Food Chemistry 155 (2014) 50–56
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
Effect of ultrasonic extraction conditions on antioxidative and immunomodulatory activities of a Ganoderma lucidum polysaccharide originated from fermented soybean curd residue Min Shi, Yingnan Yang ⇑, Xuansheng Hu, Zhenya Zhang Graduate School of Life and Environmental Science, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan
a r t i c l e
i n f o
Article history: Received 1 March 2012 Received in revised form 18 December 2013 Accepted 15 January 2014 Available online 23 January 2014 Keywords: Soybean curd residue Ganoderma lucidum Polysaccharide Ultrasonic assisted extraction Antioxidant activities Immunomodulation activities
a b s t r a c t A crude Ganoderma lucidum polysaccharide (GLPL) was extracted from fermented soybean curd residue by ultrasonic assisted extraction. The optimal extraction conditions were 30 min at 80 °C with 80 W and water to solid ratio of 10, and with this method 115.47 ± 2.95 mg/g of GLPL yield was obtained. Additionally, the antioxidant and immunomodulatory activities of GLPL were investigated. The results showed that GLPL exhibited strong antioxidant effects, which included scavenging activities against DPPH radicals, hydrogen oxide and ABTS radicals with IC50 values of 0.23, 0.48 and 0.69 mg/mL, respectively. For immunomodulatory activities, GLPL was shown to strongly stimulate the proliferation of macrophages (158.02 ± 13.12%), the production of nitric oxide and phagocytosis (21.16 ± 1.65 lM), and, at 40.00 lg/mL, protected macrophage from Doxorubicin (DOX) (0.16 ± 0.003). Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Ganoderma lucidum (G. lucidum), a medicinal fungus called ‘‘Lingzhi’’ in China is one of the most famous fungi in traditional Chinese medicine. In regions of China and other Asian countries, G. lucidum has been used as a remedy to promote health and longevity (Yuen & Gohel, 2008). Modern pharmaceutical research shows that G. lucidum polysaccharide (GLPL) has some physiological effects, including strong antioxidant, immunomodulating (Lin et al., 2006), and anti-tumour activities (Yuen & Gohel, 2008). Traditionally, polysaccharides are extracted from G. lucidum fruiting bodies; however, the incubation time of these fruiting bodies is as long as 60 days with a polysaccharide yield of 32.7 mg/g (Huanga & Ninga, 2010). In this study, GLPL was produced using the food waste from soybean curd residue (SCR), which could greatly reduce the production time. Therefore, this is a promising new technology for GLPL production. SCR, a by-product of tofu, soymilk or soy protein processing, is discharged as an agro-industrial waste and has caused severe environmental pollution. In fact, SCR is rich in fat, starch, protein and sugar and could be used as a high quality media for microbial fermentation (Shi, Yang, Guan, Wang, & Zhang, 2012a,b). However, there are few previous reports describing the production of GLPL ⇑ Corresponding author. Tel./fax: +81 29 853 4650. E-mail address: [email protected] (Y. Yang). 0308-8146/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2014.01.037
using SCR, and GLPL from natural food waste could be used as a functional food additive in the future. For the present, several conventional extraction techniques have been reported for the extraction of GLPL. Hot water technology is the main and most conventional extraction method for polysaccharides mentioned in recent studies (Guan, Zhang, Yang, Xin, & Liu, 2011; Kazahiro, Akira, Naomi, & Hidenori, 2009; Shi et al., 2012a,b). However, it is usually associated with a longer extraction time, more than 2 h, and a higher temperature (95 °C) but a lower extraction efficiency with a yield of less than 70.00 mg/g (Xiangqun, Yongde, & Hui, 2011). Recently, ultrasonic assisted extraction has been employed widely in the extraction of target compounds from different materials because of the facilitated mass transfer between immiscible phases through super agitation