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Radiation processing and functional properties of soybean (Glycine max)

Mrinal Pednekar a,n, Amit K. Das b, Rajalakshmi Va, Arun Sharma a

a Food Technology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400085, Maharashtra, Indiab Department of Food Engineering, CFTRI, Mysore 570020, Karnataka, India

a r t i c l e i n f o

Article history:

Received 25 June 2009

Accepted 28 October 2009

Keywords:

Radiation processing

Soybean

Tofu

Soya protein

Functional properties

a b s t r a c t

Effect of radiation processing (10, 20 and 30 kGy) on soybean for better utilization was studied.

Radiation processing reduced the cooking time of soybean and increased the oil absorption capacity of

soy flour without affecting its proximate composition. Irradiation improved the functional properties

like solubility, emulsification activity and foam stability of soybean protein isolate. The value addition

effect of radiation processing has been discussed for the products (soy milk, tofu and tofu fortified

patties) prepared from soybean.

& 2009 Published by Elsevier Ltd.

1. Introduction

In most developing countries, per capita consumption ofprotein is below the recommended level resulting in widespreadprotein calorie malnutrition. Soybeans with 40% protein and 20%oil have a great potential of solving the problem of protein caloriemalnutrition. Soy protein efficiently supplements cereal grainprotein, because it corrects the lysine deficiency of cereals. Insome cases, for example in corn, it also corrects tryptophandeficiency. Because of its quality, soybean protein can replaceanimal protein without a significant decrease in nutritive value(Tripathi and Misra, 2005). In fact, soy protein has an advantageover animal protein as it does not raise the serum cholesterolvalues (Fukushima, 2001) and hence, is useful for people sufferingfrom cardiovascular disorder (De Kleijn et al., 2002). Soybean alsohas a number of phytochemicals found to be effective in fightingosteoporosis (Anderson and Garner, 1997), obesity, cancer(Messina, 1999) and postmenopausal problems (Albertazzi et al.,1998). Soy milk is used in cases of lactose intolerance. However,soybean has a long cooking time, beany flavor, antinutritionalfactors like raffinose family oligosaccharides and proteaseinhibitors (Liener, 1994). Hence, processing of soybean is essentialfor better utilization. Most native proteins do not show desirablefunctional properties and modifications for improving the nutri-tional value and/or functional properties like protein solubility,foaming and gelling need to be induced (Chove et al., 2001). Suchmodifications imply changes in both protein structure andconformation at different levels by changing molecular composi-tion or size.

Radiation processing is an ecofriendly technology utilized fornutritional safety and security. It can be used for value additionsuch as elimination of flatulence factors (Machaiah et al., 1999;Machaiah and Pednekar, 2002), increase in starch and proteindepolymerization (Sharif and Farkas, 1993; Nene et al., 1975) andextractability.

We have conducted studies to evaluate the effects of radiationprocessing on functional properties like cooking time and theyield of soy milk and tofu. Potato patties prepared from tofuincorporation indicated good acceptability. Functional propertieslike solubility, foaming, emulsification capacity and gelling ofprotein extracted from soybean were also evaluated.

2. Materials and Methods

2.1. Irradiation

Soybean was purchased from a local market, cleaned andirradiated at 10, 20 and 30 kGy. Irradiation was carried out in aGamma Cell-5000 loaded with Co60(Board of Radiation and Isotope,Mumbai, India) at an effective dose rate of 152.3 Gy/min. Dose ratewas determined using standard Fricke dosimetry (Sehsted, 1970).Calibration was done by keeping the dosimeter vials in theirradiation chamber at different positions. Dosimeters wereanalyzed using a UV spectrophotometer. 9% variation in the dosedistribution was recorded.

2.2. Soybean flour

Whole soybean seeds were pulverized into fine flour (710 mm)with a mixer grinder and stored in self sealable polyethylene bags.

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Radiation Physics and Chemistry

0969-806X/$ - see front matter & 2009 Published by Elsevier Ltd.

doi:10.1016/j.radphyschem.2009.10.009

n Corresponding author. Tel.:+91 22 25595375.

