Spray drying of the soybean extract Effects on chemical properties and antioxidant activity

LWT 41(2008)1521–1527

Spray drying of the soybean extract:Effects on chemical properties

and antioxidant activity

Sandra Regina Georgetti a ,Ru bia Casagrande b ,Cla

udia Regina Fernandes Souza a ,Wanderley Pereira Oliveira a ,Maria Jose

Vieira Fonseca a,?a

Department of Pharmaceutical Science,Faculty of Pharmaceutical Sciences of Ribeira ?o Preto –USP,Av.do Cafe

′s/n,CEP 14040-903,Ribeira ?o Preto,SP,Brazil

Department of Food and Drugs Technology,Agricultural Sciences Center –UEL,Brazil

Received 19September 2006;received in revised form 1September 2007;accepted 4September 2007

Abstract

Recent ?ndings that many human chronic diseases are associated with oxidative stresses have instigated the search for dietary antioxidants.Iso?avones,a special phenolic group found in soybean,have been found to act as antioxidant in some model systems.The aim of this work was the evaluation the effects of spray drying on the chemical and biological properties of soybean extract.The total polyphenol contents in the concentrated soybean extract (CSE)and spray dried soybean extract (SDSE)were determined by Folin–Ciocalteau method and HPLC was utilized for the quanti?cation of the genistein,active compound used as a chemical marker.The evaluation of antioxidant activity of CSE and SDSE was assessed by the hydrogen-donor ability to 2,2-diphenyl-1-picrylhydrazyl radical (DPPH d ).The results indicated that the inlet gas temperature showed signi?cant effect on the total polyphenol,protein and genistein contents of the dried extracts.The SDSE obtained this work showed high antioxidant activity,opening perspectives for its use as food additive and/or ingredient.

Keywords:Soybean extracts;Spray drying;Dried extract;Antioxidant activity

1.Introduction

Free radicals other reactive oxygen species are generated by exogenous chemicals or endogenous metabolic process in food systems or the human body.The radicals may cause oxidative damage by oxidizing biomolecules and results in cell death tissue damage.However,ingestion of antioxidative supplements,or foods containing antiox-idants,may reduce the oxidative damage on the human body (Wang,Yu,&Chou,2006).

An increasing body of epidemiological research has associated high soy intake with a lower risk for certain types of cancer.Soybean is a rich source of phenolic antioxidants with iso?avonoids being major components (McCue &Shetty,2005).They are classi?ed four groups:

aglycons,glucosides,malonylglucosides and acetylgluco-sides (Kim,Kim,Hahn,&Chung,2005).The chief iso?avonoid found in soybean is the genistein (McCue &Shetty,2005).

The iso?avones have been shown to reduce risks of cancer,inhibit the activity of tyrosine kinase,and decrease the risk of cardiovascular diseases by reducing the level of total cholesterol as well as low-density lipoprotein (LDL)cholesterol.Other health bene?ts include reductions in postmenopausal symptoms and risk of osteoporosis in women.Their possible ability to reduce cancer and cardiovascular risks is partially explained by their anti-oxidant activity (Ungar,Osundahunsi,&Shimoni,2003).Several studies have been shown that the biological activity of iso?avones may depend on the type of soy food and its processing and storage conditions (Rostagno,Palma,&Barroso,2005).The temperature,during drying and extraction,affects the activity and stability of compounds due to chemical and enzymatic degradation,

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losses by volatization or thermal decomposition.These latter factors have been suggested to be the main mechanism causing the reduction in polyphenol content (Moure et al.,2001).

Nowadays,the development of new technologies to obtain standardized dried plant and food extracts is an important research subject.The advantages of the dried extract over conventional liquid forms are lower storage costs,higher concentration,and stability of active sub-stances(Oliveira,Bott,&Souza,2006).The spray drying has been adopted for manufacture of powders due to its ability to generate a product with precise quality speci?ca-tions in continuous operation(Souza&Oliveira,2006). The aim of this work is the evaluation of the effects of processing parameters on free radical-scavenging activity, polyphenol,protein and genistein contents of the spray dried soybean extract.

