Glycoside-hydrolase enzyme inhibitors

BACKGROUND OF THE INVENTION
Carbohydrates are eaten either as simple sugars or as more complex molecules called polysaccharides, which are built up from simple sugar units and of which the starch of potatoes, bread, etc., is a well-known example. The body absorbs carbohydrates from the small intestine in the form of single sugar units, or monosaccharides. Those sugars which are ingested in this form, e.g., glucose in various "energy-giving" preparations and fructose, the sugar of fruit, thus require no further treatment. Sugar molecules bigger than this require enzymic digestion.
The digestion of starch begins in the mouth. Saliva contains an enzyme, amylase, which attacks starch and similar polysaccharides, reducing the size of the molecule. Starch is made up of chains of glucose molecules linked in a particular way, and amylase attacks the links between the glucose units. The enzyme acts at many points in the chain, and the smallest units it produces are molecules of the sugar maltose. Maltose is made up of two glucose units; this sugar is thus a disaccharide. ##SPC1##
Starch also contains branched chains of glucose units, and the amylase of saliva cannot break the chains at the points where branching occurs: ##SPC2##
If the branched-chain structure of starch is thought of as resembling a bush, the enzyme can prune the outer branches until it reaches a fork and then its action stops. Thus, as well as molecules of maltose, amylase produces from starch fragments of the original molecule which are like hard-pruned bushes and which are called "limit dextrins". Glycogen, the carbohydrate storage material of animal tissues, is also made up of much-branched chains of glucose units.
The digestion of starch or glycogen by salivary amylase probably does not get very far, however. The process continues for a while after the food has entered the stomach, but as the acid of the gastric juice penetrates through the food mass the amylase action slows down and stops. The main attack on these polysaccharides takes place in the small intestine by an amylase in pancreatic juice similar to that in saliva.
The dextrins resulting from the "pruning" by amylase are attacked by an enzyme in the small intestine which can break the inter-chain links. A specific enzyme, maltase, also splits each molecule of maltose produced from starch into two molecules of glucose, which are then absorbed and metabolized by the body. Some of the sugars of food are disaccharides; common sugar (sucrose) is made up of a molecule of glucose joined to a molecule of fructose, and lactose (the sugar of milk) is also a disaccharide. These sugars are split into their component monosaccharides by specific enzymes in the intestine.
However, in the treatment of conditions in which there is an indication of obesity, adipose, hyperlipidemia (arteriosclerosis), diabetes, pre-diabetes, gastritis, gastric ulcer, duodenal ulcer, and/or caries, it is necessary that such glycosidehydrolase enzyme be inhibited or suppressed in such a manner that they cannot further catalyze the breakdown thereof, as indicated in the manner above for subsequent utilization by the body and thus further promotion or worsening of the condition being treated.
It has been known heretofore that .alpha.-amylases can be inhibited by the use of various low molecular substances, such as, for example, salicylic acid and abiscisin [T. Hemberg, J. Larsson, Physiol. Plant. 14, 861 (1961), T. Hemberg, Acta Chem. Scand. 21, 1665 (1967)]. It is further known that there are also higher-molecular substances which are capable of inhibiting the activity of certain amylases non-specifically by physical adsorption [T. Chrzaszcz, J. Janicki, Bioch. Z. 260, 354 (1933) and Bioch. J. 28, 296 (1934)] or by denaturation and precipitation of the enzyme [B. S. Miller, E. Kneen, Arch. Biochem. 15, 251 (1947), D, H. Struhmeyer, M. H. Malin, Biochem. Biophys. Acta 184, 643 (1969)]. It has also been observed that it is possible to elute a substance from wheat by means of distilled water, which lowers the dextrifying activity of salivary amylase but has little influence on the activity of pancreatic amylase [E. Kneen, R. M. Sandstedt, Arch., Bioch. 9, 235 (1946)].
It is a disadvantage of these known inhibitors that either the inhibition of the amylase is non-specific or that the inhibiting activity of the inhibitor is slight, especially on pancreatic amylases, as has been shown by our own investigations; that is to say, only at very high ratios of inhibitor: enzyme is almost complete inhibition of the amylases (up to 90% and above) attained.
An earlier proposal (United States patent application Ser. No. 110,482, filed Jan. 28, 1971, now abandoned) relates to amylase inhibitors. In fact, the older proposal shows that by means of aqueous electrolyte solutions, preferably dilute acids, or above all by means of water-alcohol (C.sub.1 -C.sub.3 -alcohols) mixtures, preferably at acid pH values, a highly active inhibitor for pancreatic amylase, which does not show the disadvantages mentioned, can be extracted in high yields from wheat (coarse ground wheat, wheat flour or wheat gluten). The substance thus obtained inhibits pancreatic amylase to the extent of more than 90% even at very low inhibitor:enzyme ratios.
THE PRESENT INVENTION
The present invention now relates to inhibitors for glycoside-hydrolases from actinomycetes, and in particular to inhibitors for glycoside-hydrolases of preferably carbohydrate-splitting enzymes of the digestive tract from actinomycetes. These inhibitors are formed by microorganisms of the order Actinomycetales, especially by those of the family of Streptomycetaceae, Thermoactinomycetaces, Micromonospora, Nocardia, and above all those of the family Actinoplanaceae. They are also formed to a particularly high degree by strains of the genera Actinoplanes, Ampullariella, Streptosporangium, Streptomyces, Chainia, Pilimelia, Planomonospora, and Actinobifida.
Accordingly, this invention provides, as a new product, a glycoside-hydrolase inhibitor of microbial origin.
This invention further provides a method for the production of a glycoside-hydrolase inhibitor comprising culturing a microbe of the order Actinomycetales and extracting the inhibitor from the resultant culture.
The preferred microorganisms are preferably of the family Streptomycetaceae or Actinoplanaceae.
A wide variety of microorganisms of the order Actinomycetales have been found to be glycoside-hydrolase enzyme inhibitors and the methods described below can be used to test the microorganisms to determine whether or not the desired glycoside-hydrolase enzyme inhibitor activity is present and the approximate relative value of this activity.
Strains of the order Actinomycetales, especially those of the families Streptomycetaceae and Actinoplanaceae, are isolated in a known manner from samples of soil or strains of those orders bought from culture collections. Culture flasks containing nutrient solutions which permit the growth of these strains are inoculated with inoculum of these strains. For example, the glycerine-glycine nutrient solution according to von Plotho, of composition 2% glycerine, 0.25% glycine, 0.1% NaCl, 0.1% K.sub.2 HPO.sub.4, 0.01% FeSO.sub.4, 0.7 H.sub.2 O, 0.01% MgSO.sub.4, 0.7 H.sub.2 O and 0.1% CaCO.sub.3 can be used. For more rapid growth, it is advisable also to add to such a nutrient solution complex sources of carbon, such as, for example, corn-steep liquor or soya flour or yeast extract or protein hydrolysates, for example NZ-amines, or mixtures of these substances. In these cases, the pH value of the solution must be adjusted. An initial pH of the nutrient solution of between 6.0 and 8.0, especially between 6.5 and 7.5, is preferred.
The glycerine of the nutrient solution can also be replaced by other sources of carbon, such as, for example, glucose or sucrose or starch or mixtures of these substances. Instead of glycine, it is also possible to use other sources of nitrogen, such as, for example, yeast extract, soya flour, NZ-amines, pharmamedia and others. The concentrations of the sources of carbon and nitrogen, and also the concentrations of the salts, can vary within wide limits. FeSO.sub.4, CaCO.sub.3 and MgSO.sub.4 can also be entirely absent. 100-200 ml, for example, of the nutrient solution are introduced into 1 liter Erlenmeyer flasks, sterilized in a known manner and inoculated with the strain to be investigated, and the flask is incubated on shaking machines at 15.degree.-60.degree.C., preferably at 24.degree.-50.degree.C. If the culture shows growth, which generally takes place after 1-10 days, and in most cases after 3-5 days, a sample of, for example 5 ml is taken and the mycelium in this sample is separated off by filtration or centrifugation. 0-100 .mu.l of the culture broths are employed in the tests described below, and the inhibiting capacity per ml is calculated.
The mycelia are extracted twice with 5 volumes (relative to the volume of mycelium) of acetone at a time, and subsequently 1 .times. 5 volumes of diethyl ether, the extracted mycelium residue is dried in vacuo at 20.degree. and the resulting dry mycelium powder is extracted with 4-8 parts by weight of dimethyl sulphoxide (DMSO).
The two acetone extracts and the ether extracts are combined and concentrated almost to dryness in vacuo. The residue from these extracts is taken up with the dimethyl sulphoxide (DMSO) extract from the dry powder and 0-100 .mu.l thereof are employed in the tests described below.
AMYLASE TEST
One amuylase inhibitor unit (1 AIU) is defined as the amount of inhibitor which inhibits two amylase units to the extent of 50%. one amylase unit (AU) is the amount of enzyme which in 1 minute, under the test conditions indicated below, splits 1 .mu. equivalent of glucoside bonds in starch. The .mu.Val of split bonds are determined colorimetrically as .mu.Val of reducing sugar formed, by means of dinitrosalicylic acid, and are specified as .mu.Val of maltose equivalents by means of a maltose calibration curve. To carry out the test, 0.1 ml of amylase solution (20-22 AU/ml) are treated with 0-400 ug of inhibitor or 0-100 .mu.l of the culture solution or mycelium extracts to be tested, in 0.4 ml of 0.02 M sodium glycerophosphate buffer/0.001 M CaCl.sub.2 pH 6.9, and the mixture is equilibrated in a waterbath at 35.degree.C. for about 10-20 minutes. It is then incubated for 5 minutes at 35.degree.C. with 0.5 ml of a 1% strength starch solution (soluble starch of Messrs. Merck, Darmstadt, No. 1252) which has been pre-warmed to 35.degree.C., and is subsequently treated with 1 ml of dinitrosalicylic acid reagent (according to P. Bernfeld in Colowick-Kaplan, Meth. Enzymol., volume 1, page 149). To develop the color, the batch is heated for 5 minutes on a boiling waterbath and then cooled and treated with 10 ml of distilled water. The extinction at 540 nm is measured against an appropriately mixed blank, without amylase. For evaluation, the amylase activity which is still active after addition of the inhibitor is read off from a previously recorded amylase calibration curve, and the percentage inhibition of the amylase employed is calculated therefrom. The percentage inhibition is plotted as a function of the quotient: ##EQU1## (+ relative to solids, ++ AU in the non-inhibited mix of the same series), and the 50% inhibition point is read off from the curve and converted to AIU/mg of inhibitor.
SACCHARASE TEST
One saccharase inhibitor unit (SIU) is defined as the amount of inhibitor which inhibits two saccharase units to the extent of 50%. One saccharase unit (SU) is the amount of enzyme which in one minute, under the test conditions indicated below, splits 1 .mu.mol of sucrose into glucose and fructose. The .mu.mol of split sucrose are determined colorimetrically as the glucose and fructose formed, by means of dinitrosalicylic acid, and calculated by means of a glucose/fructose calibration curve.
To carry out the test, 0.1 ml of saccharase solution comprising a solubilized saccharase from intestinal mucous membrane of pigs, [according to B. Borgstrom, A. Dahlquist, Acta Chem. Scand. 12, (1958) page 1997] in the amount of 0.3-0.4 SU/ml are mixed with 0-400 .mu.g of inhibitor or 0-50 .mu.l of the culture solution of the mycelium extract which is to be investigated, in 0.1 ml of a 0.1 M Na maleate buffer of pH 6.0, and the mixture is equilibrated for about 10-20 minutes in a waterbath at 35.degree.C. It is then incubated for 60 minutes at 35.degree.C. with 0.2 ml of an 0.056 M sucrose solution (sucrose: Messrs. Merck, Darmstadt, No. 7652) which has been pre-warmed to 35.degree.C., and is subsequently treated with 0.5 ml of dinitrosalicylic acid reagent (according to P. Bernfeld in Colowick-Kaplan. Meth. Enzymol., Volume 1, page 149). To develop the color, the mix is heated for 5 minutes on a boiling waterbath and then cooled and treated with 5 ml of distilled water. The extinction at 540 nm is measured against an appropriate blank value without saccharase.
For evaluation, the saccharase units which are still active after addition of inhibitor are determined from the calibration curve and the percentage inhibition of the saccharase employed is calculated therefrom. The percentage inhibition is plotted as a function of the quotient: ##EQU2## (+ relative to solids, ++ SU in non-inhibited mix of the same series), and the 50% inhibition point is read off from the curve and converted to SIU/mg of inhibitor.
MALTASE TEST
One maltase inhibitor unit (1 MIU) is defined as the amount of inhibitor which inhibits two maltase units to the extent of 50%. One maltase unit (MU) is the amount of enzyme which in 1 minute, under the test conditions indicated below, splits 1 .mu. equivalent of glucosidic bond in the p-nitrophenyl-.alpha.-D-glucopyranoside. The .mu.Val of split bonds are determined photometrically as .mu.Val of p-nitrophenolate.
