Microbiological Assay of Glucose as Applied to Starch Hydrolyzates

Preparation and Evaluation of Hydroxyethyl Starch. S. I. El-Hinnawy , A. Fahmy , H. M. El-Saied , A. F. El-Shirbeeny , K. M. El-Sahy. Starch - Stärke...
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Although t,he pyrolysis technique was developed for the qualitative analysis of explosive mixtures and the determination of thermal sensitivity and stability, it has also been used for quantitative analysis. Volatile fractions o1 decomposition products can be trapped for identification and determination by suitable means, or pyrolysis cllrve areas can be planimetered.

Sei. In&. 29; 392 (1958). '

(2) Beamish, F. E., Westland, A. D., ANAL.CHEM.30, 805 (1958). (3) Duval, C., "Inorganic Thermogravimetric Analysis," Elsevier, Amsterdam, 1953. (4) Felton, H. R.,Roc. Instrument SOC. Am., 1957 International Gas Chromatography Symposium, p. 113. (5) Felton, H. R., Buehler, A. A,, ANAL. CHEM.30, 1163 (1958). (6) Grimshaw, R. W., Heaton, E., Roberts, A. I,., Trans. Brit. Ceram. SOC.44, 76 (1945). ( 7 ) Haul. R. A. W.. Heystek, H., Am. .1952). orem; S. D., Proc. Instrument SOC. International Gas Chromatography Symposium, p, 162.

(9) Norton, F. H., J . Am. Ceram. SOC.22, 54 (1939). (10) Robertson, A. J. B., J . SOC.Chem. Znd. (London)67,221 (1948). (11) Robertson, A. J. B., Trans. Faraday Soc. 45, 85 (1949).

(12) ,Rowland, R.. A., Lewis, D. R., Am. Mzneralogist 36, 80 (1951). (13) Stone, R. L., J . Am. Ceram. SOC.35, 76 (1952). (14) Wendlandt, W. W., AXAL.CHEM.27, 1277 (1955). RECEIVEDfor review August 31, 1959. Accepted March 7, 1900. Work performed under the auspices of the U. S. Atomic Energy Commission.

Microbiological Assay of Glucose as Applied to Starch Hydrolyzates M. DOREEN SMITH, M. W. RADOMSKI, and JOHN J. KAGAN Department of Food Chemisfry, University of Toronto, Toronto, Ontario, Canada b A microbiological method for the quantitative determination of glucose in hydrolyzates of starch and related polysaccharides i s based on the ability of lactobacillus casei to utilize glucose selectively in a mixture of sugars in those hydrolyzate samples. A heavy inoculum technique shortens the incubation period to 2 hours and at the same time i s sensitive to small amounts of glucose. Response i s measured in terms of acid production. The glucose content in unknown samples i s obtained from a standard curve which i s linear up to 4 mg. of glucose. Agreement i s good when this method i s compared with physicochemical methods for determining glucose in maltose and starch acid hydrolyzates. The method is accurate and easily adapted for large scale operations.

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techniques for assaying specific biological factors are useful in analytical chemistry (4). Basic concepts, techniques, practical applications, and future possibilities have been summarized recently ( 6 ) . Of the microorganisms used, bacteria, particularly the lactobacilli, have been widely applied and are the basis of official analytical methods for riboflavin and niacin (1). The application reported here takes advantage of the ability of Lactobacillus casei to ferment glucose selectively in a mixture of sugars found in starch conversion liquors, Total response is determined by titration with a standard alkali solution, and the dose-response curve is linear up to 4 mg. of glucose. Because a n energy 678

ICROBIOLOGICAL

ANALYTICAL CHEMISTRY

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4 6 REACTION TIME, hours at 100°C. Figure 1.

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Acid hydrolysis rate curve for maltose 0

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Microbiological assay Reducing method Optical rotation

source and not a growth factor is determined, it is possible to use a heavy ;noculum with the accrued benefits of a considerably reduced incubation period and elimination of extreme sterilization and aseptic precautions. The method has been used for some time in this laboratory and is particularly adapted

for determining hydrolytic glucose yields over the wide range of concentrations encountered during acid hydrolysis of dilute polysaccharide solutions. MATERIALS

