Journal of Science of Food and Agriculture J Sci Food Agric 79 :249–252 (1999)

Fate of oligosaccharides during production ofsoya bean tempeFrancis co Ruiz-Tera� n and J David Owens *Department of Food Science and Technology, The Univers ity of Reading, Reading RG6 6AP,UK

Abstract : The concentrations of the ýatulence and possibly prebiotic oligosaccharides, stachyose and

raffinose, as well as sucrose, glucose and fructose, were monitored during the preparation of bacteria-

free tempe made with Rhizopus oligosporus NRRL 2710. The initial soya beans contained (g [kg dry

matter ]—1): stachyose, 30; raffinose, 11; sucrose, 57, glucose, Æ 0.1, and fructose, Æ 0.1. Losses of oli-

gosaccharides during preparation of tempe were due to leaching during the hydration (100ÄC for

30 min), washing (60ÄC) and cooking stages (121ÄC for 10 min). The losses were (g [kg dry initial soya

bean ]—1): hydration 45; dehulling and washing, 14, and cooking, 24. Concentrations remaining in the

cooked cotyledons were (g [kg dry weight ]—1): stachyose, 7.0; raffinose, 2.1; sucrose, 7.2; glucose, 1.8,

and fructose, 0.9, representing 17% (w/w) of the stachyose, 15% of the raffinose and 13% of the

sucrose + glucose + fructose in the initial soya beans. Within the errors of measurement, concentra-

tions of stachyose, raffinose and sucrose did not change during the fermentation but concentrations of

glucose and fructose were reduced to 0.2 and 0.3 g (kg dry weight)—1, respectively. The results are

discussed in relation to controlling the concentrations of oligosaccharides in tempe.

1999 Society of Chemical Industry(

Keywords: Glycine max ; oligosaccharide; prebiotic, raffinose; Rhizopus oligosporus ; stachyose; solid substratefermentation; soya bean; sucrose; tempe; tempeh

INTRODUCTION

Soya beans (Glycine max) contain the oligosac-charides, stachyose (a-D-galactopyranosyl-[1] 6]-a-D-galactopyranosyl-[1]6]-a-D-glucopyranosyl-[1]2]-b-D-fructofuranoside) and raffinose (a-D-galactopyranosyl-[1]6]-a-D-glucopyranosyl-[1]2]-b-D-fructofuranoside), which are not digested in thehuman small intestine. When these compounds reachthe large intestine they may be fermented by bac-teria, producing gas and giving rise to uncomfortableýatulence.1 There are suggestions that these sameoligosaccharides may also exhibit health-promotingprebiotic properties and encourage the proliferationof beneücial bacteria in the colon.2 Hence, there is aneed to understand the eþects of processing on theseoligosaccharides in order to reduce their concentra-tions to non-ýatulence-inducing levels whilstretaining sufficient concentrations for possible pre-biotic eþects.

Tempe is a traditional Indonesian product, con-sisting of cooked soya bean cotyledons boundtogether into a cake by growth of a mould,3 whichhas considerable potential as a vegetarian food ingre-dient in Western markets. It is known that the con-centrations of oligosaccharides in soya bean arereduced during the tempe process but there is con-

siderable confusion in the literature about the rela-tive contributions of the diþerent processing stages,especially regarding the role(s) of micro-organisms inthe fermentation stage. It is well established that themajor losses occur during the preparation of thecotyledons prior to the mould fermentation.4h8However, there are considerable diþerences in theextent of the reductions reported, with concentra-tions of individual oligosaccharides in the cookedcotyledons ranging from 12 to 83% of the concentra-tions in the initial soya beans.

It is also unclear as to what extent the oligosac-charide concentrations are further reduced duringthe fermentation stage. The usual moulds used intempe production are unable to utilise stachyose orraffinose as sole carbon and energy sources9,10 but anumber of authors5,7,8 have recorded changes in theconcentrations of these compounds during the fer-mentation. However, unless special precautions aretaken,11 bacterial contamination is usual intempe12,13 and its occurrence precludes the unam-bivalent attribution of observed chemical changes toactivities of the mould.