To carry out the test, 0.05 ml of maltase solution comprising a solubilized maltase from intestinal mucous membrane of pigs [according to B. Borgstrom, A. Dahlquist, Acta Chem. Scand. 12, (1958), page 1997], or maltase in the form of human pancreatic juice lyophilizate, (0.09-0.12 MU/ml) are equilibrated with 0-400 .mu.g of inhibitor or 0-20 .mu.l of the culture solution to be investigated or of the mycelium extract in 0.05 ml of 0.1 M sodium maleate buffer at pH 6.0 for about 10-20 minutes in a waterbath at 35.degree.C. The mixture is then incubated for 30 minutes at 35.degree.C. with 0.1 ml of an 0.4% strength solution of p-nitrophenyl-.alpha.-D-glucopyranoside (Messrs. Serva, Heidelberg, No. 30,716) in 0.1 M Na maleate buffer at pH 6, which has been pre-warmed to 35.degree.C., and the reaction is subsequently stopped by adding 2 ml of 0.565 M tris-buffer of pH 7.6. The extinction at 403 nm is immediately measured against an appropriately mixed blank without maltase.
For evaluation, the maltase units still active after addition of inhibitor are calculated on the basis of a molar extinction coefficient of E.sub.403 = 13.2 .times. 10.sup.3 for the p-nitrophenolate anion at pH 7.6, and from the maltase units still active the percentage inhibition of the maltase employed is calculated. The percentage inhibition is plotted as a function of the quotient: ##EQU3## (+ relative to solids, ++ MU in the non-inhibited mix) and the 50% inhibition point is read off from the curve and converted to MIU/mg of inhibitor.
Since, in this simple routine test, an artificial substrate and not the actual substrate of maltase (maltose) is used, the preparations are additionally tested for their maltase-inhibiting activity in a more involved maltase test described by Dahlquist (Enzyme. biol. clin. 11, 52/1970). Herein, the glucose produced during the action of maltase on maltose is measured enzymatically by means of colorimetry, using glucose oxidase, peroxidase and dianisidine. All the maltase inhibitors described here also inhibit in this test.
A whole series of strains of various families and genera of the order Actinomycetales was tested in accordance with the method described above. In doing so, distinct, though at times weak, glycoside-hydrolase-inhibiting activities were found in various families and genera. The strains of the family of the Streptomycetaceae and especially of the family of the Actinoplanaceae proved most advantageous as regards yield.
Within these families, particularly active strains were found in the genera Streptomyces, Actinoplanes, Ampullariellaand Streptosporangium. Of the strains tested, those listed below proved particularly active in one or more of the tests indicated.
Table 1__________________________________________________________________________ Inhibiting ActionStrain No. Name Amylase Maltase SaccharaseOwn Desig- Collec-nation tion Nos. C M C M C M__________________________________________________________________________SB 2 CBS Ampullariella +++ + 951.70 regularisSB 5 CBS " +++ 952.70SB 11 CBS Actinoplanes +++ +++ 955.70 spec.SB 12 CBS " +++ 956.70SB 18 CBS " +++ +++ + ++ ++ ++ 957.70SB 27 CBS " +++ 958.70SB 46 CBS " +++ 959.70SE 5 CBS Actinoplanes ++ ++ 960.70 spec.SE 21 CBS Ampullariella +++ 953.70 regularisSE 39 CBS " +++ 954.70SE 50 CBS Actinoplanes +++ ++ +++ ++ +++ ++ 961.70 spec.SE 55 CBS " ++ + ++ ++ ++ ++ 962.70SS 26 CBS Streptospor- ++ 963.70 angium albumSS 45 CBS " spec. ++ 964.70SS 51 CBS " ++ ++ 965.70SS 53 CBS " roseum ++ ++ 966.70St 19 ATCC Streptomyces ++ 3319 flaveolusSt 50 CBS Streptomyces + 693.69 heimiiSt 67 CBS Streptomyces ++ ++ 432.59 tendaeSt 45 CBS Streptomyces 434.51 aureofaciens +RT 36 CBS Chainia + 228.65 rubraRT 33 CBS Chainia + 295.66 poonensisST 12 NRRL Streptomyces +++ + B-2286 murinusST 51 CBS Streptomyces ++ 498.68 fradiaeST 3 NRRL Streptomyces ++ 2580 chrysomallusST 1 ATCC " ++ 11523SS 55 CBS Streptospo- ++ 624.71 rangium roseumSS 59 " " amethystogenes ++ 623.71SS 62 " " roseum ++ 625.71AT 8 KCC-A " viridalbum ++ 0027AT 11 KCC-A " album ++ 0025AT 13 CBS Ampullariella +++ 19064 campanulataAT 14 CBS Ampullariella +++ 19364 regularisSE 89 CBS Ampullariella +++ 619.71 spec.SE 100 CBS Planomono- ++ ++ 622.71 spora spec.AT 4 CBS Ampullariella + ++ 191.64 digitataAt 9 KCC-A Streptospo- + ++ ++ 0028 rangium vulgareAT 10 ATCC " indianensis ++ ++ 19190SE 103 CBS Actinoplanes ++ 616.71 spec.HN 6 CBS Actinobifida ++ 602.71 chromogenaHN 2 CBS " +++ 601.71AT 2 CBS Actinoplanes + 367.66 utahensisSE 101 CBS Planomonospora + 621.71 parontosporaSK 2 CBS Pilimelia spec. + ++ 620.71SE 82 CBS Actinoplanes +++ +++ ++ ++ ++ ++ 615.71 spec.SA 28 CBS Ampullariella + + 617.71 digitataSA 8 CBS Actinoplanes +++ 611.71 spec.AT 7 ATCC Streptosporan- ++ 12428 gium roseumSE 45 CBS Ampullariella + 618.71 regularis__________________________________________________________________________ATCC = American Type Culture CollectionCBS = Centraalbureau voor Schimmelcultures, BaarnNRRL = Culture Collection Unit, Fermentation Section, Norther Utilization Research Branch, Peoria, Illinois, USAKCC = Culture Collection, Research Division, Kaken Chemical Co., Ltd., Tokyo, JapanC = Culture solutionM = Mycelium extract+ = distinctly active++ = strongly active+++ = very strongly active
Table 2__________________________________________________________________________ Streptosporangium album Streptosporangium spec. Streptosporangium spec. SS 26 SS 45 SS 51__________________________________________________________________________Date of isolation August 2, 1967 November 8, 1968 November 9, 1968Method soil smear, plate soil smear, plate soil smear, plateOrigin Rhon, Heidelstein, turf Kenya, near Ruiru, Kenya, near Ruiru, soil between basalt coffee plantation, coffee plantation, blocks, pH 3.9 pH 5.4 pH 5.4 white--cream--yellowish white--cream--yellowishMycelium AM white or pink or pink SM 0.5-1.2 .mu., septate 0.4-1.2 .mu. 0.4-1.2 .mu., septateShape of Sporangia spherical, in part spherical, smooth spherical, smooth, some wrinkled and deformed also wrinkled and irre- gular, shriveled formsSize of Sporangia 3-7 .mu. .phi. 3-11 .mu. .phi. 3-11 .mu. .phi.Shape of Spores .+-. spherical to elongated mostly .+-. oval mostly .+-. oval, in parts sphericalSize of Spores 0.6-0.9 .times. 0.7-1.1 .times. 0.7-1.0 .times. 0.6-1.3 .mu. 1.0-1.7 .mu. 0.8-1.7 .mu.Flagellation - - -Arrangement of spiral chains spiral chains spiral chainsSpores in SporangiumConidia, Substratespores and the likeMelanine PEH - PEH - PEH - CPC - - CPC - - CPC - - Ty - Ty - Ty - Gel - Gel - Gel -Nitrate reduction + (weak) - -Gelatine liquefaction + + +Milk peptonization +Growth at 20.degree.C. + + + 26.degree.C. ++ ++ ++ 32.degree.C. ++ + +(+) 37.degree.C. - + + 42.degree.C. - - -NaCl 2% + slight growth ++ ++toleration 3% - ++ ++ 4% - + + 7% - - -Starch hydrolysis - + +__________________________________________________________________________ Streptosporangium roseum Actinoplanes spec. SS 53 SE 5 SE 55__________________________________________________________________________Date of isolation November 9, 1968 December 16, 1969 December 31, 1969Method soil smear, plate pollen pollenOrigin Kenya, near Ruiru coffee Kenya, near Ruiru Kenya, Njoro, Eldoret plantation, pH 5.7 coffee plantation, Experimental Station, pH 5.6 pH 5.1Mycelium AM pink 0.4-1.3 .mu. 0.3-1.3 .mu., septate SM 0.4-1.2 .mu.Shape of Sporangia spherical, in part irregular slightly wrinkledSize of Sporangia 3-10 .mu. .phi. about 4-12 .mu.Shape of Spores mostly .+-. oval .+-. sphericalSize of Spores 0.7-1.1 .times. 1.0-1.9 .mu. Ca. 1 .mu.Flagellation -Arrangement of spiral chains coiled chains; sporeSpores in Sporangium chains also in places running parallel and straightConidia, Substrate on CPC: substratespores and the like spores, mostly spheri- cal (.+-.), up to approx. 2 .mu. .phi. individual or several together, ter- minal or intercalarMelanine PEH - CPC - CPC - CPC + Ty - Ty - Ty + Gel -Nitrate reduction +Gelatine +liquefactionMilk peptonization + - -Growth at 20.degree.C. ++ 26.degree.C. ++ ++ 32.degree.C. ++ ++ 37.degree.C. + ++ 42.degree.C. - -NaCl 2% +(+)toleration 3% + 4% - 7% -Starch hydrolysis + +__________________________________________________________________________ Ampullariella regularis Ampullariella regularis Ampullariella regularis SB 2 SB 5 SE 21__________________________________________________________________________Date of isolation July 2, 1966 July 3, 1966 December 17, 1969Method pollen pollen pollenOrigin Neuhof, Fulda district, Neuhof, Fulda district, Kenya, near Ruiru soil under rotted straw, soil under rotted straw, coffee plantation, edge of field, pH 5.7 edge of field, pH 5.7 pH 5.3Mycelium 0.3-1 .mu., broad 0.3-1 .mu. 0.3-1 .mu.Shape of Sporangia bottle-shaped, bottle-shaped, bottle-shaped, cylindrical, "money- cylindrical, "money- cylindrical, "money- pouch-shaped" pouch-shaped" pouch-shaped"Size of Sporangia 4-10 .times. 3.5-7 .times. 3.5-9 .times. 7-18 .mu. 6-14 .mu. 6-13 .mu.Shape of Spores small rods small rods small rodsSize of Spores 0.5-0.7 .times. 0.5-0.7 .times. 0.5-0.7 .times. 1.5-2.2 .mu. 1.5-2.2 .mu. 1.5-2.2 .mu.Flagellation lophotrichous lophotrichous lophotrichousArrangement of linear parallel chains linear parallel chains linear parallel chainsSpores in SporangiumConidia, Substratespores and the likeMelanine CPC - CPC - CPC + Ty - Ty - Ty + Gel - Gel -Nitrate reduction + +Gelatine liquefaction + +Milk peptonization + + +Starch-hydrolysis + + +Growth at 20.degree.C. ++ ++ ++ 26.degree.C. ++ ++ ++ 32.degree.C. ++ ++ ++ 37.degree.C. - - ++ 42.degree.C. - - -__________________________________________________________________________ Ampullariella regularis Actinoplanes spec. Actinoplanes spec. SE 39 SB 11 SB 12__________________________________________________________________________Date of isolation December 22, 1966 November 9, 1966 November 9, 1966Method pollen pollen pollenOrigin Kenya, near Ruiru Eschwege district, Arfurt, Oberlahn district, coffee plantation, near Frankershausen soil from sunny position pH 5.6 "Hielocher", pH 7.7 on rock, pH 7.3Mycelium 0.3-1 .mu. 0.3-1.3 .mu., septate 0.3-1.2 .mu.Shape of Sporangia bottle-shaped .+-. spherical with cylindrical, "money- wrinkled surface, in pouch-shaped" part irregularSize of Sporangia 3-10 .times. 5-16 .mu. 3.5-12 .mu.Shape of Spores small rods .+-. sphericalSize of Spores 0.5-0.7 .times. approx. 1.5-2.2 .mu. 1-1.3 .mu.Flagellation lophotrichous mobile sporesArrangement of linear parallel chains coiled-up chainsSpores in SporangiumConidia, Substrate on CPC broad hyphenspores and the like (approx. 1.3 .mu.), septate at intervals of approx. 2.5-4 .mu. optically dense, permanent cells?Melanine CPC + CPC - CPC - Ty + + Ty - + Gel + Gel +Nitrate reduction + -Gelatine liquefaction + +Milk peptonization - + +Starch-hydrolysisGrowth at 20.degree.C. ++ ++ ++ 26.degree.C. ++ ++ ++ 32.degree.C. ++ ++ ++ 37.degree.C. ++ + ++ 42.degree.C. - - -__________________________________________________________________________ Actinoplanes Actinoplanes Actinoplanes Actinoplanes spec. spec. spec. spec. SB 18 SB 27 SB 46 SE 50__________________________________________________________________________Date of isolation December 4, 1966 December 8, 1966 March 3, 1967 December 22, 1969Method pollen pollen pollen pollenOrigin Hattenheim, Kuhkoph, Nature Neuhof, Fulda Kenya, near Ruiru Rheingau, potato Reserve, Altrhein, district, turf coffee plantation, field in the riverside wood, soil, edge of pH 6.2 "Sandaue", pH 8.0 under willows, path, pH 6.1 pH 7.6Mycleium 0.3-1.3 .mu. 0.2-1.1 .mu. 0.3-1.4 .mu. 0.4-1.3 .mu. septate septateShape of Sporangia irregular, .+-. club-shaped or irregular, irregular, humped, wrinkled, humped oval to cylindri- folded, humped wrinkled cal, in part irre- gular, surface wrinkledSize of Sporangia 6-16 .mu. 4-16 .mu. 6-20 .mu. 4-13 .mu.Shape of Spores .