超高压处理对牡蛎冷藏过程腐败菌群结构的影响及典型菌株——腐败希瓦氏菌致死机理研究
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摘要
为揭示超高压处理对牡蛎腐败菌群的影响及其作用机制,采用高通量测序技术分析生鲜和腐败牡蛎的细菌群落结构,并以存活率、胞外碱性磷酸酶活力、胞外还原糖含量等为指标,结合电镜观察,探讨超高压处理对牡蛎中典型致腐菌株的致死机制。结果表明:新鲜牡蛎中菌群以弧菌属、希瓦氏菌属和交替假单胞菌属为主,而腐败时交替假单胞菌属和希瓦氏菌属比例较高。超高压处理改变了牡蛎冷藏过程中的菌群结构,腐败样本中嗜冷菌属占绝对优势,而交替假单胞菌属比例仅为0.8%,希瓦氏菌属比例小于0.1%。代表性菌株腐败希瓦氏菌经100 MPa以下的压力处理后,存活率无明显变化(P>0.05),之后随处理压力的增大而迅速下降,用400 MPa及以上压力处理后未检出活菌。胞外碱性磷酸酶活力、还原糖含量均在较低压力处理条件(<100 MPa)下发生显著变化,之后随着处理压力的增大变化趋于缓和。菌体经超高压处理,细胞发生相互黏连,出现聚集成簇的现象。随着处理压力的进一步增大,菌体细胞彼此间的界限已模糊不清,直至丧失原有形态,最终死亡。
In order to reveal the effect of ultra-high pressure processing(UHPP) on the spoilage bacteria flora of oysters and its mechanism, high throughput sequencing technology was applied to study the bacterial community structure in fresh and spoiled oysters. Survival rate, extracellular alkaline phosphatase(AKP) activity, extracellular reducing sugar content and electron microscope observation were used to discuss the lethal mechanism of UHPP on typical spoilage strain(Shewanella putrefaciens). The results showed that the microbial flora of fresh oysters were dominated by Vibrio,Shewanella and Pseudoalteromonas. However, the proportion of Vibrio decreased gradually during storage, and Shewanella and Pseudoalteromonas were dominant in spoiled oysters. UHPP changed the bacteria community of oysters, and Psychrobacter took an obvious advantage in spoiled oysters, the proportion of both Pseudoalteromonas and Shewanella was less than 1%. The survival rate of Shewanella putrefaciens had no significant changes at 100 MPa pressure treatments,then decreased rapidly with the increase of treatment pressure and no viable bacteria could be detected after 400 MPa processing. The extracellular alkaline phosphatase activity and reducing sugar content changed significantly under relative lower pressure treatment(<100 MPa), however the change magnitude became smaller along with the increase of processing pressure. UHHP caused cell adhesion and clustering. Along with the increasing of pressure, the boundaries of Shewanella putrefaciens cells became blurred, and eventually led to their death.
In order to reveal the effect of ultra-high pressure processing(UHPP) on the spoilage bacteria flora of oysters and its mechanism, high throughput sequencing technology was applied to study the bacterial community structure in fresh and spoiled oysters. Survival rate, extracellular alkaline phosphatase(AKP) activity, extracellular reducing sugar content and electron microscope observation were used to discuss the lethal mechanism of UHPP on typical spoilage strain(Shewanella putrefaciens). The results showed that the microbial flora of fresh oysters were dominated by Vibrio,Shewanella and Pseudoalteromonas. However, the proportion of Vibrio decreased gradually during storage, and Shewanella and Pseudoalteromonas were dominant in spoiled oysters. UHPP changed the bacteria community of oysters, and Psychrobacter took an obvious advantage in spoiled oysters, the proportion of both Pseudoalteromonas and Shewanella was less than 1%. The survival rate of Shewanella putrefaciens had no significant changes at 100 MPa pressure treatments,then decreased rapidly with the increase of treatment pressure and no viable bacteria could be detected after 400 MPa processing. The extracellular alkaline phosphatase activity and reducing sugar content changed significantly under relative lower pressure treatment(<100 MPa), however the change magnitude became smaller along with the increase of processing pressure. UHHP caused cell adhesion and clustering. Along with the increasing of pressure, the boundaries of Shewanella putrefaciens cells became blurred, and eventually led to their death.
引文
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[2] PARK D, JUNG J, JUNG B, et al. Changes in salmon(Oncorhynchus keta)flesh quality following ultra-high pressure treatment and 30 d of chilled storage[J]. Journal of Food Science, 2015, 80(1):142-146.
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[10] CHEN H, GUAN D, HOOVER D G. Sensitivities of foodborne pathogens to pressure changes[J]. Journal of Food Protection, 2006, 69(1):130-136.