E-mail address: mrinal1854@yahoo.co.in (M. Pednekar).

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2.3. Proximate analysis

Proximate analysis was carried out by standard AOAC methods(AOAC, 2007) for all samples including control after storing atroom temperature for 15 days. Lipid content of soy milk wasdetermined by the Folch method (Folch et al., 1957).

2.4. Determination of water and oil absorption capacity

For water and oil absorption capacities of soy flour the methodof Sathe et al., (1982) was followed. Soy flour (1 g) was mixedwith 10 mL of distilled water or 10 mL of edible oil and vortexed.The samples were then allowed to stand at room temperature(21 1C) for 30 min, and centrifuged at 5000 rpm for 30 min.The volume of the supernatant was measured in a 10 mLgraduated cylinder.

Calculation:Water Absorption Capacity (g/g)=(Initial volume of the water�

Final volume of water)Oil Absorption Capacity (g/g)=(Initial volume of the oil–Final

volume of oil)�0.91n(n=specific gravity of the oil used)

2.5. Cooking time

Overnight soaked seeds (control and irradiated) were pressurecooked for 2, 4, 6, 8 and 10 min (after first pressure release).Theseseeds were then allowed to cool at room temperature. The degreeof softness of the seeds was then measured using TextureAnalyzer (TA.XT Plus, Stable Micro System, Surrey, U.K.).

2.6. Preparation of soy milk

Soybean seeds (100 g) were soaked for 16 h in 300 mL water.Excess water was decanted off and 80 mL fresh water was addedand seeds were pressure cooked for 10 min after first pressurerelease. The cooked seeds were blended in the grinder by adding300 mL of water. Further this homogenate was filtered throughmuslin cloth, while filtering 600 mL of water was added. Thefiltrate was heated in a boiling water bath for 15 min.

2.7. Preparation of tofu

Tofu was prepared by a modified method of Nong Sun andBreene (1991). Calcium sulfate (0.2 M) was added to warm soymilk in 1:10 ratio and was heated at 70 1C for 10 min forcoagulation to occur. The coagulated curd was separated usingcheese cloth and the collected mass (tofu) was weighed.

2.8. Preparation of tofu–potato patties

Potatoes (500 g) were cooked in a pressure cooker and mashedwith pre-moistened bread slices (4 slices each weighing 22 g).This preparation (100 g) was mixed with tofu (15 g). This wasmolded desirably and a layer of semolina was applied to thesurface. This was then shallow fried with vegetable oil to a goldenbrown colour.

2.9. Sensory evaluation of tofu–potato patties

Sensory evaluation of the tofu–potato patties was carried outusing a 7-point hedonic scale ranging from ‘like very much’ to‘dislike very much’, with ‘neither like nor dislike’ as the midpoint(ASTM, 1996). The taste panel consisted of 50 members from

different sections of the Food Technology Division, BARC. Thepanelists belonged to age group of 25–55. The hedonic scale usedwas as follows:

7=like very much (LVM), 6=like moderately (LM), 5=likeslightly (LS),

4=neither like nor dislike (NLND), 3=dislike slightly (DS),2=dislike moderately (DM),

1=dislike very much (DVM).

2.10. Preparation of soy protein isolate (SPI)

Soybean protein isolate was prepared by the isoelectric pointprecipitation method. A modified method of Sathe et al. (1982) wasused to extract protein from the defatted soybean. Finely ground soyflour was defatted with petroleum ether (1:20, W/V). Defattedsoy flour was treated with 0.2% NaOH for 24 h (1:5, W/V). Aftercentrifuging (10,000 rpm, 30 min), the residue was re-extractedwith 0.2% NaOH and again centrifuged. Protein from both the pooledsupernatants was precipitated by adjusting the pH to 4 with 1 N HCl.The precipitate was dissolved in a minimum amount of 0.2% NaOH.This protein solution was dialyzed against distilled water for 72 hand freeze-dried.

2.11. Protein solubility

Protein solubility of SPI was studied from pH 1–12. The sample(100 mg) was dissolved in distilled water and pH was adjusted tothe required value using 0.1 N HCl or NaOH. The volume wasmade up to 20 mL. The protein solution was centrifuged at8000 rpm for 15 min. The protein in the supernatant wasestimated using the Kjeldahl method.