+-. spherical, in .+-. spherical .+-. spherical .+-. spherical part with nose- like protuberanceSize of Spores 1-1.7 .mu. 1-1.3.mu. approx. 1.2-2 .mu. approx. 1 .mu.Flagellation bundles of flagellae mobile sporesArrangement of coiled chains slightly coiled, coiled chains, coiled chains,Spores in in part parallel in part parallel parallel in placesSporangium and straight chainsConidia, Substrate on CPC substratespores and the like spores, up to 3 .mu. .phi., singly or intercalar to give several in a rowMelanine CPC - CPC - CPC - CPC + TY - + Ty - - Ty - - Ty + + Gel + Gel - Gel -Nitrate reduction - + - -Gelatine liquefaction + + +Milk peptonization + + + +Starch-hydrolysis + +Growth at 20.degree.C. ++ ++ ++ ++ 26.degree.C. ++ ++ ++ ++ 32.degree.C. ++ ++ ++ ++ 37.degree.C. + + - ++ 42.degree.C. - - - -__________________________________________________________________________ Actinoplanes Actinoplanes Actinoplanes spec. spec. spec. SA 8 SE 82 SE 103__________________________________________________________________________Date of isolation June 26, 1966 February 26, 1971 March 2, 1971Method pollen pollen pollenOrigin Marburg, compost Ceylon, earth, Corsica, near Holy earth, botanic rubber plantation Trinity, from earth garden under cork-oaksMycelium 0.3-1.3 .mu. 0.3-1.3 .mu.Shape of Sporangia irregular irregular irregularSize of Sporangia 7-15 .times. 9-18 .mu. 5-14 .mu. about 5-13 .mu.Shape of spores .+-. spherical .+-. spherical .+-. sphericalSize of spores about 1.5 .mu. about 1-1.4 .mu. about 1 .mu.Flagellation mobile rapidly mobileArrangement of coiled chains coiled chainsSpores inSporangiumMelanine Ty + Gel 30 + CPC -Nitrate reduction + (very low)Gelatine liquefaction +Milk peptonization +__________________________________________________________________________ Streptosporangium Streptosporangium Streptosporangium roseum amethystogenes roseum SS 55 SS 59 SS 62__________________________________________________________________________Isolation date November 10, 1968 November 10, 1968 November 10, 1968Method soil smear, plate soil smear, plate soil smear, plateOrigin Kenya, near Ruiru, Kenya, near Njoro, Kenya, near Njoro, earth from coffee from field from field fieldMycelium 0.4-1.2 .mu. 0.4-1.2 .mu.Shape of sporangia spherical spherical sphericalSize of sporangia 3-10 .mu. 3-15 .mu. 3-10 .mu.Shape of spores mostly oval mostly ovalSize of spores about 0.8 .times. 1-1.2 .mu. about 0.8-1.1 .times. 1-1.5 .mu.Melanine - - -Nitrate reduction ++ - + +Gelatine liquefaction + + +Milk peptonization + + +Starch hydrolysis + + +Growth at 37.degree.C. + + + 42.degree.C. - - -NaCl 2% +toleration 3% + - + 4% - -__________________________________________________________________________ Ampullariella Ampullariella Ampullariella digitata regularis spec. SA 28 SE 45 SE 89__________________________________________________________________________Isolation date November 10, 1967 December 23, 1969 February 27, 1971Method fungus mycelium pollen pollenOrigin Amonau Krs. Marburg, Kenya, near Ruiru, Ceylon, earth earth from stubble- earth from coffee field fieldMycelium 0.3-1 .mu.Shape of sporangia very narrow and rela- bottle-shaped, .+-. cylindrical, fre- tively long, distally cylindrical, often quently also broader and in part also longitudinally narrow than long, often not laterally irregular, flat at the distal end, in part fingered, tubercular, in part coil-like or finger- also irregular and folded, shaped a few fingeredSize of sporangia 2-7 .times. 4-13 .mu. 4-10 .times. 6-17 .mu. 5-12 .times. 6-16 .mu.Shape of sporangia small rod-shaped small rods small rod-shapedArrangement of arranged in parallel in parallelSpores in sporangia rows straight rowsMelanine Ty - Ty + Gel - + CPC +Nitrate reduction +Gelatine liquefaction +Milk peptonization + +__________________________________________________________________________ Planomonospora Planomonospora Pilimelia spec. spec. parontospora SK 2 SE 100 Se 101__________________________________________________________________________Isolation date March 1, 1971 February 27, 1971 January 28, 1968Method pollen pollen pollenOrigin Corsica, northern Ceylon, lawn earth Marburg, garden earth, Algeria, earth, Schulerpark tree nurseryMycelium 0.3-0.7 .mu.Shape of sporangia longitudinally narrow, spherical, pear-shaped, stand compactly by each oval, with a columella, other in parallel double which projects, as a rows in the air mycelium, continuation of the arcuately bent air-hyphens sporangiophor, up to on the convex side di- about 1/3 of the rectly joined to their sporangia -.phi. or further base into the sporangiumSize of sporangia about 1-1.3 .times. about 7-15 .mu. 3-4.5 .mu.Shape of spores small rods often slightly warpedSize of spores 0.35-0.45 .times. 0.8-1.5 .mu.Flagellation mobile flagellar fascidesArrangement of lateral (and also sub-spores in polar), spore arrange-sporangium ment in chains, which grow tuft-like from the columellaMelanine Ty +Nitrate reduction +Gelatine liquefaction +Milk peptonization +Growth at 32.degree.C. + 37.degree.C. -__________________________________________________________________________
Table 3__________________________________________________________________________ G = growth SM = substrate mycelium SP = soluble pigmentSpg = sporangium C = colony shape AM = aerial mycelium SB 2 SB 5 SB__________________________________________________________________________ 11 1) G. very good G. good G. good 2) SM. orange SM. yellow-orange SM. beautiful orange,Casamino-peptone- bleaching 3) SP. agar yellowish SP. agar yellowish SP. agar yellowishCzapek agar 4) Spg. +++; rimy Spg. -; originally + Spg. -(CPC) 5) C. flat and smooth C. heavily veined C. with coiled ridges G. very good G. good to very good G. goodPeptone- SM. orange, rimy SM. orange-red SM. orange, bleaching SP. - SP. agar yellowish SP. agar golden yellowCzapek agar Spg. ++ Spg. - Spg. - C. flat, in part(PC) humped G. moderate G. moderate G. goodCzapek agar SM. orange SM. pale orange SM. brownish orange(Cz) SP. agar pale ochre SP. agar slightly SP. - yellowish Spg. ++ Spg. - Spg. - G. good G. very good G. very goodMilk agar SM. brownish orange SM. brownish orange SM. brownish orange, turns pale brown(Ca) SP. agar brownish SP. agar golden brown SP. agar yellow brown Spg. -; casein Spg. -; casein Spg. -; casein peptonized peptonized peptonized G. moderate-good G. slight G. bulky SM. reddish brown SM. brown SM. pale orangeTyrosine agar SP. agar reddish SP. agar brown SP. - brown, slight solution of Tyrosine crystals(Ty) solution of crystals - not dissolved crystals Spg. +; rimy Spg. - G. good G. good G. goodOat-Yeast agar SM. brownish orange SM. brownish orange SM. ochre-orange SP. - SP. - SP. -(OY) Spg. +++ Spg. + Spg. - G. bulky SM. colorless to pale ochreStarch agar SP. - Spg. -__________________________________________________________________________ SB 12 SB 18 SB 27__________________________________________________________________________ G. very good G. very good G. good to very goodCasamino-Peptone- SM. orange-brown SM. strong red-brown SM. luminous orange, later brown orangeCzapek agar SP. agar yellowish- SP. agar brown SP. agar yellow-brown brownish(CPC) Spg. + Spg. +(+) Spg. + C. flat with radial C. flat, radial C. flat, bulging in grooves, bulging grooves and the middle, locally in the middle wrinkles split open G. very good G. very good G. good to very goodPeptone-Czapek agar SM. brown red SM. brown SM. ochre orange SP. agar golden brown SP. brown SP. agar yellowish(PC) Spg. - Spg. ++ Spg. - G. good G. good G. good SM. orange to SM. red brown SM. red brown brownish orangeCzapek agar SP. agar flouresces SP. agar reddish SP. agar slightly greenish to brown ochre(Cz) yellow Spg. - Spg. ++; rimy Spg. - G. very good G. very good G. very goodMilk agar SM. brown orange SM. brown orange SM. brownish orange SP. agar golden brown SP. agar golden brown SP. agar golden brown(Ca) Spg. -; Spg. ++, Spg. +, casein peptonized not always; not always casein peptonized casein peptonized G. moderate G. moderate - good G. moderate - goodTyrosine agar SM. brown SM. reddish orange brown SM. ochre brown SP. agar brown; SP. agar orange brown; SP. -(Ty) crystal solution crystal solution ++ crystal solution ++ ++ Spg. - Spg. - Spg. - G. good G. good G. goodOat-Yeast agar SM. pale orange SM. red brown SM. orange brown SP. - SP. red brown SP. -(OY) Spg. + Spg. ++ Spg. - G. bulky to good G. bulky to good G. bulky to goodStarch-agar SM. pale brownish orange SM. pale brownish orange SM. pale brownish orange SP. - SP. - SP. - Spg. + Spg. ++ Spg. ++__________________________________________________________________________ SB 46 SE 21 SE 39 SE__________________________________________________________________________ 50 G. good G. good G. good G. very goodCasamino- SM. orange, later SM. orange brown SM. red brown SM. brown brownish orangepeptone- SP. agar pale SP. agar yellow SP. agar golden SP. agar brown to yellowish brown brown - red yellowishCzapek agar brown brown(CPC) Spg. + Spg. +; some Spg. ++; rimy Spg. - C. flat, with radial grooves G. good G. very good G. very good G. very goodPeptone- SM. orange SM. dark brown SM. reddish dark SM. brown brownCzapek agar SP. agar golden SP. brown SP. dark brown(PC) yellow Spg. - Spg. + Spg. ++ frost-like on the SM G. good G. good G. good G. very good SM. orange brown SM. orange SM. red-brown SM. red-brown SP. agar ochre- SP. - to pale SP. red SP. gold brown colored yellowish Spg. - Spg. +++ frost-like Spg. + on the SM G. very good G. good G. good G. goodMilk agar SM. orange SM. brown-orange SM. brown-orange SM. orange brown SP. agar golden- SP. brown SP. dark brown SP. dark brown(Ca) colored Spg. -; casein Spg. - casein Spg. - casein not Spg. - casein peptonized peptonized peptonized peptonized G. good G. bulky G. bulky G. bulkyTyrosine agar SM. brown SM. dark brown SM. dark brown SM. dark, like the agar(Ty) SP. agar golden SP. dark brown SP. dark brown SP. black-brown brown; crystal solution ++ Spg. - Spg. - Spg. - Spg. - tyrosine crystals tyrosine crystals tyrosine crystals not dissolved not dissolved not dissolved G. goodOat-Yeast agar SM. orange brown SP. -(OY) Spg. - Spg. ++ Spg. +"Emerson" G. goodagar E SM. brown(yeast-starch SP. brownagar) Spg. ++ Spg. - G. bulky to good G. bulky to good G. bulky to goodStarch agar SM. pale brownish SM. pale brownish SM. pale brownish orange orange SP. - SP. - starch hydrolysis - Spg. + Spg. + Spg. +__________________________________________________________________________ SE 5 SE 55 SE 103 G. good G. good G. good SM. orange SM. dark red brown SM. orangeCasamino- AM. - AM. -peptone- SP. - to pale yellowish SP. yellow brown SP. yellowish-brownishCzapek agar Spg. -(CPC) surface of colony in inclined test-tube shows slimy gloss G. good G. good G. very goodOat-Yeast SM. orange SM. orange brown SM. brownagar AM. -(OY) SP. light brown SP. - Spg. ++, frost-like G. good G. very good SM. orange SM. orange AM. - SP. - SP. - surface of colony Spg. ++, frost-like slimy G. good G. goodMilk agar SM. orange SM. orange brown(Ca) SP. - to yellowish SP. brown Casein not peptonized, Casein not peptonized, except merely negli- after about 2 months gibly immediately slightly peptonized around the mycelium G. good G. good G. very goodCzapek agar SM. orange, surface SM. orange SM. yellow-orange(Cz) damp and shiny SP. - SP. - SP. - Spg. - G. small to bulky G. bulkyTyrosine agar SM. colorless to pale SM. dark brown(Ty) orange SP. - SP. black brown tyrosine crystals tyrosine crystals not dissolved not dissolvedPeptone-Czapek G. very goodAgar SM. dark reddish brown(PC) SP. dark brown G. bulky to goodStarch agar SM. pale orange brown SP. - Spg. +__________________________________________________________________________ SS 45 SS 51 SS 