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JOURNAL OFFOOD COMPOSITION

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Journal of Food Composition and Analysis 20 (2007) 13–18

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Original Article

Optimization of extraction methods for determination of the raffinosefamily oligosaccharides in leguminous vine peas (Pisum sativum L.)

and effects of blanching

J. Ekvalla,�, R. Stegmarkb, M. Nymana

aApplied Nutrition and Food Chemistry, Department of Food Technology, Engineering and Nutrition, Center for Chemistry and Chemical Engineering,

Lund University, P.O. Box 124, SE-221 00 Lund, SwedenbFindus R & D AB, SE-267 25 Bjuv, Sweden

Received 16 January 2006; received in revised form 22 June 2006; accepted 22 June 2006

Abstract

The extraction procedure for analysis of the raffinose family oligosaccharides (RFOs) in blanched leguminous vine peas was

optimized. For this purpose, 25 pea samples from different cultivars and of different maturity were chosen at random. The effect of

blanching was also studied in four cultivars. The yield of oligosaccharides was almost double on average when extracted with 50%

ethanol as compared with 80% ethanol. This was generally valid for all three oligosaccharides, with some exceptions for verbascose,

where 80% ethanol was shown to be as effective as 50% ethanol. Extraction at room temperature and at boiling temperature gave similar

yields, while 15min of extraction gave lower yields than 30 and 60min of extraction, which were similar. The loss of oligosaccharides

following blanching was 32% on average, with verbascose showing the highest loss (56.4%), followed by raffinose (28.4%) and stachyose

(16.3%). It can be concluded that the procedure used for extraction is of great importance when analysing the RFOs in immature peas,

and that there is considerable loss of these sugars during blanching, which is the process commonly used to prepare the frozen

commercial product.

r 2006 Elsevier Inc. All rights reserved.

Keywords: Raffinose; Oligosaccharides; Blanched leguminous vine pea; Pisum sativum L.; Green pea; English pea

1. Introduction

Legume seeds, commonly used by humans in WestAfrica and South America, are a good and inexpensivesource of protein. Legume proteins have also partiallyreplaced the traditional sources of protein in feeds, forexample for poultry and pigs (Kim et al., 2003). However,the presence of the flatulence-causing raffinose familyoligosaccharides (RFOs; raffinose, stachyose and verbas-cose) in legume seeds limits the nutritional value ofsoybean meal and other legumes (Leske et al., 1993). Agreat deal of research has therefore been carried outthrough breeding and processing methods in order toreduce the content of these oligosaccharides (Graham

e front matter r 2006 Elsevier Inc. All rights reserved.

a.2006.06.010

ing author. Tel.: +46222 4727; fax: +46 222 45 32.

ess: Jimmy.Ekvall@inl.lth.se (J. Ekvall).

et al., 2002; Ibrahim et al., 2002; Queiroz Kda et al., 2002;Vijayakumari et al., 1996). On the other hand, as a result ofrecent reports of the prebiotic properties of RFOs (Liyinget al., 2003; Trindade et al., 2003; Tzortzis et al., 2004),there is reason to revalue these oligosaccharides as foodingredients for humans in affluent societies.Raffinose (a-D-galactopyranosyl-(1-6)-a-D-glucopyra-

nosyl-(1-2)-b-D-fructofuranoside) and its higher homo-logues a-(1-6)-galactosides are linked to the glucosemoiety of sucrose. The content of RFO is low in unripepeas, and in a previous study on 29 cultivars of differentmaturity it was shown that the average amount (afterblanching) was 2.54 g kg�1 DM (Ekvall et al., 2005). RFOaccumulates in legume seeds during maturation, and isused as a carbon and energy source, especially duringgermination, during which the RFO content declines.Furthermore, there are indications that RFOs contribute

ARTICLE IN PRESSJ. Ekvall et al. / Journal of Food Composition and Analysis 20 (2007) 13–1814

to the protection of seeds against desiccation (Bailly et al.,2001; Blackman et al., 1992) and damage inflicted by coldtemperatures (Bachmann et al., 1994).

