Glycoside-hydrolase enzyme inhibitors
United States Patent 1 1 Frommer et al.  
 [ 1 GLYCOSIDE-IIYDROLASE ENZYME INHIBITORS [75] Inventors: Werner Frommer; Walter Puls. both of Wuppertal-Elberfeld; Dietmar Schiifer, Neuhof bei Fulda; Delf Schmidt, Wuppertal-Vohwinkel. all of Germany [73] Assignee: Bayer Aktiengesellschaft,  
 Leverkusen. Germany [22] Filed: Dec. 28, I971 [21] Appl. No.: 213,066  
 [30] Foreign Application Priority Data Dec. 28. I970 Germany 2064092 [52] US. Cl 424/115; 195/80 R; 424/180; 424/181 [51] Int. Cl A6lk 27/00 [58] Field of Search 195/80 R; 424/l I5. 180. 424/181 [56] References Cited UNITED STATES PATENTS 3.127.315 3/1964 Tardrew et al. 424/115 Primary Examiner-Frederick E. Waddell Attorney. Agent. or FirmDepaoli &amp; O&#39;Brien [57] ABSTRACT This invention relates to inhibitors for glycoside- Apr. 8, 1975 hydrolases derived from bacteria of the order Actinomycetales. means for their production comprising cultivation of a microorganism of the order Actinomy cetales in appropriate nutrient solutions under conditions most favorable to growth and production of the enzyme inhibitor and recovering, as a new product, glycoside-hydrolase enzyme inhibitors, from the culture as well as the use of said enzyme inhibitors in pharmaceutically acceptable therapeutic compositions in the treatment of conditions indicating obesity, diabetes. pre-diabetes. gastritis, gastric ulcer, hyperlipidemia (atheriosclerosis) and the like. The invention also contemplates the provision of methods of inhibiting the reaction of carbohydrates and glycosidehydrolase enzymes and particularly carbohydratesplitting glycoside-hydrolase enzymes of the digestive tract by means of conducting said reaction of said carbohydrates and glycoside-hydrolase enzymes in the presence of a glycoside-hydrolase enzyme inhibitor for said glycoside-hydrolase enzyme derived from a strain of microorganism of the order Actinomycetales. The invention further contemplates the provision of methods for the treatment of indications of the group consisting of obesity. hyperlipidemia (atheriosclerosis). diabetes. pre-diabetes. gastritis, gastric ulcer. duodenal ulcer. and caries induced by the action of glycoside-hydrolase enzymes and carbohydrates. the improvement which comprises employing an enzyme inhibitor for glycoside-hydrolase enzymes produced by a strain of microorganism of the order Actinomycetales.  
 4 Claims. 6 Drawing Figures 1 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.  
 Ill  
 tase. 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 neces sary 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 a-amylases can be inhibited by the use of various low molecular substances. such as. for example. salicylic acid and abiscisin IT. Hemberg. J. Larsson. Physiol. Plant. 14. 86l  
 Point of action of amylase l l l Starch also contains branched chains of glucose units.  
 Maltose unit (l96l). T. Hemberg. Acta Chem. Scand. 2l. l665 and the amylase of saliva cannot break the chains at the (1967)]. It is further known that there are also higherpoints where branching occurs:  
 molecular substances which are capable of inhibiting i i k 1 Cannot be attacked 0 0 i/t; y amylase 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. malthe activity of certain amylases non-specifically by physical adsorption (T. Chrzaszcz J. Janicki. Bioch. Z. 260. 354 ([933) and Bioch. J. 28. 296 (l934)] or by denaturation and precipitation of the enzyme [8. S. Miller. E. Kneen. Arch. Bioch. Biophys. 15. 251 ([947). D. H. Struhmeyer. M. H. Malin. Biochem. Biophys. Acta I84. 643 (l969)]. 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. Kneerf. R. M. Sandstedt. Arch., Bioch. Biophys. 9. 235 (I946).  
  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 and above) attained.  
 An earlier proposal (United States patent application Ser. No. 1 10.482. filed Jan. 28. 1971. now abandoned.) relates to amylase inhibitors. In fact. the older proposals shows that by means of aqueous electrolyte solutions. preferably dilute acids. or above all by means of water-alcohol (C -C -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 extend 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 ucrinomyceles. and in particular to inhibitors for glycoside-hydrolases of preferably carbohydrate-splitting enzymes of the digestive tract from ar&#39;linamyceres. These inhibitors are formed by microorganisms of the order Actinomycerales. especially by those of the family of Srreprmnycemceae. Thernmucrr&#39;nomyceraceae. Micromonosporaceae. Nocardiaceae and above all those of the family Aclinuplanaceae. They are also formed to a particularly high degree of strains of the genera Acrinoplunes, Ampullariella. Srrepruspurangium. Strepromyces. Chainia. Pilimel&#39;iu. Plummmlmsporn. and Aclinobrfida.  
 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 Aclinomycemles and extracting the inhibitor from the resultant culture.  
 The preferred microorganisms are preferably of the family Slrepmmyceraceae or Acrinuplunuceue.  
  A wide variety of microorganism of the order Acrinumycemles have been found to make glycosidehydrolase 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 Acrinomycelales. especially those of the families Srreprrmryceraceae and Aclinoplanaceae. are isolated in a known manner from samples of soil or strains of these order 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 glycerineglycine nutrient solution according to von Plotho, of compositions 2% glycerine, 0.25% glycine, 0.1% NaCl. 0.1% K HPO4, 0.01% FeSO 7 H O, 0.01% MgSo. 7 H and 0.1% QaCo 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 80. 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.. CaCQ, and MgSO can also be entirely absent. l00200 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 in incubation on shaking machines at ]560C.. preferably at 24-50C. 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 of centrifugation. l-l00 p 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 l X 5 volumes of diethyl ether. the extracted mycelium residue is dried in vacuo at 20 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 extract 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 pl thereof are employed in the tests described below.  
