January, 1931
I N D USTRIAL L4Y D ENGIATEERIIYGCHEMIX T R Y
effluent. I n the hydrogen environment no oxidation of the deaminized acids would be expected, thus giving volatile acids in the effluent. Summary The work outlined is of a preliminary nature indicating some of the reactions that occur when tannery effluent is treated with oxygen and hydrogen. It is pointed out that oxygen promotes formation of ammonia, indicating deaminization of the amino acids, causes the effluent to become very
71
collodial in character, and retards formation of volatile sulfides. It is further shown that hydrogen environment furthers the formation of hydrogen sulfide, causes deaminization with subsequent reduction, and finally causes the formation of volatile acids, which are not found in the oxygentreated effluent. The work is being extended to include the effect of hydrogen-ion concentration with relation to the gaseous environment. Literature Cited (1) Theisand Lutz, IND.ENG.CHEM.,21, 763 (1929).
Diastatic Activity of Some American HoneyslB2 R. E. Lothrop and H. S. Paine BUREAU OF CHEMISTRY A N D SOILS, WASHINGTON, D. C.
In view of objections raised in Germany to the low diastatic activity of various importations of American honey, an investigation was made of the diastatic activity of a large number of samples of unheated American honey of a great variety of floral types. The results indicate that the natural diastatic activity of most types is sufficiently high to meet the German requirements i n this respect. Honeys of certain floral types, notably those of darker color, show characteristically high diastase values, whereas a few, among which alfalfa and orange are the best examples, generally show low diastase values. A considerable variation in the diastatic activity of various samples of the same floral type was found, however, so that careful attention to the diastase content of honeys intended for export (to Germany) should be observed in the future. The effect of even moderate heating of honey is to lower the diastatic activity somewhat; hence, great care should be exercised in the heating of honey when the preservation of its natural diastatic strength is de-
sired. The diastase is probably derived from pollen. The activity of honey diastase is greatly affected by variations in hydrogen-ion concentration, the optimum activity being attained a t a pH of about 5.3. Methods for determining diastatic activity of honey which are in vogue in Germany do not take this factor fully into account. Gothe’s method is objectionable i n that large differences in pH exist between the contents of the comparison tubes in a given determination. For honeys of medium diastatic activity Gothe’s method furnishes fairly satisfactory comparative values, since the pH values of the significant comparison liquids are near the optimum. In the case of very low or very high values, the method does not yield comparable results. A modification of Gothe’s procedure is described in which the pH of the various liquids in a comparison series is kept constant a t the optimum point for diastatic activity. The use of this modification is recommended when a higher degree of accuracy is desired.
.........e....
I
N CONNECTION with a comprehensive investigation
of the composition of gmerican honeys in relation to the suitability of honeys of different types for various industrial uses which has been undertaken recently by the Carbohydrate Division, considerable attention has been given to diastatic activity. The practical importance of a study of the diastase value of American honeys has arisen from the fact that during the past few years a number of shipments of American honey to Germany have been penalized by the German importers because of low diastatic activity, this action being based upon the position taken by the German food authorities that all genuine honeys which have not been overheated should show a t least a specified minimum diastatic activity. Diastase Value in Relation to Exportation of American Honey Since Germany has been the largest foreign consumer of American honey during recent years, this situation has seriously affected exports of honey from this country. Of some 20 million pounds imported into Germany annually, approximately one-third is American honey. This represents 1 Received September 22, 1930. Presented before the Division of Sugar Chemistry a t the 79th Meeting of the American Chemical Society, Atlanta, Ga., April 7 t o 11, 1930. Contribution 98, Carbohydrate Division, Bureau of Chemistry and
Soils.
