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Anaerobe 16 (2010) 380e386

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Anaerobe

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Clinical microbiology

Antioxidant activity in vitro of the selenium-contained protein from theSe-enriched Bifidobacterium animalis 01

Qian Shen 1, Bowen Zhang 1, Rihua Xu, Yang Wang, Xuelong Ding, Pinglan Li*

College of Food Science and Nutritional Engineering, China Agricultural University, 17# Qinghua East Road, Beijing 100083, China

a r t i c l e i n f o

Article history:Received 11 September 2009Received in revised form4 June 2010Accepted 13 June 2010Available online 23 June 2010

Keywords:Bifidobacterium animalisSelenium-contained proteinSeleniumAntioxidant activity

* Corresponding author at: College of Food SciencChina Agricultural University, No. 17 Qinghua Dong R100083, China. Tel.: þ86 10 62737664; fax: þ86 10 6

E-mail address: lipinglan420@126.com (P. Li).1 Authors Qian Shen, Bowen Zhang equally contrib

1075-9964/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.anaerobe.2010.06.006

a b s t r a c t

Several studies indicated that bifidobacteria possessed strong antioxidant activity. In present study, theantioxidant activities of Bifidobacterium animalis 01 proteins were evaluated using six assays, namely,linoleic acid preoxidation assay, erythrocyte hemolysis assay, 1,1-diphenyl-2-picrylhydrazyl (DPPH)assay, reducing power assay, hydroxyl (�OH) and superoxide radicals (�O2

�) assays, in which the last twoassays were measured by electron spin resonance (ESR). There were two kinds of B. animalis 01 proteinsin this study, the regular B. animalis 01 protein (Pro-CK) and the B. animalis 01 selenium-containedprotein (Pro-Se). Both Pro-CK and Pro-Se showed concentration dependent antioxidant activity in DPPHassay, reducing power assay and erythrocyte hemolysis assay. All results of six assays indicated that theantioxidant activity of the B. animalis 01 protein was improved remarkably after selenium was incor-porated. The antioxidant activity of Pro-Se increased with the increase of selenium content in Pro-Sesuggesting selenium played a positive role in enhancing the antioxidant activity of B. animalis 01 protein.Moreover, organic selenium was more effective than inorganic selenium on enhancing the hydroxylradical scavenging ability of B. animalis 01 protein.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Oxidative modification of DNA, proteins, lipids and small cellularmolecules caused by reactive oxygen species (ROS) played a role ina variety of common diseases and age-related degenerative condi-tions [1]. Under normal circumstances,1e5% of themolecular oxygenin the cells createsactiveOxy-free radicals inmanydifferentways, butthe human bodies possess enzymatic and non-enzymatic anti-oxidative mechanisms and minimize the generation of reactiveoxygen species to levels that are not harmful to the cells. When thegeneration of the active Oxy-free radical is overgrown or the freeradical scavenging capability is weakened for some reason, manydegenerative diseases, such as brain dysfunction, cancer, heartdiseases and declination of the immune system, could be caused bythe excessive of free radicals [2]. With the aging of the human body,the biosynthesis of enzymes which scavenge free radicals isdecreasing. Thus, excessive free radicals react with biologically activesubstances such as lipids, protein and DNA to cause cell membraneinjury, protein denaturation and wrong DNA replications.

e & Nutritional Engineering,oad, Haidian District, Beijing2737604.

uted to this work.

All rights reserved.

Selenium, akindof essentialminorelements forhumanbody,hasbiological effect in many ways. In the first half of the 20th century,selenium was considered an undesirable element in human bodydue to its toxicity. In the secondhalf of the20th century, Schwarz andFoltz reported that seleniumat very lowdietary concentrations is anessential nutrient [3]. Recently, selenium has been reported topossess strong free radical scavenging ability and canprotect the cellmembrane, preventing cells from malignant transformation [4].Furthermore, the antioxidant activity of the scavenger wasenhanced when it contained selenium. It is reported that seleniumenhanced the antioxidant activity of the protein extracted from theSe-enriched mushroom [5]. On the other hand, the carcinostaticactivities of selenium compounds have been shown concentrationdependent and several studieshave indicated that the concentrationof selenium may play an important role in selenium catalysis andtoxicity [6]. Another study reported that selenium compounds atlow concentrationmay have protective anticarcinogenic properties,whereas at higher concentration theycould bepossibly carcinogenic[7].Hence,whether selenium isbeneficial orharmful tohumanbodyis determined by its concentration.

