MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY SUMMARY OF DOCTORAL THESIS Specialization: Biotechnology Code: 94 20 201 HO THI THU BA COLLECTING, SELECTING AND THE RESEARCHING THE CULTIVATION OF A WILD EDIBLE MUSHROOM AND A WILD MEDICINAL MUSHROOM IN THE THAT SON REGION, AN GIANG PROVINCE Can Tho, 2019 THIS STUDY WAS COMPLETED AT CAN THO UNIVERSITY Scientific supervisor: Assoc Prof Dr TRAN NHAN DUNG Dr BUI THI MINH DIEU The dissertation was defended at the university examination committee At.………………………………………., University Can Tho At… hour ….… ,on date…… month… … year…… Referee 1: Referee 2: Referee 3: The dissertation is available at Libraries: Central library of Can Tho University National library of Vietnam PUBLISHED PAPERS Ho Thi Thu Ba, Tran Nhan Dung, Trinh Tam Kiet and Truong Tran Thuan, 2017 Propagation of Ganoderma applanatum mushroom originated from Tinh Bien, An Giang Journal of Vietnam Agricultural Science and Technology 102-105 (in Vietnamese) Ho Thi Thu Ba, Tran Nhan Dung and Truong Tran Thuan, 2018 Surveying subchronic toxic of meshima wild mushroom (Phellinus sp.) in white mouse Journal of Vietnam Agricultural Science and Technology 50-53 (in Vietnamese) Chapter I INTRODUCTION 1.1 Necessity of the dissertation All of useful microorganisms are used, the closest to humans being mushrooms Mushroom is an eukaryote, nonchlorophyll, heterotrophic In the five-gender classification system, fungi rank the third in line with plants and animals (Tran Van Mao, 2004) The technology of breeding, cultivating and processing mushrooms have now imported from Japan, Taiwan and other European countries In process of research and production, there have been changes to suit the natural and social conditions of Vietnam (Nguyen Huu Dong et al., 2000) Vietnam is an agricultural country, and there are some conditions for developing mushroom cultivation, especially in the southern provinces Materials source, abundant labor and weather, the climate is almost constant throughout the year, mushroom can be provided throughout the four seasons (Unesco Center for Knowledge, Culture and Community Education, 2004) In addition, Vietnam is a mountainous country with diverse biological resources In the rainy season, especially there are many kinds of mushrooms growing The Mekong Delta is blessed with That Son mountains, cooling climate through the year with many kinds of edible and medicinal mushrooms are valuable but have not been cultivated The facing global climate change, conservation of this precious resource is necessary to right Due to the above reasons, the thesis “collecting, selecting and the studying the cultivation of a wild edible mushroom and a wild medicinal mushroom in the That Son region, An Giang province” was conducted 1.2 The objective of the dissertation 1.2.1 The general objective of the dissertation: Finding how to cultivate cultivateof edible and medicinal mushroom collected in the That Son region, An Giang province 1.2.2 The specific objective of the dissertation: (1) The collection of large mushroom species in That Son region, An Giang province (2) The acute toxicity testing, fungal isolation on PDA media, molecular identification of non-toxic fungal species (3) Determination of nutrient ingredient, medicinal value of wild mushroom Analysis of semi-chromic toxicity and try out anti-cancer effect in vitro on blood cancer cells and colon cancer of new wild herb mushroom (4) The studing of complete cultivation process process for one edible mushroom and one selected medicinal mushroom 1.3 The object and limit of the study The edible and wild mushrooms have been used by local people but have not been researched and raised from That Son region, An Giang province 1.4 Time and place of study Performed from October 2013 to August 2017 at Can Tho University 1.5 The new contributions of the dissertation The novelty of the thesis is the study in two local mushroom, Lentinus Squarosolus and Phellinus sp In which, Inside Lentinus Squarosolus is a new edible mushroom that has been successfully cultivated from wild mushrooms in nature, which has led to the step by step establishment of the brand name mushroom by Vietnam The thesis also shows the successful cultivation process of the high value medicinal mushrooms without semi-chromic toxicity in test