more than half of the total quantity of honey exported (3) from the United States, Great Britain being the next largest consumer. American honey exporters have built up a substantial business during recent years, and have gained an excellent reputation for their honey, which has tended to wean the Germans away from the use of L‘Kunsthonig” and cheap marmalades as “spreads” in place of honey. It was therefore important to determine whether or not American honey of certain types is naturally deficient in diastase, or whether the diastase is partially destroyed or inactivated as a result of overheating during extraction or subsequent processing. A large proportion of the honey shipped to Germany from the United States originates in California, and a portion of this in turn comes from the Imperial Valley. The high temperatures prevailing in the Imperial Valley, especially during the summer, suggest the possibility that prolonged exposure of extracted honey to atmospheric temperature in that region, especially in direct sunlight, might cause an appreciable reduction in diastatic activity. As a rule, little attention has been given to diastatic activity in conjunction with chemical examination of honey in the United States, although in Germany (also in France and Switzerland) the subject of enzymes in honey has been studied rather extensively for a number of years. Erlenmeyer and von Planta ( 4 ) were the first to report the pres-
72
INDUSTRIAL A N D ENGINEERI,VG CHEMISTRY
ence of diastase in honey. Langer (8),Auzinger (a), Moreau @), and Gothe (6) have conducted comprehensive investigations on the subject of honey enzymes. These authors ascribe to the enzymes of honey a specific physiological value, and honey which has been heated sufficiently to destroy these enzymes is regarded as a “denatured” product and no longer “genuine.” Gothe has devised a fairly satisfactory method (6) for determining the diastase value of honey. As a basis for studying the diastatic activity of honey of a large number of different types, about three hundred samples were collected, an endeavor being made to obtain unheated honeys of as specific floral source as possible. The samples represented a great variety of floral types and were obtained from practically every honey-producing region of importance in the United States. They were kept in a constant-temperature room at 20” C. and the diastase values were determined as soon after receipt of the samples as possible, no sample being kept longer than 2 months before examination. With a few exceptions, the honeys examined had not been subjected to heating during extraction or subsequently. A few of the honeys had been heated for a short time a t moderate temperatures; these samples naturally have limited value for the purpose of the investigation and are therefore segregated in Table 111.
of the few samples which had been heated. The Fiehe test (1) was used for this purpose. The colors produced in the Fiehe test were compared with those obtained with negatively testing honey to which varying proportions of invert sugar had been added, thus giving an approximate comparison of the proportions of furfural derivatives present in the honey samples in comparison with those present in invert sugar produced by the usual method of acid inversion of sucrose. Diastase Values of Different Types of Honey I n tabulating the data obtained, the samples have been divided into several groups. Table I gives the results of examination of a group of orange honeys which had not been heated. Table I-Orange
Note-From a regulatory food law standpoint, Fiehe and Kordatzki (5) have recommended that honeys with diastase values under 17.9 on this scale should be regarded as suspicious and that those with diastase values under 10.9 should be classed as adufterated or overheated.
Each sample was also tested for hydroxymethyl furfural in order to determine whether any of the unheated honeys responded to this test, as well as to note the effect in the case
Honeys
ACIDITY SOLIDSDIAsTasK VALUE FIEHETKST (Gothe scale) as formic acid)
SAMPLE
% 726 792 847 680 721 980 627 1157
Method of Measuring Diastatic Activity For the determination of diastatic activity the method of Gothe (7) as modified by Fiehe and Kordatzki ( 5 ) was used. Later it was found that because of differences in the hydrogenion concentration of the comparison solutions the values given by the Gothe method are not strictly proportional to the actual quantities of diastase present. This observation led to a study of the influence of hydrogen-ion concentration on the activity of diastase in honey, which is discussed later in this article. I n the Gothe method a 10-gram sample of honey is dissolved in 50-75 cc. of distilled water, neutralized to phenolphthalein with 0.05 N sodium hydroxide, and diluted to 100 cc. with distilled water. A series of twelve test tubes is arranged for heating in a constant-temperature bath and in each tube are placed 0.5 cc. of 0.1 N sodium chloride, 0.5 cc. of 0.02 N acetic acid, 5 cc. of 1 per cent soluble starch solution, and portions of the honey solution varying from 10 to 1 cc. I n the first two tubes the quantity of honey solution is kept constant (10 cc.) and the proportion of starch solution is varied; in tubes 3 to 12, inclusive, the proportion of starch solution is kept constant (5 cc.), and the quantity of 10 per cent honey solution is progressively decreased. Sufficient distilled water is added to each tube to make a total volume of 16 cc. The comparison tubes are kept at a temperature of 45-50 O C. for 1 hour, then cooled immediately in ice water and a drop of 0.1 N iodine is added to each tube. The colorations produced vary from practically colorless through purple to blue. The tube which contains unchanged starch and the highest proportion of honey serves as a measure of the limit of diastatic activity. The diastase value is expressed as the number of cubic centimeters of 1 per cent starch solution converted by the diastase in 1 gram of honey. Values on this scale range from 1 to 50.