2.12. Determination of gelling capacity

Gelling was studied using a modified method of Sathe et al.(1982). SPI was dissolved in different concentrations of 10%, 12%,14% and 16% in phosphate buffer (pH 7.6) and 20% sugar solution.3 mL of the solution was dispensed in tubes and heated at 80 1Cin a water bath for 30 min. The tubes were cooled rapidly bykeeping in cold water and kept refrigerated overnight. Gelformation was observed by tilting the tubes and observing theflow of the solution.

2.13. Foaming properties

Whip ability and foam stability were studied according to themethod of Coffman and Garcia (1977) with slight modification. SPI(500 mg) was solubilized in 25 mL distilled water and pH wasadjusted to 7.0. The solution was whipped in a homogenizer(Polytron PT 2100, Kinematica, Switzerland) for 3 min at 10,000 rpmand poured into a 50 mL measuring cylinder. The total and drainagevolume were noted at 0, 1, 30 min, 1, 2, and 24 h intervals.

Whip ability and foam stability were calculated by thefollowing formula:

Whip ability=(Total volume–Drainage volume)/Initial volumeFoam stability=(Initial volume–Drainage volume)/Initial

volume�100

2.14. Emulsification

Emulsification was carried out according to the method ofBandyopadhyay and Ghosh (2002). 3 mL of 0.2% SPI solution at pH7 was homogenized with 1, 2 and 3 mL of peanut oil at12,000 rpm. 100 ml of emulsion was added to 4900 ml of 0.1%SDS and absorbance was read at 500 nm.

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2.15. Statistical analysis

All values are expressed as means and standard deviation of 6replicates with the exception of sensory evaluation. The resultsare the mean and standard deviations of the observed values.Differences were considered significant at po0.05 after perform-ing students’t’ test.

3. Results and discussion

3.1. Proximate analysis of soy flour

Proximate analysis of control and irradiated (10, 20 and30 kGy) soy flour was carried out and the results obtained arepresented in Table 1. It is evident from the table that there was nosignificant effect of radiation processing on the macronutrientcontent of the soybean.

3.2. Oil and water absorption capacity

Oil and water absorption capacity of flours prepared fromcontrol and radiation processed seeds was found to be within therange observed with various legume flours (Adebowale andLawal, 2004). Variation in the presence of polar and non-polarside chains among flour which bind to water or hydrocarbon sidechains of oil possibly make the difference in water or oil-bindingcapacity of flours. The data showed a non significant increasein water absorption capacity due to radiation processing(2.5770.082 in control to 3.0870.075 in 30 kGy treatedsamples). Water absorption capacity is important functionalproperty for flours as they swell and impart characteristics likebody thickness and viscosity. Radiation processing also led toincrease in oil absorption capacity of soy flours (1.5870.16 incontrol to 1.9770.18 in 30 kGy samples). Rahma and Mustafa(1988) have reported similar observations with irradiationof peanut flours. Dissociation and denaturation results in the fatand water absorption of treated proteins compared to nativeproteins (Siddharaju et al., 2002). Radiation processing mayhave resulted in protein unfolding leading to exposure of certainburied functional groups resulting in increased oil absorptioncapacity.

3.3. Determination of cooking time of soybean

Cooking time is a significant characteristic of legumes. It is thetime of boiling during which the legumes attain desirable softnesswherein at least 90% of the seeds are soft enough to masticate.Our results indicated a dose-dependent decrease in cooking timeof irradiated beans (Table 2). There was 30% decrease in cookingtime at 10 kGy which decreased to 60% at 30 kGy. The observedreduction in cooking time of irradiated soybean is consistent withreports of other workers (Rao and Vakil, 1985; Byun et al., 1993).

3.4. Qualitative analysis of soy milk

It was evident from the data that the protein concentration insoy milk from irradiated seeds nearly doubled (0.58270.08 incontrol soy milk to 1.04470.1 in 30 kGy treated soy milk).This can be correlated to better extractability of proteinsfrom irradiated seeds. Similar results were reported by Byunand Kang (1994).