mouse Which has great scientific and community significance, because of the wild mushroom that was first collected, isolated and studied In particular, Thuong Hoang fungus has the potential to inhibit the proliferation and death of the colon cancer cell line 1.6 The layout of the dissertation The dissertation is 193 pages, including an introduction, a review of the literature, research methods, discussion results, conclusions, recommendations and appendix The thesis has 56 tables, 43 images and 150 references Chapter RESEARCH METHODS 3.1 Experimental materials The wild mushrooms were collected from That Son region, An Giang province 3.2 Content and method research The dissertation had performed general research contents In each main contents with the small contents, every small content was corresponding to the method Details are given in below sections 3.2.1 Collection of large mushroom in the That Son mountain, An Giang province Then, a local investigate and find out which mushroom are usable and identify mushroom 3.2.1.1 The collecting large mushroom Purpose: collect over 20 species of mushrooms on The That Son mountain, An Giang province, set up a collection of varieties mushroom The primary identification is based on mushroom morphology, the basic procedure for the investigation of wild mushrooms according to international standards (Trinh Tam Kiet, 2011, Trinh Tam Kiet, 2012, Trinh Tam Kiet, 2013) *Monitoring criteria: The substratum imagine where large mushrooms are found; classification of mushrooms collected (mushrooms are classified into one of four groups: edible mushrooms medicinal mushroom, poisonous mushroom and uncategorized mushrooms) 3.2.1.2 The investigation mushroom species that people have ever used Purpose: Primary selection of new mushroom was used by local people but not yet cultivation in Vietnam 3.2.1.3 The identified based on the mushroom morphology Collecting mushrooms were identified by external morphological, the basic procedure for the investigation of wild mushrooms according to international standards (Trinh Tam Kiet, 2011, Trinh Tam Kiet, 2012, Trinh Tam Kiet, 2013) 3.2.2 Determination of acute toxicity levels, isolation and sequencing ITS of edible and medical mushrooms 3.2.2.1 Determination acute toxicity of edible or medicinal mushroom was selected by the local population The process of determining acute toxicity level was conducted according to toxicology determination procedure issued by the Institute of Medicinal Materials, Ministry of Health: methods of pharmacological effects of herbs drugs (2006) and method of determining the acute toxicity of the drug, Do Trung Dam in 1996 Selection of non-toxic mushrooms for next research 3.2.2.2 Isolation of selected non-toxic mushrooms The mushroom without acute toxicity were isolated and examining mycelium system on PDA media (Dung, 2003) 3.2.2.3 Identification of selected mushrooms by molecular biology method 3.2.3 Determination of nutrient, pharmaceutical, semi-chromic toxicity, testing the anti-cancer effects of medicinal mushrooms with blood and colon cancer cells Purpose: selecting a high nutritional value edible mushrooms and a highly medicinal mushroom All experiments in this content was conducted at the Institute of Ginseng and Medicinal Materials of Ho Chi Minh City 3.2.3.1 Determine nutritional ingredients of selected mushrooms Purpose: Determine nutritional ingredients of the four mushroom that were selected Thereby, selecting an edible and a medicinal mushroom have high nutrition Procedure: Quantify the value components in mushroom by thin layer chromatography and high pressure liquid chromatography Quantification of substances: fat, protein, vitamins A, vitamin C, amino acids and trace minerals (calcium, iron, zinc, potassium) 3.2.3.2 Determine of medical mushroom quantify Purpose: Determine medical ingredients of the two mushroom that were selected in content Thereby, selecting a medicinal mushroom have high nutrition Procedure: Quantify the value components in mushrooms such as triterpenoid, sugar-free, reducing sugars and polysaccharide Compare triterpenoie, polysaccharide, reducing sugar, sugar-free of some mushrooms to pick one with higher levels to assessment semi-chromic toxicity 3.2.3.3 Determine of mushroom semi-chromic toxicity Purpose: Determine new