Vol. 23, So. 1
%
82.6 81.6 83.3 84.0 81.9 81.9
Negative Negative Negative Negative Negative Negative Trace Negative
13.9 13.9 13.9 13.9 10.9 8.3 2.5 1.0
8i:o
0.17 0.11 0.11 0.11 0.14 0.14
0:07
Orange honey is quite deficient in diastase. Sample 1157, for instance, contained almost no diastase, although the honey had not been subjected to heating during extraction or subsequently and was of exceptionally fine quality, the delicate orange flavor being very pronounced. Table 11-Honeys
Representing a Variety of PredomJnant Floral Sources ~
~~~
~
PRE-
DIASTASE VALUE DOMISTAST SAMPLE! (Gothe scale) FLORAL EXAMINE SOURCK Max. .Win. Av.
Crowfoot Mesquite Sage White clover Tulip-poplar Eucalyptus Raspberry Clover (variety not known) Cotton Sweet clover Alsike clover Tupelo Sumac Catclaw Alfalfa Fireweed Orange Mangrove Algaroba (Hawaii) Indefinite mixtures Unknown
2 4 10 10 5 3 3 10 4 10 3 4 2 4 20 3
50.0 50.0 38 5 29.1 38.5 29.1 29.1
50.0 50 0
29.1 29.1 29.1 23.8 23.8 23.8 50.0 13.9 29.1 10.9
39 0 33.8 29.1 28.7 26.4 25.1 24.7 23.2 38.5 5.0 21.7 23.8 13.9 18.8 17.9 17.9 17.9
ACUITY (Calcd. as formic acid)
POSITIVE
FIEHE TEST
0.19 0.14 0.18 0.12 0.19 0.10
0.10 0.09 0.14 0.08 0.10 0.10 0.16 0.11 0.23 0.06 0.23 0.06 0.24 0.10 0.13 0.13 0.16 0.08
0.15 0.11 0.16 0.10 0.14 0.10 0.13 0.13 0.14 0.18 0.13 0.12
29.1 8.3 17.5 0.17 0.07 0.10 23.8 13.9 17.4 0.24 0.10 0.17 23.8 10.9 16.9 0.16 0.06 0.10
1
1 None None None None 1 (trace) 1 2 (trace) { (trace) “None None
1
2 h’one (trace) I -
None
1 (trace)
None 2 (trace) 3 (trace) None 1 (trace) 1 (trace)
S
2
7.6
3
17.9
2.5
62
50.0 50 0
2.5 21.2 2 . 5 17.8
22
SAMPLES GIVING
0.36 0.06 0.13
Table I1 contains data pertaining to honeys of a variety of floral types and derived from examination of two or more samples of each type. While considerable variation is exhibited by different samples of the same floral type, the average values show some apparently characteristic differences. Orange and alfalfa honeys are notably low in diastatic activity, whereas sage, buckwheat, tulippoplar, and some others show consistentlv high values. The clover “
Y
INDUSTRIAL AND ENGINEERING CHEMISTRY
January, 1931
honeys show medium values, and are to be regarded as average. It is noteworthy that such honeys as buckwheat, tulip-poplar, and in general those that have high diastase values are dark in color, whereas orange and alfalfa honeys (of characteristically low diastase value) are generally quite light in color. The results of investigations regarding the origin of diastase in honey are somewhat contradictory. Gothe (7) has concluded that the diastase is derived principally from the bees, However, Vansell (12) has shown recently that the diastase values and the number of pollen grains present in several honeys were roughly proportional. The great variation in diastase value shown by honeys of different floral sources in the present investigation supports Vansell’s conclusion that the diastase is primarily of plant origin. I n this connection it is interesting to note (see subsequent discussion) that honey diastase gives a pH-activity curve closely resembling that of malt diastase. Table 111-Honeys
Subjected t o Moderate Heating
ACIDITY DIASTASE VALUE (Calcd. as formic DOMINANT SAMPLES (Gothe scale) acid) FLORALEXAMIXEI SOURCE Max. Min. Av. Max. Min. Av. PRE-