Since bifidobacteria was isolated by Doctor Tissier, the researchon it has never stopped, especially on its physiological function [8].Bifidobacterium longum ATCC 15708 and Lactobacillus acidophilusATCC 4356 has been reported to have strong inhibitory ability onthe plasma lipid peroxidation [9]. Lin and Yen found out that the

Q. Shen et al. / Anaerobe 16 (2010) 380e386 381

cell-free extraction of the L. acidophilus and B. longum showedstrong inhibitory effect on linoleic acid peroxidation [10]. In addi-tion, a kind of soymilk fermented with bifidobacteria was reportedthat it could scavenge superoxide anion and possessed hydrogenperoxide scavenging ability [11]. However, few reports dealing withwhich substance in bifidobateria was responsible for the antioxi-dant activity could be found to date. In present study, we isolatedPro-CK, trying to evaluate its antioxidant activity. What is more, asa follow-up to our previous characterization on the distribution ofselenium in Bifidobacterium animalis 01 which indicated most ofthe organic seleniumwas found in the protein fraction [12], we alsoisolated Pro-Se and comspared the difference of the antioxidantactivity between Pro-CK and Pro-Se, trying to disclose the impor-tance of the selenium in the antioxidant process.

2. Materials and methods

2.1. Chemicals and reagents

Xanthine, xanthine oxidase, linoleic acid, Tween 20, trichloro-acetic acid (TCA), butylated hydroxytoluene (BHT), DPPH (1,1-diphenyl-2-picrylhydrazyl), thiobarbituric acid (TBA) and DMPO(dimethyl pyridine N-oxide) were purchased from Sigma-Aldrich(Shanghai, China). Tris was obtained from Roche (China). DETAPAC(diethylenetriamine penta-acetic acid), ascorbic acid, ferroussulfate, hydrogen peroxide, sodium hydroxide and ammoniumsulfate which were purchased from Beijing Chemicals Co. Ltd.(China) were of research purity grade.

2.2. Microbial strains and culture conditions

B. animalis 01 was obtained from College of Food Science &Nutritional Engineering, China Agriculture University, which wasisolated from a healthy centenarian in GuangXi of China. B. animalis01 was cultured on the improved MRS broth medium which wascomposed of peptone (10.0 g/L), beef extract (10.0 g/L), yeast extract(5.0 g/L), K2HPO4 (2.0 g/L), triammonium cetrate (2.0 g/L),sodium acetate (5.0 g/L), glucose, tween80 (1.0 ml/L), MgSO4$7H2O(0.58 g/L), MnSO4$H2O (0.25 g/L), corn steep liquor (3.0 g/L) andcysteine hydrochloride (0.3 g/L). After 6 h, sodium selenite ofdifferent concentration (0, 2.5, 5.0, 8.0,10.0 mg/ml) was added to themedium [12]. The Se-enriched bifidobacteria weremarked with CK,Se2.5, Se5.0, Se8.0 and Se10.0 respectively, according to the sodiumselenite concentration. The Se-enriched bifidobacteria wereremoved by centrifugation after cultured with sodium selenite foranother 12 h, and then it was preserved in the dark at roomtemperature after freeze drying (�50 �C, under vacuum) by vacuumfreeze drier (FD-1B-50, Beijing Boyikang Instruments Co. Ltd).

2.3. Preparation of B. animalis 01 proteins

The protein was isolated according to the method of Zhang [12].3.0 g of each kind of dry bacteria sample (CK, Se2.5, Se5.0, Se8.0,Se10.0) was dissolved in cold sodium hydroxide solution (0.25 M),the bacteria samples were broken by ultrasonication and incubatedat 50 �C in DK-8B water bath (Shanghai Jinghong InstrumentsCo. Ltd, China) for 2 h. The supernatant was obtained by filtrationand the residue was repeated twice with 50 ml of NaOH (0.25 M).Then ammonium sulfatewas added to the supernatant tomake 95%saturated solution which was followed by keeping it overnight at4 �C. Protein was precipitated using Thermo Scientific CL 10 centri-fuge (Thermo Fisher Scientific, US) at 6000 rpm for 30 min at 4 �C.The resulting precipitate was then dissolved in 10.0 ml of Tris-HCl(pH¼8.0, 50mM). This solutionwaspassed through0.22mmsyringefilter (13 mm, Millipore US) and dialysed against 1.0 L of Tris-HCl