 Amylase Test One amylase inhibitor unit l 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 l u equivalent of glu&#39; coside bonds in starch. The ,uVal of split bonds are determined colorimetrically as uVal of reduced sugar formed. by means of dinitrosalicylic acid. and are specified as p.Val of maltose equivalents by means ofa maltose calibration curve. To carry out the test, 0.l ml of amylase solution (20-22 AU/ml) are treated with l-400 pg of inhibitor or 1-100 pl of the culture solution or mycelium extracts to be tested. in 0.4 ml of 0.02 M sodium glycerophosphate buffer/0.001 M CaClpH 6.9, and the mixture is equilibrated in a waterbath at 35C. for about 10-20 minutes. It is then incubated for 5 minutes at 35C. with 0.5 ml of a 1% strength starch solution (soluble starch of Messrs. Merck. Darmstadt. No. 1252) which has been pre-warmed to 35C. and is subsequently treated with 1 ml of dinitrosalicylic acid reagent (according to P. Bernfeld in Colowick-Kaplan, Meth. EnzymoL. volume l. page 149). To develop the color. the batch is heated for 5 minutes on a boiling waterbath and the 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:  
 AU -H- relative to solids. 4-i- AU in the non-inhibited mix of the same series). and the 507: inhibition point is read off from the curve and converted to AlU/mg of inhibitor. Saccharase Test One saccharase inhibitor unit (STU) 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 nmol of sucrose into glucose and fructose. The nmol 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. (I958) page 1977] in the amount of 0.3-0.4 SU/ml are mixed with -400 pg of inhibitor or 0-50 t] of the culture solution of the mycelium extract which is to be investigated. in 0.l ml of a 0.l M Na maleate buffer of pH 6.0. and the mixture is equilibrated for about 10-20 minutes in a waterbath at 35C. It is then incubated for 60 minutes at 35C. with 0.2 ml of an 0.056 M sucrose solution (sucrose: Messrs. Merck, Darmstadt, No. 7652) which has been pre-warmed to 35C.. and is subsequently treated 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 Dahlquist. Acta Chem. Scand. 12. (1958). page I997], or maltase in the form of human pancreatic juice lyophilizatc. (0.09-0.12 MU/ml) are equilibrated with 0-400 p.g of inhibitor or 0-20 p.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 35C. The mixture is then incubated for minutes at C. with 0.l ml of an 0.4% strength solution of p-nitrophenyl-a-D- glucopyranoside (Messrs. Serva. Heidelberg. No. 30.7l6) in 0.1 M Na maleate buffer at pH 6. which has been pre-warmed to 35C.. 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 ofa molar extinction coefficient of E 13.2 X [0 for the pnitrophenolate 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:  
 pg of inhibitor MU -H- relative to solids, MU in the non-inhibited mix) and the 50% inhibition point is read off from the curve and converted to MlU/mg of inhibitor.  
 Since. in this simple routine test. an artificial sub- 30 calibration i l fi mhlbmlonp strate and not the actual substrate of maltase (maltose) i e P g E sacs 339 is used. the preparations are additionally tested for ca a h c peliccnlmge m I mun their maltase-inhibiting activity in a more involved malp one unctmn O t e quonem&#39; tase test described by Dahlquist (Enzyme. biol. clin. l l, a or inhibimr+ 35 5 /l970). Herein, the glucose produced during the ac L1 tron of maltase on maltose 15 measured enzymatically by means of colorimetry. using glucose oxidase. peroxil+ lati 0 lids. S n nn it d miX Of the dase and dianisidine. All the maltase inhibitors desame series). and the 50% inhibition point is read off ib d h e also inhibit in this test. m the curve n converted to l /mg of inh r- 40 A whole series of strains of various families and gen- Maltase Test era of the order Actinomycetales was tested in accor- One maltase inhibitor Unit (lMlU) is defined as the dance with the method described above. In doing so. amount of inhibitor which inhibits two maltase units to distinct. though at times weak, glycoside-hydrolase- 1118 exlenl Of 507% one maltase Unit is the inhibiting activities were found in various families and amount of enzyme i h in 1 minute. under h e genera. The strains of the family of the Streptomyceconditions indicated below, splits l p equivalent of glutaceae and especially of the family of the Actinoplacosidic bond in the p-nitrophenyl-a-D- naceae proved most advantageous as regards yield. glucopyranoside. The Val of split bonds are deter- Within these families. particularly active strains were mined photometrically as rival of p-nitrophenolate. found in the genera Streptomyces. Actinoplanes. Am-  
  To carry out the test, 0.05 ml of maltase solution pullariella and Streptosporangium. Of the strains comprising a solubilized maltase from intestinal mutested, those listed below proved particularly active in cous membrane of pigs [according to B. Borgstrom. A. one or more of the tests indicated.  
 Table l Inhibiting Action Strain No. Name Amylase Maltase Saccharasc Own Desig- C ollec nation tion Nos C M C M C M SB 2 CBS Ampullariellu -H-+ )5 l .70 regulan&#39;s SB 5 CBS -H+ 952.70 SB ll CBS Aclinnplum&#39;s -+H- i-H 955.70 spec. SB 12 CBS 956.70 88 I8 CBS -i++ +7-7- 957.70 SB 27 CBS 4-H- 958.70 SB 46 CBS +-H- 959.70 SE 5 CBS Aclinoplmmr l+ H spec.  