2.Materials and methods

2.1.Chemicals

Methanol and Folin–Ciocalteau reagent were obtained from Merck(Darmstat,Germany).Thiobarbituric acid (TBA),genistein,bovine serum albumin,gallic acid and 2,2-diphenyl-1-picrylhydrazyl were purchased from Sigma Chemical Co.(St.Louis,MO,USA),quercetin was purchased from Acros(New Jersey,USA).All other chemicals were of reagent grade and were used without further puri?cation.

2.2.Preparation of the extractive solution

The extract was prepared with dried soybeans,Doko variety,acquired from a Brazilian producer of the Parana State.The dried soybeans were ground with a knife mill until a mean diameter of0.3mm.Before the iso?avone extraction,the ground soybean was defatted by mixing the soybean?our with isopropanol(1:3)in a vortex for2h (van Ruth,Shaker,&Morrissey,2001).The extract was ?ltered and the remained solvent evaporated.The defatted soy?our was submitted a second extraction using methanol80%added with HCl3M for2h in a stirred jacketed vessel with temperature controlled at601C (Franke,Custer,Cerna,&Narala,1994).The extractive solution was?ltered and concentrated in a rotary evaporator to25%of solids contents.The extraction procedure,including the fat removal stage,extraction method and solvent type used represents optimized one from a previous study developed earlier(data not shown).

2.3.Selection of the drying excipient and spray drying of the soybean extract

Preliminary spray drying tests were carried out in order to select an adequate drying excipient to be added to the soybean extract before spray drying.Drying adjuvants generally are added in order to improve both the dryer performance and product properties.In this work it was evaluated the effect of the adjuvants colloidal silicon dioxide(tixosil333),maltodextrin and starch on chemical properties and antioxidant activity of the product.The drying was carried out in a spray dryer model SD-05(Lab-plant,UK),with con-current?ow regime.The drying chamber has diameter of215mm and height of500mm. The main components of the system were the feed system of the soy extract,constituted by a peristaltic pump,a two ?uid atomizer(inlet ori?ce diameter of0.5mm)and an air compressor;a feed system of the drying gas,constituted by a blower and a air?lter;a temperature control system of the drying gas and a product collect system(cyclone).A scheme of this system is presented elsewhere(Oliveira et al., 2006).The gas humidity was measured at inlet and at exhaust of the SD by means of a Traceable s thermo-hygrometer.The evaluation of the dryer performance and the product properties were determined as a function of the kind of drying aids in concentration of2.5%relative to solids content.The inlet drying gas temperature and feed ?ow rate were kept constant at1501C and4g/min, respectively.The?ow rate of the drying air was?xed at 0.0227kg/s.The atomizing air feed?ow rate was?xed at 15L/min at a pressure of1bar.

The results of the preliminary runs showed a better drying performance and product quality with the use of the colloidal silicon dioxide as drying aid.Therefore,this product was selected as the drying aid,to be used in the subsequent tests.After the selection of the drying aid, several spray-drying runs were carried out in order to investigate the effects of the inlet drying gas temperature; 80,115and1501C(with and without the adding of the selected adjuvant)on drying performance and on product properties.Samples of the spray dried extract were removed for the determination of polyphenol,protein and genistein content and antioxidant activity using DPPH d assay.

2.4.Particle morphology of the soybean dried extract with different adjuvants

Photomicrographs were obtained in a scanning electro-nic microscope(DSM960Zeiss),with magni?cation of 3000?,in order to assess the morphological appearance of the dried product.

2.5.Polyphenol and protein contents

About0.1g of the spray dried soybean extracts were stirred with of80%ethanol for15min.The ethanol suspensions were centrifuged at1660g for10min and supernatant fraction collected into25mL of the volumetric ?ask.The precipitate was extracted with5mL of80% ethanol.Finally,the supernatant fractions were combined and the volume adjusted to25mL with deionized water.

S.R.Georgetti et al./LWT-Food Science and Technology41(2008)1521–1527 1522

The concentrated soybean extract(CSE)was directly diluted200times with ethanol50%.

