微生物学通报 JAN 20, 2009, 36(1): 64~70 Microbiology tongbao@im.ac.cn 研究报告 2009 by Institute of Microbiology, CAS 两种包装市售冷却牛肉中微生物多样性 的比较分析 * 李正堂李柏林赵勇欧杰 ( 200090) 摘要 : 本文比较分析了相同销售条件下的未包装冷却牛肉与保鲜膜包装冷却牛肉中的微生物群落结构 采用 16S rdna 的 V3 区片段进行 PCR-DGGE( 变性梯度凝胶电泳 ), 同时利用 16S rdna 全长序列, 通过克隆文库技术对两个样品的微生物群落结构进行研究 研究发现 : 微生物群落结构表现出较大的差异 保鲜膜包装冷却牛肉共有 6 个 OTU (Operational Taxonomic Unit), 主要为 Lactococcus( 乳球菌属, 28%), Lactobacillus( 乳杆菌属, 26%), Carnobacterium( 肉品杆菌属, 18%) 和 Brochothrix( 索丝菌属, 10%); 未包装冷却牛肉则共得到 18 个 OTU, 主要是 Lactococcus( 乳球菌属, 28%), Brochothrix( 索丝菌属, 18%) 和 Acinetobacter( 不动杆菌属, 11%) 结果表明, 保鲜膜包装对于葡萄球菌属以及冷杆菌属等一些细菌都起到了一定的抑制作用 本研究为肉类加工生产中微生物的控制提供一定的理论依据 关键词 : 冷却牛肉, 16S rdna 克隆文库法, PCR-DGGE, 微生物群落结构 The Comparative Analysis of Microorganism Diversity in Two Kinds of Packaged Chilled Beef on Sale LI Zheng-Tang LI Bai-Lin ZHAO Yong OU Jie * (College of Food Science and Technology, Shanghai Ocean University, Shanghai 200090, China) Abstract: This research is for the purpose of comparative analysis of the microbial flora structure in the chilled beef with no packing and cling film, which under the same terms of sale. It was used the V3 area fragment of 16S rdna to carry on PCR-DGGE, Meanwhile used the 16S rdna sequence to analysis the microbial flora structure of the two samples, according to the technology of clone.the research discovered that the flora structure displays a biggish difference; there was 6 OTU in the chilled beef with cling film, mainly was that Lactococcus(28%), Lactobacillus (26%), Carnobacterium(18%) and Brochothrix (10%); but there was 18 OTU in the chilled beef with no packing, mainly was that Lactococcus(28%), Brchothrix(18%), Acinetobacter (11%). The result indicates that cling film played a certain inhibitory action regarding the Staphylococcus as well as the cold pole bacteria and such bacterium. And it can provide a certain theory basis for the meat processing in the department of microorganism s control. Keywords: Chilled beef, 16S rdna clone library analysis, PCR-DGGE, Microbial flora structure 基金项目 : (No. 2007BAD52B05); (No. T1102) * 通讯作者 :Tel: 86-21-65710324; : jou@shou.edu.cn 收稿日期 :2008-07-07; 接受日期 :2008-09-02
: 65,,,,,, [1,2], [3,4],,, [5],,,,, 1 材料和方法 1.1 样品采集 2007 11 1.2 主要试剂和仪器 PCR ; Biospin DNA Biospin; pgem-t Easy ; DH5α Promega ; Eppendorf AG 22331 Hamburg PCR 1.3 DNA 的提取 [6] Biospin DNA DNA 1.4 变性梯度凝胶电泳分析 1.4.1 PCR-DGGE 的 V3 可变区 PCR 扩增 : 16S rdna V3 V3-2 5 -ATTA CCGCGGCTGCTGG-3 V3-3 5 -CGCCCGCCGC GCGCGGCGGGCGGGGCGGGGGCACGGGGGGCC TACGGGAGGCAGCAG-3 25 μl 10 PCR 2.5 μl, MgCl 2 (25 mmol/l)1.5 μl, dntp 2 μl, 27f 1492r 1 μl, 1 μl, Taq (1000U, 5 U/mL)0.3 μl, 15.2 μl PCR 95 C 3 min; 95 C 1 min, 55 C 1 min, 72 C, 25 ; 72 C 10 min 1.0%, 1.4.2 变性梯度凝胶电泳 : 35%~55% V3 DGGE [7] DNA 10 μl Dcode DGGE (Bio Rad), 1 TAE, 120 V 4 h, SYB green I (1 TAE, 1 10000) 60 min,, ddh 2 O 5 min, UVP 1.5 16S rdna 克隆文库分析 1.5.1 16S rdna 的 PCR 扩增 : DNA, 16S 27f 5 -GAGAGTTTGATCCTGGCTCAG-3, 1492r 5 -CTACGGCTACCTTGTTACGA-3 PCR V3 DNA PCR 1.5.2 克隆文库构建 [8] : 16S rdna pgem-t Easy, CaCl 2 DH5α, X-gal(40 μg/ml) IPTG(40 μg/ml) Ap(50 μg/ml) LA, [9] SP6 T7 PCR, 1.6 克隆序列测定及比对, 16S rdna( 1500 bp), Blast GenBank ( http://ncbi.nlm.nih.gov /blast), 90% (OTU, Operational Taxonomic Unit) Bioedit MEGA4, 1.7 文库库容的评价, 16S rdna
66 微生物学通报 2009, Vol.36, No.1 (Library Size), Good [10] Coverage C Coverage C 16S rdna, Coverage C=[1 (n/n)] 100 (1), n, N Coverage C 2 结果 2.1 样品 DNA 的提取 DNA 1%, 23 kb ( 1), DNA 1~ 4, 1 2 图 2 两种牛肉样品的 16S rdna 电泳图谱 Fig. 2 PCR amplification of 16S rdna derived from the two samples 图 3 两种牛肉样品 V3 可变区 PCR 扩增结果 Fig. 3 PCR amplification products of V3 region from the two samples 图 1 两种牛肉样品总 DNA 电泳图谱 Fig. 1 Electrophoresis analysis of total DNA from the two simples 2.2 16S rdna 和 V3 可变区 PCR 扩增 27f 1492r PCR,, 1500 bp( 2) 16S rdna V3 ( GC ) PCR, 1%, 200 bp, 3, PCR, DGGE 2.3 PCR-DGGE 结果 DGGE DNA,, DGGE,, DGGE ( 4) DGGE,, 15, 13, 5, 4 a b c d e DGGE, 1 1, 5,
