Introduction

Increasing evidence indicates that excessive reactive oxygen species (ROS) plays an important role in damaging cellular components, causing cell membrane injury, protein denaturation and wrong DNA replications, and inducing many diseases including aging, cancer, diabetes, and rheumatoid arthritis [12, 22]. Although antioxidant and repair systems such as GSH-Px, CAT, and SOD exist in human bodies, sometimes they are not sufficient to protect human bodies from oxidative damage. Hence consuming antioxidant supplements would help to prevent human bodies from oxidative damage.

Bifidobacteria is a member of dominant microbiota in human intestinal tract. It plays an important role in promoting human health. For example, in the presence of β-galactosidase producing bifidobacteria, people suffering from lactose malabsorption could improve their lactose digestibility and tolerance [14]. Some Bifidobacterium species can produce antimicrobial compounds (bacteriocin) to control gastrointestinal infections [7, 13, 28]. In addition, it also contributes to stimulation of the immune system, anti-ulcer activity, anti-tumor activity, and gastrointestinal disorder prevention or treatment [16, 17, 27]. Various health promoting functions of bifidobacteria has enabled it to be the ideal dietary supplement.

Several studies have been conducted on the antioxidant activity of bifidobacteria. Lin and Yen [11] evaluated the inhibitory effect of Bifidobacterium longum on lipid peroxidation. Based on DPPH-free radical scavenging activity assay and oxygen tolerance assay, Kim et al. [8] isolated antioxidative Bifidobacterium species from infant feces. However, the studies mentioned above were performed without involving animal experiments which failed to reveal the antioxidative potential of bifidobacteria in vivo completely. In addition, it has been reported that fermentation products by bifidobacteria possessed strong antioxidant activity [25]. And in our previous study, we demonstrated that proteins extracted from Bifidobacterium animalis 01 cells showed evident antioxidant activity in vitro [20]. Therefore, in present study, the antioxidant activities of culture supernatant, intact cells and intracellular extracts of B. animalis 01 in vitro and in vivo were characterized, trying to explore the application potential of B. animalis 01 as dietary antioxidant supplements.

Materials and Methods

Microbial Strains

B. animalis 01 (China General Microbiological Collection Center 1353), identified by the method based on 16S rRNA sequences analysis, was isolated from the fecal samples of the healthy centenarian volunteers living in Bama of Guangxi province in China, which was listed as the fifth officially certificated longevity village by the International Society of Natural Medicine in 1991. B. animalis 01 was cultured and in MRS broth at 37°C for 24 h under anaerobic conditions.

Reagents and Materials

Xanthine, xanthine oxidase, 1,1-diphenyl-2-picrylhydrazyl (DPPH), linoleic acid, Tween 20, and 5,5-dimethyl-pyrroline-N-oxide (DMPO) were purchased from Sigma Company. Diethylenetriaminepentaacetic acid (DETAPAC), N,N,N,N-Tetramethyl ethylenediamine (TEMED), thiobarbituric acid (TBA), trichloroacetic acid (TCA), butylated hydroxytoluene (BHT), ascorbic acid, copper sulfate, and ferrous sulfate were purchased Beijing Chemicals Co. Ltd. Assay kits for malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), total antioxidant capacity (TAOC), glutathione-S-transferase (GST), and monoamine oxidase (MAO) were the products of Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The male Kunming mice were purchased from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences (Beijing, China). All other reagents were of analytical grade.

Preparation of Culture Supernatant, Intact Cells, and Intracellular Cell-Free Extracts

The preparation process was conducted basically according to the method of Lin and Yen [11]. In the preparation process, total cell numbers were adjusted to about 109 CFU/ml. Through centrifugation at 4,400 rpm for 10 min, culture supernatant was separated from intact cells. After being washed three times with deionized water, intact cells were obtained and resuspended in deionized water. For the preparation of intracellular cell-free extracts, cells were quickly washed twice with deionized water and resuspended in deionized water followed by ultrasonic disruption. Sonication was performed in an ice-water bath for 60 cycles of 5 s, with 5 s intervals until no cells could be checked under microscope. Cell debris was removed by centrifugation at 7,800 rpm for 10 min and the supernatants were collected as intracellular cell-free extracts.