To balance possible prebiotic beneüts with theneed to reduce ýatulence problems it is desirable tocontrol the concentrations of oligosaccharides in the

* Corres pondence to : J David Owens , Department of FoodScience and Technology, PO Box 226, The Univers ity of Reading,

Reading RG6 6AP, UK

Contract/grant s pons or : Univers idad Nacional Autonoma de

Mexico

(Received 26 Augus t 1997 ; revis ed vers ion received 19 February

1998; accepted 25 May 1998)

( 1999 Society of Chemical Industry. J Sci Food Agric 0022–5142/99/$17.50 249

F JD OwensRuiz-Tera� n,

ünal product. Hence, the aim of this study was toidentify the relative contributions of the hydration,dehulling, cooking and fermentation stages tochanges in the concentrations of oligosaccharidesduring the production of bacteria-free tempe.

MATERIALS AND METHODS

Micro-organism

Rhizopus oligosporus NRRL 2710 was maintained asa spore suspension frozen at [ 70¡C. Spore suspen-sions for inoculation of soya beans were prepared aspreviously.11

Preparation of tempe

Two batches of soya beans (Canadian Number 1;200g each) were hydrated in puriüed water (PuriteRO50 reverse osmosis and ion exchange unit) at100¡C for 30min, dehulled, washed free of hulls, ste-rilised and cooked by autoclaving at 121¡C for 10minin 0.11M lactic acid solution, inoculated with sporesof R oligosporus and incubated at 30¡C.11 Twobatches (250g each) were prepared similarly exceptthat they were autoclaved in puriüed water instead oflactic acid solution and for 20min instead of 10min.At each stage, the volumes of water used and recov-ered were recorded and samples retained for analysis.The pH values of the cooked soya beans prior toinoculation were 4.2 for those cooked in lactic and6.7 for those cooked in water.

The absence of bacteria in cooked cotyledons andtempe cultures was conürmed by adding cotyledonsto thioglycollate broth (Oxoid CM173) and incu-bating at 30¡C for 3 days.11 Turbidity was notobserved in any tube.

The amounts of dry matter remaining at each stagewere estimated as follows : hydrated beans dry matterwas calculated as initial beans dry matter less mea-sured total solids loss in the hydration water (73g[kg initial dry beans]~1); dry matter of dehulledbeans (hydrated cotyledons) was estimated as theweight of ünal cooked cotyledons plus the measuredtotal solubles loss in cooking water (54g [kg initialdry beans]~1); the dry matter of cooked cotyledonswas calculated from the measured wet weight ofcotyledons and their water content ; and loss of drymatter during fermentation was assumed to be 100g(kg dry cooked cotyledons)~1.11 The resultant yieldfactors obtained for each stage were (g dry matter [ginitial dry soya beans]~1): hydrated beans, 0.93;dehulled beans (hydrated cotyledons), 0.77; cookedcotyledons, 0.73 and tempe, 0.66.

Chemical and physical analyses

The pH values and dry matter contents were deter-mined as previously.11

Determination of oligosaccharides

Samples of soya beans, cotyledons and tempe werefreeze dried, ground in a coþee grinder and stored in

a desiccator at ambient temperature. Samples weredefatted11 prior to extraction of oligosaccharides.

Oligosaccharides were extracted by adding 1gdefatted soya bean ýour to 20ml puriüed water andholding at 100¡C for 30min.14 Extract (5ml) wasclariüed by twice centrifuging at 10000] g for15min and decanting the supernatant. It was thenultraültered (5000 NMWL ; Millipore UFC3LCC00) by centrifuging at 5000] g for 30min. Fil-trates were stored at [18¡C until analysed.

Stachyose, raffinose, sucrose, glucose and fructosewere determined by HPLC on a Bio-Rad AminexHPX-87N column at 85¡C with a cation Na` guardcolumn (Bio-Rad 125-0508) eluted with 0.015M

in high purity water (Purite RO50 with StillNa2SO4Plus HP unit ; conductivity ¹ 0.08kS cm~1) at aýow rate of 0.5ml min~1. The HPLC apparatuscomprised a Thermo Separation Products isocraticP100 pump, a Showdex RI-71 refractive indexdetector (Showa Denko, Japan), a Rheodyne 7125injector with a 20kl loop and a Hewlett-Packard HP3396A integrator. Peaks were identiüed by compari-son of their retention times with those of pure com-pounds and concentrations were determined fromstandards curves relating peak area to concentration.

