Change of Crude Lipides of Wheat on Storage H.
1
sul.lAvAh h h l ) (
PlHE\IOL:S p u b l i c a tions frum this lahonitory (2-5) a n1ethrrd f o r t h e e s t i m a t i o n of tile l i p i d c s of w11ent nnd it,s ~)nxluots has been gi yen arid tlv importancr of these compoinids in relation to glut.err quality has lieen stressed. 'The term "lipides" as it is used in this p a p e r i i r c l u d c s the fats,fatty their ne,ltrai esters, aiid other corri1,ounds S
.
.
CVtieut orid ils products were e+lruclud wi/h ,uriolls fat ,solcrrlt,s the pho,spilorus niIrogeIpIi corilenls of lite cslrucls masured.
Cnder different sloruge coriditions the urnourits qf erlrucls urid their phosphorus und riifrogeri s/loloed rrrarked Dlzriu,tiori.~f~~ c}larye,s
e+I,iLirled as kiry dlle lo erlzyrrric hydrolysis arid are correluled w i l h iricreesirig riroislure urid acidity .f
,.I he
the surripks. riitrogerr-pii.osf,horrls ralios of u / / lire
whicll of t'he s o l v e n t s w a s the most e f f e c t i v e in removing the c o m p o u n d s in wliicli ne were i n t e r e s t e d . At the Ijcgiririing of this \r.ork it was fiiiind difficult to secure closely a g r e e i n g results o n s o m e of tlic extracts from \vheut germ aiid bran unless the extractions were done same day, Ti,e reason f o r t h i s v a s found later. S a m p l e s ~ e r csealed
a i r e e i n g rcsiilts were difficult sterols, waxes, and plant pigments, in addition to traces of &er extraneous siilistliiiccs ti, s e c u r ~\vlwri cxtractioiis were done nt different times. having no chemical relationship to tiir Eats, arc' always present, Samples itused in the air in cotton bags, on tbc otlicr linnd, when a plant product is extracted with any of tlic so-callcd Eat slioweil iittle or no eliangc. solvents. Different solvents extract varying amounts of tlic E S I ~ I ~ I ~ I APaocmtiii~ ~~~XTAI. fats and fat-like compounds, and conseqiieritly the extracts art: A11 t.he samplns used in this investigation were iiiillwl i n genernlly named according t,o tlic solvent used instcad of tiia substance extracted, with the exception of the aleolitil-ether it :+OOO-barrelmill froin iiortliwest hard spring wlieat. The extract. The alcohol-ether extract of a plant tissue which has arialyses of the wheat and its product,s are given in Table I. been evaporated to dryness and tiien talcerr IIJI in cliloroform Total phospiiorus was determined in tlie ssh by means of has been referred t u by many writ.ers, iiichidirig t,lic aiithors, the standard gravimetric rrretliod and ealciilatecl to the as "lipoids." Since this term has been used h y different iri- prodrrct. vcstigators in such difierent and cmflict,inE riiaiiners, it was Extraetioris were coiiducted in a Goshlet extraction outfit using a n e l e c t r i c thouglit advisable hot plate and Bailey to refer to the alcoWalker condensers. hol-ether extract as C h e n i i c a l l y pure such. solvents acre used I n an e f f o r t to in e v e r y case, the isolate pure lecithin e t h e r in addition as well as to ascerbeing dried a n d tain the prescncr. kept over s o d i u m . or absence US In the case of wheat s p 11i n g o m yelin , and flour, the samcerelmsidcs, a n d ples were mixed with other lipidcs i n the pumice to p r e v e n t d i f f e r e n t milling p a c k i n g in t h e separations o l a l c o h o l - e t h e r exwheat, i t was isaction. Previous t h o u g h t advisable to extraction all the to measure first the samples were dried a m o u n t of lipide for 20 m i n u t e s a t material extracted 70°C. and 100mm. by various solvents, p r e s s u r e . All esand by determining tractions except the the n i t r o g e n and alcohol-ether we r e ph osplr o r u s conc o n d u c t e d for 16 tent of tlie crude hours. It was lipides, gain a rough FATExm.%mnR UBEUIN EXPERIMENT^ necessary to f i l t e r indication as t o
INDUSTRIAL AND ENGINEERING CHEMISTRY
JanuarJ-, 1933
all the extracts except the ether through a Bertrand asbestos filter before evaporating to dryness and constant weight at 70" C. and 100 mm. pressure. TABLEI. ANALYSES OF PRODCCTS Protein, % .ish Yo Phosphorus, %
(Results calculated to dry basis) BRAN WHEAT PATENT FLOUR 21.69 16.77 14.20 (N X 6.25) ( N X 5.7) ( N X 5.7) 6.53 2.05 0.47 1.478 0.451 0.111
GERM
33 40 i N X 6.25) 4.82 1,133
