LC-MS-based metabolomics reveals metabolite dynamic changes of beef after superchilling early post-mortem.

To explore the underlying mechanisms by which superchilling (SC, -3 °C within 5 h of slaughter) improves beef tenderness, an untargeted metabolomics strategy was employed. M. Longissimus lumborum (LL) muscles from twelve beef carcasses were assigned to either SC or very fast chilling (VFC, 0 °C within 5 h of slaughter) treatments, with conventional chilling (CC, 0 âˆ¼ 4 °C until 24 h post-mortem) serving as the control (6 per group). Biochemical properties and metabolites were investigated during the early post-mortem period. The results showed that the degradation of µ-calpain and caspase 3 occurred earlier in SC treated sample, which might be attributed to the accelerated accumulation of free Ca2+. The metabolomic profiles of samples from the SC and CC treatments were clearly distinguished based on partial least squares-discriminant analysis (PLS-DA) at each time point. It is noteworthy that more IMP and 4-hydroxyproline were found in the comparison between SC and CC treatments. According to the results of metabolic pathways analysis and the correlation analysis between traits related to tenderness and metabolites with significant differences (SC vs. CC), it can be suggested that the tenderization effect of the SC treatment may be related to the alteration of arginine and proline metabolism, and purine metabolism in the early post-mortem phase.

The Effect of the PhoP/PhoQ System on the Regulation of Multi-Stress Adaptation Induced by Acid Stress in Salmonella Typhimurium.

Acidic stress in beef cattle slaughtering abattoirs can induce the acid adaptation response of in-plant contaminated Salmonella. This may further lead to multiple resistance responses threatening public health. Therefore, the acid, heat, osmotic and antibiotic resistances of Salmonella typhimurium (ATCC14028) were evaluated after a 90 min adaption in a pH = 5.4 "mild acid" Luria-Bertani medium. Differences in such resistances were also determined between the ∆phoP mutant and wild-type Salmonella strains to confirm the contribution of the PhoP/PhoQ system. The transcriptomic differences between the acid-adapted and ∆phoP strain were compared to explore the role of the PhoP/Q two-component system in regulating multi-stress resistance. Acid adaptation was found to increase the viability of Salmonella to lethal acid, heat and hyperosmotic treatments. In particular, acid adaptation significantly increased the resistance of Salmonella typhimurium to Polymyxin B, and such resistance can last for 21 days when the adapted strain was stored in meat extract medium at 4 °C. Transcriptomics analysis revealed 178 up-regulated and 274 down-regulated genes in the ∆phoP strain. The Salmonella infection, cationic antimicrobial peptide (CAMP) resistance, quorum sensing and two-component system pathways were down-regulated, while the bacterial tricarboxylic acid cycle pathways were up-regulated. Transcriptomics and RT-qPCR analyses revealed that the deletion of the phoP gene resulted in the down-regulation of the expression of genes related to lipid A modification and efflux pumps. These changes in the gene expression result in the change in net negative charge and the mobility of the cell membrane, resulting in enhanced CAMP resistance. The confirmation of multiple stress resistance under acid adaptation and the transcriptomic study in the current study may provide valuable information for the control of multiple stress resistance and meat safety.

Effects and mechanism of sub-freezing storage on water holding capacity and tenderness of beef.

In order to explore the effect of sub-freezing storage on water holding capacity and tenderness of beef, four treatments were compared in this study: sub-freezing (-7 °C) fast sub-freezing (-38 °C until the core temperature achieved to -7 °C), superchilling (-1 °C) and fast frozen (-38 °C until the core temperature achieved to -18 °C) with the latter two treatments serving as the controls. The differences in muscle fiber structure, water distribution, protein oxidation and cytoskeletal protein degradation were studied. The results demonstrated that compared with other treatments, the fast sub-freezing treatment resulted in less structural damage to the muscle fibers and had better water holding capacity. Both sub-freezing and fast sub-freezing treatments inhibited protein oxidation compared with superchilling, but the former treatment's level of protein oxidation was higher than that in fast sub-freezing treatment during long-term storage (42 weeks). In addition, the structural proteins in the sub-freezing and fast sub-freezing treatments underwent faster degradation during long-term storage and therefore the meat was more tender compared with the fast frozen treatment. The results indicate that the fast sub-freezing treatment can be potentially applied in beef storage.