53__________________________________________________________________________ G. moderate (to good) G. moderate (to good) G. goodCasamino- SM. reddish brown SM. reddish brown SM. pale light brownpeptone- AM. - AM. - AM. whiteCzapek agar SP. - SP. - SP. brownish(CPC) colony flat, 6 mm .phi. colony flat, 6 mm .phi. colony flat, center after 9 weeks after 9 weeks without AM G. good G. good G. good to very goodOat-Yeast SM. brown red SM. brown red SM. purple brown toagar red brown(OY) AM. +, white and pink AM. +, white and pink AM. ++, dirty white and pink SP. pale yellowish to SP. pale yellowish with SP. purple-red brown yellowish-greenish greenish tinge Spg. + Spg. + Spg. ++ G. good G. good G. good"Emerson" SM. red SM. brown red SM. red brownagar AM. - AM. +, slight, white AM. ++, pink(E) SP. pale yellowish SP. - to pale yellowish SP. red brown with greenish tinge brownish Spg. ++ G. good G. good G. goodMilk agar SM. pale brownish red SM. pale brownish red SM. pale light brown(Ca) AM. - AM. - AM. - SP. agar gold-yellow SP. agar gold-yellow SP. agar yellow brown Casein peptonized Casein peptonized Casein peptonized G. good G. good G. goodYeast-glucose-soil SM. brownish red SM. brown red SM. brownish claretextract agar AM. + thin coating AM. +, white AM. ++, pink(YGS) SP. golden yellow with SP. yellow with greenish SP. reddish yellow greenish tinge tinge brownManure extract G. moderate (to good) G. moderate (to good)agar AM. ++, white and pink AM. ++, pink(Ma) Spg. + Spg. +__________________________________________________________________________ SA 8 SE 82 SS 26 G. very good G. good G. goodCasamino- SM. orange SM. brown SM. pale ochrepeptone AM. few, whiteCzapek SP. yellowish-brownish SP. brown SP. -agar Spg. + Spg. -(CPC) colony flat with a few radial grooves and con- centric humpsPeptone G. very goodCzapek agar SM. orange(PC) SP. yellowish-brownish Spg. + G. good G. very good G. good to bulkyOat-Yeast SM. brown SM. orange to brownish SM. colorlessagar orange(OY) AM. few, white SP. - SP. weak yellowish- SP. - brownish Spg. + Spg. + G. very good G. bulky"Emerson" SM. orange-brown SM. colorlessagar LM. +, white(E) SP. brown SP. - G. very good G. goodMilk agar SM. orange brown SM. colorless(Ca) SP. agar gold-brown LM. - Spg. + SP. - Casein peptonized Casein peptonizedCzapek G. good G. goodagar SM. brown-orange SM. red-orange(Cz) SP. yellow brown SP. yellowish Spg. -Tyrosine G. bulkyagar SM. black(Ty) SP. black tyrosine crystals dissolved Spg. -Yeast-glucose- G. good G. goodsoil extract SM. brown SM. colorlessagar SP. gold-brown LM +, white(YGS) Spg. + SP. -Manure extract G. bulkyagar SM. pale brown(Ma) LM. +, white Spg. +__________________________________________________________________________ SS 55 SS 59 SS 62__________________________________________________________________________Casamino- G. good, colony flat G. good, colony flatpeptone- SM. light brown SM. pale light brownCzapek AM. white AM. whiteagar (CPC) SP. weak pale brown SP. - G. good G. good G. good SM. pale light brown SM. dark brown SM. pale light brownMilk AM. - AM. - AM. -agar SP. - SP. yellow brown SP. agar yellow-brown Casein peptonized violet crystals Casein peptonized Casein peptonized G. good G. good G. good SM. brown SM. dark brown SM. brownTyrosine AM. white AM. - AM. few, whiteagar SP. - SP. brown SP. brown(Ty) tyrosine crystals tyrosine crystals tyrosine crystals dissolved dissolved dissolved G. good to very good G. good G. good to very goodOat-Yeast SM. red-brown SM. dark brown SM. red brownagar AM. dirty pink AM. few, white AM. dirty pink(OY) SP. red brown SP. brownish SP. red brown many violet crystals Spg. - G. good G. good G. good"Emerson" SM. red brown SM. dark brown SM. red brownagar AM. pink and white AM. - AM. few, thin coating,(E) in part stronger and pink SP. red brown SP. yellow brown SP. red brown violet crystals G. good G. good G. goodYeast-glucose SM. red brown SM. dark brown SM. dark redsoil extract AM. pink AM. - AM. pinkagar SP. yellow-brown SP. yellow brown SP. yellow brown(YGS) violet crystals__________________________________________________________________________ SA 28 SE 45 SE 89 G. good to very good G. good G. goodCasamino SM. initially red-brown, SM. red-brown SM. brownish orangepeptone later dark brownCzapek agar to black(CPC) SP. dark olive SP. strongly red-brown SP. - Spg. - Spg. + Spg. + G. good to very goodPeptone SM. initially red-brown,Czapek later dark brownagar to black(PC) SP. dark olive Spg. - G. bulky to good, colonies G. good G. goodCzapek agar flat(Cz) SM. orange brown SM. brown-red SM. dark-brown SP. weak ochre SP. brownish-red SP. light yellowish brown Spg. +, in part frost-like Spg. ++, frost-like Spg. ++, frost-like G. very good G. goodMilk agar SM. black brown SM. orange brown(Ca) SP. dark olive to SP. brown greenish-black Spg. - Spg. + Casein peptonized Casein peptonized G. bulky to good G. bulkyTyrosine SM. brown SM. brownagar SP. - SP. dark brown(Ty) Spg. - Spg. - tyrosine crystals tyrosine crystals dissolved under not dissolved mycelium G. good G. good G. good to very goodOat-yeast SM. dark brown Spg. ++, frost-like SM. orangeagar on the SM.(OY) SP. light brownish SP. - Spg. + Spg. ++, frost-like G. good to very good G. very good"Emerson" SM. brown to brown-black SM. orangeagar SP. ochre with greenish tint SP. -(E) Spg. + Spg. ++, frost-like__________________________________________________________________________ SE 100 SE 101 SK 2Casamino- G. good G. goodpeptone SM. pale ochre SM. redCzapek AM. ++, white AM. few, whiteagar (CPC) SP. - SP. - G. good G. bulkyCzapek SM. colorless SM. colorless to pinkagar AM. ++, white AM. +, white(Cz) SP. - G. good G. good"Emerson" SM. colorless to SM. pale red to red,agar pale ochre later paling(E) AM. ++, white AM. - SP. - SP. - G. good G. good G. bulkyOat-yeast SM. pale ochre SM. pink SM. brownagar AM. ++, white AM. ++, dirty pink(OY) SP. - SP. yellowish-brownish SP. - Spg. ++ G. good (relatively)Milk agar SM. golden yellow(Ca surface knobbyCa/2) SP. - Spg. - Casein peptonized G. goodTyrosine SM. brownish gold-yellow toagar yellow-orange(Ty) SP. golden brown to reddish brown Spg. - tyrosine crystals dissolved__________________________________________________________________________
Table 4__________________________________________________________________________Growth in Various Carbon Sources__________________________________________________________________________ SB2 SB5 SB11 SB12 SB18 SB27 SB46 SE21 SE39 SE50 SE55__________________________________________________________________________Glucose ++ ++ ++ ++ ++ ++ ++ ++ ++ ++Fructose ++ ++ ++ ++ ++ ++ ++ ++ ++ ++Saccharose ++ ++ ++ ++ ++ ++ ++ ++ ++ ++Mannitol ++ ++ ++ ++ ++ ++ ++ - ++ ++L-Arabinase ++ ++ ++ ++ ++ ++ ++ ++ ++ ++Raffinose - - ++ - - - - - + +Inositol - - ++ - ++ ++ ++ - - -D-Xylose ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++__________________________________________________________________________ ++ Growth as good as on glucose or better + Growth worse than on glucose, but better than on sugar-free mineral salt-agar - Growth as on mineral salt-agar without carbon source
The strains set forth in Table 1 were either drawn from established culture collections such as the American Type Culture Collection and the Centraalbureau voor Schimmelcultures, or deposited under the CBS-numbers set forth above in the Centraalbureau voor Schimmelcultures in Baarn, Holland.
Tables 2 and 3 list some characteristics of the strains described.
To obtain the glycoside-hydrolase inhibitors, the strains listed above are cultured in the nutrient solutions described above. In doing so it should be noted that for optimum production practically every strain requires a different nutrient solution of different qualitative and quantitative composition.
After 1 to 10 days' incubation at 15.degree.-60.degree.C., preferably 24.degree.-50.degree.C., in a shaking flask or in fermenters of different size, the mycelium is separated from the culture solution and, depending on the occurrence of the inhibitors, the active principle is concentrated using the culture solution and/or the mycelium.
The inhibitors are obtained from the culture broths by lyophilization or precipitation with salt or water-soluble organic solvents (such as, for example, lower alcohols and ketones) or by adsorption of the active substances on ion exchangers.
The inhibitors are obtained from the mycelia by extraction with organic solvents, such as, for example, alcohols, ketones, ethers, esters and sulphoxides.
For this purpose, the fermentation batch is centrifuged at 3,000-20,000 revolutions per minute, preferably 6,000-10,000 revolutions per minute, for 10-60 minutes, preferably 30 minutes, or is filtered, preferably under pressure and with the help of filter aids, such as, for example, Claricel, and is thus separated into culture broth and mycelium residue.
The inhibitor can be isolated from the particular culture broth in various ways:
a. Concentration of the culture broths under reduced pressure (10-50 mm Hg) at bath temperatures of 20.degree.-100.degree.C., preferably 40.degree.-80.degree.C., to approximately one-fifth to one-fiftieth of the initial volume. The concentrated extract is filtered or centrifuged and the clear filtrate (or the clear supernatant liquid is lyophilized, if required after prior desalination.
b. Precipitation of the inhibitors from the culture broth [or from the culture broths concentrated according to (a)] by adding water-soluble organic solvents, such as, for example, alcohols or ketones, preferably methanol, ethanol, or acetone, up to a content of 60-90%. Since inactive concomitant substances are precipitated at low concentration of solvents, this precipitation process is particularly suitable for fractional precipitation to remove undesired concomitant substances.
c. Salting-out of the inhibitors from the extracts [or from the extracts concentrated according to (a)], for example, with ammonium sulphate, sodium chloride and the like. The precipitate formed is collected by centrifuging or filtering and is either directly washed with acetone and ether and dried in vacuo or redissolved in water, dialyzed and lyophilized.
d. Adsorption of the inhibitors on ion exchangers. This process is suitable for isolating those inhibitors which, because of their chemical nature, carry a charge. The inhibitor is desorbed by changing the ionic strength or the pH value of the elution medium.
In addition to the inhibitor, undesired concomitant substances are frequently present in the culture broths. These concomitant substances can be separated off in various ways, for example, by denaturing the concomitant substances by means of heat in the case of inhibitors which are heat-stable, or by dialysis through appropriate membranes in the case of low molecular inhibitors, in which case the undesired concomitant substances are retained by the membrane, or by fractional precipitation [compare (b)] or by adsorption of the concomitant substances on ion exchangers.
The inhibitors are obtained from the mycelia by repeated extraction of the mycelium with organic solvents, preferably two extractions of 10-20 minutes with 3--5 volumes of acetone (relative to the moist mycelium volume) and subsequent single extraction of 5-10 minutes with ether. The mycelium extracted in this way is dried in vacuo and subsequently extracted for 2-8 hours with 3-10 parts by weight of dimethyl sulphoxide, while stirring, and thereafter centrifuged at 10,000 to 20,000 revolutions per minute. The acetone extracts and ether extracts are concentrated to dryness in vacuo and taken up with the dimethyl sulfoxide (DMSO) extract.
Instead of extracting the dry mycelium powder with dimethyl sulfoxide (DMSO), it can also be extracted over a longer period, preferably 12-24 hours, with water or dilute electrolyte solutions.
