Studies on the Factors Affecting Growth of Mycelium and Fruiting Body Formation, and Antioxidant Activities of the Extracts of Cordyceps militaris L. ex St. Aman

國立屏東科技大學熱帶農業暨國際合作系

Department of Tropical Agriculture and International Cooperation

National Pingtung University of Science and Technology

博士學位論文

Ph.D. Dissertation

蛹蟲草菌絲體生長與子實體形成條件及

萃取物抗氧化活性之研究

Studies on the Factors Affecting Growth of Mycelium and Fruiting

Body Formation, and Antioxidant Activities of the Extracts of

Cordyceps militaris L. ex St. Aman

指導教授 Advisors：賴宏亮 博士（Lay Horng Liang, Ph.D.）

王均琍 博士（Wang Chun Li, Ph.D.）

研究生 Student：鄧玉雄 Dang Ngoc Hung

中華民國 110 年 1 月 28 日

January 28, 2021

I

摘 要

學號：P10222014

論文題目：蛹蟲草菌絲體生長與子實體形成條件及萃取物抗氧化活性之研

究

總頁數：201 頁

學校名稱：國立屏東科技大學 系（所）別：熱帶農業暨國際合作系

畢業時間及摘要別：109 學年度第 1 學期博士學位論文摘要

研究生：鄧玉雄 指導教授：賴宏亮 博士

王均琍 博士

論文摘要內容：

蛹蟲草含有生物代謝產物，具有潛力作為中草藥。從遠古時代就有證

據顯示蛹蟲草可用於活化人體的各種系統，除了早期廣泛應用於食補，在

現代醫學蛹蟲草成分更是廣泛應用於各項研究中。目前相關研究利用蛹蟲

草潛在的有效成分促進中草藥治療之功效，並能提升綠色生技革命發展，

以建立安全、合理性之友善環境。

本研究目的在探討對蛹蟲草菌絲體、子實體與抗氧化物質產生的最佳

培養條件，進行下列試驗：（1）探討不同培養條件（培養基、溫度、碳

源、維生素源與穀物源）對蛹蟲草 Cordyceps militaris 兩個菌株 (AG-1、

PSJ-1)菌絲生長和生產的影響；（2）探討不同液態培養方法（搖動和靜態

培養）對菌絲體生產的影響；（3）探討不同菌絲體乾燥方法對生物量、

胞外和胞內多醣生產的影響；（4）探討不同的液態培養方法（PVC 培養

基）對子實體生長的影響；（5）探討不同溫度及濃度之蛹蟲粉對子實體

生長的影響；（6）探討不同的液態培養方法（搖動，靜態）和菌絲體乾

燥方法（烤箱乾燥和冷凍乾燥）對抗氧化物質性的影響。

II

結果顯示，在 MYPS 培養基和溫度 20-24 oC 下，C. militaris 兩個菌株

AG-1 和 PSJ-1 的菌絲體生長最佳。添加葡萄糖濃度為 30 g/L 及維生素 B1

濃度為 0.03 g/L 可以促進菌絲生長。以黑糯米（越南產）作為培養基，可

獲得兩種菌株的最佳產量。利用不同的液態培養基和不同培養方法（靜態，

靜態+搖動，搖動）發現 MYPS、PVC 兩種是適合培養蛹蟲草的培養基，

而靜態浸沒培養方法適合兩菌株 AG-1 和 PSJ-1 的菌絲生長。

以 C.militaris 兩個菌株的抗氧化能力來看，以 PVC 和 MYPS 培養基靜

態培養，其萃取物對 1,1-二苯基的自由基清除率較高（DPPH）。採用靜態

培養方法的液態培養基（PVC）降低 TPC，TFC，而 TPC和 TFC 降低與 C.