 Srrepmqxirmzgium album Table 2 Srrcprmporungimn spec. SS 45 Slrvpmqmraugium spec. SS 5 I Date of isolation Method Origin AM Mycelium SM Shape of Sporangia August 2. i967 soil smear. plate Rhon. Heidelstein. turf soil between basalt blocks. pH 3.9  
 white 0.5 L2 p. septate spherical, in part wrinkled and deformed November 8, i968 soil smear. plate Kenya. near Ruiru. coffee plantation.  
 pH 5.4 white-cream-yellowish or pink spherical. smooth also wrinkled and irregular. shriveled forms SizcofSporangia 3-7ptd: 3-lllJ-d) 3-llpdi Shape of Spores :t spherical to elongated mostly 1 oval mostly i oval. in parts spherical Size of Spores 0.6 0.9 x 0.7 L] x 0.7 l.0 x 0.o-l.3;t l.0l.7a 0.8-l.7  
 Flagellation Arrangement of Spores in Sporangium Conidia. Substrate spores and the like spiral chains spiral chains PEH) PEH) PEH-) Melanine CFC-l CPC) CFC-l Ty l- Ty l- Ty Gel) Gell Gel-l Nitrate reduction (weak) Gelatine liquefaction Milk peptonization Growth at 32C.  
  37C. 42C. 27: slight growth -H- H- NaCl 3% toleration 4% 7% Starch hydrolysis Srrepmsporunginm mreum Ar&#39;linuplanes spec.  
 SS 53 SE 5 SE 55 November 9. i968 soil smear. plate Kenya. near Ruiru coffee plantation. pH 5.7  
 December 16. i969 pollen Kenya. near Ruiru coffee plantation.  
 December 31. I969 pollen Kenya. Njoro. Eldoret Experimental Station.  
  pH 5.6 pH S.l Mycelium AM pink 0.4 1.3 a 0.3 [.3 a. septate SM 0.4 l.2 p.  
 Shape of Sporangia spherical. in part irregular s ightly wrinkled Size of Sporangia 3 l0 p o about 4 l2 p. Shape of Spores mostly total 1 spherical Size of Spores 0.7 1.1 x  
 ill-1.9 1. Ca.lp.  
 Flagellation Arrangement of Spores in Sporangium Conidia. Substrate spores and the like spiral chains on C PC substrate spores. mostly spherical (11. u to a prox. 2 p d: indtvidua or several together. terminal or intercalar coiled chains; s ore chains also in p aces running parallel and straight PEH Melanine CFC CFC (PC T) g Ty Gel Nitrate reduction Gelatine liquefaction Milk peptonization 20C. H 26C. H Growth at 32C. -H- 17C. 42C.  
  2% NaCl 3% toleration 4% 7% a Starch hydrolysis Continued AmpuIIur/cllu regular-ix SB 2 Ampulluriellu regulurrli SB 5 Ampulluriella rt&#39;gnlarrli&#39; SE 2 I Date ofisulation Jul\ 2. I966 July 3. [966 December I7. 1969 Method pollen pollen pollen Origin 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 5.3  
 Mycelium 0.3 l a. broad 0.3 a 0.3 l  
 Shape of Sporangia bottle-sha ed. bottle-sha d. bottle-sha d.  
 cylindrica money pouch-shaped&#34; cylindrica moneypouch-shaped&#34; cylindrica moneypouch-shaped&#34; Size of Sporangia 4 10 x 3.5 7 x 3.5 9 x 7-l8 6 -l4 6 -l3;t Shape of Spores small rods small rods small rods Size of Spores 0.5 0.7 x 0.5 0.7 x 0.5 0.7 x 1.5-2.2 l.52.2[4 l.5-2.2 Flagellation lophotrichous lophotrichous lophotrichous Arrangement of Spores linear parallel chains linear parallel chains linear parallel chains in Sporangium Conidia. Substrate spores and the like Melanirie Growth at 32C.  
 Date of isolation lliii++++ Ampullariella regulurrlr S 39 December 22. I969 l|iii++++ Avrinuplunes spec.  
 November 9. I966 CPC+ Ty Iiiii++ Actrnoplanes spec.  
 November 9. 1966 Method pollen pollen pollen Origin 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 7.3  
 Mxcelium 0.3 l p. 0.3 L3 11., septate 0.3 l.2 p.  
 Shape of Sporangia bottle shaped. cylindrical. money pouch-shaped&#34; I spherical with wrinkled surface. in pan irregular Size of Sporangia 3 l0 x 5 16 p. 3.5 12 p. Shape of Spores small rods spherical Size of Sores 0.5 0.7 x approx.  
  1.5-2.2 ll.3p Fl gellation lophotrichous mobile spores Arrangement of Spores in Sporan ium Conidia. ubstrate spores and the like linear parallel chains on C PC broad hyphen (approx. 1.3 a). septate at intervals of approx. 2.5 4 a optically dense. permanent cells? coiled-up chains Melaninc CFC PC CPC I Ty Ty Gel l Gel Nitrate reduction Gclatine liquefaction Milk peptonization Starch-hydrolysis 2 C. +9- -H- H 26C. +4- Growth at 32C. -H- -+l- +i- 37C 4-i- 42C.  
  Actinopltmes Actinoplanes Aclinnplam&#39;i&#39; Acrinuplaues sgec. s ec. Spec. 5 18 S 27 S 50 Date of isolation December 4. 1966 December 8. 1966 March 3. [967 December 22. 1969 Method pollen pollen pollen pollen Origin Hattenheim. Kuhkopf. Nature Neuhot&#39;. Fulda Kenya. near Ruiru Rheingau. potato Reserve. Altrhein. district. turf coffee plantation. field in the riverside wood. soil. ed e of pH 6.2 &#34;Sandaue&#34;. pH 8.0 under willows. path. p  
  pH 7.6 Mycelium 0.3 1.3 1.1 0.2 1.1 p. 0.3 1.4 p. 0.4 L?! septate septate Table 2 Continued Date of isolation December 4. 1966 December 8, 1966 March 3, 1967 December 22, 1969 Method pollen pollen pollen pollen Shape of Sporangia irregular. :club-shaped or irregular. irregular. humped.  
 wrinkled. humped oval to cyllndrifolded. humped wrinkled cal. in part irregular. surface wrinkled Size of Sporangia 6 lb 44. 4 6p. 6 20 p. 4 I} p. Shape of Spores spherical. in 1- spherical spherical t spherical art with noseike protuberance Size of Spores l l.7 p. l 1.3 p. approxv L2 2 p. approx. 1 p.  
