Characterisation of the bacterial flora of modified atmosphere packaged farmed Atlantic cod (Gadus morhua) by PCR-DGGE of conserved 16S rRNA gene regions

The present article describes the use of broad-range molecular analyses to characterise the microbial population of farmed Atlantic cod (Gadus morhua) packaged for the retail market. Cod was filleted post rigor, packaged in air or in modified atmosphere (MA) (50% CO(2):50% N(2) or 50% CO(2):50% O(2)) and stored at 0 degrees C for 11 days. To determine the community profiles of the samples the variable V3-region of the bacterial 16S rRNA gene were amplified by PCR, before the PCR products were separated by denaturing gradient gel electrophoresis (DGGE). From sequence analyses Pseudomonas spp. were found to be the predominant bacteria in oxygen enriched atmospheres, whereas the spoilage bacteria Photobacterium sp., Shewanella putrefaciens and Pseudomonas spp. dominated in CO(2):N(2) and air packaged samples. Additional microbial analyses by cultivation methods observed highest bacterial numbers in air stored samples, and both MA mixtures gave growth inhibition when measuring aerobic plate count, psychrotrophic bacteria and H(2)S-producing bacteria. The results show that PCR-DGGE can be applied to examine bacterial diversity and population shifts among different MA-packaged products.     

不同包装方式下冷鲜青虾的菌群多样性分析

对青虾采用两种包装方式：托盘包装和气调包装（体积分数50% CO2＋50% N2）,在4?℃条件下贮藏6?d,测定菌落总数和挥发性盐基氮含量,确定不同包装方式下青虾的货架期;随后利用16S?rDNA扩增子测序对两种包装方式下青虾微生物多样性进行研究,比较其菌相变化,确定其变化规律。结果表明：托盘包装组和气调包装组的货架期分别为2?d和4?d;且两种包装方式下变质青虾的菌群结构有较大差异,在托盘包装方式下,优势菌群主要以不动杆菌、黄杆菌和希瓦氏菌为主,是普通低温贮藏时造成青虾腐败的主要菌群;采用气调包装后,黄杆菌和希瓦氏菌数量大大降低,假单胞菌成为主要优势菌群;体积分数50%?CO2＋50%?N2气调包装能抑制托盘包装时优势腐败菌的增殖,从而延长货架期;而假单胞菌有较强的耐受力,成为新的优势腐败菌。研究结果为保障青虾的质量安全提供了理论依据。     

Estimating packaging atmosphere–temperature effects on the shelf life of cod fillets

The behaviour of the natural microflora (total bacterial count, psychrotrophic bacteria, total coliforms, and lactic acid bacteria) of fresh cod fillets packaged in air, under vacuum, in a modified atmosphere with low oxygen concentration (5%) and in a modified atmosphere with high oxygen concentration (80%) was monitored during storage at different isothermal conditions from 4 to 12 °C. The growth data of the total bacterial count and total coliforms were used to model kinetically the shelf life of the samples in order to check the effects of storage temperature and packaging atmosphere. The acceptability times were compared with the stability times to establish if the shelf lives predicted were correlated. At 4 °C, cod fillets packaged under vacuum and in a modified atmosphere with low oxygen concentration had a higher shelf life (6.00 and 5.42 days), calculated from the growth of the total bacterial count, than the samples packaged in other atmospheres (1.96 and 2.62). Kinetic modelling could be valuable for the prediction of microbial fish quality loss.     

冷藏大菱鲆细菌组成变化和优势腐败菌

对大菱鲆(Scophthalmus maximus)在0、3、7、10℃冷藏过程中的细菌菌相进行定性和定量分析。研究发现新鲜大菱鲆细菌菌相比较复杂,分离得到114株细菌,其中革兰氏阴性菌和革兰氏阳性菌分别占68.4%和21.1%,优势菌为肠杆菌、侵肺巴斯德菌、拟态弧菌、假单胞菌属和腐败希瓦氏菌,比例分别为14.9%、13.2%、12.3%、10.5%和8.8%。冷藏过程中,细菌菌相逐渐单一,腐败希瓦氏菌和假单胞菌增长显著,0、3、7、10℃冷藏至较好品质期终点时,腐败希瓦氏菌比例分别为42.2%、39.0%、57.1%和56.3%,平均比例为50.2%;其次是假单胞菌,比例分别为28.0%、23.7%、25.5%和31.0%,平均比例为27.2%。0、3、7、10℃冷藏至货架期终点时,腐败希瓦氏菌比例分别为53.9%、50.0%、57.4%和47.6%,平均比例为52.3%;假单胞菌属比例分别为25.5%、25.0%、41.0%和15.9%,平均比例为26.5%。由此可得出大菱鲆在0～10℃冷藏过程中的优势腐败菌是腐败希瓦氏菌,其次是假单胞菌。     

