Culture conditions for mycelial growth of Poria cocos

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J. Mushrooms 2016 March, 14(1):6-13 http://dx.doi.org/10.14480/JM.2016.14.1.6 Print ISSN 1738-0294, Online ISSN 2288-8853

© The Korean Society of Mushroom Science

*Corresponding author E-mail : [email protected]

Tel : +82-53-320-0321, Fax : +82-53-320-0295 Received February 29, 2016

Revised March 17, 2016 Accepted March 24, 2016

Culture conditions for mycelial growth of Poria cocos

Woo-Sik Jo¹*, Ju-Ri Park¹, So-Ra Oh¹, Min-Gu Kang¹, Woo-Hyun Kim¹ and Seung-Chun Park²

1Gyeongbuk Province Agricultural Technology Administration, Daegu 702-708, Korea

2College of Veterinary medicine Kyugpook National University, Daegu 702-701, Korea

ABSTRACT: This study was carried out to determine the basic mycelial culture conditions for Poria cocos growth. According to colony diameter and mycelial density, suitable media for mycelial growth were Malt yeast extract, Potato dextrose agar, Yeast extract agar, and Yeast malt agar. The optimum temperature for mycelial growth was between 25 and 35^oC, and the optimum pH value was between 4 and 7. Carbon and nitrogen sources were fructose and yeast extract. The optimum C/N ratio was about 10 to 1 with 2% glucose. Other minor components for optimal growth were thiamine-HCl and nicotinamide as vitamins, acetic and lactic acid as organic acids, and MgSO4·7H2O and FeSO4·7H2O as mineral salts.

KEYWORDS: Culture condition, Medicinal mushroom, Poria cocos

Introduction

The number of mushrooms on Earth is estimated at 140,000 species, yet only 10% of them are known (Kirk et al., 2001). For a long time, mushrooms have been valued as an edible and medicinal resource (Sung et al., 2000). Poria cocos is a species of medicinal Polyporales fungus found worldwide (Sung et al., 2000). The sclerotium of Poria cocos is uesd medicinally. The fruiting bodies of Poria cocos is reproductive organ and the mycelium is vegetative organ. Fruiting bodies with a diameter 0.5~2 mm, is a sawtooth-shaped (Hong et al., 1991).

Moreover, P. cocos was reported it's excellent at stomach, heart, lung and kidney as one of the crude drug. It is used that the treatment effect on anti-stomach disorder, anti-diabetes and anti-tuberculosis (Jo et al., 2013). This study has been conducted to development of mass culture system and species improvement, pharmacological

action of Poria cocos. This study was focused on culture conditions affecting the optimal mycelial growth of Poria cocos.

Materials and Methods

Fungal isolates

The isolates of P. cocos species used in this study were listed in Table 1. P. cocos ASI 13007 was obtained from Rural Development Administration. KFRI 1103, KFRI 1104, KFRI 1105, KFRI 1106, KFRI 1107 and KFRI 1108 were obtained from KOREA FOREST SERVICE. Andong 01, Andong 02 and Andong 03 were

Table 1. List of Poria cocos strains used in this study Scientific

name

Origin

culture Organ

Poria cocos Andong 01 Ryu Chunghyeon medicinal mushroom. Co., Ltd.

Poria cocos Andong 02 Ryu Chunghyeon medicinal mushroom. Co., Ltd.

Poria cocos Andong 03 Ryu Chunghyeon medicinal mushroom. Co., Ltd.

Poria cocos KFRI 1103 KOREA FOREST SERVICE Poria cocos KFRI 1104 KOREA FOREST SERVICE Poria cocos KFRI 1105 KOREA FOREST SERVICE Poria cocos KFRI 1106 KOREA FOREST SERVICE Poria cocos KFRI 1107 KOREA FOREST SERVICE Poria cocos KFRI 1108 KOREA FOREST SERVICE Poria cocos ASI 13007 Rural Development Administration,

Korea

obtained from Ryu Chunghyeon medicinal mushroom.

Co., Ltd. All isolates were maintained on Potato Dextrose Agar medium (PDA).

Culture media

Twelve different culture media were prepared to screen suitable culture media to mycelial growth of P.

cocos (Table 2). The culture media were sterilized for 20 minutes at 121^oC and aseptically poured into plastic petridish. An inoculum was removed from five days old cultures of P. cocos grown on PDA at 25^oC. Mycelial disk (5 mm in diameter) from the cultures was placed in the center of each 85 mm plastic petridish containing about 20 ml of 12 different media. The fungi were incubated under the dark condition for 5 days at 25^oC.