The extract termed "alcohol-ether" was obtained by extracting the samples with 95 per cent alcohol for 2 hours and then with ether for 2 hours, except in the case of ground bran, where it was found necessary t o reflux overnight with alcohol, as the extraction a t the end of 2 hours was not complete. The combined alcohol and ether extracts were evaporated t o dryness on a boiling water bath and the residue taken u p in chloroform, filtered, and evaporated to constant weight at 70" C. and 100 mm. pressure. Hot alcohol extracts practically all the lipide material, the ether being used merely to remove the last traces. I n all cases the alcohol-ether extract had a higher percentage of phosphorus than any of the other extracts. In order t o prove that the alcoholether extraction procedure removed the maximum amount of phosphorus-containing lipides, extractions were done with 95 per cent alcohol for 16 hours, the extracts cooled to room temperature, filtered, and evaporated to dryness a t i o " C. and 100 mm. pressure. The amount of the extract was not significant because of the contamination by gliadin, starch, and other impurities, but the total amount of phosphorus extracted was a very close check with the amount of phosphorus extracted by the alcohol-ether procedure on each product. Phosphorus and nitrogen are reported as determined in the total lipides extracted by various solvents. Phosphorus of the lipides was determined by the micromethod of Pregl (1) as follows: From 0.1 to 0.5 gram of fat (depending on the phosphorus content) is mixed thoroughly with a 1 to 1 mixture of c. P. potassium nitrate and c. P. sodium carbonate. The mixture is ignited slowly on an open flame in a covered platinum dish. This heating must be done very cautiously since the mixture has a tendency to spatter. The mixture is then ignited in a muffle a t 575' C. until white. The ignited residue is taken up in a solution of one part of nitric acid to 2 parts of water, filtered, and washed with hot water, with the volume carefullv kept as small as possible. Then 2 cc. of the sulfuric-nitric acrd mixture
101
are added, the solution heated to 97" C., and shaken vigorously. Next, 15 cc. of sulfate-molybdate solution are made up to the same volume as the volume of the sample, and are then poured into the center of the sample without shaking. The resulting temperature should be 65" to 70" C. At the end of 3 minutes the solution is shaken for 30 seconds and then allowed to stand for one hour at room temperature. A Jena glass crucible No. 10G4, which has been previously washed with ammonia, water, nitric acid, water, alcohol, and ether, and dried for 30 minutes in a vacuum desiccator, is used for the filtration. The precipitate is washed alternately with 2 per cent ammonium nitrate and alcohol, then finally with alcohol and ether, dried for 30 minutes in a vacuum desiccator, and weighed. The factor used was 0.01453 for converting the yellow precipitate to phosphorus. Experiments were conducted using different temperatures and volumes in the precipitation of the yellow precipitate. It mas found that when the above procedure was followed, the results checked closely with the standard gravimetric procedure in which the phosphorus is weighed as magnesium pyrophosphate. The solutions for the Pregl method were made up exactly as given by Pregl (1). The following procedure was used in determining the nitrogen in the lipides: From 0.2 to 0.5 gram of fat n-as taken up in ether and transferred to a Kjeldahl flask, and the ether evaporated. Nitrogen was determined according to the regular Kjeldahl method using potassium sulfate with mercury as a catalyst. Sulfuric acid and sodium hydroxide, both 0.02 N , were used for the titration with sodium alizarin sulfonate as the indicator. The official A. 0. -4.C. method mas used for the acidity determination, and the results calculated as lactic acid except on the germ. The end points given by the germ with both the official and the Greek methods were unsatisfactory, but the Greek acidity method was preferable, and results by this method, in line with common practice, were reported as sulfuric acid. Moisture was determined by the 130" C. air oven method. Wheat and samples of germ, bran, and patent flour milled from it Tvere taken from the mill on different dates in order to have fresh samples for analysis. The analyses on the original samples in each case were done as rapidly as possible to prevent any change. A part of each original sample was placed in a glass container and the stopper sealed with paraffin. Another part of each sample was stored in a cotton bag in the laboratory a t room temperature. Months later both the samples in the sealed containers and the cloth
TABLE .[I. CHANGE OF LIPIDESo x STORAGE (Results calculated t o d r y basis)
--.
