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Microdetermination of Keltrol (Xanthan Gum)
Microdetermination of Keltrol (Xanthan Gum) HORACE D. G R A H A M
Department of Chemistry, University of Puerto Rico, h4ayaguez 00708 Abstract
the ratio of 2.8: 3.0: 2.0. I t is partially acetylated and contains pyruvic acid attached to the glucose side chain residue (5 to 10). Two methods are available for detection and determination of Keltrol (1). I n the qualitative locust bean gum gel test, a gel forms when the xanthan gum is mixed with locust bean gum under appropriate conditions. This test requires 1 g of the hydrocolloid and special mixing conditions with respect to temperature and cooling. The pyruvie acid test quantitatively extracts phenylhydrazone in a sodium carbonate solution. This test requires 60 mg of the hydrocolloid, which is explicable by the small (3.0 to 3.5%) amount of pyruvic acid in the polymer. As a consequence of its approval for use in foods, and because of its unique properties (viscosity, stability to heat, acid and alkali etc.), it is highly likely that Keltrol will be used as a substitute for, or as a complement to, one or more of the hydrocolloids now being used in ice cream, chocolate milk etc. I n view of this, a rapid test, capable of measuring microgram quantities of the hydrocolloid is needed. Such a test is outlined here. I n addition, this test will serve for studying the interaction of the hydrocolloid with other components of food mixtures.
Keltrol added to milk, ice cream, chocolate milk and other products was determined colorimetrically in the presence of carboxymethylcellulose. Alginate and pectic acid were removed by precipitation as the insoluble calcium salts, while sulfated polysaccharides were removed by precipitation with cetylpyridinium chloride in 0.5 NaC1. Without locust bean gum, filtration was over Reeve angel 202 paper. With locust bean gum in the mixture, filtration was over a column of acid-washed silica (sand) instead of over filter paper. Finally, Keltrol and CMC were coprecipitated by cetylpyridinium chloride in 0.2 ~ NaC1. The mixed precipitate was trapped on a Hyfio Super Cel column, washed with 0.2 ~ Na~SO 4 and then with hot (80 C or higher) 30% (v/v) H2SO 4 to disperse the gum complexes and elute the Keltrol and CMC. Carboxymethylcellulose in the eluate was determined by the chromotropic acid method (Keltrol does not react). Carboxymethylcellulose + Keltrol was determined by the phenolH2SO 4 and the anthrone methods. The amount of Keltrol present was determined by difference, using standard curves for the hydrocolloids when reacted with the aforementioned reagents. Recoveries of 90 to 96% were achieved from milk, chocolate milk, and salad dressing. The method determined Keltrol in milk and other food products at 0.05% (w/w) or lower.
Experimental Procedures
I ntroduction
Keltrol (xanthan gum, polysaccharide B].459) is a high molecular weight linear poIysaccharide synthesized by Xanthomonas campestris. The food grade gum thickens, suspends, emulsifies, and stabilizes water-based systems. Its use in foods has been sanctioned (1) and it is used widely in pharmaceutical and cosmetic preparations (2). Having a molecular weight greater than t million, the linear polysaccharide has a fl-linked backbone and contains Dglucose, D-mannose, and D-glucuronic acid in Received for publication May 17, 1971.
Materials. The hydrocolloids, listed in Table ], were prepared as described previously (3). Acetate buffer, 0.1 ~, p H 5.5, and containing 1 g of eysteine dihydrochloride and 2 g of ethylenediaminetetraacetate p e r liter was used. The p a p a i n - E D T A - c y s t e i n e h y d r o c h l o r i d e buffer mixture contained 8 g of papain concentrate (Schwartz-Mann Biochemicals, 72 milk clotting units per g) per 100 ml. This mixture was pre-incubated at 70 C for 30 rain, before being added to the food-hydrocolloid mixture (3). Solutions of ~ CaC12, 0.01 ~ CaC12 and a mixture of 0.01% cetylpyridinium chloride (CPC) and 0.001 ~ NaC1 were prepared from commercial, reagent grade chemicals. Hereafter, the mixture of 0.01% CPC - - 0.001 NaC1 will be called solution A. Analytical methods. The protein content of the samples, sulfate content of the carrageenan, and the degree of substitution (D.S.) of the
1622
XANTHAN GU~
carboxymethylcellulose, were determined as described previously (3). Preparation of the Celite filtration column. Place a perforated disk or a 1 cm piece of glass tubing, diameter 3/~ ram, at the bottom of a 250-ml burette, and pack glass wool on top of this to a height of about 3 cm. Add 2 g of Hyflo Super Cel and wash the column with 100 ml of tap water. Then add 25 ml of concentrated sulfuric acid and allow to drain. Wash column consecutively with 200 ml of tap water, 100 ml of 10% Na2C03, and finally, with 100 ml of 1% CPC. At this point a 2-ml aliquot of the washings should give a negative test with the phenol-H2SO4 reagent. Finally, close the stopcock of the burette and add 20 ml of 1% CPC. Leave CPC in the eolmnn until ready for filtration. Isolation of the Keltrol without locust bean gum. Add 10 nag of Keltrol dispersed in 10 ml of distilled water to 10 g of each of the products listed in Table 1. Place each mixture in a 250-ml Erlenmeyer flask and add distilled water to bring the volume to about 75 ml. Add 6 g of NaC1. Heat the mixture for 10 rain in a boiling water bath, cool it to about 70 C and add 25 ml of the papain-cysteine • HCl-buffer mixture. Incubate the mixture at 70 C for 16 hr. Close each flask with a hard rubber stopper and tape on the stopper to prevent it from blowing off during incubation. At the end of the incubation period, add 10 ml of ~ CaCle, and 1 g of ttyflo Super Cel. After 5 to 10 rain, filter the mixture through Reeve angel 202 paper, rinse the flask with four 25-ml portions of 0.01 H CaCI2 and pour the rinsings over the residue on the filter paper. Collect the filtrate and washings in a 500-ml Erlenmeyer flask, add 1 g of CPC, 1 g of ttyflo Super CeI and 5 ml of ~ CaC12. Stir the mixture well and, after 10 rain, pour it through Reeve angel 202 filter paper, rinse the flask with five 100-ml portions of Solution A and pour the rinsings over the residue on the filter paper. Collect the filtrate and washings (about 750 ml) in a 1000-ml beaker, stir it well and incubate it at 35 C for 15 rain. Filter the contents of the beaker through the tIyflo Super Cel column at 15 to 20 ml per minute, and return the first 50 ml of filtrate to the column. After all liquid has passed through, wash the column with Solution A until a 2-ml aliquot of the filtrate gives no color with the phenol-l=t2S04 reagent and then wash with 200 ml of 0.2~ Na2S04t (v/v). Pour hot (80 C) 30% I-I2S04 (v/v) over the column in about 25-ml aliquots and collect 200 ml of the acidic
1623
DETERMINATION
Food sample (10 g) in 250-ml Erlenmeyer flask. Add 6 g NaC]. Heat 10 rnln at 99 ± I C. Coo] to 70 C. Add 25 mi buffer-papain-Cysteine HCiEDTA mixture. Incubate 16 hr at 70 C. Cool to 30 C. Add 10 ml 1M CaC ~ and g Hyflo Super Cel, Fiter through Reeve ange 202 paper.