RESULTS

Changes in concentrations of oligosaccharides

during preparation of cooked cotyledons

The initial soya beans contained (g [kg drymatter]~1): stachyose, 30; raffinose, 11; sucrose, 57,glucose, \ 0.1 and fructose, \ 0.1. The concentra-tions of oligosaccharides in the beans/cotyledonswere reduced progressively during processing (Fig1). Oligosaccharides in the initial soya beans wereaccounted for by oligosaccharides recovered in thehydration, washing and cooking waters, and thecooked cotyledons, with overall recoveries of (g [g

Figure 1. Concentrations of s ugars at different s tages in the

preparation of s oya bean tempe.4, s tachyos e ; raffinos e ;0=,s ucros e ; glucos e] fructos e. Error bars are es timated rangesK,for entire columns .

250 J Sci Food Agric 79 :249–252 (1999)

Oligosaccharides during production of soya bean tempe

Figure 2. Fates of s tachyos e, raffinos e and s ucros e during

production of s oya bean tempe. Fructos e and glucos e are

pres umed to derive from s ucros e. dry s oya beans ;=, Kz ,hydration water ; dehulling was h water ;4, cooking water ;0,K,cooked cotyledons . Error bars are es timated ranges for entire

columns .

initial oligosaccharide in beans]~1): stachyose, 0.93;raffinose, 0.88; and sucrose ] glucose ] fructose,1.03 (Fig 2).

The greatest losses occurred in the hydration(100¡C for 30min) and cooking (121¡C for 10min)stages. Essentially no oligosaccharides were lost withthe hulls, as the oligosaccharide content of thehydrated beans was entirely accounted for by that inthe dehulled beans and wash water. The concentra-tions in the cooked cotyledons (stachyose, 7.0, raff-inose, 2.1, sucrose, 7.2, glucose, 1.8 and fructose,0.9g [kg dry weight]~1) represented 17% (w/w) ofstachyose, 15% of raffinose and 13% ofsucrose ] glucose ] fructose present in the originalbeans.

Low concentrations of fructose and glucose weredetected only in the hydration water, cooking water

Table 1. Water s oluble los s es during preparation of s terile,

cooked s oya bean cotyledons a

Sample Water s oluble los s es

(g [kg initial dry s oya bean]É1)

Oligos accharides b Total

Hydration water 45(^4)c 73(^8)cWas h water 14(^2) 42(^7)Cook water 24(^2) 54(^9)Total 83(^8) 169(^17)

a Soya beans were hydrated in water at 100¡C for 30min,dehulled and was hed, and s terilis ed and cooked in 0.11M lactic

acid s olution at 121¡C for 10minb Stachyos e] raffinos e] s ucros e] fructos e] glucos e.Fructos e and glucos e are pres umed to derive from s ucros e

c Figures in parenthes es are ranges for duplicate batches of s oya

beans

and cooked cotyledons (Fig 2). These are presumedto derive from hydrolysis of sucrose.

Effect of cooking in water instead of lactic acid

solution

The amounts of oligosaccharides in non-acidiüedcooking water and non-acidiüed cooked cotyledonswere very similar to those for acidiüed cooking waterand acidiüed cotyledons (data not shown). Fructoseand glucose concentrations in the ünal cooked coty-ledons were lower in non-acidiüed cotyledons(glucose and fructose, \ 0.1g [kg drycotyledons]~1) than in acidiüed ones (glucose, 1.8;fructose, 0.9g [kg dry cotyledons]~1).

Total water-soluble losses

Total soluble losses in the hydration, wash andcooking waters were approximately 170g (kg initialdry soya beans)~1 of which oligosaccharides rep-resented 49% (w/w) (Table 1).

Effects of fermentation on oligosaccharides

There was no change (within the errors ofmeasurement) in either the concentrations or theamounts of oligosaccharides during the fermentation(Fig 1). There was a reduction in the concentrationsof fructose and glucose.

DISCUSSION

Reduction in oligosaccharides during preparation of

cotyledons

It is clear that essentially all the losses of oligosac-charides were due to leaching during the hydration,washing and cooking of the beans/cotyledons. Con-centrations of stachyose, raffinose andsucrose ] glucose ] fructose in the cooked cotyle-dons were 23% (w/w), 20% and 17% of the concen-trations in the dry soya beans, representing 17, 15and 13% (w/w) of the amounts present in the initialsoya beans.