militaris 的抗氧化特性相關。PVC 浸沒式液態培養可以代替兩個菌株 AG-1

和 PSJ-1 某些培養成分，改善萃取物的抗氧化能力和活性 。在浸沒的液態

培養基中培養的兩個菌株，其菌絲體以冷凍乾燥方法可提高 TFC 和抗氧化

物含量。結果顯示，在所有浸沒液態培養處理條件下，C. militaris AG-1 和

PSJ-1菌絲體都具有良好的抗氧化性能，尤其是 DPPH自由基清除試驗和脂

質過氧化作用。

初始培養基 pH 影響 C. militaris AG-1 和 PSJ-1 的生物量和多醣產生。

在 24 oC 以 PVC 培養基（pH 6.7）進行靜態培養 18 天後，具有最佳生物量

（AG：12.92±0.3 g/L，PSJ-1：9.03±0.24 g/L）和細胞外與細胞內多醣

（AG：209.70±1.56 mg/L，PSJ-1：198.16±0.85 mg/L；AG-1：32.62±0.87

mg/L，PSJ-1：30.63±1.96 mg/L）。持續搖動培養對於生物質和細胞外多

醣的產生是最佳的，而在靜態條件下的培養對於細胞內多醣的產生是最佳

的。測試不同的油脂添加對菌絲體生物量和多醣產生的影 響，結果顯示在

菌絲生物量（AG-1：8.27±0.09 g/L，PSJ-1：8.01±0.0 g/L）的生產中添加椰

子油 3.5％，胞外多醣（EPS）（AG-1：1208.00 ± 2.30 mg/L；PSJ-1：

1110.40 ± 3.16 mg/L），胞內多糖(IPS)（AG-1：23.61 ± 1.31 mg/g，PSJ-1：

20.39 ± 1.55 mg/g）迅速增加並達到最高水平。

本研究探討不同的液態培養方法、溫度、蛹蟲粉添加和光照條件，對

C. militaris (AG-1、PSJ-1)子實體生產之影響。結果顯示，子實體的菇原體

萌發時間提前 (AG-1：5.80 ± 0.58 天; PSJ-1：6.20 ± 0.37 天），產量和生物

性狀（長/寬（cm））存在明顯差異。在藍光條件下培養，AG-1 和 PSJ-1

獲得了最高產量（AG-1：14.35 ± 0.53 g/ 瓶；PSJ-1：12.54 ± 0.61 g/瓶）和

III

表現較佳之長與寬（cm）AG-1：5.04 ± 0.41，0.50 ± 0.03 cm；PSJ-1： 4.96

± 0.36，0.44 ± 0.02 cm) 。

關鍵字：抗氧化活性、黑糯米（越南產）、蛹蟲草、浸沒液態培養、維他

命 B、蛹蟲粉、發光二極體

IV

ABSTRACT

Student ID: P10222014

Title of Dissertation: Studies on the Factors Affecting Growth of Mycelium and

Fruiting Body Formation, and Antioxidant Activities of the

Extracts of Cordyceps militaris L. ex St. Aman

Total Pages: 201 Pages

Name of Institute: Department of Tropical Agriculture and International

Cooperation, National Pingtung University of Science and

Technology

Graduate Date: November 28, 2021 Degree Conferred: Doctoral Degree

Name of Student: Dang Ngoc Hung Advisors: Lay Horng Liang, Ph.D.

Wang Chun Li, Ph.D.

The Content of Abstract in This Dissertation:

Cordyceps militaris is a potential harbor of bio-metabolites for herbal drugs

and evidences are available about its applications for revitalization of various

systems of the body from ancient times. Besides their popular applications for

tonic medicine by the all stairs of the community, the constituents of C. militaris

are now used extensively in modern systems of medicine. The current survey

records the mysterious potentials of C. militaris are boosting up the present herbal

treatments, as well as gearing up the green pharmacy revolution, in order to create

a friendly environment with reasonable safety.

The objective of the study was evaluating the best culture conditions for

mycelium, fruiting body, antioxidant substance production. The item were carried

out as follows: (1) evaluate the effects of different factors (media, temperature,

carbon sources, vitamins sources, grain sources) on the mycelium growth and

production of C. militaris two strains; (2) evaluate the effect of different liquid

culture method (shake and static culture) on the mycelial production; (3) evaluate

the effects of different mycelium drying method on the biomass and extra and

intra-cellular polysaccharides production; (4) evaluate the effect of submerged

liquid culture (PVC media) with different methods on the fruiting body growth;

V

(5) evaluate the effect of temperature and pupa powder sources and concentration

on fruiting body growth of C. militaris; (6) evaluate effect of different liquid

culture methods (shake, static), and mycelium drying method (oven drying, and

freeze drying) on antioxidant compound and activity of the C. militaris.