[11] ABE F. Dynamic structural changes in microbial membranes in response to high hydrostatic pressure analyzed using time-resolved fluorescence anisotropy measurement[J]. Biophysical Chemistry, 2013, 183:3-8.
[12] MOLINA-GUTIERREZ A, STIPPL V, DELGADO A, et al. In situ determination of the intracellular pH of Lactococcus lactis and Lactobacillus plantarum during pressure treatment[J]. Applied and Environmental Microbiology, 2002, 68(9):4399-4406.
[13]江波,缪铭.高静压加工优化食品酶催化体系:现状与趋势[J].中国食品学报, 2011, 11(9):93-97.
[14] ARQU魪S J L, GARDE S, GAYA P, et al. Short communication:inactivation of microbial contaminants in raw milk La Serena cheese by high-pressure treatments[J]. Journal of Dairy Science, 2006,89(3):888-891.
[15] GANZLE M G, VOGEL R F. On-line fluorescence determination of pressure mediated outer membrane damage in Escherichia coli[J]. Systematic and Applied Microbiology, 2001, 24(4):477-485.
[16] MOHAMED H M H, DIONO B H S, YOUSEF A E. Structural changes in Listeria monocytogenes treated with gamma radiation, pulsed electric field and ultra-high pressure[J]. Journal of Food Safety,2012, 32(1):66-73.
[17] YANG B, SHI Y, XIA X, et al. Inactivation of foodborne pathogens in raw milk using high hydrostatic pressure[J]. Food Control, 2012, 28(2):273-278.
[18] WANG C Y, HUANG H W, HSU C P, et al. Inactivation and morphological damage of Vibrio parahaemolyticus treated with high hydrostatic pressure[J].Food Control, 2013, 32(2):348-353.
[19] SHARMA P, PUROHIT S D. An improved method of DNA isolation from polysaccharide rich leaves of Boswellia serrata Roxb[J]. Indian Journal of Biotechnology, 2012, 11(1):67-71.
[20] AVERSHINA E, FRISLI T, RUDI K. De novo semi-alignment of 16S rRNA gene sequences for deep phylogenetic characterization of next generation sequencing data[J]. Microbes and Environments, 2013,28(2):211-216.
[21] CAPORASO J G, KUCZYNSKI J, STOMBAUGH J,et al. QIIME allows analysis of high-throughput community sequencing data[J]. Nature Methods, 2010,7(5):335-336.
[22] EDGAR R C. UPARSE:highly accurate OTU sequences from microbial amplicon reads[J]. Nature Methods, 2013, 10(10):996-998.
[23] WRIGHT M H, MATTHEWS B, ARNOLD M S J,et al. The prevention of fish spoilage by high antioxidant Australian culinary plants:Shewanella putrefaciens, growth inhibition[J]. International Journal of Food Science&Technology, 2016, 39(3):203-220.
[24] TAMS L, HUTTOVJ, MISTRK I, et al. Effect of carboxymethyl chitin-glucan on the activity of some hydrolytic enzymes in maize plants[J]. Chemical Papers Chemicke Zvesti, 2002, 56(5):326-329.
[25] ZHANG Z, JIANG B, LIAO X, et al. Inactivation of Bacillus subtilis spores by combining high-pressure thermal sterilization and ethanol[J]. International Journal of Food Microbiology, 2012, 160(2):99-104.
[26] MOUSSA M, PERRIER-CORNET J M, GERVAIS P. Damage in Escherichia coli cells treated with a combination of high hydrostatic pressure and subzero temperature[J]. Applied and Environmental Microbiology, 2007, 73(20):6508-6518.
[2] PARK D, JUNG J, JUNG B, et al. Changes in salmon(Oncorhynchus keta)flesh quality following ultra-high pressure treatment and 30 d of chilled storage[J]. Journal of Food Science, 2015, 80(1):142-146.
[3] MARTYNENKO A, ASTATKIE T, SATANINA V,et al. Novel hydrothermodynamic food processing technology[J]. Journal of Food Engineering, 2015,152(5):8-16
[4] HE H, ADAMS R M, FARKAS D F, et al. Use of High-pressure processing for oyster shucking and shelf-life extension[J]. Journal of Food Science,2002, 67(2):640-645.
[5] L魷PEZCABALLERO M E, P魪REZMATEOS M,MONTERO P, et al. Oyster preservation by highpressure treatment[J]. Journal of Food Protection,2000, 63(2):196-201.
[6] CRUZ-ROMERO M, SMIDDY M, HILL C, et al.Effects of high pressure treatment on physicochemical characteristics of fresh oysters(Crassostrea gigas)[J]. Innovative Food Science&Emerging Technologies, 2004, 5(2):161-169.
