Mycelial fermentation characteristics and antiproliferative activity of Phellinus vaninii Ljup
Wei Hu1, Shuai Liu2, Yuexin Zhang3, Deng Xun4, Chunping Xu2
1 Branch of Forest Protection, School of Forestry, Northeast Forestry University; Heilongjiang Forest By product and Specialty Institute, Mudanjiang 157011, China
2 College of Food and Biological Engineering, Zhengzhou University of Light Industry, Zhengzhou, Henan 450002, China
3 Heilongjiang Forest By product and Specialty Institute, Mudanjiang 157011, China
4 Heilongjiang Forestry Protection Institute, Harbin 150040, China
|Date of Submission||17-Dec-2013|
|Date of Acceptance||27-Mar-2014|
|Date of Web Publication||26-Sep-2014|
College of Food and Biological Engineering, Zhengzhou University of Light Industry, Zhengzhou, Henan 450002
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: The mycelial fermentation of higher fungi were investigated to posses various bioactivities. Materials and Methods: The mycelial growth and pellet morphology in a 5-L bioreactor were investigated. The mycelial broth containing biomass and extracellular products harvested from the fermentor was tested for antiproliferative activity of colon cancer LoVo cells using 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide assay. Results: The maximum mycelial concentration in a 5-L bioreactor was 12.5 g/L after 8 days cultivation. Further investigation in the mycelial pellets during the fermentation period revealed that the mean diameter of the pellet morphology was positively correlated with mycelial biomass (R2 = 0.82, P < 0.05) and broth viscosity (R2 = 0.90, P < 0.01), significantly. The ethyl acetate extract showed the most significant effects, increasing the inhibition rate up to 87.5% after 48 h at concentration of 1000 μg/mL. Conclusion: The results demonstrated the feasibility of P. vaninii Ljup mycelial fermentation for large-scale production of bioactive and medicinal compounds.
Keywords: Antiproliferative activity, fermentation, morphology, mycelia, Phellinus vaninii Ljup
|How to cite this article:|
Hu W, Liu S, Zhang Y, Xun D, Xu C. Mycelial fermentation characteristics and antiproliferative activity of Phellinus vaninii Ljup. Phcog Mag 2014;10:430-4
|How to cite this URL:|
Hu W, Liu S, Zhang Y, Xun D, Xu C. Mycelial fermentation characteristics and antiproliferative activity of Phellinus vaninii Ljup. Phcog Mag [serial online] 2014 [cited 2021 May 11];10:430-4. Available from: http://www.phcog.com/text.asp?2014/10/40/430/141812
| Introduction|| |
Higher fungi in fruit body and mycelium forms have been increasingly applied as functional foods and a source of pharmaceutical compounds due to their well-known nutritive and medicinal properties, such as immunomodulation, anticancer, antioxidant, hypolipidemic and hyperglycemic activities.  Phellinus vaninii Ljup, is a famous Chinese medicinal fungus belonging to Basidiomycetes, Aphyllophorales, Hymenochaetacae, Phellinus.  P. vaninii Ljup was first discovered in the far East of Asian Russia and mainly distributed in the Northeast China.  Bioactive metabolites from P. vaninii Ljup can be isolated from fruiting bodies, pure culture mycelia and culture broth, including phenolic compounds, flavonoids, polysaccharides, triterpenoids, terpenes, alkaloid, etc., and polysaccharide is one of the most important bioactive compounds. , P. vaninii Ljup was found to possess a significant inhibition effect of many cancer cells and can treat for dyspepsia as well without toxicity. ,,
Many studies on fungal fermentations have been investigated for mycelial and metabolite product production in a stirred tank reactor. , However, no information is available in the literature on the characteristics and performance of P. vaninii Ljup mycelial fermentation. P. vaninii Ljup has been isolated, cultivated artificially and obtained sporocarp successfully in our Laboratory.  To evaluate the potential capability of P. vaninii Ljup mycelial liquid fermentation for commercial application, we performed this study in a 5-L stirred-tank fermenter. The mycelial morphology and broth rheology were related to the mycelial biomass production during the fermentation. The mycelial broth and biomass attained from the fermenter were applied for inhibition effects on the proliferation of colon cancer LoVo cells.
| Materials and methods|| |
Microorganism and growth conditions
Phellinus vaninii Ljup was isolated from Wanda Mountains of Heilongjiang Province and kept in the Forest Microbiological Research Center of Northeast Forestry University (deposit no. 199537). Stock cultures were maintained on potato dextrose agar slants. The liquid culture of mycelia was initiated by transferring the fungal mycelia from the stock culture on a Petri dish More Details into the seed culture medium. The seed culture was propagated in a 250 ml Erlenmeyer flask containing 50 ml of liquid medium at 26°C on a shaking incubator at 150 rpm for 4 days. The fermentation liquid medium was composed of 30 g glucose and 3 g peptone (per liter) with an initial pH about 7.0 (not adjusted).