ORIGINAL S l Y P L E -
Extract
P
N
%
%
%
Ratio N : P
13.7% MOISTURE A N D 0.111% ACIDITY AS
Germ: Alcohol-ether Ether .icetone Ethyl acetate Bran: Alcohol-ether Ether .icetone Ethyl acetate
Patent flour: Alcohol-ether Ether Acetone Ethyl acetate
Extract
P
%
%
0.518 0.114 0.164 0.268
AS
0.39 0.08
0.23 0.22
1.7:l 1.6:l 3.1:l 1.8:l
13.970 MOIBTURE A N D 0.568% ACIDITY A S LACTIC ACID
7.36
:.SO
1.05 7.26
3.16 2.50 3.19 3.40
0.464 0.091 0.150 0.248
0.31 0.13 0.19 0.18
MOISTURE A N D 0.351% ACIIIITY AS LACTIC ACID
0.481 0.145 0.188 0.301
0.38 0.33 0.27 0.32
1 3 . 6 7 0 MOISTCRE A N D 0.104% L l C T I C ACID
1.29 1.13 1.25 1.20
1.5:l 3.2:1 2.8:1 1.6:l
0,575 0.234 0.433 0.302
0.56 0.79 0.40 0.32
1.8:l 5,O:l 3.2:l 2.4:1 ACIDITY AS
2.4:l 7.4:l
2.O:l 2.3:l
N %
13.670 X O I S T U R E A N D 0.51070 ACIDITY
His04 15.24 12.05 13.06 13.16
14.8%
Wheat: Alcohol-ether Ether Acetone Ethyl acetate
S A M P L E S E l L E D I N C O N T A I N E R WITH HIGH~IOISTTJRE F O R 3 MOXTHS
13.41 12.74 14.22 14.11 13.8%
H~SOI 0.197 0.018 0,055 0.052
MOISTURE A N D 1.22% A S LACTIC h C I D
7.05 6.50 7.59 7.53
0.093 Trace 0.029 0,019
0.238 0.063 0.115 0.102
ACIDITY
0.19 Trace 0.12 0.10
0.32 0.21 0.25 0 . 2~. 4 ~
13.6% MOISTURE A N D 0.116% ACIDITY A S LACTIC ACID
1.40 1.12 1.27 1.21
0.502 0.251 0.322 0.329
P %
Extract
%
0.54 0.73 0.35 0.27
3
N
%
8.270 MOISTURE A N D 0.13970 ACIDITY
AS
0.15 0.03 0.16 0.11
13.5% MOISTURE A N D 0.49570 hCIDITY A S LACTIC ACID
2.99 2.62 3.30 3.29
S A M P L E S T O R E D IN B A G FOR LTOXTHS
14.07 12.40 13.19 13.54
H2S04 0.500 0.129 0.133 0.233
0.32 0.07 0.27 0.35
8.070 MOISTURE A N D 0.9370 ACIDITY A 8 LACTIC ACID
7.39 5.68 7.13 6.92 9.6%
0.41 0.18 0.22 0.28
MOISTURE A N D 0.345% ACIDITY A S LACTIC .ACID
3.16 2.44 3.04 3.04 6.7%
0.458 0.105 0.163 0.229
0.511 0.128 0.204 0.291
0.45 0.31 0.30 0.35
MOISTURE AND o.ils% A C I D I T Y A S LACTIC .tCID
1.37 1.03 1.23 1.10
0.572 0.241 0.382 0.277
0.71 0.73 0.45 0.34
INDUSTRIAL AND ENGINEERING CHEMISTRY
102
bags were analyzed and compared with the results on the original fresh samples. Those sealed in the glass containers maintained their original moistures very closely and the samples in the bags lost several per cent of moisture by gradual evaporation.
DISCUSSION The differences in the analyses of the samples stored under different conditions are very marked in the case of the germ, bran, and wheat, but only slight in the patent flour, as shown in Table 11. These changes on storage explain the differences in the phosphorus of the alcohol-ether extract of wheat products as previously reported ( 2 ) . The alcohol-ether extract on the germ, bran, and wheat dropped conTABLE 111.
