128-JOURNAL OF FOOD SCIENCE-Volume 33 (1968)

Lewis, M. J. and Stephanopoulos, D. 1967. Glucose-induced release of amino acids from Sacchavomyces carlsbergensis by action on the cvtonlasmic membrane. J. Bacterial. 93. 976984.

Mitchell, P. D. 1959. Biochemical cytology of microorganisms. Amt. Rev. Microbial. 13, 407-440.

Rose, A. H. 1963. On the osmotic behavior of Saccharowzyces ccrevisiac as affected bv biotin deficiencv. J. Gelt. Microbial. 31, Z-160.

Schlenk, F. and Dainko, J. L. 1965. Action of ribonuclease prepa- rations on viable veast cells and suheronlasts. J. Bacterial. 89, 428-436. -

_ _

Silver. S. and Wendt. L. 1967. Mechanism of action of nhenethvl alcohol : breakdown of the cellular permeability- barrier. J. Bacterial. 93, 560-566.

Sniecelman. S.. Halvorson. H. 0. and Ben-Ishai. R. 1955. Free I -

amino acids and the enzyme-forming mechanism. In “Amino

Acid Metabolism.” eds. McElroy, W. D. and Glass, B. Johns Hopkins Press, Baltimore, Md.

Stachiewicz, E. and Quastel, J. H. 1963. Amino acid transport in yeast and effect of nystatin. Casad. J. Biochmm. Plzysiol. 41. 397-407.

Umbreit,,W. W., Burris, R. H. and Stauffer, J. I. 1957. “Mano- metric Techniques,” 3rd ed. Burges Publishing Co., Minne- apolis, Minn.

Ms. accepted 2/l/68.

The material in this naner is nart of a thesis submitted bv T. C. Lee to the Graduati School of the University of California in partial satisfaction of the requirements for the M.S. degree in Food Science.

The authors wish to thank the Brewing Industrv Research Institute for financial support of this project-

ROBERT BECKER Western Utilization Research and Development Division, V. S. Department of Agriculture

Albany, C&o&a 94710

Identification of Some Sugars and Mannitol in Celery

SUMMARY-The carbohydrate content of celery petioles was determined using paper chromatographic techniques. Sucrose, glucose, fructose and mannitol were identified and quantita- tively determined. Mannitol crystals were isolated. Sugars chromatographed with solvents containing boric acid showed characteristic stabilities to indicators.

INTRODUCTION PROCESSING STUDIES in progress in this laboratory have

created a need for more detailed knowledge of the carbo- hydrates in celery. Obaton (1929) reviewed the properties and occurrence of mamlitol and found the polyol in all parts of the celery plant; it was thought to be synthesized in the leaves, transported through the petioles and stored in the roots. He lists Hubner ef al. (1823) as the first report of mannitol in celery. Later, Payen (1934) de- scribed the isolation of the sugar from celery juice but gave I~Q quantitative data.

Total and reducing sugars in celery petioles were deter- mined by Hall (1957, 1959) among others. Siegel et al. (1962) determined glucose, fructose and sucrose in leaves from celery plants grown in a nutrient culture. In a recent review, Crosby et al. (1963) reported that only sucrose, glucose and apiose have actually been identified in celery, but he proposed that other common sugars were undoubt- edly present. They mention one report (Anon., 1923) of mannitol having been found in celery roots and two reports of reducing sugars (Hall et al., 1961, Myers et al., 1921).

In view of the omission of the easily detectable mannitol in recent reports, it seemed prudent to reexamine the relative amounts of the predominant sugars in the com- inercially important celery petioles.

EXPERIMENTAL METHODS MATURE FRESH PASCAL CELERY petioles from three

plants were trinnned at both ends, washed, dried, and

chopped into g-inch chips. Four hundred g of the diced celery was blended with 2 g calcium carbonate and the resulting slurry was heated 1 hr on a steam bath. After filtering through a coarse paper filter with suction, the residue was mixed with 100 ml water, heated 1 hr on a steam bath and filtered. The extraction was repeated four more times and the extracts were combined.

A final filtration of the combined extracts was done with a medium porosity sintered glass filter which had been coated with an aqueous suspension of Analytical Grade Celite and washed with water. The solution was made to a known volume and deionized with Dowex 5OW-X4 and Duolite A4 ion exchange resin similar to the procedure of Partridge ( 1948). Aliquots were chromatographically analyzed for all sugars. Proteins, if extracted, did not interfere.

Whatman No. 1 chromatographic paper was used as received for the identification of the sugars. All chemicals were reagent grade unless otherwise specified, and all melting points are corrected.