Thereafter the mycelial growth, density and color of the colony were examined.

Temperature

The growth of mushroom was investigated at different temperatures in the ranges of 10~35^oC. The fungi were cultured on PDA for 4 days and the mycelial growth was determined as described above.

Effect of pH

To screen pH value necessary for a favorable growth of P. cocos species, a 5 mm diameter plug of an inoculum was removed with cork borer from 10 days old cultures of P. cocos grown on PDA, placed in the center of PDA adjusted to the range of pH 4~9 with 1 N NaOH or HCl and incubated under the dark condition for 4 days at 25^oC. The measurement of mycelial growth was performed according to the method described by Shim et al.

(1997).

Effect of favorable nutrient sources Carbon sources

The experiment was performed on the mushroom minimal media (MMM: dextrose 20 g, MgSO₄ 0.5 g, KH₂PO₄ 0.46 g, K₂HPO₄ 1 g, asparagine 2 g, thiamine- HCl 120 µg, agar 20 g, distilled water 1,000 ml) supplemented with each of 10 carbon sources. Each carbon source was added to mushroom minimal media at the concentration of 2%. The fungi were incubated under the dark condition for 10 days at 25^oC. Thereafter we examined the mycelial growth, density and color of the colony.

Table 2. Composition of media used in this study Nutritional

reagents

Media and composition (g/l)

PDA MEA YEA Czapek

dox

Glucose

peptone YMA Malt Yeast

extract Leonian MCM Henner

-berg Lilly Hoppkins

Glucose 10 10 10 25 20 50 10

Sucrose 30

Maltose 10

Peptone 3 10 5 2

Yeast extract 5 10 3 5 2

Malt extract 30 15 3 3

Potato extract 4

DL-Asparagine 2

Dextrose 20 10

NaNO3 3 2

MgSO₄·7H₂O 0.5 0.5 0.5 0.5 0.5 0.5

KCl 0.5

FeSO4·7H2O 0.01 0.02

CaCl₂·2H₂O 0.1

ZnSO4·7H2O

MnSO4·5H2O 0.01

K₂HPO₄ 1 1

KH2PO4 1 1 0.5 1 1 0.1

KNO3 2 2

Agar 15 15 20 20 20 20 20 20 20 20 20 20

Nitrogen sources

To screen nitrogen source suitable to the mycelial growth of P. cocos, the experiment was performed on the mushroom minimal media supplemented with each of 12 nitrogen sources. Each nitrogen source was added to mushroom minimal media at the concentration of 0.2%. A 5 mm diameter plug an inoculum of P. cocos cultures placed in the center of petridish and incubated under the dark condition for 5 days at 25^oC. Thereafter the mycelial growth, density and color of the colony were examined.

C/N ratio

On the mushroom minimal media which were mixed with 10, 8, 6, 4, 2, 1, 0.4 and 0.2% glucose as a carbon source and then mixed continually with 0.2% NaNO₃ as a nitrogen source, the mycelial growth of P. cocos was examined. The C/N ratio was adjusted to 50:1, 40:1, 30:1, 20:1, 10:1, 5:1, 2:1 and 1:1 in each medium.

Inoculated each media incubated under the dark condition for 7 days at 25^oC. Thereafter the mycelial growth, density and color of the colony were examined.

Vitamin

On the sterilized mushroom minimal media which were mixed with thiamine-HCl 0.1 mg/l, riboflavin 0.5 mg/l, biotin 0.005 mg/l, pyridoxine 0.5 mg/l and nicotinamide 2.0 mg/l those were filtrated by metrical membrane filter (0.2µm). Inoculated each media incubated under the dark condition for 10 days at 25^oC. Thereafter the mycelial growth, density and color of the colony

were examined.

Organic acid

On the mushroom minimal media which were mixed with acetic acid, citric acid, maleic acid, lactic acid, succinic acid and fumaric acid at the concentration of 0.1%, respectively. Inoculated each medium incubated under the dark condition for 5 days at 25^oC. Thereafter the mycelial growth, density and color of the colony were examined.

Mineral salt

To screen mineral salts suitable to the mycelial growth of P. cocos, the experiment was performed on the YM solid media (peptone 5 g, yeast extract 3 g, malt extract 3 g, dextrose 10 g, agar 20 g and DW 1,000 ml) which was supplemented with each of 9 mineral salts. Each mineral salt was added to YM solid media at the concentration of 0.1%. Inoculated each media incubated under the dark condition for 5 days at 25^oC. Thereafter the mycelial growth, density and color of the colony were examined.

