T H E J O U R N A L O F I N D U S T R I A L A h - D ELYGINEERING C H E M I S T R Y Another experiment was tried following t h e suggestion of Reynolds Green (“Soluble Ferments,” p. 2 2 7 ) . Five grams of fat-free material were extracted with IOO cc. of 5 per cent NaCl containing 0 . 2 per cent K C S for -2 hours at 3j-40’. T h e preparation v a s filtered a n d trials made ( I ) directly with t h e filtrate a n d ( - 2 ) with t h e filtrate after “actirating” b y making slightly acid with dilute acetic acid a n d allowing t o T h e unactivated s t a n d z hours longer a t 3j-40’. filtrate ( I ) showed no pon-er t o hydrolyze b u t y r i n ; t h e activated filtrate ( 2 ) was decidedly active. T w e n t y cc., equivalent t o one gram of soy bean meal, acting upon c . 2 j cc. butyrin required a t 24-, 48- a n d j z - h o u r periods 2.0: 1.7 a n d 2 . 0 cc.: total 5.7 cc.: N/ION a O H t o neutralize t h e acidity developed. T h e control in 15-hich t h e extract h a d been boiled required a t corresponding inter\-als 0 . 6 , 0.7 a n d 1.0cc., or a total of 2 . 3 cc. This activated extract also showed lipolysis of nn emulsion prcpared v i t h soy bean oil. I t appears t h a t t h e spontaneous acid production of t h e soy bean itself h a s been largely eliminated b y t h e above process. T h e amounts of acid developed b y t h e control are uniform a n d much less t h a n in previous controls, a n d t h e decomposition of butyrin is uniform a n d progressive, not diminishing as in previous trials. There is present t h e n a t r u e lipase. which is dissolved b y j per cent XaC1 a n d n-hich is either activated b y acetic acid or generated b y t h a t acid from its zymogen. co~cLusIos I n addition t o t h e urease, amylase a n d glucosidesplitting enzyme reported by other workers, t h e soy bean contains also a protease of t h e peptoclastic t y p e , a peroxidase a n d a lipase. Negative results have been obtained for sucrase a n d protease of t h e peptonizing type. I t was thought unnecessary t o examine t h e material for urease a n d no a t t e m p t n-as made t o corroborate t h e presence of t h e glucoside-splitting The presence of a n active amylase h a s enzyme. been corroborated.
Val. 7 , h-0. I O
found t h a t t h e method is altogether t o o exacting in order t o be serviceable in technical work. Unless t h e determination is carried out with t h e utmost care t h e results fail t o be reliable. Owing t o t h e varying composition of t h e gelatine employed a n d t h e exacting conditions for performing t h e experiment, such as a fixed incubation temperature at n-hich t h e solution is t o be kept for a number of hours, t h e necessity of ice for chilling t h e water, t h e special preparation of a bacterial acid solution, etc., I have found t h a t , when employing this method i n practice, considerable difficulties are encountered. As a m a t t e r of fact i t seems almost impossible for two chemists working independently a n d each employing reagents of his own preparation, t o arriT-e at similar results. Although I have h a d occasion t o experiment with almost every one of t h e known methods for determining t h e extent. or progress, or products of proteolysis in malt solutions. such as t h e Fuld-Levison-Edestin method. t h e electrical conductivity method, e t c . , I have found t h a t , aside from t h e gelatine method above mentioned. there is only one other method which is applicable t o malt solutions. This method is t h e one I have employed a n d has for its basis t h e Soerensen formaldehyde titration as originally published in Biochem. Ztschr.. 7 (1908). 4 j . It has been so modified by me as t o greatly simplify manipulation in order t o p u t t h e method within t h e reach of every one accustomed t o relatively simple laboratory work. By t h u s simplifying t h e method with only a comparatively slight sacrifice as t o accuracy, I have been able t o reduce experimental error t o a mini m u m so t h a t t h e results obtained will be of such degree of correctness as t o be equal in every respect t o t h e usual standard of accuracy maintained in technical analysis. Furthermore, t h e preparation of t h e different reagents has been greatly simplified, a n d t h e entire work so arranged t h a t t h e estimation does not require more t h a n a total of j o minutes in time. A N A L Y T I C A L L.4BORATORY T h e principle upon Thich t h e formaldehyde method C O N X E C T I C U T t \ C R l C U L T