J. Nutr. Sci. Vitaminol.,27, 563-572, 1981

Contents and Compositions of the Aroma in "Wasanbon"

Toshiyuki MATSUI1 and Shozaburo KITAOKA2 1Department of Food Science , Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa 761-07, 2Department of Agricultural Chemistry , Faculty of Agriculture, University of Osaka Prefecture, Osaka 591, Japan (Received Mareh 4, 1981)

Summary "Wasanbon" sugar is handmade sugar which has been manufactured traditionally in Japan by a unique refining procedure, and is used in the making of Japanese traditional confectionary. No reports have been published on the substances responsible for the unique aroma of "Wasanbon" sugar . In this paper, the contents and compositions of the aroma in "Wasanbon" sugar and refinery final are reported as studied by column chromatography, gas chromatography, mass spectrom etry and sensory evaluation. The samples are the first press-off molasses ("Ara-mitsu" molasses), refinery final molasses, "Shiroshita" sugar (pre refined sugar) and "Wasanbon" sugar. The summarized results are as follows: In the acidic fraction, the aroma of 3-phenylpropionic acid is similar to the stored aroma of "Wasanbon" sugar, whereas the aroma of its methyl ester was not similar to that aroma. Although aroma contents of the weakly acidic fraction in "Wasanbon" sugar and refinery final molasses are 8.5 to 8.7% of those of the acidic fraction, and their main components are cyclotene and maltol, which are formed by thermal degradation of sugar. These components show a higher preference than other weakly acidic fraction aromas, by a paired pre ference test. Cyclotene and maltol increased about 3.7 and 1.5 times, respectively, by the heating of "Shiroshita" sugar. Key Words "Wasanbon" sugar, "Shiroshita" sugar, first press-off mol asses, refinery final molasses, cyclotene, maltol, paired preference test, 3 - phenylpropionic acid

"Wasanbon" sugar is traditional sugar , widely used in preparing Japanese confections, and it is manufactured by a unique refining procedure. Despite its long usage, there have been few studies on the composition and contribution of the volatile components to the aroma of this sugar. Ito (1) separated several peaks by

1 松 井 年 行 , 2 北 岡 正 三 郎

563 564 T. MATSUIand S. KITAOKA gas chromatography after solvent extraction of sugar. Though this work included organoleptic analysis, it did not explain the correlation with the flavors of "Wasanbon" sugar . The present paper deals with isolation and identification of aroma substances in "Ara-mitsu" molasses, and the first press-off molasses, by gas chromatography-mass spectrometry (GC-MS). After identification, we determined the aroma of "Wasanbon" sugar, its refinery final molasses, and determined the major aroma compounds in "Wasanbon" sugar by sensory evaluation .

EXPERIMENTAL

1. Materials. "Wasanbon" sugar, "Ara-mitsu" molasses and refinery final molasses used in the experiments were products of Kurokawa "Wasanbon-to" Factory, Hiketa, Kagawa Prefecture, made in December, 1978. "Shiroshita"

Fig. 1. Fractionation procedures of aroma components in "Ara-mitsu" molasses.

J. Nutr. Sci. Vitaminol. AROMA IN "WASANBON" SUGAR 565

Table 1. Aroma characteristics of fractionated extracts from "Ara-mitsu" molasses, refinery final molasses and "Wasanbon" sugar.

Panel size (male 6 and female 4). a Data show rather rough numbers since each fraction was not entirely chloroform-free. sugar was the product of Yamada "Shiroshita-to" Factory, Tsuda, Kagawa

Prefecture, made in January, 1979. 2. Solvent extraction of the aroma substance. Twenty-four kg of "Ara-mitsu" molasses produced as a by-product of "Shiroshita" sugar in the refining process was treated as shown in Fig. 1. The residue extracted with hexane was extracted with 3% HCl, 5% NaHCO3 and 2% NaOH successively in the usual way, and each fraction was measured by evaporating under reduced pressure, and determined by weighing. In the case of "Wasanbon" sugar, 8kg was extracted with 45 liters of methyl alcohol and 10 liters of ethyl alcohol. Refinery final molasses (5kg) was extracted with 18 liters of methyl alcohol and 36 liters of ethyl alcohol. The measurement of each fraction was made by weighing the same as in the procedure described above. In order to investigate increasing of aroma, during heat treatment of "Shiroshita" sugar, "Shiroshita" sugar (6kg) was dissolved in 3 liters of distilled water and the solution was heated in an oil bath at 120•Ž for 30min. The solution thus obtained was extracted with the same solvents as described for the refinery final molasses. Sensory evaluation and yields of the fractions are summarized in Table 1. The acidic and weakly acidic fractions were used exclusively in this study to elucidate the unique aroma of "Wasanbon" sugar which was noted in these two fractions by sensory evaluation.

