海洋细菌P.leiognathi的发光性能研究及其在食品安全检测中的应用探索
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摘要
本文以筛选具有优良体外发光性能、并对化学污染物敏感的海洋发光细菌为出发点,研究了发光细菌的菌体性质,分离纯化了催化菌体发光的荧光素酶和FMN:NADH氧化还原酶,构建了双酶体外发光的检测体系。在此基础上,利用菌体和双酶进行了食品安全检测的探索研究。
1、对海洋发光细菌的菌体性质进行研究。从青岛海域分离纯化了五株海洋发光细菌。经16SrDNA、生理生化试验鉴定,它们分属鳆发光杆菌(P.leiognathi)、哈维氏弧菌(V.harveyi)、明亮发光杆菌(P.phosphoreum)、灿烂弧菌(V.splendidus)和费氏弧菌(V.fischeri)。对五菌株进行发光特性研究和化学污染物敏感性的筛选。结果显示,P.leiognathi YL最大发射波长471nm,持续稳定发光16h,发光性能稳定,且对抗生素和重金属最敏感。优化了P.leiognathi YL的生长发光培养基:0.5%蛋白胨;0.1%酵母浸膏;0.01% FePO4;2-4% NaCl;0.4%甘油;陈海水。最佳发光条件:初始pH 7.5;温度25-30℃;转速150r/min。
2、建立了双酶体外发光的检测体系。对五菌株进行跟踪酶活测定,筛选出产酶起始时间早、产酶活力高且稳定的P.leiognathi YL作为双酶的制备菌株。优化了P.leiognathi YL的产酶培养基:0.5%蛋白胨;0.1%酵母浸膏;0.01% FePO4;2-4% NaCl;0.4%甘油;陈海水。最佳产酶条件:初始pH 7.0;温度25-30℃;转速150r/min。建立了单、双酶体外发光检测体系,并对两个体系进行比较。荧光素酶检测体系(室温): 1mL酶液中,迅速加入底物十二烷醛100μL(27mM)、FMN-Na53μL(10mM)和Na2S2O4 200μL(34mM),测定酶活。双酶检测体系(室温):1mL酶液中,一次性快速加入底物十二烷醛100μL(27mM)、FMN-Na53μL(10mM)、NADH 200μL(0.14mM),测定酶活。双酶体系比单酶体系发光稳定性好,更适合用于检测操作。
3、对双酶的酶学性质进行初步研究。超声破碎菌体细胞,通过硫酸铵盐析、DEAE-Sepharose CL-6B离子交换柱层析得到了电泳纯的FMN:NADH氧化还原酶,测定分子量为28KDa。通过硫酸铵盐析、DEAE-Sepharose CL-6B离子交换柱层析和DEAE-Sephadex凝胶柱层析得到了电泳纯的荧光素酶,并成功分离出了酶的两个亚基,其分子量分别为41KDa和36KDa。对荧光素酶两个亚基的进行活性测定,结果发现,两个亚基单独存在时无酶活,只有两个亚基同时存在,荧光素酶才有活性。优化了双酶的最适反应条件:温度为35℃,pH 7.0,最适NaCl浓度1.5-2%。K+、Ca2+、Na+、Mg2+离子对酶活有促进作用;Fe2+、Zn2+、Hg2+、Ba2+等离子对酶活有抑制作用。抗生素对双酶基本无影响。双酶的热稳定性较好。4℃下可长时间保持酶活。随着温度升高,双酶的耐热性变差,且酶失活的时间缩短。
4、菌体在食品安全检测中的应用探索。建立了P.leiognathi YL检测水产品中氯霉素残留的方法体系。选取对数生长期的P.leiognathi YL,控制菌数为107cfu/mL,乙酸乙酯提取氯霉素,5mL菌液中加入5mL氯霉素提取液,27℃150r/min培养30min,测定发光抑制率。检测线性范围0.14-1.0μg/L,方法检出限0.14μg/kg。建立了P.leiognathi YL检测水产品中重金属的方法体系。重金属敏感性筛选结果显示,P.leiognathi YL对Hg2+、Cr6+、Cd2+最敏感。选取对数生长期的P.leiognathi YL,控制菌数为107cfu/mL,样品灰化处理后,5mL菌液中加入5mL重金属提取液,27℃150r/min培养30min,测定发光抑制率。Hg2+检测线性范围0.007-0.28μg/L,方法检出限为0.007μg/kg;Cr6+检测线性范围0.9-30.0μg/L,方法检出限为0.9μg/kg;Cd2+检测线性范围5.4-65.0μg/L,方法检出限为5.4μg/kg。
5、双酶在食品安全检测中的应用探索。建立了双酶检测水产品中重金属残留的方法体系。重金属敏感性筛选结果显示,双酶对Hg2+、Cr6+最敏感。样品灰化处理后,1mL酶液中加入500μL样品溶液,4℃放置反应15min,测定酶活抑制率。Hg2+检测线性范围0.0007-0.030μg/L,方法检出限0.0007μg/kg;Cr6+检测线性范围0.05-5.0μg/L,方法检出限0.08μg/kg。初步研究了大肠杆菌内NADH对双酶酶活的影响。测定了NADH含量对双酶酶活的影响曲线。在此基础上,对不同菌量的大肠杆菌进行超声细胞破碎,释放NADH,进行双酶酶活检测,为今后利用双酶检测大肠杆菌的菌数提供基础理论数据。
Five luminous bacterial strains were isolated from Qingdao coast and characterized by a combination of 16SrDNA sequences, the morphology observation, physiological test, molecular and biochemical analysis. In comparison with the similar strains reported, the five strains are identified as P.leiognathi, V.harveyi, P.phosphoreum, V.splendidus, V.fischeri.