L. casei (ATCC 7469) was maintained b y frequent Stock Culture.

transfers on a glucose-agar-yeast medium (9). The culture was incubated a t 37" C. until good growth occurred (18 to 24 hours), and refri rated until the next transfer. A fregly incubated stab culture was always used for preparation of heavy inoculum. Basal Medium. This medium was substantially similar t o t h a t used for riboflavin assay (1). The photolyzed peptone and yeast supplement were replaced by Bacto peptone (1.OojO) and Bacto yeast extract (0.2%), respectively. In addition to L-cystine, Lasparagine and Gglutamic acid were added in 0.025% amounts (9). Glucose and riboflavin were omitted from this medium but were added as separate solutions for the preparation of inoculum. Usually, 1-liter amounts were prepared and 100-ml. portions were poured into 125-ml. Erlenmeyer flasks. These were plugged with cotton, autoclaved a t 15 pounds for 20 minutes, and refrigerated. Buffered Saline-Phosphate Solution. Sodium chloride (8.00 grams) and anhydrous sodium acetate (1.00 gram) were dissolved in 100 ml. of distilled water. Salt solution A (0.5 ml.) (1) was added and the p H adjusted t o 6.8 with acetic acid. This solution was refrigerated under toluene. PROCEDURE

Preparation of Washed, Heavy Inoculum. T o each of four 50-ml. centrifuge tubes, add 20 ml. of basal medium, 4 ml. of glucose solution (l.OM), 0.16 ml. of riboflavin solution (25 y per ml.), and distilled water to a final volume of 40 ml. Autoclave the tubes at 15 pounds for 20 minutes, inoculate, and incubate a t 37" C. for 24 hours. Centrifuge the cell suspensions a t 3000 r.p.m. for 15 minutes and discard the supernatants. Combine and resuspend the cells into one centrifu e tube by a 25-ml. wash with sterile O.& saline and centrifuge. Discard the supernatant and wash the cells with another portion of sterile saline to ensure complete removal of lactic acid and to reduce background contamination. Centrifuge, and suspend the white cellular mass in 2 ml. of sterile 0.9% saline with a 10-ml. blowout pipet fitted with a mouthpiece and a length of rubber tubing which holds a glass-bead valve to deliver standard drops of the heavy inoculum. Approximately 25 assays can be run with this amount of inoculum. Glucose Assay. Pipet an aliquot of the sample to be analyzed, containing not more than 4 mg. of glucose, into a test tube (150 X 15 mm.). Add 1 ml. of buffered saline-phosphate solution and adjust the total volume to 10 ml. with distilled water. With each run, assay 0 to 4 mg. of glucose in duplicate to obtain a standard curve. Autoclave the tubes a t 15 pounds for 5 minutes, cool, and inoculate each tube with 2 drops of heavy inoculum. Incubate the assay tubes a t 37' C. for 2 hours in a constant temperature water bath or an incubator. After incubation, pour the contents of

each tube into a 100-ml. bea,ker with two 10-ml. rinsings of distilled water and titrate electrometrically with 0.005N sodium hydroxide to pH 6.75 i 0.05. The buffered saline-phosphate maintains a suitable p H level in the glucose assay tubes (pH was reduced to 5.40 a t the 3-mg. glucose level), and the addition of phosphate prevents excessively large variations in titratable acidity ( 8 ) - Precision of duplicates is improved by using a 5-ml. buret with 0.01-ml. divisions for the titrations. Calibration. Prepare a standard curve by plotting the average titration values for each level of glucose assayed against milligrams of glucose in the respective tubes. Obtain glucose content in assay tubes by interpolation from the standard curve or, more conveniently, by the relation

G

=

KT

where G is total glucose in milligrams, T is the average titration value in milliliters, and K is a constant derived from the curve. Average value for K obtained from 10 standard curves was 0.54 =k 0.04. Blank titrations ranged from 0 to 0.40 ml, of 0.005N sodium hydroxide. Precision and Recoveries. Analyses of 50 duplicate titrations gave an average difference of *3,0% with a standard deviation of 0.148. T h e average difference was reduced t o 1.5%, standard deviation 0.043, by titrating with the 5-ml. buret, Recoveries of1 -mg. amounts of glucose added t o starch hydrolyzate samples were close t o 1 0 0 ~ (Table o I).

