T H E J O U R N A L O F I N D C S T R I A L d.VD ELVGILVEERISG C H E M I S T R Y
148
1701. 9 , No.
2
a n d contained 300 mg. of t i n per kg., were boiled for was greatly diminished by adding it t o chopped meat about j hours with water a n d t h e three protein sol- a n d submitting i t t o a n artificial gastric digestion. vents, I O per cent NaCl solution, 7 0 per cent alcohol One mg. of mercury as bichloride will kill a 250-g. a n d 2 per cent HCI, a n d filtered through hardened guinea pig in 4 hrs. if injected subcutaneously, toxic filters. I n t h e first three cases-water, alcohol a n d symptoms beginning i n a few minutes. The same salt solutions-only 32. j , 3 2 . 8 a n d 3 j . 8 per cent, quantity of mercury, after combining i t with tissue respectively, of t h e tin was found in solution. T h e as described above, produced no toxic symptoms a n d hydrochloric acid seems t o break up t h e tin corn- death did not follow until t h e fifth day. Rabbits pound slowly on boiling a n d after j hours 2 j.4 per also were injected without apparent harmful effects. cent of t h e tin was still found combined with t h e solid R 6 S V M 6 A K D CONCLUSIONS residue. T h e question as t o whether t h e tin v h i c h It has been shown t h a t the solution of tin by canned is adsorbed by these proteins passes through the profoods is neither dependent upon, nor proportional to, cesses of digestion without being absorbed is of first t h e acidity alone also, that in the foods of relaimportance. w e have mentioned this point in retively slight acidity which dissolve large amounts of gard t o t h e tin which x i s found combined in t h e seeds tin, the greater part of the tin is in the form of an inof berries a n d in addition have performed t h e follov7soluble and stable complex. The explanation which ing experiments t o obtain further information. agrees most closely with t h e observed facts is t h a t Artificial gastric digestions were carried out upon we are dealing here with adsorption phenomena; t h a t t h e solid residue obtained b y boiling canned squash, t h e tin. after being dissolved from the lining of the which contained 300 mg. of t i n per kg. with water c a n , is being constantly removed from solution b y a n d filtering. This solid residue contained about 67 the proteins, carbohydrates and other highly porous per cent Of the tin in the 'quash The solid phases in contact with the solution. Whether gastric j u i c e , pepsin in O.35 per cent HC1* was 'Idded we regard this as an adsorption of tin ions, or whether to the squash and the mixture kept in a thermostat we consider t h e tin salt to be first hydrolyzed and t h e at 3 6 0 c. f o r 24 hrs., after which it was transferred resulting stannous hydroxide adsorbed, in either t o a dialyzer a n d t h e t i n i n solution determined i n t h e the acid would be regenerated and able to attack usual way. Less t h a n I O per cent of t h e t i n was found tin. The former explanation to be the in solution. Both gastric a n d t r y p t i c digestions were probable; i. e., the tin ions are adsorbed, since tin kindly carried out f o r us b y Dr. E. N. Harvey, of t h e is taken up equally well b y proteins even from conBiology Department, on t h e t i n Protein complex! centrated acid solution. I t will be seen f r o m t h e prepared by the protein above results t h a t while i n several respects t h e obt o s t a n d i n contact with t i n solutions, after i t h a d been served phenomena appear to be true adsorptions, allowed t o d r y , a n d in each case only a trace of t i n was i n one important respect they differ. While a true found in solution. I t appears from the above results adsorption is an equilibrium and can be approached t h a t t h e t i n protein combination which is formed is from either side, being reversible, this removal of tin . v e r y stable, and in most Of the foods containing the is not a reversible action, f o r if t h e t i n protein complex larger amounts of t i n , t h e greater p a r t is in a n insolu- is transferred to an aqueous solution containing no ble form. T h e possibility suggests itself t h a t the tin, it does not lose tin to the liquid phase. A numadsorbed be ber of cases similar t o this are known a n d have been p a r t of t h e t i n which is so Taylor, I L P S e U ~ o ~ a ~ S O r p t ~ o ~ S , eliminated directly i n t h e actual digestive processes called by a n d not figure i n t h e physiological action as deter- The removal of heavy metal salts from solution b y mined for soluble tin salts. charcoal is a n example of this t y p e of action; t h e first T h e work of J. p. Atkinson on t h e electrolYsis Of stage m a y be a n adsorption, since t h e salts of heavy metallic salt solutions t o which chipped beef h a d been metals are strongly adsorbable, but a secondary readded is of interest i n this connection. A known action have taken place and the final state a m o u n t of t h e metal i n t h e form of a soluble salt was not be put down to adsorption alone. added t o finely divided beef a n d t h e n submitted t o The author wishes t o express his appreciation a n d artificial gastric digestion for 24 hrs. a t 37' after t h a n k s for t h e very valuable assistance a n d advice which t h e solution was electrolyzed for 4 j t o j o hrs. given by Dr. G. A. Hulett in connection with this A few typical results follow: work.