PCR-DGGE指纹技术研究复合保鲜剂对冷藏带鱼贮藏期间微生物变化的影响

为探讨复合保鲜剂的作用机理和延长水产品货架期,应用聚合酶链式反应(PCR)-变性梯度凝胶电泳(DGGE)指纹技术研究复合保鲜剂对冷藏带鱼贮藏期间微生物种群多样性演替规律的影响。采用SDS(sodium dodecyl sulfate)化学裂解法,分别从对照组与复合保鲜剂处理组的冷藏带鱼中提取总细菌基因组DNA,同时进行16S rDNA的V3可变区PCR扩增,再通过DGGE得到动态变化的指纹图谱。DGGE图谱表明,产品在贮藏初期具有丰富的微生物群落,说明污染微生物的多样性,但经过一段时间后,只有少数种类细菌存活并最终成为主导菌群;通过对冷藏带鱼DGGE图谱上的主要条带进行测序分析,获得12种细菌。对照组与复合保鲜剂处理组在腐败后期的特定腐败微生物种类具有高度的相似性。嗜冷杆菌为带鱼贮藏初期的优势菌,随着贮藏时间的延长,希瓦氏菌与假单胞菌的比例逐渐增加,在贮藏过程中成为主要优势菌,荧光假单胞菌与弧菌在贮藏期间占有较大比例。同时,冷藏带鱼经复合保鲜剂处理后,其中的希瓦氏菌与假单胞菌受到明显抑制。     

气调包装的调理啤酒鲈鱼片在微冻贮藏过程中的微生物群落多样性分析

为揭示调理啤酒鲈鱼片气调包装和微冻贮藏过程中的微生物变化规律及其腐败本质,为优化产品工艺和推广产品提供理论依据,采用Illumina MiSeq测序技术解析调理啤酒鲈鱼制品气调包装和－3?℃贮藏过程中微生物群落多样性,并比较其与真空包装4?℃贮藏鲈鱼片的微生物群落多样性的区别。结果表明：鲈鱼制品在贮藏过程中的优势微生物为厚壁菌门（Firmicutes）、变形菌门（Proteobacteria）、拟杆菌门（Bacteroidetes）;真空包装的未调味鲈鱼片在4?℃贮藏过程中微生物多样性比在－3?℃贮藏的气调包装调理啤酒鲈鱼丰富,且在贮藏第4天时,开始出现了索丝菌属（Brochothrix）等常见的腐败微生物,到第10天时,索丝菌属、假单胞菌属（Pseudomonas）丰度迅速增大,出现了希瓦氏菌属（Shewanella）,造成鲈鱼片彻底腐败变质;气调包装的调理啤酒鲈鱼片在第20天时才开始出现假单胞菌属和希瓦氏菌属等腐败菌,之后在CO2的抑制作用下,腐败菌丰度逐渐减小,直到货架期终点时,假单胞菌才迅速增长,且出现了嗜冷杆菌属（Psychrobacte）,造成了鲈鱼制品的彻底腐败变质。这说明气调包装联合啤酒调理的加工方式及－3 ℃贮藏可以有效延长产品的货架期。     

养殖大黄鱼冷却链全程细菌定性和定量研究

对在闽东三都湾养殖密集海区所捕获的大黄鱼冷却链全过程的产品品质和细菌相变化进行了定性和定量研究。刚捕获的大黄鱼,其细菌总数受养殖水域卫生状况影响很大,为4.1×106cfu/g,菌群中嗜水气单胞菌(Aeromonas hydrophila)居优势,占25.2%,阴沟肠杆菌(Enterobacter cloacae)和弗氏柠檬酸杆菌(Cirtobacterfreundii)占较大比例。处理后的细菌总数和细菌相表明,加工处理过程没有明显的细菌污染。冰藏初期细菌总数明显下降,菌群中好冷的不动细菌(Acinetobactria)和摩氏杆菌(moxeralle)比例上升,其他菌群不同程度下降,出现了较多比例的假单胞菌(Pseudomonasspp.)和腐败希瓦氏菌(Shewanella putrefaciens),表明鱼体温度迅速下降对有些细菌产生了冷冲击致死作用。冷藏过程中,腐败希瓦氏菌增殖比其他细菌快,在货架期终点达到86.0%,是冷却链大黄鱼产品的优势腐败菌。货架期终点12 d时,细菌总数为6.7×107cfu/g。     

