P.K. SARKAR, L.J. JONES, G.S. CRAVEN AND S.M. SOMERSET. 1997. Oligosaccharide profiles of unfermented and fermented soybeans were determined by a ...
Letters in Applied Microbiology 1997, 24, 337–339
Oligosaccharide profiles of soybeans during kinema production P.K. Sarkar, L.J. Jones, G.S. Craven and S.M. Somerset Government Chemical Laboratory, Queensland Health, Archerfield, Queensland, Australia 1203/96: received 5 September 1996 and accepted 12 September 1996 P .K . S A RK AR , L. J. J ON ES , G. S. C RA VE N AN D S . M. SO M ER SE T . 1997. Oligosaccharide profiles of unfermented and fermented soybeans were determined by a high-performance liquid chromatographic method using a Hypersil NH2 column. In raw soybeans, mean contents of sucrose, raffinose and stachyose on a dry weight basis were 6·3, 1·9 and 4·3%, respectively (ratio of 3·3 : 1 : 2·3). Although soaking and cooking treatments caused a 73–88% reduction in oligosaccharide content, fermentation lowered these levels below the limit of detection (1·0 g kg−1 dry wt), thus eliminating a potential cause of flatulence for consumers.
INTRODUCTION
Kinema is a solid-substrate, fermented, flavoursome and alkaline food prepared from soybeans. In traditional kinema preparation, locally grown yellow-coated soybeans are washed, soaked overnight and cooked by boiling in spring water for about 90 min. After decanting the water, seeds are ground to grits, wrapped in leaves and sackcloth, and left in a warm place to ferment for 1–3 days. Optimal fermentation is indicated by the appearance of a rough, white, viscous mass on the grits and a typical kinema flavour dominated by ammonia. Fresh kinema is fried briefly in oil and cooked with vegetables, spices, salt and a little water to produce a thick curry, which is eaten with rice (Tamang et al. 1988). Bacillus subtilis is the predominant micro-organism in kinema, and is responsible for kinema production. Enterococcus faecium, Geotrichum candidum and Candida parapsilosis, occurring in 100%, 50–80% and 40–50%, respectively, of market kinema samples, make up the accompanying, probably opportunistic, microflora (Sarkar et al. 1994). The fermentation process as well as kinema quality are improved by inoculating sterilized soybeans with B. subtilis and incubating at 45°C (Sarkar and Tamang 1995). Soybeans are valued for their high oil and protein contents. However, their consumption is limited because soybeans contain high levels (120–150 g kg−1 dry wt) of a-galactosides of sucrose, causing internal gas production in humans (Rackis et al. 1970). Kawamura (1954) observed that over 90% of the sugars present in ripe soybeans comprise sucrose, raffinose Correspondence to : Dr P.K. Sarkar, Microbiology Laboratory, Department of Botany, University of North Bengal, Siliguri 734430, India. © 1997 The Society for Applied Bacteriology
and stachyose. These three oligosaccharides may be important determinants of consumer acceptance of soybean products. Minimizing factors that cause flatus may improve the acceptance of these products as an inexpensive protein source. Since kinema is an important dietary protein source in Nepal, Bhutan and parts of north-east India, quantitative changes in oligosaccharide content of soybeans during kinema production were the subject of this present investigation. MATERIALS AND METHODS Bacteria
Strains of B. subtilis DK-W1 (MTCC 1747), Ent. faecium DKC1, C. parapsilosis DK-Sm1 (MTCC 1744) and G. candidum DK-Ch1 (MTCC 1735) used in this investigation were representative of isolates from commercially prepared kinema (Sarkar et al. 1994). Kinema preparation
Soybean [Glycine max (L.) Merr. cultivar ‘local yellow’] seeds, purchased from Gangtok town market in Sikkim, were washed and soaked in deionized water (water : bean, 5 : 1 w/w) for 16 h at 22–25°C. After decanting the water, beans were mixed with fresh water (water : bean, 2 : 1 w/w), autoclaved at 121°C for 15 min and cooled to about 50°C. The beans were drained, transferred to a sterile polythene bag and ground from outside the bag to break most of the seeds to half-cotyledons. The Bacillus, Enterococcus and yeast inocula were prepared