The new substances dissolve well in water. One group of the inhibitors is heat-stable at neutral pH values, stable to acid (pH 2), stable to alkali (pH 12), and slowly dialyzable These inhibitors are not inactivated by trypsin and pepsin and, in turn, do not inhibit the enzymes mentioned. They cannot be dyed with the typical protein dyes and do not show a characteristic absorption in the UV up to 250 nm. These inhibitors cannot be inactivated with urea and .beta.-mercaptoethanol. According to estimates from gel filtration, the molecular weight of these inhibitors is above 500 but below 6,000. In hydrolytic splitting, monosaccharides, for example, glucose, are obtained. According to these findings, these inhibitors are oliogosaccharides or polysaccharides or their derivatives.
The best inhibitors of this group show inhibition activities, against amylase, of 8,000 AIU/mg.
Another group of inhibitors is heat-labile and not dialyzable, or hardly dialyzable. These inhibitors are inactivated more or less rapidly by trypsin. Urea and .alpha.-mercaptoethanol also inactivate most of these inhibitors. These inhibitors are probably substances of peptide character.
The best inhibitors of this group show inhibiting activities, against amylase, of 80 AIU/mg.
It is known that in the case of animals and man hyperglycemias arise after the intake of foodstuffs and beverages containing carbohydrates (for example, corn starch, potato starch, fruit, fruit juice or chocolate), these hyperglycemias resulting from a rapid degradation of the carbohydrates by glycoside-hydrolases (for example, salivary and pancreatic amylases, maltases and saccharases) in accordance with the following equation:
starch .sup.amylase .fwdarw. maltose .sup.maltase .fwdarw. glucose or glycogen
sucrose .sup.saccharase .fwdarw. glucose + fructose
These hyperglycaemias are of particularly strong and lasting character in the case of diabetics. In adipose cases, alimentary hyperglycemia frequently causes a particularly strong secretion of insulin, which, in turn, leads to increased synthesis of fat and reduced degradation of fat. In connection with such hyperglycemias, a hypoglycemia frequently occurs in metabolically healthy and adipose persons as a result of the insulin secretion. It is known that not only hypoglycemias but also chyne remaining in the stomach stimulate the production of gastric juice, which for its part participates or encourages the formation of a gastritis, or a gastric or duodenal ulcer.
It has now been found that inhibitors of glycosiden-hydrolases, according to the invention, obtained and isolated in accordance with the above methods, considerably reduces alimentary hyperglycemia, hyperinsulinemia and hypoglycemia after dosing rats and/or man with wheat starch or sucrose or maltose, and speed up the passage of these carbohydrates through the stomach.
Furthermore, it is known that carbohydrates, especially sucrose, are split by microorganisms in the mouth cavity and that caries formation is promoted thereby.
Inhibitors of glycoside-hydrolases are, therefore, suitable for use as therapeutic agents for the following indications: obesity, adiposit, hyperlipoidemia (arteriosclerosis) diabetes, pre-diabetes, gastritis, gastric ulcer, duodenal ulcer, and caries.
The present invention, therefore, provides a pharmaceutical composition containing as active ingredient an inhibitor of the invention in admixture with a liquid diluent other than a solvent of a molecular weight less than 200 (preferably less than 350) except in the presence of a surface active agent.
The invention further provides a pharmaceutical composition containing as active ingredient an inhibitor of the invention in the form of a sterile or isotonic aqueous solution.
The invention also provides a medicament in dosage unit form comprising an inhibitor of the invention either alone or in a admixture with a diluent.
The invention also provides a medicament in the form of tablets, (including lozenges and granules), dragees, capsules, pills, ampoules and suppositories comprising an inhibitor of the invention either alone or in admixture with a diluent.
"Medicament" as used in this specification means physically discrete coherent portions suitable for medical administration. "Medicament in dosage unit form" as used in this specification, means physically discrete coherent portions suitable for medical administration each containing a unit dose or a multiple (up to 4 times) or submultiple [down to a fortieth] of a unit dose of the inhibitor of the invention. Whether the medicament contains a unit dose or, for example, a half, a third, or a quarter of a unit dose will depend on whether the medicament is to be administered once, for example, twice, 3 times or 4 times a day, respectively.
A unit dose is the amount of inhibitor to be taken on one occasion.
The pharmaceutical compositions according to the invention may, for example, take the form of gels, pastes (e.g., toothpastes), creams, chewing-gums, suspensions, solutions and emulsions of the active ingredient in aqueous or non-aqueous diluents, syrups, granules or powders.
The diluents to be used in pharmaceutical composition (e.g., granulates) adapted to be formed into tablets, dragees, capsules and pills include the following: (a) fillers and extenders, e.g., starch, sugars, mannitol, and silicic acid; (b) binding agents, e.g., carboxymethyl cellulose and other cellulose derivatives, alginates, gelatine, and polyvinyl pyrrolidone; (c) moisturizing agents, e.g., glycerol; (d) disintegrating agents, e.g., agar-agar, calcium carbonate, and sodium bicarbonate, (e) agents for retarding dissolution, e.g., paraffin; (f) resorption accelerators, e.g., quaternary ammonium compounds; (g) surface active agents, e.g., cetyl alcohol, glycerol monostearate; (h) adsorptive carriers, e.g., kaolin and bentonite; (i) lubricants, e.g., talc, calcium, and magnesium stearate, and solid polyethylene glycols; (j) elastomeric binders such as chicle.
The tablets, dragees, capsules and pills formed from the pharmaceutical compositions of the invention can have the customary coatings, envelopes and protective matrices, which may contain opacifiers. They can be so constituted that they release the active ingredient only or preferably in a particular part of the intestinal tract, possibly over a period of time. The coatings, envelopes and protective matrices may be made, for example, of polymeric substances or waxes.
The ingredient can also be made up in microencapsulated form together with one or several of the above-mentioned diluents.
The diluents to be used in pharmaceutical compositions adapted to be formed into suppositories can, for example, be the usual water-soluble or water-insoluble diluents, such as polyethylene glycols and fats [e.g., cocoa oil and high esters (e.g., C.sub.14 -alcohol and C.sub.16 -fatty acid)] or mixtures of these diluents.
The pharmaceutical compositions which are pastes, creams, and gels can, for example, contain the usual diluents, e.g., animal and vegetable fats, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide or mixtures of these substances.
The pharmaceutical compositions which are powders can, for example, contain the usual diluents, e.g., lactose, talc, silicic acid, aluminum hydroxide, calcium silicate, and polyamide power or mixtures of these substances.
The pharmaceutical compositions which are solutions and emulsions can, for example, contain the customary diluents (with, of course, the above-mentioned exclusion of solvents having a molecular weight below 200 except in the presence of a surface-active agent), such as solvents, dissolving agents and emulsifiers; specific examples of such diluents are water, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (for example, ground nut oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitol or mixtures thereof.
For parenteral administration, the solutions and emulsions should be sterile, and, if appropriate, blood-isotonic.
The pharmaceutical compositions which are suspensions can contain the usual diluents, such as liquid diluents, e.g., water, ethyl alcohol, propylene glycol, surface-active agents (e.g., ethoxylated isostearyl alcohols, polyoxyethylene sorbite and sorbitane esters), microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth or mixtures thereof.
All the pharmaceutical compositions according to the invention can also contain coloring agents and preservatives as well as perfumes and flavoring additions (e.g., peppermint oil and eucalyptus oil) and sweetening agents (e.g., saccharin). In particular, chewing gums and toothpastes will contain flavoring agents.
The pharmaceutical compositions according to the invention preferably contain about 0.1 to 99.5, more preferably from about 0.5 to 95% of the inhibitor by weight of the total composition.
In addition to an inhibitor of the invention, the pharmaceutical compositions according to the invention can also contain other pharmaceutically active compounds. They may also contain a plurality of different inhibitors of the invention. Particular examples of such other pharmaceutically active compounds are oral antidiabetic agents such as .beta.-cytotropic sulphonyl-urea derivatives and biguanides which influence the blood sugar level.
The diluent in the medicament of the present invention may be any of these mentioned above in relation to the pharmaceutical compositions of the present invention. Such medicament may include solvents of molecular weight less than 200 as sole diluent.
The discrete coherent portions constituting the medicament according to the invention (whether in dosage unit form or net) may be, for example, any of the following: tablets (including lozenges and granules), pills, dragees, capsules, suppositories, and ampoules. Some of these forms may be made up for delayed release of the inhibitor. Some, such as capsules, include a protective envelope which renders the portions of the medicament physically discrete and coherent.
The preferred unit dose for the medicaments of the invention is 5,000-500,000 AIU, 2.5-250 MIU, or 100-10,000 SIU of inhibitor. A unit dose will be taken orally once or several times daily, usually immediately before, during, or after a meal.
It is envisaged that the inhibitor will be administered perorally. Preferred medicaments are, therefore, those adapted for peroral administration, such as tablets, dragees, and portions of chewing gum.
The toxicity of some of these inhibitors of glycoside-hydrolases is extremely low. The active substance from Examples 14 and 37 was tolerated, without symptoms, at a dosage of 5 .times. 10.sup.6 AIU/kg of mouse or rat, administered orally. In the case of intravenous injection, mice and rats tolerated 10.sup.6 AIU/kg.

The following examples illustrate the production of the inhibitor according to the invention.
EXAMPLE 1
Three 1-liter Erlenmeyer flasks, containing 200 ml of a nutrient solution of composition: 2% starch, 1% glucose, 0.5% NZ-amines, 1.0% yeast extract and 0.4% CaCO.sub.3 (sterilization: 30 minutes, 121.degree.C.; pH adjusted to 7.2 before sterilization) are each inoculated with 1 ml of a primary culture (obtained in the same nutrient solution, inoculated from sloping test-tube cultures with oatmeal agar) of the strain SB 2, and the flasks are incubated at 28.degree.C. on a rotary shaking machine. After a period of culture of 5.5 days, the contents of the three flasks are combined and the mycelium is separated off by centrifuging. 425 ml of supernatant liquor, containing 100 AIU/ml, are obtained.
The centrifuged supernatant liquor is concentrated to 60 ml on a rotary evaporator at 15-20 mm Hg and approximately 37.degree.C. waterbath temperature. The viscous solution is stirred into 8 volumes = 480 ml of ethanol, while stirring. The precipitate formed is collected by centrifuging, again dissolved in 60 ml of water, and dialyzed for 6 hours against distilled water. The dialyzate in lyophilized. Yield: 1.6 g containing 19 .times. 10.sup.3 AIU/g.
EXAMPLE 2
Using a batch according to Example 1, 440 ml of centrifuged supernatant liquor containing 100 AIU/ml are obtained.
These 440 ml of centrifuged supernatant liquor are concentrated to 100 ml on a rotary evaporator. The concentrated solution is stirred into 8 volumes = 800 ml of ethanol and the precipitate is collected by centrifuging, twice washed with acetone and once with ether and dried in vacuo at room temperature. Yield: 2.2 g containing 15 .times. 10.sup.3 AIU/g.
EXAMPLE 3
Using a batch according to Example 1, 500 ml of a centrifuged supernatant liquor containing 120 AIU/ml are obtained after 5 days' culture. These 500 ml are lyophilized directly. Yield: 7.1 g containing 8.3 .times. 10.sup.3 AIU/g.
EXAMPLE 4
Three 1-liter Erlenmeyer flasks containing 200 ml of a nutrient solution of composition: 3% glycerine, 3% soya flour, and 0.2% CaCO.sub.3 (sterilization: 30 minutes, 121.degree.C.; pH after sterilization 7.2) are each inoculated with 1 ml of a primary culture (obtained in the same nutrient solution, inoculated from sloping test-tube cultures containing Czapek-peptone-casein agar) of the strain SB 12, and the flasks are incubated for 3 days at 28.degree.C. on a rotary shaking machine. After the incubation, the contents of the flasks are combined and the mycelium is separated off by centrifuging. 500 ml of supernatant liquor containing 450 AIU/ml are obtained.
The supernatant liquor is treated with 250 g of ammonium sulphate added in portions while stirring and the mixture is subsequently centrifuged for 10 minutes at 12,000 revolutions per minute. The precipitate is dissolved in 100 ml of distilled H.sub.2 O and 4 volumes (=400 ml) of acetone are added while stirring. A precipitate which sediments well is formed. The liquid is decanted off and the precipitate is washed twice with acetone and once with ether and dried in vacuo. Yield: 13.4 g containing 10 .times. 10.sup.3 AIU/g.
EXAMPLE 5
A precipitate obtained according to Example 4 after precipitation with ammonium sulphate is dissolved in 100 ml of water and dialyzed for 6 hours against distilled water. A dialyzate is obtained, which is frozen and lyophilized. Yield: 1.5 g containing 80 AIU/mg.
EXAMPLE 6
If a 1 liter Erlenmeyer flask is inoculated with 120 ml of a nutrient solution according to Example 1, from a sloping test-tube culture of the strain St. 19, a culture solution containing 25 AIU/ml is obtained after 3 days' culture at 28.degree.C. on a rotary shaking machine.
EXAMPLE 7
If a mix according to Example 4 is incubated for 3 days at 32.degree.C., a culture filtrate containing 350 AIU/ml is obtained after filtering off the mycelium.
EXAMPLE 8
If a nutrient solution according to Example 1 is inoculated in accordance with Example 4, culture filtrates containing 270 AIU/ml are obtained after 3 days' incubation at 28.degree.C.