Due to the lack of the enzyme a-galactosidase in theupper intestinal tract of humans, RFOs escape digestion,and reach the colon as a substrate for luminal fermentationby the microbial flora. Different types of carbohydratesgive rise to different amounts and profiles of short-chain-fatty acids (SCFA) and microbial degradation of raffinosehas been reported to generate high proportions of lacticacid (Tzortzis et al., 2004) and butyric acid (Berggren et al.,1993; Goodlad and Mathers, 1990; Tzortzis et al., 2004).The latter, at least is considered to be protective againstcolonic diseases such as ulcerative colitis (Cummings, 1997;Scheppach et al., 1992) and possibly also colon cancer(Hague et al., 1993;Augeron and Laboisse, 1984; Archeret al., 1998).

During wet treatment such as blanching, boiling andautoclaving of vegetables, sugars such as sucrose, glucoseand fructose have been reported to be lost to varyingextents in different plant materials (Nyman et al., 1987).In addition, soaking and cooking for a long time havebeen shown to reduce the content of RFOs in legumeseeds (Nwinuka et al., 1997). For the processing ofleguminous vine peas (also commonly known as greenpeas or English peas) for commercial use, blanching iscommonly used to inactivate enzymes that catalyse lipidoxidation and cause unappealing flavours and changes inthe appearance and texture of the peas. To our knowledge,the extent to which this heat treatment leads to any lossesin RFO is, however, not known. Considering the lowconsumption of indigestible carbohydrates in westerncountries, a moderate intake of RFOs may be desirable.It should therefore be of interest to protect the naturalsources of RFO in our daily diet from being lost duringprocessing of foods.

For extraction of low-molecular weight carbohydrates(LMWCs) such as RFOs from plant material, the optimalsolvent would be water (Knudsen and Li, 1991). However,such a medium also facilitates interference betweencarbohydrates and other water-soluble substances such assome dietary fibre polysaccharides and proteins (Johansenet al., 1996). Thus, most of the conventional extractionmethods for LMWCs often use high concentrations ofalcohol (Dobrenz et al., 1993; Oku et al., 1998). Oneadvantage of using 80% ethanol is that polymers with anapproximate degree of polymerization (DP)420 areprecipitated and thus do not interfere with the alcohol-soluble LMWCs (Johansson, 1987). Such a treatment willalso inactivate enzymes such as invertase and a-galactosi-dase which, especially in plant material that has not beensubjected to pre-treatment such as blanching, could alterthe content of analytes present (Knudsen and Li, 1991).Previous studies, however, have shown that lower con-centrations of methanol and ethanol (50%) are betteralternatives when extracting RFOs from mature legumeseeds, and that higher concentrations (60–90%) yield less

oligosaccharides at room temperature (Johansen et al.,1996; Knudsen and Li, 1991; Kosson, 1992).Immature leguminous vine peas (Pisum sativum) are

green peas which have a low content of RFOs. These peasdiffer from mature legume seeds by their high watercontent (80% and 20%, respectively), and lower content ofstarch and RFOs (Daveby et al., 1993). The RFOs inmature peas may also be more efficiently enclosed withincells, as part of the development of cold hardiness(Kahraman et al., 2004). These differences in compositionmay alter, and in come cases obstruct, the conditions forextraction of RFOs.The aim of this work was to optimize the extraction

procedure of RFOs in order to be able to perform aquantitative measurement of the sugars present in greenpeas that have been blanched. Twenty-five samples fromdifferent cultivars and of different maturity were chosenrandomly from the sample storage at Findus R&D AB. Asblanching is the common method of producing green peasfor commercial markets, we studied the effect of thisprocess on the RFO content of four cultivars.