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APPLICATION

Increase in glucose during acid hydrolysis of maltose was followed by microbiological assay; typical results are shown in Table 11. I n this experiment, a dilute solution (approximately 1%) of crystalline maltose was hydrolyzed with 0.07N hydrochloric acid a t 100' C. Concentration in terms of glucose was 6.51, 6.42, and 6.38 mg. per ml. as determined by the anthrone-sulfuric acid reagent (8), by reduction after total acid hydrolysis (61, and by optical activity, respectively. Degrees of hydrolysis a t specific time intervals as calculated by increase in reducing activity (difference titration) and from optical rotation data agreed well with values obtained by microbiological assay (Figure 1). The method was applied to a study of specific glucose content in acid hydrolyzates of starch-type polysaccharides. I n these studies, dilute solutions of the polysaccharides were hydrolyzed with 0.07N hydrochloric acid a t 100' C. Glucose content in the neutralized hydrolyzate samples was determined by microbiological assay and by the anthrone method after separation of

Table I. Recovery of 1 Mg. of Glucose Added to Starch Hydrolyzate Samples Glucose Content, Mg. HydrolAfter yzate glucose Recovery, Sample Original addition % 1 2 3 4

0.19 0.29 0.76 1.14

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1 16

6 7

2.73 3.29

97

1.16 1.29 1.80 2.16 2 19 3.68 4.29

100

Av.

104 102 103 95 100 100.1

Table II. Increase in Glucose during Hydrolysis of Maltose with Hydrochloric Acid Time of Hydrol- HydrolGlucose yzate ysis, Titerlb Content, Sample" Hours 1\11. Mg./Ml. 0

1

2 -

0.5

3 4 5 6 7

1.0 1.5 2.0 3.0

s 9

10 11

4 0

5.0 7.0 8.0 m

0.30 2.61 4.48 3.05 3.67 4.61 5 40 5.70 5.95 6.02 6.15

0

1.36 2.33 3.21 3.87 4.86 5.69 6.00 6.27 6.33 6.48

1.0-ml. aliquots analyzed for samples 1 to 3; 0.5-ml. aliquots for all others. b Average of two titrations with 0.005N sodium hydroxide, within =kt1.5y0 of one a

another.

Table 111. Glucose Content in Acid Hydrolyzates of Various Polysaccharides Glucose Content, Mg./hll. Paper Dextrose Micro- chioPolyEquiv- biological matogsaccharide &lento assayb raphyO

Amylopectin

1.55 2.22 2.94 4.39 0.49 1.23 3.16 3.47 4.58 5.86 0.40 1.15 2.20 2.78 3.92 4.02 6.46 0.33 1.08 1.54 2.70 3.82

1.54 2.36 3.10 4.25 0.48 1.27 2.96 3.53 4.57 5.74 0.38 1.12 2.17 2.61 3.91 3.98 6.45 0.30 1.13 1.50

25.3 43.6 65.6 68.9 79.7 97.7 Starch 27.9 46.3 63.0 73.1 84.5 86.6 98.0 Mixture of 3 5 . 5 amylose and 4 8 . 9 amylopectin 5 8 . 0 2.61 71.9 3.71 87.0 0 Determined by ferricyanide-cerimetric method (6).

b Average of two determinations, results within =k3Y0of one another. e Average of at least four determinations, results within &55Z0 of one another

(3).

VOL. 32, NO. 6, MAY 1960

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glucose on paper chromatograms (8) and compared (Table 111). A statistical analysis for comparison of averages (t test) (IO) shows no significant difference between the methods at the 5% probability level, and the standard deviation of the difference is 0.069.

to correlate hydrolytic glucose yields with molecular structure in the starch family of polysaccharides (‘7). Its practical application to similar studies with other polysaccharides-for example, cellulose and dextrans-should prove useful.

DISCUSSION

LITERATURE CITED

The microbiological assay of glucose

is easily adapted for large scale operations and results are obtained from a simple acid-base titration. Forty unknowns can be analyzed in a single working day with inoculum prepared from eight centrifuge tubes. The time required for titrating can be shortened considerably by use of an automatic titrator. Because of its sensitivity, the method has been used successfully

(1) Assoc. Offic. Agr. Chemists, “Official Methods of Analysis,” 8th ed., 1955. (2) Bloore, E. A,, L‘Applicationof Microbiological Asmy to Sugar Solutions,’J M.A. thesis, University of Toronto, 1950. (3) Dimler, R. J., Schaeffer, W. C., Wise, C. S., Rist, C. E., ANAL. CHEM.24, 1411 (1952). 4) Harris, D. A,, Zbid. 27, 1690 (1955). 15) Hassid, W. Z., ~ N D . ENG. CHEM., ANAL.ED.9, 228 (1937). (6) Hutner, S. H., Cur A., Baker, H., ANAL.CHEM.30,849 0958).