at a low frequency. It offers the benefits of high reproducibility, shorter times, simplified manipulation, and lowered energy input and solvent consumption (Khan, Maryline, Fabiano-Tixier, Dangles, & Chemat, 2010). Until now, there has been no comprehensive evaluation of the antioxidant and immunomodulatory activities of GLPL. Papers report that polysaccharides from mushrooms can enhance and activate macrophage-based immune responses, leading to immunomodulation, anti-tumour activity, wound-healing and other therapeutic effects (Huanga & Ninga, 2010; Shi et al., 2012a,b). Macrophages have a significant role in host defence mechanisms. Phagocytic activity produces reactive oxygen species (ROS) and
M. Shi et al. / Food Chemistry 155 (2014) 50–56
nitric oxide (NO) in response to stimulation from a variety of agents, and it can inhibit the growth of a wide variety of tumour cells and micro-organisms (Schepetkin & Quinn, 2006). Moreover, the immunomodulatory activity not only involves effects macrophage activation but also proliferation and differentiation of these cells (Schepetkin & Quinn, 2006). Reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), hydroxyl radical (HO) and other free radicals, are byproducts of biological metabolism (Xu et al., 2009). In recent years, many studies have shown that ROS may be responsible for or contribute to human diseases (Liu, Wang, Pang, Yao, & Gao, 2010; Wu & Hansen, 2008; Xu et al., 2009). Antioxidants can scavenge free radicals and protect against diseases. Bioactive components in fruits and vegetables have been shown to have beneficial activity (Wu & Hansen, 2008; Xu et al., 2009). In recent years, there has been increasing evidence that some polysaccharides isolated from plants, herbs and fungi also have putative health benefits and low cytotoxicity (Chatchai, Saranyu, Khajeelak, Chantragan, & Rakrudee, 2011; Maja, Klaus, Dragica, Johannes, & Leo, 2011). The purpose of this study was to extract GLPL using an optimal ultrasonic assisted extraction method from fermented SCR using G. lucidum, and to verify its antioxidant activities and immunomodulatory activities in vitro.
51
for 2–3 days to reach logarithmic phase and then used for experiments. 2.4. Preparation of GLPL The fermented SCR was dried in a convection oven at 50 °C and ground to a powder. The impurities were removed from the crushed powder (1.00 g) with 80% ethanol at room temperature for 24 h. The eluent was discarded and the residue was further extracted using different experimental conditions of ultrasonic assisted extraction. Then, the extract was filtered and centrifuged at 7500 rpm for 30 min at room temperature. The supernatant was concentrated in a rotary evaporator under reduced pressure at 50 °C and free protein layer was removed using the method of Sevage (Staub, 1999). Finally, the extract was subjected to precipitation with fourfold volumes of ethanol. The GLPL curds were collected by centrifugation, washed with ethanol twice, and freeze-dried. Total GLPL was determined using the phenol–sulphuric acid method, and D-glucose was used as the standard (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956). The results were expressed as milligram of glucose equivalent per gram of fermented SCR. 2.5. Assay for antioxidant activities of GLPL
2. Materials and methods 2.1. Materials and chemicals Eagle’s minimum essential medium (MEM), 2,2-diphenyl-1picry-hydrazyl radical (DPPH), fetal bovine serum (FBS), phosphate buffered saline (PBS), trichloroacetic acid (TCA) and 2,20 -Azinobis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) were purchased from Sigma–Aldrich, Inc. (Saint Louis, MO, USA). Lipopolysaccharide (LPS) from Escherichia coli 055, ascorbic acid, hydrogen peroxide, chloride ferric, ferrous sulphate, trichloroacetic acid, sodium salicylate and ethylenediaminetetraacetic acid (EDTA) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). The SOD Assay Kit-WST and Cell Counting Kit-8 (CCK-8) were purchased from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). Doxorubicin (DOX) was purchased from TopoGEN, Inc. (Florida, USA). All other chemical reagents were of analytical grade. 