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1 微生物学通报 OCT 20, 2009, 36(10): 1542~1546 Microbiology 研究报告 2009 by Institute of Microbiology, CAS 高产糖化酶黄曲霉菌的选育及初步应用夏艳秋 1* 朱强 1 汪志君 2 ( ) ( ) 摘要 : 试验目的是选育优良黄酒糖化菌种对原始菌株 YA, 采用紫外线与 LiCl 复合诱变碳酸钡诱发及自然分离方法, 选育到一株淀粉酶活力高不产黄曲霉毒素的优良黄曲霉突变株 YA4.9 其生长繁殖快, 菌丝粗短密集最佳制曲时间为 32 h, 糖化酶和液化酶活力分别为 U/g 和 U/g, 比亲株提高 % 和 65.08%, 酿酒性能好继代培养表明其性状能稳定遗传关键词 : 黄酒, 黄曲霉, 糖化酶, 黄曲霉毒素, 选育 Breeding of High-Glucoamylase Activity Aspergilius flavus and Application XIA Yan-Qiu 1* ZHU Qiang 1 WANG Zhi-Jun 2 (1. College of Marine, Huaihai Institute of Technology, Lianyungang, Jiangsu , China) (2. College of Food Science and Technology, Yangzhou University, Yangzhou, Jiangsu , China) Abstract: In order to breed excellent diastatic strain using for Chinese Rice Wine fermentation, UV, LiCl, BaCO 3 and natural selection to mutagenize (Aspergillus flavus) YA were used. Mutant (Asp. flavus) YA4.9 was obtained with properties as follows: growing fast, mycelium dense-dumpy, not producing aflatoxins. The optimum culture time of moldy bran was 32 hours, the glucoamylase and α-amylase were U/g and U/g, comparing to the parent strain, increased by % and 65.08%, respectively, the finished Chinese Rice Wine has good quality. The inherited characters were steadily by many generations. Keywords: Chinese Rice Wine(CRW), Aspergillus flavus, Glucoamylase, Aflatoxin, Breeding,,,,,,,,,,,,,,,,,,, * 通讯作者 :Tel: ; : xyq78412@163.com 收稿日期 : ; 接受日期 :

2 : 1543,,, 1 材料与方法 1.1 菌种 1) (Asp. flavus)ya, ; (Asp. flavus)afp,, ; 2), 1.2 培养基斜面培养基 : 初筛培养基 (W/V): 1.2%, 0.8%, 2%, ph 5.0~ 复筛培养基 (W/V): 1:1, NaNO 3 1.5%, K 2 HPO %, MgSO 4 7H 2 O 0.03%, ph 4.0~ 中间培养培养基 (W/V): 5%, 1.5%, 1.5%, ph 5.0~ 花生察氏培养基 (PCD): 10%, NH 4 Cl 0.16%, ph 5.5~ HgCl 2 培养基 (SM)(W/V): 0.05%, HgCl mol/l, (NH 4 )H 2 PO 4 1%, ph 5.5~ 基础培养基 (PGAN)(W/V): 7.5 L, A B 10 ml (A: ZnSO 4 7H 2 O 2%, CuSO 4 5H 2 O 0.2%, CoCl 2 0.1%; B: CaCl 2 5%), 5%, NH 4 NO %, ph 5.5~ Pa 50 min, Pa 20 min 1.3 试验处理方法单孢子悬浮液 (MSS) 的制备 :,,,,, CFU/mL 初筛 : 0.2 ml MSS, 30 C 2 d~3 d,, (D) (d),,, d/d 复筛 :, 30 C 5 d~7 d, 2, 32 C 36 h,,, 麸曲粗酶浸提液的制备 :, ph ( ph 4.6 ; ph 6.0 ; ph 3.0 ), 30 C 1 h,, 3000 r/min 20 min, 紫外线 (UV) 和氯化锂 (LiCl) 复合诱变 : 5 ml MSS, 15 W 28 cm, 10 h~12 h,, LiCl 碳酸钡 (BaCO 3 ) 诱发试验 : 100 ml PGAN 1.0 g BaCO 3, 30 C 200 r/min,, 24 h g BaCO 3, 1 g~2 g 产黄曲霉毒素菌株的快速检测 : [1] PCD SM, 30 C 36 h~ 48 h,,, 125 W 麸曲氯仿抽提液的制备 : 3 g 7.5 ml, 5 min, 2.5 ml, 5 min,,,,, 2, 黄酒发酵工艺 : 1.4 分析方法糖化酶活力的测定 : GB/T : 1 g 40 C ph 4.6, 1 h 1 mg 液化酶活力的测定 : [2] : 1 g 60 C ph 6.0, 5 min 1 mg 酸性蛋白酶活力的测定 : SB/T : 1 g 40 C ph 3.5, 1 min 1 μg 黄酒理化指标的测定 : GB/T