[7] CRUZ-ROMERO M, KELLY A L, KERRY J P.Effects of high-pressure and heat treatments on physical and biochemical characteristics of oysters(Crassostrea gigas)[J]. Innovative Food Science&Emerging Technologies, 2007, 8(1):30-38.
[8] CRUZ-ROMERO M, KERRY J P, KELLY A L.Changes in the microbiological and physicochemical quality of high-pressure-treated oysters(Crassostrea gigas)during chilled storage[J]. Food Control, 2008,19(12):1139-1147.
[9] LINGHAM T, YE M, CHEN H, et al. Effects of high hydrostatic pressure on the physical, microbial,and chemical attributes of oysters(Crassostrea virginica)[J]. Journal of Food Science, 2016, 81(5):1158-1166.
[10] CHEN H, GUAN D, HOOVER D G. Sensitivities of foodborne pathogens to pressure changes[J]. Journal of Food Protection, 2006, 69(1):130-136.
[11] ABE F. Dynamic structural changes in microbial membranes in response to high hydrostatic pressure analyzed using time-resolved fluorescence anisotropy measurement[J]. Biophysical Chemistry, 2013, 183:3-8.
[12] MOLINA-GUTIERREZ A, STIPPL V, DELGADO A, et al. In situ determination of the intracellular pH of Lactococcus lactis and Lactobacillus plantarum during pressure treatment[J]. Applied and Environmental Microbiology, 2002, 68(9):4399-4406.
[13]江波,缪铭.高静压加工优化食品酶催化体系:现状与趋势[J].中国食品学报, 2011, 11(9):93-97.
[14] ARQU魪S J L, GARDE S, GAYA P, et al. Short communication:inactivation of microbial contaminants in raw milk La Serena cheese by high-pressure treatments[J]. Journal of Dairy Science, 2006,89(3):888-891.
[15] GANZLE M G, VOGEL R F. On-line fluorescence determination of pressure mediated outer membrane damage in Escherichia coli[J]. Systematic and Applied Microbiology, 2001, 24(4):477-485.
[16] MOHAMED H M H, DIONO B H S, YOUSEF A E. Structural changes in Listeria monocytogenes treated with gamma radiation, pulsed electric field and ultra-high pressure[J]. Journal of Food Safety,2012, 32(1):66-73.
[17] YANG B, SHI Y, XIA X, et al. Inactivation of foodborne pathogens in raw milk using high hydrostatic pressure[J]. Food Control, 2012, 28(2):273-278.
[18] WANG C Y, HUANG H W, HSU C P, et al. Inactivation and morphological damage of Vibrio parahaemolyticus treated with high hydrostatic pressure[J].Food Control, 2013, 32(2):348-353.
[19] SHARMA P, PUROHIT S D. An improved method of DNA isolation from polysaccharide rich leaves of Boswellia serrata Roxb[J]. Indian Journal of Biotechnology, 2012, 11(1):67-71.
[20] AVERSHINA E, FRISLI T, RUDI K. De novo semi-alignment of 16S rRNA gene sequences for deep phylogenetic characterization of next generation sequencing data[J]. Microbes and Environments, 2013,28(2):211-216.
[21] CAPORASO J G, KUCZYNSKI J, STOMBAUGH J,et al. QIIME allows analysis of high-throughput community sequencing data[J]. Nature Methods, 2010,7(5):335-336.
[22] EDGAR R C. UPARSE:highly accurate OTU sequences from microbial amplicon reads[J]. Nature Methods, 2013, 10(10):996-998.
[23] WRIGHT M H, MATTHEWS B, ARNOLD M S J,et al. The prevention of fish spoilage by high antioxidant Australian culinary plants:Shewanella putrefaciens, growth inhibition[J]. International Journal of Food Science&Technology, 2016, 39(3):203-220.
[24] TAMS L, HUTTOVJ, MISTRK I, et al. Effect of carboxymethyl chitin-glucan on the activity of some hydrolytic enzymes in maize plants[J]. Chemical Papers Chemicke Zvesti, 2002, 56(5):326-329.
[25] ZHANG Z, JIANG B, LIAO X, et al. Inactivation of Bacillus subtilis spores by combining high-pressure thermal sterilization and ethanol[J]. International Journal of Food Microbiology, 2012, 160(2):99-104.
[26] MOUSSA M, PERRIER-CORNET J M, GERVAIS P. Damage in Escherichia coli cells treated with a combination of high hydrostatic pressure and subzero temperature[J]. Applied and Environmental Microbiology, 2007, 73(20):6508-6518.