Results and Discussion

Screening of the suitable culture media

The mycelial growth of P. cocos was favorable in Malt Yeast Extract, PDA, YEA and YMA whereas was poor in Czapek dox, Leonian, Hennerberg, Lilly and Hoppkins medium (Table 3). The mycelial growth of ASI 13007 was less than KFRI 1103, KFRI 1104, KFRI

Fig. 1. Mycelial growth of P. cocos on PDA for 4 days at different temperatures.

Table 3. Effect of culture medium on the mycelial growth of Poria cocos at 25o C Culture media

Mycelial densitya) Colony diameter (mm/5 days) Andong 1Andong 2Andong 3KFRI 1103KFRI 1104KFRI 1105KFRI 1106KFRI 1107KFRI 1108ASI 13007Andong 1Andong 2Andong 3KFRI 1103KFRI 1104KFRI 1105KFRI 1106KFRI 1107KFRI 1108ASI 13007 PDASTSCTSCCCSCSTSTT28.3±6.8a 83.8±1.3a 30.3±9.0bc 74.7±3.4a 85.0±0.0a 82.7±1.3a 81.2±1.0a 64.8±4.3a 85.0±0.0a 39.7±1.6d MEA-STSTSTSCSCCSCSTT069.0±2.6abc 53.8±1.8a 48.3±5.2c 64.3±2.5d 67.3±2.5d 73.3±5.3b 50.2±5.2b 69.8±5.5b 39.3±1.5d YEASTSTSTSCCCCSCSTST7.7±2.9d 74.3±3.9a 32.0±23.2b 74.0±3.1a 78.5±0.5bc 81.3±1.3a 83.0±3.0a 62.7±8.9a 84.0±1.7a 15.0±4.8f Czapek dox-T-TTTT-T-026.3±1.3de 012.2±11.4d 19.3±2.0g 30.7±2.4f 33.0±3.6e 031.3±0.8e 0 Glucose Peptone-TTCSCCCSCCT016.0±27.7e 9.8±1.0d 62.5±1.5b 72.5±2.6c 74.2±1.6bc 71.8±6.4b 6.5±1.3d 73.0±2.8b 5.0±0.0g YMASTST-SCSTCCSTSTST8.5±0.9cd 12.0±0.5e 071.5±3.8a 78.3±7.5bc 82.3±1.3a 82.0±4.8a 68.5±1.7a 73.0±6.4b 11.7±1.0f Malt Yeast ExtractTCCCCCCSTSTT7.5±0.5d 82.3±1.3a 68.5±14.5a 75.8±3.8a 83.8±0.3ab 83.5±0.9a 82.3±1.3a 67.5±9.5a 83.5±0.5a 23.3±3.0e LeonianTTTTTTTTTT15.3±3.9bc 54.2±3.0c 13.0±5.0cd 45.7±0.6c 66.8±5.0d 52.8±1.5e 52.5±1.0cd 28.2±1.9c 52.2±3.3d 51.2±0.8bc MCM-SCTSTSTSTSC-ST-072.0±4.0ab 11.8±20.5d 70.3±3.4a 83.2±1.4ab 75.3±4.1b 80.3±1.0a 073.5±1.8b 0 HennerbergTT-TTTTTTT21.7±10.8ab 56.3±0.8bc 041.7±1.0c 51.8±1.4f 51.3±1.6e 50.0±0.5d 26.3±3.0c 50.3±2.6d 54.3±1.3ab LillyTST-TTTSTSTTT12.3±0.8cd 38.7±8.0d 040.2±4.3c 58.5±1.3e 71.3±4.0c 57.8±4.1c 24.8±4.5c 62.0±9.2c 57.5±3.9a HoppkinsTT-TTTTTTT13.8±3.2cd 68.3±1.3abc 046.0±3.5c 73.3±5.3c 54.0±1.0e 56.7±3.1c 24.5±1.7c 56.7±5.8cd 49.5±0.5c a) : C; Compact, SC; somewhat compact, ST; Somewhat thin, T; thin a~d : Value in the same line with different literal diffet at Duncan'smultiple range test(P<0.05) and results are mean ± standard deviation of three replicates Table 4. Effect of pH on the mycelial growth of Poria cocos at 25o C pH

