气调包装淡水鱼微生物菌群的演替及其对顶空气体变化的响应

Modified atmosphere packaging (MAP) has become an increasing popular preservation technique, bringing major changes in storage of raw products to meet consumer demands for fresh, healthy and safe foods, showing positive effect on shelf-life in cold chain. In order to overcome the setback of most existing MAP design, which has been mainly empirical with “trial and error” or “pack and pray” approach, an improved understanding of dynamic changes of headspace gas were considered to be the base for MAP design. The microbial succession in MAP aquatic product during storage and response of microflora to the headspace gas change should be monitored. In our preliminary research, specific spoilage organism (SSO) had been identified with fingerprinting molecular method (16S rDNA-RFLP) in crisped grass carp with different package during storage. It has been suggested that identification of the dominant microflora in different stage of storage was essential for developing the new technology of target inhibition on microorganism. Based on these, we select grass carp （one of most abundant freshwater fish species in China） and crisp grass carp (one of freshwater fish with special texture, cultivating in south of China) as our research subjects. The headspace gas profile in MAP fish during storage in cold chain would be plotted and mass transfer between headspace gas and fish would be modeled basing on artificial neural network (ANN). Pronounced change of microflora coincided with the change in gas profile. The succession of microflora diversity of fish in MAP during storage would be plotted by combination of polymerase chain reaction (PCR) based molecular technology-denaturing gradient gel electrophoresis (DGGE) and conventional evaluation of spoilage characteristic including physicochemical index and sensory index. Dominant microbial of different stage would be identified by high throughput sequencing technology. The spoilage potential of dominant microbial would be evaluated by inoculation into sterile raw fish followed by storage in MAP model system and the effect of their interaction were evaluated by co-culture in MAP model system. The growth curve and interaction would be modeled by applying Baranyi model, Square root model and the modified Lotka-Volterra model. Based on the interaction among microflora and the microbial succession in MAP during storage, the response of microflora to MAP would be analysis comprehensively. This study would give the significant improvement for MAP design and MAP optimization and provide the theory foundation for developing multi-target hurdle technologies.

气调包装（MAP）因能延长食品货架期且安全卫生成为最有潜力的保鲜手段之一，而确定微生物菌群在保鲜过程中的演替进程及其对环境变化的响应模式是设计和优化MAP方案的基础。本项目在确定不同包装脆肉鲩SSO的基础上，以大宗淡水鱼（鲩鱼）和特色淡水鱼（脆肉鲩）为研究材料，绘制鱼肉在MAP环境中顶空气体的变化图谱，同时建立基于ANN的气体与鱼肉样品传质模型；应用PCR-DGGE监测微生物菌群的演替规律，进而结合高通量测序识别不同阶段的优势菌；比较优势菌在纯培养、单独接种及混合接种于模拟MAP环境中的生长特性及腐败特性，探讨气体环境对微生物的影响及菌群的相互作用；结合Baranyi、平方根方程及改良Lotka-Volterra构建优势菌的生长及交互作用模型，确定微生物菌群对MAP环境的响应模式。该项目可为阐明MAP原理提供基础资料，避免MAP在方案设计及优化方面的盲目性；同时为开发靶向抑菌奠定理论基础。

本研究绘制了MAP脆肉鲩及草鱼鱼片在冷链保鲜过程中顶空气体（CO2和O2）的变化图谱并确定了不同温度下两种鱼片的Henry系数；结合理化指标、感官指标及固相微萃取SPME-GC-MS技术分析脆肉鲩及草鱼的腐败特性，构建16S rDNA基因文库并结合RFLP确定两种鱼的特定腐败菌；利用PCR-DGGE和高通量测序技术绘制不同MAP环境下脆肉鲩鱼片在冷藏过程中微生物菌群图谱结构，确定优势菌在冷藏过程中的演替及对不同气体环境的响应；从微生物生长参数及腐败产物两方面评价假单胞菌A14和B14的腐败能力，建立优势菌生长模型。结果表明：在MAP草鱼和脆肉鲩鱼片的冷藏中，顶空气体CO2逐渐降低并在8-24h与食物基质达到传质平衡，CO2在食物基质中的溶解率（S(PCO2,T)）与温度及CO2分压呈正相关，4℃下草鱼和脆肉鲩的Henry系数（Ks）分别为2292.2±211.4 Pa/mmol•g-1和2156.4±215.6 Pa/mmol•g-1；固相微萃取技术显示新鲜草鱼和脆肉鲩鱼片的主要风味物质是醛酮类、醇类和烃类，而腐败草鱼和脆肉鲩鱼片中的挥发性物质则以醛酮、胺类等为主；两种鱼在4℃和8℃的货架期分别为6d和3d，假单胞菌为脆肉鲩的特定腐败菌（SSO），气单胞菌、不动杆菌及假单胞菌是草鱼的SSOs；菌群结构图谱显示：和腐败鱼片相比，新鲜草鱼和脆肉鲩鱼片具有丰富的微生物多样性；在高CO2分压环境下（MAPa-50%CO2+20%O2+30%N2, MAPb-60%CO2+10%O2+30%N2），脆肉鲩鱼片的优势菌由原来的不动杆菌（Acinetobater）转变为肉杆菌（Carnobacterium）；与普通包装相比，真空环境对脆肉鲩鱼片优势菌的影响不大，其特定腐败菌（SSO）仍是假单胞菌（Pseudomonas）和气单胞菌（Aeromonas），不过兼性厌氧的乳杆菌（Lactococcus）在冷藏后期出现。微生物生长模型显示CO2可有效抑制特定腐败菌(SSO)假单胞菌，在高CO2环境下（CO2≥50%）其生长速率只有普通包装的6.72%和8.48%，嗜冷菌和气单胞菌也表现出在高CO2分压环境下被抑制的特性。本研究可为科学设计草鱼和脆肉鲩鱼的MAP工程提供参考和预测模型，同时为淡水鱼的冷链保鲜提供指导，为开发靶向抑菌技术打下基础。