Analysis of a Mixture of Glucose, Maltose, and Fermentable Oligosaccharides Improved Yeast Fermentation Method S. C. PAN1, L. W. NICHOLSOR’, ~ N P.4UL D KOLACHOV? Joseph E . Seagram & Sons, Znc., Louisville, K y .
A reliable and accurate method for analyzing a mixture of glucose, maltose, and dextrins is essential in studying the enzymatic hydrolysis of starch. The yeast fermentation method of Stark and Somogyi for analyzing carbohydrate mixtures, such as enzymic starch hydrolyzates, has been studied in detail and improved in accuracy and reliability. Employing an initial pH of 9.3, instead of 8.4 as in the original method, up to 400 mg. of glucose per 100 ml. can be completely fermented with 5 grams of baker’s yeast without any loss of maltose. Up to 400 mg. of maltose per 100 ml. can be completely fermented by using RlcIlvaine’s buffer at pH 4.8 in
place of the original acetate buffer. The residual carbohydrates from these fermentations can be determined with a high degree of accuracy even if they represent only small percentages of the total carbohydrates. Isomaltose, maltotriose, and panose can be completely fermented and analyzed as a separate group by using a pure culture fermentation of distiller’syeast (NRRLY-132). Thus,carbohydrate mixtures, such as enzymic starch hydrolyzates, can be differentiated quantitatively into four fractions : glucose, maltose, fermentable oligosaccharides, and unfermentable dextrins. Results of fermentations can be checked qualitatively by paper chromatography.
A
RELIABLE and accurate method for analyzing a mixture of glucose, maltose, and “dextrins” is essential in studying the problems related to the enzymic hydrolysis of starch. A number of methods have been developed for this purpose, involving the use of various strains of yeast, or other microorganisms, to ferment the sugars selectively (5-7, 9, 15, 20, 26, 27, 31). [In the absence of a more appropriate term, the word “fermentation” is used in the present report, although Harding and Sicholson (9) have questioned whether the sugars when treated with suspensions of yeast cells actually undergo anaerobic dissimilation.] I n addition, methods have recently been developed for quantitatively analyzing sugar mixtures by paper chromatography ( 4 , 11, 13, 24, 28, 34). The fermentation methods, however, offer a t least one advantage over paper chromatography in that they simultaneously reveal the fermentability of the sugars being analyzed. The Stark and Somogyi method for analyzing mixtures of sugars ( 3 1 ) is used routinely in this laboratory for analyzing starch hydrolyzates or similar samples. The method is relatively simple, inasmuch as it employs a commercial baker’s yeast. Glucose is removed by fermentation with baker’s yeast at pH 8.4,while glucose and maltose are removed by the same organism at p H 4.8. Aliquot samples of each fermentation are acidhydrolyzed and analyzed for the resulting glucose; the concentrations of glucose, maltose, and unfermentable dextrins are calculated from the initial total carbohydrate and residual carbohydrate values. Acid hydrolysis makes it possible to express all results in terms of glucose equivalents. It has been shown in recent years that oligosaccharides ( 1 4 ) , isomaltose ( I d ) , maltotriose (33, 35), and trisaccharides with maltose-isomaltose linkages (17, 23, 37) are present in starch hydrolysates or similar enzyme digests (17, 21-23, 34, 36‘). blyrback has reported that a Swedish compressed yeast J\ ill ferment maltotriose rapidly and will ferment isomaltose and the trisaccharide with maltose-isomaltose linkages a t a slower rate (16-17). As the conditions used by Myrback for these experiments were similar to those prescribed by Stark and Somogyi, 1
the question naturally arose whether these same sugars are removed by the Stark and Somogyi method. Preliminary studies indicated that the commercial baker’s yeast used in the Stark and Somogj-i method only partially ferments maltotriose under the conditions described in this report. Moreover, from experience gathered in this laboratory it was evident that the Stark and Somogyi method x a s not sufficiently accurate or reliable for quantitatively analyzing an enzymic starch hydrolyzate, and that the method would have to be modified to overcome the following limitations. The maximum sugar concentrations that can he fermented are relatively low-40 mg. per 100 ml. for maltose and 50 mg. per 100 ml. for glucose. Acid hydrolysis and subsequent neutralization cause a further dilution of the sample. When the residual carbohydrate concentration resulting from either the glucose or maltose fermentation is very low-Le., 5% or less of the total-the titration values are so small that they cannot be determined with any reasonable degree of accuracy. Increasing the concentration by evaporation of the sample is not practical, as it requires additional equipment and labor as well as many precautions (6). The concentration of yeast required for the fermentation is relatively high-10 and 15 grams per 100 ml. for glucose and maltose, respectively. Even though the baker’s yeast is thoroughly washed before it is used, small but definite amounts of reducing substances are a h ays introduced. These are detectable after acid hydrolysis and invalidate the data for samples containing minute amounts of unfermentable carbohydrates. The method prescribes the use of “fresh” yeast-Le., as fresh as can be obtained-because the fermentation power decreases rapidly upon storage. I n this laboratory, however, even fresh yeast often does not ferment completely solutions of maltose which contain an acetate buffer such as Stark and Somogyi used in some of th& experiments. Although they reported that there was an advantage in using unbuffered sugar solutions and usEd no extra buffer in their final procedure, in many instances it was found necessary to incorporate a buffer in the sugar samples to ensure a correct pH. Therefore, a study was made to determine whether the complete fermentation of maltose can be ensured by the use of a suitable buffer. The modifications of the Stark and Somogyi method and an extension of the yeast fermentation method, as described in this report, overcome these limitations of the original method and make it possible to determine glucose, maltose, fermentable
Present address, E. R. Squibb & Sons, New Brunawick, K.J. Yorth lferidian St., Indianapolis, Ind
* Present address, 4270
23 1
ANALYTICAL CHEMISTRY
232 oligosaccharides (isomaltose, maltotriose, and panose), and unfermentable dextrins quantitatively in starch hydrolyzates. MATERIALS AND TECHNIQUES
Baker’s Yeast. Fleischmann’s yeast (Standard Brands, Inc.) was purchased from a local distributor. The yeast was washed four times with distilled water by centrifugation. Distiller’s Yeast. Pure cultures of Seagram strain SC l y and Northern Regional Research Laboratory strain Y-132 were used. Paper Chromatography. The paper chromatographic method developed by Jeanes, Wise, and Dimler ( I O ) was used. The Rt values tended to decrease after repeated use of the solvent, probably because of changes in the composition of the solvent caused by evaporation and hydration of the filter paper. The exact R, values, therefore, are not given in the present report; instead, diagrams of the chromatogram are presented. I n these diagrams the circles represent spots on the original chromatogram having diameters proportional to those on the original.