Mycelial densitya) Colony diameter (mm/4 days) Andong 1Andong 2Andong 3KFRI 1103KFRI 1104KFRI 1105KFRI 1106KFRI 1107KFRI 1108ASI 13007Andong 1Andong 2Andong 3KFRI 1103KFRI 1104KFRI 1105KFRI 1106KFRI 1107KFRI 1108ASI 13007 4STSTTSTSTTTSTTT61.0±1.8a 73.3±8.6a 68.8±4.0a 58.3.0±1.0a 84.3±1.2a 65.5±1.8a 68.5±0.5a 74.7±2.0a 56.7±0.3ab 80.3±0.6a 5STSCSTSTSTSTSTSTTST57.3±4.3ab 59.0±22.6a 41.5±17.3b 56.0±6.9a 81.0±1.5b 49.8±9.0b 66.5±6.9a 77.7±4.5a 60.2±9.0a 47.7±10.9b 6SCSCSTSTSTSTSTSTSTT49.2±4.9bc 59.0±22.6a 22.0±4.0c 52.2±5.0a 81.2±1.8b 40.7±4.8b 66.2±5.3a 71.3±3.3ab 48.3±2.3bc 32.7±2.1c 7STSCTSTSTSTSTSTSTT45.0±1.7cd 24.3±2.1b 7.8±2.0d 52.0±1.8a 79.2±1.3bc 39.7±9.5b 58.5±12.7ab 66.2±2.0ab 44.5±5.1c 26.8±1.6cd 8SCSTSTSTSTSTSTSTSTST35.0±8.0d 12.7±5.8b 9.3±1.0cd 50.7±0.8a 76.8±1.5c 22.8±8.0c 49.0±8.9b 52.3±4.3b 37.8±4.2c 22.2±6.0d 9ST-TSTSTT STSTSTT19.2±9.6e 07.8±1.4d 38.3±4.6b 69.5±1.3d 9.7±0.8d 34.0±1.8c 33.7±23.9c 25.8±9.6d 5.3±4.7e a) : C; Compact, SC; somewhat compact, ST; Somewhat thin, T; thin a~d : Value in the same line with different literal diffet at Duncan'smultiple range test(P<0.05) and results are mean ± standard deviation of three replicates

Table 5. Effect of carbon sources on the mycelial growth of Poria cocos at 25o C Culture media

Mycelial densitya) Colony diameter (mm/10 days) Andong 1Andong 2Andong 3KFRI 1103KFRI 1104KFRI 1105KFRI 1106KFRI 1107KFRI 1108ASI 13007Andong 1Andong 2Andong 3KFRI 1103KFRI 1104KFRI 1105KFRI 1106KFRI 1107KFRI 1108ASI 13007 SucroseSTT-TSTTSTSTTT50.0±29.4c 10.0±0.0a 01.7±2.9bc 85±0.0a 9.2±0.3b 27.3±32.2a 54.3±7.2b 7.5±0.5bc 7.0±0.0b LactoseSTT-TSTTSTTTT75.0±2.2ab 5.2±4.8bc 03.3±2.9ab 85±0.0a 7.2±0.3cde 31.0±36.5a 50.2±1.3bc 7.5±0.5bc 6.8±0.3b DextroseSTT-TSTTSTTTT81.2±4.3a 7.3±0.6ab 05.0±0.0a 85±0.0a 10.2±0.8a 22.2±13.0a 64.3±8.9ab 7.8±0.3b 7.5±0.5b MannitolSTT-TSTTSTTTT83.3±0.8a 7.5±0.5ab 05.0±0.0a 85±0.0a 7.7±0.6cd 10.7±4.3a 32.7±10.3cd 7.0±0.5c 7.3±0.3b MaltoseSTT-TSTTTTTT75.8±4.3ab 4.0±3.5bc 04.0±3.5ab 85±0.0a 7.3±0.6cde 8.2±0.3a 34.0±17.5cd 6.8±0.3c 1.8±3.2c GlucoseSTT-TSTTTTTT57.8±7.8bc 1.8±3.2c 06.0±0.0a 85±0.0a 6.8±0.3de 7.5±0.5a 6.7±0.6e 7.0±0.5c 6.3±0.8b FructoseSTTTTSTTSTTTT56.3±6.4bc 5.5±0.0bc 7±0.5a 5.2±0.3a 85±0.0a 6.8±0.3de 15.8±6.7a 17.7±10.4de 6.8±0.3c 12.0±0.5a SorbitolSTT-TSTTSTTTT51.3±14.0c 3.3±2.9bc 05.0±0.0a 85±0.0a 7.2±0.6cde 35.8±27.3a 32.8±12.8cd 6.8±0.3c 7.2±1.8b MannoseSTT-TSTTTTTT74.5±8.3ab 10.5±0.5a 05.0±0.0a 85±0.0a 7.8±0.3c 10.7±2.3a 21.5±17.2de 7.0±0.5c 6.5±0.5b StarchSTT--STT-STT-83.3±2.9a 6.7±0.8ab 0085±0.0a 6.5±0.9e 078.5±11.3a 85.0±0.0a 0 a) : C; Compact, SC; somewhat compact, ST; Somewhat thin, T; thin a~d : Value in the same line with different literal diffet at Duncan'smultiple range test(P<0.05) and results are mean ± standard deviation of three replicates Table 6. Effect of Nitrogen sources on the mycelial growth of Poria cocos at 25o C Culture media