3.5. Preparation of tofu

The results indicated that irradiation increased the yield oftofu from 5.27% in control to 11.36% in 30 kGy treated samples.The yield increased as the dose increased. This can be correlatedto the higher concentration of protein in the milk from irradiatedsamples. Byun et al., (1993) have observed a similar pattern.

3.6. Sensory evaluation

The data of the sensory analysis was analyzed and responses(%) were plotted against hedonic ratings. As seen in Fig. 1, tofu–potato patties, a tertiary product from soybean irradiated at20 kGy dose has scored maximum (40%) as ‘Like very much’,whereas 55% of the panel members rated the same as ‘Likemoderately’. It is also noteworthy that products from soybeanirradiated with other doses (10 and 30 kGy) scored more than theproduct prepared from control tofu which showed the lowestscores among all. This can be attributed to the fact that soybeanhas a typical beany flavor and taste which are also expected to bepresent in its secondary (control tofu) as well as tertiary (tofu–potato patties) products. Irradiation may have caused reduction ofthe beany flavor, as the scores for the products prepared fromirradiated soybean were always higher than those for the controlsoybean.

3.7. Functional properties of soy protein isolate

3.7.1. Protein solubility

The solubility profiles of SPI in water at different pH values arepresented in Fig. 2. At pH 4 and 5, which encompasses the

Table 1Proximate qualities of soybean.

Sample Protein % Lipid % Moisture% Ash % Carbohydrates%

Control 36.53571.704 22.70070.582 7.35570.077 0.78770.033 32.62370.293

10 kGy 37.59071.730 22.22570.523 7.34570.190 0.82770.009 32.01370.489

20 kGy 36.10371.595 21.60071.015 6.47572.27 0.82670.139 34.99670.528

30 kGy 36.6570.826 23.05070.825 7.2270.155 0.73370.094 32.34770.386

The values presented are mean7S.D. computed from 6 replicates.

Table 2Texturo-metric analysis of cooked soybean.

Force (Kg)a

Control 10 kGy 20 kGy 30 kGy

2 min 6.04771.252 3.83470.581 2.44871.282 2.50570.781

4 min 6.64671.923 3.33270.960 3.19770.964 2.03870.364

6 min 7.14070.764 3.32371.538 3.65270.977 1.31170.551

8 min 4.58170.942 1.71970.726 2.76170.133 1.52470.658

10 min 4.06470.526 2.02870.987 2.61371.174 1.15770.748

a S.D. computed from 6 replicates.

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isoelectric point of soy proteins, nitrogen solubility wassignificantly low for all the samples. This is in concurrence withthe data obtained by McWatters and Holmes (1979) for soy flour.As the pH increased above 5, nitrogen solubility increased steeplyreaching almost a plateau around neutral pH. The solubilityremained consistently higher in the case of protein extracted fromirradiated soybean. SPI extracted from 10 kGy treated samplesshowed higher solubility (100%) at most of the pH values.However, in the range encompassing the isoelectric point dosedependency was evident. The amino-acid composition,particularly at the protein surface influences protein solubility.Higher solubility is related to the presence of low numbers ofhydrophobic residues (Moure et al., 2006). Irradiation at this dosemust have led to formation of smaller peptides exposing

hydrophilic groups which increase the interactions ofhydrophilic amino acids with water molecules. Protein from the20 kGy treated sample showed a slightly different pattern. Itshowed the least solubility up to pH 3, but then increased afterisoelectric point showing higher solubility than the control in thealkaline range. Physical treatments such as high pressure havebeen found to unfold the protein resulting in exposure ofhydrophobic sites altering the functional properties of proteins(Molina et al., 2002). Irradiation at high doses has been found toalter protein structure. Such alterations must have changed theprotein solubility pattern at different doses due to alterations inamino acids at the surface as well as length of polypeptides(Bautista et al., 2006).

