8/8/2019 J. Nutr.-1974-Fossum-930-6

1/7

Sim ple M ethod for D etecting m ylase Inhibitorsin B iological Materials1

KARE POSSUM AND JOHN R. W HITAKERD epartm ent of M icrobiology and Im munology, V eterinary C ollegeof Norway, Oslo, Norway, and Department of Food Science andT ech no lo gy , U niver sity o f C alifo rn ia, D av is, C alifo rnia 9 561 6ABSTRACT A simple semiquantitative method for detecting amylase or amylaseinhibitors in biological m aterials is described. T he substrate consists of a 0.25% starch-1.5% agar gel slab buffered at pH 6.5 with 0.1 M phosphate-0.01 M sodium chloride.Three m illimeters wide cellulose strips saturated with a solution to be examined for inhibitor are placed parallel on the gel slab for 2 hours at 37.The strips are removed andother 3-mm wide cellulose strips saturated with amylase solutions are placed at rightangles across the first strips. The system is incubated for 6 or 18 hours at 37.Afterflooding the slab w ith Lugol (an iodine containing) solution, aniylase activity is show nby clear lysis zones on a deep-purple background. Presence of inhibitors is indicated byinterruption or narrow ing of the lysis zone w here the inhibitor-containing and am ylase-containing strips crossed. A variation of this m ethod using am ylase or am ylase-inhibitorm ixtures placed into 7-m m w ells cut into the starchagar g el slab is also described. Thestarchagar gel slab m ethods w ere com pared w ith the B ernfeld m ethod of determ iningamylase activity. J. Nutr. 104: 930-936, 1974.INDEXING KEY WORDS amylase amylase inhibitor

Many raw food materials are known tocontain various types of inhibitors whichaffect nutritional quality (1). Some ofth ese in hib ito rs a re p ro te in s w hic h d ec re asespecific enzym e activities, as for exam plethe inhibitors of proteases and amylases(2).During the past 20 years remarkableprogress has been made in methods fordetecting - ( 3 ), purifying ( 4, 5 ) and characterizing (6) the protease protein inhibitors. This progress has made possible extensive studies of the structures and m echanisms of action of these inhibitors (6),including X-ray crystallography of inhibitor (7) and inhibitor-enzym e complexes(8).Sim ilar progress has not been m ade withthe amylase inhibitors even though theyappear to be nutritionally important (9,10). A lthough the presence of amylase inhibitors in beans and wheat was noted asearly as 1945 and 1946, respectively (11,12), they have received very little attention until recently3 (13-15). R ye (10, 16),rice follow ing microbial infection (17),

mangoes (18), colocasia, a tuberous foodcrop of India (19, 20), acorns (21) and theferm entation liquor from actinom ycetesproduction4 also have been shown to contain inhibitors of amylase activity. Theamylase inhibitor(s) of wheat is stable toheat during bread production ( 22 ) .The nutritional significance of am ylaseinhibitors in foods is not clear. They couldbe important in celiac disease associatedwith the gluten fraction of wheat (10).Rats fed raw white kidney beans containing high amounts of amylase inhibitor excreted undigested starch in the feces andon autopsy the rats were found to haveRec ei ve d f or p ub li ca ti on J an ua ry 1 7, 1 97 4.1 The authors are grateful to the Agricultural Research C ouncil of N orw ay for financial support andto Professor S andvik and the D epartm ent of M icrob io lo gy an d Imm un olo gy, V ete rin ary C olleg e o f N orw ay f or u se o f f ac il it ie s."Sandvik, O . (1962) Studies on casein precipita tin g e nz ymes o f a ero bi c a nd f ac ult at iv el y a na er ob icb ac te ria . T he sis , V eterin ary C olle ge o f N orw ay , O slo.a S c hm id t, D . & P uls, W . (1 97 1) Amy lase in hib ito r.G er . O ff en 2 ,0 03 ,9 34 ; C hem. Abs tr . 7 5: 9 12 90 P ( 19 71 ).'From mer, W ., Puls, W ., S chaefer. D . & Schm idt,D . (1972) G lucoside hydrolase inhibitors from actin om yce te s. G er. O ffe n 2 ,06 4,0 92 ; C hem. A bs tr. 7 7:!iim