2. Materials and methods

2.1. Materials

2.1.1. Samples and sample preparation

Twenty-five leguminous vine pea samples (Pisum sati-

vum) from different cultivars and of different maturity werechosen at random. These were provided by Findus R&DAB (Bjuv, Sweden) which, besides shelling, also blanchedthe peas in a 40-L steam-heated water tub (at 97 1C for100 s). The peas were then frozen at �20 1C in packages of75 g and stored at this temperature.The effects of blanching were studied in four randomly

chosen cultivars. These peas were divided into two batchesand the raw peas were transported on ice for immediateanalysis of RFO (within 2 h), while the other batch wasexposed to blanching as described above.All samples (except for those in the blanching study)

were freeze-dried and milled to a particle size of less than1mm (Foss-Tecator, Hoganas, Sweden) before extraction.When effects of blanching were studied, raw and blanchedsamples were homogenized instead in water before extrac-tion, to create the same experimental conditions for thesesamples. The average moisture content of the raw peas was81%.

2.1.2. Extraction conditions

Extraction was carried out with two concentrations ofethanol (50% and 80% v/v), two temperatures (roomtemperature and boiling temperature) and three differentextraction times (15, 30 and 60min).For evaluation of the effect of the concentration of

ethanol, 10 pea samples (1–10) were chosen. Each samplewas extracted with both 50% and 80% ethanol at roomtemperature (21 1C) for 30min. The effect of temperature

ARTICLE IN PRESSJ. Ekvall et al. / Journal of Food Composition and Analysis 20 (2007) 13–18 15

was studied with 10 pea samples (11–20) extracted for30min in 50% ethanol at room temperature, or at boilingtemperature (83 1C). To study the influence of time, each offive pea samples (21–25) were extracted in 50% ethanol atroom temperature for 15, 30 and 60min.

2.1.3. Treatment of samples for studying effects of blanching

Four cultivars of green peas were shelled and eachcultivar was divided into two batches. One batch wastransported on ice for immediate extraction and analysis ofRFO (within 2 h), while the other batch was subjected toblanching and freezing as described above. The raw peas,and also the blanched and newly thawed frozen peas (35 g)were homogenized in 50mL water (Ultra-Turrax) beforeextraction of the RFOs with the optimized methodology intriplicate. After homogenization, ethanol (95% v/v) wasadded to a final concentration of 50% (v/v) and the RFOswere extracted for 30min at room temperature.

2.2. Method

Freeze-dried samples of 0.2 g, or 5.0 g of the homo-genized samples, were placed in test tubes, and arabinose(1mL, 5mgmL�1) was added as internal standard.Ethanol and water were added to obtain an ethanolconcentration of either 50% or 80%. The RFOs were thenextracted for 15, 30 or 60min at room temperature orboiling temperature, while being stirred every 10min on avortex. Cellular material was then removed by centrifuga-tion at 3000 g for 10min.

Polysaccharides (with a DP420) (Johansson, 1987) andproteins in the supernatant were precipitated with ethanol(80%, v/v) for 30min at �20 1C. For separation of theLMWCs from the precipitated compounds, the sampleswere centrifuged at 3000 g and 0 1C for 15min. Onemillilitre of the supernatant was then transferred to around flask, heated to dryness by evaporation (Heidolph,Germany) at 40 1C, and redissolved in 3mL water. Thesample was finally filtered through a 0.45-mm Millex-HVfilter (Millipore Corporation, Bedford, MA, USA) and a10-mL sample was analysed by high-performance anionexchange chromatography with pulsed amperometricdetection (HPAEC-PAD). NaOH (15–200mmol) was usedas the eluent with a flow rate of 1.0mLmin�1.

In the final procedure, used to study the effects ofblanching, samples were extracted in 50% ethanol for30min at room temperature.

2.3. Statistical evaluation

All analyses for studying the effects of blanching wereperformed in triplicate. For all statistical evaluations ofresults, a general linear model (ANOVA) was used,followed by a pairwise comparison using the method ofTukey. Mean values being compared were considered to besignificantly different at a P-value of less than 0.05. All

statistical evaluation was done with the statistical softwareMINITAB 13 (Minitab, 2000).