(7) Kagan, J. J., “Studiee on the Acid

Hydro1 sia of Selected Starches and Starch %roducts,”Ph.D. thesis, University of Toronto, 1957. (8) Morris, D. L., Science 107, 254 (1948). (9) Strong, F. M., “Estimation of the Vitamins,” W. J. Dann G. H.Satterfield, eds., Biological mposia, Vol. XII, p. 143, Jaques Battell Prese, Lancaster, Pa., 1947. (10) Youden, W. J., “Statistical Methods for Chemists,’! p. 24, Wiley, London, 1951. RECEIVEDfor review October 29, 1959. Accepted January 8 1960. Based on a paper presented at the 41st Annual Conference and Exhibition of the Chemical Institute of Canada, Toronto, May 26, 1958. Research supported in grant from the Ontano Researc/%:ubnYd; tion through the Advisor Committee on Scientific Research, Bniversity of Toronto.

Determination of Chlorendic Acid in Fire-Retardant Paint G. G. ESPOSITO and

M. H. SWANN

Coating and Chemical Laboratory, Aberdeen Proving Ground, Md.

b Conventional methods of separating and measuring dicarboxylic acids in alkyd resins are unsuitable for determining chlorendic acid in alkyds used as vehicles for fire-retardant paints. A procedure is described that involves isolation ef chlorendic acid as the dipotassium salt by saponification in isopropyl alcohol, followed by acid treatment and extraction of the chlorendic acid with ethyl ether, washing free of other organic acids with water, and titration in a nonaqueous medium.

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made with chlorendic (3,6 endodichloromethylene -3,4,5,6 tetrachloro A4 tetrahydrophthalic) acid are used as vehicles for LKYD RESINS

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CI fire-retardant paints. Although a very limited number of such resins are commercially available, their durability, 680

ANALYTICAL CHEMISTRY

flexibility, rapid-drying properties, and shelf stability have attracted sufficient interest to tesult in issuance of specifications on paints incorporating this type of chlorinated binder. An analytical procedure for measuring the chlorendic acid in this class of paints was needed for quality control. Conventionally, the dicarboxylic acids in alkyd resins have been separated from other constituents as dipotassium salts obtained by saponification in anhydrous alcohol medium, followed by weighing or other means of measurement. If such a method (1-8) is applied to a chlorendic acid alkyd, some dipotassium chlorendate will precipitate, but the yield varies with sample size (Table I). If the sample size is increased sufficiently to obtain theoretical yields, the precipitated salts are contaminated with excessive entrained impurities. In addition, attempts to base the chlorendic acid content on these gravimetric fields would be affected by the presence of other dicarboxylic acids such as phthalic anhydride. Measurement of chlorendic acid by total chlorine content would make no distinction between the chlorinated alkyds and other chlorinated resins used in fire-retardant paints, but such an analysis can be useful for correlating different analytical schemes. In the method described, isopropyl

alcohol is substituted for ethyl alcohol t b obtain complete separation of chlorendic acid as the potassium salt from small samples of resin. It is then filtered, washed, dissolved in water, and transferred to a separatory funnel. After mineral acid treatment, the chlorendic acid is extracted with ethyl ether, washed free of mineral and organic acids with water, and titrated directly in nonaqueous medium (4). PROCEDURE

A sample of the isolated vehicle, representing 0.5 to 1.0 gram of nonvolatile material, is weighed into a 500ml. Erlenmeyer flask. It is dissolved in 100 ml. of benzene, and 50 ml. of IN potassium hydroxide in isopro yl alcohol (less than 1% water) are ad&d. A condenser is attached and the sample is refluxed in a water bath for 1 hour. On cooling, it is filtered through a Gooch crucible, using a mixture of 1 volume of isopropyl alcohol with 2 volumes of benzene for transferring and washing the precipitated salts. After a final washing with 25 ml. of ethyl ether, air is drawn through the crucible for 1 minute. The salts are dissolved from the crucible with a small amount of water, transferred to a 500-ml. separatory funnel, and diluted to 50 ml. Sulfuric acid (1 to 1) is added in small portions until a permanent cloud forms and then I-ml, excess is added.