Sensitivity screening examination indicated that P.leiognathi YL was most sensitive to the antibiotics and heavy metals among the five strains. YL strain has a maxium wavelength for emission at 471nm and the strong light lasted for 16h. The optimum growth and luminescence conditions of YL were determined as: Initial pH 7.5;temperature 25-30℃;shaking speed 150r/min。The medium was composed of 0.5% peptone, 0.1% yeast extraction, 0.01% FePO4, 2-4% NaCl, 0.4% glycerin and seawater.
P.leiognathi YL was selected to produce luciferase and FMN:NADH oxidoreductase(the couple enzymes) for its stable product ability. The optimum enzyme-producing conditions were determined : Initial pH 7.5;temperature 25-30℃;shaking speed 150r/min。
Luciferase was assayed at room temperature by measuring the light emission upon the rapid injection of 1.0mL luciferase mixture, 53μL FMN-Na(10mM), 100μL Na2S2O4(34mM), 100μLdodecanal(27mM).FMN:NADH oxidoreductase was assayed at room temperature by measuring the light emission upon the rapid injection of 1.0mL oxidoreductase mixture, 53μL FMN-Na(10mM), 200μL NADH(0.14mM), 100μL dodecanal(27mM). The luminescence of the coupled enzyme system was more stable than luciferase system.
The couple enzymes were released from the cells by sonicate and purified followed the procedures: precipitated by solid ammonium sulfate; elution on DEAE-Sepharose CL-6B and DEAE-Sephadex columns. The molecular weights of oxidoreductase is 28KDa. Luciferase has two subunits and their molecular weights are 36KDa and 41KDa respectively. No activity was found when either of the isolated subunits is absent. The optimum reaction conditions of the couple enzymes are: Initial pH 7.5;temperature 25-30℃;shaking speed 150r/min. K+、Ca2+、Na+、Mg2+ promoted the activity of the couple enzymes while Fe2+、Zn2+、Sn2+、Hg2+、Ba2+ inhibited the activity. Antibiotics had no effect on the activity of the couple enzymes. The couple enzymes had a good activity at 4℃and the activity decreased with the temperature raised over 40℃.
The concentration of chloramphenicol in shrimp and turbot tissues was detected with P.leiognathi YL. The chloramphenicol in the tissues was extracted by ethyl acetate. The optimal test inoculum is 107cells/mL. 5mL chloramphenicol solution was added into 5mL inoculum. The mixture solution reacted for 30min at 27℃with the shaking speed 150r/min. The concentration of chloramphenicol ranging from 0.14 to 1.0μg/L was determined by the luminescence inhibition rate of P.leiognathi YL. The detection limit is 0.14μg/kg. The concentration of heavy metals(Hg2+、Cr6+、Cd2+)in shrimp tissues was also detected with P.leiognathi YL. The ions of heavy metal in the tissues were extracted by cineration. The detection procedure of heavy metals(Hg2+、Cr6+、Cd2+)was the same as the chloramphenicol. The concentration of Hg2+ ranging from 0.007μg/L to 0.28μg/L was detected, the detection limit was 0.007μg/kg.The detection concentration of Cr6+ is 0.9-30.0μg/L and the detection limit was 0.9μg/kg. The detection concentration of Cd2+ was 5.4-65.0μg/L and the detection limit was 5.4μg/kg.
Sensitivity screening examination indicated that the couple enzymes were most sensitive to Hg2+and Cr6+. The concentrations of Hg2+and Cr6+ in shrimp tissues were detected with the couple enzymes respectively. The ions of heavy metal in the tissues were extracted by cineration. 500μL ion solution was added into the enzyme mixture to react for 15min at 4℃, and the activity of the couple enzymes was detected. The detection concentration of Hg2+ was 0.0007-0.030μg/L and the detection limit was 0.0007μg/kg. The detection concentration of Cd2+ was 0.05-5.0μg/L and the detection limit was 0.08μg/kg. NADH in E.coli cells was released by sonicate and promoted the luminescence of the couple enzymes. The results provided data to detect the total number of E.coli with the couple enzymes.