L
Filtrate
~
Residue
'-~ornL ~k,~"
Wash with four 25-ml portions I of 0.I M CaCl2
Add I g CPC, 5 rnl of 1 M CaCl2, and I g Hyflo Super Cel. Mix well. Incubate 15 mln at 4SC, Cool to 30C. Filter over Reeve angel 202 paper. Residue
Filtrate
ash with 400 ml of Solution A
-~" ' / ~
J Washings--~/
Residue (Discard)
Make up to 750 ml
well, incubate 15 rain
at 35 C, pour over colI urnn, f
Residue
Filtrate
(discard) Wash with 200ml of 0.2 M Na.aS04. Wash wffh Solution A till a 2-ml aliquot is negative to the phenoI-H2SO~ test.
1
1
Residue on coiurnn
Washings (discard)
Wash with ten 20-ml aliquots of hot 30% H2SO,
Residue (discard)
Filtrate
Use alfquots to test for Keltrol and CMC
FIG. 1. Flow sheet for ~solation of Keltrol in absence of locust bean gum. Procedure A. eluate in a 200-ml volumetric flask. Cool the flask and contents to 30 C and make the volume up to the mark with 30% 1=[2S04, if necessary. Determination of Keltrol in the presence of locust bean gum. After digestion with papain, add 10 ml of ~ CaCI2 to the flask. Mix contents well and, after about 10 rain pour the mixture over the sand-glass wool column and collect the filtrate in a 300-ml Erlenmeyer flask. Wash the column 4 times with 25-ml portions of 0.01 ~ CaCI2, collect the washings and combine them with the filtrate. Add one gram of Food sample (10 g) in 250-Erlenmeyer flask. Digest with I papain as in Procedure A. Add 10 rnl of I M CaCl=. Filter over glass wool-sand column. Filtrate
Residue
~.~6/~_
J
--~;~,
j
Wash with four 25-ml allquots
of hot 001 M Cats,.
~vasnings
Kesiaue (discard)
Add ! g of CPC and 5 ml of 1 M CaCl~. Incubate for 15 rain at 4S C. FiJter over glass woot-sand column. I Residue
Filtrate
Wash with 400 ml of Solution A
Residue (discard)
x X ~ .b"-~. -~'~" .o~/ ~o~O~
~ Washings
Make volume up to 750 m] with distilled H=O. Stir well and I incubate IS at 35 C, Pour over Hyflo Super Cel column.
Proceed as in Procedure A.
FIG. 2. Flow sheet for separation of Keltrol in presence of locust bean gum. Procedure B. JOURNAL OF DAIRY SOIENCE ~OL. 54, NO. 11
1624
~RA~,AM
0.7
0.6
0.5
/
0.4
~ 0.3 [9
0.2
O.l
0
25
50
75
100
0 MC O Y
25
50
75 ~ a
I00
125
150
175
200
225 250
HYDROCOLLOID PER REACTION TUBE
:Fie. 3. Standard curves for determination of earboxymethycellulose and Keltrol. 1 = CMC -- Chromotropic acid (570 m/~) 2 = CMC + Keltrol (1:1 mixture) Phenol-H=SO4 (490 m/z) 3 = C M C - Phenol-H2S04 (490 m#) 4 ---- CMC -- Anthrone (620 m#) 5 = CMC + Ke]trol (1:1 mixture) Anthrone (620 m/~) 6 = Keltrol -- Phenol -- H=SO4 (490 m/~) 7 = Keltrol -- Anthrone (620 nqz) 8 = Intersecting line illustrating calculation of Keltrol in CMC -- Keltrol mixture. cetylpyridininm chloride and 5 ml of ~ CaCI2 to the filtrate and washings. Stir the mixture well. Incubate it f o r 15 min at 45 C and then pour it over the sand-glass wool column, return the filtrate to the column and filter it again. Finally, wash the column with 250 ml of 0.1% CPC - - 021 )~ NaC1, collect the filtrate and washings in a 1,000-ml beaker. Dilute the liquid to 750 ml with distilled water, incubate it for 15 rain, at 35 C and filter it. Elute the precipitate and determine the K e l t r o l as in Procedure A. The entire procedure for this operation and that in the absence of locust bean gum are summarized in Figures 1 and 2. Aliquots of the eluate were tested with following reagents: (a) The anthrone reagent. 2 g of anthrone in 100-ml of concentrated H2SO 4. ]]eat 3 ml of the eluate in a boiling J O U R N A L OF I)AII~Y S C I E N C E V 0 L .
54,
l~0. 11
water bath with 5 ml of the reagent for exactly 10 min. Cool to 30 ± 1 C and measure the absorbance of the color developed at 620 m/~. (b) The phenol-H2SO 4 reagent. Add 1 ml of 5% phenol in water to 2 ml of the eluate. Add rapidly to the mixture 5 ml of concentrated }I2SO 4. Leave the mixture at 29 ± 1 C for 10 rain, cool it to 29 ± 1 C and measure the color developed at 490 m/~. (c) The chromotropic acid reagent. Dissolve 150 mg of chromotropie acid (1,8-dihydroxynaphthalene3-6-disulfonic acid) in 100 ml of concentrated H2SO 4. Heat 1 ml of the sample and 9 ml of the reagent in a water bath for 90 rain, cool it, and measure the color developed at 570 m/~. Prepare standard curves for each reagent after reaction with CMC and Keltrol (Fig. 3).