% Sag! Tullp-poplar Clover (variety not known) Sweet clover Orange Alfalfa Algaroba (Hawaii) Indefinite mixtures Unknown
%
SAMPLES GIVISG POSITIVE FIEHB TEST
%
2 3
None 38.5 17.9 26 2 0.13 0.09 0.11 17.9 17.9 17.9 0.20 0.19 0.20 2 (trace)
2 5
29.1 23.8
2
ii:9
1
..
1 5 5
2.5 15.8 0.10 0.07 0 08 0.0 13 1 0.10 0.06 0.08 0.11 10 9 i:o 9.4 o:ig o:i7 o 18
,.
2.5
50.0 10.9 20 7 17.9 2.5 1 2 . 9
,.
..
1 2 None None 1
0.07
0 16 0.06 0 10 0.19 0.10 0.15
None 5
73
garded as particularly characteristic of the diastatic activity of the types represented since, as has been pointed out, rather large differences may be exhibited by individual samples. Several samples of honey collected during the years 1923, 1924, and 1926 and held in storage since that time were examined for diastase. The diastase values were still appreciable, but all samples gave distinct tests for furfural derivatives. Influence of Hydrogen-Ion Concentration on Diastatic Activity
During the course of the investigation it was observed that considerable differences in hydrogen-ion concentration exist in the various tubes of a diastase determination as conducted according to Gothe’s method. I n order to activate the diastase and produce an optimum pH value, 0.5 cc. of 0.1 N sodium chloride and 0.5 cc. of 0.02 N acetic acid are added to each tube. The larger quantities of honey in the first few tubes produce an increased buffer action, so that the pH values of the solutions in these tubes are higher than those in tubes containing the smaller proportions of honey. This buffer action decreases progressively in the series of tubes as the proportion of honey decreases. h
‘0
f , 2 3
8
L ’
a6
c5
2 3 U
Table I11 gives the diastase values of a few honey samples that had been subjected to carefully controlled heating which was not sufficient to alter materially the appearance and flavor. These diastase values are, on the whole, somewhat lower than those of unheated samples of the same type, and a large proportion of the samples showed the presence of furfural derivatives resulting from decomposition of levulose. Table IV-Honeys
Aster Spikeweed Firtree Wild pea vine Lima bean Wild coffee Snowberry Heartsease Vervain Lippia Tarweed Manzanita Phacetia Salal Sourwood Ti ti White cedar Willow Milkweed Deerweed Tulip Fruit bloom Honeydew Dandelion Huajilla MilstarA . .-..- -
Representing a Variety of P r e d o m i n a n t Floral Sources (One sample each)
50.0 38.5 38.5 38.5 38.5 38.5 38.5 38.5 29.1 29.1 29.1 29.1 29.1 23.8 23.8 23.8 23.8 17.9 17.9 17.9 13.9 13.9 13.9 10.9 8.3 0 0
0.10 0.28 0.10 0.11 0.16 0.06 0.05 0.14 0.27 0.22 0.06 0.17 0.16 0.13 0.16 0.10 0.14 0.24 0.10 0.10 0.17 0.10 0.19 0.08
0.10 0.13
Negative Negative Negative Negative Negative Negative Negative Negative Positive Positive Negative Negative Negative Negative Negative Negative Negative Positive Trace Negative Negative Negative Negative Negative Negative Positive
I n Table I V are presented the diastase values of honeys of several floral types, only a single sample of each type being available for examination. These values cannot be re-
z 2 $
1
ri 0
2
3
4
5
6
7
8
PH
Figure 1-Influence of pH on Diastatic Activity (Composite of Four Curves)