2.2. Microorganism and culture conditions The A50 strain of G. lucidum was purchased from Agriculture and Forestry Strains in Kaishas, Japan. The strain was maintained on potato dextrose agar (PDA) at 4 °C. 15 mL of the liquid culture was added to a 50-mL flask containing one unit of mycelia agar, which was a 5 mm 5 mm square that was obtained using a self-designed cutter. The initial pH was from 5.0 to 5.5, and the culture was incubated on a rotary shaker at 100 rpm and 25 °C for 6 days. The seed for the solid culture was obtained from the liquid culture. Solid-state fermentation was performed in a 200-mL flask with wet SCR in optimal culture conditions and incubated at 25 °C. All of the media were autoclaved at 121 °C for 20 min prior to use. 2.3. Cell lines The murine macrophage cell line, RAW 264.7 was obtained from the Riken Cell Bank (Tsukuba, Japan) and maintained in MEM medium containing 10% fetal bovine serum, 100 U/mL penicillin and 100 lg/mL of streptomycin at 37 °C in a humidified 5% CO2 atmosphere (ESPEC CO2 Incubator). The cells were cultured
HO scavenging activity, the ferrous metal ion-chelating activity, the reducing power and the ability of GLPL to quench H2O2 were measured according to a literature procedure with certain modifications (Liu et al., 2010). DPPH radical-scavenging activities and the levels of SOD-like activity of GLPL were measured according to the manufacturer’s instructions (Shi et al., 2012a,b). ABTS radical-scavenging activity of GLPL was measured according to the method of Trishna et al. (2011) with slight modifications. 2.6. Immunomodulation activities of GLPL 2.6.1. Bioactivity assay The effect of GLPL on the proliferation of RAW 264.7 cells was estimated using CCK-8, and the method described by Shi et al. (2012a,b). The data were expressed as percentages of the control. 2.6.2. Measurement of the production of the nitric oxide The nitrite accumulation was measured using the Griess reagent and used as an indicator of nitric oxide (NO) production in the medium (Shi et al., 2012a,b). NaNO2 was used as a standard to calculate the nitrite concentrations. 2.6.3. Phagocytosis assay The phagocytic ability of the macrophages was measured using a neutral red uptake assay (Wei, Zhao, Shi-Fei, & Yong-Quan, 2008). 2.6.4. Protective activity RAW 264.7 cells were cultured in a 96-well plate at a density of 5 104 cells/mL for 24 h at 37 °C in a 5% CO2 atmosphere. Then the cells were incubated with DOX (5.00 lmol/L) in the presence or absence of various concentrations of GLPL for 24 h. After drug exposure, 10.00 lL of CCK-8 solution was added and incubated at 37 °C for 4 h. The cell numbers were quantified by reading the absorbance at 450 nm. The data were expressed as the percentages of the control. 2.7. Statistical analysis The factors affecting GLPL extraction by ultrasonic assisted extraction are shown in Table 1. According to the orthogonal
52
M. Shi et al. / Food Chemistry 155 (2014) 50–56
Table 1 Levels and factors affecting the yield of GLPL. Level
1 2 3 a
Factors Factor A time (min)
Factor B temperature (°C)
Factor C power (W)
Factor D liquid: solida
10 20 30
30 50 80
50 80 110
10: 1 20: 1 30: 1
Liquid: solid was liquid (mL): solid (g).
experiment design L9 (34), four of these factors, each at three different levels, were selected for the study. All experiments were performed at least in duplicate, and analyses of all samples were done in four replicates and averaged. Statistical analyses were done using the DPS statistical analysis (DPS, version 13.5) software package (Hangzhou city, Zhejiang province, China). The results were presented as means of three determinations ± SD (standard deviation). Significant differences of GLPL between two means were determined by Duncan multiple-range tests. Means were compared by the least significant difference test at 0.05 significance levels.