Fermentations in bioreactors
Four percent (v/v) of mycelial suspensions was carried out in a 5-L stirred-tank reactor with sixblade Rushton turbine impeller (Infors, Switzerland). Unless otherwise specified, fermentations were performed under the following conditions: 28°C, agitation speed 160 rpm, aeration rate 2 vvm, working volume 3 L, cultivation time 12 days. Samples were taken aseptically from the bioreactor at 2-day intervals for analyses.  All experiments were performed in triplicate to ensure the trends observed were reproducible.
Measurements of the viscosity and morphology
The viscosity measurements were performed on samples collected from bioreactor at regular intervals using a using a Brookfield programmable LVDVII + digital viscometer (Brookfield Engineering Laboratories Inc., Stoughton, MA, USA) fitted with a small sample adapter. The morphological properties of the samples collected were evaluated using an image analyzer (DT2000 System, China) with software linked to a light microscope (Nikon, Japan) through a CCD camera. Samples were fixed with an equal volume of ﬁxative (13 ml of 40% formaldehyde, 5 ml glacial acetic acid, and 200 mL of 50% ethanol). An aliquot (0.1 mL) of each ﬁxed sample was transferred to a slide, air dried, and then stained with methylene blue (0.3 g of methylene blue, 30 ml of 95% ethanol in 100 mL water). For each sample, the morphology of pellet was characterized by measuring the area and perimeter of the pellet core and the maximum diameter of the pellet. Normally, a 40-fold magniﬁcation was used. The morphology of the pellets was characterized by their mean diameter, circularity, roughness, and compactness. The circularity was estimated as the ratio of the Fieret's minimum diameter to the Fieret's maximum diameter of the pellets or aggregates. The compactness was estimated as the ratio of the projected area of the hyphae in a clump to the projected convex area of that clump, the latter being the area after filling internal voids and concavities in the clump's external perimeter. In addition, the roughness (R) was measured using the following equation:
R = (pellet/aggregateperimeter) 2/(4π× pellet area) 
Processing of mycelial fermentation broth for antiproliferation measurements
The mycelial fermentation broth was centrifuged at 8000 g for 15 min to obtain the mycelium biomass. The mycelial pellet was washed thoroughly with distilled water and dried at 60°C in an oven till constant weight for measurement of the dry weight.  The liquid broth containing mycelial biomass and extracellular metabolite products from the fermentors was processed in a batch by spray-drying into dried powder with an inlet air temperature 185-190°C. The power was divided into four equal portions and extracted with reflux for 3 times (1 h and each) by the following solvents (solid/liquid ratio 1:3) and methods: Boiling water (WE), WE with ultrasonication (30 min, 100 W) (WUE), butanol (BE) and ethyl acetate extract (EAE), respectively. The liquid extract was concentrated by evaporation and then freeze-dried. The WE, WUE, BE, and EAE were applied to the antiproliferation activity tests.
Evaluation of antiproliferation activity
For the antiproliferation effect study, LoVo cells of colon cancer were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum in a humidified 5% CO 2 atmosphere at 37°C.  The proliferation of LoVo cells was determined using the colorimetric 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay described by Mosmann.  Briefly, cells were seeded at a density of 3 × 10 4 cells/well in a 100 μL volume of the medium in 96-well plates and allowed to attach for 24 h. The dosages of extract were 1, 10, 100, and 1000 μg/mL while distilled water instead of the extract was used for the negative controls. Ten microliter MTT (0.4%) was added after 48 h. After incubated at 37°C for 4 h, the supernatant was aspirated and then 150 μL dimethyl sulfoxide was added to each well. Absorbance was measured at 490 nm by a 96 well microplate reader (Tecan, GENios ELIASA Co., Austria). All results in vitro were expressed as the inhibition rate of tumor cell proliferation as follows:
Inhibition rate (%) = (1−A sample /A control )×100% (1)
Where A sample and A control were defined as absorbances of the sample, control (without extract). And IC 50 was determined by nonlinear regression analysis using the GraphPad PRISMe statistics software package (Version. 2.0;
San Diego, CA, USA).