Determinations by the quinhydrone electrode method showed differences in hydrogen-ion concentration greater than one unit on the pH scale in tubes of the same series; for instance, the solution in tube No. 3, containing the most honey, had a pH value of 5.6, whereas the solution in tube No, 12, containing the least honey, showed a pH value of 4.3. The investigations of Ohlsson (IO) and of Sherman (11) indicate that the activity of malt diastase is greatly affected by changes in hydrogen-ion concentration. Ohlsson’s results show that the activity of malt diastase at pH 4.5 is only 50 per cent as great as at pH 5.5, the optimum pH for this particular amylase. The influence of hydrogen-ion concentration on the diastatic activity of honeys of four floral types (white clover, gallberry, wild buckwheat, and sage) was studied by means of a method somewhat similar to that used by Ohlsson. The pH value of all solutions in the same series was kept constant by use of a buffer mixture of M / 1 5 phosphate solutions consisting of disodium phosphate, monopotassium phosphate, and phosphoric acid, the total phosphate molar concentration being constant at the various pH values. The difference in pH between the contents of tubes in the same series was in no case greater than 0.03, which is negligible. The diastatic activity-pH curves for the four honeys are very similar, showing nearly the same maximum point and the same general shape and indicating that the diastases of these honeys are identical, or a t least very similar. Figure 1 is a composite curve for the four honeys, showing graphically the relationship between diastatic activity and pH.
Vol. 23, KO.1
INDUSTRIAL A 6 D ENGINEERTNG CHEMISTRY
74
The curve for malt diastase (dextrinogen amylase), as obtained by Ohlsson, is of the same general shape as the curve for honey diastase, the principal difference being in the optimum point, which is a t pH 5.6 for malt diastase and at about pH 5.3 for honey diastase. Since it is shown that small differences in hydrogen-ion concentration materially affect the values obtained in a diastase determination, a study was made to determine the effect that differences in pH have on the diastase values obtained by the Gothe method. The pH values of the members of a Gothe series were determined and compared with those of a similar series in which the liquid in each tube was buffered to optimum pH with a M/15 phosphate buffer solution. Table V shows the considerable variation in pH in the tubes in a Gothe series as compared with the practically constant pH values in the buffered series. Table V-Comparison of pH Values of a Gothe Series with Those of a Buffered Series TUBE
GOTEESERIES
BUFFERED SERIES
I n order to obtain an indication of the probable range of variation in the pH of the members of a Gothe series, as shown by different honeys, the pH values of the first and last members of the series were determined in the case of five honeys of different floral types. These data are shown in Table VI. Table VI-Maximum SAMPLE
860 862 865 870 872
Variation in pH of a Gothe Series Shown by Several Honeys TUBE1
TUBE12
DIFFERENCE
fiH
QH
fiH
5.38 5.70 5.51 5.40 5.48
4.31 4.48 4.26 4.31 4.30
1.07 1.22 1.25 1.09 1.18
are lower than the optimum. This accounts for the variation in the ratio of diastase values by the two methods. Table VII-Comparison of Diastase Values by Gothe Method w i t h Those Obtained bv DH-Controlled Method
I
SAMPLE 1063 1070 901 947 953 845
I
DIASTASE VALUE Gothe method 29.1 29.1 17.9 12.4 10.9 10.9
pH-controlled method
70 70 35 28 17 20
I
I
APPROX.RATIO
- -...--.