XANTHAN
1625
GU~[ DETERMINATION
Results and Discussion
Based on the procedure outlined, the following formulations may be written: Let OD at 570 m~ (chromotropic acid) = CMC in sample Let OD at 490 mt~ (phenol-H2S04) -CMC + Keltrol in sample Let OD at 620 mtL (Anthrone) : CMC + Keltrol The amount of CMC in the sample is determined with a standard curve prepared by reacting varying amounts of CMC with the chromotropic acid reagent (4). Since optical density is additive, and the amount of CMC in the mixture is known from the chromotropic acid test, optical density contributed by CMC in the phenol-H2SO4 test or the anthrone test can be readily determined from standard curves. I n the phenol-H~S04 test:
reacts with locust bean to form a characteristic gel. Such gel formation is used as one of the official tests for the detection of Keltrol (1). Salts, especially, potassium and calcium chloride, increase the viscosity of Keltrol considerably (2). I n Procedure A, (Fig. 1), when cetylpyridinium chloride is added to a mixture containing NaCI, CaCl~, locust bean gum, and Celite, the gum could not be recovered from such a mixture, by direct filtration over paper or by centrifugation and pouring off the supernatant. Apparently, the resulting highly viscous mixture is adsorbed onto the Celite and is lost in the preliminary filtration steps. I t was necessary, therefore, to develop a special process for the isolation and determination of Keltrol in the presence of locust bean gum. (
LIQUID
IN
Let the OD contributed by CMC = B Let the total OD (OD CMC + OD Keltrol) = A Then OD contributed by Keltrol only = A--B--C F r o m the phenol-I~I2S0 4 standard curve for Keltrol, the amount of Keltrol corresponding to an optical density equal to C, can be readily determined. An example will suffice: 10 mg of CMC plus 10 mg of Keltrol were added to 10 g of milk and the gums separated and determined by the suggested method. The 30% tt2SO 4 eluate = 200 ml. The optical density (of CMC) by the ehromotropic acid test was found to be = 0.35 = 50 #g of CMC (1 ml used). The optical density (of CMC + Keltrol) by the phenol-H2SO 4 = 0.62 (A) (2 ml of filtrate used). F i f t y micrograms of CMC will give an OD of 0.18 : (B) by the pheno]]=t2SO4. Therefore, since optical density is additive, the OD contributed by Keltrol = A -B = 0.62-0.18 = 0.44. Now, from the standard curve for Keltrol by the phenol-H2SO 4 test, OD of 0.44 ~- 82.5 tLg of Keltrol. The totM amount of Keltrol present = 82.5 × 100 (dilution factor of 100) = 8,250 ~g = 82.5 mg : 82.5% recovery.
Similarly, the amount of Keltrol present and the per cent recovery could be calculated, if the anthrone test were used. Determination of Keltrol in the presence of locust bean gum, needs some comment. Keltrol
GLASS C O L U M N (Length 40 cm, Diameter 2.5 cm}
GLASS WOOL ACID-WASHED SAND
m
GLASS WOOL
.-.~--- -_-~--.: Noo 4 HARD RUBBER STOPPER WITH HOLE
GLASS TUBING
BEAKER
FIG. 4. Sand-glass wool column for filtering mixture containing Keltrol and locust bean gum. JOUEI~AL OF DAIRY SCIENCE V0L. 54, NO. Ii
1626
aRAHA~
Here filtration had to be done over a glass woolsand column shown in Figure 4. Powered glass may be substituted for sand but the rate of filtration is slower. Table 1 shows the recovery of Keltrol in the presence of several other food gums. I n the presence of higher (0.2% or more) concentrations of starch, recovery was somewhat higher than with other thickeners, due probably to the strong adsorption of the starch to the column, or to its occlusion by the precipitates of Xeltrol and CMC. Pectic acid and alginate were removed as the insoluble calcium salts. Carrageenan was removed as the highly insoluble cetylpyridinium complex in the presence of 0.5 ]~ or greater NaC1. When the filtrate is diluted to 750 ml both I(eltrol and carboxymethylcellulose will precipitate, leaving in solution, if present, pectin, the soluble portions of gum arabic and the phosphomannans. Quince seed mucilage, if present, will be removed along with the sulfated hydrocolloids and with alginate. Should any of these gums remain on the column in the free state, they will be removed during the washing. I f perchance, traces of the cetylpyridinium complexes of any
of these other gums are formed and are trapped on the Celite column, they will be removed by the 0.2 ~ sodium sulfate. Recovery of Keltrol from various food products is shown in Table 2. Generally, considering the excessive washing necessary, such recoveries may be considered as good. I n the determination, it is not necessary to know the degree of substitution of the earboxymethylcellulose since, irrespective of the color variations due to this characteristic of commercial samples, the chromotropic acid will determine the total amount present and the amount of I~eltrol determined by difference will not be affected. A major advantage of the method is that the reaction of Keltrol with the phenol-H2SO 4 and the anthrone methods depends on its major sugar and uronic acid components rather than on the content of pyruvic acid, s. minor component (3.0 to 3.5%). Variations in the pyruvic acid content, even i f very great, will not severely affect the results. I t is tempting to propose that the demonstration of pyruvic acid, along with this method, would constitute good proof and quantitative determination of the gum in
TABLE 1. Recovery of Keltrol from milk in the presence of other food gums. (10 mg of Keltrol and 10 mg of each gum added to 10 g of milk). Keltrol recovered a Average
Recovery
Gum
Range
A1
B2
(rag)
(rag)
(%)
(%)
None Carrageenan Furcellaran Alginate Pectic acid Carboxymethylcellulose Quince seed mucilage Locust bean gum Starch Pectin Gum arabic Gum tragacanth Gum ghatti Gum karaya Gum guar Agar Gelatin Phosphomannan Y-2154 Phosphomannon Y-2448
8.7-9.5 8.4-9.0 8.5-9.2 8.7-9.3 8.4-9.2 8.6-9.2 8.5-9.3 8.4-9.3 8.0-9.1 8.6-9.5 8.5-9.1 8.7-9.3 8.6-9.0 8.3-9.1 8.5-9.2 8.5-9.3 8.4-9.4 8.6-9.2 8.5-9.1
8.9 8.6 8.7 8.9 8.7 8.8 8.7 8.6 8.4 8.8 8.7 8.9 8.5 8.7 8.8 8.7 8.8 8.9 8.8
89 86 87 89 87 88 87 86 84 88 87 89 85 87 88 87 88 89 88
94.5 91.0 93.0 94.5 93.0 93.5 93.0 91.0 90.0 93.5 93.0 94.5 90.4 93.0 93.5 93.0 93.5 94.5 93.5
a Average of 5 different samples. 1 Based on 10 mg added to milk. 2 Based on recovery of 10 mg of Keltrol added to water and passed through the entire process. JOURNAL OF DAIRY SCIENCE VOL. 54, NO. 11
X A N T H A N GUM D E T E R M I N A T I O N
1627
TABLE 2. Recovery of Keltrol from various products. Keltrol
Rec°verye
Product
added
Range
(rag) Water
5 nag 10 mg
4.4-4.9 9.3-9.6
Milk
5 mg 10 mg
4.2-4.5 8.7-9.3
Average
Recovery A
B
(rag)
(%)
(%)
4.6 9.4
92.0 94.0
...... ......