Data were expressed as mean ± standard deviation. The results were analyzed for statistical significance by one-way analysis of variance test using the Statistical Package of the Social Science (SPSS) version 11.0 (SPSS Inc., Chicago, IL, USA). The statistical differences were considered significant at P < 0.05. Using the Pearson coefficient analysis was used to analyze the coefficient of determination (R2 ) and evaluate the relationship among mycelial production, mycelial morphology, and broth viscosity.
| Results and discussion|| |
Fermentation result in a stirred-tank reactor
[Figure 1] shows the typical time courses of mycelial culture in a 5-L stirred-tank bioreactor, including biomass growth, nutrient consumption and medium pH change. The mycelial biomass grew rapidly (exponentially) from day 2 to 6 and reached a maximum biomass (12.5 g/L) in day 8 [Figure 1]a]. As expected, the concentration of residual sugar dropped to below 5 g/l at day 8 and to nearly zero at day 12 [Figure 1]b]. However, no drastic change in pH was recorded during the course of fermentation [Figure 1]c].
|Figure 1: Time profiles of (a) mycelial biomass, (b) residual sugar, and (c) pH in submerged culture of Phellinus vaninii Ljup in a stirred-tank fermenter|
Click here to view
Characterization of broth viscosity and mycelial morphology
The apparent viscosity of the whole broth according to the fermentation period is depicted in [Figure 2]. The viscosity of the fermentation broth at 300 rpm increased rapidly as the cells entered their exponential growth. This continued up to day 8 (64.5 mPa.S), which microorganisms entered their stationary phase and the viscosity of the broth, which was proportional to the cell concentration, declined accordingly. Between two typical fungal morphologies (e.g. filamentous and pelleted), pellet morphology was cited as one of the key factors directly affecting fermentation productivity.  [Figure 3] shows the typical morphological changes (2 days interval) during the entire fermentation period (0-12 days). The cells were observed to form mainly pellets during the entire culture period. The pellet diameter increased rapidly, and the outer hairy regions of the pellets became fluffier. [Figure 4] shows the mean diameter (a), circularity (b), compactness (c), and roughness (d) of the pellets during cultivation of P. vaninii Ljup. The compactness, mean diameter and roughness of the pellets increased during the 4, 10, and 8 days of fermentation, respectively, and then decreased, but no drastic changes in circularity were revealed during the course of fermentation. Further investigation in the mycelial pellets during the fermentation period revealed that mean diameter of the pellet was positively correlated with mycelial biomass (R2 = 0.82, P < 0.05) and broth viscosity (R2 = 0.90, P < 0.01), significantly. These results clearly show that the mycelial morphological change was coincidental with fungal biomass production and viscosity change. Our findings were consistent with Hwang et al., who reported Phellinus baumii showed the largest pellet size, which accordingly resulted in extremely high apparent viscosity.  It is also found that the mycelial biomass was positively correlated with broth viscosity (R2 = 0.96, P < 0.01) during the course of fermentation. It is similar to the finding by Hwang et al., which indicated that the morphological variation of the three Phellinus species was closely linked to the apparent viscosity of the whole broth. 
|Figure 2: Time profile of apparent viscosity of fermentation broth of Phellinus vaninii Ljup in a stirred-tank fermenter|
Click here to view
|Figure 3: The morphological changes in Phellinus vaninii Ljup in a stirred-tank fermenter. Representative images were taken at 40-fold magnification. One to 9 day means fermentation period in day|
Click here to view
|Figure 4: Mean diameter (a), circularity (b), compactness (c), and roughness (d) of Phellinus vaninii Ljup pellets growing in a stirred-tank fermenter|
Click here to view
Antiproliferative effects of mycelial fermentation products
Colon cancer is the third most commonly diagnosed cancer in the world; but, it is more common in developed countries.  In this work, to evaluate the effect of growth inhibition, various concentration of extracts from P. vaninii Ljup (i.e. WE, WUE, BE and EAE) (1 μg/L-1000 μg/L) was added to the culture medium of colon cancer LoVo cells. It shows that all extracts have the inhibition effect on LoVo cell proliferation [Table 1]. The inhibition effects of BE and EAE are better than that of WE and WUE, and the effects were significantly dose-dependent. At 1000 μg/ml and 48 h after medicating, the inhibition rate of BE on colon cancer LoVo cell was 86.8%, the inhibition rate of EAE was 87.5%. Though the inhibition effects of BE and EAE are similar, taking into account that the evaporating temperature of ethyl acetate is lower than that of BE under vacuum, ethyl acetate is regarded as a desirable solvate. And the mean IC 50 of EAE against LoVo cells was 53.459 μg/mL. Similar to our results, Ajith and Janardhanan investigated the antitumor activities of ethyl acetate, methanol and aqueous extracts from the sporocarp of Phellinus rimosus (Berk) Pilat and it showed that extract of ethyl acetate was the most effective than others to inhibit of solid tumor induced by Dalton's lymphoma ascites cell line.  The incubation time of EAE (100 μg/mL) on inhibition effects of colon cancer LoVo cells was investigated and showed in [Figure 5]. The inhibition rate is enhanced corresponding to increase with the incubation time, and under the incubation time of 120 h, the inhibition rate on cancer cells reaches almost 100%.