Mycelial densitya) Colony diameter (mm/5 days) Andong 1Andong 2Andong 3KFRI 1103KFRI 1104KFRI 1105KFRI 1106KFRI 1107KFRI 1108ASI 13007Andong 1Andong 2Andong 3KFRI 1103KFRI 1104KFRI 1105KFRI 1106KFRI 1107KFRI 1108ASI 13007 Yeast Extract-TTSTSTSTSTSTSTT070.5±4.8a 47.5±23.4a 73.2±2.0a 85.0±0.0a 85.0±0.0a 85.0±0.0a 85.0±0.0a 79.5±2.2a 52.5±13.7b Malt ExtractTTTTTTSTTTT22.5±13.9ab 31.5±4.3c 2.2±3.8a 7.5±0.5de 57.5±17.8d 59.5±5.4d 37.5±29.8d 43.5±1.8def 48.0±5.2cd 35.0±1.3c PeptoneTTTTTSTTSTTT15.5±15.3ab 47.2±10.5b 5.7±5.5a 50.2±6.7b 76.0±0.5ab 76.7±4.5b 44.5±13.5cd 53.2±3.0cd 47.8±2.0cd 42.8±4.0bc Urea----------0000000000 Ammonium nitrateT--TTT-TTT12.0±10.4ab 002.7±4.6e 59.3±3.6d 60.3±4.0cd 09.5±0.5g 4.8±8.4f 34.7±1.0c Ammonium chloride-TTTTTTTTT06.0±0.0e 11.7±0.6a 4.8±4.2de 76.8±3.8ab 53.0±3.8de 55.3±1.0bc 40.2±4.5ef 47.3±3.4cd 51.5±5.8b Ammonium acetate--T-T-----001.8±3.2a 06.0±0.5e 00000 Ammonium sulfate-TT-TTTTTT05.5±0.0e 7.5±0.5a 067.5±6.4bcd 40.2±4.2f 52.2±1.6cd 36.8±2.8f 39.3±3.8e 38.2±6.4c Potassium nitrateTTTTTTTTTT23.0±23.5ab 35.8±1.5c 16.2±8.1a 12.0±9.6d 59.2±3.8d 46.2±5.0ef 45.8±2.8cd 40.0±12.3ef 51.8±5.1c 35.5±5.4c Sodium nitrateTTTTTT-TTT11.7±6.8ab 25.2±1.6d 2.3±23.4a 7.3±8.4de 57.2±3.3d 48.0±9.1e 046.3±8.3def 43.5±4.8de 33.2±1.9c Calcium nitrateTTT-TTTTTT24.3±9.8a 5.8±0.3e 23.8±20.6a 0.0±0.0e 58.7±5.3d 7.0±0.5g 46.0±4.0cd 59.7±2.4bc 66.3±2.8b 51.8±7.3b L-glutamic acidTTTTTTTTTT15.0±26.0ab 34.2±5.2c 39.8±4.5a 25.7±1.0c 70.2±1.5bc 67.3±4.2c 57.2±4.9bc 63.7±1.5b 73.2±1.5a 67.5±2.2a L-arginineTTTTTTTTTT6.3±11.0ab 5.5±0.0e 32.2±8.1a 8.2±0.8de 64.8±0.8cd 39.2±3.3f 70.3±3.8ab 47.8±10.4de 6.0±0.5f 38.3±3.4c a) : C; Compact, SC; somewhat compact, ST; Somewhat thin, T; thin a~d : Value in the same line with different literal diffet at Duncan's multiple range test (P<0.05) and results are mean ± standard deviation of three replicates

Table 7. Effect of C/N ratio on the mycelial growth of Poria cocos at 25o C C/N ratio