4.3 8.8
86.0 88.0
93.5 94.0
Chocolate milk (1 gram cocoa added to commercial milk)
5 mg
4.0-4.5
4.3
86.0
93.5
10 mg
8.2-9.0
8.4
84.0
90.0
Ice cream (vanilla flavor)
5 mg 10 nag
4.2-4.7 8.4-9.3
4.4 8.7
88.0 87.0
95.7 92.6
Ice cream (chocolate flavor)
5 mg 10 nag
4.0-4.5 8.4-9.1
4.2 8.6
84.0 86.0
91.3 91.5
Cream cheese
5 mg 10 nag
4.2-4.7 8.5-9.1
4.2 8.7
84.0 87.0
91.3 92.6
Salad dressing
5 mg 10 nag
4.1-4.5 8.5-9.1
4.3 8.7
86.0 87.0
93.5 92.6
A -~ Recovery based on 10 mg of IKeltrol added to product itself. B ---- Recovery based on 10 mg of Keltrol added to water and passed through the entire process. c Average of 5 different determinations. preparations of any kind. The method of separation can be readily applied to pharmaceutical and other preparations. The proposed method requires very simple equipment and reagents which are easy to procure and to prepare. Ten to twelve samples can be handled at the same time by a single technician. W i t h most products, incubation for 4 hr may be sufficient to degrade the proteins with papain. However, it is convenient to digest overnight so that the samples may be filtered and prepared for colorimetric determination the next morning. Dilution of the filtrate and washings to 750 ml (approximately equivalent to 0.2 ~ NaC1) assures the co-precipitation of Keltrol and CMC in the presence of gum arabic, the phosphomannans, pectin, and the soluble portions of gum tragacanth, ghatti, and karaya. However, if pectin is absent, this volume may be exceeded by as much as 400 ml or more without affecting the final results. I n this case, the other gums will co-precipitate also. However, their precipitates will be dispersed and eluted by the 0.2 ~ Na2SO 4. The pectin - - CP precipitate adsorbs strongly on the Hyflo Super
Cel and is not eluted by 0.2 ~ NaeS04. When present, its co-precipitation must be avoided. The Keltrol - - CP precipitate, trapped on the Hyflo Super Cel column is not dispersible by even molar cold Na2S04. However, as the concentration of l~a2SO 4 increases beyond 0.2 ~I, filtration becomes measurably slower. Chocolate, which usually interferes severely in the phenol-H2S04 test, caused much less problem here in Procedure A. Apparently, during the preliminary removal of sulfated polysaccharides and of alginate and pectic acid, most of the chocolate and its interfering ingredient(s) were removed. Lower reagent blanks were obtained with the anthrone reagent than with the phenol-H2SO 4 reagent, although the latter is more stable and is easier to work with. W i t h Procedure B, the greater interference from chocolate in the phenol-H2S04 test, occurs because less is removed during the preliminary steps. The anthrone test is preferable here since lower blanks will be obtained. Charcoal may be used to remove the chocolate and other interfering colors incorporated into certain food products. However, when charcoal is used, filtration is extremely slow, even with J0~RNAL OF DAffY SCl~NC~ "VOL. 54, NO. 1i
1628
aRX~A~
Acknowledgments the sand-glass wool or ground glass column. Keltrol does not produce the typical pink Thanks are due to the Kelco Company, San color with the chromotropic acid reagent as Diego, California, for the Keltrol and to the does CMC. However, due to the relatively long Hercules Powder Company, Wilmington, Delaware heating period and most likely to its decom- for the carboxymethy]cellulose. This investigation was supported by U.S. Public position by the hot acid, the decomposition Health Service Grant FD-00083-07. products absorb at 570 m~ to about 0.05 to 0.07 and this increases the blank value to about two References times the normal reading. For accurate deter(1) Federal Register. 1969. Xanthan gum. ruination, therefore, a reagent blank should be Section 121.1224 5376-5377, March 19. run with every batch being heated and the (2) Glicksman, M. 1969. Gum Technology in the value obtained multiplied by 2 and substraeted Food IndustlT. Academic Press, New York. from the sample reading. (3) Graham, H. D. 1969. Determination of The determination of carboxymethylcellulose alginate iu dairy products. J. Dairy Sci., by chromotropic acid in the presence of Keltrol 52 : 443. is highly specific, since Keltrol does not produce (4) Graham, It. D. 1971. Reaction of carboxymethylcellulose with the chromotropic acid the typical purple color (4). Pectin and pectic reagent. J. Dairy Sci., 54: 761. acid, if present, will give color but these are (5) Jeanes, A. R., J. E. Pitts]ey, and F. R. removed prior to the colorimetric determination. Senti. 1961. B-1459. A new hydrocolloid Since both Keltrol and sodium carboxymethylpolyelectrolyte produced from glucose by cellulose react in the phenol-H2S04 and the bacterial fermentation. J. Appl. Polymer anthrone methods, the proposed quantitative Sci., 5 : 519. determination of Keltrol by difference is valid (6) Kelco Co. 1969. Keltrol. Tech. Bull., DB and the proposed method for the isolation of 18, Kelco Co., New Jersey. the polysaccharide/colloid is highly specific. The (7) Rocks, J. K. 1971. Xanthan gum. Food colorimetric methods used for the quantitative Technol., 25: 476. (8) Rogovin, S. P., R. F. Anderson, and M. G. determination of the gums are very sensitive. Cadmus. 1961. Production of polysacThus, the proposed method, as far as is known, eharide with Xanthomonas campestris. J. may be the first one to describe the isolation Bioehem. Microbiol. Technol., ]Eng., 3: 51. of such small quantities of Keltrol from food (9) Sloneker, J. H., D. G. 0rentas, and A. products as complex as those listed in Table 1 Jeanes. 1964. Extracellular polysaccharide and which contain such high contents of protein. from Xanthoqnonas caqnpestris ~RRLBAs far as is known, it is the most sensitive 1459. III. Structure. Canadian J. Chem., method yet proposed since in both the locust 42 : 1261. (10) Smiley, K. L. 1969. Microbial polysacbean gum gel test and the pyruvic acid tests, charides--a review. Food Technol., 20: 112. considerably higher quantities are needed.