8/8/2019 J. Nutr.-1974-Fossum-930-6

2/7

DETECTION OF AM YLASE INHIBITORS 931bloated, white intestines (9). Am ylase inhibitory activity was detected in the fecesof these rats. Oral administration of purified amylase inhibitors from wheat had amarked effect on the rate of digestion ofstarch in tests on dogs, rats and man (23).We believe that the lack of research,both nutritionally and biochemically, onamylase inhibitors is due in part to lack ofsim ple m ethods for detecting these inhibitors. The present paper describes a sim ple,semiquantitative method for detectingam ylase inhibitors in biological m aterialswhich is ideal in screening large numbersof samples. The method can be usedequally w ell for detectin g am ylase activity .It is hoped the availability of this simplem ethod w ill stim ulate nutritional as w ell asother studies on this group of inhibitors.The quantitative potentialities of the simple method are compared with a morequantitative, but laborious, m ethod of m easu rin g in hib itio n o f amy la se a ctiv ity .

M ATERIALS AND M ETHODSM aterials. The two crude preparationso f tt-amy la se ( -1 ,4 -g lu ca n 4 -g lu ca no hy -drolase; EC 3.2.1.1) used were from swinepancr ea s 3 and Baci ll us sub ti li sInh ib it ingantibodies against sw ine pancreas a-am yl-ase were elicited by injecting a rabbit w itha suspension consisting of equal am ountsof a 10% a-amylase solution in water andFreund's adjuvant." Four-m illiliter injec

tions w ere given subcutaneously and intra-cutaneously at 7-day intervals. On day 28the rabbit was bled, the blood allowed tocoagulate, and the serum collected aftercentrifugation. T he blo od serum w as storedfrozen until used. Lugol solution was prepared by dissolving 1.0 gram of Iu. and 2.0gram of KI in 100 ml of water.Methods. The starch agar plates conta ined (final concen tration) agar7 (1.5% ),starch (0.25% ), m erthiolate (1:10,000),0.1 M phosphate buffer, pH 6.5, and 0.01 MNaCl. The agar and starch were dissolvedseparately in 0.1 M phosphate buffer, pH6.5, containing 0.01 M NaCl. The agar wasmelted by heating for 20 to 30 minutes inan autoclave, m ixed im mediately with thestarch solution and the m ixture poured intoglass trays (12 by IS cm) to a thickness of2 mm. The thickness was standardized by

pouring a fixed volum e into each tray. Theplates were allowed to stand until the agarhad so lidi fied .For detection of inhibitors againsta-am ylase, 3-mm w ide cellulose strips w eresaturated with a solution of the materialexpected to contain inhibitor, and placedin parallel on a starch-agar gel plate. Theplate was covered with a sheet of glass, andthe solution allowed to migrate into thestarch-agar gel for a period of 2 hours at37.The paper strips were rem oved, andother strips of 3 mm wide cellulose paper,saturated with enzyme solution to be examined, were placed on the agar gel atright angles to the first strips. The plateswere again covered with a sheet of glass,and incubated at 37for 6 or 18 hours.The strips were then removed, and thesurface was flooded with Lugol solution.Am ylase activity was shown by clear lysiszones. P resence of inhibitors w as in dicatedby interruption of the lysis zone, or by narrowing of the lysis zone depending uponthe strength of both the inhibitor-containing solution and the enzyme solution, atthe point where the two paper stripscrossed. We shall designate this as thecrossw ise paper strip m ethod.A sem iquantitative determ ination of inhibitory activity by the crosswise paperstrip method was performed on a serialdilution of the inhibitor-containing m aterial in order to find the highest dilutionw hic h resulte d in inhibition.Starch-agar plates were also used in asecond way for detection and sem iquantitative determ ination of a-amylase and inhibitor activities. For this purpose, w ells 7mm in diameter were made in the starch-agar gel (prepared as above) with a corkborer. A fixed volum e (25 / I)of an a-amyla se solution , or a serially diluted solution,to be tested was introduced into the wells,then the plate was covered with a tight fitting glass plate and incubated for a standard time (6 or 18 hours) at 37.At theend of incubation, the starch-agar platewas flooded w ith Lugol solution and theexcess solution poured off. The presence of