|Figure 5: Effect of incubation time of ethyl acetate extract on tumor LoVo cells|
Click here to view
|Table 1: Antiproliferative activity of WE, WUE, BE, and EAE on tumor LoVo cells |
Click here to view
| Conclusions|| |
In this work, mycelial fermentation characteristics of
P. vaninii Ljup were evaluated in the fermentor for the first time. Our results indicated that the pellet formation and viscosity of P. vaninii Ljup were closely correlated during the fermentation. The EAE of P. vaninii Ljup exhibited the highest activity in the antiproliferative effect in vitro. In the future, pharmacological and biochemical investigations, especially acute toxicity, are needed to analyze the active compounds (could be polysaccharides) from P. vaninii Ljup.
| References|| |
|1.||Wasser SP. Medicinal mushrooms as a source of antitumor and immunomodulating polysaccharides. Appl Microbiol Biotechnol 2002;60:258-74. |
|2.||Dai YC. Hymenochaetaceae (Basidiomycota) in China. Fungal Divers 2010;45:131-343. |
|3.||Dai YC. A new kind of medicinal fungi-Phellinus vaninii0 (Phellinus spp). Chin Edible Fungi 2003;22:7-8. |
|4.||Cheng XY, Bao HY, Ding Y, Kim YH. Free radical scavenging activities of phenolic and flavonoid compounds from fruiting body of Phellinus vaninii. Mycosystema 2011;30:281-7. |
|5.||Li G, Kim DH, Kim TD, Park BJ, Park HD, Park JI, et al. Protein-bound polysaccharide from Phellinus linteus induces G2/M phase arrest and apoptosis in SW480 human colon cancer cells. Cancer Lett 2004;216:175-81. |
|6.||Cheng KC, Demirci A, Catchmark JM. Evaluation of medium composition and fermentation parameters on pullulan production by Aureobasidium pullulans. Food Sci Technol Int 2011;17:99-109. |
|7.||Chen W, Zhao Z, Chen SF, Li YQ. Optimization for the production of exopolysaccharide from Fomes fomentarius in submerged culture and its antitumor effect in vitro. Bioresour Technol 2008;99:3187-94. |
|8.||Hu W, Wang CS, Zhang J. The preliminary study on the cultivation of Phellinus spp. Substitute materials. Chin Forest Prod 2007;86:12-3. |
|9.||Bae JT, Sinha J, Park JP, Song CH, Yun JW. Optimization of submerged culture conditions for exo-biopolymer production by Paecilomyces japonica. J Microbiol Biotechnol 2000;10:482-7. |
|10.||Park JP, Kim YM, Kim SW, Hwang HJ, Cho YJ, Lee YS, et al. Effect of agitation intensity on the exo-biopolymer production and mycelial morphology in Cordyceps militaris. Lett Appl Microbiol 2002;34:433-8. |
|11.||Park JP, Kim SW, Hwang HJ, Yun JW. Optimization of submerged culture conditions for the mycelial growth and exo-biopolymer production by Cordyceps militaris. Lett Appl Microbiol 2001;33:76-81. |
|12.||Zhang H, Zhang M, Yu L, Zhao Y, He N, Yang X. Antitumor activities of quercetin and quercetin-5',8-disulfonate in human colon and breast cancer cell lines. Food Chem Toxicol 2012;50:1589-99. |
|13.||Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55-63. |
|14.||Fazenda ML, Seviour R, McNeil B, Harvey LM. Submerged culture fermentation of "higher fungi": The macrofungi. Adv Appl Microbiol 2008;63:33-103. |
|15.||Hwang HJ, Kim SW, Xu CP, Choi JW, Yun JW. Morphological and rheological properties of the three different species of basidiomycetes Phellinus in submerged cultures. J Appl Microbiol 2004;96:1296-305. |
|16.||Haggar FA, Boushey RP. Colorectal cancer epidemiology: Incidence, mortality, survival, and risk factors. Clin Colon Rectal Surg 2009;22:191-7. |
|17.||Ajith TA, Janardhanan KK. Cytotoxic and antitumor activities of a polypore macrofungus, Phellinus rimosus (Berk) Pilat. J Ethnopharmacol 2003;84:157-62. |
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]