GLUCOSE
0
MALTOSE
O
BOMALTOSE MALTOTRIOSE PANOSE MALTOTETRAOSE
0
0
00
0
0
0
0
000
0
00
0
0 0 0
0
000
R ” HA
Figure
1.
G
Paper
Me
MI
PE
PA
D
Chroniatogram Samples Used
T
HA
of
hT
PA
Carbohydrate
Partial hydrolyzate of amylose Commercial C.P. glucose Mg. Maltose recrystallized four times M I . Commercial C.P. maltose PB. Preparation B PA. Preparation A D. Dextrin T. Crystalline trisaccharide, panose H T . Partial hydrolyzate of panose
HA. G.
Carbohydrate Samples. All the carbohydrate samples used in this study were identified on chromatograms (see Figure 1). PARTIAL HYDROLYZATE OF AMYLOSE.Amylose, prepared from cornstarch according to the method of Schoch (N), was hydrolyzed in 0.35 N hydrochloric acid for 1 hour in a boiling water hath. The acid mas removed by passing the solution through Amberlite IR-4B (Research Products & Chemical Co.). This hydrolyzate was used as a reference for determining the position of glucose, maltose, and higher saccharides on chromatograms and is designated as sample H A (Figure 1). GLCCOSE.As shown in Figure 1, the commercial C.P. grade of glucose used in these experiments was free from all other saccharides and is designated as sample G. MALTOSE.Commercial C.P. grade maltose was found to contain a significant amount of higher saccharides as impurities. When recrystallized four times from aqueous methanol, a product was obtained which was practically free of these impurities. The unpurified maltose sample is referred to as maltose I ( X I , Figure l), while the recrystallized product is designated as maltose I1 ( M 2 ,Figure 1). MALTOTRIOSE.Commercial C.P. maltose was treated with baker’s yeast; the fermentation residue was maltose-free and contained all of the higher saccharides which were present as impurities in the maltose. This residue was purified by precipitating with basic lead acetate and by passing the filtrate through Amberlite IR-100 and IR-4B. The resulting solution was designated as preparation B ( P B , Figure 1) and contained a series of saccharides with maltotriose as the lowest member (topmost spot in the chromatogram). CRYSTALLINE TRISACCHARIDE OR PANOSE (36). This is the sugar isolated from the unfermentable carbohydrate synthesized from maltose by the enzymes of Aspergzllics niqer S R R L 337
( 2 2 ) . French ( 8 ) and Wolfrom et al. (36) have shown that this sugar is 4 [+( a-D-glucopyranosy1)-a-D-glucopyranosyl1-D-glucose. The position of this sugar on the paper chromatograms is shown in Figure 1, sample 2’. ISOMALTOSE. Panose was partially hydrolyzed and used as a source of isomaltose. A 3% solution of panose was hydrolyzed in 0.3 N sulfuric acid for 1hour in a boiling water bath; the acid was subsequently removed with Amberlite IR-4B. As French previously reported ( 8 ) , this hydrolyzate contains glucose, maltose, and isomaltose in addition to some unhydrolyzed panose, and is shown in Figure 1 as sample H T . Glucose and maltose were removed from the hydrolyzate with baker’s yeast and the residue was purified mith basic lead acetate and Amberlite resins as described under maltotriose. The resulting solution is a mixture of isomaltose and panose and is designated as preparation -4( P A , Figure 1). Preparation A was used as a mixture of isomaltose and panose and preparation B as a source of maltotriose. The chromatograms for these two preparations can be compared in Figure 1. Obviously, the R/ value for isomaltose is just slightly higher than that for maltotriose. (A mixture of isomaltose and maltotriose shows only one spot on a paper chromatogram.) Samples of pure isomaltose and maltotriose obtained from M. L. Wolfrom of the Ohio State University show spots identical with those shown here. The fact that commercial maltose is prepared from barley malt hydrolysis of starch (3) justifies the conclusion that maltotriose but not isomaltose is the principal contaminating sugar (33.35). DEXTRIKSAMPLE.This is the dextrin prepared from the barley malt hydrolysis of cornstarch as described by Back, Stark, and Scalf ( 2 ) (sample D, Figure 1). Its reducing power tonard sodium hypoiodite indicates that it has an average chain length of 13 glucose units. GLUCOSE FERMENTATION
Effect of pH on Rate of Glucose Fermentation. One of the purposes of, this study Kas to increase the sugar concentration during fermentation using as little yeast as possible. Naturally, the higher the initial glucose concentration the lower the final pH after fermentation; therefore, it would be necessary t o raise the initial pH above 8.4 ( S I ) to avoid having maltose fermented. T o establish this pH, glucose solutions containing the same carbonate-bicarbonate buffer used by Somogyi in his oi iginal studies (69) were fermented a t different alkaline pH’s. The buffer was prepared by mixing 1 JI sodium carbonate and 1 31 sodium bicarbonate in different proportions and determining t h e resulting pH with a Beckman p H meter. The results are presented graphically in Figure 2. Solutions of glucose, carbonate-bicarbonate buffer, and yeast slurry were pipetted into 25-ml. volumetric flasks to give the following final concentrations: glucose, 400 mg. per 100 ml.; buffer, 0.2 11; and yeast, 5 grams per 100 ml. The mixture was 100
90 80
/?e;-
70 60
t
DH
-
B
FINAL -
0
85
!? E‘
71
a
90
79
93
(18
9.7
9.1
/
: I
40
/ / /
0
IO
20
30 TIME
Figure 2.