The results showed that the mycelium of C. militaris two strains AG-1 and

PSJ-1 present the best growth with MYPS media and at 20-24 oC, Addition of

glucose at 30 g/L, vitamin B1 at 0.03 g/L concentration could promote the

mycelium growth. Black glutinous rice (Vietnam) was the best grain source to

produce spawn of C. militaris two strains. Using different liquid culture media

and different methods (static, static+shake, shake), MYPS, PVC were the suitable

meida and static culture were suitable submerged method for mycelium growth

of C. militaris two strains AG-1 and PSJ-1.

Regarding to antioxidant properties and contents of C. militaris two strains

AG-1 and PSJ-1, the results showed that submerged liquid culture containing

higher contents of PVC and MYPS media by static culture reached the higher

values of Scavenging on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical,

Chelating on ferrous ions, Hydroxyl radical scavenging assay, Scavenging

activity of ABTS.+ radical cation, Lipid peroxidation as well as higher value

efficiency of total phenolic contents (TPC), total flavonoids content (TFC).

Whereas, submerged liquid culture medium (PVC) with static culture method

reduced TPC, TFC that directly linked to a decreased antioxidant properties of C.

militaris. These results suggested that PVC submerged liquid culture can be used

to replace some parts for C. militaris two strains AG-1 and PSJ-1 cultivation,

which also improved antioxidant properties and antioxidant activity of C. militaris,

extracts. With freeze, drying method of C. militaris two strains AG-1 and PSJ-1

cultivated in almost submerged liquid culture showed efficiency in improving the

TFC as well as antioxidant contents in comparison with oven drying method. In

general, C. militaris two strains AG-1 and PSJ-1 had good antioxidant properties,

especially DPPH radical scavenging assay and Lipid peroxidation at all

submerged liquid culture treating conditions.

VI

Cultivation conditions (initial medium pH) affect biomass and

polysaccharide production in C. militaris two strains AG-1 and PSJ-1. The static

culture with PVC media (pH 6.7) at 24 oC, after 18 days obtained the best biomass

(AG: 12.92±0.3, PSJ-1: 9.03±0.24 g/L) and extra- and intra-cellular

polysaccharide (AG: 209.70±1.56, PSJ-1: 198.16±0.85 mg/L; AG-1: 32.62±0.87,

PSJ-1: 30.63±1.96 mg/L, respectively). Submerged liquid culture constant

aeration was optimal for biomass and extracellular polysaccharide production,

whereas cultivation under static conditions was the best for intracellular

polysaccharide production. In this research, different oils addition was studied on

the production of mycelial biomass and polysaccharides of C. militaris strains

AG-1 and PSJ-1. The results showed that with coconut oil 3.5 % addition in the

production of mycelial biomass (AG-1: 8.27±0.09, PSJ-1: 8.01±0.0 g/L), extra￾cellular polysaccharide (EPS) (AG-1: 1208.00±2.30; PSJ-1: 1110.40±3.16 mg/L),

and the IPS (AG-1: 23.61±1.31, PSJ-1: 20.39±1.55 mg/g) increased rapidly and

reached the maximum level.

Different the liquid culture methods, temperatures, pupa powder conditional,

and light conditions were studied on the fruiting body of C. militaris two strains

AG-1 and PSJ-1. The results indicated that the yield and biological properties

(length/ width (cm)) of fruiting body exist distinct differences. The period of

primordia appearance days of C. militaris two strains AG-1 and PSJ-1 had been

shortened (AG-1: 5.80±0.58 days; PSJ-1: 6.20±0.37 days), yield (AG￾1:14.35±0.53 g/bottle; PSJ-1: 12.54±0.61 g/bottle), and biological (length and

width (cm)) AG-1: 5.04±0.41, 0.50±0.03 cm; PSJ-1: 4.96±0.36, 0.44±0.02 cm)

were better under blue light conditions.