EXAMPLE 9
Six 1-liter Erlenmeyer flasks, each containing 100 ml of a nutrient solution consisting of 3% glucose, 3% soya flour, and 0.2% CaCO.sub.3 (sterilization: 30 minutes, 121.degree.C.; pH after sterilization 7.2) are each inoculated with 1 ml of primary culture (obtained as in Example 1) of the strain SB 5, and are incubated for four days at 28.degree.C. on rotary shaking machines. The contents of the flasks are combined and the mycelium is separated off by centrifuging. The resulting 500 ml of supernatant liquor containing 150 AIU//ml are frozen and lyophilized. Yield: 6.9 g containing 11 .times. 10.sup.3 AIU/g.
EXAMPLE 10
If 24 flasks of a mix according to Example 4, each containing 120 ml, are inoculated with a primary culture of the strain SB 11 (obtained according to Example 4) and incubated for 5 days at 28.degree.C., 2.0 liters of a supernatant liquor containing 1.1 MIU/ml are obtained after centrifuging. These 2 liters are concentrated to 200 ml on a rotary evaporator. The 200 ml of concentrate are dialyzed for 24 hours in a Visking dialysis tube (type 27/100 FT, Union Carbide Corporation) against 2 liters of distilled water, at room temperature. The outer medium, containing inhibitor, is concentrated to 100 ml on a rotary evaporator and is added dropwise to 900 ml of absolute ethanol, while stirring. The almost inactive precipitate which separates is centrifuged off and discarded, and the alcoholic supernatant liquor is concentrated to 30 ml on a rotary evaporator.
1 ml of this concentrate contains 70 MIU/ml.
For further purification, this solution is passed over an anion exchage column (Amberlite IRA 410, acetate form, in 0.05 M NH.sub.4 -acetate, pH 7, 2.5 .times. 20 cm column) and the active fractions are combined and gel-filtered on Sephadex .sup.R G 75 in H.sub.2 O. The active fractions from the gel filtration are concentrated to 12 ml. 1 ml of this solution contains 150 MIU/ml.
The mycelium (.about.500 ml) was twice extracted with 1 liter of acetone and once with 1 liter of ether and the extracts were combined and evaporated to dryness on a rotary evaporator in vacuo. The mycelium residue was dried in vacuo at 20.degree.C. and the resulting dry mycelium powder (.about.51 g) was subsequently extracted for 2 hours at room temperature with 150 ml of dimethyl sulfoxide (DMSO). After centrifuging (30 minutes, 15,000 r.p.m.) the acetone/ether extract which has been concentrated to dryness is taken up with the dimethyl sulfoxide (DMSO) supernatant liquor from the dry mycelium powder. Yield: 120 ml containing 3 MIU/ml.
EXAMPLE 11
If a 1 liter Erlenmeyer flask containing 120 ml of a nutrient solution according to Example 1 is inoculated according to Example 9 and incubated for 6 days at 28.degree.C. on a rotary shaking machine, a culture filtrate containing 0.9 MIU/ml is obtained.
EXAMPLE 12
If five 1-liter Erlenmeyer flasks, each containing 120 ml of a nutrient solution of composition: 2.5% starch, 0.5% glucose, 0.5% NZ-amines, 1.0% yeast extract and 0.4% CaCO.sub.3 (sterilization: 30 minutes, 121.degree.C.; pH adjusted to 7.2 before sterilization) are each inoculated with 2 ml of a primary culture (obtained according to Example 1) of the strain SB 27 and these flasks are incubated for 4 days at 28.degree.C. on a rotary shaking machine, 500 ml of supernatant liquor containing 70 AIU/ml are obtained after combining the flasks and centrifuging off the mycelium. The centrifuged supernatant liquor is frozen and lyophilized. Yield: 7.7 g containing 3.5 .times. 10.sup.3 AIU/g.
EXAMPLE 13
If a mix according to Example 1 is inoculated with 2 ml of a primary culture of the strain SB 18 (obtained according to Example 1) and incubated for 3 days at 28.degree.C., a culture broth containing 1100 AIU/ml, 0.17 MIU/ml and 1.0 SIU/ml is obtained.
EXAMPLE 14
If five experimental fermenters each containing 8 liters of culture solution according to Example 1 are each inoculated with 120 ml of a primary culture (obtained according to Example 1) and incubated for 65 hours at 28.degree.C. while stirring and aerating, 30 liters of culture broth are obtained after combining the fermentation broths and separating off the mycelium. These 30 liters of centrifuged culture broth (0.57 MIU/ml, 6.2 SIU/ml, and 8,000 AIU/ml) are concentrated to 5 liters in vacuo at 20 mm Hg and 100.degree.C. bath temperature, 4 volumes (=20 liters) of acetone are added to the concentrate while stirring, and the black smeary precipitate which forms is collected by centrifuging at 6,000 r.p.m. for 30 minutes; the precipitate is dissolved in 2.5 liters of water and the black-colored solution is stirred for 60 minutes with 500 g of moist Amberlite [IRA 410 (acetate form, pH 7)]. The mixture is separated into supernatant liquor and Amberlite sediment by centrifuging for 10 minutes at 6,000 r.p.m. In the same way, the supernatant liquor is further stirred 3 times, in each case with 500 g of Amberlite for 60 minutes, and subsequently with a further 500 g of Amberlite overnight (.about.15 hours). After this treatment, the supernatant liquor shows a light yellow coloration, while the black concomitant dyestuffs were bonded to the ion exchanger. The collected Amberlite residues are twice washed with 1.5 liters of water and these wash waters are combined with the supernatant liquor containing inhibitor. The supernatant liquor, combined with the wash water, is concentrated to 1 liter in a rotary evaporator at 15 mm Hg and 80.degree.C. bath temperature and subsequently added dropwise to 10 liters of acetone, with vigorous stirring. Hereupon, a white flocculent precipitate results, which is filtered off, washed with acetone and ether and dried in vacuo. Yield: 150 g of a white powder containing 1 .times. 10.sup.6 AIU/g and 450 SIU/g.
EXAMPLE 15
If, in a nutrient solution according to Example 1, the glucose is replaced by other sugars or sugar alcohols and shaking flasks each containing 120 ml of culture solution are each inoculated with 1 ml of a primary culture of the strain SB 18 (manufactured according to Example 1), culture solutions containing the following amylase inhibitor concentrations are obtained after 3 or 4 days' culture at 28.degree.C. on rotary shaking machines:
Additive AIU/ml AIU/ml1% Each after 3 days after 4 days______________________________________sucrose 5,400 10,500lactose 9,100 4,600maltose 7,500 13,600galactose 9,100 10,200glucose 7,000 9,700sorbitol 2,600 10,200mannitol 8,500 8,700inositol 4,800 5,600starch 6,400 10,200______________________________________
EXAMPLE 16
If a nutrient solution consisting of 3% soya flour, 2% starch, 1% glucose, and 0.2% CaCO.sub.3 is inoculated and incubated in accordance with Example 15, a culture broth containing 5,600 AIU/ml is obtained after 4 days' fermentation.
EXAMPLE 17
If a mix according to Example 13 is inoculated and incubated with a morphological variant of SB 18, the strain SB 18/5, a culture broth containing 27,400 AIU/ml is obtained after 4 days' fermentation.
The strain SB 18/5 was deposited at the Central Bureau voor Schimmelcultures in Baarn, Holland, under CBS No. 613.71.
EXAMPLE 18
If a mix according to Example 13 in inoculated and incubated with a morphological variant of SB 18, the strain SB 18/4, a culture broth containing 13,900 AIU/ml is obtained after 4 days' fermentation.
The strain SB 18/4 was deposited at the Central Bureau voor Schmmelcultures in Baarn, Holland, under CBS No. 612.71.
EXAMPLE 19
If a nutrient solution of composition: 2% starch, 1% glucose, 0.3% glycine, 0.25% corn-steep liquor, 0.4% soya flour, 0.1% NaCl, 0.1% K.sub.2 HPO.sub.4, 0.01% FeSO.sub.4, and 0.01% CaCO.sub.3 is inoculated and incubated in accordance with Example 12, a culture broth containing 2,900 AIU/ml is obtained after 3 days' incubation.
EXAMPLE 20
If two flasks of a mix according to Example 1 are inoculated with 1 ml of a primary culture of the strain SB 46 and incubated for 4 days at 28.degree.C. on a rotary shaking machine, 250 ml of supernatant liquor containing 250 AIU/ml are obtained after centrifuging.
These 250 ml of supernatant liquor are treated with 150 g of ammonium-sulphate added in portions while stirring and the mixture is subsequently centrifuged for 15 minutes at 10,000 r.p.m. The residue is dissolved in 12 ml of H.sub.2 O and dialyzed for 3 hours against distilled water. The 20 ml of dialysate are precipitated with 6 volumes (= 120 ml) of acetone in an ice bath and the precipitate is filtered off, washed with acetone and ether and subsequently dried in vacuo. Yield: 0.28 g containing 220 .times. 10.sup.3 AIU/g.
EXAMPLE 21
If a 1 liter Erlenmeyer flask containing 120 ml of a nutrient solution according to Example 1 is inoculated with 2 ml of a primary culture of the strain SE 5 (produced according to Example 4) and incubated for 7 days on a rotary shaking machine at 28.degree.C., a culture broth containing 0.09 MIU/ml is obtained.
The mycelium is treated with 50 ml of acetone, homogenized for one minute on an Ultraturrax homogenizer (Messrs. Janke and Kunkel, Staufen, Breisgau) and the mixture is subsequently centrifuged for 10 minutes at 3,000 r.p.m. The residue is again extracted, in the same way, with 50 ml of acetone and subsequently extracted once with 50 ml of ether, and the three extracts are combined and concentrated almost to dryness in a rotary evaporator at approximately 10-20 mm Hg and a waterbath temperature of 37.degree.C. The mycelium residue is dried in vacuo and subsequently treated with 15 ml of dimethyl sulphoxide (DMSO), homogenized for two minutes by means of the Ultraturrax and extracted for 2 hours while stirring (magnetic stirrer). Thereafter the mixture is centrifuged for 30 minutes at 20,000 r.p.m. The dimethyl sulfoxide (DMSO) extract is then decanted from the extracted mycelium, added to the residue of the acetone/ether extraction, and the mixture stirred for approximately 30 minutes (magnetic stirrer), and again centrifuged for 10 minutes at 20,000 r.p.m., and the clear supernatant liquor is tested as mycelium extract. 0.14 MIU/ml.
EXAMPLE 22
If two shaking flasks according to Example 1, each containing a charge of 160 ml, are each inoculated and incubated with 1 ml of a primary culture (according to Example 1) of the strain SE 21, 200 ml of a supernatant liquor containing 360 AIU/ml are obtained after centrifuging.
The centrifuged supernatant liquor is treated with 100 g of ammonium-sulphate added in portions while stirring and is centrifuged, the precipitate is dissolved in 20 ml of water and the solution is precipitated with 2 volumes (ca 40 ml) of acetone. The precipitate is washed with acetone and ether and dried in vacuo. Yield: 2 g containing 25 .times. 10.sup.3 AIU/g.
EXAMPLE 23
If a mix according to Example 14 is inoculated and incubated with primary cultures of the strain SE 21, culture broths containing 140 AIU/ml are obtained after 65 hours' fermentation.
EXAMPLE 24
If a 1 liter Erlenmeyer flask containing 120 ml of a nutrient solution of the composition according to Example 4 is inoculated with 1 ml of a primary culture, according to Example 1, of the strain SE 39 and incubated in accordance with Example 1, a culture solution containing 50 AIU/ml is obtained after only 3 days' fermentation.
EXAMPLE 25
If glycerine is replaced by glucose in a mix according to Example 24, culture solutions containing 60 AIU/ml are obtained after 3 days.
EXAMPLE 26
If in a mix according to Example 24 the nutrient solution according to Example 1 is modified and the starch replaced by glucose, culture solutions containing 140 AIU/ml are obtained after 3 days' incubation.
EXAMPLE 27
If five 1-liter Erlenmeyer flasks each containing 120 ml of nutrient solution of composition: 3% glucose, 0.5% NZ-amines, 1.0% yeast extract and 0.4% CaCO.sub.3 (sterilization: 30 minutes, 121.degree.C.; pH before sterilization 7.2) are inoculated with 1 ml of a primary culture of the strain SE 39 (manufactured according to Example 4) and incubated for 3 days on a rotary shaking machine at 28.degree.C., 500 ml of a supernatant liquor containing 160 AIU/ml are obtained after centrifuging.
The 500 ml of the supernatant liquor are precipitated with 6 volumes (= 3 liters) of acetone while stirring and the light brown precipitate is filtered off, washed with acetone and ether and dried in vacuo. Yield: 3.5 g containing 13.5 .times. 10.sup.3 AIU/g.
EXAMPLE 28
If an Erlenmeyer flask containing 120 ml of a nutrient solution according to Example 4 is inoculated with 1 ml of a primary culture of the strain SE 50 (primary culture manufactured according to Example 4) and incubated, again in accordance with Example 4, a culture solution containing 1.0 SIU/ml is obtained after 5 days.