1、对海洋发光细菌的菌体性质进行研究。从青岛海域分离纯化了五株海洋发光细菌。经16SrDNA、生理生化试验鉴定,它们分属鳆发光杆菌(P.leiognathi)、哈维氏弧菌(V.harveyi)、明亮发光杆菌(P.phosphoreum)、灿烂弧菌(V.splendidus)和费氏弧菌(V.fischeri)。对五菌株进行发光特性研究和化学污染物敏感性的筛选。结果显示,P.leiognathi YL最大发射波长471nm,持续稳定发光16h,发光性能稳定,且对抗生素和重金属最敏感。优化了P.leiognathi YL的生长发光培养基:0.5%蛋白胨;0.1%酵母浸膏;0.01% FePO4;2-4% NaCl;0.4%甘油;陈海水。最佳发光条件:初始pH 7.5;温度25-30℃;转速150r/min。
2、建立了双酶体外发光的检测体系。对五菌株进行跟踪酶活测定,筛选出产酶起始时间早、产酶活力高且稳定的P.leiognathi YL作为双酶的制备菌株。优化了P.leiognathi YL的产酶培养基:0.5%蛋白胨;0.1%酵母浸膏;0.01% FePO4;2-4% NaCl;0.4%甘油;陈海水。最佳产酶条件:初始pH 7.0;温度25-30℃;转速150r/min。建立了单、双酶体外发光检测体系,并对两个体系进行比较。荧光素酶检测体系(室温): 1mL酶液中,迅速加入底物十二烷醛100μL(27mM)、FMN-Na53μL(10mM)和Na2S2O4 200μL(34mM),测定酶活。双酶检测体系(室温):1mL酶液中,一次性快速加入底物十二烷醛100μL(27mM)、FMN-Na53μL(10mM)、NADH 200μL(0.14mM),测定酶活。双酶体系比单酶体系发光稳定性好,更适合用于检测操作。
3、对双酶的酶学性质进行初步研究。超声破碎菌体细胞,通过硫酸铵盐析、DEAE-Sepharose CL-6B离子交换柱层析得到了电泳纯的FMN:NADH氧化还原酶,测定分子量为28KDa。通过硫酸铵盐析、DEAE-Sepharose CL-6B离子交换柱层析和DEAE-Sephadex凝胶柱层析得到了电泳纯的荧光素酶,并成功分离出了酶的两个亚基,其分子量分别为41KDa和36KDa。对荧光素酶两个亚基的进行活性测定,结果发现,两个亚基单独存在时无酶活,只有两个亚基同时存在,荧光素酶才有活性。优化了双酶的最适反应条件:温度为35℃,pH 7.0,最适NaCl浓度1.5-2%。K+、Ca2+、Na+、Mg2+离子对酶活有促进作用;Fe2+、Zn2+、Hg2+、Ba2+等离子对酶活有抑制作用。抗生素对双酶基本无影响。双酶的热稳定性较好。4℃下可长时间保持酶活。随着温度升高,双酶的耐热性变差,且酶失活的时间缩短。
4、菌体在食品安全检测中的应用探索。建立了P.leiognathi YL检测水产品中氯霉素残留的方法体系。选取对数生长期的P.leiognathi YL,控制菌数为107cfu/mL,乙酸乙酯提取氯霉素,5mL菌液中加入5mL氯霉素提取液,27℃150r/min培养30min,测定发光抑制率。检测线性范围0.14-1.0μg/L,方法检出限0.14μg/kg。建立了P.leiognathi YL检测水产品中重金属的方法体系。重金属敏感性筛选结果显示,P.leiognathi YL对Hg2+、Cr6+、Cd2+最敏感。选取对数生长期的P.leiognathi YL,控制菌数为107cfu/mL,样品灰化处理后,5mL菌液中加入5mL重金属提取液,27℃150r/min培养30min,测定发光抑制率。Hg2+检测线性范围0.007-0.28μg/L,方法检出限为0.007μg/kg;Cr6+检测线性范围0.9-30.0μg/L,方法检出限为0.9μg/kg;Cd2+检测线性范围5.4-65.0μg/L,方法检出限为5.4μg/kg。
5、双酶在食品安全检测中的应用探索。建立了双酶检测水产品中重金属残留的方法体系。重金属敏感性筛选结果显示,双酶对Hg2+、Cr6+最敏感。样品灰化处理后,1mL酶液中加入500μL样品溶液,4℃放置反应15min,测定酶活抑制率。Hg2+检测线性范围0.0007-0.030μg/L,方法检出限0.0007μg/kg;Cr6+检测线性范围0.05-5.0μg/L,方法检出限0.08μg/kg。初步研究了大肠杆菌内NADH对双酶酶活的影响。测定了NADH含量对双酶酶活的影响曲线。在此基础上,对不同菌量的大肠杆菌进行超声细胞破碎,释放NADH,进行双酶酶活检测,为今后利用双酶检测大肠杆菌的菌数提供基础理论数据。
Five luminous bacterial strains were isolated from Qingdao coast and characterized by a combination of 16SrDNA sequences, the morphology observation, physiological test, molecular and biochemical analysis. In comparison with the similar strains reported, the five strains are identified as P.leiognathi, V.harveyi, P.phosphoreum, V.splendidus, V.fischeri.