3. Results and discussion

3.1. Effect of extraction conditions on blanched peas

3.1.1. Ethanol concentration

The yield of RFOs from immature green peas was 97%higher on average with 50% ethanol than with 80%ethanol (Fig. 1a). The results were quite consistent for allsamples, and valid for all the oligosaccharides. In somecases, however, extraction of verbascose was almost asefficient with 80% ethanol. Higher yields have also beenachieved with lower ethanol concentrations in earlierstudies performed on plant materials containing highquantities of RFO, such as soybean concentrates (Shukla,1987), mature legume seeds (Kosson, 1992) and pea seeds(Johansen et al., 1996). The effectiveness of 50% ethanol asan extraction solvent for RFOs can most likely be ascribedto their higher polarity at the lower concentration ofethanol. In accordance with this, methanol, which is morehydrophilic than ethanol, has been reported to be an evenmore efficient solvent (Johansen et al., 1996). Furthermore,certain proteins may be denaturated by 80% ethanol, andit is possible that any precipitates that are formed mightsterically hinder RFOs from diffusing into the alcoholsolution. Since 80% ethanol is generally used as adeproteinization medium and also for precipitation ofpolysaccharides, it would be favourable to use this highethanol concentration during extraction as well, as thiswould be less time consuming. Based on the results ofprevious work performed on legume seeds with high RFOcontent (Johansen et al., 1996), it is probable that the yieldof extraction in 80% ethanol would be higher at anelevated temperature.

3.1.2. Temperature

Temperature had little or no effect on the extractionyield of RFOs in 50% ethanol. Although the average yieldof stachyose was slightly higher with extraction at boilingtemperature (4.76 g kg�1) than at room temperature(4.61 g kg�1) (Fig. 1b), the difference was not statisticallysignificant (P ¼ 0:068). This indicates that extraction ofRFOs, at room temperature, is complete after 30min in50% ethanol. A higher temperature will most likely speedup the extraction of RFOs in 50% ethanol. The problem ofcombining boiling temperature with ethanol as a solvent isthat the concentration of ethanol will be affected, anddifficult to adjust for upon precipitation of proteins.

3.1.3. Time

The average amount of the different oligosaccharidesextracted was lower in samples extracted for 15min than inthose extracted for 30 or 60min (Fig. 2), but only thedifference in amount of extracted verbascose was statisti-cally significant. No differences in yield were observed

ARTICLE IN PRESS

0

5

10

1550%80%

g/kg

DM

11 12 13 14 15 16 17 18 19 20

BPRT

g/kg

DM

5.0

10.050%80%

g/kg

DM

0.0

2.5

5.0

7.5

10.0 BPRT

g/kg

DM

50%80%

g/kg

DM

BPRT

g/kg

DM

1 4 7 9 10Sample

2 3 5 6 8

Verbascose

Sample

11 12 13 14 15 16 17 18 19 201 4 7 9 10Sample

2 3 5 6 8Sample

11 12 13 14 15 16 17 18 19 201 4 7 9 10Sample

2 3 5 6 8Sample

StachyoseStachyose

RaffinoseRaffinose

Verbascose

7.5

2.5

0.0

1.5

1.0

0.5

0.0

10.0

7.5

5.0

2.5

0.0

0.5

0.4

0.3

0.2

0.1

0.0

(a) (b)

Fig. 1. Content of raffinose, stachyose and verbascose (g kg�1 DM, corrected for losses of dry substance) in 2� 10 pea samples, either as (a) related to the

ethanol concentration (50% or 80%, v/v) when extracted for 30min at room temperature, or as (b) related to temperature (boiling point, BP, or room

temperature, RT) when extracted in 50% ethanol for 30min.

15.0 60.00

1

2

3

4

5

6 RaffinoseStachyoseVerbascose

g/kg

30.0Time (min)

Fig. 2. Average RFO content (g kg�1 DM, corrected for losses of dry

substance) in 3� 5 pea samples extracted in 50% ethanol for 15, 30 or

60min at room temperature.