w. w.
must
METAL Mercury . . . . . . . . . . . . Mercury.. , , , . , , , , , . Tin ....... , , , . . . . . . . Tin ,,,, ,,,, Zinc . . . . . . . . . . . . . . . . Nickel.. .. , ..... , , .. Iron . . . . . . , , .. , , . . . .
.......
.
.
Added Gram 0.0500 0,0500 0.0330 0,0330 0.0500 0,0492 0,0500
Recovered Gram 0.0121 0.021i 0.0051 0.0063 0.0561 0.0497 0.049i
DitTerence Gram -0.0379 -0.0283 -0.02i9
-0.0267 f0.0061 +0.0005 -0.0003
Per cent Recovered 24.1 43.4 15.5 19.1 100.0 100.0 99.7
I t appears t h a t t h e metals of relatively low toxicity are least firmly bound and he suggests 'Ghat this may offer an explanation of t h e relative toxicity of metals, i n t h a t t h e y interfere with t h e metabolism of t h e cell. Iron, being so easily separated, adds t o this view. I t was found t h a t t h e toxicity of mercury as the bichloride
LABORATORY OF PRYSICAL CHEMISTRY, PRINCETON, N . J. AND BUREAU OF CHEMISTRY, WASHINGTOK, D. C.
THE UTILIZATION OF AMMONIUM CHLORIDE BY YEAST . .
.
H. HOFFMAN By CHARLES Received January 6 , 1917
The economy resulting f r o m the use of certain mineral salts, especially calcium a n d ammonium salts, as yeast foods in baking, has been shown in a previous paper2 by Dr. H . A. ~~h~~~ and the writer. ~t was
, %, w, Taylor, ',Chemistry of Colloids,,, p, 252, 2
THISJOURiY.4L: 8 (1916), 78.1.