(pH ¼ 8.0, 50 mM) using a membrane (Shanghai OuweiDa Instru-ments Scientific Co. Ltd, China)with3500molecularweight cutoff at4 �C three times to remove ammonium sulfate. Finally, the solutionin dialysis bagwas lyophilised using vacuum freeze drier (FD-1B-50,Beijing Boyikang Instruments Co. Ltd). Each kind of protein wasmarked with Pro-CK, Pro-Se2.5, Pro-Se5.0, Pro-Se8.0 and Pro-Se10.0. The selenium content in each protein sample has beendetermined to be 0, 0.269, 0.325, 0.498 and 0.641 mg/L respectivelyby hydride generation-atomic absorption spectrometry (HG-AAS,haiguang analytical Co., China) in our previous study [12].

2.4. Hydroxyl radical scavenging assay

The hydroxyl radical scavenging activity was measured by themodified method of Liu et al. [13]. Hydroxyl radical was generatedby the addition of ferrous ion to a reaction mixture which containsphosphate buffer. The adduction activity of the hydroxyl radicalcaused the decrease of absorbance at 510 nmmeasured by UV-2100spectrophotometer (UNICO, US). If hydroxyl radical scavenger wasadded to the mixture, part of the hydroxyl radical can be scavengedand the absorbance decrease at 510 nm will reduce. In order toconduct the rest of assays with a proper sample concentration,two concentration groups were involved in this assay-the highconcentration group (5.00 mg/L) and the low concentration group(1.00 mg/L). 1.0 ml sample was added to tubes containing 1.0 mlphosphate buffer (pH ¼ 7.4, 0.75 M), 0.5 ml phenanthroline(1.0 mM), 3.5 ml deionized water, 1.0 ml ferrous sulfate (7.5 mM)and 1.0 ml H2O2 (0.01%, v/v). The tube was incubated at 37 �C for90 min. In the H2O2 control, the sample was substituted withdeionized water. In the normal control, both the sample and theH2O2 were substituted with deionized water. In the positivecontrol, the sample was substituted with ascorbic acid. The capa-bility of scavenging hydroxyl radical was calculated using thefollowing equation:

Scavengingeffectð%Þ ¼h�

Asample�Anormal

�.�AH2O2

�Anormal

�i

�100

2.5. Inhibition of linoleic acid peroxidation

The inhibitoryeffect of theB. animalis 01 proteinswas determinedaccording to the thiobarbituric acid method [14]. Linoleic acid wasprepared in Tween 20, a slightmodification according to themethodof Grossman and Zakut [15]. In brief, the linoleic acid emulsion waspreparedbyaddition of 0.1ml linoleic acid to 19.7ml deionizedwatercontaining 0.2 ml Tween 20. In 1.0 ml linoleic acid emulsion, 0.2 mlferrous sulfate (0.01%,w/v), 0.2mlH2O2 (0.02%, v/v) and1.0ml sample(1.00 mg/L) was dissolved followed by incubation at 37 �C for 12 h.After that, 0.2 ml trichloroacetic acid (4.0%, w/v) was added toterminate the reaction, and then the solution was heated at 100 �Cwith2.0ml TBA (0.8%,w/v) and 0.2ml BHT (0.4%,w/v) for 30min. Theabsorbance was measured by UV-2100 spectrophotometer (UNICO,US) at 532 nm. In the control, the samples were substituted withphosphate buffered saline (PBS) buffer (pH¼ 7.4, 0.02 M) which wascomposedofNa2HPO4 (0.02M) andNaH2PO4 (0.02M). The capabilityof inhibitory effect was calculated using the following equation:

Inhibitory effectð%Þ ¼�1� Asample532=Acontrol532

�� 100

2.6. DPPH radical scavenging activity assay

DPPH radical scavenging activity of the B. animalis 01 proteinswas evaluated as described by Blois with some modifications [16].