Photochemical Transformation of Dissolved Organic Matter in Surface Water Augmented the Formation of Disinfection Byproducts.

Sunlight may lead to changes in disinfection byproducts (DBPs) formation potentials of source water via transforming dissolved organic matter (DOM); however, the underlying mechanisms behind these changes remain unclear. This work systematically investigated the effect of photochemical transformation of DOM from reservoir water (DOMRe) and micropolluted river water (DOMRi) after 36 h of simulated sunlight irradiation (equivalent to one month under natural sunlight) on DBPs formation. Upon irradiation, high molecular weight (MW) and aromatic molecules tended to be mineralized or converted into low-MW and highly oxidized (O/C > 0.5) ones which might react with chlorine to generate high levels of DBPs, resulting in an elevation in the yields (µg DBP/mg C) of almost all the measured DBPs and the quantities of unknown DBPs in both DOM samples after chlorination. Additionally, DOMRi contained more aromatic molecules susceptible to photooxidation than DOMRe. Consequently, irradiated DOMRi exhibited a greater increase in the formation potentials of haloacetonitriles, halonitromethanes, and specific regulated DBPs, with nitrogenous DBPs being responsible for the overall rise in the calculated cytotoxicity following chlorination. This work emphasized the importance of a comprehensive removal of phototransformation products that may serve as DBPs precursors from source waters, especially from micropolluted source waters.

Effects of CO2 on the physicochemical, microbial, and sensory properties of pork patties packaged under optimized O2 levels.

The storage quality characteristics of fresh pork patties were investigated under 80% O2 modified atmosphere packaging (MAP80:20 = 80% O2/20% CO2) and 40% O2 MAP with various CO2 levels (MAP40:20 = 40% O2/20% CO2/40% N2; MAP40:40 = 40% O2/40% CO2/20% N2; MAP40:60 = 40% O2/60% CO2). Packaged patties were stored for 16 days at 4 °C to monitor their physicochemical (pH, instrumental color, oxidative stability, and fatty acid profile), microbial, and sensorial changes. Results suggested that decreasing O2 levels from 80% to 40% significantly inhibited the lipid oxidation of patties but led to a lower (P < 0.05) color stability. Elevating CO2 levels from 20% to 60% in combination with 40% O2 significantly suppressed bacterial growth and total volatile basic nitrogen production, and thus rendered patties with a better sensory quality and a similar meat color to 80% O2. However, increased CO2 levels promoted lipid oxidation through reducing the antioxidant capacity of patties, which was attributed to a CO2-induced reduction in superoxide dismutase and glutathione peroxidase activities during storage rather than a pH reduction or changes in fatty acid composition. Overall, 40% O2/40% CO2/20% N2 is a realistic alternative for pork patties to improve meat quality and extend the shelf-life to over 16 days.

Study of the transfer of Shiga toxin-producing Escherichia coli during the slaughter of cattle using molecular typing combined with epidemiologic data.

Investigation on the distribution and biological characteristics of Shiga-toxin producing Escherichia coli (STEC) during beef processing is essential for in-plant critical control points and food safety risk assessment. Serogroups and subtypes of stx genes of STEC strains isolated from beef processing lines were first investigated. Identification to cross-contamination among different sampling sites was further conducted by combining multilocus sequence typing (MLST) with the previous distribution and characterization data. The PCR-positive rate for STEC in 435 samples from two slaughter plants in China was 14.3% and the isolation rate for the 62 PCR positive and the entire set of 435 samples were 26% and 3.68% respectively. The existence of serotype O157:H7 (33%) and serogroups O121 (42%) and O26 (21%) as well as the high detection rate of high pathogenic gene stx2a (68%) in these serogroups indicated potential risk to the safety of beef. Traceability analysis showed that hide plays a critical role in cross-contamination between feces, lairage pens and post-washing carcasses from a molecular perspective. Intervening measures revolves around de-hiding should be involved in the in-plant safety control policy according to the tracing analysis.

Effects of different protein phosphorylation levels on the tenderness of different ultimate pH beef.

