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中國精品科技期刊2020

銅綠假單胞菌噬菌體K4的性質及其在食品防腐方面的應用

龐文靜 韓慶竹 尤甲甲 李東航 李佩澤 李玥瑩 楊洪江

龐文靜,韓慶竹,尤甲甲,等. 銅綠假單胞菌噬菌體K4的性質及其在食品防腐方面的應用[J]. 食品工業科技,2022,43(16):130?139. doi:  10.13386/j.issn1002-0306.2021110193
引用本文: 龐文靜,韓慶竹,尤甲甲,等. 銅綠假單胞菌噬菌體K4的性質及其在食品防腐方面的應用[J]. 食品工業科技,2022,43(16):130?139. doi:  10.13386/j.issn1002-0306.2021110193
PANG Wenjing, HAN Qingzhu, YOU Jiajia, et al. Properties of Pseudomonas aeruginosa Phage K4 and Its Applications in Food Preservation[J]. Science and Technology of Food Industry, 2022, 43(16): 130?139. (in Chinese with English abstract). doi:  10.13386/j.issn1002-0306.2021110193
Citation: PANG Wenjing, HAN Qingzhu, YOU Jiajia, et al. Properties of Pseudomonas aeruginosa Phage K4 and Its Applications in Food Preservation[J]. Science and Technology of Food Industry, 2022, 43(16): 130?139. (in Chinese with English abstract). doi:  10.13386/j.issn1002-0306.2021110193

銅綠假單胞菌噬菌體K4的性質及其在食品防腐方面的應用

doi: 10.13386/j.issn1002-0306.2021110193
基金項目: 國家自然科學基金(31970150)。
詳細信息
    作者簡介:

    龐文靜(1996?),女,碩士研究生,研究方向:微生物,E-mail:wenjingpang@yeah.net

    通訊作者:

    楊洪江(1966?),男,博士,教授,研究方向:噬菌體制劑及應用,E-mail:hongjiangyang@tust.edu.cn

  • 中圖分類號: Q939.48

Properties of Pseudomonas aeruginosa Phage K4 and Its Applications in Food Preservation

  • 摘要: 目的:為研究噬菌體在控制細菌污染方面的應用,本文對銅綠假單胞菌噬菌體K4的生物學性質及抑菌活性進行了探究。方法:包括一步生長曲線、穩定性研究、基因組測序、比較基因組學分析以及抑菌曲線等方法。結果:K4潛伏期約為15 min,釋放量約為85 PFU/infection center,具有較強的感染性;在不同感染復數(MOI)下,噬菌體K4對宿主菌均有明顯的抑制效果;基因組分析顯示,K4基因組長度為50358 bp,編碼77個蛋白和1個tRNA-Arg;比較基因組學分析發現,噬菌體K4與Paundecimvirus屬的Pseudomonas virus PA11基因組序列的一致性達95.08%,證實噬菌體K4是該屬的新成員。在應用實驗中,噬菌體K4能夠顯著抑制牛奶和午餐肉等樣品中宿主菌的生長,同時噬菌體的數量也有顯著增加。本研究結果顯示,噬菌體K4生長速度快,具有較強的殺菌活性,基因組不攜帶整合酶、抗性以及毒力因子等編碼基因。結論:噬菌體K4可以用于控制食品中銅綠假單胞菌的污染。
  • 圖  1  噬菌體K4的穩定性分析

    Figure  1.  Stability analysis of phage K4

    注:A:噬菌體K4的氯仿穩定性;B:噬菌體K4的熱穩定性;***:P<0.001。

    圖  2  噬菌體K4基因組序列的注釋

    Figure  2.  Annotation of the phage K4 genome

    圖  3  噬菌體比較基因組學分析

    Figure  3.  Comparative genomic analysis of phage

    圖  4  噬菌體K4生物學特性

    Figure  4.  Biological characteristics of phage K4

    注:A:噬菌體K4的一步生長曲線;B:噬菌體K4的抑菌曲線。

    圖  5  在4和25 °C條件下不同MOI的噬菌體在牛奶中的細菌濃度和噬菌體滴度

    Figure  5.  Colony forming unit and titer of phage with different MOI in milk at 4 and 25 °C

    注:A:在4 °C條件下,在噬菌體作用下宿主菌濃度的變化;B:在4 °C條件下,噬菌體K4效價的變化;C:在25 °C條件下,在噬菌體作用下宿主菌濃度的變化;D:在25 °C條件下,噬菌體K4效價的變化;*:P<0.05;***:P<0.001。

    圖  6  在4和25 °C條件下不同MOI的噬菌體在午餐肉中的細菌濃度和噬菌體滴度

    Figure  6.  Colony forming unit and titer of phage with different MOI in Luncheon meat at 4 and 25 °C

    注:A:在4 °C條件下,在噬菌體作用下宿主菌濃度的變化;B:在4 °C條件下,噬菌體K4效價的變化;C:在25 °C條件下,在噬菌體作用下宿主菌濃度的變化;D:在25 °C條件下,噬菌體K4效價的變化;*:P<0.05;***:P<0.001。

