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1 7 8 Vol. 7 No Journal of Food Safety and Quality Aug., 2016 邢良英, 陈 * 琳, 侯保朝, 李言郡 (,, ) 摘要 : 目的 方法 45, 104, 16S rdna 结果 8, 4 Lactobacillus plantarum(whh727 WHH730 WHH777 WHH1118) 3 Lactobacillum fermentum(whh734 WHH778 WHH868) 1 Lactobacillus acidophilus(whh729), L. fermentum WHH734, ph h, 86.32%, ph h, 96.85% 结论, 关键词 : ; ; 16S rdna ; ; ; Isolation, identification and acid tolerance of acid-tolerant lactic acid bacteria from traditional milk product in Inner Mongolia XING Liang-Ying, CHEN Lin, HOU Bao-Chao, LI Yan-Jun * (Key Laboratory of Food and Biological Engineering of Zhejiang Province, Hangzhou Wahaha Group Co., Ltd., Hangzhou , China) ABSTRACT: Objective To screen acid tolerant lactic acid bacteria strains, which can tolerate gastroenteric environment and survive in human intestine. Methods A total of 104 strains of lactic acid bacteria were isolated and purified from 45 samples of traditional milk products in Inner Mongolia, which were conducted morphological, physiological, biochemistry test, 16S rdna sequencing analysis, acid-tolerant test and artificial gastric juice resistance test. Results Totally 8 strains of lactic acid bacteria were screened to have excellent resistance in artificial gastric juice, in which 4 strains were identified as Lactobacillus plantarum (WHH727, WHH730, WHH777 and WHH1118), 3 strains were identified as Lactobacillum fermentum (WHH734, WHH778 and WHH868) and one strain was identified as Lactobacillus acidophilus (WHH729). Among that, L. fermentum WHH734 was the best strain for tolerance of artificial gastric juice. The survival rates were 86.32% in the screening experiment of resistance to the artificial gastric juice (ph 1.5, 2 h), and 96.85% at ph 2.5. Conclusion The isolation and identification of strains and screening of high acid-tolerant strains have very important significance to the preservation and exploitation of probiotic resource. 基金项目 : (2015C02SA100006) Fund: Supported by the Key Research Projects of Science and Technology Department of Zhejiang Province (2015C02SA100006) * 通讯作者 :,, lyj@wahaha.com.cn *Corresponding author: LI Yan-Jun, Senior Engineer, Hangzhou Wahaha Group Co., Ltd., Hangzhou , China. lyj@wahaha.com.cn

2 8, : 3127 KEY WORDS: lactic acid bacteria; identification; 16s rdna sequence analysis; high-throughput; acid tolerance; artificial gastric juice 1 引言 Metchnikoff,, [1,2], [3] [4] [5],,, [6],,,,,, [7] 45, 104,,, 16S rdna,, 2 材料与方法 2.1 材料 7 45, , 1 ml 1 ml 50% 2 ml, ;, 10 g,,,,, 1 g 1 ml 50% 2 ml, 仪器与试剂 MRS (Oxoid); Elliker (Oxoid); PCR ; API 50 CHL ( ); DNA (); Premix Taq( ); ( 1:10000, Sigma ) SG403A-HE-INT ( Baker ); Binder kb400 ( Binder ); Forma702 ( Thermo Scientific ); OLYMPUS BX41 ( OLYMPUS ); Bio-Rad 680 ( ); Eppendorf 5417R ( ); HIRAYMA HV-110 ( HIRAYMA ); Eppendorf Masfercy cler PROS PCR ( ); BIO-RAD GELDOC XR + () 2.3 实验方法 乳酸菌的分离与纯化 0.85% 10, , 100 μl 1.5% MRS 1.5% Elliker, 37 36~48 h,, MRS Elliker,,,, 25%, -80 [9-11,15,16] 高通量耐酸性优良菌株筛选试验 104 WHH544 MRS, 2%, h, 2, 6000 r/min 10 min,, 2, CFU/mL 1 ml 1.5 ml, 3, 6000 r/min 1 min,,, 1 ml ph 2 MRS, ph 3 MRS, ph 6.5 MRS, 37 2 h; r/min 1 min,, 2, 1 ml ph 6.5 MRS, h, 100 μl 96, 600 nm OD OD(pH2.2h 16h) OD0 ph2 存活率 % 100% OD(pH6.5MRS2h 16h) OD0 OD 0 : MRS OD ; OD (ph 2,2 h+16 h) : ph 2 MRS 2 h ph 6.5 MRS 16 h OD ( );