"icilia Chemical Co., St. Louis. Mo. The losim iicn-ns a-innylntip was type VI-A, lot 71C-0210 andHni-illHx militili a-ninylnxe was type III-A, lotS IM ' 1 17 O." liltc o I, al> .m il.id es, D etro it. M ie li.; Sec footnote fi. N eutral agar.

8/8/2019 J. Nutr.-1974-Fossum-930-6

3/7

93 2 KARE FOSSUM AND JOHN R. W HITAKERa-amylase activity was indicated by clearzones around the wells because of hydrolysis of starch. The highest dilution ofan enzym e resulting in clear zones is takenas one diffusion unit. This m ethod has beenextended to perm it detection and estimation of inhibitors of amylase by adding toeach well a fixed amount of amylase butvarying amounts of inhibitor (total combined volume of 25 /J). The diameter ofthe lysis zones decreased proportionally asthe inhibitor concentration w as increased.Quantitative determ ination of am ylaseand amylase inhibitory activity was doneby the Bernfeld method (24). Solublestarch 8 (0.50 g), dispersed in 50 ml of 0.1M phosphate, pH 6.5, containing 0.01 MNaCl, was dissolved by heating in a boiling water bath with constant stirring forexactly 10 m inutes. This solution was prepared fresh each day. The 3,5-dinitrosali-cylic acid9 reagent was prepared as described by Bernfeld (24). For activity determ ination, 0.20 ml of starch solution at25was added at zero time to 0.20 ml of asolution, also at 25 ,contain ing am ylase in0.1 M phosphate buffer, pH 6.5, with 0.01M NaCl. W hen inhibitor activity was to bedeterm ined, the am ylase and inhibitor w ereincubated together at 25for 30 m inutesbefore adding the starch in order to perm itmaxim um complex formation as shown byprior experim ents. A fter adding the starchto initiate reaction, the solution was incubated at 25for exactly 10 minutes. Reaction was term inated by addition of 0.40ml of dinitrosalicylic acid reagent. Forcolor development, the term inated reactions were heated for exactly 5 minutesin a vigorously boiling water bath, removed, cooled, diluted with 4 ml of waterand the absorbance read at 540 nm againsta blank prepared in an identical fashion asthe reaction except that the enzyme wasadded after the dinitrosalicylic acid reagent. A pp ropriate controls contain ing 0 .20ml of starch and various amounts of inhibitory extract (total volume 0.40 m l)were also run. The concentration of amylase should be low enough to give a linearactivity response with time for at least 15minutes. Absorbance at 540 nm can be related to concentration of reducing groupsformed by means of a standard curve; prepared w ith m altose. The stock m altose solu

