Letters in Applied Microbiology ISSN 0266-8254
ORIGINAL ARTICLE
Bioactivity of proteins isolated from Lactobacillus plantarum L67 treated with Zanthoxylum piperitum DC glycoprotein S. Song, S. Oh and K.-T. Lim Division of Animal Science, Chonnam National University, Gwangju, Korea
Significance and Impact of the Study: This study demonstrated that Lactobacillus plantarum L67 possesses anti-oxidative activity. This strain of lactic bacteria has been known to have various probiotic uses, such as yogurt starters and dietary additional supplements. We found, through this experiment, that the protein has a strong anti-oxidative character, and the activity can be enhanced by treatment with Zanthoxylum piperitum DC (ZPDC) glycoprotein. This study may be application of Lact. plantarum L67 treated by ZPDC glycoprotein in yogurt fermentation. It could be one of the avenues of minimizing yogurt postacidification during storage. In addition, it can be manufactured and incorporated in food products without losing viability and functionality of Lact. plantarum L67.
Keywords antioxidant, ATPase activity, betagalactosidase, CAT, GPx, Lactobacillus plantarum L67, Raw 264.7 cells, SOD, Zanthoxylum piperitum DC glycoprotein. Correspondence Kye-Taek Lim, Division of Animal Science, Chonnam National University, 77 Yongbongro, Buk-gu, Gwangju 500-757, Korea. E-mail: [email protected] 2014/2106: received 13 October 2014, revised 5 March 2015 and accepted 6 March 2015 doi:10.1111/lam.12416
Abstract Lactobacilli in the human gastrointestinal tract have beneficial effects on the health of their host. To enhance these effects, the bioactivity of lactobacilli can be fortified through exogenous dietary or pharmacological agents, such as glycoproteins. To elucidate the inductive effect of Zanthoxylum piperitum DC (ZPDC) glycoprotein on Lactobacillus plantarum L67, we evaluated the radicalscavenging activity, anti-oxidative enzymes (SOD, GPx and CAT), growth rate, ATPase activity and b-galactosidase activity of this strain. When Lact. plantarum L67 was treated with ZPDC glycoprotein at different concentrations, the intensities of a few SDS-PAGE bands were slightly changed. The amount of a 23 kDa protein was increased upon treatment with increasing concentrations of ZPDC glycoprotein. The results of this study indicate that the radical-scavenging activity for O 2 and OH¯, but not for the DPPH radical, increased in a concentration-dependent manner after treatment with ZPDC glycoprotein. The activation of anti-oxidative enzymes (SOD, GPx and CAT), growth rate and b-galactosidase activity also increased in a concentrationdependent manner in response to ZPDC glycoprotein treatment, whereas ATPase activity was decreased. In summary, ZPDC glycoprotein stimulated an increase in the bioactivity of Lact. plantarum L67.
Introduction Lactobacilli and bifidobacteria are known as the most safe and frequently used probiotics, even for children and immunocompromised individuals (Borriello et al. 2003). Lactobacillus plantarum exhibits various biological effects, including antitumor, anticoagulant, antiviral, immunomodulatory, anti-inflammatory, antidiabetic, antioxidant and free radical-scavenging activity (Andersson et al.
2010; Li et al. 2012; Giardina et al. 2014; Kassayova et al. 2014). It is possible to maintain a healthy intestinal tract and to prevent colon cancer by augmenting or fortifying the bioactivity of Lact. plantarum L67 through exogenous dietary or pharmacological means, such as phytoglycoprotein. Most glycoproteins isolated from herbal plants can scavenge reactive oxygen radicals, preventing inflammation and colon cancer both in vitro and in vivo (Lee and Lim 2008; Oh and Lim 2011; Noysang et al. 2014).