 Flagellation Arrangement of bundles of flagellae coiled chains mobile spores slightly coiled.  
 coiled chains. in part parallel coiled chains. parallel in places Spores in in part parallel Sporangium and straight chains Conidia. Substrate on C PC substrate spores and the like 5 res. up to 3 p. singly or intercalar to give several in a row Melanine CFC- CFC-l CFC-l CPC+)+ Ty T Tyl- Ty+l Gel+) Gel-l Gel-) Nitrate reduction Gelatine liquefaction Milk peptonization Starch-hydrolysis 20C. -+l- *H- H 4-i- Z6C. i-i- V H H H Growth at 32C. +0- ifi. 37C.  
  Aciinuplanex Arlinoplanes Actinuplanes spec. s spec. SA 8 S 82 SE 103 Date of isolation June 26. 1966 February 26. 197i March 2. 197i Method pollen pollen pollen Origin Marburg. compost Ceylon. earth. Corsica. near Hol earth. botanic rubber plantation Trinity. from eart garden under cork-oaks Mycelium 0.3 L3 p 0.3 L3 p. Shape of Sporangia irregular irregular irregular Size of Sporangia 7-l5 X 9-18 [.L 5 14 p. about 5 l3 p Shape of spores spherical spherical :spherical Size of spores about 1.5 p. about I 1.4 11- about l p. Flagellation mobile rapidly mobile Arrangement of coiled chains coiled chains Spores in Sporangium Ty Melanine Gel CFC Nitrate reduction (very low) Gclatine liquefaction Milk peptonization S! repmsporangium S lrepwspuran gium S lrepmrpnran gi um roseum amelhyxmgt&#39;m&#39;s msvum SS 55 SS 59 SS 62 Isolation date November l0. I968 November [0. I968 soil smear. plate Method soil smear. plate soil smear. plate Origin Ken a. near Ruiru. Kenya. near Njoro. Kenya. near Njoro.  
 can from coffee from field from field field Mycelium 0.4 L2 1.1. 0.4 1.2 p.  
 Shape of sporangia spherical spherical spherical Size of sporangia 3 [0 p 3 IS a 3 10 p.  
 Shape of spores mostly oval mostly oval Size of spores about 0.3 x l 1.2 a  
 about 0.8 L] X l Melanine Nitrate reduction 4+ Gelatine liquefaction Milk peptonization Starch hydrolysis Growth at 37C.  
 NaCl 3% toleration 4&#39;1:  
 Table 2 Continued Ampullurivllu Ampullurir l&#39;lu Ampull&#39;ariella digituta regulartli s cc.  
  SA 28 SE 45 S 8) Isolation date November 10. I967 December 23. l969 February 27. 1971 Method fungus mycclium pollen pollen Origin Amonau Krs. Marburg, Kenya. near Ruiru. Ceylon. earth earth from stubble earth from coffee field field Mycelium 0.3 l .1  
 Shape of sporangia very narrow and relatively long. distally and in part also laterally irregular.  
 in part fingered. coil-like or fingerbottle-shaped. cylindrical. often longitudinally narrow I cylindrical. frequently also broader than long. often not flat at the distal end. tubercular. in part also irregular and folded.  
  shaped a few fingered Size of sporangia 2-7 X 4-l3 4 4l0 6-l7 p. 5-l2 X 6-l6 p. Shape of sporangia small rod-shaped small rods small rod-shaped Arrangement of arranged in parallel in parallel spores in sporangia rows straight rows Ty l Melanine Gel l C PC I Nitrate reduction Gelatinc liquefaction Milk pcptonization Plummmnox mm Plannnmnospm&#39;u Pilimelia spec.  
 spec. pumutuspom SK 2 SE I00 SE 101 Isolation date March 1. I971 February 27. l97l January 23. l968 Method pollen pollen hair of mice Origin Corsica. northern Ceylon. lawn earth Marburg. garden earth.  
 Algeria, earth. Schulerpark tree nursery Mycelium 0.3 0.7 p.  
 Shape of sporangia longitudinally narrow. stand compactly by each other in parallel double rows in the air mycelium. arcuately bent air-hyphens on the convex side directly joined to their base spherical. pear-shaped. oval, with a columella. which projects. as a continuation of the sporangsiophor. up to about of the sporangia -0 or further into the sporangium Size of sporangia about I 1.3 X about 7 15 u 3 4.5 p. Shape of spores small rods often slightly warped Size of spores 0.. 5 0.45 X  
  0.8 L5 [1. Flagellation mobile l&#39;lagellar fascidcs Arrangement of lateral (and also subspores in polar). spore arrangesporangium ment in chains. which grow tuft-like from the columella Melanine Ty Nitrate reduction Gclatine liquefaction Milk pcptonization Growth at 32C. 37C.  
 Table 3 G growth SM substrate mycelium SP soluble pigment Spg sporangium C colony shape AM aerial mycelium SB 2 SB 5 SB ll 1 1 G. very good G. good G. nod 21 SM. orange SM. yellow-orange SM. eautiful orange. C asamino-pcptonebleaching 3) SP. agar yellowish SP. agar yellowish SP. agar yellowish Czapek agar 4) Spg. -+H-; rimy Spg. originally Spg. (C PC 5) C. flat and smooth C. heavily veined C. with coiled ridges G. very good G. good to very good G. Peptone SM. orange, rimy SM. orange-red SM. orange, bleaching SP. SP. agar yellowish SP. agar golden yellow Czapek agar Sp 4+ Spg. Spg.  
