol -D-glu< L-rha L-rna Leptine I 0(23) - -D-glu< Acetylleptinidine L-rha D-glu Leptine II 0(23) - -D-glu< Acetylleptinidine L-rha L-rna Leptininine I Leptinidine -D-glu< L-rha D-glu eptinine II Leptinidine -D-glu< L-rha D-glu Commersonine Demissidine -D-gal-D-glu< D-glu D-glu Demissine Demissidine -D-gal-D-glu< D-xyl - 68 - Table 12 : composition of the feeds (%) ' & < && * % * > > '- Fa& S; 61.5 51 50 0 10 10 19 19.5 20 13 13 13 4.5 4.5 5 1 1 1 1 1 1 Feed 1 was a conventional feed for rainbow trout and contained no potato protein. Feed 2 and 3 were the test feeds . Feed 2 contained the untreated potato protein, Feed 3 contained potato protein treated according to the invention. The analyzed chemical composition of the experimental diets is listed m Table 12. Table 13: Analyzed chemical composition of the diets (%) '■. 7 w^?- Fe& X I€SβCi f 44.8 45.1 44.4 26 25.2 26.1 14.3 15 15.6 ' Λ'" "7yfcr7y>' , ■ ■. ' T?g_* -ma zft-ψ ' - ' 92.2 91.7 92.2 7.1 6.4 6.1 23.4 23.3 23.6 * Nitrogen Free Exact = dry matter- (protem+fat+ash) ** GE (gross energy) is calculated based on an energy content of 23.66 KJ/g for protein, 39.78 KJ/g for fat and 17.7 KJ/ g for carbohydrates. - 69 - The water quality was good throughout the trial. None of the measurements were on levels that might have had a negative influence on the results of the trial. The results regarding acceptance, specific growth rate and feed conversion ratio of the fish in this trial are listed in Tables 14 and 15. Table 14 g s^ι«si«^ ,,, ^ ^g^^'ϊΛα«Moβ * scsaβapsfce® 11 204,143,121 319,370,410 63,74,78 358,374,374 120, 138,156 28,30,33 30 158, 158,150 355,360,338 71,71,71 71 The results in Table 14 show that Feed 2 (untreated potato protein) has the lowest acceptance. The fish fed with the potato protein treated according to the invention accepted their feed very well, as shown by the high percentage of 71, which is nearly as good as the acceptance of the control feed. The data suggest that removing the 'bitter' glycoalkaloids according to the invention improves the palatability of potato protein significantly. Table 15: SGR & FCR results for the trial given as an average for each code. Sd values are given. A different letter within a column indicates significance (P<0.01) sm {%) wen 1.53 (0.18) a 0.81 (0.02) a 0.07 (0.03) b 6.49 (2.63) b ■ i ' .y f ' y<fct* χ & 7yy i 1.50 (0.14) a 0.79 (0.08) a SGR (i Specific Growth Ratio = 100 x ( (Wt/WO) 1/t -l) Wt = biomass at the end of the trial WO = biomass at the start of the trial t = time of the trial (feeding days) - 70 - FCR = Feed Conversion Ratio = kg feed eaten / kg gross growth . Feed 2, containing the untreated potato protein, showed the poorest SGR and the highest FCR, compared to both the control feed and the feed containing the treated potato protein. This difference appeared to be highly significant (P<0.01). Feed 1 (the control feed) and Feed 3, the feed containing 10% potato protein, treated according to the invention, were very similar. The difference between the two was not significant (P>0.05) . In summary: removal of glycoalkaloids out of potato protein according to the invention improved the acceptance of the feed, raised SGR and lowered FCR, all very significantly. Potato protein treated in the way according to the invention could easily be included in 10% of the diet for rainbow trout, replacing LT fish meal, without any decreases in acceptance, SGR or FCR. - 71 - Legends to the figures Fig. 1. Conversion of SGA by (A) culture 13A and (B) culture 10B. Represented are the curves of the carbohydrate concentration (Δ) , the TOC content (o) , the solanine content (A) and the chaconine content (•) . Fig. 2. Chromatograms of solanine (t r = 8.6 min.) and chaconine (t r = 9.6 