U R A L E X P E R I M E N T STATIOX depends is t h e estimation of reacting carboxylic-acid h-i~rnHAVEN groups present in a digestion mixture. An amino . ACID RATIO : A NEW METHOD FOR DETERMINING THE acid, owing t o its amphoteric character, cannot be directly titrated by a s t a n d a r d alkali solution in t h e PROTEOLYTIC STRENGTH OF GERMINATED presence of a n indicator. If, however, formaldehyde GRAIN 1N TECHNICAL ANALYSIS in excess be added before t h e titration, t h e amino B y CARL A. SOWAK Received bray 10, 1915 groups are converted into methyleneimino groups, T h e proteolytic activity of malts has been studied losing their amphoteric character, whereupon t h e y can extensively b y R . W a h l , t h e results of whose experi- be directly t i t r a t e d with standard alkali in t h e presence ments were published in t h e P i o c e e d i i z g s of t h e 8 t 1 ~ of phenolphthalein. T h e result of this titration is usually expressed as I n i e r n a t i o ? z a l C o i i g y e s s of A p p l i e d C h e m i s t r y , 14, 21j--220. I n this paper D r . Wahl gives t h e t h e percentage a m o u n t of total nitrogen in a mixture details of a method devized by him, based upon t h e which enters into reaction with formaldehyde. For principle first made use of by Schidrowitz; namely, t h e purpose of malt valuation this is, however, not t h a t t h e proteolytic enzymes exert a liquefying action necessary, as we are not so much concerned with abupon semi-solid gelatine a n d t h a t t h e relative pro- solute values as with relative values. VV7hat we wish teolytic strength can be measured b y t h e extent of t o determine is which malt or t y p e of malt yields t h e this liquefying action. B y using this method Wahl greatest a m o u n t of amino acids, a n d also t h e extent has arrived a t a series of very valuable results regarding t o which this degradation of t h e proteids proceeds t h e peptic powers of various malts prepared from dif- within a given time in different types of malt. I t is, ferent t y p e s of barleys. T17hile I have made use of therefore, equally serviceable a n d far more convenient Wahl’s method on a number of occasions, I have t o make use of t h e titration figures a s t h e y are ob-
Oct., 1 9 1 j
T H E J O U R N A L 0 F I.1' D US T RI A L A N D E S G I L\TEE RI dVG CH E M I S1 'R Y
tained, rather t h a n t o first convert these into percent- of t h e filtrate remaining from t h e 2 0 cc. used for t h e ages of nitrogen, a n d t h e n t o make t h e comparison. above determination is permitted t o s t a n d a t room A big saving in time a n d labor is t h u s effected. temperature for a given time, preferably over night T h e manner in which I operate is as follows: I for a period of 16 hours, after which time a second prepare a cold water extract of malt with distilled water acid ratio determination is made. Malts showing t h e a t room temperature in t h e proportion of I p a r t of greatest increase i n t h e acid ratio are those having t h e malt t o 3 of water, using 5 0 grams of malt finely greatest peptic strength. This increase :n t h e acid ground. T h e water a n d t h e malt are shaken at room ratio m a y amount t o from j t o 30 or even more. temperature for a period of 30 minutes b y means of T h e reagents employed in t h e above determinations some convenient shaking device, a n d t h e n promptly aside from t h e t e n t h normal alkali found in every Iaborafiltered into a clean, d r y flask. About I O cc. are first tory are prepared as follows: permitted t o pass through t h e filter a n d are t h e n 0.5 p e r ceizt solzrtioii of p h e u o l p h t h a l e i n in soyGalcohol returned t o t h e filter again, this procedure being re- t o which is added a sufficient amount of t e n t h normal peated a second time. Exactly 4 j minutes after t h e soda t o impart t o i t a slightly pink color. t i m e of starting t h e extraction or 1 5 minutes after re40 p e r c e n t j o r m a l d e i i y d e solzilioii (technical strength) moving t h e solution from t h e shaking machine, a por- t o which is added j per cent by volume of t h e above tion of t h e filtrate is titrated in t h e following manner: phenolphthalein solution a n d a sufficicnt amount of T w e n t y cc. of t h e solution are placed in a beaker of j o o t e n t h normal soda t o impart t o it a slight b u t decc. capacity a n d about 2 0 cc. of distilled water a n d 0 . j cc. cided pink color. In conclusion, I wish t o point out t h a t t h e acid ratio of standard phenolphthalein solution are t h e n added, method developed b y me can be used t o advantage whereupon t h e mixture is titrated with T h e number of cc. of t h e .!