This study investigated the relationship between tenderness and protein phosphorylation levels of normal ultimate pH (pHu, 5.4-5.8, NpHu), intermediate pHu (5.8-6.2, IpHu) and high pHu (≥6.2, HpHu) Longissimus lumborum from beef. During 21 d of ageing, the HpHu group had the lowest Warner-Bratzler shear force (WBSF) values, while the IpHu group showed the highest and even after 21 days of ageing still had high levels. In the late stage of the 24 h post-mortem period the faster degradation rate of troponin T and earlier activation of caspase 9 in the HpHu group were the key reasons for the lower WBSF compared with the NpHu and IpHu groups. The activity of caspase 3 cannot explain the tenderness differences between IpHu and HpHu groups, since their activities did not show any difference. At 24 h post-mortem, 17 common differential phosphorylated peptides were detected among pHu groups, of which nine were associated with pHu and WBSF. The higher phosphorylation level of glycogen synthase may have caused the delay of meat tenderization in the IpHu group.

Sous vide cooking improved the physicochemical parameters of hot-boned bovine semimembranosus muscles.

The physicochemical parameters of hot-boned bovine semimembranosus muscles after sous vide cooking were investigated. Hot-boned or wet-aged steaks were collected, and cooked by different cooking methods, including sous vide (57 °C, 11 h, SV), grilling (at 200 °C to the central temperature of 72 °C, GR) or boiling (100 °C, 2 h, BO). The meat color, tenderness, water-holding capacity, degree of oxidation, myoglobin denaturation and sensory quality traits were determined, as well as the changes in the microstructure. Compared to other cooking methods, SV reduced the degree of oxidation and muscle shortening, and significantly improved the water holding capacity (WHC), tenderness, connective tissue content and overall acceptability for both hot-boned and wet-aged steaks. The oxidation and muscle shortening were reduced in hot-boned SV steaks (P < 0.05), and the water-holding capacity and sensory scores for juiciness, connective tissue content and overall acceptability were increased (P < 0.05) compared to the wet-aged steaks. The combination of hot-boning and SV cooking resulted in an acceptable tenderness, better overall sensory acceptability and higher WHC than other combinations of muscle states and cooking methods. Therefore, it is a good choice to cook hot-boned semimembranosus muscles using SV to improve the eating quality, which can eliminate the need for aging, benefiting the beef industry.

Effects of dissolved organic matter removal and molecular transformation in different water treatment processes on formation of disinfection byproducts.

Alterations in molecular composition of dissolved organic matter (DOM) during water treatments can influence the composition and toxicity of disinfection by-products (DBPs) in subsequent chlorination disinfection process. In this study, the impacts of DOM composition after various water treatment techniques (coagulation, adsorption, nanofiltration, biological aerated filter (BAF), and their integrated processes) on the generation mechanisms of DBPs were comprehensively explored by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) in combination with GC-MS and LC-MS analysis. The results indicated that coagulation preferentially removed unsaturated (low H/C) and oxidized (high O/C) compounds, whereas adsorption was prone to remove the reduced (low O/C) component that was more reactive with chlorine, leading to lower yields (µg DBP/mg DOC) of trihalomethanes (THMs) and haloacetic acids (HAAs) during subsequent chlorination. The coagulation-adsorption technique exhibited a relatively high removal of both known and unknown DBPs, demonstrating that coagulation and adsorption were complementary for DOM removal at the molecular level. Nanofiltration selectively removed molecules with relatively high O/C, however, those with very low O/C that were more reactive with chlorine could pass through the nanofiltration membrane, resulting in the highest yields of THMs and HAAs. Although BAF was inefficient in removing DBPs precursors, it could convert molecules with low degree of oxidation and unsaturation into highly oxidized and unsaturated ones, thereby significantly enhancing the removal of DBPs precursors in the subsequent coagulation-adsorption process. These findings are instrumental in developing and selecting more effective techniques to minimize the formation of DBPs in water treatment.

Effect of resveratrol on skeletal slow-twitch muscle fiber expression via AMPK/PGC-1α signaling pathway in bovine myotubes.