40
50
VALUES
INITIAL
50
60
70
80
90
- MINUTES
Effect of pH upon Rate of Glucose Fermentation
Initial eoncentration of glucose, 400 mg. per 100 ml.; yeast, 5 grams per 100 m l . Age of yeast, 13 days; incubation temperature, 30’ C.
V O L U M E 25, NO. 2, F E B R U A R Y 1 9 5 3
233
100
90 80
0.03 M MclLVAINE'S 8 U F F E R f l
0025 M 8 $ g ; V /
70 2
60 4 2
a
50
Y
d
40
30
I
//
f,'
0 05 M ACETATE BUFFER?
20 IO
20
40
60 TIME
Figure 3.
80
-
100
150
120
MINUTES
Effect of Buffer Salts upon Rate of Maltose Fermentation
Initial concentration of maltose, 800 mg. per 100 ml.; yeast, 5 grams per 100 ml. Age of yeast, 7 days, initial pH 4.8; incubation temperature, 30° C.
made to volume, emptied into a 40-ml. centrifuge tube, and incubated in a 30" C. water bath; the mixture was stirred a t 20- to 30-minute intervals. A number of flasks, varying in initial pH, were set up in this way and analyzed a t various intervals. In each case the yeast was centrifuged off and an aliquot of the centrifugate was acid-hydrolyzed; hydrolysis was applied even when pure glucose was tested, in order to simulate the conditions under which sugar mixtures were analyzed. The size of the aliquot taken for hydrolysis and the final volume to which it was diluted after hydrolysis and neutralization depended upon the concentration of residual sugars. When the Concentration was very low, dilution was held to a minimum as follows: A 15-ml. aliquot sample was placed in a 25-ml. volumetric flask and acidified with 2.7 ml. of 6 Ar hydrochloric acid, giving a final acid concentration of 0.7 N . The flask was heated in a boiling water bath for 2.5 hours; the contents were first partially neutralized ( t o about 90%) with 6 N sodium hydroxide and then completely neutralized with 1 N sodium hydroxide and made to volume. Five milliliters of this solution was used for determining the glucose value by the method of Somogyi (30). Using this procedure, a residual sugar equivalent to 1% of the original glucose used would produce a titration difference of 0.9 ml. of O.OO5N sodium thiosulfate, thus ensuring a high degree of accuracy. A yeast blank containing no added sugar was analyzed simultaneously and applied as a correction. Another control flask containing no yeast was siniilarly analyzed for total sugar. Quantitative techniques were thus ensured throughout the analysis. As shown in Figure 2, the rate of fermentation decreased as the initial pH increased, yet 400 mg. of glucose per 100 ml. could be completely fermented by 5 grams of yeast per 100 ml. in 1.5 hours a t all the pH's tested; final pH values are also given in this figure. F h e n the initial p H was below 9.0 the p H dropped rapidly to values below 8.0, which would allow maltose to be fermented; nhen the initial pH was above 9.3, however, the pH remained n ell above 8.5, which would preclude maltose fermentation. .A number of tests were made in which the concentrations of glucose and yeast were varied while the initial p H was kept constant a t 9.3. I t was found that a t glucose concentrations greater than 400 mg. per 100 ml. the pH eventually dropped below 8.0. The rate of fermentation decreased and fermentation was incomplete if less than 5 grams of yeast per 100 ml. was used. Therefore, an initial p H of 9.3 (obtained by using a buffer consisting of approximately 80 ml. of 1 3f sodium carbonate and 20 ml. of 1 .TI sodium bicarbonate with the exact p H determined by
means of a pH meter), 5 grams of yeast per 100 ml., a maximum of 400 nig of glucose per 100 ml. , and a fermentation period of 90 minutes are recommended for the analysis. Recovery of Maltose after Glucose Fermentation. The procedure described above was applied to maltose I and to a mixture of maltose I and glucose. The data obtained, summarized in Table I, show that maltose is not attacked within 90 minutes a t any pH above 8.4 and that it can be recovered quantitatively after glucose fermentation if the initial p H is 9.3. Recovery values in excess of 100% obviously are due to 3 reduction in the volume of the solution caused by the presence of the yeast cells. The recovery values for maltose in this experiment offer a ready means for determining the correction factor for yeast displacement; the average value for this correction factor is 3.2%, as shown in the last column of Table I. This factor must be applied to the values determined for maltose or other residual sugars. Replicate results were obtained using yeast which had been stored for 7 and 21 days, showing that the age of the yeast is not a significant factor in this procedure. MALTOSE FERMENTATIOK
Effect of Buffer Salts upon Rate of Maltose Fermentation. I t was pointed out by Stark and Somogyi (31) that the rate of maltose fermentation was lower in acetate buffer than in unbuffered water solutions. ( I t is understood that when maltose is fermented, glucose necessarily must also be fermented.) Therefore, in attempting to increase the maltose concrntration the effect of different buffers was tested first. Alaltose I1 was used in all these tests and the details of the procedure were the same as those for glucose fermentation, except for the buffers that were used. The data in Figure 3 demonstrate that by using either IlcIlvaine's citrate-phosphate buffer ( 5 ml. per 25 nil ) or 0.W JI citrate-citric acid buffer, 5 grams of yeast pcr 100 nil. could conipletely ferment 800 mg. of maltose per 100 ml. i n 2 hours. Frrmentation was barely complete in 2.5 hours n-hr,n 0.025 J1 :iwtate buffer was used and was far from complete i n this length of time n-hen 0.05 JI acetatc huffer was used.