Feb., I 91 7
T H E JOL-RA\TA4L O F I X D I - S T R I A L i l N D E A V G I S E E R I S G C H E M I S T R Y
there s h o m i t h a t the use of ammonium chloride or other ammonium salts, lb. per 1.000lhs. f o u r used n i t h the water in the dough batch, produces a saving of 3 0 per cent of t h e usual quantity of yeast and a t t h e same time decidedly improves t h e quality of the bread. When calcium sulfate and potassium bromate are used in addition t o t h e ammonium chloride a saving of j o per cent in yeast results. I t was also stated. in this paper, t h a t t.hc ammonium chloride disappeared during t h e fermentation and t h a t it cotild not be recovered as ammonia from the bread. The present work substantiates our previous finding in t h a t i t shows t h a t t h e ammonium chloride disappears during the fermentation and as a result i t must be changed into albuminous substances [yeast protein) b y t h e yeast. I t further shows a t exactly what stage of t h e fermentation it is consumed b y t h e yeast. The results of these experiments show positively t h a t ammonium chloride added t o t h e dough batch is a yeast food, and during t h e progress of t h e fermentation is utilized b y t h e yeast for building new yeast cells. T h e fact t h a t t h e ammonium salt disappears most readily during t h e 3rd. 4th and j t h hours of t h e fermentation, just a t the time when t h e yeast shows its most active reproduction in t h e dough, a n d t h e fact t h a t no ammonia from t h e added ammonium salt is recoverable as such from t h e bread (properly fermented) upon distillation with magnesium oxide, lead t o the conclusion t h a t the yeast consumes i t a n d changes i t into a yeast protein. Evidence, too, t h a t i t is a yeast food and not a mere enzyme stimulant or catalyst is shown b y t h e experiment in which no increased gas production results when ammonium chloride is added t o a cane sugar solution, using distilled water, whereas, when adcfed t o a dough where me have t h e necessary elements for yeast production a significant increase, 3 5 . 3 per cent in t h e quantity of gas produced, is noted. Nor can t h e disappearance of the ammonium salt be attributed t o enzymes of t h e flour or t o bacterial action, for in a dough made of flour. water, salt, sugar and ammonium chloride. t h e amounts of ammonia recoverable a t t h e time of mixing and after standing j hours was identical. The added ammonium salt t h e n functions as a food f o r t h e yeast which is converted b y it into albuminous matter and does not serve as a gluten conditioner. and if such action is noted it is a secondary one, being t h e result of t h e decomposition of the ammonium chloride. The idea of econoniically using ammonium salts t o stimulate t h e growth of yeast in t h e dough, and thereby saving a large portion of t h e yeast otherwise necessary t o leaven t h e bread satisfactorill-. is no\-el. Hon-e\-er, other writers haT-e found and agree t h a t ammonium salts used in t h e fermentation of n.ine musts. hcer worts, distillery mashes and synthetic media, serve as yeast foods. Pasteur used ammonium t a r t r a t e in what is known as Pasteur's fluid for gro\Ting yeast. Jagol agrees t h a t ammonium salts are consumed b y t h e yeast for building u p albuminous substances. Ehrlich? finds t h a t ammonium salts are 1
"The Technology of Bread-Making," by William Jago, p 159. 32, 603-10; Biochein. Z.,18: 391-423.
M o i i . sci.,
I49
readily transformed into albuminous material by yeasts, and their presence in a fermenting media pre\Tents the formation of fu+sel oils and succinic acid which he finds are formed from such amino products as valine, leucine, isoleucine and glutaminic acid. T h a t these higher alcohols ifusel oil) and succinic acid are formed in the dough is then self-eTitlent since we have an abundance of these b:ises, especially glutaminic acid. which forms nearly 30 per cent of the protein present in t h e flour. The writer has added fusel oil t o a dough batch and obtained a flavor a n d odor not a t all unlike many commercial breads that v e r e made with excessive yeast or were not properly fermented. When using ammonium chloride, these higher alcohols and succinic acid t h a t are formed. and remain in t h e bread when baked. are much diminished in quantity, and as a