Q. Shen et al. / Anaerobe 16 (2010) 380e386382

0.1 mM of DPPH in 95% EtOH (v/v) was prepared and 1.0 ml sample(1.00 mg/L) was added to 3.0 ml of such solution. The mixture wasallowed to stand in dark for 30 min. The absorbance was measuredat 517 nm. In the blank control, the sample was substituted withdeionized water. In the positive control, the samplewas substitutedwith BHT. The DPPH scavenging activity was calculated by thefollowing equation:

Scavenging effectð%Þ ¼�1� Asample517=Acontrol517

�� 100

2.7. Reducing power assay

The reducing power of B. animalis 01 proteins was determinedaccording to the method reported by Yen and Chen [17]. 1.0 ml ofsample (1.00mg/L) in PBS buffer (pH¼ 7.4, 0.02M) was mixed with1.0 ml potassium ferricyanide (1.0%, w/v). After this, the mixturewas incubated at 50 �C for 20 min. Then 1.0 ml of trichloroaceticacid (10.0%, w/v) was added to the mixture to terminate the reac-tion. After that, the solution was mixed with 0.4 ml ferric chloride(0.1%, w/v) for 10 min. The absorbance was measured at 700 nm.Increased absorbance of the reaction mixture indicated increasedreducing power.

2.8. ESR measurements

2.8.1. ESR measurement parameterMeasurement of superoxide anion and hydroxyl free radical

scavenging activity was based on the ESR techniques. Measure-ments were performed at room temperature on an ER200D-SRCspectrometer (Bruker, Germany). When it was used for superoxideanion scavenging assay, the ESR parameters were set at frequency,9.4386 GHz; modulation frequency, 100 KHz; width 0.79 � 0.1 mT;power, 10 mW; time constant, 0.1 s; sweep time, 2 min; amplitude,200. When it was used for hydroxyl radical scavenging assay, theESR parameters were set at frequency, 9.4386 GHz; modulationfrequency, 100 KHz; width, 0.2 � 1 mT; power, 10 mW; timeconstant, 0.1 s; sweep time, 2 min; amplitude, 50.

2.8.2. Superoxide anion assayDMPO was used as a spin trap, the typical reaction between

DMPO and superoxide anion forms into a stable and ESR-detectableradical complex [18]. If superoxide anion scavenger was addedbefore adding DMPO, the typical ESR spectrum of DMPOeOOHadducts would be reduced. The reaction of xanthine and xanthineoxidase was used to produce superoxide anion [19]. The reactionmixture contains 5 mL DMPO (1.0 M), 5 mL DETAPAC (4.0 mM), 5 mLxanthine (0.75 mM), 5 mL xanthine oxidase (0.75 U/ml) and 5 mLsample (1.00 mg/L). In the control, the sample was substituted withPBS buffer. The capability of scavenging superoxide anion wascalculated using the following equation:

Scavenging effectð%Þ ¼ ½ðHo �HsÞ=Ho� � 100

Where Ho was the amplitute of the second peak in the ERS spec-trum for the control, Hs was the amplitute of the second peak in theERS spectrum for the sample.

2.8.3. Hydroxyl radical assayThe hydroxyl free radical detection was based on the specific

reaction between �OH and DMPO, which forms into a stable andESR-detectable DMPOeOH adduct. The reaction mixture contains5 mL DMPO (1.0 M), 5 mL FeSO4 (0.1 mM), 5 mL H2O2 (10 mM) and5 mL DETAPAC (4.0 mM) in the presence of samples (1.00 mg/L) ina final volume of 25 mL. In the control, the sample was substituted

with PBS buffer. The capability of scavenging superoxide anion wascalculated using the following equation:

Scavenging effectð%Þ ¼ ½ðHo � HsÞ=Ho� � 100

Where Ho was the amplitute of the second peak in the ERS spec-trum of the control, Hs was the amplitute of the second peak in theERS spectrum of the sample.