Table I.
Initial
Recovery of 3Ialtose from Glucose Fermentation llaltose Maltose Glucose after 40 . Used Minutes hIg. per 100 inl.
PIT 8.4 174.0 8.9 174.0 9.3 174.0 9.3 972 9.3 48.6 9.3 194.4 9,3 97.2 9.3 48.6 Yeast, 5 grariis per 100
.. . .. ... ...
180 2 178 0 178.8
ilib
...
,
.....
...
400 ... 400 , . . nil., 12 day- old.
Correction Maltose Recovered Factor f o r after 90 ~ o l u m eof Minutes yeast cells, 3Ip./100 Recovery, % ml. 181.0 178 0 179.2 99.3 50.3 200.2 101.2 50.4
5%
4.0 2.5 3.0 2.2 3.4 2.9 4.3 3 6
104.0
102.5 103.0 102.2 103.4 102.9 104.3 108.6 AV.
3.2
Khen 0.2 M acetate buffer iT-as used [the same concentration that Stark and Somogyi used in some of their experiments (SI)], even 15 grams of yeast per 100 ml. failed to ferment completely 40 mg. of maltose per 100 ml. (This is especially true with yeast obtained during the summer. Past experience in this laboratory indicates that complete fermentation does occur in most cases.) A fairly high fermentation rate could be obtained in unbuffered n-ater solutions; hoxever, the p H dropped during the fermenta-
ANALYTICAL CHEMISTRY
234 tion to 3.8 in 40 minutes and the fermentation was not complete in 2.5 hours. The residual carbohydrate after the fermentation of maltose IT in McIlvaine's buffer amounted to 0.6 to 0.9%of the initial maltose used. Paper chromatograms were prepared with the residue after it had been purified by basic lead acetate precipitation and treatment with Amberlite resins. These chromatograms showed only spots which corresponded to those for the contaminating saccharides found in the original commercial maltose (sample PB, Figure l), but no maltose spot. Therefore, the complete fermentation of maltose was ensured by using McIlvaine's buffer. Other Factors Affecting Rate of Maltose Fermentation. Experiments in which the pH and concentrations of maltose and yeast were varied showed that within a pH range of 3.6 to 5.5, 5 grams or more of yeast was sufficient to ferment completely 800 mg. of maltose per 100 ml. in 2.5 hours. Therefore, a pH of 4.8 (using McIlvaine's buffer), a yeast concentration of 5 grams per 100 ml., and a fermentation period of 150 minutes were adopted as appropriate values to be used.
Table 11. Recovery of Maltotriose during Fermentation of Glucose and Maltose Maltose (or GluTest cose)Used.
KO. AIg./lOO All.
1
2 3 4 B
6
i
9 10 11 12
13 14 15
0
20 40 80 160 400 800 400 400 400 400 400 0 400 (glucose)
Preparation BO Used, Mg./100 Ml. b 43 6 43 6 43 6 43 6 43 6 43 6 43 6 15 6 38.7 77.4 193.5 387.0 193.5 36.7
Errors Introduced t o Recovery of Car- Maltose (or Glucose) hohydrates Con- Values Due t o Fertained in Prep. B, mentation of Malto"cc triose, 70 98 8 0
101 99 97 95 88
2 (97 81d 5 (96 0)d 8 0 (78 2j4
80 8 94 2 92 6 91 0 86 0 80 5 99 2 95 0
1 13 1 3 1 (2 3 8 ) l 1 03 0 22 0 72 1 75 6 87
18 9
0 45
2 23 (glucose) a Containing maltotriose as major component. h Values refer to total amount of carbohydrate contained in preparation B. C Corrected for volume occupied b y yeast cells. d Values in parentheses refer t o experiments with pure maltotriose pH 4.8 with hlcIlraine's buffer, yeast 5 ? 7 , 8 days old. 400
178 5
2 2
0 88 (2 4 0 j d I 36 (2 18jd
95 0
EFFECT OF STORING YEAST
Yeast which had been stored in the refrigerator for periods not exceeding 3 weeks could be used without showing any effect on the rate of fermentation. Even when yeast which had been stored for 5 weeks and had become slightly moldy was used, the fermentation was 98.6% complete in 2.5 hours. The age of the veast, therefore, is not a significant factor in the modified procedure. However, the use of fresh yeast is always to be preferred. RECOPERY OF OLIGOSACCH4RIDES OTHER THAN MALTOSE
The procedure for maltose fermentation described above u-as applied to preparation -4,which was a mixture of isomaltose and panose; neither of the sugars \Tas attacked and thus both could be recovered quantitatively from maltose fermentations. The data are very similar to those for the recovery of maltose from glucose fermentation and, therefore, are not presented here. For all practical purposes, maltotriose (preparation B), in the absence of maltose (or glucose), was not fermented by baker's yeast (see Table 11, tests 1 and 13). In the presence of maltose, however, maltotriose partially disappeared. The recovery of maltotriose decreased as either the maltose or the maltotriose concentration increased, as sho5-n in Table 11. As much as 20% of the carbohydrates contained in preparation B was lost a t the highest concentrations tested (Nos. 7 and 12). This apparently agrees with Myrback's findings that maltotriose is more readily fermented than either isomaltose or the trisaccharide with one maltose and one isomaltose linkages (18). Tests with pure maltotriose showed similar but higher losses (values in parenthesis, Table 11); this was to be expected, since only a part of the carbohydrate in preparation B was maltotriose. The loss of maltotriose was far less in the presence of glucose, as shown in tests 14 and 15, Table 11. Attempts t o eliminate this loss of maltotriose by using pH's other than 4.8 for the yeast treatment were unsuccessful. This loss can be avoided, however, by limiting the size of the sample used for analysis. When the total carbohydrate concentration of a mixture of pure maltose and maltotriose was not greater than 100 mg. per 100 ml., the errors introduced were within 370,as shown by tests 1 to 4, Table 11; this would be satisfactory for most purposes. However, when the maltotriose concentration of a maltose sample is very small-Le., 5 % or less of the total carbohydrate-a total carbohydrate content of 100 mg. per 100 ml. nil1 be too low for an accurate determination of the residual sugar (maltotriose), and a larger sample must be used. As shown in test 6, Table 11, when the total carbohydrate concentration is increased to 400 mg. per 100 ml., the error in maltose calculation (2.4%) is still small enough for most purposes. In that case, of course, the maltotriose value will become only a rough approxi-