60Coγ-射线及电子束辐照对冷鲜鸡菌群多样性的影响

为探明γ-射线与电子束辐照对冷鲜鸡微生物含量和微生物群落多样性的影响,采用2.5 kGy剂量的γ-射线和电子束辐照处理真空包装冷鲜鸡,对其贮藏期内菌落总数、群落结构进行测定分析。结果表明,两种射线均能显著(P<0.05)降低冷鲜鸡菌落总数;高通量测序后得到了9门,62属的菌群结构,在门水平上,变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)、梭杆菌门(Fusobacteria)、拟杆菌门(Bacteroidetes)为优势菌群;在属水平上,贮藏0 d时,对照组内环丝菌属(Brochothrix)、希瓦氏菌属(Shewanella)、不动细菌属(Acinetobacter)为优势菌群;贮藏5 d时,对照组内希瓦氏菌属(Shewanella)、γ-射线组内嗜冷菌属(Psychrobacter)、电子束处理组内希瓦氏菌属(Shewanella)为优势菌群;贮藏10 d时,对照组内嗜冷菌属(Psychrobacter)、γ-射线组不动细菌属(Acinetobacter)、电子束处理组内希瓦氏菌属(Shewanella)为优势菌群;贮藏15 d时,对照组内嗜冷菌属(Psychrobacter)、γ-射线组希瓦氏菌属(Shewanella)、电子束处理组内希瓦氏菌属(Shewanella)成为优势菌群。由此可知,γ-射线与电子束辐照均对冷鲜鸡微生物数量和群落结构产生显著(P<0.05)影响。     

Microbiological spoilage of vacuum and modified atmosphere packaged Vietnamese Pangasius hypophthalmus fillets

This study investigated the identity, growth and metabolite production of micro-organisms causing spoilage of Pangasius hypophthalmus fillets packaged in air, vacuum and modified atmospheres (MAP) (MAP 1: 50%CO(2)-50%N(2) and MAP 2: 50%CO(2)-50%O(2)) during storage at 4 °C. Based on the time it took for psychrotrophic total colony counts to exceed 7 log cfu g(-1), the shelf life of the fillets packaged in air, vacuum, MAP 1 and MAP 2 was estimated to be 7, 10, 12 and 14 days respectively. The longest lag phases were observed in the samples packaged in MAP 2 (50%CO(2)-50%O(2)). In the fillets packaged in air and under vacuum, the dominant flora identified by partial 16S rDNA sequencing at the end of the shelf life generally consisted of Gram-negative bacteria mostly belonging to the genera Serratia and Pseudomonas. In contrast, lactic acid bacteria (Carnobacterium maltaromaticum and Carnobacterium divergens) and Brochothrix thermosphacta were identified as the dominant spoilage flora in the samples packaged under the two MAPs investigated. By means of solid-phase microextraction gas chromatography mass spectrometry (SPME GC-MS) analysis, volatile organic compounds in the headspace of the samples at the end of the shelf life were identified for each packaging condition. Based on these results, a selective ion flow tube mass spectrometry (SIFT-MS) method was developed to quantify the production of volatile metabolites during storage of the fillets. The results of these analyses indicated that several compounds contributed to the bacterial spoilage of Pangasius fillets e.g., ethanol, 2,3-butanediol, diacetyl, acetoin, ethyl acetate, acetic acid and sulfur compounds. It also emerged that the production of these compounds was dependent on the packaging condition applied.     