result. we get a sweeter loaf, and one of a flavor more like the natural flavor of wheat. The work of G. Heinzelmann and Joh. Dehnickel strongly supports t h e writer's v i e w in this matter. They find the production of higher alcohols t o be greatest in grain mashes, less in molasses and least in potato mashes. Their work shows t h a t the nitrogenous yeast food in t h e mashes is of t h e utmost influence on t h e production of higher alcohols. They find t h a t if asparagine a n d ammonium salts are added t o the mash a diminution of higher alcohols results and t h a t their production m a y even be almost completely inhibited b y a sufficient quantity of asparagine or ammonium sulfate. T h e more recent work of Delbrfick and Classen,2 in which they utilize ammonium salts as t h e main source of nitrogen for growing yeast on a commercial scale, shows how readily available t h e ammonium salts are for yeast food. The work of volt^,^ moreover, shows t h a t yeast grown from ammonium salts is alike in composition t o a n y other grown yeast, and t h a t its physiological action and nutritive value is the same. The most convincing proof. however! t h a t t h e ammonium chloride is a source of nitrogenous food for t h e yeast is shown b y t h e experiments performed in the laboratory in which yeast vas grown in a solution containing cane sugar, ammonium chloride and calcium sulfate together with the necessary inorganic phosphates. The yeast thus grown was then utilized for baking a batch of bread. The yeast functioned lilie the regular compressed yeast and maintained a \-igorous a n d healthy fermentation in the dough. The accompanying photograph shows the bread made from the yeast grox-n on mineral sal's. having n m monium chloride as its sole source of nitrogen. Since there is so much evidence in fa\-or of :ill the ammonium chloride being changed into protein by the yeast, we can calculate how much protein we gain, in the form of yeast protein, from adding 0 . j lh. of ammonium chloride in a dough batch containing 1.000lbs. flour, and h o much ~ protein we lose by Z. .S#iYi/u.sind., 38, 316, 328, 34;. "A l i e w Method for Increasing t h e Production of Yeast," Z . I-ev. detrt. Ing., 59 (1915). 844. 3 "Utilization by the Animal Organism of \*east Produced from Sucrose and Nutritive Mineral Salts," Z . S p i r i i u s i n d . , 38 (1915), 235-6. 1
?
Banan BAXBOw i l i i Y X A S ?GxuY;~. O N MWSR~(I. SALTS HAVIYE AVMONIIIYC i i i o ~ ~ oAaS Irs SOL= SOUXCBOP ~ - ~ K O C . I I N
r y for the production of t h e compressed yeast t h a t is eliminated by t h e use of yeast foods. For determining ammonia in this n.ork, 50 grams of moisture-free brcnd mere extracted with joo cc. distilled water for 3 hours, with shaking at frequent intervals. Tlic extract vas t h e n filtered a n d 300 cc. taken and rlistillcri with j grains of MgO. I n t,hc experiments using dough. enough dough was usccl t o give j o firtinis of ilry material and distillcd water added t o make t h e totid water j o o cc. T h e dough was then carefully broken u p by hand, working it in t h e water until all tlie starch was washed o u t and t h e gluten collected into a ball. After settling, t h e ext r a c t was filtered and 300 cc. used as above. Tlie ammonia wits collected in N / i o acid, using incthyl orange for a n indicator. Very good results are ohtaincd and no trouhle cxpcricnced i n getting dupliby t h i s method if t h e r a t e oi distillntion a n d t h e riuaiitity of distillate are t h e same in each case. Table I shows Lhc ammonia, figured RS ammoniuni chloride obtained hy t h e distilhtion oi extracts oE dough and bread made with a n d without nmmnnium
,_ I lie total :~nimoni:i as ;ammonium chloride wiis delei-inincd at various stages of fermentation f r o m tlie dough extracts from t h e t w o formulas. I t is seen t h a t in E s p t . I t h e amount of ammonia obinincd on distill:%tion, though considerable, did not inaterinlly v a r y during t h e progress of t h e iermentation. 13rc:~d was IxAcd :it various interviils of fermentation m i l the ammonium chloride equirnlent for t h e totzl batch determined. The ammonium chloride equivalent obtained f r o m t h e dough in Z x p t . I1 at various stages in t h e fernieni:rt.ion jiroccss, decreased most markedly during 4 t h a n d jtli hours. ammonium chloride equivalent of a dough williout added ammonium chloride is o. 135. If we deiluct t,his from 0.7 2 5 we get o . 