冷鲜牛肉贮藏中菌群结构及优势菌致腐性的分析

Bacterial Community Structure and Spoilage Potential of Dominant Bacteria in Chilled Beef

为探究牛肉在0℃冷藏下微生物菌群变化和优势腐败菌,采用培养依赖的16S rRNA结合高通量测序技术分析牛肉样品的微生物多样性变化。通过定期测定接种牛肉汁的生长、感官、蛋白酶活性、pH值和挥发性盐基氮(total volatile basic nitrogen,TVB-N)值,分析分离株的致腐特征。结果表明,牛肉冷藏中感官品质保持良好,15 d出现异味,18 d有腐臭味。而样品中菌落总数第3天快速上升,15 d菌落总数为8.73(lg(CFU/g)),之后呈现稳定,假单胞菌(Pseudomonas spp.)、热死环丝菌(Brochothrix thermosphacta)、肠杆菌(Enterobacter)和乳酸菌4种分离培养基中细菌与菌落总数增长趋势相似,其中假单胞菌增长最快,肠杆菌数和乳酸菌数生长最慢。两种菌群鉴定结果显示,冷鲜牛肉初始微生物构成复杂,包括热死环丝菌、不动杆菌属(Acinetobacter spp.)、气单胞菌属(Aeromonas spp.)和假单胞菌等多种菌属构成,而腐败末期菌群构成趋于单一,假单胞菌和热死环丝菌为优势腐败菌,特别是莓实假单胞菌(P.fragi)。将腐败分离株10株假单胞菌、4株热死环丝菌和1株蜂房哈夫尼菌(Hafnia alve)接种于冷藏的牛肉汁,发现假单胞菌和蜂房哈夫尼菌的接种组感官评分、pH值和TVB-N值高于热死环丝菌,且假单胞菌有较强的蛋白酶活性。研究表明,结合感官和微生物评价冷鲜牛肉贮藏期为15 d,且菌群多样性下降,假单胞菌和热死环丝菌是优势腐败菌,其中假单胞菌致腐性较强。

To investigate the changes in the bacterial community and the dominant spoilage bacteria in chilled beef stored at 0 ℃, culture-dependent 16S rRNA gene sequencing and culture-independent high-throughput sequencing were carried out to detect the changes in bacterial diversity during the storage. The sensory score, protease activity, pH, and total volatile base nitrogen (TVB-N) in sterile beef extract inoculated with the isolates from spoiled beef were assessed for spoilage potential. The results showed that chilled beef maintained good sensory quality during the early period of storage, and it smelled unpleasant and rotten on the 15th and 18th day, respectively. Total viable count (TVC) rapidly increased during the first 15 days, reaching 8.73 (lg(CFU/g)), and then remained steady. The growth rates of Pseudomonas, Brochothrix thermosphacta, Enterobacter and lactic acid bacteria (LAB) exhibited similar trends to TVC, among which Pseudomonas grew fastest, while Enterobacter and LAB showed the slowest growth rate. The bacterial composition in chilled beef was complex and diverse, including B. thermosphacta, Acinetobacter spp., Aeromonas spp. and Pseudomonas spp., while it became simpler after spoilage. Pseudomonas especially P. fragi and B. thermosphacta were the dominant spoilage bacteria in spoiled beef stored at 0 ℃. Furthermore, these spoilage isolates, including 10 strains of Pseudomonas, 4 strains of B. thermosphacta and 1 strain of Hafnia alve, were separately inoculated to sterile beef juice. The samples inoculated with Pseudomonas and H. alvei exhibited higher sensory score, pH value and TVB-N content, revealing stronger spoilage potential, compared to B. thermosphacta. Moreover, Pseudomonas had stronger protease activity. Thus, the shelf-life of chilled beef was about 15 d based on sensory and microbial evaluation. During the storage, the microbial diversity decreased, and Pseudomonas and B. thermosphacta were the dominant spoilage bacteria in chilled beef. Pseudomonas presented stronger spoilage ability in vitro than B. thermosphacta.