The purpose of this study was to evaluate the impact of resveratrol on slow-twitch muscle fiber expression in bovine myotubes. The results revealed that resveratrol enhanced slow myosin heavy chain (MyHC) and suppressed fast MyHC protein expression, accompanied by increased MyHC I/IIa and decreased MyHC IIx/IIb mRNA levels in bovine myotubes (P < 0.05). Resveratrol also enhanced the activities of succinic dehydrogenase (SDH), malate dehydrogenase (MDH) and the mitochondrial DNA (mtDNA) content, but reduced lactate dehydrogenase (LDH) activity (P < 0.05). Meanwhile, the protein and gene expression of AMPK, SIRT1 and PGC-1α were upregulated by resveratrol (P < 0.05). Furthermore, PGC-1α inhibitor SR-18292 could attenuate resveratrol-induced muscle fiber conversion from fast-twitch to slow-twitch. These results suggest that resveratrol might promote muscle fiber type transition from fast-twitch to slow-twitch through the AMPK/PGC-1α signaling pathway and mitochondrial biogenesis in bovine myotubes.

Effects of dietary resveratrol supplementation in cattle on the anti-oxidative capacity and meat quality of beef steaks under high­oxygen packaging.

The effects of dietary resveratrol supplementation on beef quality and antioxidant capacity under high­oxygen packaging were studied. Twelve cattle were selected and fed a total mixed ration (Control, CON) or supplemented with resveratrol (5 g/cattle/day, RES) for 120 days. The antioxidant capacity and meat quality of beef under high­oxygen modified atmosphere packaging (HiOx-MAP, 80%O2/20%CO2) and overwrap packaging (OW) were evaluated during storage. Compared to the CON, RES enhanced antioxidant enzyme activity in serum and muscle, and increased the expression of Nrf2 and its downstream target genes (P < 0.05), which decreased the lipid and protein oxidation of steaks during storage (P < 0.05). The RES resulted in a* values increasing throughout storage (P < 0.05) and lower MetMb% than CON steaks (P < 0.05) in HiOx-MAP. The water-holding capacity (WHC) was improved and Warner-Bratzler shear force (WBSF) was reduced (P < 0.05) in RES steaks during storage. Thus dietary resveratrol increased beef antioxidant capacity under HiOx-MAP and improved meat quality, and can be used as a potential method to elevate beef quality and reduce the oxidation under HiOx-MAP.

Potential inhibitory effect of carbon dioxide on the spoilage behaviors of Pseudomonas fragi in high-oxygen packaged beef during refrigerated storage.

Pseudomonas fragi is a dominant meat spoilage organism under high-oxygen modified atmosphere packaging (HiOx-MAP). This work investigated the effects of CO2 on P. fragi growth and the related spoilage phenomena of HiOx-MAP beef. Minced beef incubated with P. fragi T1, a strain owning the strongest spoilage potential among isolates, was stored under CO2-enriched HiOx-MAP (TMAP; 50% O2/40% CO2/10% N2) or non-CO2 HiOx-MAP (CMAP; 50% O2/50% N2) at 4 °C for 14 days. Compared to CMAP, TMAP maintained sufficient O2 levels to endow beef with higher a* values and meat color stability due to lower P. fragi counts from day 1 (P < 0.05). TMAP samples also showed lower (P < 0.05) lipase activity and protease activity within 14-days and 6-days than CMAP samples respectively. TMAP delayed the significantly increased pH and total volatile basic nitrogen contents occurred in CMAP beef during storage. Despite TMAP markedly promoted the lipid oxidation associated with higher concentrations of hexanal and 2,3-octanedione than CMAP (P < 0.05), TMAP beef retained an acceptable organoleptic odor due to a CO2-inhibition on the microbial-induced 2,3-butanedione and ethyl 2-butenoate formation. This study provided a comprehensive insight into the antibacterial mechanism of CO2 on P. fragi in HiOx-MAP beef.

The biological effect of a beef-derived Latilactobacillus sakei on beef steaks during chilled storage.