Vol. 25, No. 1
moisture continued to increase owing to respiration changes. The sample which had been in a bag for 3 months a t 9.5 per cent moisture was divided, part stored a t that moisture in a sealed container, and the other part placed over water to increase its moisture content several per cent and it, too, stored in a sealed container. At the end of 4 months the sample stored in a sealed container with 9.5 per cent moisture showed only a slight decrease in the phosphorus contents of all the extracts, but the one with 12.2 per cent moisture showed a very decided drop in phosphorus. This indicates that the higher moisture content rather than the anaerobic condition is responsible for the observed changes. In order to obtain more evidence that the anaerobic condition was not responsible for the changes, a fresh sample
ANALYSES OF WHEAT STORED UNDER VARIOUS CONDITIONS (Results caloulated to dry basis)
SAMPLESTORED I N BAGAS GIVENI N TABLE11 A T SOLVENTS 9.5% MOISTURE Extract
t % .-
3.16 2.44 3.04 3.04
0.511 0.128 0.204 0.291
I -
Alcohol-ether Ether Acetone Ethyl acetate
P
t %
s.4MPLE
DIVIDED AND
S T O R E D AT DIFFERENT hfOISTURE8 S E A L E D CONT.4INERJ
FOR 4 MOWrHs I N At 9.5% moisture Extract P Tfi T " % % ," 2.94 0.507 2.40 0.143 2.92 0.167 3.04 0.264
GROUNDSAMPLEI N SEALEDCONTAINER WHOLEKERNELS I N SEALED FOR 4 MONTES C O N T A I N E R FOR 7 MONTHS (RESPIRATION) AT AT
At 12.2y0 moisture Extract P
,I
siderably on the samples stored in the sealed containers a t or near their original moisture content-as much as 2 per cent in the case of the germ. The phosphorus and nitrogen content of all these alcohol-ether extracts decreased, the phosphorus dropping to less than half the original amount. However, the ether, acetone, and ethyl acetate extracts increased in amount, although the phosphorus and nitrogen in all cases decreased as in the alcohol-ether extracts. It will be noted that on the germ, bran, and wheat the acetone and ethyl acetate extracts increased to such an extent that they became higher in amount than the alcohol-ether extracts, although the material extracted by acetone and ethyl acetate always contained less phosphorus. The analyses of the bran and wheat samples stored in bags gave almost perfect checks throughout with the original samples when all results were calculated to dry basis. The germ, however, showed a slight change in the same direction as the sample which had been kept near its original moisture by storage in the sealed container. The changes taking place in the germ were rapid, it being difficult to obtain closely agreeing results even a few days after the original test because of the activity of the enzymes in germ a t the relatively high moisture content before natural evaporation could take place. The sample of patent flour showed little change in 3 months' time, although the tendency of the changes was in the same direction as those of the other products. For example, the phosphorus content of the alcohol-ether extract of the flour stored at its original moisture was 0.502 per cent, compared with the phosphorus on the original of 0.572 per cent. Table 111 gives results on the wheat stored under different conditions. Analysis is given for the wheat stored for several months as whole kernels in a sealed container with the original moisture, whereas the sealed sample in Table I1 was a ground sample, and shows the same change with a decided drop in phosphorus content of the extracts throughout. Quite by accident one sample of ground wheat sealed with the original moisture started respiration when it was opened, and its moisture content increased rapidly. The fat extracted by all the solvents showed a marked decrease, and the phosphorus content of the fat likewise decreased. The acetone and ethyl acetate extracts were considerably higher than the ether and the alcohol-ether extracts because the last two were done a week later, during which time the
% 2.96 2.61 3.02 3.04
% 0.357 0.097 0.132 0.177
14.1%
14.8% MOISTURE
MOISTURE
Extract
P
Extract
P
%
%
%
%
2.86 2.42 3.16 3.36
0.225 0.053 0.143 0.069
1.44 0.98 2.41 2.16
0.195 0.034 0.086 0.050
of germ was taken. Table IV gives the analysis of this original germ, the same germ after 6 months' storage in a sealed container at the original moisture, and the same germ dried in a vacuum desiccator over Desicchlora down to 4 per cent moisture before storing in a sealed container for 6 months. The sample stored with the higher moisture showed a very marked decrease in the alcohol-ether extract, an increase in the ether and acetone extraots, and a decided drop in both phosphorus and nitrogen content, just as the germ sample in Table 11. But the sample sealed with low moisture showed a relatively slight change in the phosphorus and nitrogen contents of the extracts, as compared with the sample stored a t a higher moisture. These results indicate again that the moisture content is the predominant factor in causing a change in the amount and composition of the lipides extracted by different solvents.