tion should contain 0.20 mg maltose permilliliter.RESULTS AND D ISCUSS IONSince introduction of the casein-agarplate method in 1962 for detecting pro-teolytic activity ,2 its use h as been e xtendedto id en tific atio n o f v ario us m ic ro org an isms -(25-27), determ ination of the heterogeneity of crude protease preparations (28)and for detection and estimation of protease inhibito rs in biolo gical m ate rials (3).The principles of this valuable techniquecan be extended to include other enzymesand their inhibitors as shown here fora-am ylase and its inhibitors.The crosswise paper strip m ethod is particularly useful for detecting amylase activity and inhibitors of am ylase activity.Amylase activity is readily observed bythe presence of a clear lysis zone follow ingtreatment of the starch-agar plate withLugol solution (fig. 1). In the absence ofam ylase activity the starch is stained dark-purple by the iodine-containing Lugolso lu tio n. P re se nc e o f in hib ito rs is in dic ate dby a narrowing of the lysis zone at pointsw he re am ylase- and am ylase inhibito r-containing paper strips cross. The extent ofnarrow ing of the lysis zone is a function ofboth the amylase and amylase inhibitorc on centra tio ns . Amyla se in hib ito r p re pa re din rabbit against hog pancreas a-amylasedid not inhibit B acittus subtilis a-am ylase(fig. 1). Inhibitory activity of as many asfive to eight bio logical extra cts against fivedifferent amylase preparations can be determ in ed sim ultaneously on a single starch-agar gel plate (12 by 18 cm). Thus, themethod is ideal for screening a large number of samples for amylase or amylase inh ib ito r ac tivi ti es .The crossw ise paper strip method canalso be used to estimate the amount ofinhibitor present. For this purpose theam ylase concentration is kept constant buttwofold serial dilutions of the inhibitor-containing solution are made. Extent ofnarrowing of the lysis zone at point ofcrossing of the amylase- and amylase in

hib itor-containing strips is pro portional tothe concentration of inhibitor (fig. 2). Bydefinition, the lowest concentration of in-" MI II IIi 's i'i in -li L almn iti ir ii 's , I nr .. NVw Yor k.* E astman Kodak Co.. U ocliester, N . V.

8/8/2019 J. Nutr.-1974-Fossum-930-6

4/7

DETECTION OF AMYLASE INHIB ITOR S 933

S WINE P ANCREAS AMYLAS E

BACILI US S UBI II IS AMVLAS L

Fig. 1 Crosswise paper strip method for demonstration of a-amylase inhibitors. From left toright, the inhibitory m aterials w ere: 1, undilutedand unheated serum from rabbit immunized withswine pancreas a-amylase; 2, the same serum diluted 1:10; 3, the same serum heated to 70for5 m inutes undiluted; and 4, the heated serumdiluted 1:10. The two enzymes used were swinepancreas o-amylase (top), and a-amylase fromBacillus sub-ais a t 0.1 mg per ml. The plate wasincubated for 6 hours at 37from the time whenthe enzym e-containing strips w ere applied.hibitor giving detectable inhibition contains one unit of inhibitor per milliliter.This would be the 1:160 dilution (no. 6)in figure 2 since inhibition w as no t detectedat a 1:320 dilution (no. 7). Therefore, theundiluted sample contained 160 units ofi nh ib it or p er mi ll il it er .Detection and estimation of amylaseand/or amylase inhib itor activ ities can alsobe made by a modification of the crosswise paper strip method. In this m odified"well" method uniform size plugs ofstarch-agar gel are rem oved from the slaband a fixed volum e of solution containingamylase or amylase-amylase inhibitor is

Fig. 2 Semiquantitative determination of inhibitors by the crosswise paper strip method. Different dilutions of immune serum against swinepancreas a-am ylase, heated at 70 for 5 m inutes,were used. The serum dilutions were (from leftto right): 1, undiluted; 2, diluted 1:10; 3, 1:20;4, 1:40; 5, 1:80; 6, 1:160 and 7, 1:320. Theenzyme preparations used were swine pancreasa-amylase (top, 0.1 mg per m l and bottom, 0.01mg per ml). The plate was incubated at 37for1 8 hou rs .