Inhibition of Linoleic Acid Peroxidation

The anti-lipid peroxidation activity of B. animalis 01 was carried out by the thiobarbituric acid (TBA) method, according to the method of Grossman and Zakut [6] with some modifications. Linoleic acid emulsion (20 ml) was composed of 0.1 ml linoleic acid, 0.2 ml Tween 20, and 19.7 ml deionized water. 0.2 ml phosphate buffer solution (0.02 M, pH 7.4), 1 ml of linoleic acid emulsion, 0.2 ml FeSO4 (0.01%, w/v), 0.2 ml H2O2 (0.02%, v/v), and 0.4 ml sample (MRS broth, culture supernatant, intact cells or intracellular cell-free extracts) were mixed and then incubated at 37°C for 12 h. Afterwards 2.0 ml reaction solution was mixed with 0.2 ml TCA (4%, w/v), 2 ml TBA (0.8%, w/v), and 0.2 ml BHT (0.4%, w/v). The mixture was incubated at 100°C for 30 min and then allowed to cool. The absorbance was measured at 532 nm. In the blank control, samples were substituted with deionized water. The inhibition rate was calculated as follows:

$$ {\text{Inhibition effect }}\left( \% \right) = \left[ { 1- A_{{ 5 3 2({\text{sample}})}} /A_{{ 5 3 2({\text{blank}})}} } \right] \times 100 $$

DPPH-free Radical Scavenging Activity

Scavenging capacity of DPPH-free radical was evaluated by the method of Blois [3]. The assay mixture was composed of 2.2 ml DPPH-free radical (0.1 mM) and 0.8 ml sample. The mixture was shook up vigorously and allowed to stand in dark room temperature for 30 min. The absorbance was measured at 517 nm. In the blank control, the sample was substituted with deionized water. The scavenging effect was calculated using the following equation:

$$ {\text{Scavenging effect }}\left( \% \right) = \left[ { 1- A_{{ 5 1 7\left( {\text{sample}} \right)}} /A_{{ 5 1 7\left( {\text{blank}} \right)}} } \right] \times 100 $$

Hydroxyl Radical Scavenging Assay

Hydroxyl radical scavenging activity was performed at room temperature on an ER200D-SRC spectrometer (Bruker, Germany). The Fenton reaction system containing ferrous ion and H2O2 was used as hydroxyl radical generating system. DMPO was used as a spin trap reagent [5]. The reaction mixture consisted of 5 μl FeSO4 (0.1 mM), 5 μl of DETAPAC (4 mM), 5 μl DMPO (0.25 M), and 5 μl sample. The reaction was started in the presence of 5 μl H2O2 (10 mM). In the blank control, the sample was substituted with deionized water.

$$ {\text{Scavenging effect }}\left( \% \right) = \left[ {\left( {H_{\text{o}} - H_{\text{s}} } \right)/H_{\text{o}} } \right] \times 100 $$

where H o was the amplitude of the second peak in the ERS spectrum for the control, H s was the amplitude of the second peak in the ERS spectrum for the sample. The ESR instrument conditions were as follows: freq, 9.438350 GHz; field center, 336.00 mT; Mod, 100 Hz; width 0.2 × 1 mT; time constant, 0.1 s; power, 10 mw; sweep time, 2 min; amplitude, 50.

Superoxide Anion Assay

The xanthine/xanthine oxidase system was used to generate superoxide anion [29]. The reaction mixture was composed of 5 μl xanthine (0.75 mM), 5 μl DETAPAC (4 mM), 5 μl DMPO (1 M), and 5 μl sample. The reaction was started by the addition of 5 μl xanthine oxidase (0.71 U/ml). The ESR signal obtained for the DMPO-OH adduct was measured in 50 s. In the blank control, the sample was substituted with deionized water.

$$ {\text{Scavenging effect }}\left( \% \right) = \left[ {\left( {H_{\text{o}} - H_{\text{s}} } \right)/H_{\text{o}} } \right] \times 100 $$

where H o was the amplitude of the first peak in the ERS spectrum for the control, H s was the amplitude of the first peak in the ERS spectrum for the sample. The ESR instrument conditions were as follows: freq, 9.4386 GHz; field center, 336.0 mT; Mod, 100 Hz; width, 0.79 × 0.1 mT; time constant, 0.1 s; power, 10 mw; sweep time, 2 min; amplitude, 200.

