International Food Research Journal 20(5): 2301-2307 (2013) Journal homepage: http://www.ifrj.upm.edu.my Molecular phylogenetic analysis of wild Tiger’s milk mushroom (Lignosus rhinocerus) collected from Pahang, Malaysia and its nutritional value and toxic metal content 1*Lai, W.H., 1Loo, S.S., 1Rahmat, N., 1Shaharuddin, S., 2Daud, F., 2Zamri, Z. and 1Saleh, N.M. 1Agro-Biotechnology Institute, Ministry of Science, Technology and Innovation, c/o Malaysian Agricultural Research and Development Institute, 43400, Serdang, Selangor Darul Ehsan, Malaysia 2School of Biosciences and Biotechnology, Faculty of Science and Technology, National University of Malaysia, 43600, Bangi, Selangor Darul Ehsan, Malaysia Article history Abstract Received: 4 October 2012 Tiger’s Milk mushroom has been used for medicinal purposes by local aborigines to treat Received in revised form: asthma, breast cancer, cough, fever and food poisoning. Molecular phylogenetic analysis 1 April 2013 utilizing RNA polymerase II, second largest subunit (RPB2) gene, identified the wild Tiger’s Accepted: 5 April 2013 Milk mushrooms collected from the state of Pahang in Malaysia for this study as Lignosus rhinocerus in the order Polyporales. The tuber, stipe and pileus of this mushroom were analyzed Keywords for their basic nutritional composition (fat, protein, and carbohydrate) and toxic metal content profile (Cadmium, Lead and Mercury). The moisture content of these mushroom parts varied Lignosus rhinocerus from 32.22% (pileus) – 46.31% (stipe). The dry matter of the mushrooms contained 2.76% Medicinal mushroom (stipe) – 6.60% (pileus) proteins, 0.21% (pileus) – 0.30% (tuber) fat, 1.76% (stipe) – 4.38% RPB2 gene (tuber) ash and 38.47% (stipe) – 56.30% (pileus) carbohydrates. The toxic metal content of the Phylogenetic mushroom samples ranged from 0.03–0.12 mg/kg for Cd, 0.80–1.94 mg/kg for Pb and 0.05– Proximate analysis 0.10 mg/kg for Hg. The present study demonstrated that L. rhinocerus is a potential source of Toxic metals food due to its high carbohydrate content. In addition, the trace levels of toxic metals in this mushroom are within the safe level for consumption. © All Rights Reserved Introduction et al., 2009). In a recent report, the phylogenetic of wild Tiger’s Tiger’s Milk mushroom (Lignosus spp.) are Milk mushroom collected from Perak was studied native to tropical parts of the world and has been using the RNA polymerase II second largest subunit utilized as traditional medicine to treat asthma, breast (RPB2) gene (Sotome et al., 2008). The RPB2 gene is cancer, cough, fever and food poisoning (Lee et al., an excellent tool for phylogenetic studies (Sun et al., 2012). Lignosus spp. belong to the Polyporaceae 2008) and this gene, which encodes the second large family in the phylum Basidiomycota and comprise subunit of RNA Polymerase II, is highly conserved of six species; L. dimiticus, L. ekombitii, L. goetzii, among eukaryotes and the Archae group (Matheny L. rhinocerus, L. sacer and L. hainanensis (Ryvarden et al., 2007). It encodes a protein with a modest rate and Johnson, 1980; Núñez and Ryvarden, 2001; of evolutionary change and its polypeptide sequence Douanla-Meli and Langer, 2003; Cui et al., 2010). has been used for phylogenetic studies in green plants Tiger’s Milk mushroom has three distinct parts: cap and Ascomycetes (Malkus et al., 2006). In addition, (pileus), stem (stipe), and tuber (sclerotium). They Reeb et al. (2004) also highlighted the importance of are polypores as the stem is centrally joined to the protein-coding genes in resolving deep phylogenetic brownish woody cap that grows from a tuber in soil in relationships. humid environment. The irregularly shaped tuber has Currently, local herbalists are collecting Tiger’s leathery flesh consisting of whitish mycelia (Chroma Milk mushroom from the remote regions of the states meter values are L* = 65.73, a* = 3.76, b* = 10.79). of Pahang and Perak in Malaysia to accommodate the In Malaysia, Lignosus rhinocerus is the most popular growing demand. Although this mushroom has been and specifically sought after medicinal mushroom widely used and consumed as traditional medicine, by the Semai aborigine upon request by urban information on its nutritional properties and toxic middlemen consisting mostly of local herbalists (Lee metals contents are scarce. Hence, this study is *Corresponding author. Email: [email protected] 2302 Lai et al./IFRJ 20(5): 2301-2307 essential to mushroom growers and enthusiasts to 3’) were designed based on known RPB2 sequences understand the nutritional values and toxic metals in GenBank using Primer Premier (version 5.0) content of wild Tiger’s Milk mushroom. Raised software. The protocols for polymerase chain reaction awareness of the nutritional content and safety of (PCR) were 1 cycle of 95ºC for 15 min followed by this mushroom will promote the cultivation and 35 cycles of 94ºC for 1 min, 53ºC for 1 min, 72ºC for consumption of this mushroom. 