JOURNAL OF DAIRY SOIEI~CE VOL. 54, NO, 11
Department of Chemistry, University of Puerto Rico, h4ayaguez 00708 Abstract
the ratio of 2.8: 3.0: 2.0. I t is partially acetylated and contains pyruvic acid attached to the glucose side chain residue (5 to 10). Two methods are available for detection and determination of Keltrol (1). I n the qualitative locust bean gum gel test, a gel forms when the xanthan gum is mixed with locust bean gum under appropriate conditions. This test requires 1 g of the hydrocolloid and special mixing conditions with respect to temperature and cooling. The pyruvie acid test quantitatively extracts phenylhydrazone in a sodium carbonate solution. This test requires 60 mg of the hydrocolloid, which is explicable by the small (3.0 to 3.5%) amount of pyruvic acid in the polymer. As a consequence of its approval for use in foods, and because of its unique properties (viscosity, stability to heat, acid and alkali etc.), it is highly likely that Keltrol will be used as a substitute for, or as a complement to, one or more of the hydrocolloids now being used in ice cream, chocolate milk etc. I n view of this, a rapid test, capable of measuring microgram quantities of the hydrocolloid is needed. Such a test is outlined here. I n addition, this test will serve for studying the interaction of the hydrocolloid with other components of food mixtures.
Keltrol added to milk, ice cream, chocolate milk and other products was determined colorimetrically in the presence of carboxymethylcellulose. Alginate and pectic acid were removed by precipitation as the insoluble calcium salts, while sulfated polysaccharides were removed by precipitation with cetylpyridinium chloride in 0.5 NaC1. Without locust bean gum, filtration was over Reeve angel 202 paper. With locust bean gum in the mixture, filtration was over a column of acid-washed silica (sand) instead of over filter paper. Finally, Keltrol and CMC were coprecipitated by cetylpyridinium chloride in 0.2 ~ NaC1. The mixed precipitate was trapped on a Hyfio Super Cel column, washed with 0.2 ~ Na~SO 4 and then with hot (80 C or higher) 30% (v/v) H2SO 4 to disperse the gum complexes and elute the Keltrol and CMC. Carboxymethylcellulose in the eluate was determined by the chromotropic acid method (Keltrol does not react). Carboxymethylcellulose + Keltrol was determined by the phenolH2SO 4 and the anthrone methods. The amount of Keltrol present was determined by difference, using standard curves for the hydrocolloids when reacted with the aforementioned reagents. Recoveries of 90 to 96% were achieved from milk, chocolate milk, and salad dressing. The method determined Keltrol in milk and other food products at 0.05% (w/w) or lower.
Experimental Procedures
I ntroduction
Keltrol (xanthan gum, polysaccharide B].459) is a high molecular weight linear poIysaccharide synthesized by Xanthomonas campestris. The food grade gum thickens, suspends, emulsifies, and stabilizes water-based systems. Its use in foods has been sanctioned (1) and it is used widely in pharmaceutical and cosmetic preparations (2). Having a molecular weight greater than t million, the linear polysaccharide has a fl-linked backbone and contains Dglucose, D-mannose, and D-glucuronic acid in Received for publication May 17, 1971.
Materials. The hydrocolloids, listed in Table ], were prepared as described previously (3). Acetate buffer, 0.1 ~, p H 5.5, and containing 1 g of eysteine dihydrochloride and 2 g of ethylenediaminetetraacetate p e r liter was used. The p a p a i n - E D T A - c y s t e i n e h y d r o c h l o r i d e buffer mixture contained 8 g of papain concentrate (Schwartz-Mann Biochemicals, 72 milk clotting units per g) per 100 ml. This mixture was pre-incubated at 70 C for 30 rain, before being added to the food-hydrocolloid mixture (3). Solutions of ~ CaC12, 0.01 ~ CaC12 and a mixture of 0.01% cetylpyridinium chloride (CPC) and 0.001 ~ NaC1 were prepared from commercial, reagent grade chemicals. Hereafter, the mixture of 0.01% CPC - - 0.001 NaC1 will be called solution A. Analytical methods. The protein content of the samples, sulfate content of the carrageenan, and the degree of substitution (D.S.) of the
1622
XANTHAN GU~
carboxymethylcellulose, were determined as described previously (3). Preparation of the Celite filtration column. Place a perforated disk or a 1 cm piece of glass tubing, diameter 3/~ ram, at the bottom of a 250-ml burette, and pack glass wool on top of this to a height of about 3 cm. Add 2 g of Hyflo Super Cel and wash the column with 100 ml of tap water. Then add 25 ml of concentrated sulfuric acid and allow to drain. Wash column consecutively with 200 ml of tap water, 100 ml of 10% Na2C03, and finally, with 100 ml of 1% CPC. At this point a 2-ml aliquot of the washings should give a negative test with the phenol-H2SO4 reagent. Finally, close the stopcock of the burette and add 20 ml of 1% CPC. Leave CPC in the eolmnn until ready for filtration. Isolation of the Keltrol without locust bean gum. Add 10 nag of Keltrol dispersed in 10 ml of distilled water to 10 g of each of the products listed in Table 1. Place each mixture in a 250-ml Erlenmeyer flask and add distilled water to bring the volume to about 75 ml. Add 6 g of NaC1. Heat the mixture for 10 rain in a boiling water bath, cool it to about 70 C and add 25 ml of the papain-cysteine • HCl-buffer mixture. Incubate the mixture at 70 C for 16 hr. Close each flask with a hard rubber stopper and tape on the stopper to prevent it from blowing off during incubation. At the end of the incubation period, add 10 ml of ~ CaCle, and 1 g of ttyflo Super Cel. After 5 to 10 rain, filter the mixture through Reeve angel 202 paper, rinse the flask with four 25-ml portions of 0.01 H CaCI2 and pour the rinsings over the residue on the filter paper. Collect the filtrate and washings in a 500-ml Erlenmeyer flask, add 1 g of CPC, 1 g of ttyflo Super CeI and 5 ml of ~ CaC12. Stir the mixture well and, after 10 rain, pour it through Reeve angel 202 filter paper, rinse the flask with five 100-ml portions of Solution A and pour the rinsings over the residue on the filter paper. Collect the filtrate and washings (about 750 ml) in a 1000-ml beaker, stir it well and incubate it at 35 C for 15 rain. Filter the contents of the beaker through the tIyflo Super Cel column at 15 to 20 ml per minute, and return the first 50 ml of filtrate to the column. After all liquid has passed through, wash the column with Solution A until a 2-ml aliquot of the filtrate gives no color with the phenol-l=t2S04 reagent and then wash with 200 ml of 0.2~ Na2S04t (v/v). Pour hot (80 C) 30% I-I2S04 (v/v) over the column in about 25-ml aliquots and collect 200 ml of the acidic
1623
DETERMINATION
Food sample (10 g) in 250-ml Erlenmeyer flask. Add 6 g NaC]. Heat 10 rnln at 99 ± I C. Coo] to 70 C. Add 25 mi buffer-papain-Cysteine HCiEDTA mixture. Incubate 16 hr at 70 C. Cool to 30 C. Add 10 ml 1M CaC ~ and g Hyflo Super Cel, Fiter through Reeve ange 202 paper.