  C flat. in art (PCl hump G. moderate G. moderate G. good Table 3 Continued G growth Spg sporangium SM substrate mycelium C colony shape SP soluble pigment AM aerial mycelium yellow SB 2 SB 5 SB l l (zapek agar SM. orange SM. pale orange SM. brownish orange (C1) SP. agar pale ochre SP. agar slightly SP.  
  yellowish P2 psus G. good G. very good G. very good Milk agar SM. brownish orange SM. brownish orange SM. brownish orange, turns pale brown (Cal SP. agar brownish SP. agar golden brown SP. agar yellow brown Spg. casein Spg. casein Spg. casein peptonized pcptonized peptonized G. moderate-good G. slight G. moderate SM. reddish brown SM. brown SM. pale orange Tyrosine 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 ood G. good Oat-Yeast aqar SM. brownish orange SM. brownish orange SM. ochre-orange SP. SP. SP. (OY) Spg. -ll-+ Spg. Spg.  
 G. moderate-good SM. colorless to pale ochre Starch agar SP. ns-  
 SB l2 SB 18 SB 27 G. very good G. very good G. ood to very good C asamino-Peptone- SM. orangebrown SM. strong red-brown SM. uminous orange.  
 later brown orange C zapek agar SP. agar yellowish- SP. agar brown SP. agar yellow-brown brownish (CPC) Spg. Spg. Sp  
 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 good Peptone-Czapek agar SM. brown red SM. brown SM. ochre orange SP. agar golden brown SP. brown SP. agar yellowish (PC) Spg. Spg. -H- Spg.  
 G good G good G. good SM. orange to SM. red brown SM. red brown brownish orange Czapek agar SP. agar fluoresces SP. agar reddish SP. agar slightly greenish to brown ochre (Cl) yellow Spg. Spg. rimv Spg. G. very good G. very good G. very good Milk agar SM. brown orange SM. brown orange SM. brownish orange SP. agar golden brown SP. agar golden brown SP. agar golden brown lCa) Spg. Spg. -il-. Spg.  
 casein peptonized not always: not always:  
  casein peptonized casein peptonized G. moderate G. moderate good G. moderate good Tyrosine agar SM. brown SM. reddish orange brown SM. ochre brown SP. agar brown; SP. agar orange brown: SP. (Ty) crystal solution crystal solution llcrystal solution -H- 4+ a p p 6. good G. good 0. good Oat-Yeast agar SM. pale orange SM. red brown SM. orange brown SP. SP. red brown SP. (OY) Spg. Spg. -H- Spg.  
  G. moderate to good G. moderate to good G. moderate to good Starchagar 2M. pale brownish orange pale brownish orange pale brownish orange P. Spg. Spg. Spg. H-  
 58 46 SE 2| SE 39 SE 50 G. good G. good G. good G. very good C asamino- SM. orange. later SM. orange brown SM. red brown SM. brown brownish orange peptone- SP. agar pale SP. agar yellow SP. agar golden SP. agar brown to yellowish brown brown red yellowish Czapek agar brown brown (CPC Spg. Spg. some Spg. +-+1 rimy Spg.  
 C. flat. with radial grooves G. good G. \er ood G. very good G. very good Peptone- SM. orange SM. dar rown SM. reddish dark SM. brown Czapek agar SP. agar golden SP. brown SP. dark brown SP dark brown Table 3 Continued G growth Spg sporangium SP= soluble pigment AM aerial mycelium SB 46 SE21 SE39 SE50 Spg. Spg. Spg. frost-like on the SM Gzapek agar G. good G. good G. good G. very good SM. orange brown SM. orange SM. red-brown SM. red-brown (C 2] 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. good Milk agar SM. orange SM. brown-orange SM. brown-orange SM orange brown SP. agar golden- SP. brown SP. dark brown SP. dark brown (Cal colored Spg. casein Spg. casein Spg. casein not Spg. casein pepronized pcplonized peptonized peplonized G. good (i. bulky G. hulls G. bulky Tyrosine 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 -H&#39; P2 p p vsl)l0SlIlC crvstals )TUSIHC crystals t rosinc or sials noi dissolved no! dissolved not dissolved G. good Oat-Yeasl agar SM. orange brown SP. (OY) Spg. Spg. 4-i- Spg. Emerson&#34; G. good agar E SM. brown (yeast-starch SP. brown agar) Spg. Spg.  
 G. moderate to good G.  
 moderate to good G.  
 moderaie to good Starch agar SM. pale brownish SM. pale brownish SM. pale brownish orange orange SP. SP. 7 starch hydrolysis Spg. Spg. Spg.  
 SE 5 SE 55 SE l03 G good G good G good SM. orange SM. dark red brown SM. orange Casamino- AM. AM. peptone- SP. to pale yellowish SP. yellow brown SP. yellowish-brownish Czapek agar Spg. 1C PC 1 surface of colony in inclined lest-tube shows slimy gloss G. good 0. good G. very good Oat-Yeast SM. orange SM. orange brown SM. brown a at AM. Y] SP. light brown SP.  
 Spg. -H  
 frostlike G. good G. very good Emerson agar SM. orange SM. orange AM. lEl SP. SP.  
  surface of colony Spg. H. frost-like slim G. good G. good Milk agar SM. orange SM. orange brown 1C a) SP. lo yellowish SP. brown Casein not peptonized. Casein not peptonized. except merely negliafter aboui 2 months gihly immeditel slightly peplonized around the mycelium G. good G. good G. very good Czapck agar SM. orange, surface SM. orange SM. yelloworangc ((zl damp and sliny SP. SP. SP. Spg. G. small to bulky G. bulky Tyrosine agar SM. colorless to pale SM. dark brown (TY) orange SP. SP. black brown tyrosine crystals tyrosine crystals not dissolved not dissolved Pcpione-Czapek G. veri good Agar SM. dar reddish brown (PC) SP. dark brown G. moderate to good Starch agar SM. pale orange brown Spg.  