3. Results and discussion 3.1. Optimisation of GLPL extraction The results from optimisation of GLPL extraction are showed in Table 2. These results show the range of GLPL yield varied from 37.35 to 115.47 mg/mL, and the values therein were taken as the original data to be used in the range analysis and the analysis of variance (ANOVA). For each factor, a higher mean value (Kji) indicates the ‘factor’ increased yield. Therefore, the best level for each factor can be determined according to the highest mean value of the experimental condition (Kji). Based on data shown in Table 2, the highest GLPL yield was achieved with: extraction time 30 min (73.46), extraction temperature 80 °C (90.13), ultrasonic power 80 W (71.09) and a 10:1 ratio of liquid to solid (71.59). The best combination was A3B3C2D1 (number 9, Table 2). The range value (Rj) indicates the significance of the effect and a larger Rj means the factor increased yield. Therefore, comparison of Rj for the different factors, suggests the significance of the factors on yield were: extraction temperature (42.00) > extraction time (17.83) > ultrasonic power (12.99) > ratio of liquid to solid (11.06). Extraction temperature was the best indicator of
significant for the yield of GLPL. The product yield of GLPL changed only slightly with changes in the ratio of liquid to solid, because the range value of the ratio of liquid to solid was small. The result of the orthogonal experiment also showed that all of the single factor effects on the yield of GLPL were significant (p < 0.01) (Table 3). The mean yield of GLPL under optimum extraction conditions was 113.59 ± 3.78 mg/g. This proves the response model constructed was adequate in predicting optimal conditions that are achievable in laboratory settings. The industrial settings will be optimised in the future. In this study, soybean curd residue was used as a solid substrate to produce GLPL by G. lucidum for the first time. After fermentation, GLPL yield was increased as much as sixfold compared with unfermented SCR; additionally, the total phenolics, proteins and amino acids were increased significantly compared to unfermented SCR (Shi et al., 2012a,b). As the quantity of active substances in SCR was apparently raised after fermentation, fermented SCR could be a potential nutritious (protein) and functional food (phenolics). Using ultrasound irradiation (20–100 kHz) in otherwise conventional extraction methods can reduce or eliminate structural changes and degradation of polysaccharides (Khan et al., 2010); thus, ultrasonic-assisted extraction may be an effective and advisable technique for the extraction of polysaccharides. Our results showed that GLPL yield was increased significantly, by more than twofold, using ultrasonic extraction compared hot water extraction (48.14 mg/g) (Shi et al., 2012a,b).
3.2. Antioxidant activities of GLPL 3.2.1. Scavenging activity of hydroxyl radicals by GLPL As shown in Fig. 1A, GLPL could scavenge HO (98.83%) at 5.00 mg/mL. This compares well with ascorbic acid (positive control), which exhibited 99.28% hydroxyl radical scavenging activity at 1.25 mg/mL. Liu et al. (2010) showed that 8 mg/mL of
Table 2 Experimental design and results of the orthogonal experiment L9 (34).
a b c d
Run no.
Factor A time (min)
Factor B temp (°C)
Factor C power (W)
Factor D liquid: solida
Polysaccharide (mg/g)
1 2 3 4 5 6 7 8 9 K1c K2 K3 Rd Optimal level
10 10 10 20 20 20 30 30 30 63.68 ± 3.62 53.65 ± 3.91 73.46 ± 4.66 17.83 ± 1.03 3
30 50 80 30 50 80 30 50 80 43.50 ± 2.35 57.17 ± 3.44 90.13 ± 5.09 42.00 ± 2.72 3
50 80 110 80 110 50 110 50 80 56.67 ± 2.45 71.09 ± 4.37 63.03 ± 1.90 12.99 ± 1.06 2
10: 1 20: 1 30: 1 30: 1 10: 1 20: 1 20: 1 30: 1 10: 1 71.59 ± 2.89 59.90 ± 1.57 59.30 ± 2.84 11.06 ± 1.03 1
44.64 ± 1.30b 60.46 ± 1.15 85.95 ± 4.31 37.35 ± 1.40 54.65 ± 1.48 68.96 ± 2.40 48.49 ± 0.62 56.41 ± 2.96 115.47 ± 2.95
Liquid: solid was liquid (mL): solid (g). Values were mean of three determinations with standard deviation (±). P K Ai ¼ polysaccharide yield at Ai . Values were mean of three determinations with standard deviation (±). RA ¼ maxfK Ai g minfK Ai g. Values were mean of three determinations with standard deviation (±).
M. Shi et al. / Food Chemistry 155 (2014) 50–56 Table 3 Variance analysis of the orthogonal experiment. Variation source
d.f.