min.). Represented are a control incubation in the absence of bacteria after 168 hours (A) ; culture 10B after 24 hours (B) ; culture 10B after 48 hours (C) ; culture 10B after 168 hours (D) and culture 13A after 24 hours (E) . Fig. 3. Chromatograms of the aglycon of SGA. Represented are: a solanidine .HCI standard (A) with t r -solanidine = 17.74 min. and t r -solanthrene = 14.62 min.; aglycon from culture 6B after 168 hours of incubation (B, peak with t r = 20.14 min.) Fig. 4. SGA-degradation compared with the % T20.1. Relation between SGA-degradation (solanine and chaconine) and the proportion of a component with a retention time of 20.1 min. (T20.1) within the total aglycon (o-o) . In addition, the proportion of the free aglycon (•--•) is represented. Fig. 5. Chromatograms of the aglycon of SGA. Represented are: total aglycon from culture 10B after 168 hours of incubation (A, with t r - solanthrene = 14.62 min.) and the free aglycon after 168 hours of incubation (B, t r -solanidine = 17.79 min.). Fig. 6. Conversion of SGA with cultures 6B, 13A and after addition of physiological salt solution (BL) . Represented are: SGA (+) , solanine (Δ) , chaconine (O) , total aglycon (A), free aglycon (•) and 4-solanidine- 3-one (■) . - 72 - Fig. 7. Curve of pH and dry matter contents upon incubation of about 10% potato protein. Represented are: pH-control (+) , dry matter control (Δ) , pH-6B (o) , dry matter-6B (A) , pH-13A (•) and dry matter-13A (■) . Fig. 8. Growth of Arthrobacter 6B in protamylasse. A: 0.5% (Δ) , 1.0% (O) , 1.5% (A) and 2.0% (<■) protamylasse. B: 0.5% protamylasse with 0.1% (Δ) and 0.2% (O) glucose. C: 2.0% protamylasse with 0.1% (Δ) , 0.2% (O) and 0.5% (A) glucose . Fig. 9. Conversion of SGA in 2% protamylasse. Represented are the conversion in the absence of glucose (O) and in the presence of 0.5% glucose (•) . Fig. 10. Conversion of SGA in protein by R. erythropolis Fig. 11. Molecular structure of α-solanine and α-chaconine . Fig. 12. Structure of some aglycons. Fig. 13. Removal of alkaloid from a buffer solution with 0.2 % SGA after incubation with chaconine deglycosylating enzyme preparation. Effect pH. Dark bars: % α-solanine present in supernatant after centrifugation (100% = 756 mg/l); light bars: idem solanidine (100% = 328 mg/l); ■: pellet obtained after centrifugation expressed as g dry matter per 1. Fig. 14. Removal of alkaloid from potato liquor with 0.2% SGA after incubation with chaconine deglycosylating enzyme. Effect pH. A: Alkaloid concentration in supernatant and mass pellet after centrifugation; B: idem after temperature treatment followed by centrifugation; C: idem - 73 - after filtration. Dark bars: % α-solanine (100% = 552 mg/l); light bars: idem solanidine (100% = 240 mg/l); ■: pellet expressed as g dry matter per 1. Fig. 15. Effect treatments from Fig. 14 on protein concentration in supernatant/filtrate (A) and ratio alkaloid/protein in supernatant (B) and obtained pellet (C) . Light bar: centrifugation; horizontally hatched bar: temperature treatment/centrifugation; checkered bar: filtration; black bar in 15B,C: untreated AVW. Fig. 16. Possible reactions in conversion of solanidine by Aspergillus solanidine oxidase as present in Hemicellulase F35000, by analogy of the mechanism for Pseudomonas fluorescens cholesterol oxidase presented by J.I. Teng and L.L. Smith (Steroids 61 (1996) 627-633). 1: solanidine (22R, 25S-Solanide-5-ene-3β-ol) ; 2: solanide- 3 , 5-diene-3-ol ; 3: 6β-hydroperoxysolanide-4-ene-3 -one; 4: solanide-4-ene-3-one (4-solanidine-3-one) ; 5: solanide-4- ene-3 , 6-dione (keto-4-solanidine-3 -one) ; 6 : 6β-hydroxysolanide-4 -ene-3 -one . Fig. 17. 13 C-NMR spectrum of solanidine. Fig. 18. ^-N R (top) and 13 C- MR (bottom) spectrum of AG-Xl.