Animal and Experimental Design

Male Kunming mice (8 weeks old, 25 ± 2 g) were kept in plastic cages under a controlled atmosphere (Temperature 22 ± 2°C; humidity 50 ± 5%). They were kept on a 12 h light/dark cycles and were free to access to food and water during the experiments. After adaptation for 1 week, the mice were randomly divided into seven groups (6 mice per group): normal control group (NCG), d-galactose model control group (MCG), ascorbic acid positive control group (PCG), MRS broth treatment group, culture supernatant treatment group, intact cell treatment group, and intracellular cell-free extracts treatment group. Mice in NCG were given 0.5 ml physiological saline solution (0.85%, w/v) per day via gastric gavage. Mice in MCG were fed with 0.5 ml physiological saline solution (0.9%, w/v) via gastric gavage and 0.2 ml (20 mg per mouse) d-galactose through subcutaneous injection per day. Mice in PCG were given 0.5 ml (5 mg per mouse) ascorbic acid via gastric gavage and 0.2 ml d-galactose (20 mg per mouse) through subcutaneous injection per day. The mice in sample treatment groups were given 0.5 ml MRS broth, culture supernatant, intact cell suspension or intracellular cell-free extracts via gastric gavage and 0.2 ml d-galactose (20 mg per mouse) through subcutaneous injection per day. All groups were performed once daily for 30 consecutive days.

Assays of SOD, CAT, GSH-Px, TAOC, and MDA in Mice Serums

Twenty-four hours after the last drug administration, mice were killed. Blood samples were collected and centrifuged at 4,000 rpm for 10 min at 4°C to afford the serums. The activities of SOD, CAT, and GSH-Px and levels of MDA and TAOC in serum were measured according to the instructions on the kits.

Assays of MDA and GST in Mice Livers

The liver was removed, washed, and homogenized in ice-cold physiological saline to prepare 10% (w/v) homogenate. Then the homogenate was centrifuged at 4,000 rpm for 10 min at 4°C to remove cellular residue. Its supernatant was collected to measure the GST activity and MDA level. Both assays were conducted according to the instructions on the kits.

Assays of MAO and Lipofuscin in Mice Brains

The brain was removed, washed, and homogenized in ice-cold physiological saline to prepare 10% (w/v) homogenate. The homogenate was centrifuged at 4,000 rpm for 10 min at 4°C. Its supernatant was collected to measure MAO activity and lipofuscin level.

The MAO activity was measured according to the instructions on the kits. The lipofuscin level was determined by previous extraction method with slight modifications [15, 23]. Briefly, 2 ml 10% (w/v) brain tissue homogenate was mixed into 4 ml chloroform–methanol solution (2:1, v/v). After centrifugation at 2,000 rpm for 10 min, the lipofuscin levels were evaluated in the chloroform phase using a RF-510 fluorescence spectrophotometer (Shimadzu, Japan). The luminescence of the sample was determined at an emission maximum of 450 nm. The concentrations of lipofuscin were determined using quinine (0.1 μg/ml) as a standard.

Statistical Analysis

All experiments were performed at least three times. Results were expressed as the mean ± standard deviation. The Student–Newman–Keuls test and one-way analysis of variance (ANOVA) were used for multiple comparisons by the SPSS 11.5 software package. Difference was considered to be statistically significant if P < 0.05.

Results

Inhibition of Linoleic Acid Peroxidation

Culture supernatant, intact cells and intracellular cell-free extracts of B. animalis 01 all exhibited evident inhibitory effect on the inhibition of linoleic acid peroxidation (Table 1). The inhibitory effect was 30.48, 41.12, and 71.02% for culture supernatant, intact cells, and intracellular cell-free extracts, respectively. MRS broth showed certain inhibitory effect, but the inhibitory effect of culture supernatant was significantly (P < 0.05) higher than that of MRS broth.