Fig. 3 A mylase activity determ ined on starch-agar gel plates by the "w ell" m ethod. T he am ountsof swine pancreas a-amylase preparation were:1, 25.0 Mg; 2, 12.5 Mg; 3, 6.25 Mg; 4, 3.12 Mg;5, 1.56 Mg; 6, 0.78 Mg; 7, 0.39 Mg; 8, 0.20 Mg;9, 0.10 M g; 10, 0.050 M g; 11, 0.025 M g; 12, 0.012M g. The volume of enzyme solution applied intoeach well was 25 pi. The plate was incubated at3 7for 6 hou rs .added to each well. After incubation for6 hours (or 18 hours) at 37and treatm ent w ith Lugol solution, am ylase activityis indicated by a lysis zone around the well(fig. 3). D iameters of the lysis zones are

j if f HO UR S IN CUB ATIO N\ 5 HOURS INCU BATION

O 1.0 .O 3.0i. O G fo i D IF FU SI ON U NI TS /2 St tl Fig. 4 Standard curves for swine pancreasa-amylase activity by the "well" method, pre

pared from the data of figure 3 as indicated bythe legend. The abscissa units are the logarithmsof the number of diffusion units, and the zonediam eters are show n on the ordinate. O ne diffusionunit is the highest dilution of enzyme whichgives a clear zone. This would be well no. 10 containing 0.05 M g enzyme in figure 3.

8/8/2019 J. Nutr.-1974-Fossum-930-6

5/7

93 4 KARE POSSUM AND JOHN R. WHITAKERproportional to amylase concentration aswell as incubation time (fig. 4). In experim ents for detection and sem iquantitativeestim ation of am ylase inhibitor activity,each well contained a fixed am ount of am ylase and variable amounts of inhibitor(prepared as tw ofold serial dilutions). Thediam eters of the lysis zones w ere inverselyproportional to the concentration of inhibitor (data not shown). This modifiedmethod is not as useful as the crossw isepaper strip method for detecting the presence of amylase and amylase inhibitor inbiological materials as it requires muchm ore work and can lead to misinterpretation (3). Furthermore, it is less sensitivethan the crosswise paper strip method.H ow ever, it is m ore useful for sem iquantita tiv e w ork .Amylase and am ylase inhibitor activ itiescan be determ ined quantitatively by appropriate modifications of the Bernfeldprocedure (24) as shown by the data infigures 5 and 6. The change in absorbanceis converted to am ount of reducing groupsproduced by use of a standard curve pre-

f ig H-A MY I.A SE PER R EA CTIO N

Fig. 5 Relationship between amylase concentration and concentration of reducing groups, asmeasured by change in absorbance at 540 nm ,produced from starch as determined by the di-nitrosalicylic acid m ethod. Various amounts of astock solution of swine pancreas a-am ylase werediluted to a total of 0.20 m l with 0.1 M phosphate,pH 6.5 containing 0.01 M NaCl and brought to25. At zero time 0.20 ml of \% starch in thesame buffer equilibrated at 25 w as added toeach tube and the tubes incubated at 25 forexactly 10 minutes. Remainder of procedure asd es cribe d in tex t.

J

--r-ju l B LOOD S ER UM PER R EA CTION

Fig. 6 Relationship between inhibitor concentration and percentage inhibition of am ylase activity. Each reaction contained 20 fig of swine pancreas a-amylase, various amounts (0 to 15 n\) ofinhibitor (present in blood serum ) and sufficientbuffer to give a volume of 0.20 ml. After incubation at 25 for 30 m inutes for com plex form ationbetween enzyme and inhibitor, 0.20 ml of 1%starch in the same buffer was added. Exactly 10m inutes later the reaction w as term inated by addition of 0.40 ml of dinitrosalicylic acid reagent.Remainder of the procedure was as described infigure 5 and text.pared w ith maltose (Fig. 7). The methodis not ideal for screening large num bers ofsamples because of the large amount ofwork involved in running all the appropriate controls. Proper controls must be runon the starch and on each concentration ofamylase and amylase inhibitor used so asto assess the am ount of substances capableo f re du cin g 3 ,5 -d in itro sa lic yl ic a cid in it ia llypresent. These controls are not needed inthe starch-agar gel methods. The sensitivity of the starch-agar gel methods isapproximately 10-fold greater than thequantitative B ernfeld m ethod prim arily because of the longer incubation tim e.Regardless of the method selected, dete ction and e stim ation o f am ylase inh ibitorin crude biological extracts is com plicatedby the presence of endogenous amylase.This is demonstrated in figure 1 whereamylase activity from the extract showedup in the sample applied vertically on the