Total polyphenol content was determined by Folin–Cio-cateau colorimetric method(Kumatzawa,Hamasaka,& Nakayama,2004).About0.5mL of the concentrated extract and of the spray-dried soybean extract were mixed with0.5mL of the Folin–Ciocalteau reagent and 0.5mL of10%Na2CO3,and the absorbance was measured at760nm after1h incubation at room temperature.Total polyphenol contents were expressed as mg/g(gallic acid equivalents).

The total protein present in CSE and SDSE was determinate by the method of Lowry,Brough,Farr,and Randall(1951).Bovine serum albumin was used as standard.The concentration of total protein in the extracts was calculated using the following linear equation based on the calibration curve(R?0.996).

C?0:2151:At0:00446.(1) 2.6.HPLC determination of the genistein content

Genistein content in CSE and SDSE was determined by reversed-phase HPLC analysis.The CSE was diluted?ve times in DMSO and,after,?ve times in the mobile phase.About0.1g of SDSE was dissolved with 5mL of DMSO and after,diluted more?ve times with the mobile phase.Aliquots of20m L were injected into the HPLC system.Genistein separation was performed em-ploying the SuperPac Sephasil C18(5m m)column, 250?4mm—attached to a pre-column.The mobile phase consisted of0.1%acetic acid in acetonitrile,0.1%acetic acid(35:65),1mL/min.Eluted iso?avonoid was detected by their absorbance at250nm.Quantitative data for genistein was obtained comparing with their standard (Georgetti,Casagrande,Vicentini,Verri-Jr,&Fonseca, 2006).

2.7.DPPH d assay

Besides the free radical scavenging activity tests per-formed for different SDSEs and CSE,the same test was also performed for quercetin.Quercetin was used as antioxidant reference with the purpose of comparing CSE and SDSEs activities.The choice was due to the potent antioxidant activity of this?avonoid(Marquele,Stracieri, Fonseca,&Freitas,2006),that shows a scavenging activity of manner dose dependent,which permitted to calculate the IC50.

2.7.1.Sample preparation

Quercetin was diluted to give media concentrations of 0.4,1.0,2.0,4.0and10.0m g/mL.About125.0mg of SDSE sample was diluted in DMSO to yield in the reaction medium a?nal concentration of 1.0mg/mL.The CSE was also weighted and diluted2,5,10and20times with DMSO.2.7.2.Hydrogen-donor ability to2,2-diphenyl-1-pcrylhydrazyl radical(DPPH d)

DPPH d is a stable free radical that reacts with compounds,which are able to donate a hydrogen atom. Thus,the hydrogen donating ability of the CSE and SDSEs,to DPPH d was determined from the change in absorbance at517nm,according to Blois(1958)method. For radical scavenging measurements aliquots of each sample were added in the reaction mixture containing1mL of0.1M acetate buffer pH 5.5,1mL of ethanol,and 0.5mL of100m M DPPH d in ethanolic solution.The change in absorbance was measured after15min at room temperature.All measurements were made in triplicate (Georgetti et al.,2006;Rodrigues et al.,2002).

2.8.Statistical analysis

One-way analysis of variance(ANOVA)followed by the Bonferroni’s test was used to examine the signi?cance of the different drying aids(colloidal silicon dioxide,mal-todextrin and starch)used in the preliminary tests.Results were presented as mean7standard error(SE)and con-sidered signi?cantly different when P o0.05.

The statistical signi?cance of the spray drying tempera-ture and presence(or absence)of colloidal silicon dioxide on the properties of the spray dried soybean extract (SDSE)(polyphenol,protein and genistein content and antioxidant activity)were tested by performing an ANO-VA,using the Fisher’s F-distribution.

3.Results and discussion

Several drying techniques can be utilized including freeze drying,spray drying,and spouted beds,with spray dryers being the most commonly used in the herbal processing industries.One of main reasons why spray drying has been adopted for manufacture of powders is its ability to generate a product with precise quality speci?cations in continuous operations(Souza&Oliveira,2006).

In the pharmaceutical and food industries,the correct selection of the drying excipients is an important step to guarantee the stability and the quality of?nished product. Thus,we evaluated the chemical properties and antiox-idant activity of the SDSEs added with different adjuvant, in order to analyze the adequacy of these excipients to generate a product with good chemical and biological properties.