Mycelial densitya) Colony diameter (mm/7 days) Andong 1Andong 2Andong 3KFRI 1103KFRI 1104KFRI 1105KFRI 1106KFRI 1107KFRI 1108ASI 13007Andong 1Andong 2Andong 3KFRI 1103KFRI 1104KFRI 1105KFRI 1106KFRI 1107KFRI 1108ASI 13007 1 : 1TTTTTTTTTT16.2±0.3a 64.7±7.8bc 29.8±2.5a 29.0±1.8c 85.0±0.0a 54.5±1.8b 49.5±16.3a 48.8±4.0cd 67.0±1.5b 40.3±5.1b 2 : 1TTTTTTTTTT8.7±2.5c 70.3±1.6ab 32.5±0.9a 32.3±3.3c 85.0±0.0a 56.7±4.9ab 51.7±1.3a 62.3±6.3ab 76.8±4.3a 51.2±6.1a 5 : 1TTTTTTTTTT11.2±0.8b 72.7±7.1a 33.5±1.5a 41.8±2.5b 85.0±0.0a 60.7±2.4a 53.5±2.6a 53.5±8.2bcd 72.5±5.3ab 56.0±1.0a 10 : 1TTTTTTTTTT10.8±0.3b 72.8±4.3a 35.7±2.3a 47.3±0.8a 85.0±0.0a 59.8±2.1a 58.5±2.8a 67.5±3.9a 70.3±4.9ab 57.2±0.8a 20 : 1TTTTTTTTTT8.7±1.6c 59.3±1.5c 30.0±3.0a 48.5±3.5a 71.3±0.6b 56.8±0.6ab 55.8±0.8a 58.2±9.1abc 66.3±4.5b 53.5±2.8a 30 : 1TTTTTTTTTT9.8±0.6bc 59.8±1.0c 30.0±2.3a 47.5±0.5a 68.5±1.8c 55.3±0.6b 54.2±3.1a 57.8±1.6abc 70.2±9.1ab 57.2±6.9a 40 : 1TTTTTTTTTT11.0±0.5b 60.2±1.5c 31.3±6.7a 48.8±3.3a 67.8±1.5c 53.3±1.3b 54.8±3.8a 50.0±1.8cd 66.7±0.6b 56.7±1.2a 50 : 1TTTTTTTTTT10.2±0.3bc 57.0±1.3c 20.2±6.3b 43.7±5.0ab 65.2±0.8d 52.8±1.6b 54.5±0.5a 47.0±2.8d 56.5±0.9c 54.8±1.0a a) : C; Compact, SC; somewhat compact, ST; Somewhat thin, T; thin a~d : Value in the same line with different literal diffet at Duncan'smultiple range test(P<0.05) and results are mean ± standard deviation of three replicates Table 8. Effect of vitamins on the mycelial growth of Poria cocos at 25o C Temp.

Mycelial densitya) Colony diameter (mm/10 days) Andong 1Andong 2Andong 3KFRI 1103KFRI 1104KFRI 1105KFRI 1106KFRI 1107KFRI 1108ASI 13007Andong 1Andong 2Andong 3KFRI 1103KFRI 1104KFRI 1105KFRI 1106KFRI 1107KFRI 1108ASI 13007 Thiamine HClSTTTTSTT-TT-28.2±10.7b 5.3±0.3a 5.0±0.0a 5.7±0.3a 85.0±0.0a 19.0±32.9a 063.7±6.7bc 79.0±10.4a 0 RiboflavinTTTTST-TSTT-9.0±4.8b 5.5±0.5a 5.2±0.3a 5.7±0.8a 85.0±0.0a 05.8±0.3a 85.0±0.0a 85.0±0.0a 0 BiotinTTTTT--TT-31.7±7.5b 5.0±0.0a 5.8±0.3a 5.0±0.0a 85.0±0.0a 0058.8±8.9c 56.3±43.3a 0 PyridoxineSTTTTST--TT-21.2±26.7b 5.8±0.6a 6.5±1.7a 5.2±0.3a 85.0±0.0a 0065.2±2.3bc 85.0±0.0a 0 NicotinamideSTTTTST--TT-73.2±14.4a 5.7±0.8a 6.3±1.9a 5.3±0.3a 85.0±0.0a 0072.8±6.1b 74.7±4.5a 0 a) : C; Compact, SC; somewhat compact, ST; Somewhat thin, T; thin a~d : Value in the same line with different literal diffet at Duncan'smultiple range test(P<0.05) and results are mean ± standard deviation of three replicates