不同温度条件下养殖大菱鲆菌群变化和货架期预测

对低温(0~10℃)、室温(25℃)和变温贮藏条件下养殖大菱鲆的细菌种群变化进行研究,确定不同温度条件下养殖大菱鲆的优势腐败菌和货架期,并采用Exponential、School-field和Square-root方程构建货架期预测模型,并对货架期预测模型的适用性进行评价和验证。结果显示,低温贮藏大菱鲆货架期为6.1~32.6 d,优势腐败菌为腐败希瓦氏菌(40.3%)和假单胞菌属(27.4%),室温时货架期为1.3 d,优势腐败菌为气单胞菌(47.1%)和腐败希瓦氏菌(29.4%);构建的Exponential、School-field和Square-root货架期模型参数分别为温度特征系数a=0.12,T_(min)=-6.6℃,表观活化能(E_a)为81.4 k J/mol,采用均方根误差、残差平方和、偏差度、准确度对模型的拟合优度进行比较,Square-root模型预测性能最优,用恒温和变温对3种货架期预测模型进行验证,显示Square-root货架期模型误差最小,可有效预测恒温和变温条件下大菱鲆的货架期。     

真空包装冷藏大菱鲆鱼片的货架期及其优势腐败菌群体感应检测

本研究以真空包装冷藏大菱鲆鱼片为研究对象,通过感官、微生物和化学指标评价其货架期,利用16Sr DNA方法对其贮藏初期和末期的微生物分布进行鉴定,并采用报告菌株法检测大菱鲆鱼片贮藏过程中两种类型群体感应信号分子(AHLs和AI-2)的产生情况,检测其优势腐败菌的群体感应类型。结果显示,大菱鲆鱼片在真空包装冷藏环境下可以贮藏9 d,此时细菌总数达6.87 logCFU/g,挥发性盐基氮含量为28.70 mg/100 g。对比初始和末期微生物种类变化,真空包装大菱鲆鱼片冷藏过程中的优势腐败菌主要为希瓦氏菌,其次为肠杆菌科细菌。群体感应信号分子AHLs和AI-2含量随贮藏时间延长而增加。贮藏末期分离的希瓦氏菌均能分泌AI-2信号分子,分泌能力因菌株而异。利用基因手段可从希瓦氏菌中扩增到AI-2的分泌基因lux S,表明lux S在产AI-2菌株中具有一定保守性,可能参与希瓦氏菌的腐败进程。     

Effect of Modified Atmosphere Packaging and Superchilled Storage on the Microbial and Sensory Quality of Atlantic Salmon (Salmo salar) Fillets

ABSTRACT: Fresh Atlantic salmon fillets packaged under modified atmosphere (MA) (CO²:N² 60:40) and air was stored at superchilled (-2 °C) and chilled (+4 °C) temperatures. Changes in sensory scores, microbial growth, headspace gas composition, water loss, and pH were monitored during 24 d of storage. The superchilled MA packaged salmon maintained a good quality, with negligible microbial growth (<1000 colony-forming units [CFU]/g) for more than 24 d based on both sensory and microbial analyses (aerobic plate count, H2S-producing, and psychrotrophic bacteria). Superchilled salmon in air had a 21-d sensory shelf life, whereas MA and air-stored fillets at chilled conditions was spoiled after 10 d and 7 d, respectively.     

Effects of modified atmosphere storage on colour and microbiological shelf life of normal and pale, soft and exudative pork

Pale, soft and exudative (PSE) and normal pork loins (n = 9 of each) were deboned, divided and packaged in modified atmospheres (MA) of 100% carbon dioxide containing 0, 0.5 and 1.0% residual oxygen (O(2)). The meat was stored at 3 °C, first in MA for 21 days, followed by 5 additional days under retail display conditions with access to air. Before packaging, PSE loins were more light and less red than those of normal meat. The drip loss after MA storage was twice as high from PSE meat as from normal meat. PSE meat was not more discoloured after MA storage than normal meat. The level of residual O(2) in the MA had a significant impact on the colour of the two types of meat. Discoloration was observed on both PSE and normal meat with 0.5% O(2) and even more clearly with 1.0% O(2), as demonstrated by instrumental and visual colour analyses. After MA storage, the microbiological shelf life and flora were not affected by the type of meat, or level of residual O(2).     

The effects of cultivar and physiological age on quality and shelf-life of coleslaw mix packaged in modified atmospheres

Two cultivars of ‘Dutch’ cabbage were used to determine conditions whereby coleslaw made from them could be stored under modified atmospheres (MA) with minimal deterioration. The cabbage was sliced and mixed (4:1) with shredded carrot. The effects of storing the cabbages prior to processing, the cultivar identity, and use of low temperatures and MA storage on the quality and storage life were investigated. Quality in terms of appearance and aroma were measured as well as bacterial contamination before and during storage. Cultivar (cv.) Marathon rather than cv. Lennox gave a product of higher quality, with a better shelf‐life, particularly when packaged under micro‐perforated PA‐160 film rather than oriented polypropylene (OPP). Long‐term storage of cabbage prior to processing had detrimental effects on the acceptability of MA packaged coleslaw. Attempts were made to explain the findings in terms of differences in microbial counts. Guidelines were established that could be used in the production of high quality coleslaw mix packaged in MA.     