Claims: - 74 - CLAIMS 1. An isolated or recombinant micro-organism capable of deglycosylating steroid glycoalkaloids (SGA) , preferably solanine, and converting same to solanidine. 2. A micro-organism according to claim 1, further capable of deglycosylating chaconine and converting same to solanidine. 3. A micro-organism according to claim 1 or 2 , capable of deglycosylating α-solanine, β-solanine and/or γ-solanine. 4. A micro-organism according to claim 3, further capable of deglycosylating α-chaconine, β-chaconine and/or γ-chaconine. 5. A micro-organism according to any one of claims 1 through 4, characterized in that it is capable of detoxifying solanine and/or chaconine. 6. An isolated or recombinant micro-organism, characterized in that it is capable of detoxifying solanine and/or chaconine . 7. A micro-organism according to any one of claims 1 through 6, characterized in that it is capable of converting solanidine to 4-solanidine-3-one and/or to keto- 4-solanidine-3-one . 8. An isolated or recombinant micro-organism, characterized in that it is capable of converting solanidine to 4-solanidine-3 -one and/or to keto-4-solanidine-3-one . 9. An isolated or recombinant micro-organism, characterized in that it is capable of detoxifying solanidine. 10. A micro-organism according to any one of claims 1 through 9, characterized in that it is an Arthrobacter or a micro-organism with related functional properties with regard to SGA. 11. A micro-organism according to claim 10, characterized in that it is A. hi stidinolovorans /nicotinovorans , - 75 - preferably characterized in that it is isolate 6B, deposited under CBS 100108. 12. A micro-organism according to any one of claims 1 through 9, characterized in that it is an Aureobacterium or a micro-organism with related functional properties with regard to SGA. 13. A micro-organism according to claim 12, characterized in that it is isolate 2B1, deposited under CBS 100107. 14. A micro-organism according to any one of claims 1 through 9, characterized in that it is capable of converting 4-solanidine-3 -one . 15. An isolated or recombinant micro-organism, characterized in that it is capable of converting 4- solanidine-3-one . 16. An isolated micro-organism, characterized in that it is capable of detoxifying 4-solanidine-3-one . 17. An isolated micro-organism, characterized in that it is capable of mineralizing 4-solanidine-3 -one . 18. A micro-organism according to any one of claims 14 through 17, characterized in that it is a Rhodococcus or a micro-organism with related functional properties with regard to SGA. 19. A micro-organism according to claim 18, characterized in that it is R. erythropolis, preferably characterized in that it is isolate AVB-sol 6, deposited under CBS 100109. 20. A micro-organism according to any one of claims 1 through 9, characterized in that it is a Paenibacillus pabuli or a micro-organism with related functional properties with regard to SGA. 21. A micro-organism according to any one of claims 1 through 9, characterized in that it is an Enterococcus flavescens or a micro-organism with related functional properties with regard to SGA. 22. A micro-organism according to any one of claims 1 through 21 which displays SGA and/or SGA-derivative - 76 - conversion in a culture medium which in addition to SGA also contains simple and/or complex carbohydrates and/or proteins . 23. A micro-organism according to claim 22, which preferentially displays SGA and/or SGA-derivative conversion. 24. A microbial culture comprising at least one microorganism according to any one of claims 1 through 23. 25. A culture according to claim 24, in which the culture medium is protamylasse. 