The sucrose, glucose and fructose bands were identified by their colors and reaction times when sprayed with $-anisidine hydrochloride (Hough et al., 1950) and aniline hydrogen phthalate (Partridge, 1949). Sucrose was con- firmed by its reaction with invertase (Williams et al., 1951) and glucose, by elution, oxidation to gluconic acid and isolation of the potassium salt (m.p. 180°C) (Moore et al., 1940).

Mannitol, sorbitol, and in some cases fructose will react feebly or not at all with silver nitrate after being developed in solvents containing water saturated with boric acid. Dedonder ( 1952) showed that n-butanol : ethanol : water (40 : 11 : 19 j will separate glucose, fructose and sucrose. However, in this solvent, mannose and fructose had nearly the same Ri. They were distinguished by developing in n-butanol : ethanol : aqueous saturated boric acid (40 : 11 :

19) and using silver nitrate as the indicator. Fructose was confirmed by-its stability to silver nitrate when developed in this solvent. Similarly, the polyol spots in n-butanol: acetic acid : water (4 : 1 : 5) were stabilized to the silver nitrate indicator by using aqueous boric acid.

Schleicher and Schuell 20431, paper was used for the quantitative determinations of the sugars. The trailing edge of the paper was serrated and the paper was washed overnight in the solvent before being used. The chromato- grams were developed 18-20 hr by descent in a closed tank at room temperature. The spots were detected by dipping the paper in an acetone solution of silver nitrate, drying, dipping in ethanolic sodium hydroxide, drying and dipping in saturated 60% ethanolic thiosulfate (McCready et al., 1966). The concentrations were determined with a Photovolt Densitometer and deionized standards, using the method of McFarren et al. (1951).

Of the sugars tested, only mannitol and sorbitol did not react with silver nitrate when a chromatogram was de- veloped in the solvent used by Rees (1958) (2-butanone : acetic acid :aqueous saturated boric acid, 9 :l : 1). This stability persisted when ammoniacal silver nitrate (Part- ridge, 1948) or alkaline permanganate (Pacsu et al., 1949) were sprayed, but the sugar alcohols did react if the dried chromatogram was heated to 100-110°C for lo-15 min and immediately immersed in the acetone-silver nitrate solution.

To isolate mannitol, dried celery (cu. 3% moisture) was blended with petroleum ether (Skelly B) and filtered. The residue was refluxed 2 hr with 95% ethanol and filtered hot. On cooling, grayish crystals of mannitol pre- cipitated. The mannitol was recrystallized three times by dissolving it in a minimum amount of water, filtering, and adding two volumes of 95% ethanol.

The identification of the crystalline isolate as mannitol was based on the following observations. The dried color- less crystals melted at 166°C. The hexa-acetate prepared by the method of Shriner et al. (1956) melted at 119- 120°C and had (a) D2j +25.2” in CHC&. The melting points and specific rotation agree with those stated by Heilbron et al. ( 1953). Crystallographic comparison by Dr. F. T. Jones of this laboratory of the unknown and its acetate with authentic samples confirmed the identification.

RESULTS THE WATER EXTRACT of celery was chromatographed

against known standards and the following average con- centrations were determined (g/l00 g fresh celery) : su- crose 0.488, glucose 0.309, fructose 0.314 and mannitol 0.114. The results are the average of three determinations. Maximum variation between determinations of a sample was 4.1%. The sensitivity of the experimental procedures precluded detection of sugars such as apiose which are present in small amounts.

As mentioned earlier, the sugars were extrac!ed from the petioles with water. Although this solvent insured the solubilization of mannitol, it may have permitted invertase activity. Therefore, the sucrose value might be low even though it is higher than some of the literature reports. The high solubility of mannitol in hot alcohol would have complicated the use of boiling alcohol for enzyme inactiva- tion before the quantitative determination.

IDENTIFYING SUGARS AND MANNITOL IN CELERY-129

Obaton (1929) determined mannitol, reducing sugars and sucrose at 10 maturity levels in the variety “Celeri rave de Paris amPliorC.” He found the following concen- tration ranges (g/100 g fresh weight) : mannitol 0.75X.93, reducing sugars 0.21-l. 10, and sucrose 0.1-0.4.

The results of Myers et al. (1921) for reducing and non-reducing sugars are within the above ranges.

Since different varieties would not necessarily contain similar sugar concentrations, the results mentioned previ- ousiy might not be comparable to the more recent work.

The results of Hall et al. (1961) are typical of some of the more recent sugar determinations. He examined Flor- ida Summer Pascal celery petioles at different maturity levels and found the following concentration ranges (g/100 g fresh weight) : inner petioles, reducing sugars OS-l.3 and total sugars 0.9-1.6 ; outer petioles, reducing sugars OS-l.2 and total sugars 0.9-1.5. The chromato- graphically determined sugar concentrations are within this range. The tests used for reducing sugars would not be expected to detect the polyols.