3. Fractionation by Florisil column chromatography. In order to purify the fractions obtained by solvent separations, the acidic and weakly acidic fractions (1g each) were fractionated by Florisil column chromatography: column, 2cm i.d.•~5cm; solvents of stepwise elution were as follows: (1) 100ml of hexane containing 1.5ml methyl alcohol and 30ml ethyl ether, (2) 200ml of a mixture of 3 parts benzene and 2 parts ethyl acetate, (3) 50ml of ethyl acetate, and (4) 50ml of methyl alcohol (flow rate, 30ml/hr; collected in 10ml portions).

4. Identification of the aroma compounds. 1) Conditions of gas chromatography (GC). The acidic fraction was methy

Vol. 27, No. 6, 1981 566 T. MATSUI and S. KITAOKA

lated with diazomethane (2) using the apparatus described by Usui and

Yoshitomi (3) before applying to GC, whereas the weakly acidic fraction was not methylated because the chromatogram of the latter was identical before and after

methylation. The acidic and weakly acidic fractions were analyzed by a Hitachi Model 063 gas chromatograph, fitted with a hydrogen flame ionization detector.

The columns were: 2m•~3mm i.d. stainless steel containing 3% Silicon SE-30 on 60/80 mesh Chromosorb W (AW-DMCS): 2m•~3mm i.d. stainless steel containing

5% Carbowax 20M on 60/80 mesh Chromosrb W (AW-DMCS). The temperature of the column was programmed from 70 to 190•Ž at the rate of 5•Ž/min, with the nitrogen flow rate 30ml/min. The injection port and detector oven temperatures

were 220•Ž.

2) Conditions of GC-MS. The GC-MS system consisted of a Hitachi M 5201

gas chromatograph coupled with a Hitachi Model RMG mass spectrometer. The

gas chromatographic separation was made on a glass column containing 3% Silicon SE-30 on 60/80 mesh Gaschrom-Q (2m•~3mm i.d.). The column temperature was

programmed from 70 to 190•Ž at the rate of 5•Ž/min and the carrier gas was helium with an inlet pressure of 0.8kg/cm2. The spectrometer was operated under the

conditions of 70eV ionization voltage, 3kV ion accelerating voltage, 60ƒÊA of ion current, and 250•Ž of ionization chamber temperature.

3) Peak assignment and quantitative determination. Each compound in the gas chromatogram was identified by comparison of retention time with that of the

authentic compound. Each mass spectrum was also compared with that of the

authentic substance or with that found in the literature. After 250mg each of the acidic and weakly acidic fraction was charged to the column packed with Florisil, the solvent of (1) to (4) described above was eluted and the total mixed eluate except the last solvent (4) was evaporated under reduced

pressure at 18 to 20•Ž so as to analyze by GC. To an aliquot of the acidic fraction was added 0.5mg of levulinic acid as the internal standard, it was methylated with

diazomethane, and was made up to 2ml with methyl alcohol. To the weakly acidic

fraction was added 0.1ml of methyl benzoate as the internal standard and it was filled to 2ml with methyl alcohol. Next, the recorder response (the ratio of each

peak height for the internal standard) for each component, compared with the internal standard, was calculated by using a calibration curve made from the chromatograms of the mixtures belonging to approximately 0.5, 1, 1.5 and 2 times

the amount of each authentic weakly acidic compound. As for the acidic com

pounds, the mixed authentic compounds were derived into methyl esters by the same treatment described above and used for determination of the standard curves on the basis of recorder response. Contents of aroma components in refinery final molasses, "Wasanbon" sugar, "Shiroshita" sugar were determined from the

standard curves. 4) Sensory evaluation. The panel consisted of 10 persons familiarized with

sensory evaluation. The panel assessed the strength and nature of the aroma of a 10-2mg authentic sample in a stoppered flask and that of 10mg of the acidic or

J. Nutr. Sci. Vitaminol. AROMAIN "WASANBON"SUGAR 567 weakly acidic fraction in a similar flask. The paired preference test of the aroma was carried out with three ranks: +, likeness and -, unlikeness (based on similarity to the authentic aroma, was contained in the paired fraction of "Ara-mitsu" molasses): 0, cannot define likeness or unlikeness.