Sum of squares
F value
p (t-test)
Time (min) Temp (°C) Power (W) Liquid (mL): solid (g)a Error Sum
2 2 2 2 2 16
1764.48 10342.13 940.32 863.39 94.83 14009.04
148.86 872.48 79.33 72.84
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
Effect of ultrasonic extraction conditions on antioxidative and immunomodulatory activities of a Ganoderma lucidum polysaccharide originated from fermented soybean curd residue Min Shi, Yingnan Yang ⇑, Xuansheng Hu, Zhenya Zhang Graduate School of Life and Environmental Science, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan
a r t i c l e
i n f o
Article history: Received 1 March 2012 Received in revised form 18 December 2013 Accepted 15 January 2014 Available online 23 January 2014 Keywords: Soybean curd residue Ganoderma lucidum Polysaccharide Ultrasonic assisted extraction Antioxidant activities Immunomodulation activities
a b s t r a c t A crude Ganoderma lucidum polysaccharide (GLPL) was extracted from fermented soybean curd residue by ultrasonic assisted extraction. The optimal extraction conditions were 30 min at 80 °C with 80 W and water to solid ratio of 10, and with this method 115.47 ± 2.95 mg/g of GLPL yield was obtained. Additionally, the antioxidant and immunomodulatory activities of GLPL were investigated. The results showed that GLPL exhibited strong antioxidant effects, which included scavenging activities against DPPH radicals, hydrogen oxide and ABTS radicals with IC50 values of 0.23, 0.48 and 0.69 mg/mL, respectively. For immunomodulatory activities, GLPL was shown to strongly stimulate the proliferation of macrophages (158.02 ± 13.12%), the production of nitric oxide and phagocytosis (21.16 ± 1.65 lM), and, at 40.00 lg/mL, protected macrophage from Doxorubicin (DOX) (0.16 ± 0.003). Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Ganoderma lucidum (G. lucidum), a medicinal fungus called ‘‘Lingzhi’’ in China is one of the most famous fungi in traditional Chinese medicine. In regions of China and other Asian countries, G. lucidum has been used as a remedy to promote health and longevity (Yuen & Gohel, 2008). Modern pharmaceutical research shows that G. lucidum polysaccharide (GLPL) has some physiological effects, including strong antioxidant, immunomodulating (Lin et al., 2006), and anti-tumour activities (Yuen & Gohel, 2008). Traditionally, polysaccharides are extracted from G. lucidum fruiting bodies; however, the incubation time of these fruiting bodies is as long as 60 days with a polysaccharide yield of 32.7 mg/g (Huanga & Ninga, 2010). In this study, GLPL was produced using the food waste from soybean curd residue (SCR), which could greatly reduce the production time. Therefore, this is a promising new technology for GLPL production. SCR, a by-product of tofu, soymilk or soy protein processing, is discharged as an agro-industrial waste and has caused severe environmental pollution. In fact, SCR is rich in fat, starch, protein and sugar and could be used as a high quality media for microbial fermentation (Shi, Yang, Guan, Wang, & Zhang, 2012a,b). However, there are few previous reports describing the production of GLPL ⇑ Corresponding author. Tel./fax: +81 29 853 4650. E-mail address: [email protected] (Y. Yang). 0308-8146/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2014.01.037
using SCR, and GLPL from natural food waste could be used as a functional food additive in the future. For the present, several conventional extraction techniques have been reported for the extraction of GLPL. Hot water technology is the main and most conventional extraction method for polysaccharides mentioned in recent studies (Guan, Zhang, Yang, Xin, & Liu, 2011; Kazahiro, Akira, Naomi, & Hidenori, 2009; Shi et al., 2012a,b). However, it is usually associated with a longer extraction time, more than 2 h, and a higher temperature (95 °C) but a lower extraction efficiency with a yield of less than 70.00 mg/g (Xiangqun, Yongde, & Hui, 2011). Recently, ultrasonic assisted extraction has been employed widely in the extraction of target compounds from different