J. Ekvall et al. / Journal of Food Composition and Analysis 20 (2007) 13–1816

between 30 and 60min of extraction. The study onextraction time supports the hypothesis that RFOs willbe fully extracted in 50% ethanol after 30min.

3.2. Effect of blanching on four cultivars of green peas

The loss of RFOs during blanching was on average24.2% (77.6), based on the dry weight content (Table 1).However, as there is a loss of approximately 10.4% dryweight in peas during the blanching procedure (Nymanet al., 1987), these values are underestimated. Thus, to beable to compare results with those obtained in fresh peas,the values for the blanched samples were compensated forthis loss. Taking this into consideration, the total loss ofRFOs increased to about 32% (range 27–42%, Fig. 3).Interestingly, the loss was unevenly distributed between

the different oligosaccharides. The average losses in percent of raffinose, stachyose and verbascose were 27.1

ARTICLE IN PRESS

Table 1

Average content of RFO (g kg�1 DM) in four cultivars of raw and

blanched peas. The third column gives the content per DM, when

compensated for losses of dry substance (10.4%) during blanching

Fresh Blancheda

Raffinose 2.95 2.18***

Stachyose 1.44 1.17

Verbascose 1.29 0.57***

Total RFO 5.67 3.92***

*Po0:05, **Po0:01, ***Po0:001.aCorrected for losses of dry substance into the blanching water.

Significantly different from the fresh pea samples according to Tukey’s

test.

Raw Blanch Raw Blanch Raw Blanch Raw Blanch0

1

2

3

4Raffinose

Stachyose

Verbascose

6 AD 6 Z 8 N 8 X

g/kg

DM

Fig. 3. RFO content (g kg�1 DM, corrected for losses of dry substance) in

four cultivars of raw and blanched peas.

J. Ekvall et al. / Journal of Food Composition and Analysis 20 (2007) 13–18 17

(74.3), 14.8 (78.9) and 55.6 (75.4), respectively. Con-trary to what was found, verbascose, being the largestmolecule, should have the lowest diffusion rate. The loss ofverbascose was, however, consistently higher than the lossof raffinose (P ¼ 0:0022) and stachyose (P ¼ 0:0003). Inaddition, the loss of stachyose was surprisingly low. Oneexplanation for this could be that verbascose is decom-posed to stachyose and galactose by residual enzymaticactivity, or by the elevated temperatures used duringblanching. However, there have been no reports ofverbascose being unstable at 97 1C, and if this were thecase, boiling would not be an efficient method forquantitative extraction of RFOs from legume seeds. Seedsof Dolichos lablab var. vulgaris have been reported to losemore verbascose than raffinose and stachyose uponsoaking at room temperature (Vijayakumari et al., 1995).This was explained by the authors as an indication of adifferent distribution of verbascose in the plant material.Furthermore, Conkerton et al. (1983) have identified amajor component of defatted soybean and peanut flourscomposed of glucose, fructose and galactose. After storageat 27 1C for several weeks, the compound was decomposedto a confirmed mixture of stachyose and sucrose, suggest-ing that the initial compound was a stachyose–sucrose

complex. To our knowledge, no such compound has beenreported in green peas, but could, if the compound isdecomposed upon blanching, explain the results of low lossof stachyose during blanching in the present study. Theextraction conditions were optimized on blanched peas,and although it seems unlikely, one cannot rule out that theraw peas may not be fully extracted. Assuming that, theloss of RFO following blanching would be even higher.

4. Conclusion

This study shows that RFOs can be extracted completelyfrom immature green peas at room temperature after30min in 50% ethanol. Thus, a higher temperature will notincrease the yield of RFOs when extracted in 50% ethanol.An increase in ethanol concentration to 80% reduced theyield at room temperature. One-third of the RFO contentis lost when green peas are blanched. The proportion ofverbascose lost following blanching was higher than forraffinose and stachyose.

Acknowledgements

This work was supported by the Swedish Agency forInnovation Systems (project no. P13652-1) and the SwedishResearch Council for Environment, Agricultural Sciencesand Spatial Planning (Project No. 50.0298).

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