3 OD (ph 6.5MRS,2 h+16 h) : ph 6.5 MRS 2 h ph 6.5 MRS 16 h OD ( ) 模拟人工胃液试验 2010 [8] 8 MRS, 2%, h, 2, r/ min 10 min,, 2, 2% ph 1.5 ph 2.5 [9-11], h,, [12] 碳水化合物代谢分析 8 MRS, 2%, h, API 50 CHL, 24 h 48 h,, S rdna 序列分析 8 10 ml MRS, h, 2 DNA, DNA DNA [13] 27f/1492r (27f, 5 -AGA GTT TGATCCTGGCTCAG-3 ; 1492r, 5 -GGT TACCTTGTTACGACTT-3 ), 16S rdna, 1.5 kb PCR (50 μl), : 1 μl (10 μmol/l), DNA 1 μl, 25 μl, 22 μl, PCR : 95, 5 min; 98, 30 s, 55, 30 s, 72, 100 s, 30 ; 72, 10 min, 4 PCR 1% 1500 bp 3 结果与分析 3.1 乳酸菌的分离纯化 45, 104, 74, ,, 24 h, 10 8 CFU/mL, 10 9 CFU/mL 3.2 高通量耐酸性优良菌株筛选试验 Berrada [14] 1~2 h, ph, 1.5, 1.5, ph 3.0 ph 1.5,,,, ph 2 ph 3,,,, [9-11,15,16],,, MRS, 16 h OD,,,,, , ph 2 MRS 2 h, WHH544 27, 25.96%; ph 3 MRS 2 h, WHH544 17, 16.34%;, ph 2 2 h, 90%, ph 3 2 h, 100% 8, 8,, ph 2 2 h, 70%, ph 3 2 h, 80% 3.3 模拟人工胃液试验 [17], [18] 2, 8, ph 1.5, WHH729 3 lg, 0.1%(10-3 ); ph 2.5, WHH729 WHH778 2 lg, 1%(10-2 ); 8 WHH734, ph %, ph % 3.4 乳酸菌的碳水化合物代谢分析, API 50 CHL 8, 3, 6, WHH727 WHH730 WHH734 WHH778, WHH729, WHH868 ;, WHH777 WHH1118,,, WHH1118,

