| Beef was the second largest meat consumption in China.In 2017,domestic consumption reached 7.89 million tons,which has a good industry prospect.Chilled beef is more effective on maintaining meat quality and safety.Chilled beef has gradually been the consuming mainstream.Beef spoilage was associated with enzymatic catalysis and microbial activity.Only few of these bacteria as specific spoilage organisms(SSO)led to meat spoilage.SSOs produced off flavours,off odour metabolites,such as aldehydes,ketones,amines,organic acids,causing the sensory spoilage characteristics of meat.To assure the food safety and to extend the shelf life of food products,application of plant extracts into food products have been widely reported.Cinnamaldehyde was recognized as Generally Recognized as Safe(GRAS)by UnitedStates Food and Drug Administration.Trans-cinnamaldehyde(TC)and cinnamaldehyde(CD)have been shown to antibacterial and antibiofilm activity against both Gram-positive and Gram-negative bacteria.Various studies focused on microbial quality of beef and inhibition of food-borne pathogenic bacteria,however,few study investigated the spoilage potential by SSOs and control their spoilage ability.In this study,the shelf life of beef tenderloin and development of microbial communities in beef were analyzed.Sensory,physical and chemical parameters in sterile beef extract and beef inoculated with the dominant spoilage bacteria were assessed to spoilage potential.The biofilm formation of dominant spoilage bacteria were comparatively characterized.Finally,the inhibitory activity of TC and CD on spoilage and biofilm formation of the SSO PS28 was investigated.The main results are as follows:Chilled beef had good sensory quality in the beginning storage,and appeared rotten smell for 18d at 0 ?.The TVB-N and TBA exceeded the upper limit at 15d and 18d,respectively.Total viable counts(TVC)of chilled beef rapidly increased at the initial 3d,and increased to 8.73 log cfu/g at 15 d,then keep steady.The TVC of the beef was 6.75 log CFU/g on the 9d,which exceeded the value of 6 log CFU/g as the upper limit for fresh beef.Growth of bacteria of Pseudomonas,Brochothrix thermosphacta,Enterobacter and Lactic acid bacteria(LAB)exhibited similar trend with TVC,in which Pseudomonas increased the fastest,and Enterobacter and LAB grew the slowest.chemical parameters and TVC had a significant positive correlation with sensory score.Two identification of microbial diversity indicated that the initial microbial composition of beef was complex.Predominant bacteria of fresh beef were Brochothrix thermosphacta,Aeromonas and Acinetobacter for cultural methods,while Psychrobacter,Pseudomonas and Lactobacillus for high-throughput sequencing technique.Pseudomonas and B.thermosphacta were the dominant spoilage bacteria in spoiled beef stored at 0?.By sequencing gyrb gene of Pseudomonas,the majority of Pseudomonas were identified as P.fragi(50%),P.lundensis(30%)and P.fluorescen(20%).Spoilage potential and biofilm formation of the dominant spoilage bacteria were comparatively characterized.The spoilage potential of 15 isolates was measured in sterilizedbeef juice and sterilized beef.The biofilm forming ability and adhesion of these isolates were measured by using crystal violet staining,phenol sulfuric acid assay and bead vortexing method.The results showed that three isolates,PS 16,PS17 and PS28,had large decomposition zone diameters(DZDs)among the dominant spoilage bacteria.Fifteen isolates exhibited similar growth,and Hafnia alvei was highest cell density at 28? and 0?,and Pseudomonas grew rapidly at 0?.Pseudomonas and H.alvei exhibited higher sensory score,pH value and TVB-N inoculated in beef juice,revealing the stronger spoilage ability of two species compared to B.thermosphacta.Moreover,Pseudomonas PS26,PS20,PS 16,PS27,PS28,PS29 and PS30 presented high protease activity decomposing marcoplasmic and