Hall (1959) suggested that the sugar content appears to be an important part of the total flavor of celery petioles. Since Carr et al. (1936) and Schutz et al. (1957) found d-mannitol to be about 60-70s as sweet as sucrose, or about as sweet as glucose, and the polyol is present in large amounts, it appears that mannitol is an important constituent of celery petioles.

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Lorraine 50, 134. Carr, C. J., Beck, F. F. and Krantz, J. C. Jr. 1936. Sugar alcohols.

V. Chemical constitution and sweet taste. J. Am. Chew SOL. 58, 1394.

Crosby, D. G. and Anderson, L. J. 1963. The organic constituents of food. II. J. Food Sci. 28, 640.

Dedonder. R. 1952. Les Glucldes du Topinambour. Bull. Sot. Chim: Biol. 34, 144.

Hall, C. B. 1957. Composit ion and organoleptic evaluation of uortions of celery stalks. Proc. Florida St. Hart. Sot. 70, 204.

Hal< C. B. 1959. -Compositional and organoleptical differences between celery grown in Florida and California. Proc. Florida St. Hort. Sot. 72, 142

Hall, C. B., Burdine, H. W. and Guzman, V. L. 1961. The com- position of three celery varieties at several stages of maturity. Proc. Am. Sot. Hort. Sci. 78, 361.

Heilbron, I. and Bunbury, H. M. 1953. “Dictionary of Organic Compounds.” p. 215. Eyre and Spottiswoods, London.

Hough, L., Jones. J. K. N. and Wadman, W. H. 1950. Quantfta- tive analysis of mixtures of sugars by the method of partition chromatography. J. Chem. Sot., 1702.

Hubner et vbg& 1823. Ueber den Mannazucker des Sellerie- Wiirzel. Repertoriwn fiir die Pharmacie 15, 276.

McCready, R. M. and Goodwin, J. C. 1966. Isolation of I-kestose and nystose by chromatography on a cation exchange resin. J. Chrowatog. 22, 195.

McFarren, E. F., Brand, K. and Rutkowski, H. R. Quantitative determination of sugars on filter paper chromatograms by direct photometry. Anal. Chew. 23, 1146.

Moore, S. and Link, K. P. 1940. Carbohydrate characterization. J. Biol. Chem. 133, 293.

Myers, V. C. and Croll, H. M. 1921. The determination of carbo- hydrates in vegetable foods. /. Biol. Chem. 46, 537.

Obaton, F. 1929. Evaluation de !a mannite (mannitol) chez les vegetaux. I. Rev. gcn. botan. 41, 555.

Pacsu, E., Mora, T. P. and Kent, P W. 1949. General method for paper chromatographic analysis of reducing and nonre- ducing carbohydrates and derivatives. Science 110, 446.

Partridge, S. M. 1948. Filter paper partition chromatography of sugars. Biochem. J. 42, 238.

Partridge, S. M. 1949. Aniline hydrogen phthalate as a spraying reagent for chromatography of sugars. Nature 164, 443.

130-JOURNAL OF FOOD SCIENCE-Volume 33 (1968)

Payen, M. 1934. Darstellung des Mannits aus dem Saft des Selleri’s (Celeri-rave). Justus Liebig’s Annalen der chemie 12, 60.

Rees, W. R. and Reynolds, T. 1958. A solvent for the paper chromatographic separation of glucose and sorbitol. Nature 181, 767.

Schutz, H. G. and Pilgrim, F. J. 1957. Sweetness of various compounds and its measurement. Food Res. 22, 206.

Shriner, R. L., Fuson, R. C. and Curtin, D. Y. 1956. “The Syste- matic Identification of Organic Compounds,” p. 212, 4th ed. John Wiley and Sons, New York.

Siegel, 0. and Bjarsch, H. J. 1962. Uber die Wirkung von Chlorid- und Sulfatronen auf den Stoffwechsel von Tomaten, Sellerie und Reben II. Gartenbauzeissenschaft 27 (2)) 103.

Williams! K. T. and Bevenue, A. 1951. A simple technique for the identrfication of raffinose and sucrose by enzymatic hydrolysis on paper chromatograms and the subsequent separation of the hydrolyzed products by paper chromatography. Science 113, 582.

Ms. accepted 12/18/67.

The author expresses his appreciation to Dr. Rolland M. McCready for his interest and suggestions, and to Dr. F .T. Jones for confirming the mannitol identification.

Reference to a company or product name does not imply ap- proval or recommendation of the product by the U. S. Department of Agriculture to the exclusion of others that may be suitable.

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