 Table 3 -Contmued G growth M substrate mycelium SP soluble pigment Spg sporangium C colony shape AM =aerial myclium SS 45 SS 51 SS 53 G. moderate (to good) G. moderate (to good) G. good Casamino- SM. reddish brown SM. reddish brown SM. pale light brown peptone- AM. AM. AM. white Czapek agar SP. SP. SP. brownish (CPC) colony flat. 6 mm di colony flat. 6 mm d: colony flat. center after 9 weeks after 9 weeks without AM G. good G. good G. good to very good Oat-Yeast SM. brown red SM. brown red SM. purple brown to agar red brown (OY) AM. white and pink AM. white aand pink AM. H. diny while and pink SP. pale yellowish to SP. pale yellowish with SP. purple-red brown yellowish-greenish greenish tinge Spg. Spg. Spg.  
 G. good G. ood G. good Emerson&#34; SM. red SM. rown red SM. red brown agar AM. AM. slight white AM. H. pink (E) SP. pale yellowish SP. to pale yellowish SP. red brown with greenish tinge brownish Spg. H- G. good G. good G. good Milk agar SM. pale brownish red SM. pale brownish red SM. pale light brown (C a) AM. AM. AM.  
 SP. agar goldyellow SP. agar gold-yellow SP. agar yellow brown Casein peptonized Casein peptonized Casein peptonized G. good G. good G. good Ycast-glucose-soil SM. brownish red SM. brown red SM. brownish claret extract agar AM. thin coating AM. white AM. -H-. pink (YGS) SP. golden yellow with SP. yellow with greenish SP. reddish yellow greenish tinge tinge brown Manure extract G. moderate (to good) G. moderate (to good) agar AM. -H-. white and pink AM. -H-. pink (Ma) Spg. Spg.  
 SA 8 SE 82 SS 26 G. very good G. good G. good C asamino- SM. orange SM. brown SM. pale ochre peptone AM. few. white Czapek SP. yellowish-brownish SP. brown SP.  
 a ar Spg. Spg.  
 ( PC colony flat with a few radial grooves and concentric humps Peptone G. very good C zapck agar SM. orange (PC SP. yellowish-brownish Spg. G. good G. very good G. good to moderate Oat-Yeast SM. brown SM. orange to brownish SM. colorless a air orange AM. few. white SP. SP. weak yellowish- SP.  
  brownish p Spg.  
 G. very good G. moderate &#34;Emerson&#34; SM. orange-brown SM. colorless agar LM. white l E SP. brown SP.  
  G. very good G. good Milk agar SM. orange brown SM. colorless (C a) SP. agar gold-brown LM. Spg. SP.  
 Casein peptonized Casein peptonizcd C zapck G. good G. good a air SM. brown-orange SM. red-orange 2) SP. yellow brown SP. yellowish Spg.  
 Tyrosine G. moderate agar SM. black (Ty) SP. black tryosine crystals dissolved p Yeast-glucosc- G. ood G. good soil extract SM. rown SM. colorless a an SP. gold-brown LM. white GS) Spg. SP.  
 Manure extract G. moderate a at SM. pale brown :1) LM. white Spg.  
 Table 3 Continued G growth SM substrate mycelium SP soluble pigment Spg sporangium C colony shape AM aerial mycelium SS 55 SS 59 SS 62 (asamino G. good. colony flat G. good. colony flat peptone- SM. ight brown AM. white Czapek AM. white AM. white agar (CFC! SP. \vezik pale brown SP.  
  G. good G good G. good SM. pale light brown SM. dark brown SM. pale light brown Milk AM. AM. AM. agar SP. e SP. yello brown SPv agar yellow-brown Casein pcptonizcd violet crystals Casein peptonized Casein pcptonized G ood G good G ood SM. rown SM. dark brown SM. rown Tyrosine AM. white AM. AM. few, white agar SP. SP. brown SP. brown (Ty) tvrosine crystals t\&#39;rosine crystals t &#39;rosine crystals ssolved &#39;ssolved issolved G. good to very good G. good G. good to very good Oat-Yeast SM. red-brown SM. dark brown SM. red brown a ar AM. dirty pink AM. few. white AM. dirty pink (GY) SP. red brown SP. brownish SP. red brown many violet crystals Spg. G. good G. good G. good &#34;Emerson&#34; SM. red brown SM. dark brown SM. red brown agar AM. pink and white AM. AM. few. thin coating. (E1 in part stronger and pink SP. red brown SP. yellow brown SP. red brown violet crystals G. good G. good G. good Yeast-glucose SM. red brown SM. dark brown SM. dark red soil extract AM. pink AM. AM. pink 2! ar SP. yellovv-bronn SP. yellow brown SP. yellow brown GS) violet crystals SA 28 SE 45 SE 89 G. good to very good G. good G. good C asamino SM. initially redbrown. SM. redbro\vn SM. brownish orange peptone later dark brown (zapck agar to black (CFC) SP. dark olive SP. strongly red-brown SP. Spg. Spg. Spg. G. good to very good Peptone SM. initially red-brown. C&#39;zapek later dark brown agar to black (PC 1 SP. dark olive ps- G. moderate to good, G. good G. good (zapek agar colonies flat (CZ) SM. orange brown SM. brown-red SM. darkhro\vn SP. weal; ochre SP. brownish-red SP. light yellowish brown Sp in part frost-like Spg. 4+. frost-like Spg. -H-. frost-like G. very good G. good Milk agar SM. black brown SM. orange brown (Cal SP. dark olive to SP. brown greenish-blzlck Spa v Casein pcptonized Casein peptomzed G. moderate to good 0. moderate Tyrosine SM. brown SM. brown agar SP. SP. dark brown [Ty] Spg. Spg.  
  tvrosine crystals tyrosine crystals dissolved under not dissolved mycclium G. good G. good G. good to very good Oat-yeast SM. dark brown Spg. -l+-. frost-like SM. orange agar on the SM. (OY) SP. light brownish SP.  
  Spg. Spg. frost-like G. good to very good G. very good Emerson&#34; SM. brown to brown-black SM. orange agar SP. ochre with greenish tint SP. (El Spg. Spg. -H-. frost-like SE IOU SE ltll SK 2 C asam ino- G. good G. good pcptone SM. pale ochre SM. rc Czapek AM. white AM. few. white agar (CPC) SP. SP.  