materials because of the facilitated mass transfer between immiscible phases through super agitation at a low frequency. It offers the benefits of high reproducibility, shorter times, simplified manipulation, and lowered energy input and solvent consumption (Khan, Maryline, Fabiano-Tixier, Dangles, & Chemat, 2010). Until now, there has been no comprehensive evaluation of the antioxidant and immunomodulatory activities of GLPL. Papers report that polysaccharides from mushrooms can enhance and activate macrophage-based immune responses, leading to immunomodulation, anti-tumour activity, wound-healing and other therapeutic effects (Huanga & Ninga, 2010; Shi et al., 2012a,b). Macrophages have a significant role in host defence mechanisms. Phagocytic activity produces reactive oxygen species (ROS) and
M. Shi et al. / Food Chemistry 155 (2014) 50–56
nitric oxide (NO) in response to stimulation from a variety of agents, and it can inhibit the growth of a wide variety of tumour cells and micro-organisms (Schepetkin & Quinn, 2006). Moreover, the immunomodulatory activity not only involves effects macrophage activation but also proliferation and differentiation of these cells (Schepetkin & Quinn, 2006). Reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), hydroxyl radical (HO) and other free radicals, are byproducts of biological metabolism (Xu et al., 2009). In recent years, many studies have shown that ROS may be responsible for or contribute to human diseases (Liu, Wang, Pang, Yao, & Gao, 2010; Wu & Hansen, 2008; Xu et al., 2009). Antioxidants can scavenge free radicals and protect against diseases. Bioactive components in fruits and vegetables have been shown to have beneficial activity (Wu & Hansen, 2008; Xu et al., 2009). In recent years, there has been increasing evidence that some polysaccharides isolated from plants, herbs and fungi also have putative health benefits and low cytotoxicity (Chatchai, Saranyu, Khajeelak, Chantragan, & Rakrudee, 2011; Maja, Klaus, Dragica, Johannes, & Leo, 2011). The purpose of this study was to extract GLPL using an optimal ultrasonic assisted extraction method from fermented SCR using G. lucidum, and to verify its antioxidant activities and immunomodulatory activities in vitro.
51
for 2–3 days to reach logarithmic phase and then used for experiments. 2.4. Preparation of GLPL The fermented SCR was dried in a convection oven at 50 °C and ground to a powder. The impurities were removed from the crushed powder (1.00 g) with 80% ethanol at room temperature for 24 h. The eluent was discarded and the residue was further extracted using different experimental conditions of ultrasonic assisted extraction. Then, the extract was filtered and centrifuged at 7500 rpm for 30 min at room temperature. The supernatant was concentrated in a rotary evaporator under reduced pressure at 50 °C and free protein layer was removed using the method of Sevage (Staub, 1999). Finally, the extract was subjected to precipitation with fourfold volumes of ethanol. The GLPL curds were collected by centrifugation, washed with ethanol twice, and freeze-dried. Total GLPL was determined using the phenol–sulphuric acid method, and D-glucose was used as the standard (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956). The results were expressed as milligram of glucose equivalent per gram of fermented SCR. 2.5. Assay for antioxidant activities of GLPL
2. Materials and methods 2.1. Materials and chemicals Eagle’s minimum essential medium (MEM), 2,2-diphenyl-1picry-hydrazyl radical (DPPH), fetal bovine serum (FBS), phosphate buffered saline (PBS), trichloroacetic acid (TCA) and 2,20 -Azinobis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) were purchased from Sigma–Aldrich, Inc. (Saint Louis, MO, USA). Lipopolysaccharide (LPS) from Escherichia coli 055, ascorbic acid, hydrogen peroxide, chloride ferric, ferrous sulphate, trichloroacetic acid, sodium salicylate and ethylenediaminetetraacetic acid (EDTA) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). The SOD Assay Kit-WST and Cell Counting Kit-8 (CCK-8) were purchased from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). Doxorubicin (DOX) was purchased from TopoGEN, Inc. (Florida, USA). All other chemical reagents were of analytical grade. 