2.9. Inhibition of erythrocyte hemolysis

The in vitro inhibition of rat erythrocyte hemolysis by B. animalis01 proteinswas evaluated according to the procedures described byNg, Liu and Wang with slight modifications [20]. The blood samplewas obtained frommice carotid and collected in a tubewith sodiumcitrate (3%, w/v). Then it was centrifuged using Thermo Scientific CL10 centrifuge (Thermo Fisher Scientific, US) at 3000 g for 10 min toseparate erythrocytes from the plasma. After being washed threetimes by centrifugation with PBS buffer (pH ¼ 7.4, 0.02 M), theerythrocytes were made into a suspension (1.25%, w/v) for the test.The erythrocyte hemolysis induced absorbance increase of thehemoglobin at 540 nm. Pro-CK and Pro-Se8.0 were dilutedrespectively to 2.00, 1.00, 0.50 and 0.25 mg/mL with Tris buffer(pH ¼ 7.4, 0.05 M). The sample was incubated at 37 �C for 30 minwith 2 ml erythrocyte suspension and 1.0 ml H2O2 (2.5 mM). Later,the mixture was incubated in icewater bath (0 �C) to terminate thereaction immediately. The absorbance of the supernatant wasmeasured at 540 nm. In the normal control, the H2O2 and samplewas substituted with Tris buffer. In the H2O2 control, the samplewas substituted with Tris buffer.

The hemolysis extent of the erythrocyte was calculated usingthe following equation:

Hemolysis extentð%Þ ¼�AH2O2

or Asample=AH2O2

�� 100

The inhibition rate of the protein was calculated using thefollowing equation:

Inhibition rateð%Þ ¼h�

AH2O2� Asample

�.�AH2O2

� Anormal�i

� 100

2.10. Statistical analysis

Every experiment was conducted in triplicate. All statisticalanalyses were performed with SPSS10.0 (SPSS Inc., Chicago, IL). Thesignificance was established at p < 0.05.

3. Results

3.1. Hydroxyl radical scavenging assay

Pro-Se2.5, Pro-CK and sodium selenite with Pro-CK exhibitedevident hydroxyl free radical scavenging activity (Table 1). Thescavenging effects of Pro-Se2.5 and Pro-CK were 28.1% and 14.6%respectively for low concentration group. The scavenging activity ofthe high concentration group was significantly (p < 0.05) higherthan that of the low concentration group. The Pro-Se showeda significantly (p< 0.05) higher scavenging ability than Pro-CK bothin low concentration group and high concentration group. It indi-cated that the hydroxyl free radical scavenging ability of the proteinwas improved after it contained selenium. Moreover, the scav-enging ability of the Pro-Se2.5 was higher than that of sodiumselenite with Pro-CK. It suggested that organic selenium was more

Table 1The activity of scavenging hydroxyl radical of B. animalis 01 protein.

Sample Absorbance at 510 nm Scavenging effect of �OH (%)

Normal Control 0.386 � 0.014 N/AH2O2 Control 0.215 � 0.011 N/A

Low concentration group Vc (0.1 mg/L) 0.246 � 0.009 18.1 � 0.9 fPro-CK (1.00 mg/L) 0.240 � 0.011 14.6 � 0.2 gNa2SeO3 (0.269 mgSe/L) 0.219 � 0.004 2.34 � 0.11 iPro-CK (1.00 mg/L)þNa2SeO3 (0.269 mgSe/L) 0.252 � 0.007 21.6 � 0.5 ePro-Se2.5 (0.269 mg/L of Se; 1.00 mg/L of protein) 0.263 � 0.010 28.1 � 0.7 d

High concentration group Vc (0.5 mg/L) 0.315 � 0.011 58.5 � 0.4 bPro-CK (5.00 mg/L) 0.286 � 0.008 41.5 � 0.9 cNa2SeO3 (1.35 mgSe/L) 0.222 � 0.016 4.09 � 0.75 hPro-CK (5.00 mg/mL)þNa2SeO3 (1.35 mgSe/L) 0.287 � 0.005 42.1 � 0.3 cPro-Se2.5 (1.35 mg/L of Se; 5.00 mg/mL of protein) 0.328 � 0.009 66.1 � 0.6 a

Values are the means � standard deviations (n ¼ 3). Values followed by different letters in same column are significantly different (p < 0.05).

Q. Shen et al. / Anaerobe 16 (2010) 380e386 383

effective than inorganic selenium on enhancing the hydroxyl freeradical scavenging ability of the protein.