4 8, : 3129 Table 1 表 1 乳酸菌耐酸性实验结果 (n=3) Screening experiment of lactic acid bacteria for acid tolerance (n=3) ph 2, 2 h ph 3, 2 h ph 2, 2 h ph 3, 2 h ph 2-2 h ph 3-2 h WHH ± ±8.25 WHH ± ±2.25 WHH ± ±5.25 WHH ± ±5.45 WHH ± ±5.33 WHH ± ±4.25 WHH ± ±2.25 WHH ± ±2.23 WHH ± ±5.86 WHH ± ±1.55 WHH ± ±2.58 WHH ± ±3.25 WHH ± ±4.58 WHH ± ±5.04 WHH ± ±4.12 WHH ± ±6.28 WHH ± ±4.21 WHH ± ±4.44 WHH ± ±1.05 WHH ± ±1.23 WHH ± ±1.02 WHH ± ±3.55 WHH ± ±1.24 WHH ± ±3.55 WHH ± ±7.88 WHH ± ±2.58 WHH ± ±3.22 WHH ± ±6.55 WHH ± ±5.55 WHH ± ±4.12 WHH ± ±8.89 WHH ± ±3.59 WHH ± ±4.55 WHH ± ±7.22 WHH ± ±5.12 WHH ± ±1.02 WHH ± ±3.55 WHH ± ±2.25 WHH ± ±5.22 WHH ± ±6.22 WHH ± ±0.55 WHH ± ±4.33 WHH ± ±3.24 WHH ± ±1.55 WHH ± ±1.85 WHH ± ±5.25 WHH ± ±1.02 WHH ± ±1.45 WHH ± ±7.88 WHH ± ±5.89 WHH ± ±8.78 WHH ± ±3.22 WHH ± ±6.23 WHH ± ±5.22 WHH ± ±5.56 WHH ± ±1.88 WHH ± ±3.66 WHH ± ±6.25 WHH ± ±3.58 WHH ± ±4.21 WHH ± ±7.23 WHH ± ±1.25 WHH ± ±1.56 WHH ± ±8.25 WHH ± ±8.99 WHH ± ±7.88 WHH ± ±8.99 WHH ± ±4.78 WHH ± ±5.66 WHH ± ±7.86 WHH ± ±5.98 WHH ± ±4.56 WHH ± ±5.89 WHH ± ±4.32 WHH ± ±4.25 WHH ± ±8.56 WHH ± ±8.27 WHH ± ±3.85 WHH ± ±7.88 WHH ± ±6.22 WHH ± ±2.55 WHH ± ±6.88 WHH ± ±8.99 WHH ± ±5.98 WHH ± ±6.25 WHH ± ±2.33 WHH ± ±5.12 WHH ± ±5.86 WHH ± ±5.25 WHH ± ±3.58 WHH ± ±5.66 WHH ± ±5.23 WHH ± ±2.55 WHH ± ±2.55 WHH ± ±2.22 WHH ± ±1.22 WHH ± ±8.99 WHH ± ±3.25 WHH ± ±4.33 WHH ± ±7.88 WHH ± ±1.09 WHH ± ±4.22 WHH ± ±8.21 WHH ± ±3.15 WHH ± ±3.56

5 Table 2 表 2 乳酸菌在 ph 1.5 和 ph 2.5 的人工胃液中处理不同时间的活菌数及存活率 (lg(cfu/ml)) The viable count and survival rate of lactic acid bacteria in ph 1.5 and ph 2.5 artificial gastric juice at different treatment time (lg(cfu/ml)) ph 1.5 ph h 0.5 h 1 h 2 h 2h lg( ) 0 h 1 h 2 h 2 h lg( ) WHH WHH WHH WHH WHH WHH WHH WHH Table 3 表 3 高耐酸性乳酸菌的生理生化特性 Physiological and biochemical characteristics of lactic acid bacteria with high acid tolerance WHH 727 WHH 729 WHH 730 WHH 734 WHH 777 WHH 778 WHH 868 WHH 1118 L- - * + # D D D D L L αd N ARBULIN D D D D D D

6 8, : 3131 续表 3 WHH 727 WHH 729 WHH 730 WHH 734 WHH 777 WHH 778 WHH 868 WHH D D D D D D L L / / 99.10% 99.90% 99.90% 99.80% / 99.90% 97.50% / - ; 乳酸菌的分子鉴定 27f/1492r 8 DNA PCR, 16S rdna/rrna GenBank, 4, 3, WHH730 WHH868,,,, 4 3 1, 表 4 乳酸菌同源性分析 ( % ) WHH727 Lactobacillus plantarum (100%) WHH729 Lactobacillus acidophilus (100%) WHH730 Lactobacillus plantarum (100%) WHH734 Lactobacillus fermentum (100%) WHH777 Lactobacillus plantarum (100%) WHH778 Lactobacillus fermentum (100%) WHH868 Lactobacillus fermentum (100%) WHH1118 Lactobacillus plantarum (99%) 4 结论,,,, [9-11,15,16],,,,,,, 104, 8,, 7 ph h 0.1% 8 Lactobacillus fermentum WHH734, ph %, ph %, 参考文献 [1] Yu J, Wang M, Zha SM, et al. Molecular identification and quantification of lactic acid bacteria in traditional fermented dairy foods of Russia [J]. J Dairy Sci, 2015, 98(8): [2] Huang RH, Tao XY, Wan CX, et al. In vitro probiotic characteristics of Lactobacillus plantarum ZDY 2013 and its modulatory effect on gut microbiota of mice [J]. J Dairy Sci, 2015, 98(9): [3],,. [J]., 2005, 17(1): Dong K, Liu JX, Guo XK The molecular mechanism of probiotics to

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