Table 1 Anti-lipid peroxidation effect and DPPH-free radical scavenging effect of B. animalis 01
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DPPH-Free Radical Scavenging Activity

DPPH-free radical scavenging effects of culture supernatant, intact cells and intracellular cell-free extracts of B. animalis 01 were presented in Table 1. In this assay, the culture supernatant of B. animalis 01 exhibited the highest scavenging effect (73.11%), which was significantly (P < 0.05) higher than that of MRS broth. The scavenging effect of intact cells on DPPH-free radicals was the lowest among the groups.

Scavenging Effect on Hydroxyl Radical and Superoxide Anion

Culture supernatant, intact cells and intracellular cell-free extracts of B. animalis 01 exhibited excellent scavenging activity on both hydroxyl radical and superoxide anion (Table 2). In both assays, the culture supernatant of B. animalis 01 showed the strongest scavenging ability, which was significantly (P < 0.05) higher than that of MRS broth, whereas intact cells exhibited weak scavenging effect.

Table 2 Scavenging effect of B. animalis 01 on oxygen-free radicals
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Activities of Antioxidant Enzymes in Mice Serums

As shown in Table 3, compared with NCG, significant (P < 0.05) increase in MDA level, decrease of antioxidative enzymes activities (SOD, GSH-Px, and CAT) and TAOC level were observed in serums of MCG. Culture supernatant, intact cells, intracellular cell-free extracts, and ascorbic acid significantly (P < 0.05) enhanced the activities of antioxidant enzymes (SOD, CAT, and GSH-Px) and the TAOC level in mice serums, reduced the MDA content in mice serums. In addition, results showed that culture supernatant, was significantly (P < 0.05) more effective than MRS broth on enhancing activities of antioxidant enzymes and reducing MDA content in mice serums.

Table 3 Effect of B. animalis 01 on the activities of SOD (U/ml), CAT (U/ml), GSH-Px (U/ml), TAOC (U/ml), and levels of MDA (nmol/ml) in mice serums
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GST Activity and MDA Level in Mice Livers

Each group’s GST activity and MDA level in mice livers were presented in Table 4. The MDA level and GST activity of MCG were significantly (P < 0.05) higher than that of NCG. Compared with MCG group, culture supernatant, intact cells, intracellular cell-free extracts, and ascorbic acid significantly (P < 0.05) reduced GST activity and MDA level in mice livers. In addition, the GST activity of MRS broth treatment group was significantly (P < 0.05) higher than that of culture supernatant treatment group.

Table 4 Effect of B. animalis 01 on GST activity (U/mg protein) and MDA level (nmol/mg protein) in mice livers, lipofusion level (μg/g) and MAO activity (U/h/mg protein) in mice brains
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MAO Activity and Lipofuscin Level in Mice Brains

Compared with NCG group, there was a significant (P < 0.05) increase of MAO activity and lipofuscin level in MCG group (Table 4). The results showed that culture supernatant, intact cells, and intracellular cell-free extracts significantly (P < 0.05) reduced MAO activity and lipofuscin level in mice brains. Moreover, MAO activity and lipofuscin level of MRS broth treatment group were significantly (P < 0.05) higher than that of culture supernatant treatment group.

Discussion

The antioxidative potential of bifidobacteria has been reported by several studies. Most of them involved antioxidant assays in vitro such as DPPH-free radical scavenging assay and inhibition of lipid peroxidation assays. In our study, the results indicated that intact cells and intracellular cell-free extracts of B. animalis 01 showed strong antioxidant activity in vitro, which was similar to the antioxidant activity of Bifidobacterium longum ATCC 15708 described by Lin and Chang [10]. Moreover, intracellular cell-free extracts exhibited stronger antioxidant activity than intact cells in vitro. This was probably because most antioxidative compounds of bifidobacteria existed in vivo and the reaction time in vitro was too short for intact cells to exhibit antioxidant activity. This result also can be supported by our previous research which revealed that protein extracted from B. animalis 01 cells possessed evident antioxidant activity in vitro [20]. Culture supernatant exhibited stronger scavenging effect than MRS broth, which may result from hydrolyzed medium composition. It has been reported that the antioxidant activity of fermented milk by lactic acid bacteria may came from hydrolysed components [24]. On the other hand, MRS broth also exhibited certain antioxidant activity in vitro. It could be attributed to the components of MRS medium such as glucose, peptone, and yeast extracts.