    表  1  本研究中所用菌株、噬菌體和質粒

    Table  1.   Strains, phages and plasmids used in this study

    菌株、噬菌體、質粒描述菌株、質粒、噬菌體來源
    P. aeruginosa
    PAKLaboratory strain, serotype O6[21]
    M4wbpR::GmR mutant of PAK, resistant to phage O4[13]
    M19wzy::GmR mutant of PAK, resistant to phage O4[13]
    M25wzy::GmR mutant of PAK, resistant to phage O4[13]
    M30wzy::GmR mutant of PAK, resistant to phage O4[13]
    M39wbpL::GmR mutant of PAK, resistant to phage O4[13]
    RC11-2BN889_05221:: GmR mutant of PAK, resistant to phage C11[12]
    SK45wbpR::GmR mutant of PAK, resistant to phage K8[11]
    SK16wbpV::GmR mutant of PAK, resistant to phage K8[11]
    SK75Y880_RS05480 ::GmR mutant of PAK, resistant to phage K8[11]
    SK98ssg::GmR mutant of PAK, resistant to phage K8[11]
    Plasmid
    pZM1504wbpR gene with its own promoter cloned in pUCP18, ApR[13]
    pLLY1501wzy gene with its own promoter cloned in pUCP18, ApR[13]
    pZM1502wbpL gene with its own promoter cloned in pUCP18, ApR[13]
    pXL1502BN889_05221 gene driven by Plac promoter cloned in pUCP18, ApR[12]
    pXL1503wbpR gene with its own promoter cloned in pUCP18, ApR[11]
    pFJ1501wbpV gene with its own promoter cloned in pUCP18, ApR[11]
    pLY1201Y880_RS05480 gene with its own promoter cloned in pUCP18, ApR[11]
    pXW1501ssg gene driven by Plac cloned in pUCP18, ApR[11]
    Phage
    K4lytic phage specific to the strain PAK[20]
    K5lytic phage specific to the strain PAK[14]
    K8lytic phage specific to the strain PAK[11]
    O4lytic phage specific to the strain PAO1[13]
    C11lytic phage specific to the strain TJC422[12]
    下載: 導出CSV

    表  2  噬菌體K4的宿主范圍分析

    Table  2.   Host range of phage K4

    K4K5K8C11O4
    M4?????
    M4/pZM1504+++++
    M19?????
    M19/pLLY1501+++++
    M25?????
    M25/pLLY1501+++++
    M30?????
    M30/pLLY1501+++++
    M39?????
    M39/pZM1502+++++
    RC11-2++??+
    RC11-2/pXL1502+++++
    SK16?????
    SK16/pFJ1501+++++
    SK45?????
    SK45/pXL1503+++++
    SK75?????
    SK75/Ply1201+++++
    SK98?????
    SK98/pXW1501+++++
    下載: 導出CSV

    表  3  噬菌體K4基因組注釋

    Table  3.   Annotation of the phage K4 genome

    基因起始終止基因方向同源噬菌體覆蓋率(%)功能
    gp0933713871+vB_PaeM_MIJ350.30possible DNA binding protein
    gp1350945167+tRNA-Arg-TCT
    gp2174538073+O4100.00putative amidotransferase
    gp24890510176+O4100.00putative amidotransferase
    gp251018611046+O4100.00putative COOH-NH2 ligase
    gp271159412544+O4100.00putative amidoligase
    gp281254413293+O4100.00putative ATP-grasp protein
    gp311383014129+O490.91DNA-binding protein
    gp351486116576+O4100.00DNA primase/helicase, phage-associated
    gp361657617082+O498.19RNA polymerase sigma factor
    gp391739519272+O499.84Phage DNA polymerase I
    gp452077621651+O4100.00putative 5'-3' exonuclease
    gp462163022130+O4100.00putative serine/threonine protein phosphatase
    gp472210522527+O4100.00putative terminase small subunit
    gp502311323880+O4100.00putative 3'-5' exonuclease
    gp512388224109+TC691.30DUF3310 domain-containing protein
    gp522409024731+O4100.00putative thymidylate synthase
    gp542525325900+O4100.00putative PhoH-like protein
    gp552588426258+O4100.00Phage thioredoxin or glutaredoxin (Nrd)
    gp562624227210+O4100.00putative ribonucleotide reductase
    gp572720428898+O4100.00putative ribonucleotide reductase
    gp582925128973?PA11100.00Phage capsid and scaffold
    gp613019729760?O4100.00putative endolysin
    gp623196230184?IME18098.99putative hemagglutinin protein
    gp653978037465?O4100.00putative head closure protein
    gp674076539962?TC699.25putative adaptor protein
    gp694170741441?O4100.00putative head fiber protein
    gp714302842087?O4100.00putative capsid protein
    gp724379743039?O4100.00putative scaffold protein
    gp744590944155?O4100.00putative portal protein
    gp754752945922?GP10074.48Phage terminase, large subunit
    gp774948348089?O499.78putative pectate lyase
    注:identity根據氨基酸水平計算。
    下載: 導出CSV
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  • 收稿日期:  2021-11-17
  • 網絡出版日期:  2022-08-11
  • 刊出日期:  2022-08-11

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