L
Filtrate
~
Residue
'-~ornL ~k,~"
Wash with four 25-ml portions I of 0.I M CaCl2
Add I g CPC, 5 rnl of 1 M CaCl2, and I g Hyflo Super Cel. Mix well. Incubate 15 mln at 4SC, Cool to 30C. Filter over Reeve angel 202 paper. Residue
Filtrate
ash with 400 ml of Solution A
-~" ' / ~
J Washings--~/
Residue (Discard)
Make up to 750 ml
well, incubate 15 rain
at 35 C, pour over colI urnn, f
Residue
Filtrate
(discard) Wash with 200ml of 0.2 M Na.aS04. Wash wffh Solution A till a 2-ml aliquot is negative to the phenoI-H2SO~ test.
1
1
Residue on coiurnn
Washings (discard)
Wash with ten 20-ml aliquots of hot 30% H2SO,
Residue (discard)
Filtrate
Use alfquots to test for Keltrol and CMC
FIG. 1. Flow sheet for ~solation of Keltrol in absence of locust bean gum. Procedure A. eluate in a 200-ml volumetric flask. Cool the flask and contents to 30 C and make the volume up to the mark with 30% 1=[2S04, if necessary. Determination of Keltrol in the presence of locust bean gum. After digestion with papain, add 10 ml of ~ CaCI2 to the flask. Mix contents well and, after about 10 rain pour the mixture over the sand-glass wool column and collect the filtrate in a 300-ml Erlenmeyer flask. Wash the column 4 times with 25-ml portions of 0.01 ~ CaCI2, collect the washings and combine them with the filtrate. Add one gram of Food sample (10 g) in 250-Erlenmeyer flask. Digest with I papain as in Procedure A. Add 10 rnl of I M CaCl=. Filter over glass wool-sand column. Filtrate
Residue
~.~6/~_
J
--~;~,
j
Wash with four 25-ml allquots
of hot 001 M Cats,.
~vasnings
Kesiaue (discard)
Add ! g of CPC and 5 ml of 1 M CaCl~. Incubate for 15 rain at 4S C. FiJter over glass woot-sand column. I Residue
Filtrate
Wash with 400 ml of Solution A
Residue (discard)
x X ~ .b"-~. -~'~" .o~/ ~o~O~
~ Washings
Make volume up to 750 m] with distilled H=O. Stir well and I incubate IS at 35 C, Pour over Hyflo Super Cel column.
Proceed as in Procedure A.
FIG. 2. Flow sheet for separation of Keltrol in presence of locust bean gum. Procedure B. JOURNAL OF DAIRY SOIENCE ~OL. 54, NO. 11
1624
~RA~,AM
0.7
0.6
0.5
/
0.4
~ 0.3 [9
0.2
O.l
0
25
50
75
100
0 MC O Y
25
50
75 ~ a
I00
125
150
175
200
225 250
HYDROCOLLOID PER REACTION TUBE
:Fie. 3. Standard curves for determination of earboxymethycellulose and Keltrol. 1 = CMC -- Chromotropic acid (570 m/~) 2 = CMC + Keltrol (1:1 mixture) Phenol-H=SO4 (490 m/z) 3 = C M C - Phenol-H2S04 (490 m#) 4 ---- CMC -- Anthrone (620 m#) 5 = CMC + Ke]trol (1:1 mixture) Anthrone (620 m/~) 6 = Keltrol -- Phenol -- H=SO4 (490 m/~) 7 = Keltrol -- Anthrone (620 nqz) 8 = Intersecting line illustrating calculation of Keltrol in CMC -- Keltrol mixture. cetylpyridininm chloride and 5 ml of ~ CaCI2 to the filtrate and washings. Stir the mixture well. Incubate it f o r 15 min at 45 C and then pour it over the sand-glass wool column, return the filtrate to the column and filter it again. Finally, wash the column with 250 ml of 0.1% CPC - - 021 )~ NaC1, collect the filtrate and washings in a 1,000-ml beaker. Dilute the liquid to 750 ml with distilled water, incubate it for 15 rain, at 35 C and filter it. Elute the precipitate and determine the K e l t r o l as in Procedure A. The entire procedure for this operation and that in the absence of locust bean gum are summarized in Figures 1 and 2. Aliquots of the eluate were tested with following reagents: (a) The anthrone reagent. 2 g of anthrone in 100-ml of concentrated H2SO 4. ]]eat 3 ml of the eluate in a boiling J O U R N A L OF I)AII~Y S C I E N C E V 0 L .
54,
l~0. 11
water bath with 5 ml of the reagent for exactly 10 min. Cool to 30 ± 1 C and measure the absorbance of the color developed at 620 m/~. (b) The phenol-H2SO 4 reagent. Add 1 ml of 5% phenol in water to 2 ml of the eluate. Add rapidly to the mixture 5 ml of concentrated }I2SO 4. Leave the mixture at 29 ± 1 C for 10 rain, cool it to 29 ± 1 C and measure the color developed at 490 m/~. (c) The chromotropic acid reagent. Dissolve 150 mg of chromotropie acid (1,8-dihydroxynaphthalene3-6-disulfonic acid) in 100 ml of concentrated H2SO 4. Heat 1 ml of the sample and 9 ml of the reagent in a water bath for 90 rain, cool it, and measure the color developed at 570 m/~. Prepare standard curves for each reagent after reaction with CMC and Keltrol (Fig. 3).
