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1 5 12 Vol. 5 No Journal of Food Safety and Quality Dec., 2014 D * 王乐, 惠明, 尹艳丽, 黄继红 (, ) 摘要 : 目的 D- 方法,,, D- 结果 200 g/l, 30, 120 h, D g/l,,, b-1 Candida parapsilosis 结论 b-1 D-,, 关键词 : D-; ; ; ; Isolation and screening of a strain for high D-arabitol production WANG Le, HUI Ming, YIN Yan-Li, HUANG Ji-Hong * (College of Biological Engineering, Henan University of Technology, Zhengzhou , China) ABSTRACT: Objective To isolate and screen a high yield strain for D-arabitol production. Methods With the determination of paper chromatography and high performance liquid chromatography (HPLC), the fine strain for high concentration yield of D-arabitol from glucose had been separated and screened from high glucose environment, such as raisins, sweet potato to soil, honey, pollen samples, after the first and second screening. Results At the initial glucose concentration of 200 g/l on the culture medium, 30, D-arabitol yield was 82.4 g/l after the fermentation of 120 h. According to the conventional morphological identification, determination of physiological and biochemical experiment, and the nucleic acid sequence homology comparison analysis, the high-producing strain b-1 was confirmed as Candida parapsilosis. Conclusion The yield of D-arabitol is high, the by-products are low in the fermentation by the screened strains b-1, which has a certain application prospect of industrialization. KEY WORDS: D-arabitol; glucose; yeast; screening; identification 1 引言 D-,,,,, [1,2], D- [3] 基金项目 : ( ), ( ), () (CRE-2014-C-301) Fund: Supported by the National Natural Science Foundation of China ( ), Natural Science Foundation of Science and Technology Agency of Henan Province ( ) and (Effective Utilization of Chemical Resources) State Key Laboratory Project (CRE-2014-C-301) * 通讯作者 :,,, Huangjihong1216@126.com *Corresponding author: HUANG Ji-Hong, Doctor, Senior Engineer, Henan University of Technology Institute of Biological Engineering, Zhengzhou , China. Huangjihong1216@126.com

2 12, : D- 4019,, [4-6] D-,,,,,, [7] D- (EMP) 6- (PPP) [8] D- [9] D- (Aspergillus) [10] (pichia) [11] (Debaryomyces) [12] (Hansenula) [13] D-, D- 1, D- [14] D- [15-17], VHb Trition X-100 D- [18] Tokuoka [7,9] D-, D-, [19], D-, D-,,,,, D- 2 材料与方法 2.1 样品来源,, 2.2 实验试剂及设备主要试剂 D-,,,,,,,,,, DNS(3,5- ),,,,,,,,,,,, D-,,,,,, (1) - A, 0.1 ml,, 20 ml B, NaOH 0.5 mol/l (2) : : : (v:v:v)=40:10:10 (3) DNS [20] 主要设备 JY2502 ( ); (); ( ); HZQ-X100 ( ); WAY-2S ( ); BH-2 Olympus ( Table 1 表 1 发酵产 D- 阿拉伯醇菌株及发酵结果 Results of the strains for D-arabitol production and fermentations D- (g/l) (%) chodati Endomycopsis rouxii NRRL Zygosaccharomyces ohmeri Kodamae anomala Hansenula hansenii ATCC Debaryomyces polymorpha DSM Hansenula

3 ); T-6 ( ); Agilent 1200 ( ) 2.3 培养基富集培养基 (1 L) 50 g, 10 g, 4 g, 3 g, 0.5 g, 10 μg/ml ( ), 1 L, min AYC 培养基 (1 L) 150 g, 20 g, 6 g, 4 g, 0.5 g, 20 g, 1 L, min 种子培养基发酵培养基 (1 L) 200 g/l 酵母鉴定培养基 (1) (1 L): 5 g, 0.2 g, 5 g, 5 g, 0.5 g 250 m L, min (2) (1 L): 20 g, 0.2 g, 1.5 g, 0.5 g, 20 g 250 ml, min 2.4 产 D- 阿拉伯醇酵母菌种的分离与筛选方法菌种分离, 30, 150 r/min, 2 d,,,,,,,,, 摇瓶初筛, 3 d, 30, 150 r/min, D-,, D-,,, 摇瓶复筛, 30, 150 r/min, 3 d, D-, D- 2.5 菌种鉴定菌落形态观察 ACY, 30 3 d 细胞形态观察,,,, 生理生化实验, L- D- [21] S rdna 的 PCR 扩增及序列分析 2.6 发酵液成分鉴定菌体浓度 (100 ) 640 nm 50 ml, 10 min, 2500 r/min,,, 2-3, h,, 可溶性固形物 50 ml, ( 3500 r/min), 20, 2.7 产物鉴定 [13] D- 阿拉伯醇纸层析法分析高效液相色谱法测定发酵液中葡萄糖 D- 阿拉伯醇和甘油含量 : Agilent 1200 ( ( ) ), Sugar-pak 6.5 mm 300 mm, 5 μm ( ( ) ), 80,, 0.5 ml/min, 15 μl, (RID, ShodexRI100) 3 结果与讨论 3.1 产 D- 阿拉伯醇菌株的筛选富集培养 (a-b- c-