The aim of this study was to investigate the biological inhibiting effect of a beef-derived Latilactobacillus sakei (RS-25) on the spoilage of beef steaks in overwrapped packaging during the 12 days of storage at 4°C. Beef quality as well as microbial indicators were determined at different intervals during the storage after the inoculation of RS-25 at the 6 log CFU/g, and the high-throughput sequencing was applied to investigate the changes of microbial community structure during the storage. The inoculation of RS-25 on beef had no effect (p > .05) on pH, TBARS, and TVB-N during storage indicating the weak effect of such strain on the eat quality. Furthermore, the rise of L* and the delayed decline of a* and b* reveal the protection effect of RS-25 on the meat color. RS-25 reduced the re-contaminated Salmonella typhimurium by 1.16 log CFU/g (p < .01), and the growth of Brochothrix thermosphacta was also inhibited but no inhibition was found on the Pseudomonas spp. at the first 6 days of storage. The inhibiting effect of RS-25 was covered by the rapid growth of other microorganism during the following 6 days of storage. Consistent with the microbial counts results, high-throughput sequencing analysis confirmed that the inoculated L. sakei RS-25 was dominant at first 6 days, and then replaced by Pseudomonas spp. The findings obtained from the current study may provide basic information for the further application of bioprotective bacteria in preservation of beef steaks in the overwrapped packaging.

The retail color characteristics of vacuum-packaged beef m. longissimus lumborum following long-term superchilled storage.

This study investigated whether beef m. longissimus lumborum (LL) can be merchandised under retail conditions, following long-term superchilled storage (-1 °C). At 24 h post-mortem, the LL from left side of beef carcasses (n = 5) were fabricated into vacuum packaged beef thick-cuts, and then stored for 0, 2, 4, 8, 12, 16, or 20 weeks under superchilled conditions (-1 °C). Following storage, beef cuts were fabricated into steaks and aerobically displayed (0- 4 °C) for 5 days. Instrumental color, percentage of myoglobin redox forms, metmyoglobin reducing activity, oxygen consumption, and lipid oxidation were evaluated. After 4 weeks, the steaks had the highest a*, b* and chroma values between 1 and 3 days of display. Longer superchilled storage resulted in a rapid increase in discoloration and lipid oxidation which were observed in samples during display. Specifically, the a* values of steaks superchilled for 16 and 20 weeks approached the unacceptability threshold (a* ≥ 14.5) after 3 days of display.

Exploratory study on the potential regulating role of Peroxiredoxin 6 on proteolysis and relationships with desmin early postmortem.

The objective of the present study was to explore the effect of Peroxiredoxin 6 (Prdx 6) on beef tenderization during the early postmortem period. The longissimus lumborum (LL) were obtained at 45 min postmortem from 6 beef carcasses and then incubated with or without the inhibitor of Prdx6 (NSC348884) for different times, followed by incubation with or without the H2O2 (simulation of oxidative stress). The expression of Prdx6, proteolysis indicated by desmin degradation, cell apoptosis rate and expression of caspases were measured. The results indicated that the inhibitor significantly reduced the Prdx6 level, while the cells adaptively increased Prdx6 expression to resist the oxidative stress caused by H2O2. Moreover, the samples in which Prdx6 was inhibited demonstrated more severe desmin degradation accompanied by a higher apoptosis rate which was induced by the increase in caspase degradation as well as the ratio of Bax/Bcl-2. These results demonstrated that inhibiting Prdx6 could promote cell apoptosis and further accelerate beef tenderization.

Effect of dietary resveratrol supplementation on muscle fiber types and meat quality in beef cattle.

In order to investigate the effect of dietary resveratrol supplementation on muscle fiber types and meat quality in beef cattle, a feeding experiment was undertaken. Longissimus lumborum, Psoas major and Semitendinosus muscles were collected 24 h post-mortem from two groups of cattle, which were fed with a total mixed ration (Control - CON) or supplemented with resveratrol (5 g/animal/day, RES) for 120 d before slaughter. The results showed that dietary resveratrol increased the gene expression of MyHC I and enhanced the proportion of type I fibers in three muscles. The cooking loss and Warner-Bratzler shear force of all muscles during aging for 21 days were decreased. However, the increased proportion of type I fibers resulted in a darker initial color, but did improve color stability, as the a* value of RES samples was lower initially but higher in the later stage of aging. This study indicates the supplementation potential of resveratrol for beef cattle for tenderness and color stability.

