【作者】 杨琴；

【导师】 曾雪峰；

【作者基本信息】 贵州大学 ， 食品科学与工程， 2020， 硕士

【摘要】 生物胺形成是酸鱼重要的安全隐患,肠细菌是非常重要的产生物胺菌,其分泌多种氨基酸脱羧酶脱羧对应氨基酸产生相应的生物胺。目前国内外对鲜鱼中产胺微生物研究较多,但对于发酵鱼制品中发酵过程中产胺微生物研究较少。本研究通过聚合酶链氏反应(PCR)对中国传统发酵酸鱼中肠细菌进行氨基酸脱羧酶基因多样性检测,利用反相高效液相色谱(RP-HPLC)检测生物胺含量;研究了pH对肠细菌产生物胺类型和能力的影响,以及酸胁迫下各产胺途径的关系;研究K.aerogenes 10C2组氨酸脱羧酶的酶学性质;研究K.aerogenes10C2组氨酸脱羧酶基因克隆和生物信息学分析。具体内容及结果如下:(1)本研究测定了酸鱼中97株肠细菌的生物胺形成能力。研究结果表明,97株菌产大量腐胺、尸胺、组胺。将97株菌按产生物胺能力的强弱分为4个等级,大肠菌产腐胺最多,其次是组胺、尸胺。在产腐胺方面,产腐胺含量大于900 mg/l的占33.0%(32/97),产腐胺含量大于500 mg/l的占81.4%(79/97)。产组胺含量在100-500 mg/L之间占41.2%(40/97)。然而,没有一个产尸胺大于500 mg/L的,产尸胺含量在100-500 mg/L的占16.5%(16/97),低于100 mg/L的占83.5%(81/97)。对27株菌的16S rDNA序列比对,比对结果得2株Enterobacter asburiae,2株Klebsiella aerogenes,3株Klebsiella oxytoca,1株Klebsiella pneumoniae,5株Enterobacter hormaechei,3株Enterobacter ludwigii,7株Morganella morganii,1株Citrobacter youngae,2株Enterobacter cloacae,1株Enterobacter sp。检测了27株高产生物胺的氨基酸脱羧酶基因型27株菌携带odc、speA、speB、adiA和ldc基因,13株携带hdc基因。(2)不同pH对27株菌产胺类型及能力影响。研究结果表明,pH显著影响了产胺菌的产胺类型和能力。odc、adiA、spe B、ldc、和hdc在细胞抵抗酸胁迫中扮演重要角色,各产胺途径相互协作抵抗酸环境。在pH 6.70,pH 6.00时大部分肠细菌以产腐胺、尸胺、组胺去调节细胞外酸环境以适应生长;在pH 5.00,pH 4.50时产腐胺和尸胺为主去调节细胞外酸环境以适应生长,也有少部分菌以腐胺和组胺为主去调节细胞外酸环境。酸胁迫会造成微生物生长滞后但是会增加腐胺、组胺、尸胺的含量。氨基酸脱羧酶具有菌株特异性而不是菌种特异性。(3)研究了K.aerogenes 10C2组氨酸脱羧酶酶学性质。该组氨酸脱羧酶最适温度是45℃,最适pH是6.00,保持组氨酸脱羧酶高酶活性热稳定性范围在4-30℃,pH稳定性范围是p H 5.00-8.00。Mg2+可以提高该菌的组氨酸脱羧酶活,相对酶活106%,Ca2+、Mn2+、K+、Pb2+对该酶的酶活基本没有影响,Fe2+、Fe3+降低了部分酶活性,但酶活性仍然保持在89%以上。而Zn2+、Cu2+极大地抑制了酶活性,剩余相对酶活分别为28%、1%。(4)基因克隆和生物信息学分析。编码该酶的核酸序列长度为1137 bp,蛋白质大小约为44 KDa。该酶位于细胞质,分子式为C1905H2914N510O558S17,分子质量为42434.20,该蛋白等电点理论值为5.86<7.0,因此该蛋白为酸性蛋白,脂肪系数是84.68。总平均亲水性为-0.176<0,由此可知该蛋白易溶,具有较强的亲水性。该酶由378个氨基酸组成的,包含了20中氨基酸。其中丙氨酸(Ala)、异亮氨酸(Ile)在该组氨酸脱羧酶的编码氨基酸中使用最多,均占氨基酸总数的8.7%,其次是丝氨酸(Ser)7.7%,天冬氨酸(Asp)7.4%。吡咯赖氨酸(Pyl)、硒代半胱氨酸(Sec)这两种特殊的氨基酸不被包含在内。该酶有11个激酶磷酸化位点、无信号肽、无跨膜结构。二级结构预测可知,该蛋白主要包含有Alpha helix(Hh)α-螺旋、Extended strand(Ee)延伸链、Beta turn(Tt)β-转角、Random coil(Cc)无规则卷曲。α-螺旋、延伸链、β-转角、无规则卷曲结构贯穿整个氨基酸链,且均衡分布。其中最多的是α-螺旋(Hh)和无规则卷曲结构(Cc),Hh有151个氨基酸,占39.95%,Cc有150个氨基酸,占39.68%。其次是延伸链(Ee),有59个氨基酸,占15.61%。最少的是β-转角(Tt),只有18个氨基酸,占4.76%。更多还原