Keywords: Antioxidant activity, black glutinous rice Vietnam, Cordyceps

militaris, submerged liquid culture, vitamin B, pupa powder, LED￾light

VII

LIST OF ABBREVIATION

ABTS.+ The 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)

AG-1 C. militaris strain

BE Biological efficiencies

BHA Butylated hydroxyanisole

BHT butylated hydroxytoluene

DETBA 1,3-diethyl-2-thiobarbituric acid

DPPH 1,1-diphenyl-2-picrylhydrazyl

EDTA Ethylenediaminetetraacetic acid

EPS Extra cellular polysaccharides

FD Freeze drying

GAE Gallic acid equivalents

HWE Hot water extraction

IC50 Half maximal inhibitory concentration

IPS Intra cellular polysaccharides

LED Light Emitting Diode

MYPS media Maltose yeast extract peptone sucrose

OD Oven drying

PDA media Potatoes dextrose sucrose agar powder

PSJ-1 C. militaris strain

TPC Total phenolic contents

TFC Total flavonoid contents

VIII

ACKNOWLEDGEMENTS

I wish to express my sincere appreciation to NPUST Scholarship,

Department of Tropical Agriculture and International Cooperation, and

Departmant of Plant Industry for providing a good study condition. Espercially, I

would like to give my sincere thanks to my super advisor, Prof. Dr. Lay Horng

Liang and Prof. Dr. Wang Chun Li for their guidance, help, and supervision

throughout this study.

I am grateful to respectable and intellectual professors in my Ph. D first year

valuation and graduate committees for their suggestions and comments.

I would like to give my gratitude to professors, staffs, and students of

NPUST, OIA, DTAIC, Department of Plant Industry, Plant Physiology and Value

Added Microorganisms Laboratory (AG-204), and Medicinal Plant Laboratory

(AG-104) for their help.

Finally, I would like to thank my family members, and my friend for their

help and encouragement.

Taiwan, Juanary, 2021

Dang Ngoc Hung

IX

TABLE OF CONTENTS

摘 要.......................................................................................................................I

ABSTRACT........................................................................................................IV

LIST OF ABBREVIATION ..............................................................................VII

ACKNOWLEDGEMENTS ............................................................................. VIII

Table OF CONTENTS........................................................................................IX

LIST OF TABLES .............................................................................................XV

LIST OF FIGURRES.................................................................................... XVIII

CHAPTER 1.......................................................................................................... 1

INTRODUCTION................................................................................................. 1

1.1. Introdution (general introduction).................................................................. 1

1.2. Fundamentals of medicinal mushrooms ........................................................ 4

1.3. Applications of medicinal mushrooms .......................................................... 4

1.4. Antioxidant effect of rebeal herbs.................................................................. 6

1.5. Advances in research of polysaccharides in Cordyceps Species.................. 7

1.6. Cordycepin and Adenosine ............................................................................ 8

CHAPTER 2........................................................................................................ 11

LITERATURE REVIEW.................................................................................... 11

2.1. Introduction Cordyceps ............................................................................... 11

2.2. The description and distribution of Cordyceps samples............................. 14

2.2.1. Culture information................................................................................ 14

2.2.1.1. Culture type...................................................................................... 14

2.2.1.1.1. Storage culture/stock culture ..................................................... 14

2.2.1.1.2. Pre-culture (slant and plate culture)........................................... 14

2.2.1.1.3. Popular/indigenous culture ........................................................ 14

2.2.1.1.3.1. Spawn production................................................................ 15

2.2.1.1.3.2. Husked rice culture.............................................................. 15

2.2.1.1.3.3. Shaking culture.................................................................... 15

2.2.1.1.3.4. Surface liquid culture. ......................................................... 15

2.2.1.1.3.5. Continuous culture or repeated batch culture...................... 16

X

2.2.1.1.3.6. Culture media ...................................................................... 17

2.2.2. Chemical constituents............................................................................ 18

2.2.3. Biological activities of C. militaris........................................................ 21

2.3. Medicinal uses/clinical applications of Cordyceps including C. militaris.. 22

2.4. Current state, limitations and remedies........................................................ 23

2.5. Future prospects nature is the source of all the raw materials that we

need. .................................................................................................................... 24

2.6. Polysaccharide content in the cultured Cordyceps ..................................... 24

2.6.1. Polysaccharide content........................................................................... 24

2.6.2. Differences in polysaccharide CP content ............................................. 25

2.6.3. Differences in Cordyceps polysaccharide (CP) under different cultural

conditions......................................................................................................... 26