2.2. Microorganism and culture conditions The A50 strain of G. lucidum was purchased from Agriculture and Forestry Strains in Kaishas, Japan. The strain was maintained on potato dextrose agar (PDA) at 4 °C. 15 mL of the liquid culture was added to a 50-mL flask containing one unit of mycelia agar, which was a 5 mm 5 mm square that was obtained using a self-designed cutter. The initial pH was from 5.0 to 5.5, and the culture was incubated on a rotary shaker at 100 rpm and 25 °C for 6 days. The seed for the solid culture was obtained from the liquid culture. Solid-state fermentation was performed in a 200-mL flask with wet SCR in optimal culture conditions and incubated at 25 °C. All of the media were autoclaved at 121 °C for 20 min prior to use. 2.3. Cell lines The murine macrophage cell line, RAW 264.7 was obtained from the Riken Cell Bank (Tsukuba, Japan) and maintained in MEM medium containing 10% fetal bovine serum, 100 U/mL penicillin and 100 lg/mL of streptomycin at 37 °C in a humidified 5% CO2 atmosphere (ESPEC CO2 Incubator). The cells were cultured
HO scavenging activity, the ferrous metal ion-chelating activity, the reducing power and the ability of GLPL to quench H2O2 were measured according to a literature procedure with certain modifications (Liu et al., 2010). DPPH radical-scavenging activities and the levels of SOD-like activity of GLPL were measured according to the manufacturer’s instructions (Shi et al., 2012a,b). ABTS radical-scavenging activity of GLPL was measured according to the method of Trishna et al. (2011) with slight modifications. 2.6. Immunomodulation activities of GLPL 2.6.1. Bioactivity assay The effect of GLPL on the proliferation of RAW 264.7 cells was estimated using CCK-8, and the method described by Shi et al. (2012a,b). The data were expressed as percentages of the control. 2.6.2. Measurement of the production of the nitric oxide The nitrite accumulation was measured using the Griess reagent and used as an indicator of nitric oxide (NO) production in the medium (Shi et al., 2012a,b). NaNO2 was used as a standard to calculate the nitrite concentrations. 2.6.3. Phagocytosis assay The phagocytic ability of the macrophages was measured using a neutral red uptake assay (Wei, Zhao, Shi-Fei, & Yong-Quan, 2008). 2.6.4. Protective activity RAW 264.7 cells were cultured in a 96-well plate at a density of 5 104 cells/mL for 24 h at 37 °C in a 5% CO2 atmosphere. Then the cells were incubated with DOX (5.00 lmol/L) in the presence or absence of various concentrations of GLPL for 24 h. After drug exposure, 10.00 lL of CCK-8 solution was added and incubated at 37 °C for 4 h. The cell numbers were quantified by reading the absorbance at 450 nm. The data were expressed as the percentages of the control. 2.7. Statistical analysis The factors affecting GLPL extraction by ultrasonic assisted extraction are shown in Table 1. According to the orthogonal
52
M. Shi et al. / Food Chemistry 155 (2014) 50–56
Table 1 Levels and factors affecting the yield of GLPL. Level
1 2 3 a
Factors Factor A time (min)
Factor B temperature (°C)
Factor C power (W)
Factor D liquid: solida
10 20 30
30 50 80
50 80 110
10: 1 20: 1 30: 1
Liquid: solid was liquid (mL): solid (g).
experiment design L9 (34), four of these factors, each at three different levels, were selected for the study. All experiments were performed at least in duplicate, and analyses of all samples were done in four replicates and averaged. Statistical analyses were done using the DPS statistical analysis (DPS, version 13.5) software package (Hangzhou city, Zhejiang province, China). The results were presented as means of three determinations ± SD (standard deviation). Significant differences of GLPL between two means were determined by Duncan multiple-range tests. Means were compared by the least significant difference test at 0.05 significance levels.