P.PTUTRPN
DIASTASE VALUES 2.4:l 2.4:l 2:l 2.2:l 1.6:l 1.8:l
Although rather large differences in pH are exhibited progressively by the contents of the twelve tubes in a Gothe determination, only the contents of those tubes corresponding to relatively low or relatively high diastase values have pH values distinctly unfavorable to maximum diastatic activity. For mean diastase values, more nearly optimal H-ion concentrations exist, so that the Gothe values for different honeys in this range would be fairly comparable. For honeys of very low or very high diastatic activity, values obtained by the unmodified Gothe method are not proportional to the actual amounts of diastase present. I n view of the objections raised in Germany to the low diastase activity of certain shipments of American honeys, and the commercial importance of this situation, it was considered desirable, for the purpose of comparison, that determination of the diastatic activity of the approximately three hundred samples examined by the writers should be made by the Gothe method, since this method is probably fairly typical of the method or methods used by the German chemists who have examined importations of American honey. However, it is believed that the pH-controlled modification of Gothe's method which has just been described is distinctly preferable and should be used in cases where a greater degree of accuracy of comparison is desired. Acknowledgment
Grateful acknowledgment is made of the invaluable cooperation of Jas. I. Hambleton, of Bee Culture Investigations, Bureau of Entomology, U. S. Department of Agriculture, in obtaining the samples used in this investigation. Literature Cited
The maximum differences in pH do not vary greatly from the maximum difference of 1.30 shown by the complete series in Table V. Determinations of the diastase values of six honey samples of various floral types were made by both methods. The samples were chosen so as to give a considerable range of diastatic activity. The results are shown in Table VII. It will be observed that in general the ratio of the diastase values determined by the two methods decreases as the values decrease. I n Table V it is seen that when tube 4 or 5 is the significant member of the series the pH for the significant tube is near the optimum, whereas for the higher diastatic activities the pH values for the significant tubes
(1) Assocn. Official Agr. Chem., Methods, p. 201, 94 (1925). (2) Auzinger, 2. Nahr. Genussm., 19, 65-83, 353-62 (1910). (3) Commerce, U. S. Dept. of, Foreign Commerce and Navigation of United States, 1928. (4) Erlenmeyer and von Planta, Bienen Ztg., 34, 181 (1878); 35, 155 (1879); 36, 2 (1880). ( 5 ) Fiehe and Kordatzki, 2. Untersuch. Lebensrn., 55, 162 (1928). (6) Gothe, 2. Nahr. Genussm., 28, 273-285; 286-321 (1914). (7) Gothe, Ibid., 28, 286-321 (1914). (8) Langer, Ibid., 5, 1204 (1902); 20, 696 (1910); Schweis. Wochschr., 41, 17 (1903). (9) Moreau, Ann. f a t s . , 4, 65, 145 (1926). (10) Ohlsson, Compt. rend. trow. 2ab. Cartsberg, 16, 1-68 (1926). (11) Sherman, Thomas, and Baldwin. J . Am. Chem. Soc., 41, 231 (1919). (12) Vansell, J . Econ. Entomot., 22, 922 (1929).
Spoiling of Fats Accelerated by Light-That light plays an important part in the development of the spoilage of fats has been determined after careful study of the subject by C. H. Lea, of the Low Temperature Station, Cambridge, England, says the Science News-Letkr for December 20. It was found that under ordinary conditions fresh beef kidney fat keeps well for a certain initial period and then quickly becomes
rancid. When the fat is kept in darkness, there is a longer initial safe period before it begins to spoil. Bright light has the opposite effect for, if the fat is exposed to direct sunlight on a hot day, rancidity sets in very rapidly, and the initial safe period may be almost completely eliminated. If the fat is placed in sunlight long enough for spoilage to start, even subsequent removal to a dark place will have little effect.
.