Oligosaccharides during tempe fermentation

The observations here of the non-utilisation ofsucrose, raffinose or stachyose in assured bacteria-free tempe is in agreement with observations that, invitro, tempe moulds do not use these compounds assole sources of carbon and energy.9,10

The conýicting reports in the literature on theproduction of oligosaccharidases and the utilisationof oligosaccharides during tempe fermentation arepossibly a consequence of bacterial contamination,or, in some cases, could be due to the use of diþerentmould strains. Certainly, the usual procedures formaking tempe allow the survival of bacterial sporesand post-heating contamination by non-spore-forming bacteria. A basic microbiological tenetrequires the use of axenic cultures to avoid confusionregarding the physiological capabilities of microbialspecies, and it can be suggested that future studieswith tempe should use either axenic mould culturesor deüned mixed cultures of known organisms.

J Sci Food Agric 79 :249–252 (1999) 251

F JD OwensRuiz-Tera� n,

Strategies for controlling concentrations of

oligosaccharides in tempe

The reduction of raffinose and stachyose to lowlevels is undoubtedly a desirable feature of soya beanprocessing and it is clear that in the production oftempe this is achieved during the processing stepsprior to the fungal fermentation.

Possible prebiotic eþects of soya bean oligosac-charides have not yet been clearly established, and,hence, it is not possible to suggest what concentra-tions of these oligosaccharides might be desirable intempe. It is well established that maximal leachinglosses occur at temperatures of 100¡C or more15 andthe present observations suggest that the concentra-tions of oligosaccharides in tempe could be con-trolled relatively easily by the choice of conditionsfor hydrating and cooking the beans. This control isfacilitated by the fact that the oligosaccharides arenot utilised in tempe by the mould.

ACKNOWLEDGEMENT

One of the authors (FR-T) thanks the UniversidadNacional Autonoma de Mexico for ünancial support.

REFERENCES1 Cummings JH and MacFarlane GT, The control and conse-

quences of bacterial fermentation in the human colon. J

Appl Bacteriol 70:443–459 (1991).2 Crittenden RG and Playne MJ, Production, properties and

applications of food-grade oligosaccharides. Trends Food Sci

Technol 7:353–361 (1996).

3 Nout MJR and Rombouts FM, Recent developments in temperesearch. J Appl Bacteriol 69:609–633 (1990).

4 Wang HL, Swain EW, Hesseltine CW and Heath HD, Hydra-tion of whole soybeans aþects solids losses and cookingquality. J Food Sci 44:1510–1513 (1979).

5 Van der Riet WB, Wight AW, Cilliers JJL and Datel JM,Food chemical analysis of tempeh prepared from SouthAfrican-grown soybeans. Food Chem 25:197–206 (1987).

6 Mulyowidarso RK, Fleet GH and Buckle KA, Changes in theconcentration of carbohydrates during the soaking of soy-beans for tempe production. Int J Food Sci Technol 26:595–606 (1991).

7 Nowak J and Szebiotko K, Some biochemical changes duringsoybean and pea tempeh fermentation. Food Microbiol

9:37–43 (1992).8 Steinkraus KH, Indonesian tempe and related fermentations,

in Handbook of Indigenous Fermented Foods, 2nd edn, Ed bySteinkraus KH, Marcel Dekker, New York, USA, pp 22, 39(1996).

9 Sorenson WG and Hesseltine CW, Carbon and nitrogen uti-lisation by Rhizopus oligosporus. Mycologia 58:681–689(1966).

10 Graþham AJ, Gordon MH, Westby A and Owens JD, Nutri-tion of tempe moulds. Lett Appl Microbiol 21:223–227(1995).

11 Ruiz-Tera� n F and Owens JD, Chemical and enzymic changesduring the fermentation of bacteria-free soya bean tempe. J

Sci Food Agric 71:523–530 (1996).12 Sudarmadji S and Markakis P, Lipid and other changes

occurring during the fermentation and frying of tempeh.Food Chem 3:165–170 (1978).

13 Samson RA, Van Kooij JA and De Boer E, Microbiologicalquality of commercial tempeh in The Netherlands. J Food

Protect 50:92–94 (1987).14 Kennedy IR, Mwandemele OD and McWhirter KS, Estima-

tion of sucrose, raffinose and stachyose in soybean seeds.Food Chem 17:85–93 (1985).

15 Abdel-Gawad AS, Eþect of domestic processing on oligosac-charide content of some dry legume seeds. Food Chem

46:25–31 (1993).

252 J Sci Food Agric 79 :249–252 (1999)