3.7.2. Gelation

In order to form gels, partial denaturation is desirable, sinceunfolding of the tertiary structure gives long chains withoutbreakage of covalent bonds. At the lowest concentration tried(12%), proper gels could not be obtained with control proteinwhile SPI from irradiated soybean showed gelling at 12%concentration. It is a known fact that factors like pH, ionicstrength, reducing agents and the presence of non-proteincompounds (carbohydrates) affect gelling. Sucrose is a mainingredient in commercial gel mixes and when gelling was done in20% sugar solution, even the control sample showed gelling at 12%concentration. The gels were of coagulant type indicating thepresence of more non-polar residues (Moure et al., 2006).Moreover the gels from treated samples were observed to behomogenous and smooth, when compared with control gels.

3.7.3. Foaming properties

There was no significant difference observed between controland irradiated sample at the concentration tried (2%). It is aknown fact that flexible protein molecules can have goodfoamability by reducing the surface tension.

3.7.4. Emulsification

It can be seen from Fig. 3 that the emulsification activity aswell as the stability of protein extracted from treated seeds wassignificantly higher compared to the control. Qi et al., (1997)observed an increased emulsification activity index (EAI) afterpancreatic hydrolysis of soy protein isolate, reaching maximum at15% degree of hydrolysis (DH). It indicates that at 15% DH, thehydrophilic and hydrophobic groups are well balanced. We

0

20

40

60

80

100

Appea

rance

Colour

Odour

Taste

After T

aste

Textur

e

Overal

l Acc

eptab

ility

Attributes

% A

ccep

tabi

lity

Product A

Product B

Product C

Product D

Product E

Fig. 1. Sensory evaluation of Tofu fortified potato patties. A–Control Product

(without Tofu); B–Control Tofu (mixed with Tofu obtained from un irradiated

Soya); C–Mixed with Tofu obtained from Soybean irradiated with 10 kGy dose;

D–Mixed with Tofu obtained from Soybean irradiated with 20 kGy dose; E–Mixed

with Tofu obtained from Soybean irradiated with 30 kGy dose.

0

20

40

60

80

100

% S

olub

ility

pH

Control 10 kGy 20 kGy 30 kGy

2 4 6 8 10 12

Fig. 2. Protein solubility of SPI.

0

100

200

300

400

500

600

700

0min

1 ml oil

EA

I val

ue

Control

10kGy

20kGy

30kGy

5min 0min 5min 0min 5min

2 ml oil 3 ml oil

Fig. 3. Emulsifying activity index (EAI) of SPI.

M. Pednekar et al. / Radiation Physics and Chemistry 79 (2010) 490–494 493

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observed that with 1 mL of oil, 10 kGy treated samples showedthe highest activity. With 2 mL oil, a significant dose-dependentincrease was observed (pr0.05). With 3 mL oil, the increase wasnot significant, however in all the irradiated samples theemulsification stability index (ESI) was significantly higher(pr0.05) than the control. Venkatesh and Prakash (1993) havereported increased emulsification activity after autoclavingof sunflower protein. A good correlation between surfacehydrophobicity and emulsifying property is reported (Wagnerand Gueguen, 1999). Emulsifying property depends on initialsolubility of the protein. As more protein dissolves in the system,more protein will be at the interface between the oil phaseand the continuous phase during emulsification. Our resultsindicated increased solubility of protein after irradiation. A closerelationship between emulsifying properties and solubility of soyproteins has been reported by McWatters and Holmes (1979).Formation of longer polypeptides are essential for this purpose.Our studies indicated that among the samples the 10 kGy sampleswere found to form more stable emulsions, suggesting dose-dependent breakdown of polypeptide chain to smaller units.

4. Conclusion

The results indicate that radiation processing up to 30 kGyreduced the cooking time without affecting the proximatecompositional properties. It was also helpful in increasing theprotein concentration in soy milk as well as tofu yield. Theacceptability of potato patties containing tofu from radiationprocessed seeds was better than the control. The functionalproperties like protein solubility, gelling and emulsificationwere found to be better in protein extracted from irradiatedbeans. These results clearly indicate the value addition effect ofradiation processing.

Acknowledgement

The authors are grateful to Dr. Sahayog N Jamdar (BARC) forhis invaluable help and expertise rendered in the preparation ofmanuscript and also for his technical suggestions.

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