EXAMPLE 29
If Example 28 is followed but with a nutrient solution according to Example 1, a culture solution containing 26,000 AIU/ml and 0.18 MIU/ml and 1.8 SIU/ml is obtained after 5 days' incubation.
EXAMPLE 30
If Example 29 is followed but with a flask charge of 200 ml, a culture solution containing 24,500 AIU/ml and 0.18 MIU/ml and 2.1 SIU/ml is obtained after 5 days' incubation.
EXAMPLE 31
If Example 28 is followed, but with a nutrient solution of composition: 2% starch, 1% glucose, 0.3% glycine, 0.25% cornsteep liquor, 0.4% soya flour, 0.1% NaCl, 0.1% K.sub.2 HPO.sub.4, 0.01% MgSO.sub.4, 0.01% CaCO.sub.3 and 0.01% FeSO.sub.4, a culture solution containing 12,900 AIU/ml is obtained after 5 days' fermentation.
EXAMPLE 32
If, in a nutrient solution according to Example 1, the glucose is replaced by other sugars or sugar alcohols and the solution inoculated and incubated in accordance with Example 28, culture solutions containing the following amylase inhibitor concentrations are obtained:
Additives AIU/ml AIU/ml (1%) after 3 days after 5 days______________________________________sucrose -- 23,000lactose 24,000 --maltose 23,500 --galactose 31,300 --glucose 41,000 --sorbitol 24,000 --mannitol 33,000 --inositol 21,000 --starch 33,900 --______________________________________
EXAMPLE 33
If, in a mix according to Example 28, the glycerine is replaced by glucose, culture solutions containing 330 AIU/ml are obtained.
EXAMPLE 34
If, in a mix according to Example 28, the starch is replaced by glucose, culture solutions containing 660 AIU/ml are obtained.
EXAMPLE 35
If, in mixes according to Example 29, NZ-amines are replaced by other complex sources of nitrogen, culture solutions containing the following amylase inhibitor units are obtained after 3 days' fermentation:
Substitute AIU/ml______________________________________soya flour 13,500fish solubles 16,800tryptone 25,200meat extract 32,700pharmamedia 23,100______________________________________
EXAMPLE 36
If a mix according to Example 29 is inoculated with a morphological variant of the strain SE 50, namely strain SE 50/12, culture broths containing 51,500 AIU/ml are obtained after 3 days' fermentation.
EXAMPLE 37
If five experimental fermenters each containing 8 liters of culture solution according to Example 1, but with 0.1% by volume of Bayer E 100 anti-foaming agent, are each inoculated with 120 ml of a primary culture (obtained according to Example 1) of strain SE 50 and incubated for 65 hours at 28.degree.C. while stirring and aerating, the fermentation mixes are combined and the mycelium is separated off, 24 liters of culture broth containing 13,000 AIU/ml and 6 SIU/ml are obtained. The culture broth is concentrated to 1 liter by evaporating in vacuo at 20 mm Hg and 100.degree.C. bath temperature. The black-colored concentrate was centrifuged for 60 minutes at 15,000 r.p.m., the dark supernatant liquor was applied to an Amberlite IRA 410 column (9 cm .phi., 50 cm height, Amberlite IRA 410 acetate, pH 7 in H.sub.2 O), the column was eluted with 50 ml of H.sub.2 O/hour and 20 ml of fractions were collected in the fraction collector. The light yellow-colored fractions containing inhibitor (a total of 300 = 6 liters) were combined, concentrated to a 1 liter on a rotary evaporator and added dropwise, with vigorous stirring, to 10 liters of absolute alcohol, whereupon a flocculent, almost white precipitate separates out. The precipitate is filtered off, washed with absolute alcohol and thereafter with ether and dried in vacuo. Yield: 60 g of a white powder containing 3 .times. 10.sup.6 AIU/g and 400 SIU/g.
EXAMPLE 38
The bulk of the inactive concomitant substances, especially the dark dyestuff, can also be separated off by precipitation with an equal volume of methanol. For this purpose, 26 liters of the centrifuged culture broth (produced according to Example 37, but with 0.02% by volume of Bayer Silicone E antifoaming agent) (43,000 AIU/ml) are concentrated to 1.2 liters by evaporation in vacuo at 20 mm Hg and 100.degree.C. bath temperature. The deep black solution is treated with an equal volume (1.2 liters) of methanol while stirring, whereupon a flocculent black precipitate forms. It is filtered through a fluted filter and the brown filtrate (2.2 liters) is added dropwise to 10 liters of the dry spirit, while stirring vigorously. A light brown precipitate forms, which is filtered off, washed with acetone and ether and dried in vacuo. Yield: 185 g of a light ochre-colored powder containing 3,700 AIU/mg.
For further purification, the entire precipitate (185 g) is dissolved in 250 ml of water and the dark brown solution is passed over a 5 .times. 75 cm column of Amberlite IRA 410 (acetate form, pH 7). The column is eluted with water, the first 500 ml of eluate are discarded as first runnings and the next 5,000 ml of almost colorless eluate contain the inhibitor activity. They are concentrated to 200 ml on a rotary evaporator and the inhibitor is obtained therefrom by adding the solution dropwise to 2 liters of absolute ethyl alcohol, filtering off the precipitate, washing with ethanol and ether and drying in vacuo. Yield: 80 g of a white powder containing 8,000 AIU/mg.
EXAMPLE 39
If a 1 liter Erlenmeyer flask containing 120 ml of a nutrient solution according to Example 4 is inoculated with 2 ml of a primary culture of the strain SE 55 (manufactured according to Example 4) and the culture is incubated for 6 days on a rotary shaking machine at 28.degree.C., a culture broth containing 30 AIU/ml and 0.15 MIU/ml and 0.5 SIU/ml is obtained after separating off the mycelium.
If the mycelium is extracted in accordance with Example 20, an extract containing 15 AIU/ml, 0.17 MIU/ml and 0.5 SIU/ml is obtained.
EXAMPLE 40
If Example 39 is followed, but using a nutrient solution according to Example 1, a culture solution containing 60 AIU/ml, 0.16 MIU/ml and 0.65 SIU/ml and a mycelium extract containing 30 AIU/ml and 0.16 MIU/ml and 0.33 SIU/ml are obtained.
EXAMPLE 41
If a nutrient solution according to Example 39 is inoculated from a sloping test-tube culture on oatmeal agar of the strain SS 26, and the culture is incubated in accordance with Example 40 for 5 days, an extract containing 0.14 MIU/ml is obtained after separating off the culture solution after extraction of the mycelium according to Example 21.
EXAMPLE 42
If Example 41 is followed using a culture of the strain SS 45, a mycelium extract containing 0.1 MIU/ml is obtained.
EXAMPLE 43
If Example 41 is followed using a culture of the strain SS 53, a culture broth containing 0.05 MIU/ml and a mycelium extract containing 0.07 MIU/ml is obtained.
EXAMPLE 44
If a 1 liter Erlenmeyer flask is inoculated with 120 ml of a nutrient solution of composition: 0.5% Bactopeptone, 0.5% meat extract, 0.2% yeast extract, 0.03% casein hydrolysate, 1% i-inositol, 1% sorbitol, 1% D-mannitol, 1% glucose, 0.1% K.sub.2 HPO.sub.4, 0.05% MgSO.sub.4, 0.05% KCl and 0.01% FeSO.sub.4, pH adjusted to 7.2, is inoculated from a sloping agar culture (manufactured according to Example 1) of the strain SS 51, a culture solution containing 0.072 MIU/ml and a mycelium extract (manufactured according to Example 21) containing 0.054 MIU/ml are obtained after culture on a rotary shaking machine at 28.degree.C. for 7 days.
EXAMPLE 45
If a nutrient solution according to Example 4 is inoculated from a sloping test-tube culture on oatmeal agar of the strain St 19 and is incubated for three days in accordance with Example 4, a culture solution containing 20 AIU/ml is obtained after separating off the mycelium.
EXAMPLE 46
Experimental technique for demonstrating the action of glycoside-hydrolase inhibitors in rats and man
To produce alimentary hyperglycemia and hyperinsulinemia after feeding with carbohydrate, rats (n = 6) are given 2.5 g of starch or maltose or sucrose as a solution or suspension/kg, administered orally (control animals). Batches of six other rats receive, additionally to one of the above-mentioned carbohydrates, a glycoside-hydrolase inhibitor in the dosage indicated, administered orally. The blood sugar in the blood from the retro-orbital venous plexus is determined at short intervals after the administration of the carbohydrate by means of the Auto-Analyser [Technicon, according to Hoffman: J. biol. Chem. 120, 51 (1937)].
To produce an alimentary hyperglycemia in man, 50 g of starch/Vp. are administered orally as an aqueous suspension. The blood sugar is determined immediately before the start of the experiment, and at short intervals thereafter, in the capillary blood from the fingertip, in the way described above. In a further experiment, the active substance is added to the starch suspension.
The insulin is determined at short intervals after the administration of the carbohydrate, in the serum of batches of six rats of which one group is given 2.5 g of starch/kg, administered orally (control) and the other groups are additionally given a glycoside-hydrolase inhibitor.
The determination of insulin in the serum is carried out by radio-immunology, based on the double antibody method of Hales and Randle [Biochem. J. 88, 137 (1963)].
Determination of starch in the gestrointestinal tract of rats was carried out at short intervals after oral administration of 300 mg starch/rat. To this purpose, the individual sections of the digestive tract were, after sacrifice of the animal, prepared and washed, and the undigested starch contained in them determined as glucose after acid hydrolysis.
Table 1 (Example 46)
Blood glucose in mg% (mean value .+-. 1s) of fasting rats at various times after oral administration of starch .+-. active substance from Example 5.
______________________________________ 15 60 mins.______________________________________control without starch 61 .+-. 16 70 .+-. 3.4control with starch 140 .+-. 11 145 .+-. 15starch + 25,000 AIU/kg 106 .+-. 20 128 .+-. 11______________________________________
Table 2 (Example 46)
Blood glucose in mg% (mean value .+-. 1s) of fasting rats at various times after oral administration of starch .+-. active substance from Example 14.
__________________________________________________________________________ 15 30 60 mins.__________________________________________________________________________control without starch 72 .+-. 8.5 78 .+-. 12 69 .+-. 12control with starch 159 .+-. 28 153 .+-. 36 164 .+-. 7.9starch + 25,000 AIU/mg 95 .+-. 3.7 95 .+-. 8.7 96 .+-. 7.2 ========= =========__________________________________________________________________________
Table 3 (Example 46)
Blood glucose in mg% (mean value .+-. 1s) of fasting rats at various times after oral administration of starch .+-. active substance from Example 37.
__________________________________________________________________________ 15 30 60 mins.__________________________________________________________________________control without starch 72 .+-. 8.5 78 .+-. 12 69 .+-. 12control with starch 159 .+-. 28 153 .+-. 36 164 .+-. 7.9starch + 25,000 AIU/kg 83 .+-. 6.5 105 .+-. 5.2 104 .+-. 6.5 ========= ========= against control P < 0.01 ====== P < 0.001 with carbohydrate__________________________________________________________________________
Table 4 (Example 46)
Blood glucose in mg% of a healthy test person at various times after oral administration of starch .+-. active substance from Example 37.
__________________________________________________________________________ 0 15 30 60 90 mins.starch without active substance 106 128 150 146 136starch + 60,000 AIU/Vp 100 112 108 110 104__________________________________________________________________________
Table 5 (Example 46)
Blood glucose in mg% (mean value .+-. 1s) of fasting rats at various times after oral administration of maltose .+-. active substance from Example 10.
__________________________________________________________________________ 15 30 60 mins.__________________________________________________________________________control without maltose 70 .+-. 6.7 81 .+-. 15 72 .+-. 9.6control with maltose 160 .+-. 13 153 .+-. 11 172 .+-. 13maltose + 1.5 MIU 120 .+-. 13 124 .+-. 12 134 .+-. 9.4 ========= ========= =========__________________________________________________________________________
Table 6 (Example 46)
Blood glucose in mg% (mean value .+-. 1s) of fasting rats at various times after oral administration of sucrose .+-. active substance from Example 14.
__________________________________________________________________________ 15 30 60 mins.__________________________________________________________________________control with sucrose 61 .+-. 16 77 .+-. 4.7 70 .+-. 3.4control with sucrose 125 .+-. 14 129 .+-. 7.9 135 .+-. 8.5sucrose + 50 SIU/kg 104 .+-. 4.1 116 .+-. 8.4 113 .+-. 3 ======== against controlP < 0.05 P < 0.01 ===== P < 0.001 with carbohydrate__________________________________________________________________________
Table 7 (Example 46)
Serum insulin in .mu.U/ml (means value .+-. 1s) of fasting rats at various times after oral administration of 2.5 g of starch .+-. active substance from Example 14; n = 6.
The serum insulin of fasting rats without being given starch was 6 .+-.1.mu.U/ml in this experiment.