  G. good G. moderate Czapck SM. colorless SM. colorless to pink Table 3 Continued G growth SM substrate mycelium SP soluble pigment Spg= sporangium C colony shape AM aerial mycelium SE l SE I01 SK 2 agar AM. white AM. white (C2 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 6. good 6. moderate Oat-yeast SM. pale ochre SM. pink SM. brown agar AM. -l+. white AM. -ll-. dirty pink (OY) SP. SP. )cllowishbrownish SP.  
 Spg. -tl- G. good (relatively) Milk agar SM. golden yellow surface knobby Cat/2) SP.  
 Spa  
  Casein peptonized G. good Tyrosine SM. brownish gold-yellow to agar yellow-orange (Ty) SP. golden brown to reddish brown P2 The strains set forth in Table l 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 l0 days incubation at l5-60C.. preferably 2450C.. 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 inhibitorsthe 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 salts or watersoluble 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-l0,000 revolutions per minute. for -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 cul&#39; ture 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 l0-50 mm Hg) at bath temperatures of -100C.. preferably 40-80C., to approximately tyrosine crystals dissolved 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 (all 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 pre cipitation 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 clution 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 sub stances are retained by the membrane. or by fractional precipitation [compare (b)] 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-l0 min utes with ether. The mycelium extracted in this way is dried in vacuo and subsequently extracted for 2-8 hours with 3-l0 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 by 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 l2-24 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 l2). 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. The inhibitors cannot be inactivated with urea and B-mercaptoethanol. According to estimates from gel filtration. the molecular weight of these inhibitors is above 500 but below 6.000. On 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 AlU/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 a-mer captoethanol 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 AlU/mg.  
  It is known that in the case of animals and man hyperglyccmias 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 glycosidehydrolases (for example. salivary and pancreatic amylases. maltases and saccharases) in accordance with the following equation:  
  amylase maltasc starch maltose glucose or or glycogen saccharase sucrose glucose fructose These hypcrglycaemias 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 glycosidehydrolases. according to the invention. obtained and isolated in accordance with the above methods. considerably reduce 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. hyperlippidemia (atheriosclerosis] 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 pres ence 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.  
  &#34;Medicament&#34; as used in this specification means physically discrete coherent portions suitable for medical administration. Medicament in dosage unit form&#34; as used in this specification. means physically discrete coherent portions suitable for medical administration each containing a unit dose or a multiple (up to four 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. three times or four times a day. respectively.  
  A unit dose is the amount of inhibitor to be taken on one occasion.  
  The pharmaceutical composition 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. gran ules 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&#39;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 abovementioned diluents.  
  The diluents to be used in pharmaceutical composi tions 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., -alcohol and C 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 20 except in the presence of a surfaceactive 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, bloodisotonic.  
  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 B-cytotropic sulphonyl-urea derivatives and biguanides which influence the blood sugar level.  
  The diluent in the medicament of the present inven tion 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 from 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 5,0005,000,000 AlU, 2.5250 MlU, or l-l0,000 SlU 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 glycosidehydrolases is extremely low. The active substance from Examples 14 and 37 was tolerated, without symptoms, at a dosage of X AlU/kg of mouse or rat, administered orally. In the case of intravenous injection, mice and rats tolerated l0 AlU/kg.  
  The following examples illustrate the production of the inhibitor according to the invention.  
 Example 1 Three l-liter Erlenmeyer flasks, containg ml of a nutrient solution of composition: 2% starch, l7c glucose, 0.5% NZ-amines, l.07c yeast extract and 0.4% CaCO (sterilization: minutes, 121C; pH adjusted to 7.2 before sterilization) are each inoculated with 1 ml of a primary cultrue (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 28C. 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 AlU/ml, are obtained.  
  The centrifuged supernatant liquor is concentrated to ml on a rotary evaporator at 15-20 mm Hg and approximately 37C. waterbath temperature. The viscous solution is stirred into 8 volumes 480 ml of ehtanol, while stirring. The precipitate formed is collected by centrifuging, again dissovled in 60 ml of water, and dialyzed for 6 hours against distilled water. The dialyzate in lyophilized. Yield: 1.6 g containing 19 X It) AlU/g.  
 Example 2 Using a batch according to Example 1,440 ml of centrifuged supernatant liquor containing [00 AlU/ml are obtained.  
  These 440 ml of centrifuged supernatant liquor are concentrated to I 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: 22 g containing l X l0 AlU/g.  
 Example 3 Using a batch according to Example 1. 500 ml of a centrifuged supernatant liquor containing 120 AlU/ml are obtained after 5 days culture. These 500 ml are lyophilized directly. Yield: 7.l g containing 8.3 X &#34;Al- U/g.  
 Example 4 Three l-liter Erlenmeyer flasks containing 200 ml of a nutrient solution of composition: 3% glycerine. 3% soya flour. and 0.2% CaCO (sterilization: 30 minutes. 121C; pH after sterilization 7.2) are each inoculated with one ml ofa primary culture (obtained in the same nutrient solution. inoculated from sloping test-tube cultures containing Czapek-peptone-casein agar) of the strain SB l2. and the flasks are incubated for 3 days at 28C. 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 AlU/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 lOO ml of distilled H 0 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 X l0 AlU/g.  
 Example 5 A precipitate obtained according to Example 4 after precipitation with ammonium sulphate is dissolved in l00 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 AlU/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 AlU/ml is obtained after 3 days culture at 28C. on a rotary shaking machine. Example 7 If a mix according to Example 4 is incubated for 3 days at 32C.. a culture filtrate containing 350 AlU/ml is obtained after filtering off the mycelium.  
 Example 8 lfa nutrient solution according to Example I is inoculated in accordance with Example 4. culture filtrates containing 270 AlU/ml are obtained after 3 days incubation at 28C.  