XANTHAN
1625
GU~[ DETERMINATION
Results and Discussion
Based on the procedure outlined, the following formulations may be written: Let OD at 570 m~ (chromotropic acid) = CMC in sample Let OD at 490 mt~ (phenol-H2S04) -CMC + Keltrol in sample Let OD at 620 mtL (Anthrone) : CMC + Keltrol The amount of CMC in the sample is determined with a standard curve prepared by reacting varying amounts of CMC with the chromotropic acid reagent (4). Since optical density is additive, and the amount of CMC in the mixture is known from the chromotropic acid test, optical density contributed by CMC in the phenol-H2SO4 test or the anthrone test can be readily determined from standard curves. I n the phenol-H~S04 test:
reacts with locust bean to form a characteristic gel. Such gel formation is used as one of the official tests for the detection of Keltrol (1). Salts, especially, potassium and calcium chloride, increase the viscosity of Keltrol considerably (2). I n Procedure A, (Fig. 1), when cetylpyridinium chloride is added to a mixture containing NaCI, CaCl~, locust bean gum, and Celite, the gum could not be recovered from such a mixture, by direct filtration over paper or by centrifugation and pouring off the supernatant. Apparently, the resulting highly viscous mixture is adsorbed onto the Celite and is lost in the preliminary filtration steps. I t was necessary, therefore, to develop a special process for the isolation and determination of Keltrol in the presence of locust bean gum. (
LIQUID
IN
Let the OD contributed by CMC = B Let the total OD (OD CMC + OD Keltrol) = A Then OD contributed by Keltrol only = A--B--C F r o m the phenol-I~I2S0 4 standard curve for Keltrol, the amount of Keltrol corresponding to an optical density equal to C, can be readily determined. An example will suffice: 10 mg of CMC plus 10 mg of Keltrol were added to 10 g of milk and the gums separated and determined by the suggested method. The 30% tt2SO 4 eluate = 200 ml. The optical density (of CMC) by the ehromotropic acid test was found to be = 0.35 = 50 #g of CMC (1 ml used). The optical density (of CMC + Keltrol) by the phenol-H2SO 4 = 0.62 (A) (2 ml of filtrate used). F i f t y micrograms of CMC will give an OD of 0.18 : (B) by the pheno]]=t2SO4. Therefore, since optical density is additive, the OD contributed by Keltrol = A -B = 0.62-0.18 = 0.44. Now, from the standard curve for Keltrol by the phenol-H2SO 4 test, OD of 0.44 ~- 82.5 tLg of Keltrol. The totM amount of Keltrol present = 82.5 × 100 (dilution factor of 100) = 8,250 ~g = 82.5 mg : 82.5% recovery.
Similarly, the amount of Keltrol present and the per cent recovery could be calculated, if the anthrone test were used. Determination of Keltrol in the presence of locust bean gum, needs some comment. Keltrol
GLASS C O L U M N (Length 40 cm, Diameter 2.5 cm}
GLASS WOOL ACID-WASHED SAND
m
GLASS WOOL
.-.~--- -_-~--.: Noo 4 HARD RUBBER STOPPER WITH HOLE
GLASS TUBING
BEAKER
FIG. 4. Sand-glass wool column for filtering mixture containing Keltrol and locust bean gum. JOUEI~AL OF DAIRY SCIENCE V0L. 54, NO. Ii
1626
aRAHA~
Here filtration had to be done over a glass woolsand column shown in Figure 4. Powered glass may be substituted for sand but the rate of filtration is slower. Table 1 shows the recovery of Keltrol in the presence of several other food gums. I n the presence of higher (0.2% or more) concentrations of starch, recovery was somewhat higher than with other thickeners, due probably to the strong adsorption of the starch to the column, or to its occlusion by the precipitates of Xeltrol and CMC. Pectic acid and alginate were removed as the insoluble calcium salts. Carrageenan was removed as the highly insoluble cetylpyridinium complex in the presence of 0.5 ]~ or greater NaC1. When the filtrate is diluted to 750 ml both I(eltrol and carboxymethylcellulose will precipitate, leaving in solution, if present, pectin, the soluble portions of gum arabic and the phosphomannans. Quince seed mucilage, if present, will be removed along with the sulfated hydrocolloids and with alginate. Should any of these gums remain on the column in the free state, they will be removed during the washing. I f perchance, traces of the cetylpyridinium complexes of any
of these other gums are formed and are trapped on the Celite column, they will be removed by the 0.2 ~ sodium sulfate. Recovery of Keltrol from various food products is shown in Table 2. Generally, considering the excessive washing necessary, such recoveries may be considered as good. I n the determination, it is not necessary to know the degree of substitution of the earboxymethylcellulose since, irrespective of the color variations due to this characteristic of commercial samples, the chromotropic acid will determine the total amount present and the amount of I~eltrol determined by difference will not be affected. A major advantage of the method is that the reaction of Keltrol with the phenol-H2SO 4 and the anthrone methods depends on its major sugar and uronic acid components rather than on the content of pyruvic acid, s. minor component (3.0 to 3.5%). Variations in the pyruvic acid content, even i f very great, will not severely affect the results. I t is tempting to propose that the demonstration of pyruvic acid, along with this method, would constitute good proof and quantitative determination of the gum in
TABLE 1. Recovery of Keltrol from milk in the presence of other food gums. (10 mg of Keltrol and 10 mg of each gum added to 10 g of milk). Keltrol recovered a Average
Recovery
Gum
Range
A1
B2
(rag)
(rag)
(%)
(%)
None Carrageenan Furcellaran Alginate Pectic acid Carboxymethylcellulose Quince seed mucilage Locust bean gum Starch Pectin Gum arabic Gum tragacanth Gum ghatti Gum karaya Gum guar Agar Gelatin Phosphomannan Y-2154 Phosphomannon Y-2448
8.7-9.5 8.4-9.0 8.5-9.2 8.7-9.3 8.4-9.2 8.6-9.2 8.5-9.3 8.4-9.3 8.0-9.1 8.6-9.5 8.5-9.1 8.7-9.3 8.6-9.0 8.3-9.1 8.5-9.2 8.5-9.3 8.4-9.4 8.6-9.2 8.5-9.1
8.9 8.6 8.7 8.9 8.7 8.8 8.7 8.6 8.4 8.8 8.7 8.9 8.5 8.7 8.8 8.7 8.8 8.9 8.8
89 86 87 89 87 88 87 86 84 88 87 89 85 87 88 87 88 89 88
94.5 91.0 93.0 94.5 93.0 93.5 93.0 91.0 90.0 93.5 93.0 94.5 90.4 93.0 93.5 93.0 93.5 94.5 93.5
a Average of 5 different samples. 1 Based on 10 mg added to milk. 2 Based on recovery of 10 mg of Keltrol added to water and passed through the entire process. JOURNAL OF DAIRY SCIENCE VOL. 54, NO. 11
X A N T H A N GUM D E T E R M I N A T I O N
1627
TABLE 2. Recovery of Keltrol from various products. Keltrol
Rec°verye
Product
added
Range
(rag) Water
5 nag 10 mg
4.4-4.9 9.3-9.6
Milk
5 mg 10 mg
4.2-4.5 8.7-9.3
Average
Recovery A
B
(rag)
(%)
(%)
4.6 9.4
92.0 94.0
...... ......