338 P .K . S A RK AR E T A L.
Sample preparation and oligosaccharide determination
Soaked and cooked beans, both unfermented and fermented, were blended (Bamix, model 122, Switzerland) for 1 min to a smooth paste. These were frozen at −20°C, freeze-dried (Dynavac, model FD12, Australia) and ground to a fine powder. Raw soybeans were dried in a hot-air oven at 105°C for 24 h and ground to a fine powder. All of these preparations were defatted with distilled petroleum ether (Recochem, Australia) in Soxhlet extractors. The ether extract was evaporated under a stream of nitrogen and quantified gravimetrically. The value was used for fat correction during calculation of the oligosaccharide content of defatted samples on a dry weight basis. The procedures for oligosaccharide extraction and deproteination were based on those of Knudsen (1986). A defatted sample (approx. 1 g) was mixed with 10 ml water and brought just to boiling. Then the mixture was shaken in a 60°C water bath for 5 min, made up to 10 ml with water and centrifuged (1100 g for 10 min). The supernatant fluid (3·5 ml) was mixed thoroughly with 6·5 ml acetonitrile (Labguard, Australia) and left overnight at 4°C. After filtering (Millipore, 0·45 mm), an aliquot of the filtrate was placed in a 5-ml glass vial for highperformance liquid chromatographic (HPLC) analysis. The chromatographic system consisted of System Gold Pumps (Beckman), a 5-mm particle size Hypersil NH2 column (4·6 mm i.d.×20 cm) (Shandon, UK) with a Waters mBondapak NH2 guard column (Millipore), a Waters model 410 differential refractometer detector and a model C-R6A Chromatopac integrator (Shimadzu, Japan). The mobile phase used for elution was acetonitrile–water (65 : 35, v/v) and the flow rate was constant at 1·0 ml min−1. The column and
detector were maintained at 31°C. A 5-ml sample was introduced into the column using a Waters WISP 710B automatic sample injector. The run time was maintained for 15 min. Sugar standards were sucrose (BDH, Australia) and D(¦) raffinose and stachyose (Sigma, USA). Each sample was analysed in triplicate. Oligosaccharides were identified by comparing retention times with standards and quantified by measuring peak heights. Data were analysed statistically using the methods of Snedecor and Cochran (1989). RESULTS AND D ISCUSSION
Figure 1a shows the separation of oligosaccharide standards on a Hypersil NH2 column. Sample peaks were obtained for each compound that resolved according to molecular weight. Under the chromatographic conditions of the present study, stachyose, the highest molecular weight oligosaccharide present, was eluted after 9 min. The separation of these oligosaccharides was similar to that reported by Knudsen (1986) on a LiChrosorb NH2 column. The reproducibility of the analysis of standards was high, with a coefficient of variation of approximately 2% between repeated injections of the (a)
Su
(b)
Su
St St Refractive index
by introducing 5 ml of sterile water onto 18 h slant cultures (incubated at 37°C) on plate count agar (Difco), 24 h slant cultures (37°C) on all purpose Tween (APT) agar (Difco) and 24 h slant cultures (28°C) on malt extract agar (Oxoid), respectively. Growth was scraped off into tubes and agitated for 30 s on a Vortex mixer (Scientific Instruments). Cell numbers were determined using a Neubauer counting chamber and a phase-contrast microscope. The suspensions were used as inocula, with concentrations of Bacillus, Enterococcus and yeast cells being 106–7, 105–6 and 103–4, respectively, per gram of soybean grits. After mixing, the beans were distributed in approximately 50 g (wet wt) amounts in sterile 250-ml Erlenmeyer flasks plugged with cotton wool, and incubated at 95% relative humidity in an environmental chamber. The process variables included unfermented beans or beans fermented in the presence of B. subtilis, B. subtilis plus Ent. faecium, or a mixture of B. subtilis, Ent. faecium, C. parapsilosis and G. candidum at a fermentation temperature of either 37 or 45°C.
Ra
Ra
15 min
15 min
Fig. 1 High-performance liquid chromatogrammes of
oligosaccharide standards (a) and an extract of raw soybeans (b). Su, sucrose ; Ra, raffinose ; St, stachyose.
© 1997 The Society for Applied Bacteriology, Letters in Applied Microbiology 24, 337–339
S OY BE A N O LI G OS AC C HA RI D ES 339
Table 1 Oligosaccharide content of soybeans before and after
various treatments to produce kinema — ––––––––––––––––––––––––––––––––––––––––––––––––––––– Oligosaccharide (g kg−1 dry wt)* — ––––––––––––––––––––––––––––– Sample Sucrose Raffinose Stachyose — ––––––––––––––––––––––––––––––––––––––––––––––––––––– Raw beans 62·820·2 19·221·0 43·220·6 Soaked and cooked beans 17·120·2 2·320·1 10·620·4 Fermented beans (kinema)† ³ DL ³ DL ³ DL — ––––––––––––––––––––––––––––––––––––––––––––––––––––– * Each value is the mean2SE of a triplicate set. † All four treatments (B. subtilis at 37°C for 48 h ; B. subtilis and Ent. faecium at 37°C for 48 h ; B. subtilis, Ent. faecium, G. candidum and C. parapsilosis at 37°C for 48 h and B. subtilis at 45°C for 18 h) gave similar results. DL, Detection limit (1·0 g kg−1 dry wt).