In the present study, the antioxidant activities of culture supernatant, intact cells, and intracellular cell-free extracts of B. animalis 01 in vivo were also characterized. SOD, GSH-Px, and CAT are regarded as major antioxidant enzymes in vivo to eliminate ROS. SOD transformed superoxide radicals to form hydrogen peroxide, which then was degraded into water and oxygen, thus preventing organisms from oxidative damage [26]. MDA could induce cell damage in various ways and its level in mice body reflects the content of free radicals produced by lipid peroxidation. Our results indicated that culture supernatant, intact cells, and intracellular cell-free extracts enhanced activities of antioxidative enzymes in serums, and reduced MDA content both in serums and livers significantly (P < 0.05) (Tables 3, 4). In addition, the TAOC level of aging mice’s serums was enhanced, which might come from the increase of non-enzymatic antioxidants, such as glutathione, thiols, and some vitamins [18]. The GST activities of all sample treatment groups were significantly (P < 0.05) lower than that of MCG group, which was in consistent with recent Tanas et al.’s [21] results, suggesting that GST activity of paw tissues decreased by administration of lichen extracts which was characterized to be an effective antioxidant. MAO activity and lipofuscin content increases with the increase of body age, thus they are regarded as important index of body aging [1, 19]. We found that administration of culture supernatant, intact cells, and intracellular cell-free extracts reduced MAO activity and lipofuscin content significantly (P < 0.05) in aging mice brains (Table 4). Furthermore, results showed that intact cells and intracellular cell-free extracts possessed similar antioxidant activities in vivo, which was in contrast to the results in vitro. The reason could be that intact cells were broken in mice body and antioxidative compounds in vivo were released, or intact cells produced bioactive metabolites in mice body.

The purpose of this study was to explore the potential of application of B. animalis 01 as dietary antioxidant supplements. Thus, antioxidant activity assays in vivo were conducted, which was different from previous studies that only involved assays in vitro [8, 10, 11]. Some investigation suggested that milk or soymilk fermented by bifidobacteria enhanced their antioxidant activities [24, 25]. In addition, bioactive compounds such as exopolysaccharide produced by bifidobacteria may contribute to its antioxidant activity [2, 9]. Hence, culture supernatant was involved in this study to determine whether B. animalis 01 was able to improve the antioxidant activity of MRS broth, and MRS broth group was a control to culture supernatant. Our results revealed that the antioxidant activity of MRS broth was enhanced significantly (P < 0.05) after the cultivation of B. animalis 01. Thus, it is entirely possible that after B. animalis 01 is consumed by humans, it takes advantage of the substances in human intestinal tract to produce antioxidative compounds to benefit the host. Compared with intracellular cell-free extracts, although intact cells showed relatively lower antioxidant activity, it could compete with potential pathogenic gas producing and putrefactive bacteria, thereby increasing colonic bifidobacteria populations. It is supported by Bouhnik et al.’s [4] conclusion that the bifidobacteria populations increased in human body as long as exogenous bifidobacteria was consumed.

According to the results obtained, culture supernatant, intact cells, and intracellular cell-free extracts of B. animalis 01 exhibited excellent antioxidant activity in vitro and in vivo. It indicated that as long as B. animalis 01 was consumed, it will benefit the host in any forms: if cells are alive, they can improve the intestinal tract microflora and produce antioxidative compounds; if cells are broken after they are ingested by human beings, the antioxidative compounds in vivo will be released to benefit the host. Besides its health promoting functions, bifidobacteria is safe for human to ingest which stems from the long historical consumption of fermented milk. Therefore, B. animalis 01 is suitable to be developed into a novel antioxidant supplement for human beings.