mation. When the sample contains sugars other t h a n maltose and maltotriose, as is usually the case, and the total carbohydrate concentration is the same as that reported here, the errors naturally are much smaller. FERMENTATION OF OLlGOSACCHARlDES
Khen tested under pure culture conditions in the synthetic medium of Olson and Johnson (19), the distiller's yeast SC 1y was found capable of fermenting maltotriose but not isomaltose and panose. The medium contains per liter: ammonium dihydrogen phosphate 6 grams, potassium dihydrogen phosphate 0.2 grams, magnesium sulfate heptahydrate 0.25 gram, sodium citrate 1 gram, Lasparagine 2.5 grams, biotin 2 micrograms, calcium pantothenate 0.5 mg., inositol 10 mg., thiamine 4 mg., pyridoxine 1 mg., zinc (as sulfate) 400 micrograms, iron (as ferrous ammonium sulfate) 150 micrograms, copper (as sulfate) 25 micrograms, and glucose 10 grams; pH 5.0. A number of other yeasts, however, were found to be capable of fermenting all three sugars. Distiller's yeast NRRL Y-132 was especially efficient in removing these three oligosaccharides, as judged on the basis of rate and completeness of fermentation. Fermentation by this yeast \\as studied in detail to determine its use for quantitatively removing these three oligosaccharides. No attempt was made to differentiate among these three sugars, inasmuch as sufficient quantitieq of pure maltotriose and isomaltose were not available.
Table 111. Fermentation of Panose by Pure Culture of Yeast NRRL Y-132 panoseUsed in ~l~~~~~Used in Residual Sugar in 7 Mi. of Medium Test 7 M1. of Me- 7 M1. of Me- 2 days, 3 days, 6 days, mg. mg. '36" dium, hlg. No. dium, Mg. mg. 2 4 101.5 31.5 3.50 .. 146,O 2 7 15.2 4.9 1.96 73.0 3 2 10.5 0.75 1.5 .. 23.3 11.4 1.25 2.93 0.86 .. 7.47 0 . 8 4 34 9 2.20 1.05 .. 2.39 2 5 1.30 0.58 11.8 70 23.3 9 1 3.10 0.68 1.12 70 7.47 As % of original trisaccharide used.
..
@
Panose was available in pure form and, therefore, was used i n the following tests as a representative of these three fermentahle oligosaccharides. Preliminary tests showed that yeast S R R L Y-132 fermented preparations A and B as rapidly as it did panose. Removal of panose with a cell suspension of yeast YRRL 1*-132 was unsatisfactory because the fermentation of the last 10% of
V O L U M E 25, NO. 2, F E B R U A R Y 1 9 5 3
235
the sugar was slow and incomplete. ri feasible alternative seemed to he fermentation under pure culture conditions. Figure 4 shows the progress of the fermentations of glucose, maltose, and panose by yeast XRRL Y-132 in the synthetic medium of Olson and Johnson. The fermentation of panose was very slow when compared with that of a glucose or maltose, hut vas 98.3% complete in 4 days. To avoid introducing errors through the use of bacteriological techniques, the following procedure was adopted for quantitative tests on panose fermentation:
3.0
g
I
28
0
-
2
20
L
‘5
s IO
L OLUCOSl
05
IO
20
30
40 TIYE
50
-
e0
70
e0
80
100
HOURS
Figure 4. Fermentation of Glucose, Maltose, and Cr) stalline Trisaccharide Panose by Pure Culture of Yeast NRRL Y-132 Lsing s)nthetic medium of Olson and Johnson. perature, 30’ C.
Incubation tem-
The carbohydrate samples to be analyzed were buffered a t pH 4.8 to 5.0 with one tenth their volumes of McIlvaine’s buffer and sterilized in test tubes. It was found necessary to buffer the samales a t this pH level to minimize the destruction of sugars and the ydrolysis of higher saccharides (the dextrin sample was used in this test) during autoclaving. After cooling, an equal volume of a sterilized solution containing all of the nutrients of the synthetic medium of Olson and Johnson, but without glucose and a t twice their usual concentrations, was added aseptically. The resulting solution was inoculated with an actively growing 24- to 48-hour culture of yeast S R R L Y-132 grown in the same medium, but u i t h glucose as the source of carbohydrate; the mixture was incubated a t 30” C. A total of 7 ml. of medium per test tube was found convenient for shaking. After fermentation, the contents of the test tubes were diluted to 25 ml. in a volumetric flask and centrifuged. An aliquot sample of the centrifugate was taken for analvsis of residual sugars. Results from fermentations of samples containing different amounts of panose and glucose are summarized in Table 111. \Vhen the sample contained more than 20 mg. of panose per tube ( 7 ml.), 97y0 or more of the trisaccharide was removed in 6 days (tests 1, 2, 3, and 6). When less than 20 mg. of panose per tube ( 7 ml.) was present, a much higher percentage of the trisaccharide u a s left unfermented (tests 4 and 5). When these experiments
Table 17’.