4.3 8.8
86.0 88.0
93.5 94.0
Chocolate milk (1 gram cocoa added to commercial milk)
5 mg
4.0-4.5
4.3
86.0
93.5
10 mg
8.2-9.0
8.4
84.0
90.0
Ice cream (vanilla flavor)
5 mg 10 nag
4.2-4.7 8.4-9.3
4.4 8.7
88.0 87.0
95.7 92.6
Ice cream (chocolate flavor)
5 mg 10 nag
4.0-4.5 8.4-9.1
4.2 8.6
84.0 86.0
91.3 91.5
Cream cheese
5 mg 10 nag
4.2-4.7 8.5-9.1
4.2 8.7
84.0 87.0
91.3 92.6
Salad dressing
5 mg 10 nag
4.1-4.5 8.5-9.1
4.3 8.7
86.0 87.0
93.5 92.6
A -~ Recovery based on 10 mg of IKeltrol added to product itself. B ---- Recovery based on 10 mg of Keltrol added to water and passed through the entire process. c Average of 5 different determinations. preparations of any kind. The method of separation can be readily applied to pharmaceutical and other preparations. The proposed method requires very simple equipment and reagents which are easy to procure and to prepare. Ten to twelve samples can be handled at the same time by a single technician. W i t h most products, incubation for 4 hr may be sufficient to degrade the proteins with papain. However, it is convenient to digest overnight so that the samples may be filtered and prepared for colorimetric determination the next morning. Dilution of the filtrate and washings to 750 ml (approximately equivalent to 0.2 ~ NaC1) assures the co-precipitation of Keltrol and CMC in the presence of gum arabic, the phosphomannans, pectin, and the soluble portions of gum tragacanth, ghatti, and karaya. However, if pectin is absent, this volume may be exceeded by as much as 400 ml or more without affecting the final results. I n this case, the other gums will co-precipitate also. However, their precipitates will be dispersed and eluted by the 0.2 ~ Na2SO 4. The pectin - - CP precipitate adsorbs strongly on the Hyflo Super
Cel and is not eluted by 0.2 ~ NaeS04. When present, its co-precipitation must be avoided. The Keltrol - - CP precipitate, trapped on the Hyflo Super Cel column is not dispersible by even molar cold Na2S04. However, as the concentration of l~a2SO 4 increases beyond 0.2 ~I, filtration becomes measurably slower. Chocolate, which usually interferes severely in the phenol-H2S04 test, caused much less problem here in Procedure A. Apparently, during the preliminary removal of sulfated polysaccharides and of alginate and pectic acid, most of the chocolate and its interfering ingredient(s) were removed. Lower reagent blanks were obtained with the anthrone reagent than with the phenol-H2SO 4 reagent, although the latter is more stable and is easier to work with. W i t h Procedure B, the greater interference from chocolate in the phenol-H2S04 test, occurs because less is removed during the preliminary steps. The anthrone test is preferable here since lower blanks will be obtained. Charcoal may be used to remove the chocolate and other interfering colors incorporated into certain food products. However, when charcoal is used, filtration is extremely slow, even with J0~RNAL OF DAffY SCl~NC~ "VOL. 54, NO. 1i
1628
aRX~A~
Acknowledgments the sand-glass wool or ground glass column. Keltrol does not produce the typical pink Thanks are due to the Kelco Company, San color with the chromotropic acid reagent as Diego, California, for the Keltrol and to the does CMC. However, due to the relatively long Hercules Powder Company, Wilmington, Delaware heating period and most likely to its decom- for the carboxymethy]cellulose. This investigation was supported by U.S. Public position by the hot acid, the decomposition Health Service Grant FD-00083-07. products absorb at 570 m~ to about 0.05 to 0.07 and this increases the blank value to about two References times the normal reading. For accurate deter(1) Federal Register. 1969. Xanthan gum. ruination, therefore, a reagent blank should be Section 121.1224 5376-5377, March 19. run with every batch being heated and the (2) Glicksman, M. 1969. Gum Technology in the value obtained multiplied by 2 and substraeted Food IndustlT. Academic Press, New York. from the sample reading. (3) Graham, H. D. 1969. Determination of The determination of carboxymethylcellulose alginate iu dairy products. J. Dairy Sci., by chromotropic acid in the presence of Keltrol 52 : 443. is highly specific, since Keltrol does not produce (4) Graham, It. D. 1971. Reaction of carboxymethylcellulose with the chromotropic acid the typical purple color (4). Pectin and pectic reagent. J. Dairy Sci., 54: 761. acid, if present, will give color but these are (5) Jeanes, A. R., J. E. Pitts]ey, and F. R. removed prior to the colorimetric determination. Senti. 1961. B-1459. A new hydrocolloid Since both Keltrol and sodium carboxymethylpolyelectrolyte produced from glucose by cellulose react in the phenol-H2S04 and the bacterial fermentation. J. Appl. Polymer anthrone methods, the proposed quantitative Sci., 5 : 519. determination of Keltrol by difference is valid (6) Kelco Co. 1969. Keltrol. Tech. Bull., DB and the proposed method for the isolation of 18, Kelco Co., New Jersey. the polysaccharide/colloid is highly specific. The (7) Rocks, J. K. 1971. Xanthan gum. Food colorimetric methods used for the quantitative Technol., 25: 476. (8) Rogovin, S. P., R. F. Anderson, and M. G. determination of the gums are very sensitive. Cadmus. 1961. Production of polysacThus, the proposed method, as far as is known, eharide with Xanthomonas campestris. J. may be the first one to describe the isolation Bioehem. Microbiol. Technol., ]Eng., 3: 51. of such small quantities of Keltrol from food (9) Sloneker, J. H., D. G. 0rentas, and A. products as complex as those listed in Table 1 Jeanes. 1964. Extracellular polysaccharide and which contain such high contents of protein. from Xanthoqnonas caqnpestris ~RRLBAs far as is known, it is the most sensitive 1459. III. Structure. Canadian J. Chem., method yet proposed since in both the locust 42 : 1261. (10) Smiley, K. L. 1969. Microbial polysacbean gum gel test and the pyruvic acid tests, charides--a review. Food Technol., 20: 112. considerably higher quantities are needed.
JOURNAL OF DAIRY SOIEI~CE VOL. 54, NO, 11