3.2. Inhibition of linoleic acid peroxidation

Lipid peroxidation is an oxidative deterioration process ofpolyunsaturated fatty acids which is induced by free radical [14]. Inthis assay, all the protein samples showed evident effect on inhi-bition of linoleic acid oxidation. The highest inhibitory effect was64% created by Pro-Se10.0 (Fig. 1). The same concentration of theprotein with different selenium content give rise to the inhibitoryability increased with the increase of selenium content in theprotein. That is to say, the inhibitory ability of the protein on linoleicacid peroxidation was improved after it contained selenium.

3.3. DPPH radical scavenging activity assay

Pro-CK and Pro-Se2.5 exhibited concentration dependent scav-enging effect on DPPH radical, but lower than that of BHT (Fig. 2).The scavenging effect increased with the increase of concentrationup to 0.4 mg/L and then leveled off with further concentrationincrease. The highest DPPH radical scavenging effects were 17% and21% for Pro-CK and Pro-Se2.5 respectively. At the same concen-tration, Pro-Se2.5 showed a higher scavenging effect than that ofPro-CK, but the difference was nonsignificant. The significant(p < 0.05) difference of the scavenging effect between the Pro-Seand Pro-CK was presented in Fig. 3. In addition, the scavengingability increased with the increase of the selenium content in theprotein.

Pro-Se5.0

Pro-CK

Pro-Se2.5

Pro-Se8.0

Pro-Se10.0

0

20

40

60

80

0.0 0.2 0.4 0.6 0.8Se Concentration (mg/L)

In

hib

itio

n o

f P

ero

xid

atio

n (%

)

Fig. 1. Effect of Se concentration on the inhibitory ability of B. animalis 01 proteins.

3.4. Reducing power assay

In the reducing power assay, the presence of antioxidants in thetested samples would cause reducing ferricyanide complex into theferrous form, turning the yellow color of test solution into variousshades of green and blue colors which depends on the reducingpower of antioxidant samples [21]. The reducing power of theprotein exhibited a dose-dependent manner within the testconcentration range of 0.1e1.0 mg/L, but lower than that of BHT(Fig. 4). At concentrations of 0.1, 0.2 and 0.4 mg/L, the reducingpower values of the B. animalis 01 proteins were 0.25, 0.27 and 0.34respectively. While a solution of BHT at the same concentration hadreducing power values of 0.85, 1.48 and 1.73. Moreover, at the sameconcentration, the reducing power of the Pro-Se was higher thanthat of Pro-CK, but the difference was nonsignificant. In Fig. 5, thedifference of the reducing power between the Pro-Se and Pro-CKwas significant (p < 0.05) and the reducing power of the proteinincreased with the increase of the selenium content in the protein.

3.5. ESR measurements

The spectrum of DMPOeOOH adducts generated using thexanthineexanthine oxidase system was presented in Fig. 6, theprotein sample reduced the typical ESR spectrum of DMPOeOOHadducts. As shown in Table 2, Bifidobacterium protein was a stronghydroxyl radical scavenger and a relatively weaker superoxideradical scavenger. Moreover, the scavenging ability of the Pro-Sewas significantly (p < 0.05) higher than that of Pro-CK and thescavenging ability increased with the increase of the seleniumcontent in the protein. It indicated that the hydroxyl radical andsuperoxide anion scavenging ability of the protein was improvedafter it contained selenium, this result was in accordance with allresults presented above.

Fig. 2. DPPH scavenging effect by Pro-CK and Pro-Se of B. animalis 01.

Pro-CK

Pro-Se2.5

Pro-Se5.0

Pro-Se8.0

Pro-Se10.0

0

10

20

30

40

50

0.0 0.2 0.4 0.6 0.8Se Concentration (mg/L)

Sc

av

en

gin

g E

ffe

ct (%

)

Fig. 3. Effect of Se concentration on the DPPH scavenging ability of B. animalis 01proteins.

Pro-CK

Pro-Se2.5

Pro-Se5.0

Pro-Se8.0

Pro-Se10.0

0.0

0.2

0.4

0.6

0.8

0.0 0.2 0.4 0.6 0.8Se Concentration (mg/L)

Red

ucin

g P

ow

er (A

bso

rb

an

ce at

700 n

m)

Fig. 5. Effect of Se concentration on the reducing power of B. animalis 01 proteins.