\'/IO sodium hydrate re- in determining whether a certain malt has been prequired t o produce a faint pink coloration are noted. pared from a specific t y p e of barley or whether i t repreT h i s titration represents t h e natural acidity a n d can, sents a mixture. Aside from its great simplicity it if so desired, be figured over t o a percentage basis a n d has t h e further ad\-antage t h a t one single determination b y multiplication with t h e factor 0.009 be stated in suffices for t h e estimation of both t h e preformed amino acids present in t h e malt a n d t h e proteolytic strength, terms of lactic acid. However, this is not necessary, inasmuch as t h e titration figures themselves serve a n d furthermore, t h a t all of t h e work can be carried out a t room temperature and will yield reliable resrilts equally well for purposes of comparison. Ten cc. of without requiring special sliill and a degree of accuracy t h e standard formaldehyde are next added a n d owing t o t h e reaction Kith amino groups the mixture becomes which, owing t o t h e time required, would render the strongly acid. T h e titration with ~ V / I O hydrate is method unsuitable for technical work. t h e n continued until t h e solution has assumed a deC H E M I C A L LABORATORY, THE \VX. R A I I R SOKS' C O X I ' A N Y ~IAKITOW \Vrscossrs OC, cided pink color. The additional amount of acid necessary t o neutralize this increased acidity gives t h e measure of t h e amino groups which have entered into A MODIFIED METHOD FOR DETERMINING CARBONFREE ASH I N PLANT SUBSTANCES t h e reaction. For brewing purposes a malt having t h e By GEOKGBE. BOI.TZ greatest amount of amino groups is t o be preferred, b u t Received May 7 , 191.5 only provided t h e original acidity has been fairly In t h e usual method for determinirg ash in plant high, a n d t h e relation or ratio between t h e formol acidity a n d t h e natural acidity, obtained b y dividing substances, where no pro\-ision is made for purifying t h e number of cc. representing t h e amino acids by t h e t h e ash, the error due t o t h e presence of carbon dinumber of cc. representing t h e natural acidity, is as oxide in combination with bases present is, in many I : 1.00 or greater. This ratio which I designate as cases, quite large. T h e percentage of error is in prot h e a c i d r a t i o o,f m a l t is a n e n t i r e l y new ,factor in malt portion t o t h e amount of basic material t h a t is free valuntioiz. A really good malt should be high in original t o unite with carbon dioxide during combustion. acidity a n d should shon- a n acid ratio of about I : 1.10, Ash containing large amounts of calcium, magnesium which for convenience sake I express simply as 110. a n d potassium should he corrected for t h e carbon diThere are some very good malts, especially low dried oxide i t contains, mhile in most determinations of ash distillers' malts, which will have a n acid ratio as'high a correction for unburned carbon, a n d in some cases, as 130, although I ha\-e failed t o find a n y having a a correction for sand, is necessary. -4 procedure which has proved t o be very satisfcichigher ratio. Large berried Western barleys (Bay Brewing, White Club, etc.) yield malts having a low tory in determining t h e real ash in plant substances acid ratio (about 88) ; two-rowed barley malts have a n is as follows: Veigh from z t o I O grams, depending upon t h e maacid ratio of a b o u t 90 t o 100,a n d regular six-rowed malts prepared from Manchuria barley about I I O t o terial, into a platinum dish. Ignite over a low flame until most of t h e carbon is burned off. Cool, cover 120. It is quite likely, however, t h a t these standards for t h e different types of malts are subject t o change, t h e dish with a watch glass a n d add through t h e lip like all other malt characteristics, from season t o of t h e dish about 2 0 cc. of hot distilled viater. Filter into a weighed zoo cc. Erlenmeyer flask, wash season. I n order t o obtain comparative figures as t o t h e t h e residue 3 or 4 times xvith hot water, replace t h e proteolytic strength of different malts, t h e balance filter paper 1%-ithresidue in t h e platinum dish. dry ~
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