myofibrillar protein.Pseudomonas had higher biofilm biomass and adhesion than H.alvei,whereas B.thermosphact almost had no adhesion and biofilm ability.In addition,the biofilm formed by PS28,PS27 and PS 16 had the strongest biofilm biomass,production of extracellular polysaccharides and adhesion at 28 and 0 ?.Among the 15 spoilage bacteria,the largest swimming zones was observed in PS 17,PS27 and PS28,indicating that three Pseudomonas had the strongest mobility.Pseudomonas formed slowly biofilm 0 ?,however,the maturing biofilm was 2.88?10.99 fold at 0? than those at 28 ?,indicating the effect of temperature on biofilm.Additionally,PS28 had higher sensory score and TVB-N in sterilized beef than PS 16 and PS 17.Flavor compounds of sterile beef inoculated with PS28 was ethyl acetate.Thus,PS28 with the strongest spoilage ability and biofilm formation,was identified as a SSO,which was as a representative strain for subsequent studies.Effect of cinnamaldehyde on the spoilage and biofilm formation of P.lundensis PS28 was further assessed at 28?.TC and CD exhibited the inhibition of growth aganist P.lundensis,and the minimal inhibitory concentrations(MIC)of TC and CD were 200 and 225 ?g/mL,respectively.TC and CD decreased(P<0.05)P.lundensis PS28 biofilm formation on well plates and stainless steel coupons.Compared to the control,the biofilm of PS28 by crystal violet staining method decreased by 32.88%,47.95%and 60.27%,and by 26.03%,41.10%and 52.05%,when supplemented with exogenous 1/8?1/4 and 1/2 MIC TC or CD,respectively.The 1/8?1/4 and 1/2 MIC of TC decreased the bacterial adhesion in stainless steel coupons by 30.82%,39.74%and 56.35%,and only 1/2 MIC of CD dropped by 61.10%.CLSM observation revealed that biofilm thickness in the PS28 was 40.67?m,and there were dense uniform layers of live cells on the coverslip surface.The biofilm thickness of PS28 adding with 1/8?1/4 and 1/2 MIC of TC were 25.33?m,17.67?m and 10.50?m,and the biofilm thickness of PS28 with three sub-MIC concentration of CD were 25.52?m,18.67?m and 14.00?m,respectively.Furthermore,the biofilm exposed to aldehyde treatments resulted in dead cells(stained red by PI dye)in biofilm.The swimming moility was strongly inhibited by cinnamaldehyde,and the inhibitory rates were 58.08%and 42.29%by 1/2 MIC of TC and CD,respectively.The addition of TC repress significantly biofilm formation(P<0.05),whereas weaker inhibitory effect was found by the addition of CD.Quantification of siderophore production showed that the inhibiting ratewas in the range of 36.84%?73.68%for TC and 10.52%?63.16%(CD),respectively.Furthermore,protease activity of P.lundensis decreased in the range of 11.90%?61.90%and 28.60%?76.19%,while lipolytic activity inhibited in the range of 12.83%?40.17%and 18.80%-47.01%in the presence of sub-MIC TC and CD,respectively,indicating that sub-MIC concentrations of TC and CD markedly repressed the protease and lipolytic activity.The inhibitory effect of CD on extracellular enzyme of P.lundensis was slightly stronger than TC.RT-PCR data revealed that great down-regulation of lapA,fliC,pvdH,aprX and lip were observed in PS28 treated with TC and CD(p<0.05),except no significant change in lapA expression when treated with 1/8 MIC of CD.The expressions of lapA,fliC,pvdH,aprX and lip decreased to 0.05,0.11,0.11,0.16,0.10 fold and 0.02,0.12,0.03,0.10,0.04 fold in P.lundensistreated treated with 1/2 MIC of TC and CD(P<0.05),respectively.Thus,the shelf-life of chilled beef was about 12d,and Pseudomonas and B.thermosphacta were the dominant spoilage bacteria in chilled beef,in which Pseudomonas lundensis PS28 was the SSO and high biofilm forming ability.TC or CD at the sub-MIC concentrationinhibited significantly extracellular enzyme and biofilm of P.lundensis.Two cinnamaldehyde down-regulated critical biofilm and spoilage associated genes in P.lundensis. |
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