25 sec and 1 cycle of 72ºC for 10 min. PCR products were then purified using Wizard®SV Gel and PCR Materials and Methods Clean-Up System (Promega Corporation, Madison, USA). Gel slice containing targeted amplicon was Mushroom materials dissolved by adding 10 µL of Membrane Binding Matured Tiger’s Milk mushrooms were collected Solution per 10 mg of gel slice and incubated at from forest in Kuala Lipis, Pahang, Malaysia. Fresh 50-65oC until gel slice is completely dissolved. Tiger’s Milk mushrooms were divided into tubers, Then, SV Minicolumn was placed in collection tube pilei and stipes and these parts weighed between and dissolved gel mixture was transferred to the 350–500 g. The separated tubers, pilei and stipes Minicolumn assembly and incubated for 1 min at were then washed several times, blotted dry, sliced, room temperature. Next, the Minicolumn assembly oven-dried at 60oC, powdered in a blender and kept was centrifuged at 16000 x g for 1 min and flow in airtight containers at 4oC prior to proximate and through was discarded. Afterward, Minicolumn was toxic metal analysis. DNA was extracted from the reinserted into the collection tube and 700 µL of tuber using NucleoSpin® Plant II (Macherey-Nagel, Membrane Wash Solution was added and centrifuged GERMAN). A hundred milligrams of mycelial cells at 16000 x g for 1 min. Then, flow through was again were washed using ethanol. Three hundred micro discarded. Next, collection tube was emptied and liter of PL1 buffer was added into the sample and recentrifuged for 1 min to remove residual ethanol. homogenized followed by incubation for 10 min Subsequently, Minicolumn was transferred to new at 65oC. Then, 100 µL of chloroform was added tube and added with 50 µL of Nuclease-Free Water to followed by centrifugation for 15 min at 20,000 x g. the center of the column. Minicolumn was incubated The top aqueous layer was pipetted into a new 1.5 for 1 min followed by centrifuged at 16000 x g for 1 ml tube. The lysate then filtered using NucleoSpin® min. Then, Minicolumn was discarded and DNA was Filter and centrifuged at 11 000 x g for 2 min. DNA stored at -20°C and sent to sequence commercially at binding condition was adjusted by adding 450 µL 1st BASE Pte Ltd (SINGAPORE). of PC buffer after removing NucleoSpin® Filter. NucleoSpin® Plant II Column was placed in collection Phylogenetic analysis tube and 700 µL of sample was centrifuged at 11 000 Multiple orthologous RPB2 sequences were x g for 1 min. Then, 400 µL of PW1 buffer was added retrieved using BLASTx (Altschul et al.,1990) into the column and centrifuged for 1 min at 110,000 with a minimum match cut-off value of E ≤ 10-107. x g. Flow through was discarded and 700 µL of A total of 10 unique taxa containing 215 characters PW2 buffer was added into column and centrifuged were selected for further analyses. Sequences were for 1 min at 110,000 x g. Flow through was again aligned using CLUSTAL W (Thompson et al., 1994) discarded and 200 µL of PW2 buffer was added into followed by visual inspection and manual adjustment. column and centrifuged for 2 min at 110,000 x g to Phylogenetic analyses were conducted using MEGA remove wash buffer and dry the silica membrane version 5 (Tamura et al., 2011). Phylogenetic trees completely. Next, NucleoSpin® Plant II Column was were constructed using the Neighbor-Joining (NJ) placed into a new tube. Fifty micro liter of PE buffer method from distance matrixes calculated by the was pipetted onto the membrane and incubated for Jones Taylor-Thornton model. The robustness of the 1 min at 65oC and centrifuged for 1 min at 110,000 trees was evaluated by bootstrap analysis of 1000 x g to elute the DNA. Further elution of DNA was random iterations. All trees were then viewed and performed by adding another 50 µL of PE buffer onto edited using TREEVIEW 1.6.6 (Page, 1996) with the membrane and incubated for 1 min at 65oC and Termitomyces sp. and Tricholoma subareum being centrifuged for 1 min at 110,000 x g. rooted as outgroups. DNA amplification and sequencing Proximate and toxic metal analysis The forward primer (LRRPB2C-F: 5’ AGA Proximate composition which included CCC ACAAGGAGTTCA A 3’) and reverse primer percentage moisture, fat, crude protein, fibre, (LRRPB2C-R: 5’ TGAGGTTGT CCC AGTTGT AT carbohydrate and ash was determined according Lai et al./IFRJ 20(5): 2301-2307 2303 to the standard method of the Association of 10 ml of 0.5 N NaOH was gradually added through Official Analytical Chemists (AOAC, 2003). All the similar approach. Distillation was continued for proximate components were analyzed in triplicate at least 10 min and NH3 produced was collected as and reported as mean on percentage of dry weight NH4OH in a conical flask containing 20 ml of 4% basis.