RESULTSAND DISCUSSION

1. Acidic fraction Figure 2 shows a typical gas chromatogram of the methylated acidic fraction of "Ara -mitsu" molasses , and Table 2 shows the mass pattern of identified com ponents of the acidic fraction. Four fractions were obtained from Florisil column chromatography. The first elution contained the lowest boiling compounds, and represented major peaks from 1 to 9 in Fig. 2. The second and the third elutions showed major peaks from 10 to 24 in Fig. 2. The last elution contained the highest boiling and polar compounds, but did not concern the aroma by sensory evaluation. Of 24 compounds identified in the acidic fraction, 13 could be considered as being principle components of the acidic fraction by gas chromatography and mass spectrometry. These compounds were previously reported as volatiles from mol asses (4, 5) (Table 2). Furfural and ƒÀ-phenylethyl alcohol were detected in the acidic fraction, but this detection seems to be caused by contamination of the neutral fraction by the solvent extraction (6). The first, second and third eluting solutions of the column chromatography were used to determine major aroma compounds in refinery final molasses and "Wasanbon" sugar. Table 3 shows the contents of major aroma compounds in refinery final molasses and "Wasanbon" sugar. Methyl

Fig. 2. Gas chromatogram of the methylated acidic fraction of "Ara-mitsu" molasses.

Column: 3% Silicon SE-30 on Chromosorb W (DMCS), 3 mm i.d.•~3m, inject, and

detect. temp., 220•Ž, column temp., 70-190•Ž (5•Ž/min). Carrier: N2, 30ml/min, FID.

Vol. 27, No. 6, 1981 568 T. MATSUI and S. KITAOKA

Table 2. Identified compounds in the acidic fraction of "Ara-mitsu" molasses.

Table 3. Contents of major aroma compounds in refinery final molasses and "Wasanbon" sugar .

J. Nutr. Sci. Vitaminol. AROMA IN "WASANBON" SUGAR 569 phenylacetate was the predominant acidic aroma compound followed by methyl 3 phenylpropionate and methyl benzoate. Ethyl caproate was the scantiest aroma compound of the acidic fraction detected on gas chromatogram. The volatile substances which had lower boiling points than methyl benzoate seemed to vaporize during the refining step, when the components of "Wasanbon" sugar and refinery final molasses were considered.

2. Weakly acidic fraction Figure 3 shows a typical gas chromatogram of a weakly acidic fraction of "Ara -mitsu" molasses and Table 4 shows the mass pattern which was obtained by GC-MS. The elution pattern by Florisil column chromatography showed a tendency similar to that of the acidic fraction. Of 18 compounds 7 were identified in the weakly acidic fraction by gas chromatography and mass spectrometry. In addition, these compounds were previously reported (4, 5), similar to those in the

Fig. 3. Gas chromatogram of the weakly acidic fraction of "Ara-mitsu" molasses.

Table 4. Identified compounds in the weakly acidic fraction of "Ara-mitsu" molasses.

Vol. 27, No. 6, 1981 570 T. MATSUI and S. KITAOKA

acidic fraction described above.