冷藏卵形鲳优势腐败菌的分离鉴定及致腐能力分析

为研究卵形鲳鲹冷藏末期的优势腐败菌及其致腐能力,采用传统选择性培养结合16S rDNA序列分析法,对卵形鲳鲹4℃贮藏期间腐败菌分别进行分离纯化、菌相分析及菌属确定,并以腐败物质的产量因子Y_TVB-N/CFU为评价标准,定量分析了贮藏末期优势菌的致腐能力。结果得出,卵形鲳鲹4℃贮藏过程中共分离纯化出16株细菌,其中革兰氏阴性菌10株,革兰氏阳性菌6株,其分属6个菌属。贮藏初期（0 d）时菌相丰富,希瓦氏菌属（Shewanella sp.）、假单胞菌属（Pseudomonas sp.）、葡萄球菌属（Staphylococcus sp.）、芽孢杆菌属（Bacillus sp.）、乳酸杆菌属（Lactobacillus sp.）与肠杆菌属（Enterobacter sp.）等均有出现;中期（3 d）菌群种类有所减少,以希瓦氏菌属（Shewanella sp.）与假单胞菌属（Pseudomonas sp.）所占比例较高,后期（6 d）种类进一步减少,只有3株菌占较高,分别是2号腐败希瓦氏菌（Shewanella putrefaciens 48%）、5号奥奈达希瓦氏菌（Shewanella oneidensis 26.6%）与16号霍氏肠杆菌（Enterobacter hormaechei 19.4%）;致腐能力由高至低依次为腐败希瓦氏菌 > 奥奈达希瓦氏菌 > 霍氏肠杆菌,其中腐败希瓦式菌致腐能力显著强于另两株菌;综合贮藏末期所占比例及致腐能力结果,最终确定腐败希瓦氏菌为4℃贮藏下卵形鲳鲹的优势腐败菌。     

PCR结合生理生化鉴定对冷藏带鱼主要细菌菌相组成分析

将传统的微生物生理生化鉴定技术与16S rDNA分子生物学方法相结合,并通过系统发育树分析,对冷藏带鱼贮藏期间的主要细菌组成比例进行研究。样品在(4±1)℃贮藏,分别于不同贮藏时间取样,对各个阶段的微生物菌落进行分离纯化、形态观察和部分生理生化鉴定,对最终得到的细菌纯培养物直接提取DNA,作16SrDNA PCR扩增,产物经测序后于NCBI与已知序列进行相似性比对,最终得到13株具有典型特征的纯菌株。结果得出,冷藏带鱼在货架期终点时,其主要微生物的种类与所占比例依次为:腐败希瓦氏菌(Shewanella putrefaciens 34.7%)、荧光假单胞菌(Pseudomonas fluorescens 14.5%)、松鼠葡萄球菌(Staphylococcus sciuri 10.2%)、嗜水气单胞菌(Aeromonas hydrophila 8.2%)、弧菌(Vibrio rumoiensis 6.1%)、恶臭假单胞菌(Pseudomonas putida6.1%)、绿色气球菌(Aerococcus viridans 4.1%)、金黄色葡萄球菌(Staphyloccocus aureus 4.1%)、蜡样芽孢杆菌(Bacillus cereus 4.0%)、铜绿假单胞菌(Pseudomonas aeruginosa 2.0%)、嗜冷杆菌(Psychrobacter sp.2.0%)、成团肠杆菌(Enterobacter agglomerans 2.0%)、约氏不动杆菌(Acinetobacter johnsonii 2.0%),其中腐败希瓦氏菌为带鱼冷藏期间的主要优势腐败菌,假单胞菌在带鱼冷藏过程中的所占比例大小依次为荧光假单胞菌>恶臭假单胞菌>铜绿假单胞菌。     

Changes in the composition of the bacterial flora on tray-packaged pork during chilled storage analyzed by PCR-DGGE and real-time PCR