4 12, : D ),, 2,, 平板筛选,, 3,,,, Table 2 表 2 富集培养筛选结果 Results of the enrichment culture screening +,,,,,, Table 3 表 3 涂布培养筛选结果 Results of the coated culture screening, ( ),,, ( ),,, ( ),, 摇瓶发酵初筛, 1, 2 D-, 4 Fig. 1 1 Results of cell concentrations after fermentation 2 (Brix) Fig. 2 Results of glucose and soluble solids content in the fermented liquid, b-1 b-3 b-4 c-2, 15 g/l

5 Table 4 表 4 D- 产阿拉伯醇菌株的初筛结果 Results of first screening of strains for D-arabitol production * (g/l) (g/l) D- a a a a b b b b c c c c *, ( 1), D-,, b-1 b-3 b-4 c-2, ( 2) b-1 c-2, 150 g/l, 15 g/l, b-1 c-2, D-, b-1 b-3 b-4, 59%, 52% 68%,, 15~17 g/l( 2) b-1 b-3 b-4 D- 3,, 4 4, b-1 b-4, D-, 3.2 D- 阿拉伯醇菌株的定性分析菌株发酵培养后主要成分的纸层析鉴定 b-1 b-4, D-,, -,, 3 3 b-1 b-4 D- Fig. 3 Results of D-arabitol in fermented liquid by the strains of b-1, b-4 with the paper chromatography test : D- 4. b-1 5. b-4, [15] 3, b-1 b-4 D 菌株主要代谢产物的定性分析, 4, 5 ( 7.3 min); D ( 9.2 min); ( 9.9 min) 4, D-, D-, 5, b-1 D-,,

6 12, : D D-, b-1 D- b 目的菌株形态观察鉴定菌株显微镜观察 6,,, 7, b-1,,,, 4 b-1 HPLC Fig. 4 HPLC image of fermented liquid by the strain b-1 6 Fig. 6 Image of the colony 7 b-1 Fig. 7 Microphotograph of the strain b-1 5 D- HPLC Fig. 5 HPLC image of fermented liquid added D - arabitol standard 3.3 D- 阿拉伯醇菌株的定量分析 b-1 b-4, 3 d,, D-, 5 5, b-1 D-,, 生理生化特征,, D-, 6, b-1 ; D-, Table 5 表 5 高效液相测定发酵 3d 后发酵液成分含量 The component content of the fermented liquid after 3 d by HPLC (g/l) (g/l) (g/l) D-(g/L) (g/l) (%) b b b b