Letters in Applied Microbiology 60, 597--604 © 2015 The Society for Applied Microbiology
597
Bioactive proteins from Lact. plantarum
S. Song et al.
Zanthoxylum piperitum DC (ZPDC) has traditionally been used as a spice to produce a fresh flavour, as an ingredient in spice mixes or to suppress unpleasant fishy and meaty odours (Jiang and Kubota 2001). In Korea, it has also traditionally been used to treat vomiting, diarrhoea and abdominal pain (Kim et al., 1984). Recently, we isolated a glycoprotein (24 kDa) from the ZPDC fruit which has pharmacological activities, such as antioxidative and hepato-protective effects (Lee and Lim 2008; Lee et al. 2014). On the basis of these findings, we speculated that the ZPDC glycoprotein might increase the activity of Lact. plantarum L67, as it does not have any cytotoxicity and can be used as a nutrient for growing Lact. plantarum L67. In addition, to date, there have been no reports on enhancing the bioactivity of Lact. plantarum via treatment with natural compounds. Therefore, the objective of this study was to investigate whether the bioactivity of Lact. plantarum L67 can be enhanced by treatment with ZPDC glycoprotein or compounds of similar molecular weights (24 kDa). Results and discussion The inductive effect of Lactobacillus plantarum L67 with ZPDC treatment Here, we studied the influence of ZPDC glycoprotein treatment on the activity of Lactobacillus plantarum L67. When Lact. plantarum L67 was treated with ZPDC glycoprotein of various concentrations, the intensities of a few SDS-PAGE bands were slightly changed. One band, corresponding to a molecular weight of 23 kDa, was increased in intensity concomitant with an increase in ZPDC glycoprotein concentration (Fig. 1a). Protein extracted from Lact. plantarum L67 was treated by ZPDC (P23 kDa). It should be noted that this protein is called the iL67 protein (induced Lact. plantarum L67 protein) in this study. As shown in Fig. 1b, the relative intensities were 108, 108, 112 and 114 at 5, 20, 40 and 100 lg ml1, respectively, compared with the control. These results indicated that proteins isolated from Lact. plantarum L67 increased in both amount and bioactivity following treatment with ZPDC. The possible explanation about increasing bioactivity might be due to interactions between the Lact. plantarum L67 protein and natural compounds with similar molecular weights. Anti-oxidative activities of iL67 protein For DPPH radicals, it was reduced by 03, 32, 45, 46 and 57 by 100 lg ml1 of iL67 protein treated with 0, 5, 20, 40 and 100 lg ml1 ZPDC glycoprotein, respectively, compared with the control (Fig. 2). L-Ascorbic acid values 598
(a) kDa 210 125 101 56·2 35·8
29 23 21 6·9 C
(b)
5
20
40
100 ZPDC (µg ml–1)
23 1
1·08
1·08
1·12
C
5
20
40
1·14 Relative intensity (folds) 100 ZPDC (µg ml–1)
Figure 1 Inductive protein isolated from Lactobacillus plantarum L67 with Zanthoxylum piperitum DC (ZPDC) glycoprotein treatment. (a) Proteins isolated from Lact. plantarum L67 grown in media treated with various concentrations of ZPDC glycoprotein. (b) The 23 kDa protein isolated from Lact. plantarum L67 grown in media treated with various concentrations of ZPDC glycoprotein. SDS-PAGE was performed using 14% acrylamide gels containing 1% sodium dodecyl sulphate. Gels were stained by Coomassie Blue.