In this study, a polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) was used to investigate the changes in the composition of the bacterial population of tray-packaged pork during chilled storage. Relative quantitative real-time PCR was further used to evaluate the predominant spoilage bacteria obtained from DGGE analysis for their relative amount to the total bacteria in meat samples. DGGE analysis of the V3 and V6-V8 regions of the 16S rRNA gene showed that Pseudomonas were the predominant bacterial species at the end of the monitoring period. Real-time PCR expressed as the ΔΔC(T) method showed that the average 2(-ΔΔC)(T) values increased continually during the storage period from less than 0.001 at day 0 to 4.438 at the end of the monitoring, which indicated that the proportions of Pseudomonas within the total bacteria in meat samples increased. Both methods confirmed that Pseudomonas was the predominant spoilage bacteria. Practical Application: This study uses new techniques to identify bacteria in fresh retail pork and to follow changes in the bacterial population during 12 d refrigerated storage. Pseudomonads were found to increase with storage time, becoming the dominant flora after 12 d.     

Effect of packaging atmosphere on the microbial attributes of pearlspot (Etroplus suratensis Bloch) stored at 0-2 degrees C

Effect of packaging atmosphere (air and under different modified atmospheres (MAs), 40% CO2/60% O2, 50%/50% O2, 60% CO2/40% O2, 70% CO2/30% O2 and 40% CO2/30% O2/30% N2) on the microbial and biochemical attributes of fresh pearlspot (Etroplus suratensis Bloch) stored at 0-2 degrees C was investigated. Trimethylamine nitrogen (TMA-N) and thiobarbituric acid (TBA) values remained lower than the proposed acceptability limits throughout the storage period. Results demonstrated that storage of pearlspot under air and MA 40% CO2/30% O(2)/30% N(2) resulted in growth of Enterobacteriaceae, Aeromonas and H(2)S-producing bacteria including Shewanella putrefaciens, while all other packaging atmospheres did not allow multiplication of Enterobacteriaceae and Aeromonas within 3 weeks. Aeromonas spp. identified were Aeromonas hydrophila, Aeromonas veronii biovar sobria and A. veronii biovar veronii. Significant reduction (p<0.01) was noticed in Aeromonas population of pearlspot stored under MA 60% CO2/40% O2 and 70% CO2/30% O2. A delay of growth of Pseudomonas below 5.0log(10)cfug(-1) was observed during the 15th day of storage at 0-2 degrees C under modified atmosphere packaging (MAP) conditions. Growth of faecal streptococci was significantly inhibited in all the packaging atmospheres at 0-2 degrees C during the entire storage period. Survival of coagulase positive Staphylococci (<50cfug(-1)) in low numbers was noticed during storage in all the packaging atmospheres. Clostridium botulinum toxin was not detected. All the packaging atmospheres did not allow multiplication of sulphite-reducing clostridia at 0-2 degrees C during the entire storage period. Packaging in MA 60% CO2/40% O2 resulted in the inhibition of growth of Aeromonas and Enterobacteriaceae, and the slowest growth of psychrotrophic bacteria, H(2)S-producing bacteria, including Shewanella putrefaciens and Pseudomonas and extended microbiological shelf life to 9-10 days. This study confirms the survival of potentially pathogenic A. hydrophila, A. veronii biovar sobria and A. veronii biovar veronii capable of growth at low temperature in pearlspot stored under MA.     

Characterisation of the dominant bacterial population in modified atmosphere packaged farmed halibut (Hippoglossus hippoglossus) based on 16S rDNA-DGGE

It is not well understood why Atlantic halibut (Hippoglossus hippoglossus) has longer shelf-life than most other white fish species. Our approach was to examine the microbiological diversity of the spoilage microbiota during modified atmosphere (MA) packaging of farmed Atlantic halibut. Portions were packaged with gas mixtures of CO(2):N(2) and CO(2):O(2) (50%:50%) and with air as a reference. The packages were stored at 4 degrees C and samples were taken 6 times during the 23 days of storage. Analyses with molecular techniques (PCR-DGGE) determined profiles of the bacterial populations in the various samples and sequencing detected the bacterial species present. In addition, samples were analysed for microbial, chemical and sensory parameters. The shelf-life was 10-13 days when stored in air and between 13 and 20 days for MA packages, with oxygen-enriched packages suggested as the better gas mixture, based on microbial growth and sensory scores. From sequence analyses of the bacterial population Photobacterium phosphoreum and Pseudomonas spp. were found to dominate in the halibut. Brochothrix thermosphacta was found in most samples at the end of the storage period. Shewanella putrefaciens was found sporadically and in low concentrations based on microbial methods, but not detected by PCR-DGGE.