 Example 9 Six l&#39;liter Erlenmeyer flasks. each containing 100 ml ofa nutrient solution consisting of 3% glucose. 3% soya flour. and 0.2% CaCO (sterilization: minutes. [2 lC.&#39;. pH after sterilization 7.2) are each inoculated with 1 ml of primary culture (obtained as in Example I l of the strain SB 5. and are incubated for four days at 28C. 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 AlU/ml are frozen and lyophilized. Yield: 6.) g containing ll X 10 AlU/g. Example 10 If 24 flasks of a mix according to Example 4. each containing I20 ml. are inoculated with a primary culture of the strain SB 1] (obtained according to Example 4] and incubated for 5 days at 28C.. 2.0 liters of a supernatant liquor containing l.l MlU/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 con centrated to l00 ml on a rotary evaporator and is added dropwise to 900 ml of absolute ethanol. while stirring. The almost inactive precipitate which sepa&#39; rates is centrifuged offand discarded. and the alcoholic supernatant liquor is concentrated to 30 ml on a rotary evaporator.  
 1 ml of this concentrate contains MlU/ml.  
  For further purification. this solution is passed over an anion exchage column (Amberlite IRA 410. acetate form. in 0.05 M NH -acetate. pH 7, 2.5 X 20 cm col umn) and the active fractions are combined and gelfiltered on Sephadex G in H O. The active fractions from the gel filtration are concentrated to 12 ml. 1 ml of this solution contains 150 MlU/ml.  
  The mycelium (-500 ml) was twice extracted with l 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 20C. and the resulting dry mycelium powder (-51 g) was subsequently extracted for 2 hours at room temperature with l50 ml of dimethyl sulfoxide (DMSO). After centrifuging (30 minutes. [5.000 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: [20 ml containing 3 MI- U/ml.  
 Example ii If a 1 liter Erlenmeyer flask containing [20 ml of a nutrient solution according to Example l is inoculated according to Example 9 and incubated for 6 days at 28C. on a rotary shaking machine. a culture filtrate containing 0.9 MlU/ml is obtained.  
 Example l2 If five l-liter Erlenmeyer flasks. each containing 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 (sterilization: 30 minutes. l2 lC.; pH adjusted to 7.2 before sterilization) are each inoculated with 2 ml ofa primary culture (obtained according to Example 1) of the strain SB 27 and these flasks are incubated for 4 days at 28C. on a rotary shaking machine. 500 ml of supernatant liquor containing 70 Al&#39; U/ml are obtained after combining the flasks and centrifuging off the mycelium. The centrifuged superna tant liquor is frozen and lyophilized. Yield: 7.7 g con raining 3.5 X it) AlU/g.  
 Example 13 if a mix according to Example I 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 28C.. a culture broth containing 1100 AlU/ml. 0.]7 MIU/ml and [.0 SlU/ml is obtained.  
 Example 14 If five experimental fermentcrs 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 28C. 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 MlU/ml. (1.2 SlU/ml. and 8.000 AlU/ml) are concentrated to 5 liters in vacuo at 20 mm Hg and 100C. 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 6000 rpm. 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 {1RA 410 (acetate form. pH 7)]. The mixture is separated into supernatant liquor and Amberlite sediment by centrifuging for 10 minutes at 6000 rpm. 1n the same way. the supernatant liquor is further stirred three times. in each case with 500 g of Amberlite for 60 minutes. and subsequently with a further 500 g of Amberlite overnight (-1S 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 mm Hg and 80C. 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 \acuo. Yield: 150 g of a white powder containing 1 X 10&#34; AlU/g and 450 SlU/g. Example 15 If. in a nutrient solution according to Example I. 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 28C. on rotary shaking machines:  
  lfa nutrient solution consisting of 371 soya flour. 27: starch. 1% glucose. and 0.271 CaCO is inoculated and incubated in accordance with Example 15. a culture broth containing 5.600 AlU/ml is obtained after four 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 Al- U/ml is obtained after 4 days fermentation.  
  The strain SB 18/5 was deposited at the Centraal- 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 Al U/ml is obtained after 4 days fermentation.  
  The strain SB 18/4 was deposited at the Centraal Bureau voor Schimmelcultures in Baarn. Holland. under CBS no. 612.71.  
 Example 19 If a nutrient solution of composition: 2% starch. 1&#39;72 glucose. 0.371 glycine. 0.25% corn-steep liquor. 0.471 soya flour. 0.171 NaCl. 0.1% K- .HPO,. 0.01% FeSO and 0.017: CaCO is inoculated and incubated in accordance with Example 12. a culture broth containing 2.900 AlU/ml is obtained after 3 days incubation. Example 20 If 2 flasks ofa 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 28C. on a rotary shaking machine. 250 ml of supernatant liquor containing 250 AlU/ml are obtained after centrifuging.  
  These 250 ml of supernatant liquor are treated with 150 g of ammoniumsulphate added in portions while stirring and the mixture is subsequently centrifuged for 15 minutes at 10,000 rpm. The residue is dissolved in 12 ml of H 0 and dialyzed for three hours against distilled water. The 20 ml of dialysate are precipitated with 6 volumes m1) 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 X 10&#34; AIU/g.  
 Example 21 1f a 1 liter Erlenmeyer flask containing 120 m1 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 28C.. a culture broth containing 0.09 MlU/ml is obtained.  
  The mycelium is treated with 50 ml of acetone. homegenized for 1 minute on an Ultraturrax homogenizer (Messrs. Janke and Kunkel. Staugen. Breisgau) and the mixture is subsequently centrifuged for 10 minutes at 3.000 rpm. 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 37C. 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 two hours while stirring (magnetic stirrer). Thereafter the mixture is centrifuged for 30 minutes at 20.000 rpm. 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 rpm. and the clear supernatant liquor is tested as mycelium extract. 0.14 MlU/ml.