7 Table 6 表 6 菌株的生理生化特征 Physiological and biochemical characteristics of the strain D : + ; S rdna 序列分析 PCR 26S rdna D1/D2, 511 bp BLAST ClustalX GenBank 26S rdna D1/D2,,, b-1 26S rdna D1/D2 Candida parapsilosis 99% b-1 Candida parapsilosis 4 结论, 12, D- b-1 HPLC, D- 200 g/l, h, D g/l, 44.5%,, 8.7 g/l,,, b-1 Candida parapsilosis 参考文献 [1],,,. 1 D- [J]., 2009, 35(1): Cai L, Zhang Y, Zhu HY, et al. Isolation andidentification of a novel D-arabitol-producing strain [J]. Food Ferment Ind, 2009, 35(1): [2],. [J]., 2004, 12(1): Yang YZ, Yang HJ. Functional sugar alcohol [J]. Fine Specialty Chem, 2004, 12(1): [3]. [J]., 2003: Zou RY. The domestic and overseas market and application of polyols [J]. Chem Intermediat, 2003: [4]. D- [D]. :, 2009: Zhang L L. Screening of yeast strains and fermentation conditions for hight yield production of D-arabitol[D]. Wuxi: Jiangnan University, 2009: [5],,,. :, CN C[P]. 2003: Jiang N, He P, Lu DJ, et al. The preparation of D-arabitol by yeast from glucose: Chinese patent, CN C [P]. 2003: [6]. D- [D]. :, 2011: 2 3 Song WB. Screening of a novel D-arabitol-producing strain, optimization of D-arabitol production conditions and clone and expression of related key enzyme gene[d]. Hangzhou: Zhejiang University, 2011: [7] Toyoda T, Ohtaguchi K. Selection of Kluyveromyces yeasts for the production of D-arabitol from Lactose [J]. J Chem Engin Japan, 2009, 42: [8],. [J]., 1999, 19(5): Wang ZX, Zhuge J. Osmotic regulation and glycerol metabolism in yeast [J]. Biol Engin Progress, 1999, 19(5): [9],,. [J]., 2001, 31(1): 1 3. Fan GX, Zhuge J, Xu C. Effects of high concentration of salts on polylol production by osmophilic yeasts [J]. Ind Microbiol, 2001, 31(1): 1 3. [10],,. Ⅱ Hansenula arabitolgenes [J]., 1963, 9(2): Zhang SZ, Wang HL, Yang LW. Studies on the production of glycerol and arabitol by osmophilic yeastsⅡarabitol production by Hansenula arabitolgenes fang [J]. Microbiology, 1963, 9 (2): [11] Ruijter GJG, Visser J, Rinzema A. Polyol accumulation by aspergillus oryzae at low water activity in solid-state fermentation [J]. Microbiology, 2004, 150: [12] Bernard EM, Christiansen KJ, Tsang SF, et al. Rate of arabitol

8 12, : D production by pathogenic yeast species [J]. J Clin Microbiol, 1981, 14: [13]. D- [D]. :, Li Z. Screening of yeast strains and fermentation conditions for the production of D-arabitol[D]. Jinan: Shandong Institute of Light Industry, [14] Escalante J, Caminal G, Figueredo M, et al. Production of arabitol from glucose by Hansenula polymorpha [J]. J Ferment Bioeng, 1990, 70(4): [15] Ingram JM, Wood WA. Enzymatic basis for D-arabitol production by saccharomyces rouxii [J]. J Bacteriol, 1965, 89: [16] Caminal B, Yoshikiyo S, Michael S. Production of D-arabitol by a newly isolated zygosaccharomyces rouxii [J]. Soc Ind Microbiol, 2007, 34: [17] Povelainen M, N. Miasnikov A. Production of D-arabitol by a metabolic engineered strain of Bacillus subtilis [J]. J Biotechnol, 2006, 1: [18],,. D- [J]., 2011, 9(2): You CP, Zhang L, Ding CY. Effect of surfactants on production of D-arabitol by Debaryomyces hansenii [J]. Biological Process, 2011, 9(2): [19],. [J]., 2003, 23(2): Yu BQ, Zhuge J. Hyper osmoadaptation of yeast cells and intracellular accumulation of glycerol [J]. Chin J Biotechnol, 2003, 23(2): [20] Wang L, Tang P, Yuan QP, et al. Multi-batches fermentation of xylitol production by Candida tropicalis immobilized in polyurethane foam as carrier [J]. Carbohyd Polym, 2012, 90(2): [21],,,. [J]., 2011, 30(3): Lv AJ, Gu D, Hu XC, et al. Identification and phylogenetic analysis of a Yeast strain from the intestinal tract of zebrafish Danio rereio [J]. J Fungus, 2011, 30(3): 作者简介 ( 责任编辑 : 白洪健 ) 王乐, 讲师, 博士, 主要研究方向为食品微生物与发酵工程 wanglely1984@163.com 黄继红, 教授级高级工程师, 主要研究方向为农产品发酵工程 Huangjihong1216@126.com

380 研究论文发酵天数双乙酰测定 : 参照 GB4927-4928-2001 标准发酵液中的化学成分的测定 : 采用 GC-8A 型气相色谱测定 1.5 离子注入方法 [6] 把待处理的菌株细胞均匀涂

380 研究论文发酵天数双乙酰测定 : 参照 GB4927-4928-2001 标准发酵液中的化学成分的测定 : 采用 GC-8A 型气相色谱测定 1.5 离子注入方法 [6] 把待处理的菌株细胞均匀涂中国细胞生物学学报 Chinese Journal of Cell Biology 2011, 33(4): 379-384 http://www.cjcb.org 全小麦啤酒酵母菌株的诱变育种及应用研究袁仲 1* 张百胜 1 张慎举 1 马绮云 2 陈柯羽 2 ( 1 商丘职业技术学院园林食品加工系,