______________________________________Mins. after admini- Starch +stration of starch Starch control 10,000 AIU/kg______________________________________5 52 .+-. 29 17 .+-. 810 72 .+-. 20 15 .+-. 4 =====20 44 .+-. 28 16 .+-. 730 25 .+-. 8 17 .+-. 345 30 .+-. 5 13 .+-. 6 =====60 21 .+-. 8 13 .+-. 8120 9 .+-. 3 10 .+-. 3180 8 .+-. 2 9 .+-. 2______________________________________
Table 8 (Example 46)
Serum insulin in .mu.U/ml (mean value .+-. 1s) of fasting rats at various times after oral administration of 2.5 g of starch .+-. active substance from Example 37; n=6.
The serum insulin of fasting rats without being given starch was 6 .+-. 1.mu.U/ml in this experiment.
______________________________________Mins. after admini- Starch +stration of starch Starch control 10,000 AIU/kg______________________________________5 52 .+-. 29 18 .+-. 810 72 .+-. 20 24 .+-. 11 ======20 44 .+-. 28 11 .+-. 530 25 .+-. 8 13 .+-. 345 30 .+-. 5 18 .+-. 760 21 .+-. 8 15 .+-. 5120 9 .+-. 3 13 .+-. 5180 8 .+-. 2 11 .+-. 5P < 0.05 P < 0.01 =====P < 0.001 against control with carbohydrate______________________________________
FIGS. 1 to 3 (Example 46)
FIGS. 1 to 3 show the average content of starch in mg (ordinate)/rat (n = 6) in the stomach (FIG. 1), small intestine (FIG. 2) and large intestine ((FIG. 3) at different times (abscissa), after oral application of 300 mg raw wheat starch/rat by a throat probe. In FIGS. 1 to 3 curves 1 demonstrate the result for the controls (Group 1), which were given starch without amylase inhibitor, while the curves 2 demonstrate the result for animals which received an equal dose of starch with addition of 1000 AIU amylase inhibitor (Group 2).
Result:
The content of wheat starch is significantly smaller in the stomach (FIG. 1) 15 + 60 minutes after application (P<0.001 to P <0.02) in Group 2 with amylase inhibitor than in Group 1. The quicker passage of starch through the stomach after addition of amylase inhibitor can also be seen from FIG. 2. In this case the content of starch in the small intestine of Group 2 is significantly (P<0.001) raised after 150-180 minutes. FIG. 3 shows that starch reaches the large intestine of rats undigested, if amylase inhibitor is added to the starch in the stated dose.
FIGS. 4 to 6 (Example 46)
In FIGS. 4 to 6 are shown the alteration of blood glucose in mg/100 ml (ordinate) (FIG. 4), the concentration of immunologically reacting insulin in .mu.U/ml serum (ordinate) (FIG. 5), and the concentration of unesterified fatty acids in micro equivalents/liter of plasma (ordinate) (FIG. 6), against the initial value (0) as dependent on time (abscissa), after oral application of 60 g cooked starch for a human experimental subject. Curve 1 shows the course of the stated parameters (blood sugar, serum insulin, plasma UFA) in the control experiment. Curve 2 shows the change of these parameters after administration of the same dose of starch with the addition of 0.25 mg. AIU amylase inhibitor, curve 3 the changes in the stated parameters after addition of 0.5 mega AIU amylase inhibitor to 60 g cooked starch per person.
Results:
After temporary hyperglycemia between 0 + 30 minutes in FIG. 4 the blood glucose falls after 45-180 minutes to significantly under the initial value. As a result of the initial hyperglycemia the concentration of insulin in the serum in the control experiment (curve 1) climbs steeply. The addition of 0.25 (curve 2) or 0.5 mega AIU (curve 3) to the starch has the effect that the hypoglycemia (FIG. 4) as well as the hyperinsulemia (FIG. 5) is quickly weakened. The ratio of unesterified fatty acids in the plasma is substantially equal in all three experiments.
EXAMPLE 47
Erlenmeyer flasks containing the medias given in the Table, are inoculated with the corresponding strains and shaken at the stated temperature on a rotary shaker. Thus are obtained, after several days, culture solutions, and, after working up according to Example 21, mycelium extracts, which have the activities given in the Table.
Table (Example 47)__________________________________________________________________________Strain Number Culture Solutions and Conditions of__________________________________________________________________________ GrowthLaboratory Collection Strain Name See Quantity In Erlenmeyer Growth CultureReference No. Example ml Flask Temperature Period__________________________________________________________________________ (Days)ST 50 CBS 693.69 Streptomyces 4 30 200 28 4 heimiiST 67 CBS 432.59 Streptomyces 44 30 200 28 4 tendaeST 45 CBS 434.51 Streptomyces 4 30 200 28 4 aureofaciensRT 36 CBS 228.65 Chainia rubra 44 30 200 28 4RT 33 CBS 295.66 Chainia poonensis 44 30 200 28 5ST 12 NRRL B-2286 Streptomyces 1 30 200 28 3 murinusST 51 CBS 498.68 Streptomyces 44 30 200 28 4 fradiaeST 3 NRRL 2580 Streptomyces 4 30 200 28 5 chrysomallusST 1 ATCC 11523 Streptomyces 4 30 200 28 5 chrysomallusSS 55 CBS 624.71 Streptosporan- 1 120 1000 28 6 gium roseumSS 59 CBS 623.71 Streptosporan- 1 120 1000 28 6 gium amethy- stogenesSS 62 CBS 625.71 Streptosporan- 1 120 1000 28 6 gium roseumAT 8 KCC-A 0027 Streptosporan- 4 120 1000 28 4 gium viri- dalbumAT 11 KCC-A 0025 Streptosporan- 4 120 1000 28 4 gium albumAT 13 CBS 190.64 Ampullariella 4 120 1000 28 6 campanulataAT 14 CBS 193.64 Ampullariella 4 120 1000 28 4 regularisSE 89 CBS 619.71 Ampullariella 1 120 1000 28 6 spec.SE 100 CBS 622.71 Planomonospora 1 120 1000 28 6 spec.__________________________________________________________________________Strain Number Activity of Culture Filtrates U/ml__________________________________________________________________________Laboratory Collection Strain Name Amylase Inhibition Maltase Inhibition Saccharase InhibitionReference No.__________________________________________________________________________ST 50 CBS 693.69 Streptomyces 0.06 heimiiST 67 CBS 432.59 Streptomyces 0.020 0.25 tendaeST 45 CBS 434.51 Streptomyces 0.002 AureofaciensRT 36 CBS 228.65 Chainia rubra 0.005RT 33 CBS 295.66 Chainia poonensis 0.003ST 12 NRRL B-2286 Streptomyces 500 0.008 murinusST 51 CBS 498.68 Streptomyces 70 fradiaeST 3 NRRL 2580 Streptomyces 20 chrysomallusST 1 ATCC 11523 Streptomyces 20 chrysomallusSS 55 CBS 624.71 Streptosporan- gium roseumSS 59 CBS 623.71 Streptosporan- gium amethy- stogenesSS 62 CBS 625.71 Streptosporan- gium roseumAT 8 KCC-A 0027 Streptosporan- gium viri- dalbumAT 11 KCC-A 0025 Streptosporan- gium albumAT 13 CBS 190.64 Ampullariella 2000 campanulataAT 14 CBS 193.64 Ampullariella 500 regularisSE 89 CBS 619.71 Ampullariella spec.SE 100 CBS 622.71 Planomonospora 50 spec.__________________________________________________________________________Strain Number Activity of the Mycelium Extract According to Example 21 (U/ml)__________________________________________________________________________Laboratory Collection Strain Name Amylase Inhibition Maltase Inhibition Saccharase InhibitionReference No.__________________________________________________________________________ST 50 CBS 693.69 Streptomyces heimiiST 67 CBS 432.59 Streptomyces tendaeST 45 CBS 434.51 Streptomyces aureofaciensRT 36 CBS 228.65 Chainia rubraRT 33 CBS 295.66 Chainia poonensisST 12 NRRL B-2286 Streptomyces murinusST 51 CBS 498.68 Streptomyces fradiaeST 3 NRRL 2580 Streptomyces chrysomallusST 1 ATCC 11523 Streptomyces chrysomallusSS 55 CBS 624.71 Streptosporan- 100 gium roseumSS 59 CBS 623.71 Streptosporan- 70 gium amethy- stogenesSS 62 CBS 625.71 Streptosporan- 150 gium roseumAT 8 KCC-A 0027 Streptosporan- 150 gium viri- dalbumAT 11 KCC-A 0025 Streptosporan- 100 gium albumAT 13 CBS 190.64 Ampullariella campanulataAT 14 CBS 193.64 Ampullariella regularisSE 89 CBS 619.71 Ampullariella 2000 spec.SE 100 CBS 622.71 Planomonospora 50 spec.__________________________________________________________________________Strain Number Culture Solutions and Conditions of__________________________________________________________________________ GrowthLaboratory Collection Strain Name See Quantity In Erlenmeyer Growth CultureReference No. Example ml Flask Temperature Period__________________________________________________________________________ (Days)AT 4 CBS 191.64 Ampullariella 4 120 1000 28 6 digitataAT 9 KCC-A 0028 streptosporan- 4 120 1000 28 5 gium vulgareAT 10 ATCC 19190 Streptosporan- 1 120 1000 28 4 gium india- nensisAT 10 ATCC 19190 Streptosporan- 44 120 1000 28 5 gium india- nensisSE 103 CBS 616.71 Actinoplanes 1 120 1000 28 4 spec.HN 6 CBS 602.71 Actinobifida 44 120 1000 50 4 chromogenaHN 2 CBS 603.71 Actinobifida 44 120 1000 50 5 chromogenaAT 2 CBS 367.66 Actinoplanes 4 120 1000 28 4 utahensisSE 101 CBS 621.71 Planomonospora 1 120 1000 28 6 parontosporaSK 2 CBS 620.71 Pilimelia spec. 1 120 1000 28 7SE 82 CBS 615.71 Actinoplanes 4 120 1000 28 5 spec. 1 120 1000 28 5SE 45 CBS 618.71 Ampullariella 4 120 1000 28 4 spec.SA 28 CBS 617.71 Ampullariella 1 120 1000 28 5 digitataSA 8 CBS 611.71 Actinoplanes 4 120 1000 28 5 spec.AT 7 ATCC 12428 Streptosporan- 4 120 1000 28 4 gium roseum__________________________________________________________________________Strain Number Activity of the Culture Filtrate__________________________________________________________________________ U/mlLaboratory Collection Strain Name Amylase Inhibition Maltase Inhibition Saccharase InhibitionReference No.__________________________________________________________________________AT 4 CBS 191.64 Ampullariella 0.003 0.5 digitataAT 9 KCC-A 0028 Streptosporan- 0.4 gium vulgareAT 10 ATCC 19190 Streptosporan- 0.5 gium india- nensisAT 10 ATCC 19190 Streptosporan- 0.3 gium india- nensisSE 103 CBS 616.71 Actinoplanes spec.HN 6 CBS 602.71 Actinobifida chromogenaHN 2 CBS 603.71 Actinobifida chromogenaAT 2 CBS 367.66 Actinoplanes 0.003 utahensisSE 101 CBS 621.71 Planomonospora parontosporaSK 2 CBS 620.71 Pilimelia spec.SE 82 CBS 615.71 Actinoplanes 200 0.010 0.8 spec. 3000 0.008 0.8SE 45 CBS 618.71 Ampullariella spec.SA 28 CBS 617.71 Ampullariella 0.002 digitataSA 8 CBS 611.71 Actinoplanes spec.AT 7 ATCC 12428 Streptosporan- gium roseum__________________________________________________________________________Strain Number Activity of Mycelium Extract According to Example 21 (U/ml)__________________________________________________________________________ Laboratory Collection Strain Name Amylase Inhibition Maltase Inhibition Saccharase InhibitionReference No.__________________________________________________________________________AT 4 CBS 191.64 Ampullariella digitataAT 9 KCC-A 0028 Streptosporan- 0.004 1.5 gium vulgareAT 10 ATCC 19190 Streptosporan- gium india- nensisAT 10 ATCC 19190 Streptosporan- 0.3 gium india- nensisSE 103 CBS 616.71 Actinoplanes 0.5 spec.HN 6 CBS 602.71 Actinobifida 1.5 chromogenaHN 2 CBS 603.71 Actinobifida 3.0 chromogenaAT 2 CBS 367.66 Actinoplanes utahensisSE 101 CBS 621.71 Planomonospora 0.006 parontosporaSK 2 CBS 620.71 Pilimelia spec. 0.8SE 82 CBS 615.71 Actinoplanes 2000 0.010 0.6 spec. 35000 0.003 0.5SE 45 CBS 618.71 Ampullariella 0.2 spec.SA 28 CBS 617.71 Ampullariella 0.003 digitataSA 8 CBS 611.71 Actinoplanes 200 spec.AT 7 ATCC 12428 Streptosporan- 50 gium roseum__________________________________________________________________________

Number	Name	Date	Kind
3127315	Tardrew et al.	Mar 1964
3629404	Florent et al.	Dec 1971

Glycoside-hydrolase enzyme inhibitors

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