Q. Shen et al. / Anaerobe 16 (2010) 380e386384

3.6. Inhibition of erythrocyte hemolysis

The Fe2þ bound to the ferroheme could be a catalyst to theFenton reaction. H2O2 was added to the erythrocyte suspension,inducing chain peroxidative reaction in the erythrocyte membrane,leading to the protease in the membrane to lose biological activity,then the permeability of the membrane increased, ultimately theerythrocyte hemolysis started. As shown in the Table 3, there wasa significant (p < 0.05) absorbance difference between the normalcontrol and the H2O2 control which indicated that the H2O2 in thereaction mixture induced the erythrocyte hemolysis. When thesample was added to the reaction mixture, the absorbancedecreased, exhibiting a concentration dependent activity withinthe tested concentration (0.25e2.00mg/ml). In addition, the Pro-Seshowed a significantly (p < 0.05) higher inhibition rate than that ofPro-CK which indicates that selenium enhanced the inhibitoryeffect of the protein on the erythrocyte hemolysis.

4. Discussion

Bifidobacteria are a normal part of the gut microbiota of healthyhumans and they create a healthy balance between beneficial andpotentially harmful microorganisms in the gut ecosystem. Severalstudies indicated that bifidobacteria possessed strong antioxidantactivity. Two strains of B. longum exhibited an inhibitory effect onlinoleic acid peroxidation. The inhibitory rates of 1.0 mL of intra-cellular cell-free extract on linoleic acid peroxidation were 33.1%and 43.0% respectively [10]. It is reported that B. longum ATCC15708 had strong antioxidant activity, including inhibiting linoleic

0.0

0.5

1.0

1.5

2.0

2.5

0.0 0.2 0.4 0.6 0.8 1.0 1.2Concentration of Samples (mg/L)

Re

du

cin

g P

ow

er (A

bs

orb

an

ce

a

t

70

0 n

m)

Fig. 4. The reducing power of B. animalis 01 proteins.

acid oxidation (inhibitory effect: 28e48%) and scavenging DPPHfree radical (scavenging effect: 21%e52%) [9]. However, they bothfailed to point out which substance in bifidobacteria was respon-sible for the antioxidant activity. There are many substances inbifidobacteria could be the source of the antioxidant activity.NADH-oxidase, NADH-peroxide and superoxide dismutase (SOD)can be detected in bifidobacteria [22]. Once the bacteria werebroken, these enzymes were released to show antioxidant activityin vitro, that could be one possible reason why the bifidobacteriapossessed antioxidant activity. In addition, bacteria produced otherbioactive compounds such as exopolysaccharide and organic acidwhich possessed strong antioxidant activity [23e25]. Thesecompounds may exist in the metabolites of bifidobacteria. Ourresults have demonstrated that B. animalis 01 protein was effectiveon scavenging hydroxyl radical, superoxide anion, DPPH freeradical, and it was also effective on inhibiting linoleic acid perox-idation and erythrocyte hemolysis. In our study, we at least pointedout one substance which was responsible for the antioxidantactivity in bifidobacteria.

Selenium is considered to be a kind of element possessingantioxidant activity. Its antioxidant activity was expressed bycombining with GSH-Px, scavenging free radical, cutting off thechain reaction induced by the lipid peroxidation, analyzingperoxide, protecting the cell membrane [26]. In our research, allresults indicated that selenium enhanced the antioxidant activity ofthe B. animalis 01 proteins. Our results were in agreements withothers’ results which showed that the antioxidant activity of greentea and phycocyanin was enhanced after selenium was incorpo-rated [27,28]. However, in the DPPH assay and reducing power

Fig. 6. ESR spectra of the DMPOeOOH before (A, Control) and after (B) adding proteinsample.

Table 2The activity of scavenging superoxide anion and hydroxyl radical of B. animalis 01 proteins.

Sample EPR amplitudefor DMPOeOH

EPR amplitudefor DMPOeOOH

Scavengingeffect of OH (%)

Scavengingeffect of O2

�$(%)

Control 982 � 19 662 � 11 N/A N/APro-CK 821 � 22 589 � 9 16.4 � 0.9 e 11.0 � 0.6 ePro-Se2.5 764 � 16 537 � 13 22.2 � 0.5 d 18.9 � 0.5 dPro-Se5.0 681 � 21 499 � 18 30.7 � 0.4 c 24.6 � 0.6 cPro-Se8.0 597 � 15 418 � 6 39.2 � 0.7 b 36.9 � 0.9 bPro-Se10.0 469 � 11 388 � 10 52.2 � 0.4 a 41.4 � 0.4 a

Values are the means � standard deviations (n ¼ 3). Values followed by different letters in same column are significantly different (p < 0.05).