8/8/2019 J. Nutr.-1974-Fossum-930-6

6/7

DETECTION OF AMYLASE INHIB ITOR S 93 5

Fig. 7 Relationship between concentration ofreducing groups and absorbance at 540 run afterrea ctio n w ith d initro sa lic ylic a cid rea ge nt. V ariou samounts (0 to 0.8 ing) of maltose (2 mg/m l stocksolution) and water to a total of 0.40 ml werem ixed w ith 0.40 m l dinitrosalicylic acid reagent,heated for exactly 5 m inutes in a boiling waterbath, cooled and diluted with 4.0 ml water. Theabsorbance was read at 540 nm against a blankcontaining no maltose. Assays were performed inOslo () and in Davis (O).left (no. 1). Fortunately, this difficultycan be overcome by heat denaturation ofthe endogenous am ylase at 60 to 70 priorto assay for the inhibitor. In our case,amylase activity was destroyed in 5 to 20minutes at 60 to 70with no detectableloss in amylase inhibitor activity. Theexact conditions of heat treatm ent m ust bedeterm ined for each different extract. R ateof destruction of amylase activity can befollowed readily by either of the starch-agar ge l m etho ds.

LITERATURE CITED1. Strong, F. (ed.) (1973) Toxicants Occurring Naturally in Foods. Nat. Acad. Sci., Nat.Res. Council, W ashington, D . C.2. Whitaker, J. R . & Feeney, R. E. (1973)Enzyme inhibitors in foods. In: ToxicantsOccurring Naturally in Foods (Strong, F.,ed.), pp. 276-298, Nat. Acad. Sci., Nat. Res.C ouncil, W ashington, D . C .3. Fossum , K. (1970) Proteolytic enzymes andbiological inhibitors. I. N aturally occurring inhibitors in sera from different species andtheir effect upon proteolytic enzym es of various origin. Acta Pathol. M icrobiol. Scand.S ect. B . 78 , 3 50 -3 62 .4. Feinstein, G . ( 1971 ) Isolation of chickovoinhibitor by affinity chromatography on

chym otrypsin-sepharose. B iochim . B iophys.A cta 236, 74-77.5. Chauvet, J. & Acher, R . (1972) Isolation ofa trypsin inhibitor (Kunitz inhibitor) frombovine ovary by affinity chromatographythrough trypsin-sepharose. FEES Letters 23,317-320.6. Fritz, H . & Tschesche, H . (eds.) (1971)Proceedings of the International C onferenceon Proteinase Inhibitors, Munich, 1970,W alter de G ruyter, B erlin.7. H uber, R ., K ukla, D ., R hlm ann,A . & Steigemann, W . ( 1971 ) The atomic structure ofthe basic trypsin inhibitor of bovine organs(Kellikrein inactivator). In: Proceedings ofthe International Conference on ProteinaseInhibitors (Fritz, H . & Tschesche, H ., eds.),pp. 56-64, M unich, 1970, W alter D . Gruyter,Berlin.8. Blow, D. M ., W right, C . S., Kukla, D ., Rhlm ann, A., Steigemann, W . & Huber, A . ( 1972)A model for the association of bovine pancreatic trypsin inhibitor with chym otrypsinand trypsin. J. M ol. Biol. 69, 137-144.