Recoverj of Dextrin from Panose Fermentation Residual Carbohydrate after Fermentation Corrected for rrsid. Apparent ual c a r b o h y - - Corrected recovery drate introduced dextrin of dextrin from panose, recovery, xtg,a % mg.a % 4.76 97.3 ... ,.. 9.55 97.7 ... ,.. 19 8 101.0 ... ... ~~~~
Glucose Used, Mg.0
Panose Used, 31g.n
Dextrin Used,
..
.. ..
4 9 9.8 19.6
..
io
zi:o 0.70 21.0 419 5 . 7 6 iij:3 21.0 9.8 10.85 110.5 21.0 19.6 20.7 105.5 21.0 39.2 39.6 101.0 hlg of carbohydrate per tube (7 ml.).
70 70 70 70 a
11g.a
...
5.06 10.15 20.0 38.9
103:2 103.5 102.0 99.2
were repeated in the presence of glucose fermentation, there was very little difference (tests 6 and 7 ) . Apparently, fermentation virtually stops when the trisaccharide concentration falls below 1 mg. per tube (7 ml.). Paper chromatography showed that the residue consisted primarily of panose plus traces of higher saccharides. This explains why there was more than 1 mg. of residual sugars per tube when the initial panose concentration was 70 mg. or more per tube (tests 1 and 2). Without evaluating the exact amount of higher saccharides present, it is to be emphasized that for quantitative determinations of these fermentable oligosaccharides sufficient sample should he used to ensure an initial concentration of more than 20 mg. of these sugars per 7 ml. of medium (at least 3 mg. per ml.), and a fermentation period of 6 days is required. Oligosaccharides not fermented by yeast NRRL Y-132 are not so well defined. The dextrin sample, representative of much higher saccharides, was chosen arbitrarily to test its recovery from the fermentation of fermentable oligosaccharides. The results of these tests are summarized in Table IV. The residual sugar derived from the fermentation of panose naturally introduces an error in the recovery of the dextrins. In the column marked “Apparent Recovery” it is shown that with a constant amount of panose this error u-ould he espected to decrease as the concentration of dextrin increased. When Forrections for these residual Carbohydrates were made, fairly accurate recoveries were obtained (last column, Table IV). APPLICATION OF MODIFIED METHOD TO CARBOHYDRATE MIXTURES
The method developed was then applied to the analysis of the follox-ing materials: 1. Corn mash converted hy barley malt (52). 2. Corn mash converted by submerged culture fungal enzymes (1). 3. The reaction mixture in which maltose was converted to other oligosaccharides by fungal enzymes (21 ).
Table 7’.
Samples
zyme converted corn mash Mixture of maltose plus fungal enzyme
.4nalytical Results of Carbohydrate Mixtures Total Carbohydrates
Fermentable OligosacGlucose Maltose charides Milligrams per Milliliter
Unfermentable Residue, (Dextrins)
120.9
11.1
42.6
27.0
40 2
94 5
12 3
56.0
22.6
3.6
Four samples were taken of each material and analyzed for: (a) total carbohydrates after acid hydrolysis; ( b ) residual carhohydrate after glucose fermentation a t pH 9.3; (c) residual carbohydrates after glucose and maltose fermentations a t p H 4.8; and ( d ) residual carbohydrates after glucose, maltose, and oligosaccharide fermentations with pure culture of yeast XRRL Y-132. From these data the concentrations of glucose] maltose, fermentable oligosaccharides, and unfermentahle dextrins mere calculated and are presented in Table V. Each of these materials contained fairly large amounts of fermentable Oligosaccharides, as shown in Table V. There was no difficulty, therefore, in adjusting the size of the samples so that the medium would contain more than 20 mg. of fermentable oligosaccharides per 7 ml. of medium for fermentation with yeast S R R L Y-132. The residue from each fermentation was purified by precipita-
ANALYTICAL CHEMISTRY
236
Funpol Enzyme Converted tion with basic lead acetate, folloLved by treatC a n Mesh ment with Amberlite resins; the solutions were then concentrated. Paper chromatograms of these concentrates are represented diagrammatirally in Figure 5. It can be seen that eachyeast 111 GLUCOSE fermentation removed one spot corresponding to the sugar to be fermented, leaving the other sugars intact. i The fermentability of some common sugarsI21 MALTOSE 12’]0 viz., sucrose, fructose, mannose, galactose, lac1s) ISOMALTOSE 0 0 0 tose, xylose, and arabinose-also has been tested n ith the method described. An initial concencilMuToTRloSE tration of 100 mg. of sugar per 100 ml. \vas used for all fermentation tests with baker’s yeast and 10 mg. per ml. for pure culture fermentations nith yeast S R R L Y-132. These fermentation tests made it possible to classify the sugars into Figure 5 , Paper Chromatogram of Carhoh>-drateMixtures before and four fractions: Fructose, sucrose, and mannose after Fermentation (91%) were fermented in the glucose fraction; 1. Original sample mannose was completely fermented in the maltose 2. Residue after removing glucose 3. Residue after removing glucose a n d maltose fraction; galactose was in the fermentable oligo4. Residue after removing all fermentable sugars saccharide fraction; and xylose, arabinose, and lactose were unfermentable and therefore were classified in the defrin fraction. In addition to revealing the (McIlvaine’s buffer) can be conipletely fermented by 5 grams of varied