3 1544 微生物学通报 2009, Vol.36, No.10 2 结果与分析 2.1 菌株筛选结果,, 4 1 d/d 1 Table 1 Glucoamylase Results of screening α-amylase YA YA YA YA , YA4 d/d,,, [2],,,,,,, E. brlich,, YA4, 2.2 菌株育种结果 1, YA4 LiCl, UV 3 min 4 min, 37.44% 88.26%; 0.6% LiCl, 73.63% 99.91%, LiCl,, [3], LiCl, UV YA4, 图 1 紫外线与 LiCl 复合诱变 YA4 的致死曲线 Fig. 1 Lethal curves of YA4 UV-LiCl mutagenized, 70%~80%,, UV 3 min, 0.6% LiCl YA4 Adye [4],,, Ba 2+ YA 1 UV 2 UV LiCl BaCO 3 4 (ns), YA 图 2 菌株选育谱系 Fig. 2 Breeding hierarchy of strains ns UV ns UV+LiCl YA YA4 UV UV14-12 Table 2 ns UV+LiCl BaCO 3 ns UV YA4.9 表 2 选育过程中菌株酶活变化 Changes of enzyme of strains in breeding d/d Glucoamylase α-amylase YA YA UV UV UV YA ,,, YA, YA % 65.08%, YA4.9,,,,,, 2.3 突变株 YA4.9 遗传稳定性 YA4.9, 3 3, YA4.9 15,,

4 : 1545 表 3 Table 3 Generation 传代对 YA4.9 酶活力的影响 Effect of passage on YA4.9 enzymes Glucoamylase α-amylase 突变株 YA4.9 黄曲霉毒素的鉴定, 222 nm 265 nm 362 nm,,, B 2 [1], YA 表 4 PCD 和 SM 培养基上各菌株的荧光现象 Table 4 Fluorescence of strains on PCD and SM Culture medium PCD SM Culture time (d) YA YA(CK ) + AFP(CK + ) YA4.9 YA(CK ) AFP(CK + ) :, ++++: ; +++: ; ++: ; +: ; :. Note: Fluorescence strength, ++++: Best strong; +++: Strong; ++: Better strong; +: Weak; : No fluorescence., 4, PCD, AFP,, YA4.9 YA 4 6, HgCl 2 [1], SM, YA4.9 YA 6 d, AFP, YA4.9,, 3, YA4.9 YA,, 2.5 突变株 YA4.9 生长与产酶时效 4, YA, YA4.9, 20 h,, 32 h~36 h,,,, YA h,,, 4 YA4.9 YA - Fig. 4 Time-efficiency curve of YA4.9 and YA 2.6 突变株 YA4.9 用于黄酒酿造结果 5, YA, YA4.9,,,,,, 图 3 各菌株麸曲氯仿抽提液的紫外吸收光谱 Fig. 3 UV spectrometries of moldy bran abstractions by CHCl 3 3 讨论 YA4.9

5 1546 微生物学通报 2009, Vol.36, No.10 Alcohol (%Vol) Table 5 Residul sugar 5 YA4.9 YA Finished CRW properties of YA4.9 and YA moldy bran Total acid α-aminontrogen Volatile acid Volatile ester Fusel oil Flavour YA YA U/g U/g,,,,,,,, ,,,,, [5 10], ( )YA4.9 ( 6),,, YA4.9,,, ( ) 6 YA4.9 Table 6 Comparison of the glucoamylase of YA4.9 with those reported Glucoamylase Asp. flavus SJM-4 [8] Asp. flavus Su-16 [9] 1120 Japanese Asp. flavus 1# [9] 960 Asp. Flavus YA , ( UV) [11], 参考文献 [1] Hare S, Fennell DI, Hesseltine CW, et al. Aflatoxin-producing strains of Asp. flavus detected by fluorescence of agar medium under ultraviolet light. Appl Microbiol, 1974, 27(7): [2],. a-., 1995, 22(1): [3].., 2000, 22(6): [4] Adye J, Matele RI. Incorporation of labelled compounds into aflatoxins. Biochem Bioph Acta, 1964, 86: [5],,.., 2004, 5: 1 3. [6],,,. (Aspergillus flavus)., 2003, 5: [7],,.., 2003, 3: [8],,.., 2003, 3: [9]. 1#-16., 2004, 6: [10],,,.., 2008, 29(3): [11] Kole MM, Altosaar I. Increased chitinase production by a non-pigmented mutant of Serratia marcescens. FEMS Microbiol Letters, 1985, 26(3):

: 307, [], [2],,,, [3] (Response Surface Methodology, RSA),,, [4,5] Design-Expert 6.0,,,, [6] VPJ33 ph 3,, ph, OD, Design-Expert 6.0 Box-Behnken, VPJ3

: 307, [], [2],,,, [3] (Response Surface Methodology, RSA),,, [4,5] Design-Expert 6.0,,,, [6] VPJ33 ph 3,, ph, OD, Design-Expert 6.0 Box-Behnken, VPJ3 微生物学通报 FEB 20, 2008, 35(2) 306~30 Microbiology 2008 by Institute of Microbiology, CAS tongbao@im.ac.cn 生物实验室响应面分析法优化副溶血性弧菌生长条件刘代新宁喜斌 * 张继伦 2 (. 200090) (2. 20202) 摘要