TABLEIV. ANALYSES OF GERM STOREDUNDER
VARIOUS
CONDITIONS
(Results calculated t o dry basis) SAMPLE SEALED IN
Per cent moisture: 13.55 Per cent acidity: 0 . 0 8 4 as HzSOt SOLVENTSExtract P N
%
%
%
CONTAINER
6 MONTHS
ORIGINALSAMPLE
SAMPLE SEALED IN
12.30
4.03
1.27 as HzSOd Extract P N
%
%
CONTAINER
6 MONTHS
%
Alcohol-ether 15.62 0 . 5 0 3 0 . 4 1 1 2 . 8 1 0 . 0 3 0 Trace Ether 1 1 . 8 1 0 . 1 4 2 0 . 1 2 1 3 . 0 0 Trace 0 . 0 4 1 2 . 5 8 0 . 1 2 8 0 . 3 4 14.60 0 . 0 4 0 0 . 0 5 Acetone
0.161 as HzSOd Extract P N
%
%
%
13.68 0.560 0 . 3 5 11.54 0.085 0 . 0 4 12.23 0.094 0 . 2 3
The above brief discussion of results and the tables containing the complete data indicate that the changes observed are due to various fat-splitting enzymes, for example, lecithinase. Such enzymes are able to show greater activity a t the higher moisture levels. The results show that the changes observed are due largely to hydrolysis rather than oxidation. When lecithin is split, fatty acids, such as stearic, palmitic, and oleic, are formed, together with choline and glycerolphosphoric acid. Pure lecithin is soluble in ether, but in wheat it is held with proteins and carbohydrates as a complex which is not completely soluble in ether until it has been split by treatment with alcohol. Hence in the original samples the amount of the ether extract is considerably lower than the alcohol-ether extract, and its phos-
January, 1933
I N L, U S T It I A I.. A 5 1) I': i G I N E E R I N G C 11 E M I S T It Y
phww content is also inuch less. But. the fatty acids wliiali are split off upon ensymic hydrolysis of the lecithin are soluble in ether, increasiiig the amount of the ether extract. However, tlie glycerolphosphoric acid, or its cleavage products, ltnd choline are insoluble in ether, and the amount of the phosphorus and nitrogen-coiitaiming material extracted by thc ether is therefore less after hydrolysis has taken place. 1,ecitliin is reported in tlie literature as being insoluble in acetone. It is vel1 recognized, homver, that. a considerable amount of the lecithin of natural products dissolves in acetone in the presence of other fats and fatty acids. The acetone and ethyl aeeta.te, as well as the ether, extract inore material after ensyrriic liydrnlysis has taken place, oiving to their grcater irbility t.o dissolve the fatty acids fomied 011 hydrolysis thari to disxdve the original lecithin. On the other hand, the alcohol-ether extraction removes all the lecithin of the original samples, bot upon enzymic hydrdysis, although the fatty acids are still soluble, the choline and the g1yceml~ilios~~lioric acid are. less soluhle in the other and chloroform, both or which solvents give the vaine results when used in the last step in the alcohol-ether extraction. This derreascs both the amount of t,he extract and its phosphorus and nitrogeii content. An increase in acidity is correlated rery definitely with increasing moisture content and with corresponding decreases in the phosphorus and nitrogen contents of the lipides. This increase in acidity is caused Loth by t.he fatty acids and the phosphoric acid liberated liy the hydrolysis of the lipides. Since in the case of the lipide material extracted by all the solvents t.he riitrogen-ptiosphorus ratio is more than
105
1 to 1, wliicli is the ratio of these elements in lecithin, there must Be some nitrogen-containing compound other than lecithin prosent in the extracts, such as sphingomyelin and galactolipides. The nitrogen-phosphorus ratios in the ether extracts of the original samples illcrease progressively from the germ to the patent flour, whereas there is no such marked difference in the nitrogen-plrosphorus rat.ios on the other extracts. 111 the saniples in which hydrolysis had taken place, the figures for pliosphorus and nitrogen were in some cases so low that the ratios arc not significant. As might be expected, the changes observed were most marked in those parts of the wheat highest in fat content and in enzymic activity (germ and bran). Wheat, which contains roughly 25 per cent of germ and bran, likewise exhibit;. theseme cha~igesbut, not to the same degree. Flours, both patent and straight, shoived only very slight changes in tlie amount of the lipides extracted by various solvents mid in the phosphorus and nitrogen contents of tlie lipides regardless of storage conditions. This investigation is being continued in an effort to isolate certain of t.hc individual lipides of wheat and its product.s.
LITEnATUKE
CIT?;Ii
(1) I'rcxl, " D i e Quantitative Organisclie Mikroanxlyae," 2nd e d , PI). 151. 8. Springer, (2) Sullivan and New, Cera1 Ckhem.. 5, llY3-X (1028). (3) Sullivan and Near, 1x0. END.C H ~ M19, . , 159-61 ( I w a i j . (4) Sullivan and Near, rbid., 19. 49&501 (1927). (5) Sullivan and Near, J . Am. ChGn. Soc.. 49, 467-72 (1W7).
iw.
K s c s ~ ~ a ;July o 12, 1032.
Le .leune Chimisle by Davtu TENIERS,
the Younger
Our sixth l'cniers re-
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