【Abstract】 The formation of biogenic amines is an important safety hazard for fermented fish.Enterobacteria are very important amine producing bacteria,which secrete a variety of amino acid decarboxylases,and the corresponding amino acids are produced by decarboxylation.At present,there are many researches on amine producing microorganisms in fresh fish at home and abroad,but few studies on amine producing microorganisms in fermented fish products.In this study,the diversity of amino acid decarboxylase gene was detected by polymerase chain reaction（PCR）and biogenic amine content was detected by RP-HPLC;the effect of pH on the type and capacity of intestinal bacteria producing amines and the relationship among the amine producing pathways under acid stress were studied;K.aerogenes was also studied objective to study the enzymatic properties of histidine decarboxylase;to study the cloning and bioinformatics analysis of K.aerogenes 10C2 histidine decarboxylase gene.The specific contents and results are as follows:（1）In this study,the biogenic amines formation ability of 97 strains of intestinal bacteria in Suan yu was determined.97 strains were divided into four grades according to their ability to produce amines.Enteric bacteria produced particularly high amounts of putrescine,followed by histamine and cadaverine.About 33.0%（32/97）of the strains produced more than 900 mg/L putrescine.Meanwhile,The content of histamine was between 100 and 500 mg/L,accounting for41.2%（40/97）.However,no strain produced cadaverine content higher than 500 mg/L.The percentage of cadaverine content between 100 and 500 mg/L was 16.5%（16/97）,and 83.5%（81/97）of the strains were below 100 mg/L.Twenty-seven strains of high-yield putrescine,histamine,and cadaverine were selected from 97 strains for follow-up study.The 16S rDNA of 27strains was amplified with the universal primers 27F/1492R,and these sequences were analyzed with the BLAST（NCBI）.Strains were assigned to the different species:2 strains of Enterobacter asburiae,2 strains of Klebsiella aerogenes,3 strains of Klebsiella oxytoca,1 strain of Klebsiella pneumoniae,5 strains of Enterobacter hormaechei,3 strains of Enterobacter ludwigii,7 strains of Morganella morganii,1 strain of Citrobacter youngae,2 strains of Enterobacter cloacae,and 1strain of Enterobacter sp.27 strains carried odc,speA,speB,adiA,and ldc genes.13 carried hdc gene.（2）The effect of different pH on the type and ability of amine production of 27 strains.The results indicated that p H can considerably affect the BA-producing ability of the tested bacteria.odc,adiA,speB,ldc,and hdc were important for enteric bacteria survival under acidic conditions,and these pathways should be collaborative.At pH 6.70 and 6.00,the putrescine-,histamine-production and cadaverine-production dominated in the regulation of extracellular acid environment in most strains of enteric bacteria.At pH 5.00 and 4.50,the the putrescine-and cadaverine-production in the regulation of extracellular acid environment in most strains of enteric bacteria and the putrescine-and histamine-production are the main pathways in a few enteric bacteria.Decarboxylase was strain specific rather than species specific（P<0.01）Acid stress caused the growth delay but can increase the contents of putrescine,histamine,and cadaverine.（3）The enzymatic properties of histidine decarboxylase were studied.The results showed that the optimum temperature was 45℃and the optimum pH was 6.00.The temperature stability range of histidine decarboxylase was 4-30℃and p H was 5.00-8.00.Mg²⁺could increase the activity of the enzyme,and the relative activity was 106%.Ca²⁺,Mn²⁺,K⁺,Pb²⁺had no effect on the enzyme activity.Fe²⁺and Fe³⁺reduced part of the enzyme activity,but the enzyme activity remained above 89%.However,Zn²⁺and Cu²⁺greatly inhibited the enzyme activity,and the residual relative enzyme activity was 28%and 1%respectively.（4）Gene cloning and bioinformatics analysis.The length of the nucleic acid sequence encoding the enzyme was 1137 bp.The protein size is about 44 kDa.The enzyme was located in the cytoplasm with the molecular formula of C₁₉₀₅H₂₉₁₄N₅₁₀O₅₅₈S₁₇,and the molecular weight was42434.20.The theoretical value of isoelectric point of the protein was 5.86<7.0,so the protein was acidic protein with fat coefficient of 84.68.The total average hydrophilicity was-0.176<0,which indicated that the protein was soluble and had strong hydrophilicity.The enzyme is composed of 378 amino acids,including 20 amino acids.Among them,alanine（Ala）and isoleucine（Ile）were the most commonly used amino acids in the histidine decarboxylase,accounting for 8.7%of the total amino acids,followed by serine（Ser）7.7%,aspartic acid（Asp）7.4%.Pyrrolysine（Pyl）and selenocysteine（Sec）,two special amino acids,were not included.The enzyme has 11 kinase phosphorylation sites,no signal peptide and no transmembrane structure.According to the secondary structure prediction,the protein mainly contains alpha helix（Hh）α-helix,extended strand（Ee）extension chain,beta turn（Tt）β-turn and random coil（Cc）random coil.α-helix,extended chain,β-turn and random coil structure run through the whole amino acid chain and are evenly distributed.The most common amino acids wereα-helix（Hh）and random coil structure（Cc）.There were 151 amino acids（39.95%）in Hh and 150 amino acids（39.68%）in Cc.The second was the extended chain（Ee）,with 59 amino acids,accounting for15.61%.The least is theβ-turn（Tt）,only 18 amino acids,accounting for 4.76%.更多还原