The morphological analyses were carried out for the dried extracts obtained at a temperature of1501C added with colloidal silicon dioxide,maltodextrin or starch.Fig.1 presents typical SEM photomicrographs of the dried product magni?ed3000times.The photomicrographs show that the product is composed by irregular particles with wide size distribution,evidencing an agglomerating tendency.However,no signi?cant differences due the drying aid used could be detected by the SEM.micro-graphs.

S.R.Georgetti et al./LWT-Food Science and Technology41(2008)1521–15271523

Furthermore,the dried extracts with different excipients were characterized by the evaluation of the polyphenol,protein and genistein contents and their degradation (Table 1),and,also,by their antioxidant activity through DPPH d assays (Table 2).

The one-way analysis followed by the Bonferroni’s test showed signi?cant differences between the results obtained with different excipients (P o 0.05).The SDSE added with colloidal silicon dioxide showed lower degradation of the polyphenol content and lower reduction of the antioxidant activity unit (UA).The difference detected in the poly-phenol content and to UA of the product added with silicon dioxide was respectively 1.05and 1.11times higher than the product containing maltodextrin and starch.Moreover,the products obtained with colloidal silicon dioxide presented a better ?ow characteristic and lower agglomerating tendency (data not shown).Therefore,it was decided to investigate the effects of the spray drying temperature on total protein,polyphenol and genistein contents (with and without the colloidal silicon dioxide as drying excipient).Table 3presents the results obtained.ANOVA was performed for the experimental results presented in Table 3,in order to identify the variables presenting signi?cant effect on the polyphenol,protein and genistein content and antioxidant activity of the dried product.The ANOVA results revealed a signi?cant effect of the spray drying temperature on the measured responses;at a signi?cance level lower than 0.05for the protein and genistein contents.For the total polyphenol content,the temperature showed signi?cance only at a signi?cance level of 0.1.The increase in the spray drying

Fig.1.Morphology of the soybean dried extract with colloidal silicon dioxide (A),with maltodextrin (B)and with starch (C).

Table 1

Total polyphenol (Tph),protein (Tpr)and genistein (PA)contents in the CSE and SDE/adj and their degradations (D)Extracts

Tph (mg/g)D (%mg/g)Tpr (mg/g)D (%mg/g)PA (m g/g)D (%m g/g)CSE

31.3070.006–146.4770.002–41.2372.3–SDSE/colloidal silicon 11.0570.003*64.7045.9270.002*68.6511.3872.65*73SDSE/maltodextrin 10.8070.004*,]65.544.3770.004*,]69.7010.4372.19*74.77SDSE/starch

9.8770.004*,]

68.47

55.2270.004*,]

62.0

10.2072.23*

75.30

CSE:concentrated soybean extract,SDSE/adj:spray-dried soybean extract with aids (2.5%).*Signi?cant statistical difference compared to CSE (P o 0.05).#

Signi?cant statistical difference compared to SDSE/colloidal silicon (P o 0.05).

Table 2

Free radical scavenging activity measured by the DPPH d assay and antioxidant activity units (UA)of the CSE and SDSE/adj Extracts

DPPH d activity (%)UA

%Activity loss CSE

59.571.24793.3371.55–SDSE/colloidal silicon 55.770.78456.5570.89*38.80SDSE/maltodextrin 52.870.97*432.7871.15*#41.98SDSE/starch

50.370.89*,]

412.3071.10*#

44.73

CSE:concentrated soybean extract,SDSE/adj:spray-dried soybean extract with aids (2.5%).*Signi?cant statistical difference compared to CSE (P o 0.05).#

Signi?cant statistical difference compared to SDSE/colloidal silicon (P o 0.05).

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temperatures leads to a product with lower concentration of genistein and polyphenol contents.On the other hand, the total protein content was slight higher for the product dried at higher temperature.In general,the addition of the colloidal silicon dioxide to caused a slight reduction on the total protein and genistein contents,respectively at an alpha level of0.05and0.1.

For the extracts containing iso?avones the temperature may be one of the most important problem during food processing,as well as in sample handling and storage (Rostagno et al.,2005).The effect of the temperature on water activity of the dried product is also very important, but insuf?ciently understood yet.Although various oxida-tion reactions show a minimum rate of reaction at a certain water activity,in general,chemical reactions are slower as the water activity decreases(Goula,Adamopoulos,Chat-zitakis,&Nikas,2006).