wild mushroom was selected without semi-chromic toxicity to study the culture process Procedure: Determine basic nutritional and pharmacological criteria according to “Methods of studying the effects of drugs from pharmacological Herbs” (Institute of 4.1.2 Investigation of selected mushroom samples 28 collected wild mushroom samples were investigate from local people Five edible/medicinal mushrooms is Ganoderma apllanatum, Trametes sp., Phellinus sp., Lentinus squarosolus and Moc Ba Hue 4.1.3 Identification of mushroom using morphological characters 4.1.3.1 Ganoderma apllanatum Local Name: Linh Chi Tang Live on trunk, the filamentous system grows in fresh dead plant tissue, was found in the Cam Mountain The spore is slightly rounded egg-shape Figure 4.1: Spore and Linh Chi Tang mushroom (code 20) Linh Chi Tang is a mushroom has some layers, fruiting body has fan-shape or circle, diameter about 6-100 cm, 3-8 cm thick The mushroom has no gloss bark on the pileus surface, no stem, it has brown or dark brown color, concentric striated lines can be prominent clearly or not, forming rough protrusions on pileus surface Ganoderma apllanatum is wood mushrooms, keratinized, ruffled, the edge has the same color as pileus and is curved with thickness about 0.5-1 cm 4.1.3.2 Trametes sp 10 Local name: Van Chi mushroom Van Chi is a mushroom without stem, growing on one side Young fruiting bodies are tumor rounded up shape, forming a rim with creamy white canopy Developed mushroom has accessory fruiting form Fruiting body has kidney-shape and concentric striated lines which are stacked alternately like roofing tiles, pileus is thin, smooth or lightly wizen, grows in clusters which are about 40-50cm wide There are no fungal fimbriae and concentric opalescent rings on cap surface Lamella has tiny tubes whose density is about 4-5 tube/mm The tubes are round or lightly round Figure 4.2: Van Chi mushroom (code 10) 4.1.3.3 Phellinus sp Local name: Thuong Hoang Thuong Hoang mushroom grows on alive tree trunk, at a height of about 5-10m It only grows deep in the forest and o high up in mountain where the temparature is about 24-27 C Fruiting body has tube layers, tissue, rusty iron-color tube There is no stem attach to fruiting body Figure 4.3: Thuong hoang mushroom and its spore (code 22) 11 The mushroom doesn’t form a cluster and it’s soft wood mushroom Upper surface of the mushroom is dark brown or black, has drain, hard bark with dark brown color and some deep crack The cap is initialy covered by a yellow velvet coat, becoming darker to black with many small bulges, cracking into small areas.Edge of pileus is yellow-brown, and young fruiting body has yellow color inside, becoming brown rustyiron-color latterly Based on the external characteristics, the species is similar to Phellinus linteus, Phellinus igniarius and Phellinus lamaensis 4.1.3.4 Lentinus squarosolus Local name: Xoai Figure 4.4: Dai mushroom (code 14) and its spores Dai mushroom has white funnel-shaped cap, whose diameter is about 2-15cm, its upper surface is covered by light brown bristly coat The mushroom’s white inside The mushrooms’ white gills embraces thin stems The stems are 35cm long, opaque white, covered by scale, the mushroom doesn’t have ring and volva L squarosolus usually appears in from March to November This mushroom grows individually or in large clusters on the trunk or punk mango The mushrooms grow year-round, especially after rain, thrive in the summer with wet weather The spore has stick-shaped 4.1.3.5 Moc Ba Hue Local name: Moc Ba Hue 12 Moc Ba Hue grow on plant roots, stems is in soil, yellow heart-shaped fruiting bodies form a cluster on lie ground There are black dots on fruiting body Mushroom mycelium is also yellow This is a relatively new species of mushroom, there is no any description of the external morphology as well as identification of the mushroom Figure 4.5: Moc Ba Hue mushroom (code 27) 4.2 Acute toxicity study, isolation of the mushrooms onf PDA medium, identification of no-toxic mushroom species by molecules technique 4.2.1 Acute toxicity study of valuable collected mushrooms Table 4.1: Maximum dose of mushrooms Name g dry mushroom/kg weight of mice Dmax(g) Water extract Alcohol extract Linh chi tang 23,41 28,66 137,7 391,9 Thuong hoang 22,86 27,97 134,5 383,4 Van