3. Sensory evaluation

The results of sensory evaluation are summarized in Table 5. Although aroma content of the weakly acidic fraction was approximately 10% that of the acidic fraction, based on the sensory evaluation the weakly acidic fraction appeared to contribute to the unique aroma of "Wasanbon" sugar. According to Underwood (7), cyclotene has the characteristic aroma that would help to build a pleasing maple flavor. Cyclotene and maltol showed as the main aroma compounds of "Wasanbon" sugar, because these compounds received higher similarity than others. Based on the evaluation, acetylfuran, ƒÂ-valerolactone and furfuryl alcohol showed a higher similarity than the rest of the weakly acidic fraction, except cyclotene and maltol. Guaiacol, which was contaminated in other fractions, showed the lowest preference rank; it seems to be one of the ill-smelling compounds of "Wasanbon" sugar. Phenylacetic acid and its esters have strong aromas and esters of benzoic acid have warm, sweet and fruity nutlike aromas (5) . However, these esters were not the main aroma compounds because of low preference. Of the acidic fraction, 3-phenylpropionic acid had an aroma similar to

Table 5. Sensory evaluation of "Ara-mitsu" molasses compared with authentic samples. The panel (male 6 and female 4) started randomly by assessing pairs consisting of 10-2mg authentic samples and 10mg acidic or weakly acidic fraction of "Ara-mitsu" molasses. Benzoic acid and phenylacetate were excluded from the sensory evaluation owing to the lack of aroma.

J. Nutr. Sci. Vitaminol. AROMA IN "WASANBON" SUGAR 571

Table 6. Aroma compound contents in "Shiroshita" sugar before and after heat treatment.

that of stored "Wasanbon" sugar aroma, but its methyl ester did not have a similar aroma. The increase of weakly acidic aroma compounds after heating treatment of "Shiroshita" sugar is shown in Table 6 . Cyclotene and maltol increased about 3.7 and 1.5 times, respectively, during the treatment of "Shiroshita" sugar. Acetylfuran, b-valerolactone and furfuryl alcohol showed less increased amounts than cyclotene and maltol. Cyclotene was a highly important aroma component of "Wasanbon" sugar by sensory evaluation. Walter and Fargerson (8) reported the presence of b valerolactone in the volatile fraction from the pyrolysis of , and this compound was detected in the volatiles from cane molasses by Yokota and Fargerson (4). Cyclotene, maltol, acetylfuran and b-valerolactone formed by ther mal degradation of sugar were the major aroma compounds in "Wasanbon" sugar by sensory evaluation, and ƒÂ-valerolactone among them was concluded to increase by heat treatment of the acidic and weakly acidic fractions. Thus, "Wasanbon" aroma was not formed during the refining process, but it was produced during the evaporation process of cane juice, or it was contained in molasses remaining as impurities in the product, which seemed to contribute to the unique aroma of this traditional sugar.

The authors wish to thank Prof. Sin'itiro Kawamura of Kagawa-ken Meizen Junior College, and Prof. Hiromichi Kato of Tokyo University, for their suggestions, and also Dr. Kisaburo Samukawa, Dr. Akira Hirota and Dr. Kazutaka Miyatake of the University of Osaka Prefecture for mass spectrometric analysis. We are grateful to Associate Prof. Dr. Itsuo Ichimoto of the University of Osaka Prefecture for gifts of cyclotene.

REFERENCES

1) Ito, H. (1975): Flavor of sugar. Proc. Res. Soc. Jpn. Sugar Refin. Technol. (in Japanese), 25, 10-16. 2) Fieser, L. F., and Fieser, M. (1967): Reagents for Organic Synthesis. John Wiley and Sons, Inc., New York, Vol. 1, pp. 191-195. 3) Usui, H., and Yoshitomi, K. (1971): Fatty acid esters, in Modern Gas Chromatography, Theory and Applications (in Japanese), ed. by Funasaka, W., and Vol. 27, No. 6, 1981 572 T. MATSUI and S. KITAOKA

Ikekawa, H., Hirokawa Publishing Co., Tokyo, 6th Ed., Vol. 2, pp. 645-667. 4) Yokota, M., and Fargerson, I. F. (1971): The major volatile components of cane molasses. J. Food Sci., 36, 1091-1094. 5) Ito, H. (1976): Isolation and identification of the characteristic sweet aroma compounds in refinery final molasses. Agric. Biol. Chem., 40, 827-832. 6) Ito, H., and Deki, M. (1978): Aroma components of heated liquid sugar. J. Soc. Food Sci. Technol. Jpn. (in Japanese), 25, 549-555. 7) Underwood, J. C. (1971): Effect of heat on the flavoring components of maple . J. Food Sci., 36, 228-230. 8) Walter, R. H., and Fargerson, I. F. (1971): Volatile compounds from heated glucose . J. Food Sci., 33, 294-299.

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