Soybean and soybean products,containing various amounts of phenolic compounds,have been shown to possess antioxidant ability(Lin,Wei,&Chou,2006).They are important antioxidants agents,since they have hydro-xyl groups in rings A and/or B,and are thus capable of donating hydrogen to free radicals.The antioxidant activity of iso?avonoids may be related to the number and position of hydroxyl groups(Rostagno,Palma,& Barroso,2003).The genistein is a very potent antioxidant because it have a hydroxyl group in position5of the ring A and a free40-hydroxyl group in ring B(Ruiz-Larrea et al., 1997).However,the degradation of the phenolic sub-stances,including the iso?avone genistein may be asso-ciated with the occurrence of oxidative reactions or decomposition of thermolabile compounds induced by heat and also with the possibility of losses of volatile substances during spray drying.These results are agree-ment with Ungar et al.(2003)that indicated that daidzein and genistein are degraded when exposed to high temperature.

The antioxidant activity of the CSE and SDSE extracts were evaluated by the hydrogen-donor ability to2,2-diphenyl-1-picrylhydrazyl radical(DPPH d)method.The DPPH d assay is widely used for the measurement of free radical scavenging capacity in the food and phytopharma-ceutical products,and in the pharmacology and toxicology ?elds as well.Polyphenols have been reported to be potent hydrogen donators to the DPPH d radical because of their ideal chemical structure(Rice-Evans,Miller,&Paganga, 1997).

Signi?cant free radical scavenging capacity compared to the positive control(absent sample)was presented by the CSE tested in this work.The antioxidant activity showed a dose-dependent response,being the maximum antioxidant activity close to75%,in which a plateau effect was observed.

The hydrogen-donor ability to DPPH d radical was also evaluated for the spray-dried extracts obtained at tempera-tures of80,115and1501C,with and without addition of 2.5%of the drying carrier(tixosil333).Figs.2A and B present the experimental results.All dried extracts showed strong hydrogen donor capacity to DPPH d radical.The hydrogen sequestering capacity tended to be lower for the extracts added with tixosil333obtained at spray drying temperatures of115and1501C comparatively to product dried at801C(P p0.05).However,for the spray dried extract without addition of tixosil333,a statistically reduction in the product activity was only observed for the drying temperature of1501C.

In order to evaluate the losses of the antioxidant activity during spray drying,the units of antioxidant activity(UA) were calculated for the concentrated and dried extracts(dry basis).The UA was estimated in reference to the hydrogen sequestering activity of quercetin,used as a reference substance.One unity of antioxidant activity was de?ned as the amount of quercetin corresponding to a50%of activity through of the dose–response curve obtained with different concentrations(data not shown).The correlation coef?-cient,R,between quercetin concentration and%DPPH d reduced is of0.994and the IC50founded was of1m g/mL. Table4presents the results of UA and the corresponding activity losses for all soybeans extracts evaluated.ANOVA was also carried out for the UA results,in order to detect signi?cant effects of the spray drying parameters.As expected,ANOVA results showed a signi?cant effect of the spray drying temperature on UA,presenting an alpha level of0.05.The effect of the addition of colloidal silicon dioxide was signi?cant only at an alpha level of0.1.In general,lower UA values were obtained for the extracts

Table3

Total polyphenol,protein and genistein contents in the CSE,SDSE/adj and SDSE

Extracts Total polyphenol(mg/g)Total protein(mg/g)Genistein content(m g/g) CSE35.5070.06189.5570.00543.8373.10

SDSE/adj.801C11.7570.03051.9170.00411.1372.78

SDSE/adj.1151C11.7370.03351.8770.00311.0773.01

SDSE/adj.1501C10.9670.0555.4570.00510.2072.79

SDSE801C12.3070.0752.8970.00411.9772.89

SDSE1151C11.9270.0453.6770.00611.4372.91

SDSE1501C10.4970.03556.3870.00510.7472.95

CSE:concentrated soybean extract,SDSE/adj:spray-dried soybean extract with colloidal silicon dioxide(2.5%),SDSE:spray-dried soybean extract without colloidal silicon dioxide.