590 gram of ammouium chloride actually i n t h e dough a n d this agrees almost pcrleci,iy wiih t h e quant.ity 0.5 9 2 t h a t is added in the lormula. At t h e end of j hours fermentation t h e quantity of added ammonium chloride left in t h e dough is t h e difference between 0 . 1 6 9 a n d 0.r 3 j gram or o.o,z4 gram. Since most doughs are fermented 4 . 5 ti) j hours, some even 6 and 7 hours i n practice before scaling t h e loaves, a n d a n additional z hours during molding a n d proofing before t h e bread is baked, t h e consumption of t h e salt is complete when t h e yeast food is used i n baking arid t h e results of t h e analysis on t h e bread bear this out. 111 E s p t . 11, 5 hours fermentation yielded t h e best bread, under t h e conditions of t h e experiment, a n d therefore represents t h e time best suited f o r properly maturing a n d conditioning t h e dough. The bread without a n y added ammonium chloride yielded i n some cases a trifle more, a n d in others a trifle less, ammonia t h a n t h e bread with j hours fernientation a n d made with tlie ammonium salt. There is, therefore, no material difference i n t h e a m monia content as measured b y MgO distillation of bread made with a n d without ammonium chloride. T h e I m a d having ; hours fermentation and having yeast food added t o t h e dough when tested with slcr's reagent, showed no greater coloration t h a n bread which h a d no ammonium salt added t o t h e dough. T h e writer has been unable t o distinguish, b y t h e Xessler test or by distilling with MgO, commercial brcads made with t h e yeast food from those
Feb., 1917
T H E JOURNAL OF INDUSTRIAL A N D ENGINEERING CHEMISTRY
made without. I n fact, all breads, whether made with a m m o n i u m salts or n o t , give a slight; coloration if Nessler solution is applied t o t h e crumb. It was found, however, t h a t cheese, eggs, cakes, salt-rising bread, meat and meat extracts, a n d particularly saliva, gave very positive tests for ammonia with Nessler's reagent. E v e n compressed yeast gives a n u n mistakable test for ammonia. Tests for ammonia in a commercial bread made with t h e yeast food containing ammonium chloride a n d other breads bought in t h e market were made according t o t h e directions given b y Prof. J. C. Summers' i n a paper before t h e members of t h e National Association of Master Bakers at Salt Lake City a n d b y distilling a water extract from these breads with MgO. T h e results are given in Table 11. TABLE 11-TEST
AMMONIA O X V A R I O y S BREADS Per cent Ammonia Per cent Ammonia NO. (Nessler Solution) (Distilled with MgO) 0.0040 0.0045 3 (made with yeast food).. . . . 0.00008 0.0045 0,00016 0,0040 0.00012 0.0040 0.0051 7 Salt-rising.. . . . . . . . . . . . . . . 0.00024 0.0153 8 Yeast.. . . . . . . . . . . . . . . . . . . 0.00024 0.0085 FOR
Bread
.. .. .. ..
Although t h e method given b y Prof. Summers is not t o be recommended for determining t h e absolute q u a n t i t y of ammonia i n foods, i t nevertheless gives valuable comparative results. T h e results from this laboratory show t h a t t h e bread made with a yeast food containing ammonium chloride gave t h e lowest value for ammonia when comparing t h e color produced with Nessler solution against t h e color produced i n a s t a n d a r d ammonium chloride solution. 0 . n t h e other hand, i t will be seen t h a t salt-rising bread a n d compressed yeast give high results for ammonia by both tests. E v e n when a short process of fermentation is used t h e quantity.of ammonium chloride left in t h e bread is insignificant. Experiments were made using increasing quantities of yeast a n d decreasing periods of fermentation. I n only t h e most extreme case, where t h e dough was fermented only I hour, could traces of t h e ammonium chloride be foKnd. Table I11 shows t h e q u a n t i t y of yeast used, with t h e time of fermentation given t h e dough a n d t h e ammonia recovered from t h e baked bread. I n each case j 6 0 grams of flour were used a n d 4 grams of yeast food containing 0 . 3 2 1 gram NHdC1. T h e ammonia added i n t h e form of ammonium chloride a n d figured on t h e dry materials of t h e bread was 0 . 0 1 9 1 per cent.