26. An enzyme (or enzyme system or enzyme combination) isolated from a micro-organism according to any one of claims 1 through 23, which displays SGA and/or SGA- derivative conversion. 27. An enzyme system or enzyme combination and/or an enzyme capable of converting solanine to solanidine. 28. An enzyme system or enzyme combination and/or an enzyme according to claim 27, further capable of converting chaconine to solanidine . 29. An enzyme system or enzyme combination and/or an enzyme capable of converting solanidine to 4-solanidine-3-one . 30. An enzyme system or enzyme combination and/or an enzyme capable of converting solanidine to keto-4-solanidine-3 - one . 31. An enzyme system or enzyme combination and/or an enzyme capable of converting 4-solanidine-3-one and metabolites thereof . 32. A (recombinant) nucleic acid molecule coding for an enzyme according to any one of claims 26 through 30. 33. A vector comprising at least one nucleic acid according to claim 32. 34. A host cell or recombinant micro-organism comprising a nucleic acid according to claim 32 or a vector according to claim 33. 35. An enzyme produced by a cell according to claim 34. - 77 - 36. A method in which a micro-organism according to any one of claims 1 through 23 and 34, or an enzyme system or enzyme combination and/or an enzyme according to any one of claims 26 through 31 and 35 is used for converting, detoxifying and/or removing SGA and/or SGA derivatives. 37. A method according to claim 36 for use in the potato and/or tomato processing industry. 38. A method according to claim 36 or 37 for detoxifying or removing taste-affecting components from plant material. 39. Food comprising plant material treated according to a method according to claim 38. 40. A method according to claim 36 or 37 for the production of raw material for and/or the synthesis of (bioactive) steroids . 41. A test method wherein the content of SGA and/or SGA derivatives in a sample is determined through an enzymatic reaction in which at least one enzyme according to any one of claims 26 through 31 or 35 is used. 42. A test kit comprising requisite means for carrying out a method according to claim 41. 43. A crop potato, preferably selected with a method according to claim 41, having an average SGA content higher than 200 mg/kg, preferably higher than 200-250 mg/kg, preferably higher than 250-500 mg/kg, or preferably higher than 500 mg/kg of fresh harvest weight. 44. A crop potato comprising a host cell according to claim 34. 45. A method wherein a potato according to claim 43 or 44 is selected for processing in the potato processing industry. 46. A method according to claim 45, wherein during the processing a method according to claim 36 is used. 47. An agricultural method wherein a potato according to claim 43 or 44 is grown. - 78 - 48. An agricultural method according to claim 47, wherein a potato with a high resistance to one or more diseases and plagues and/or a high productivity is grown. 49. An agricultural method, preferably according to claim 47 or 48, wherein potatoes are produced for processing according to a method according to claim 36.

Inventors: BINNEMA DOEDE JACOB, BOS HARMANNUS THEODORUS PETRUS, VEEN ANKO, WIJBENGA DIRK-JAN WIJNZEN, BINNEMA, DOEDE, JACOB, BOS, HARMANNUS, THEODORUS, PETRUS, VEEN, ANKO, WIJBENGA, DIRK-JAN, WIJNZEN
Applicants: AVEBE COOP VERKOOP PROD, BINNEMA DOEDE JACOB, BOS HARMANNUS THEODORUS PETRUS, VEEN ANKO, WIJBENGA DIRK JAN WIJNZEN, BINNEMA, DOEDE, JACOB, BOS, HARMANNUS, THEODORUS, PETRUS, COOEPERATIEVE VERKOOP- EN PRODUCTIEVERENIGING VAN AARDAPPELMEEL EN DERIVATEN AVEBE B.A., VEEN, ANKO, WIJBENGA, DIRK-JAN, WIJNZEN
Application year: 1999
Main classification: C12N15
IPC codes: C12N15