: 67 图 4 两种牛肉样品变性梯度凝胶电泳图谱 Fig. 4 DGGE profile of the two samples DGGE,, DGGE, 41%~49%, 45% bandscan, a b d e,, c, 2.4 未包装冷却牛肉与保鲜膜包装冷却牛肉的微生物群落结构特征 100, 100, Blast 97% (OTU) ( M ) 18 OTU, OTUM1 OTUM18, ( N ) 6 OTU, OTUN1 OTUN6 Good, M Coverage C 82%, N Coverage C 88%, OTU NCBI Blast, ( 6), (Lactococcus, 11%), (Brochothrix, 18%), (Acinetobacter, 9%), (Pseudomonas, 8%), (Carnobacterium), (Lactobacillus), (Shewanella), 图 5 两种牛肉样品群落多样性分析 Fig. 5 Community diversity of the two samples A: ; B:. Note: A: Chilled beef with no packing; B: Chilled beef with cling film.
68 微生物学通报 2009, Vol.36, No.1 Fig. 6 图 6 两种牛肉样品克隆的整体分布 Percentage of clones in the two samples and their relationship to different genera (Staphylococcus), (Rahnella), (Kocuria), (Bacillus subtilis), (Psychrobacter), (Microbacterium), (Stenotrophomonas), (Streptococcus) (Vagococcus salmoninarum), (Lactococcus, 28%), (Lactobacillus, 26%), (Carnobacterium, 18%), (Brochothrix, 16%), (Streptococcus) (Lactococcus), (Brochothrix), (Carnobacterium), (Streptococcus) (Lactobacillus), [12] (Lactococcus), (Lactobacillus) (Brochothrix),, [11] 2.5 系统发育分析 OTU NCBI Blast, ( 1), NCBI GenBank, M EU826652- EU826669 N EU826670-EU826675 1 M BLAST, OTUM6 OTUM7 OTUM11 OTUM14 OTUM18 N BLAST ; OTUN1 (Lactobacillus sakei), OTUN2 (Streptococcus agalactiae), OTUN6 (Brochothrix thermosphacta) 3 讨论 Lactobacillus Lactococcus Streptococcus Carnobacterium,,,, [12],,,,, (Brochothrix),, (Pseudomonas) (Shewanella),,, ;,, H 2 S TMAO,, (Shewanella) SSO(Specific Spoilage Organisms) [3],
: 69 Table 1 表 1 未包装冷却牛肉和保鲜膜包装冷却牛肉 16S rdna 文库 OTU 的系统发育分析 Information of 16S rdna clone library constructed from chilled beef with cling film and no packing Type of OTU Number of Clone Accession No. Most closest sequences Accession number of most closest sequence Similarity (%) OTUM1 9 EU826652 Uncultured bacterium AY958853 99 OTUM2 2 EU826653 Vagococcus salmoninarum Y18097 99 OTUM3 9 EU826654 Acinetobacter sp. AB365066 99 OTUM4 18 EU826655 Brochothrix sp. AM 409367 99 OTUM5 7 EU826656 Psychrobacter EU196312 99 OTUM6 4 EU826657 Bacillus subtilis EF032688 99 OTUM7 8 EU826658 Pseudomonas fluorescens AF094730 99 OTUM8 7 EU826659 Carnobacterium AY543016 99 OTUM9 5 EU826660 Staphylococcus sp. AB167054 99 OTUM10 5 EU826661 Shewanella sp. AF140016 98 OTUM11 6 EU826662 Lactobacillus sake AB362606 99 OTUM12 1 EU826663 Exiguobacterium DQ351341 99 OTUM13 1 EU826664 Rahnella sp. AJ415576 99 OTUM14 3 EU826665 Streptococcus agalactiae AE014210 92 OTUM15 11 EU826666 Lactococcus sp. AY762111 99 OTUM16 1 EU826667 Stenotrophomonas sp. AM403589 99 OTUM17 2 EU826668 Microbacterium EU086800 99 OTUM18 1 EU826669 Kocuria rosea EF522129 95 OTUN1 26 EU826670 Lactobacillus sakei AB362609 99 OTUN2 6 EU826671 Streptococcus agalactiae AE014207 92 OTUN3 28 EU826672 Lactococcus sp. AY762105 99 OTUN4 18 EU826673 Carnobacterium EU304249 93 OTUN5 6 EU826674 Uncultured bacterium EU024337 95 OTUN6 16 EU826675 Brochothrix thermosphacta AY543027 100 (Staphylococcus), (Psychrobacter) (Microbacterium) [13],,,, [14] (Shewanella) (Staphylococcus) (Psychrobacter) (Microbacterium),,, [10], 4 结论 1), 2) Lactococcus Brochothrix Acinetobacter Psychrobacter Pseudomonas, Lactococcus Lactobacillus Carnobacterium Brochothrix 3) Lactobacillus Lactococcus Carnobacterium Streptococcus Brochothrix
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