ing 5 0 grams of d r y material was broken u p under water immediately when mixed, a n d another after standing 5 hours, t h e a m o u n t of ammonia as ammonium chloride recovered i n each case being 0.723 gram. Deducting from this 0.135 gram, t h e equivalent of ammonium chloride obtained from a dough containing no added ammonium salt, we have 0 . 5 8 8 gram, or almost t h e complete quantity, 0 . 5 9 2 gram added. T h e following experiment shows t h a t ammonium chloride does not stimulate t h e gas production of yeast in a cane sugar solution, using distilled water. If t h e ammonium chloride merely accelerated t h e gas production this experiment would demonstrate t h a t fact. We were unable, however, t o obtain a n y increase i n total gas production repeating this experiment a number of times. This would indicate t h e n t h a t t h e ammonium chloride is utilized only a n d exercises a favorable increase in gas production only when there is a reproduction of new yeast cells. Under t h e conditions of this experiment there can be only a very limited reproduction since t h e necessary elements-phosphorus, potassium, calcium a n d magnesium-are lacking. However, in t h e dough we have these elements present a n d therefore get a tremendous increase in t h e q u a n t i t y of gas produced as t h e experiment below indicates. EXPERIUEKT
16
32 40
Fermentation of Dough 5 hrs. 3 18/4
1
Min. in Proof Box 51 41 33 33
Weekly, 18, 37.
THE
EFFECT
OF
A31XOKIUM
SGGAR SOLCTIOK
For t h e t e s t , zoo cc. distilled water, 4 grams yeast a n d 2 0 grams of sugar were 'used. Temperature, 28'
c.
......
Grams Ammonium Chloride.. 0 Cc. Gas Produced (61/2 hre.). . . . . . . . 830
0.16 815
0.32 800
0.64 800
E X P E R I M f i N T S H O W I N G T H E I N C R E A S E I N GAS P R O D U C -
TION IN D O U G H , USING Fermentation Hrs.
NH4Cl
Control Dough Cc.
Dough containing 0.2 g. DIFFERENCEN H L l per 100 Flour DIFFERENCE 0 110 cc. 0 80 80 180 105 280 100 105 390 110 80 500 110 100 640 140 95 785 145 955 125 170 90 1105 150 100 1260 155 100 1400 140 80 1520 120 70 1625 105 1120 cc. 1 5 1 5 cc. ammonium chloride.. . . . . . . . . . . . . . 35 3 CHEMICAL LABORATORY WARDBAKINGCOMPANY NEW YORK CITY
.......
SYRUPS FOR CANNING AND PRESERVING Per cent Ammonia Dry Bread 0.0045 0.0051 0.0051 0.0062
To show t h a t t h e ammonium chloride is not consumed b y t h e enzymes of t h e flour or bacterial action, t h e following experiment was carried o u t : A dough was made of t h e same formula as previously used except no yeast was added. This dough contained 0 . j g 2 g r a m ammonium chloride. A piece of dough contain-
' Bakers'
SHOWIXG
C H L O R I D E O N T H E GAS P R O D U C T I O S IS A CANE
TABLE I11 Yeast Grams 8
151
By JAMES
B. M C N A I R
Received October 30, 1916
I n t h e canning of most fruits, syrups are used of 20, 30, 40, 5 0 or 60' Balling or Brix. Some packers, however, use about 2' less for each grade, while others use s y r u p of 15, 2 j a n d 5 5 ' . T h e degree of syrup is arbitrary with t h e packer a n d is not indicated on t h e can label. T h e strength of syrup used is governed b y four main factors, viz., t h e acidity of t h e fruit, t h e q u a n t i t y of t h e fruit, fruit shrinkage or shriveling, and t h e flavor IO,