were reduced 70 and 90% compared with DPPH radicals in response to 20 and 40 lg ml1 of ZPDC glycoprotein respectively. These results indicate that Lact. plantarum L67 has no significant DPPH radical-scavenging activity. In the G/GO- or HX/XO-, when Raw 264.7 cells were treated with 04 mmol hypoxanthine (HX)/40 mU ml1 xanthine oxidase (XO) or 05% glucose (G)/40 mU ml1 glucose oxidase (GO), viability was reduced by 4, 56, 82, 97 and 97% or by 13, 54, 75, 95, and 95% for 1, 3, 6, 12 and 24 h, respectively, compared with the control (Fig. 3a). However, when cells were co-treated with a cell extract of L67 grown in media treated with ZPDC glycoprotein in the HX/XO and G/GO systems leading to
ORIGINAL ARTICLE
Bioactivity of proteins isolated from Lactobacillus plantarum L67 treated with Zanthoxylum piperitum DC glycoprotein S. Song, S. Oh and K.-T. Lim Division of Animal Science, Chonnam National University, Gwangju, Korea
Significance and Impact of the Study: This study demonstrated that Lactobacillus plantarum L67 possesses anti-oxidative activity. This strain of lactic bacteria has been known to have various probiotic uses, such as yogurt starters and dietary additional supplements. We found, through this experiment, that the protein has a strong anti-oxidative character, and the activity can be enhanced by treatment with Zanthoxylum piperitum DC (ZPDC) glycoprotein. This study may be application of Lact. plantarum L67 treated by ZPDC glycoprotein in yogurt fermentation. It could be one of the avenues of minimizing yogurt postacidification during storage. In addition, it can be manufactured and incorporated in food products without losing viability and functionality of Lact. plantarum L67.
Keywords antioxidant, ATPase activity, betagalactosidase, CAT, GPx, Lactobacillus plantarum L67, Raw 264.7 cells, SOD, Zanthoxylum piperitum DC glycoprotein. Correspondence Kye-Taek Lim, Division of Animal Science, Chonnam National University, 77 Yongbongro, Buk-gu, Gwangju 500-757, Korea. E-mail: [email protected] 2014/2106: received 13 October 2014, revised 5 March 2015 and accepted 6 March 2015 doi:10.1111/lam.12416
Abstract Lactobacilli in the human gastrointestinal tract have beneficial effects on the health of their host. To enhance these effects, the bioactivity of lactobacilli can be fortified through exogenous dietary or pharmacological agents, such as glycoproteins. To elucidate the inductive effect of Zanthoxylum piperitum DC (ZPDC) glycoprotein on Lactobacillus plantarum L67, we evaluated the radicalscavenging activity, anti-oxidative enzymes (SOD, GPx and CAT), growth rate, ATPase activity and b-galactosidase activity of this strain. When Lact. plantarum L67 was treated with ZPDC glycoprotein at different concentrations, the intensities of a few SDS-PAGE bands were slightly changed. The amount of a 23 kDa protein was increased upon treatment with increasing concentrations of ZPDC glycoprotein. The results of this study indicate that the radical-scavenging activity for O 2 and OH¯, but not for the DPPH radical, increased in a concentration-dependent manner after treatment with ZPDC glycoprotein. The activation of anti-oxidative enzymes (SOD, GPx and CAT), growth rate and b-galactosidase activity also increased in a concentrationdependent manner in response to ZPDC glycoprotein treatment, whereas ATPase activity was decreased. In summary, ZPDC glycoprotein stimulated an increase in the bioactivity of Lact. plantarum L67.
Introduction Lactobacilli and bifidobacteria are known as the most safe and frequently used probiotics, even for children and immunocompromised individuals (Borriello et al. 2003). Lactobacillus plantarum exhibits various biological effects, including antitumor, anticoagulant, antiviral, immunomodulatory, anti-inflammatory, antidiabetic, antioxidant and free radical-scavenging activity (Andersson et al.
2010; Li et al. 2012; Giardina et al. 2014; Kassayova et al. 2014). It is possible to maintain a healthy intestinal tract and to prevent colon cancer by augmenting or fortifying the bioactivity of Lact. plantarum L67 through exogenous dietary or pharmacological means, such as phytoglycoprotein. Most glycoproteins isolated from herbal plants can scavenge reactive oxygen radicals, preventing inflammation and colon cancer both in vitro and in vivo (Lee and Lim 2008; Oh and Lim 2011; Noysang et al. 2014).