fermentability of the sugars used in these tests, the washed baker’s yeast per 100 nil. within 90 and 150 minutes, respectively. The residual “unfermentable” carbohydrates can be method should also be applicable to other carbohydrate mixtures from sources surh as wood hydrolyzates and sulfite waste liquors. determined with a high degree of accuracy even if they represent only a small percentage (5% or less) of the total carbohydrate. DISCUSSION AKD CONCLUSIONS Isomaltose, maltotriose, and panose can be removed completely by employing a pure culture fermentation with a distiller’s yeast The modifications of the Stark and Somogyi method for Thus, a carbohydrate mixture, such as an enzymic starch hyanalyzing fermentable sugars described in this report make it drolyzate, can be quantitatively differentiated into four fracpossible to analyze samples containing eight times as much glutions: glucose, maltose, fermentable oligosaccharides, and uncose and ten times as much maltose (if maltotriose is not present) fermentable residue (dextrins). Results of the analysis can he as is possible with the original method. Even small amounts of checked qualitatively by paper chromatography. unfermentable residue can be determined with a high degree of
0
00
accuracy. In addition, the modifications ensure the complete feinientation of maltose even n ith relatively old yeast. Thus, the original Stark and Somogyi method has been improved. The ready fermentability of maltotriose and the incomplete fermentation of panose introduce errors in the values for maltose and fermentable oligosaccharides. These errors can be minimized to within 375,however, by properly adjusting the size of the samples. Errors from other sources are avoided by using quantitative techniques throughout the procedure. It is recognized, however, that the following disadvantage$ cannot be eliminated by the modified method described: Khen only a small amount of maltotriose is present i n a maltose samplr, the concentration of maltotriose can only be approximated. Khen the concentration of fermentable oligosaccharides is less than 3 mg. per ml., the errors in the values for this fraction are high. The incomplete fermentation of the fermentable oligosaccharides will also affect the values for unfermentable dextrins, especiallv when these dextrins are present in small amounts. The modified method described shares the advantages of the original method of Stark and Somogyi-namely, that only baker’s yeast, which is readily available, is used for the determination of glucose and maltose and that a fairly large number of samples can be handled conveniently. Only very simple bacteriological techniques are involved in determining the fermentable oligosaccharide fraction; homver, a little practice is required to enable one to perform the analysis competently. SUMM4RY
The Stark and Somogyi method for analyzing a mixture of sugars has been modified to improve its reliability and accuracy. Vp to 400 mg. of glucose per 100 ml. a t pH 9.3 (carbonate-bicarbonate buffer) and 400 mg. of maltose per 100 ml. at pH 4.8
0
ACKNOWLEDGMENT
The authors wish to thank 11.L. Wolfrom and his associates of the Ohio State University for the samples of pure maltotriose and isomaltose. LITERATURE CITED
(1) Adams. S. L.. Balankura. B.. Andreasen. A. -4.. and Stark. It’ H , > , I n d . Eng. Chem., 39, 1615 (1947). (2) Back, T. M., S t a r k , IT. H , and Scalf, R. E , A N ~ L CHEM, 20, 56 (1948). (3) Bates, F. J., a n d associates, L. S.B u r . S t a n d a r d s . Circ. C440, 470 (1942). (4) Blass, J., Rlacheboeuf, XI., and S u n e z , G., BuZL S O C . chim. biol.. 32, 130-5 (1950). (5) Blom, J., and Rosted, C. O., Acta Chem. Scand., 1, 233 (1947). (6) Browne, C. A,, a n d Zerban, F. IT,,“Physical a n d Chemical Methods of Sugar Analysis,” pp. 893, 984, 1000, New Y o r k , J o h n Wiley & Sons, 1951. (7) Doudoroff, M., Hassid, W. Z., P u t m a n , E. IT., P o t t e r , A. L., a n d Lederberg, J., J. Biol. Chem., 179, 921 (1949). (8) French, D . , Science, 113, 352 (1951). (9) Harding, V. J., a n d Nicholson, T . F., Biochem. J . , 27, 1082 (1933). (10) Jeanes, A,, Wise, C. S., a n d Dimler, R. J., ANAL.CHEM.,23, 415 (195 1). (11) M c F a r r e n , E. F., B r a n d , K., a n d Rutkowski, H. R . , -4bstracts of P a p e r s , 119th meeting, AM..CHEM.SOC., p. 41C, 1951. (12) Montgomery, E. M., TTeakley, F. B., a n d Hilbert, G. E., J . An:. Chem. Soc., 71, 1682 (1949). (13) Montreuil, J e a n , Bull. SOC. chim. biol., 31, 1639 (1919). (14) h l y r b a c k , K., Adcances in Carbohydrate Chem., 3, 251 (1948). (15) M y r b a c k , K., Biochem. Z.,304, 147 (1940). (16) M y r b a c k , K., Suensk Kern. Tidskr., 53, 67 (1941). (17) h l y r b a c k , K., a n d Leissner, E., h k i v Kemi M i n e r a l . Geol.,l7A, No. 18 (1943). (18) Myrback, K., a n d Neumuller, G., in “ T h e Enzymes, Chemistry a n d Mechanism of Action,” edited b y S u m n e r , J. B.. and MyrbKck, K., Vo1. I. Pt. 1. p. 671, New York. Academic Press, 1950.
V O L U M E 25, NO. 2, F E B R U A R Y 1 9 5 3 (19) (20) (21)
Olson, B. H., and Johnson, M. J., J . Bact., 57, 235 (1949). Ortenbald, B., and Myrback, K., Biochem. Z . , 315, 233 (1943). Pan, S. C., Andreasen, A. A,, and Kolachov, Paul, Science, 112, 115 (1950).