2.6.4. Isolation and purification of the polysaccharide.................................... 28

2.6.4.1. Isolation and purification ................................................................ 28

2.6.4.2. Monosaccharide composition and structure analysis..................... 29

2.6.4.3. Other aspects ................................................................................... 32

2.6.5. Pharmacological effects of the Cordyceps polysaccharides CP........... 32

2.6.5.1. Toxicological experiments............................................................... 32

2.6.5.2. Antitumour activity Cordyceps polysaccharides (CP) ................... 33

2.6.5.3. Antioxidative activity ....................................................................... 34

2.7. Cordyceps – A medicine mushroom and another fungal therapeutic

biofactory............................................................................................................. 36

2.8. Preparations.................................................................................................. 44

2.8.1. Pure compounds Data ............................................................................ 45

2.8.2. Polysaccharides and fractions................................................................ 52

2.8.4. Lectin...................................................................................................... 56

2.8.5. Extracts................................................................................................... 56

2.8.5.1. Water extract.................................................................................... 56

2.8.5.2. Methanol extract .............................................................................. 60

2.8.5.3. Ethanol extract................................................................................. 63

2.8.5.4. Ethyl acetate..................................................................................... 64

XI

2.8.5.5. Various............................................................................................. 64

2.8.6. Mycelium study...................................................................................... 65

2.9. Commercial preparations............................................................................. 66

2.10. Compounds of fungi isolated from Cordyceps ......................................... 68

2.11. What about the insect ................................................................................. 69

2.12. Profiling chemical constituents.................................................................. 70

CHAPTER 3........................................................................................................ 70

MATERIAL AND METHODS .......................................................................... 70

3.1. Flowchart of the study.................................................................................. 71

3.2. Materials....................................................................................................... 73

3.2.1. Sources of materials............................................................................... 73

3.2.2. Sources of instruments........................................................................... 75

3.3. Methods........................................................................................................ 77

3.3.1. Apparatus ............................................................................................... 77

3.3.2. Reagents................................................................................................. 77

3.4. Evaluating the factors on the mycelium growth of C. militaris strains AG-1

and PSJ-1 with solid medium.............................................................................. 77

3.4.1. Experimental design............................................................................... 77

3.4.2. Evaluating the culture media on the mycelium growth ......................... 77

3.4.3. Evaluating the temperatures on mycelium growth ................................ 78

3.4.4. Evaluating the carbon sources and concentrations on the mycelium

growth .............................................................................................................. 78

3.4.5. Evaluating the different vitamins B sources and concentrations on

mycelium growth ............................................................................................. 78

3.4.6. Evaluating the different grain sources on the mycelium growth........... 78

3.5. Evaluating the different submerged liquid cultures media and culture

methods on the C. militaris two strains AG-1 and PSJ-1 biomass production... 79

3.6. Evaluating the on the factors on the growth, yield of extrac-cellular and

intra-cellular polysaccharides after submerged culture of C. militaris............... 80

XII

3.6.1. Effect of different pH on biomass and on the growth, yield of extrac￾cellular and intra-cellular polysaccharides after submerged culture of C.

militaris............................................................................................................ 81

3.6.2. Extraction of extra-cellular (EPS) and intra-cellular polysaccharides

(IPS). ................................................................................................................ 80

3.6.3. Effect of oils on biomass and EPS and IPS yield after submerged culture

of C. militaris. .................................................................................................. 81

3.7. Evaluating the factors on antioxidant compound and antioxidant activity of

mycelium............................................................................................................. 81

3.7.1. Sample preparation and extraction for antioxidant................................ 81

3.7.2. Evaluating the factor on antioxidant activities and antioxidant content of

mycelial extracts of C. militaris two strains grown under different submerged

culture condition............................................................................................... 81

3.7.2.1. The drying method of mycelial......................................................... 82

3.7.2.2. Mycelial extracts methods ............................................................... 82

3.7.2.2.1. Methanol extraction of mycelia (Fractionation of the methanol

crude extract) ............................................................................................. 83

3.7.2.2.2. Hot water extraction of mycelial (Fractionation of boiling

distilled water crude extract) ..................................................................... 83

3.7.2.2.3. Ethanol extraction of mycelial (Fractionation of the ethanol

crude extract) ............................................................................................. 84