Letters in Applied Microbiology 60, 597--604 © 2015 The Society for Applied Microbiology
597
Bioactive proteins from Lact. plantarum
S. Song et al.
Zanthoxylum piperitum DC (ZPDC) has traditionally been used as a spice to produce a fresh flavour, as an ingredient in spice mixes or to suppress unpleasant fishy and meaty odours (Jiang and Kubota 2001). In Korea, it has also traditionally been used to treat vomiting, diarrhoea and abdominal pain (Kim et al., 1984). Recently, we isolated a glycoprotein (24 kDa) from the ZPDC fruit which has pharmacological activities, such as antioxidative and hepato-protective effects (Lee and Lim 2008; Lee et al. 2014). On the basis of these findings, we speculated that the ZPDC glycoprotein might increase the activity of Lact. plantarum L67, as it does not have any cytotoxicity and can be used as a nutrient for growing Lact. plantarum L67. In addition, to date, there have been no reports on enhancing the bioactivity of Lact. plantarum via treatment with natural compounds. Therefore, the objective of this study was to investigate whether the bioactivity of Lact. plantarum L67 can be enhanced by treatment with ZPDC glycoprotein or compounds of similar molecular weights (24 kDa). Results and discussion The inductive effect of Lactobacillus plantarum L67 with ZPDC treatment Here, we studied the influence of ZPDC glycoprotein treatment on the activity of Lactobacillus plantarum L67. When Lact. plantarum L67 was treated with ZPDC glycoprotein of various concentrations, the intensities of a few SDS-PAGE bands were slightly changed. One band, corresponding to a molecular weight of 23 kDa, was increased in intensity concomitant with an increase in ZPDC glycoprotein concentration (Fig. 1a). Protein extracted from Lact. plantarum L67 was treated by ZPDC (P23 kDa). It should be noted that this protein is called the iL67 protein (induced Lact. plantarum L67 protein) in this study. As shown in Fig. 1b, the relative intensities were 108, 108, 112 and 114 at 5, 20, 40 and 100 lg ml1, respectively, compared with the control. These results indicated that proteins isolated from Lact. plantarum L67 increased in both amount and bioactivity following treatment with ZPDC. The possible explanation about increasing bioactivity might be due to interactions between the Lact. plantarum L67 protein and natural compounds with similar molecular weights. Anti-oxidative activities of iL67 protein For DPPH radicals, it was reduced by 03, 32, 45, 46 and 57 by 100 lg ml1 of iL67 protein treated with 0, 5, 20, 40 and 100 lg ml1 ZPDC glycoprotein, respectively, compared with the control (Fig. 2). L-Ascorbic acid values 598
(a) kDa 210 125 101 56·2 35·8
29 23 21 6·9 C
(b)
5
20
40
100 ZPDC (µg ml–1)
23 1
1·08
1·08
1·12
C
5
20
40
1·14 Relative intensity (folds) 100 ZPDC (µg ml–1)
Figure 1 Inductive protein isolated from Lactobacillus plantarum L67 with Zanthoxylum piperitum DC (ZPDC) glycoprotein treatment. (a) Proteins isolated from Lact. plantarum L67 grown in media treated with various concentrations of ZPDC glycoprotein. (b) The 23 kDa protein isolated from Lact. plantarum L67 grown in media treated with various concentrations of ZPDC glycoprotein. SDS-PAGE was performed using 14% acrylamide gels containing 1% sodium dodecyl sulphate. Gels were stained by Coomassie Blue.
were reduced 70 and 90% compared with DPPH radicals in response to 20 and 40 lg ml1 of ZPDC glycoprotein respectively. These results indicate that Lact. plantarum L67 has no significant DPPH radical-scavenging activity. In the G/GO- or HX/XO-, when Raw 264.7 cells were treated with 04 mmol hypoxanthine (HX)/40 mU ml1 xanthine oxidase (XO) or 05% glucose (G)/40 mU ml1 glucose oxidase (GO), viability was reduced by 4, 56, 82, 97 and 97% or by 13, 54, 75, 95, and 95% for 1, 3, 6, 12 and 24 h, respectively, compared with the control (Fig. 3a). However, when cells were co-treated with a cell extract of L67 grown in media treated with ZPDC glycoprotein in the HX/XO and G/GO systems leading to