chi 23,42 28,80 132,9 388,2 Dai 23,51 29,72 111,1 338,9 Moc ba hue 23,26 29,27 111,3 332,1 13 Water extract Alcohol extract Treated of mushrooms did not show any visual symptoms of toxicity after using these extracts in 72 hours Observing mice in 14 day following, no unusual symptoms of the mice has been noted The results show that high concentrated extracts of Linh Chi Tang, Van Chi, Dai, Moc Ba Hue mushroom are devoid of any adverse effects in mice Next, we isolated these mushroom in order to preserve their breed 4.2.2 Isolation of selected mushroom in 4.2.1 PDA medium was used for isolation of mushroom in the same temperature and time conditions The result indicated that PDA medium has potential for isolating Linh Chi Tang, Thuong Hoang, Van Chi, Dai mushroom, but the medium isn’t appropriate for isolation of Moc Ba Hue mushroom We sequenced ITS to identify four species correctly in next experiment 4.2.3 ITS sequencing, species identification 4.2.3.1 Van Chi mushroom The ITS sequence of Van Chi mushroom, which has length 666 bps, share 93% of the DNA sequence with some mushroom speices in Tramates genus according to datebase on NCBI According to Trinh Tam Kiet’s morphological description (2012) Initial evaluation of the collected mushroom sample is a species in Trametes genus 4.2.3.2 Dai mushroom The 567bps ITS sequence of Dai mushroom is 96% similar to IST sequence of Lentinus squarrosulus, according to database on NCBI Initial evaluation of the mushroom collected was one of the Lentinus squarrosulus species 14 According to Trinh Tam Kiet’s morphological description (2012), we suggested that the collected mushroon in An Giang is Lentinus squarrosulus 4.2.3.3 Linh Chi Tang mushroom The 631bps ITS sequence of Linh Chi Tang mushroom is 97% similar to IST sequence of Ganoderma applanatum, according to database on NCBI According to morphological description of Trinh Tam Kiet (2012), we suggested the collected mushroom sample in An Giang province is Ganoderma applanatum speices 4.2.3.4 Thuong Hoang mushroom The ITS sequence of Thuong Hoang mushroom, which has length 666 bps, share 92% of the DNA sequence with Phellinus linteus according to datebase on NCBI Combine with morphological analysis, we suggested that the collected mushroom sample is a species belong to Phellinus genus, the mushroom is temporarily called Phellinus sp Based on two researches, four species of mushrooms were selected for further study: Dai mushroom - Lentinus squarrosolus, Linh Chi Tang mushroom Ganoderma applanatum, Thuong Hoang mushroom Phellinus sp and Moc Ba Hue mushroom 4.3 Determination of nutrient composition, pharmacology, subchronic toxicity and anticancer effect of these mushrooms on blood cancer and colon cancer cell line 4.3.1 Determination of nutrient pharmocology of selected mushroom composition and We used HPLC for determining chemical compounds in these selected mushrooms 15 Table 4.2: Nutrient composition and pharmocology of mushroom Composit ion Name h m l ng x c nh g mushroom Moc ba hue Dai Thuong hoang Linh chi tang Lipid 7,01 vt indefined indefined mg/kg Protein 0,79 0,75 indefined indefined mg/kg 4Hydroxyprolin 193 undetected 376 186 mg/kg Acid aspartic 624 695 2510 891 mg/kg Acid glutamic 759 693 1780 767 mg/kg Alanin 68,8 72,8 1460 623 mg/kg Cystine 1470 undetected 355 338 mg/kg Glycin 419 331 1810 566 mg/kg Histidin 176 169 520 259 mg/kg Isoleucin 106 113 829 421 mg/kg Leucin 439 432 1480 671 mg/kg Lysin 172 309 540 349 mg/kg Methionine 237 262 undetected 103 mg/kg Phenylalanin 244 245 658 470 mg/kg Prolin 649 495 1470 550 mg/kg Serin 245 313 1550 623 mg/kg Threonin 507 537 1610 563 mg/kg Tyrosin 117 165 158 193 mg/kg Valin 298 248 1190 494 mg/kg 16 Tro tổng 3,31 0,28 5,78 2,20 mg/kg Canxi 216 602 5370 2060 mg/kg Sắt 312 13,5 1650 2210 mg/kg Kali 3880 255 indefined indefined mg/kg Kẽm 20,8 23,4 25,9 36,4 mg/kg Vitamin A indefined indefined 1,04 0,91 mg/kg 4.3.2 Determination of pharmacological compounds in Linh Chi Tang and Thuong Hoang mushroom Table 4.3: Pharmocology composition of mushroom Name Linh chi tang Thuong hoang Content of triterpenoid in sample (%) Polysaccharid content (%) Average free sugar content (%) Average reducing sugar content (%) 1,10 0,46 29,42 22,07 2,11 4,0 40,17 57,14 We compared some of the medicinal values, combined with the