3.7.2.3. Determination of the antioxidance activity...................................... 84

3.7.2.3.1. Scavenging on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical.84

3.7.2.3.2. Chelating on ferrous ions........................................................... 85

3.7.2.3.3. Hydroxyl radical scavenging assay ........................................... 85

3.7.2.3.4. Scavenging activity of ABTS+ radical cation........................... 86

3.7.2.3.5. Antioxidant activity against lipid peroxidation ......................... 87

3.7.2.3.6. IC50 values in antioxidant properties........................................ 87

3.7.3. Determination of antioxidant contents................................................... 88

3.6.3.1. Total phenolic content (TPC) .......................................................... 88

3.7.3.2. Total flavonoid content (TFC)......................................................... 88

XIII

3.8. Evaluating the factors on the C. militaris strains AG-1 and PSJ-1 fruiting

body production................................................................................................... 89

3.8.1. Experimental design............................................................................... 89

3.8.2. Evaluating the liquid culture media on the mycelium groth of C.

militaris............................................................................................................ 89

3.8.3. Evaluating the liquid culture method on the C. militaris fruiting body

growth. ............................................................................................................. 89

3.8.3.1. Evaluating the liquid culture method on the C. militaris fruiting

body growth................................................................................................... 90

3.8.3.2. Evaluaton the various temperatures, the C. militaris fruiting body

growth. .......................................................................................................... 90

3.8.3.3. Evaluation the pupa powder condition and consentation on the C.

militaris two strains AG-1 and PSJ-1 fruiting body growth......................... 91

3.8.3.4. Evaluating the different LED lights on the C. militaris two strains

AG-1 and PSJ-1 fruiting body growth. ......................................................... 91

CHAPTER 4........................................................................................................ 93

RESULTS AND DISCUSSION ......................................................................... 93

4.1. Microscopic characteristics of Cordyceps species...................................... 93

4.1.1. Morphological characteristics of C. militaris........................................ 93

4.1.2. Observation under microscope .............................................................. 94

4.1.3. Microscopic characteristics of mycelium of Cordyceps species.......... 95

4.2. Effect of different culture media on the mycelium growth.......................... 96

4.3. Effect of temperature on mycelium growth of C. militaris in two strains

AG-1 and PSJ-1................................................................................................. 100

4.4. Effect of carbon sources on mycelium growth of C. militaris................... 103

4.5. Effect of vitamins sources on mycelium growth of C. militaris................ 106

4.6. Effect of different grain sources in myedia on mycelium growth of C.

militaris ............................................................................................................. 109

4.7. Effect of different liquids culture media and culture methods on the biomass

of mycelia growth of C. militaris...................................................................... 112

XIV

4.8. The effect of different media and drying methods on the mycelial dry

weight of C. militaris ........................................................................................ 116

4.9. Antioxidant properties................................................................................ 120

4.9.1. Scavenging activity on DPPH radicals................................................ 120

4.9.2. Chelating effect on ferrous ions........................................................... 123

4.9.3. Hydroxyl radical scavenging ............................................................ 127

4.9.4. Scavenging activity of ABTS.+ radical cation .................................... 132

4.9.5. IC50 values in antioxidant activity ...................................................... 137

4.10. Determination of antioxidant contents..................................................... 140

4.10.1. Total phenolic contents...................................................................... 140

4.10.2. Total flavonoid contents..................................................................... 143

4.11. Effect of pH on the mycelial C. militaris biomass and extra and intra

cellular polysaccharides production.................................................................. 147

4.12. Effect of different oils on biomass and on extra-cellular and intra-cellular

polysaccharide production after submerged culture of C. militaris strains AG-1

and PSJ-1........................................................................................................... 150

4.13. Evaluating the liquid culture on the C. militaris two strains AG-1 and PSJ￾1 fruiting body growth....................................................................................... 152

4.14. Evaluating the temperatures on the fruiting body growth of C. militaris

two strains AG-1 and PSJ-1. ............................................................................. 155

4.15. Evaluating the pupa powder media on fruiting body growth, yield, and

biological characters of the C. militaris two strains AG-1 and PSJ-1. ............. 157

4.16. Evaluating the lights wavelength sources on the fruiting body growth of C.

militaris two strains AG-1 and PSJ-1. .............................................................. 160

CHAPTER 5...................................................................................................... 164

GENERAL CONCLUSION ............................................................................. 164

REFERENCES.................................................................................................. 169