9. Jaffa, W . G. & Vega Lette, C . L . (1968)H eat-labile grow th inhibiting factors in beans(P haseolus vulgaris). J. N utr. 94, 203-210.10. Strumeyer, D . H. (1972) Protein amylaseinhibitors in the gliadin fraction of w heat andrye flours. Possible factors in celiac disease.Nutr. Rep. Int. 6, 45-52.11. Bowman, D. E. (1945) Amylase inhibitorof navy beans. S cience 102, 358-359.12. Sandstedt, R . M . & Beckord, O . C. (1946)P hotom icrographic studies of w heat starch. II.A mylolytic enzym es and the am ylase inhibitorof the developing w heat kernel. Cereal C hem .23, 548 -558 .13. Shainkin, R . & Birk, Y . (1970) a-Amylaseinhibitor from w heat, isolation and characterization. B iochim . B iophys. A cta 221, 502-513.14. Saunders, R. M . & Lang, J. A . (1973)a-Am ylase inhibitors in Triticum aestivum:p urific ation a nd ph ysico -ch em ica l p ro pertie s.P hy to ch em is tr y 1 2, 1 23 7- 12 41 .15. Jaffa, W . G., M oreno, R. & W allis, V . (1973)Amylase inhibitors in legume seeds. Nutr.Rep. Int. 7, 169-174.16. Kneen, E. & Sandstedt, R . M . (1946) Distribution and general properties of an am ylaseinhibitor in cereals. Arch. Biochem. 9, 235-247.17. Shishiyam a, J., Oguchi, T . & Akai, S . (1968)B io chemica l p ro pertie s of th e p ro te in frac tio nsin diseased leaves of rice plants in the earlystages of H elm inthosjw riitm oryzae infection.Nippon Shoktibutsu Byoir Gakkaiko 34, 23-27; Chem . Abstr. 69: 57506y (1968).18. Mattoo, A . K. & Modi, V . V. (1970) Partialpurification and properties of enzym e inhibitors from unripe mangoes. Enzymologia 39,237-247.19. Narayana Rao, M ., Shurpalekar, K . S. &Sundaravalli, O . E. (1967) An amylase inhibitor in Colocaste esculenta. Indian J. Biochem . 4, 185.20. Narayana Rao, M ., Shurpalekar, K . S. &Sundaravalli, O . E. (1970) Purification and

8/8/2019 J. Nutr.-1974-Fossum-930-6

7/7

936 KA RE POSSUM A ND JOHN R . W HITA KERproperties of an am ylase inhibitor f rom colo-casia (Colocaste esculenta) tubers. Indian J.B io ch em . 7 , 2 41 -2 43 .21. S tank ov ic, S . C . & M arkov ic, N . D . (1960-61) A study of am y lase inhibitors in theacorn. G lasnik H em . D rustv a, B eograd 25-26,519-525; Chem . A bstr. 59: 3084d (1963).22. B essho, H . & K urosaw a, S . (1967) Enz ym einhibitors in foods. III. Ef fect of cook ing onthe am y lase inhibitor in f lour. Eiy o T oShokury o 20, 317-319; Chem . A bstr. 68;1 13 47 4e (1 96 8).23. Puls, W . & K eup, U . (1973) Inf luence ofan a-am y lase inhibitor on blood glucose,serum insulin and N EFA in starch loadingtests in rats, dog and m an. Diabetologia 9,97-101.

24. B ernfeld, P. (1955) A m y lases, a and .M ethods in Enz ym ol. 1, 149-158.25. S andv ik , O . (1967) Identif ication of m ouldsby serologie dif f erentiation of their prote-o ly tic en zy m es. A c ta Path ol. M icro bio l. S can d.7 1, 3 33 -3 38 .26. S andv ik , O . & Hagen, O . (1968) S erologi-cal studies on proteinases produced byAeromona s salmoni cida and o the r a eromonads .A cta V et. S cand. 9, 1-9.27. Dahle, H . K . & N ordstoga, K . (1968) Identif ication of aerom onads in furred anim als.A cta V et. S cand. 9, 65-70.28. Dahle, H . K . (1970) Z ym ogram s in agar gelof som e anim al and bacterial proteinases.A cta Pathol. M icrobiol. S cand. S ec. B 78,575-580.