Sensitivity screening examination indicated that P.leiognathi YL was most sensitive to the antibiotics and heavy metals among the five strains. YL strain has a maxium wavelength for emission at 471nm and the strong light lasted for 16h. The optimum growth and luminescence conditions of YL were determined as: Initial pH 7.5;temperature 25-30℃;shaking speed 150r/min。The medium was composed of 0.5% peptone, 0.1% yeast extraction, 0.01% FePO4, 2-4% NaCl, 0.4% glycerin and seawater.
P.leiognathi YL was selected to produce luciferase and FMN:NADH oxidoreductase(the couple enzymes) for its stable product ability. The optimum enzyme-producing conditions were determined : Initial pH 7.5;temperature 25-30℃;shaking speed 150r/min。
Luciferase was assayed at room temperature by measuring the light emission upon the rapid injection of 1.0mL luciferase mixture, 53μL FMN-Na(10mM), 100μL Na2S2O4(34mM), 100μLdodecanal(27mM).FMN:NADH oxidoreductase was assayed at room temperature by measuring the light emission upon the rapid injection of 1.0mL oxidoreductase mixture, 53μL FMN-Na(10mM), 200μL NADH(0.14mM), 100μL dodecanal(27mM). The luminescence of the coupled enzyme system was more stable than luciferase system.
The couple enzymes were released from the cells by sonicate and purified followed the procedures: precipitated by solid ammonium sulfate; elution on DEAE-Sepharose CL-6B and DEAE-Sephadex columns. The molecular weights of oxidoreductase is 28KDa. Luciferase has two subunits and their molecular weights are 36KDa and 41KDa respectively. No activity was found when either of the isolated subunits is absent. The optimum reaction conditions of the couple enzymes are: Initial pH 7.5;temperature 25-30℃;shaking speed 150r/min. K+、Ca2+、Na+、Mg2+ promoted the activity of the couple enzymes while Fe2+、Zn2+、Sn2+、Hg2+、Ba2+ inhibited the activity. Antibiotics had no effect on the activity of the couple enzymes. The couple enzymes had a good activity at 4℃and the activity decreased with the temperature raised over 40℃.
The concentration of chloramphenicol in shrimp and turbot tissues was detected with P.leiognathi YL. The chloramphenicol in the tissues was extracted by ethyl acetate. The optimal test inoculum is 107cells/mL. 5mL chloramphenicol solution was added into 5mL inoculum. The mixture solution reacted for 30min at 27℃with the shaking speed 150r/min. The concentration of chloramphenicol ranging from 0.14 to 1.0μg/L was determined by the luminescence inhibition rate of P.leiognathi YL. The detection limit is 0.14μg/kg. The concentration of heavy metals(Hg2+、Cr6+、Cd2+)in shrimp tissues was also detected with P.leiognathi YL. The ions of heavy metal in the tissues were extracted by cineration. The detection procedure of heavy metals(Hg2+、Cr6+、Cd2+)was the same as the chloramphenicol. The concentration of Hg2+ ranging from 0.007μg/L to 0.28μg/L was detected, the detection limit was 0.007μg/kg.The detection concentration of Cr6+ is 0.9-30.0μg/L and the detection limit was 0.9μg/kg. The detection concentration of Cd2+ was 5.4-65.0μg/L and the detection limit was 5.4μg/kg.
Sensitivity screening examination indicated that the couple enzymes were most sensitive to Hg2+and Cr6+. The concentrations of Hg2+and Cr6+ in shrimp tissues were detected with the couple enzymes respectively. The ions of heavy metal in the tissues were extracted by cineration. 500μL ion solution was added into the enzyme mixture to react for 15min at 4℃, and the activity of the couple enzymes was detected. The detection concentration of Hg2+ was 0.0007-0.030μg/L and the detection limit was 0.0007μg/kg. The detection concentration of Cd2+ was 0.05-5.0μg/L and the detection limit was 0.08μg/kg. NADH in E.coli cells was released by sonicate and promoted the luminescence of the couple enzymes. The results provided data to detect the total number of E.coli with the couple enzymes.
引文
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