Q. Shen et al. / Anaerobe 16 (2010) 380e386 385

assay, the difference of the antioxidant activity was nonsignificantbetween Pro-CK and Pro-Se2.5, so we conducted another twoassays increasing the selenium content in the B. animalis 01proteins, the results were presented in Figs. 3 and 5 which bothindicated that between the Pro-Se and Pro-CK, therewas significant(p < 0.05) difference in DPPH scavenging ability and reducingpower.

The essential micronutrient selenium occurs in the form ofselenocysteine in selenoprotein which exert various effects, main-taining the cell reduction-oxidation balance [29]. In our previousstudy, we have already found out that selenomethionine (SeMet)was the major selenocompound in the protein [12]. According tothe theory of Shimazu and Tapple [30], as a free radical scavenger,selenoamino acids react with the hydrated free radicals (�OH,�H) toform stable compounds before they destroy the biological macro-molecules. Although the mechanism on how selenium enhancesthe antioxidant activity of the protein is unclear, one possibleexplanation for the enhancing effect is the unique atom structure ofselenium. The ionization energy of the 4 p electron in selenium ismuch lower than that of the 3 p electron in sulphur which makesselenoamino acids much easier than sulfoamino acids becomepositive ion free radical, like CH3 � Se,ðþÞ

,, � R. In addition, theselenoamino acid radical is more stable than that of sulfoaminoacid because of the existence of the empty 4F orbit. When thesulphur in B. animalis 01 proteins is replaced by selenium or somenew selenoamino acids are synthesized, the selenoamino acids aremuch easier to form stable positive ion free radicals which possessstrong antioxidant activity. Moreover, from Table 1, we wereinformed that organic selenium was more effective than inorganicselenium on enhancing hydroxyl radical scavenging activity. It isreported that goats being fed on the diet containing organic sele-nium showed a significant increase in GSH-Px and GST activities inthe gastrocnemius muscle compared with those fed on inorganicselenium [31]. Another study reported that organic selenium wasmore effective than inorganic selenium on inhibiting the mono-amine oxidase activity in the rats’ livers [32]. These results sup-ported our results again that organic selenium is more effective

Table 3Effect of B. animalis 01 proteins on hemolysis of rat erythrocytes induced by H2O2.

Sample A540 Hemolysisextent (%)

Inhibitionrate (%)

Normal 0.099 � 0.001 h 9.39 h N/AH2O2 1.05 � 0.01 a 100 a N/A

Pro-CK 0.25 mg/mL 0.99 � 0.01 b 94.0 b 6.6 g0.50 mg/mL 0.91 � 0.03 c 86.3 c 15.1 f1.00 mg/mL 0.83 � 0.01 de 78.7 de 23.5 d2.00 mg/mL 0.79 � 0.05 e 75.0 e 27.5 c

Pro-Se8.0 0.25 mg/mL 0.89 � 0.09 cd 84.4 cd 17.2 e0.50 mg/mL 0.84 � 0.04 de 79.7 de 22.4 d1.00 mg/mL 0.67 � 0.06 f 63.6 f 40.2 b2.00 mg/mL 0.51 � 0.07 g 48.4 g 57.0 a

Values are the means � standard deviations (n ¼ 3). Values followed by differentletters in same column are significantly different (p < 0.05).

than inorganic selenium on enhancing the antioxidant activity ofthe protein to some extent, but the specific mechanism needs to beelucidated in further research.

In conclusion, the antioxidant activity of B. animalis 01 proteinswas demonstrated by using a variety of testing systems. When theprotein was combined with selenium, its antioxidant activity wasimproved remarkably. In addition, the organic selenium possessedhigher enhancing effect than the inorganic selenium. As for themechanisms, they are the objects of our further research work.

Acknowledgements

This research was funded by China National 863 Program(2008AA10Z324 and 2006AA10Z343) and Beijing Natural ScienceFoundation (5072025).

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