S.R.Georgetti et al./LWT-Food Science and Technology41(2008)1521–15271525

processed at higher drying gas temperature,with and without the addition of the adjuvant.Moreover,it can be observed that the extracts added with 2.5%of colloidal silicon dioxide presented higher UA,indicative of their possible protective effect against the degradation of the antioxidants compounds presented in the extracts during spray drying.

The experimental values of the DPPH d scavenging activity (%)and the antioxidant activity unit (UA)of the CSE and SDSE were ?tted as a function of the total polyphenol contents,in order to detect any relationship between the antioxidant activities of phenolics compounds.The correlation coef?cient between the DPPH d scavenging activity and total polyphenol contents of SDSE is R 2?0.67.A R 2of 0.588was observed for the correlation between UA and the total phenolic compounds (data not shown).These results suggest that approximately 60%of the antioxidant capacity of the spray dried soy extracts may be attributed partly to the contribution of the phenolic compounds.This unclear relationship between the anti-oxidant activity and the total phenolics may be explained in numerous ways.In fact,the total phenolics content does not incorporated all antioxidants present in the extract (Djeridane et al.,2006).This is the reason why samples with similar concentration of the total polyphenol,may vary in their scavenging activity.In addition,the occur-rence of synergism between the chemical compounds in the whole extract makes the antioxidant activity dependent of the chemical structure of the antioxidant substance and interaction between them,besides its concentration.This is the reason why plant extracts with similar concentrations of total phenolics,may vary signi?cantly in their antiox-idant activities.Therefore,in some cases the combined effect of soy iso?avones might be better than the effect of any single iso?avone compound (Sharma &Sultana,2004).4.Final remarks

Spray drying is the most popular drying technology used within the food,chemical and pharmaceutical industries for production of dry particles form liquids.The modern industries empathises the production of high-quality product at minimum cost (low investment and running cost).Due to the complexity of the drying mechanisms and the diversity of materials intended for drying,new drying technologies have been developed.In recent paper,Mujundar and Huang (2007)summarize some recent advances and general trends in drying technologies of industrial interest as well as the potential application areas for the emerging hybrid drying technologies.The versatility and importance of hybrid drying technologies is also commented by Chou and Chua (2001).However,the comparison of the results reported in this paper with these emerging drying technologies is not possible hitherto,mainly due to distinct processing conditions used,remain-ing as a fascinating subject for a future research.

The results reported in this article demonstrated that the correct selection of the drying excipient is important to guarantee the chemical and nutritional quality of the spray dried soybean extract.In this study was demonstrate that the SDSE obtained with colloidal silicon dioxide showed high polyphenol and genistein contents and antioxidant properties,measured through its H-donating capacity to free radical stable DPPH d .The chemical and antioxidant

D P P H ? r e d u c e d (%)

samples

Fig.2.Free radical scavenging activity of the soybean spray dried extracts

with (A)and without (B)the drying carrier:

?801C;?1151C,and ?1501C (*signi?cant,P p 0.05comparatively to product dried at 801C).

Table 4

Antioxidant activity units by CSE and SDSE Extracts UA %Activity loss CSE

813.4071.60–SDSE/adj 801C 541.6470.4527.48SDSE/adj 1151C 494.6770.5034.06SDSE/adj 1501C 463.4471.037.92SDSE 801C 514.4070.9030.65SDSE 1151C 484.0071.1234.95SDSE1501C

448.8071.08

39.78

CSE:concentrated soybean extract,SDSE/adj:spray-dried soybean extract with colloidal silicon dioxide (2.5%),SDSE:spray-dried soybean extract without colloidal silicon dioxide.

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activities of SDSE showed to be affected by the spray drying temperature.However,the effect was lower than the losses on total polyphenol and genistein contents(67%loss of the polyphenol and75%of the initial genistein contents).Nevertheless,the SDSE obtained this work showed high antioxidant activity,opening perspectives for its use as a natural and multifunctional dietary food additive or supplements.

Acknowledgment

We would like to express our gratitude to FAPESP and CNPq for the?nancial support.

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