same dilution. Figure 1b shows a separation of oligosaccharides from an extract of raw soybeans. The chromatogram contains a number of unidentified components. The concentrations of sucrose, raffinose and stachyose in soybeans at different processing steps of kinema-making are listed in Table 1. The coefficients of variations for triplicate analyses were 0·4%, 7·4% and 2·1% in raw soybeans, and 1·9%, 7·1% and 4·8% in soaked and cooked beans for sucrose, raffinose and stachyose, respectively. To ensure that no oligosaccharides were lost during extraction and deproteination, a sample was spiked with xylose before extraction. The recoveries of xylose were in the range of 85–93%. The sum of raffinose plus stachyose (sugars not metabolized by humans) comprised 50% of the oligosaccharides present in raw soybeans. When beans were soaked, drained, cooked and again drained, there was 73–88% reduction of the oligosaccharide content of raw beans. While sucrose, raffinose and stachyose were in proportions of 3·3 : 1 : 2·3 in raw beans, the proportion changed to 7·4 : 1 : 4·6 in soaked and cooked beans, due to a marked decrease in raffinose. This loss cannot be explained solely on the basis of its solubility or molecular weight, since the loss of sucrose (73%) was less than that of raffinose (88%) although sucrose is more soluble and has the lowest molecular weight of the three oligosaccharides studied. Our observations suggest that, in addition to simple factors such as solubility and molecular size, the location and characteristics of the natural molecular structure of the sugar within the cell also play an important role in affecting the rate as well as the extent of extraction. The present findings are in agreement with those reported by Silva and Braga (1982) and Abdel-Gawad (1993) on several beans. Cooking also has an effect on oligosaccharide content. A greater decrease in disaccharide content occurred when soaked legume seeds were cooked, compared with unsoaked but cooked seeds (Abdel-Gawad 1993). The remaining quan-
tities of oligosaccharides fell to below the limit of detection (1·0 g kg−1 dry wt) after fermentation. Several processes, such as boiling and fermentation, to reduce the oligosaccharide content in legumes has been advocated by Puwastien and King (1984). Bacillus subtilis, the micro-organism responsible for kinema formation, can hydrolyse oligosaccharides (Sarkar and Tamang 1995). This bacterium alone can completely degrade the oligosaccharides. The accompanying flora had apparently no effect on this degradation by B. subtilis. On the basis of our data, we conclude that the traditional kinema-making process successfully degrades the compounds responsible for flatus resulting from soybean ingestion. ACKNOWLEDGEMENTS
The financial support from the Crawford Fund for International Agricultural Research, Melbourne, Australia, and the International Foundation for Science, Stockholm, Sweden, is greatly appreciated. The authors are indebted to Dr J.H. Bradbury, Coordinator of Asia Pacific Food Analysis Network at the Australian National University, Canberra, Australia, for co-ordination of this study. REFERENCES Abdel-Gawad, A.S. (1993) Effect of domestic processing on oligosaccharide content of some dry legume seeds. Food Chemistry 46, 25–31. Kawamura, S. (1954) Studies on soybean carbohydrates. IV. Determination of oligosaccharides in soybeans. Nippon Nogei Kogaku Kaishi 28, 851–852. Knudsen, I.M. (1986) High-performance liquid chromatographic determination of oligosaccharides in leguminous seeds. Journal of the Science of Food and Agriculture 37, 560–566. Puwastien, P. and King, R.D. (1984) Changes in raffinose, stachyose, verbascose and a-galactosidase activity in germinating winged beans (Psophocarpus tetragonolobus (L.) DC). Lebensmittel-Wissenschaft und-Technologie 17, 336–338. Rackis, J.J., Honig, D.H., Sessa, D.J. and Steggerda, F.R. (1970) Flavor and flatulence factors in soybean protein products. Journal of Agricultural and Food Chemistry 18, 977–982. Sarkar, P.K. and Tamang, J.P. (1995) Changes in the microbial profile and proximate composition during natural and controlled fermentations of soybeans to produce kinema. Food Microbiology 12, 317–325. Sarkar, P.K., Tamang, J.P., Cook, P.E. and Owens, J.D. (1994) Kinema—a traditional soybean fermented food : proximate composition and microflora Food Microbiology 11, 47–55. Silva, H.C. and Braga, G.L. (1982) Effect of soaking and cooking of dry beans (Phaseolus vulgaris L.). Journal of Food Science 47, 924–925. Snedecor, G.W. and Cochran, W.G. (1989) Statistical Methods, 8th edn. Ames : Iowa State University Press. Tamang, J.P., Sarkar, P.K. and Hesseltine, C.W. (1988). Traditional fermented foods and beverages of Darjeeling and Sikkim—a review. Journal of the Science of Food and Agriculture 44, 375–385.
© 1997 The Society for Applied Bacteriology, Letters in Applied Microbiology 24, 337–339