237 (31) (32) (33)
Pan, S. C., Nicholson, L. W., and Kolachov, Paul, J.Am. Chem. SOC.,73, 2547 (1951). (23) Parur, J. H., and French, D., Ibid., 73, 3536 (1951). (24) Parur, J: H., and French, D., paper presented before Division of Biological Chemistry, a t 118th Meeting, AM. CHEM.Soc., (22)
3357 (1949).
Tsuchiya, H. M., Borud, 0. J., and Corman, J., paper presented before Division of -4gricultural and Food Chemistry, Fermentation Subdivision, a t 118th Meeting, AM. CHEM.SOC.,1950. (35) Wolfrom, X I . L., Georges, L. W., Thompson, Aha, and Miller, (34)
I. L., J . Am. Cham. SOC., 71, 2873 (1949).
1950.
Schoch, T. J., Adiances in Cnrbohydrnte Chem., 1 , 247 (1945). Schultz, A. S., Fisher, R. A,, ;2tkin, L., and Frey, C. N., IND. ENG.C H E Y . , AK.4L. ED., 15, 496 (1943). (27) Schultr, A4.S., and Kirby, G. IT., Cereal Chem., 10, 149 (1933). (28) Yhu, P., Can. J . Research, 28B, 527 (1950). (29) Somogyi, A I , , J . Biol. Chem , 119, 741 (1937).
(25) (26)
(30) Ibid., 160, 61 (1945).
Stark,I. E., and Somogyi, M., Ibid., 142, 579 (1942). Stark, W. H., Adams, 9.L.,Scalf, R. E., and Kolachov, Paul, IND.ENG.CHEM.,ANAL.ED.,1 5 , 4 4 3 (1943). Sugihara, J. M., and Wolfrom, M. L., J . Am. Chem. Soc., 71,
(36)
Wolfrom, 31.L.,vhompson, illva, and Galkomski, T. T., Ihzd.,
73, 4093 (1951). (37) Ibid., p . 5849.
RECEIVED for review June 27, 1952. Accepted November 13, 1952. Presented before the Division of Agricultural and Food Chemistry at the 119th Meeting of the AUERICANCHExIcAL SOCIETY,Cleveland, Ohio.
Combustion Techniques for the Determination of Residues of Highly Chlorinated Pesticides By Total Chlorine Analysis E. J. AGAZZI, E. D. PETERS, AND F. R. BROOKS Shell Development Co.,Emeryrille, Calif. In the absence of specific analytical methods, trace quantities of highly chlorinated pesticides can sometimes be determined in agricultural materials by applying sufficiently sensitive total chlorine methods. Although this approach is not specific, it yields useful results when preceded by appropriate clean-up procedures to remove extraneous chlorine compounds and interfering impurities. Accurate and reliable methods have been developed for determination of the chlorine content of residues from such clean-up operations. Three methods are described which involve combustion of the residue, either in a horizontal tube or in a wick-type lamp, followed by absorption of the chlorine from the combustion products by passage through dilute caustic solution. The chloride ion content of this solution is then determined by a sensitive amperometric titration, employing 0.001 ,V silver nitrate solution as the titrant. Data are presented to illustrate the accuracy and precision of these methods when applied to the determination of the highly chlorinated pesticides, aldrin and dieldrin.
I
S RECEST years agricultural chemists have been confronted
with the problem of developing suitable analytical methods for determining trace amounts of pesticides associated with agricultural materials (6). Saturally, wherever possible specific methods have been developed but, lacking these, it has sometimes proved feasible to use nonspecific methods. Thus, for example, total chlorine analysis has been used successfully for the determination of highly chlorinated pesticides in some circumstances (3,4). Bioassay has also constituted a useful approach in other instances ( 7 ) . The present paper is concerned with the development and application of several techniques for the determination of total chlorine in connection Tvith the problem of analyzing for the pesticides aldrin and dieldrin (IO). These insecticides contain 58.3 and 56.1% chlorine, respectively, and, hence, it was felt that a sensitive total chlorine method ~vould have considerable utility. The method which has been most commonly used for this
determination involves reduction of the sample by rpfluxing u ith metallic sodium in isopropyl alcohol, followed by titration of the resulting chloride ion with silver nitrate solution (3, 4, 15). Preliminary tests by the authors indicated that this approach suffered from tR o inherent limitations: 1. Blank values were obtained which \\ere too high to allow the advantageous use of a highly sensitive chloride determination and were attributed to chloride introduced bv the large amounts of reagents required and/or interference of fatty acids from saponification. 2. The large quantities of salts resulting from the sodium decomposition process complicated the application of sensitive chloride determinations. Therefore, attention lias directed toward the development of a method for sample decomposition which would be free from these objections and would permit the full utilization of sensitive methods for the deterniination of chloride. From past extensive experience in the Shell laboratories with the application of combustion methods to the determination of chlorine in materials of petroleum origin, it appeared likely that similar combustion techniques would be well suited to the decomposition of organic agricultural materials containing chlorine Furthermore, it appeared that combustion methods would be free from the objections inherent in the sodium reduction method. The application of combustion techniques to agricultural products is not a new concept. I n 1928 McClendon (22) proposed combustion in a quartz tube in connection with the determination of iodine in agricultural materials. Godfrey, Parker, and Quackenbush recently made use of combustion in a quartz tube to determine iodine in plant materials ( 5 ) . Krause et nl. (8) employed microcombustion to determine hexachlorocyclohexane in flour. Investigation of this approach by the authors has led to the development of three useful cornbustion techniques, each with its om-n realm of applicability. Each of these combustion techniques is followed by an amperometric titration of chloride ion, a t the rotating platinum electrode, using 0.001 A’ silver nitrate solution as the titrant. 4 s with all total chlorine determinations, these methods are limited in application by their lack of specificity. Useful results can be obtained only n hen either the samplr is known to be free