3. Results and discussion 3.1. Optimisation of GLPL extraction The results from optimisation of GLPL extraction are showed in Table 2. These results show the range of GLPL yield varied from 37.35 to 115.47 mg/mL, and the values therein were taken as the original data to be used in the range analysis and the analysis of variance (ANOVA). For each factor, a higher mean value (Kji) indicates the ‘factor’ increased yield. Therefore, the best level for each factor can be determined according to the highest mean value of the experimental condition (Kji). Based on data shown in Table 2, the highest GLPL yield was achieved with: extraction time 30 min (73.46), extraction temperature 80 °C (90.13), ultrasonic power 80 W (71.09) and a 10:1 ratio of liquid to solid (71.59). The best combination was A3B3C2D1 (number 9, Table 2). The range value (Rj) indicates the significance of the effect and a larger Rj means the factor increased yield. Therefore, comparison of Rj for the different factors, suggests the significance of the factors on yield were: extraction temperature (42.00) > extraction time (17.83) > ultrasonic power (12.99) > ratio of liquid to solid (11.06). Extraction temperature was the best indicator of
significant for the yield of GLPL. The product yield of GLPL changed only slightly with changes in the ratio of liquid to solid, because the range value of the ratio of liquid to solid was small. The result of the orthogonal experiment also showed that all of the single factor effects on the yield of GLPL were significant (p < 0.01) (Table 3). The mean yield of GLPL under optimum extraction conditions was 113.59 ± 3.78 mg/g. This proves the response model constructed was adequate in predicting optimal conditions that are achievable in laboratory settings. The industrial settings will be optimised in the future. In this study, soybean curd residue was used as a solid substrate to produce GLPL by G. lucidum for the first time. After fermentation, GLPL yield was increased as much as sixfold compared with unfermented SCR; additionally, the total phenolics, proteins and amino acids were increased significantly compared to unfermented SCR (Shi et al., 2012a,b). As the quantity of active substances in SCR was apparently raised after fermentation, fermented SCR could be a potential nutritious (protein) and functional food (phenolics). Using ultrasound irradiation (20–100 kHz) in otherwise conventional extraction methods can reduce or eliminate structural changes and degradation of polysaccharides (Khan et al., 2010); thus, ultrasonic-assisted extraction may be an effective and advisable technique for the extraction of polysaccharides. Our results showed that GLPL yield was increased significantly, by more than twofold, using ultrasonic extraction compared hot water extraction (48.14 mg/g) (Shi et al., 2012a,b).
3.2. Antioxidant activities of GLPL 3.2.1. Scavenging activity of hydroxyl radicals by GLPL As shown in Fig. 1A, GLPL could scavenge HO (98.83%) at 5.00 mg/mL. This compares well with ascorbic acid (positive control), which exhibited 99.28% hydroxyl radical scavenging activity at 1.25 mg/mL. Liu et al. (2010) showed that 8 mg/mL of
Table 2 Experimental design and results of the orthogonal experiment L9 (34).
a b c d
Run no.
Factor A time (min)
Factor B temp (°C)
Factor C power (W)
Factor D liquid: solida
Polysaccharide (mg/g)
1 2 3 4 5 6 7 8 9 K1c K2 K3 Rd Optimal level
10 10 10 20 20 20 30 30 30 63.68 ± 3.62 53.65 ± 3.91 73.46 ± 4.66 17.83 ± 1.03 3
30 50 80 30 50 80 30 50 80 43.50 ± 2.35 57.17 ± 3.44 90.13 ± 5.09 42.00 ± 2.72 3
50 80 110 80 110 50 110 50 80 56.67 ± 2.45 71.09 ± 4.37 63.03 ± 1.90 12.99 ± 1.06 2
10: 1 20: 1 30: 1 30: 1 10: 1 20: 1 20: 1 30: 1 10: 1 71.59 ± 2.89 59.90 ± 1.57 59.30 ± 2.84 11.06 ± 1.03 1
44.64 ± 1.30b 60.46 ± 1.15 85.95 ± 4.31 37.35 ± 1.40 54.65 ± 1.48 68.96 ± 2.40 48.49 ± 0.62 56.41 ± 2.96 115.47 ± 2.95
Liquid: solid was liquid (mL): solid (g). Values were mean of three determinations with standard deviation (±). P K Ai ¼ polysaccharide yield at Ai . Values were mean of three determinations with standard deviation (±). RA ¼ maxfK Ai g minfK Ai g. Values were mean of three determinations with standard deviation (±).
M. Shi et al. / Food Chemistry 155 (2014) 50–56 Table 3 Variance analysis of the orthogonal experiment. Variation source
d.f.
Sum of squares
F value
p (t-test)
Time (min) Temp (°C) Power (W) Liquid (mL): solid (g)a Error Sum
2 2 2 2 2 16
1764.48 10342.13 940.32 863.39 94.83 14009.04
148.86 872.48 79.33 72.84