nutritional assessment table (Table 4.2) present in the sample, we can select the Dai mushroom and the Thuong Hoang mushroom to carry out further experiments 4.3.3 Determination of semi-chronic toxicity of Thuong Hoang mushroom Tested mice were female and male, six-weeks old, in growth and developing stage Mice were treated with oral dose of 0.4g/kg of Thuong Hoang mushroom extract everyday in month The result showed that both of control mice and tested mice gained body weight Concomitantly, there was no significant difference of weight changes between two groups of 17 mice This was asserted that Thuong Hoang mushroom extract didn’t effect on mice development during tested period Haematological parameters and biochemistry parameter of mice before and after one month testing and result of microbodies in liver and kidney tissue surgery in two group did not have any unusualness 4.3.4 Determination of anticancer effect of Thuong Hoang mushroom Phellinus sp Figure 4.6: The response of K562 cells to the extracts of Phellinus sp Observing on the 10X magnification by inverted microscope a) K562 cells, b) Disk containing K562 added with concentration of 500 µg/mL of Phellinus sp extract c) 250 µg/mL d) 125 µg/mL e) 62 µg/mL After 72 hours of treating with Thuong Hoang mushroom extract, leukemia K562 had no response to the extract High concentration of the extracts were used in the experiment were 5000 µg/ml; 2500 µg/ml, 1250 µg/ml; 625 µg/ml; 312 µg/ml; 156 µg/ml and 78 µg/ml At all concentrations of extracts, K562 cells treated with the extracts 18 were nearly as normal as the control group This result showed that Phellinus sp., did not inhibit the proliferation of K562 cells Figure 4.7: Response of HCT 116 to extracts of Phellinus sp Mushroom, being observing by inverted microscope in 10X magnification a) control HCT116 cell b) HCT116 cells added a concentration of 16 μg ml of the extract c) 31 μg ml d) 62.5 e) 125 μg mL f) 250 μg mL g) 500 μg mL h) of 1000 μg mL For colon cancer cell HCT116, after 72 hours of treatment with Phellinus sp mushroom extracts, the cells 19 proliferation was inhibited in dose-dependent manner Result showed that inhibition effect of Phellinus sp extracts were observed clearly at concentration of 16 µg/mL Concentration of 250 µg/mL and higher concentration showed significantly inscreasing anticancer effect of the mushroom Confluence of HCT 116 in medium containing concentrations of 16 µg/mL; 31 µg/mL; 62.5 µg/mL; 125 µg/mL; 250 µg/mL; 500 µg/mL and 1000 µg/mL of the extracts were 50%, 40%, 45%, 40%, 20%, 15% and 10%, alternately The results of this study were consistent with those of other researcher with effect of the mushroom on leukemia cell lines K562 and colon cancer cell line HCT116 (Alla, 2010; Ji Hun 2015) The different changes caused by Phellinus sp mushroom depend on type of cell treated Phellinus sp impact can cause apoptosis (case of HCT116) or support apoptosis in cells (case K562) 4.4 Research for complete procedure of growing selected mushroom The research on mycelium speard on culture medium was performed to determine how large mycelium speard in 3, and days in agar slant tubes On propagation and growing medium, we determined how long the mycelium of Lentinus squarrosulus and Phellinus sp spread for 50% and 100% in the medium Measure data was processed by SAS 9.1.3 softwave and average results were performed in table using DUNCAN method with 95% confidence intervals 4.4.1 Research for procedure of growing Dai mushroom Lentinus squarrosolus PDD (PDA add water coconut) medium was selected for subculture of Dai mushroom 20 Brown rice and rice containing 5% of rice bran medium was suitable for growing Dai mushroom Sawdust added 5% of cornmeal 5% of rice bran medium gave the fastest mycelium spreading day, about 70 days After three experiment of selection for growing Dai mushroom, the procedure of domestication and growing Dai mushroom was sumarized in the diagram below Figure 4.8: Procedure of growing Dai mushroom Lentinus squarrosolus 4.4.2 Establishment of Thuong (Phellinus sp.) cultivation procedure Hoang mushroom PDA medium was selected for subculture of Thuong Hoang mushroom 21 Brown rice and rice containing 5% of rice bran medium was suitable for growing Thuong Hoang mushroom Sawdust added 5% of cornmeal 5% of rice bran medium gave the fastest mycelium spreading day, about 50 days and 70 days for harvesting of fruit bodies After research, we sumarize procedure of growing and plainization of wild Thuong Hoang mushroom Phellinus sp on the diagram below Hình 4.9: Procedure of growing Thuong Hoang mushroom (Phellinus sp.) 22 Chapter 5: CONCLUSIONS AND RECOMMENDATIONS 5.1 Conclusions In the thesis, we collected 28 wild mushroom samples from nature including five edible mushrooms; kinds of medicinal mushrooms (two kinds of Thuong Hoang mushroom, kinds of lingzi); poisonous mushrooms and 11 unknown species According to the testimony of local people, mushroom which are used and sold by native people, are Dai, Linh Chi Tang, Thuong Hoang, Van Chi and Moc Ba Hue We selected and identified the mushrooms for studies All of the mushroom extract did not cause any toxicity synonym or mortality on mice models We isolated four mushroom, they are Thuong Hoang, Dai, Linh Chi Tang and Van Chi Next, we sequenced ITS to determine scientifice name of the mushroom The result was Thuong Hoang Phellinus sp., Linh Chi Tang Ganoderma applanatum, Van Chi Trametes sp., Dai mushroom Lentinus squarrosolus Four mushroom spices for next experiment were Thuong Hoang Phellinus sp., Dai Lentinus squarrosolus, Linh Chi Tang Ganoderma applanatum and Moc Ba Hue, Moc Ba Hue is a new spices, have great potential to be commercialized After determining nutrient composition of four mushroom spices, we chose mushroom to continue studing, they are Thuong Hoang Phellinus sp Dai Lentinus squarrosolus Thuong Hoang mushroom were chosen for further studies after comparing medical components between Linh Chi Tang and Thuong Hoang mushroom Mice treated with Thuong Hoang extracts in semi-chronic toxicity test weren’t noted any unusual phenomenon Thuong Hoang mushroom extract showed great anti-cancer effect on colon cancer cell line HCT116, when the cell line propagation was inhibited, and apoptosis was induced, but the extract did not 23 show any observable effect on leukemia cell K562 Study on the cultivation of edible mushroom Lentinus squarrosolus, the Harvested prediod was about 120 days and the medicinal mushroom Phellinus sp approximately 150 days after passing 5.2 RECOMMENDATIONS Moc Ba Hue is a new mushroom spices, it hasn’t been identified and have great potential The mushroom need to be researched to determine scientific mane, suitable isolated medium, nutrient components… in the mushroom) Particularly, Linh Chi Tang, which is a perennial lingzi mushroom, live, is also a valuable medicinal mushroom that needs further investigation, such as developing a culture process, identifying some important pharmaceutical ingredients to supplement It need to be added to diversity collections of Lingzhi mushrooms in Vietnam Lentinus squarrosolus, a wild mushroom that has been successfully cultivated, should have propagation strategy, which is widely used by local farmers to supply the variety of local and thoroughbred mushroom species in VietNam Thuong Hoang mushroom Phellinus sp is a rare medicinal mushroom that is present in the locality, so propagation and breeding are very easy to implemt because it is suitable for the climate and soil of the area, therefore, it is necessary to cultivate in a large amount Research on preparation of anti colon cancer drug from Thuong Hoang should be carried out We should made further experiments such as model animal testing and clinical trials of Thuong Hoang mushrooms The final issue is that the mushroom resources in That Son, An Giang are extremely rich with many rare and rare mushrooms that need to be preserved in the face of urbanization of mountainous areas and global climate change 24 ... Thi Thu Ba, Tran Nhan Dung, Trinh Tam Kiet and Truong Tran Thuan, 2017 Propagation of Ganoderma applanatum mushroom originated from Tinh Bien, An Giang Journal of Vietnam Agricultural Science and... are Thuong Hoang, Dai, Linh Chi Tang and Van Chi Next, we sequenced ITS to determine scientifice name of the mushroom The result was Thuong Hoang Phellinus sp., Linh Chi Tang Ganoderma applanatum,... suggested the collected mushroom sample in An Giang province is Ganoderma applanatum speices 4.2.3.4 Thuong Hoang mushroom The ITS sequence of Thuong Hoang mushroom, which has length 666 bps, share