The underlying mechanisms of the effect of superchilling on the tenderness of beef Longissimus lumborum.

This study investigated the effect of superchilling (-30 °C until the core temperature achieved -3 °C, then stored at -1 °C until 24 h, SC) on the tenderness of hot boned beef M. longissimus lumborum (LL), with very fast chilling (-30 °C until the core temperature achieved 0 °C, then stored at -1 °C until 24 h, VFC) and conventional chilling (0- 4 °C for 24 h, CC) as the controls. The lowest initial shear force values were obtained in SC samples compared to those from the VFC and CC treatments (P < 0.05). Clear freezing damage of muscle fibers and more myofibril fragmentation were found in SC samples compared with the other samples early post-mortem. Moreover, SC samples showed the highest level of inosine 5-monophosphate at 3 h post-mortem (P < 0.05). A reduced glycolysis rate (as evidenced by lactate content) was also found in SC treated samples suggesting little contribution of glycolysis on the tenderization of SC.

Effect of the combination of superchilling and super-chilled storage on shelf-life and bacterial community dynamics of beef during long-term storage.

This study investigated the effect of superchilling (-30 °C until the core temperature achieved -3 °C, and - 1 °C until 24 h, SC) on shelf-life and bacterial community dynamics of beef loins, with a typical very fast chilling (-30 °C until the core temperature achieved 0 °C, and - 1 °C until 24 h, VFC) and conventional chilling (0- 4 °C for 24 h, CC) as controls. The super-chilled storage (-1 °C) was adopted after each chilling procedure, and physicochemical traits and microbiological quality were evaluated during a long-term storage. No remarkable adverse impact on meat color and lipid oxidation were observed in SC treatment. The bacterial composition results showed that Carnobacterium spp. were the main bacteria in SC treatment in the late storage period (63- 84 days). The loss of Lactobacillus spp., due to the "ultra-low temperature" during the superchilling, might be the reason that the SC did not result in a longer shelf-life compared with CC samples.

Molecular transformation of dissolved organic matter and the formation of disinfection byproducts in full-scale surface water treatment processes.

Dissolved organic matters (DOM) have important effects on the performance of surface water treatment processes and may convert into disinfection by-products (DBPs) during disinfection. In this work, the transformation of DOM and the chlorinated DBPs (Cl-DBPs) formation in two different full-scale surface water treatment processes (process 1: prechlorination-coagulation-precipitation-filtration; process 2: coagulation-precipitation-post-disinfection-filtration) were comparatively investigated at molecular scale. The results showed that coagulation preferentially removed unsaturated (H/C < 1.0 and DBE > 17) and oxidized (O/C > 0.5) compounds containing more carboxyl groups. Therefore, prechlorination produced more Cl-DBPs with H/C < 1.0 and O/C > 0.5 than post-disinfection. However, the algal in the influent produced many reduced molecules (O/C < 0.5) without prechlorination, and these compounds were more reactive with disinfectants. Sand filtration was ineffective in DOM removal, while microorganisms in the filter produced high molecular weight (MW) substances that were involved in the Cl-DBPs formation, causing the generation of higher MW Cl-DBPs under post-disinfection. Furthermore, the CHO molecules with high O atom number and the CHON molecules containing one N atom were the main Cl-DBPs precursors in both surface water treatment processes. In consideration of the putative Cl-DBPs precursors and their reaction pathways, the precursors with higher unsaturation degree and aromaticity were prone to produce Cl-DBPs through addition reactions, while that with higher saturation degree tended to form Cl-DBPs through substitution reactions. These findings are useful to optimize the treatment processes to ensure the safety of water quality.

Investigation of the relationship between microbiota dynamics and volatile changes in chilled beef steaks held under high-oxygen packaging enriched in carbon dioxide.

Carbon dioxide (CO2) plays an important bacteriostatic role in high-oxygen modified atmosphere packaging (HiOx-MAP). This study aimed to explore the influence of CO2 on the volatile organic compounds (VOCs) profile in HiOx-MAP beef steaks and assess the changes in VOCs associated with microbiota dynamics during 20-days of chilled storage. Compared to control MAP (CMAP: 50% O2/50% N2), CO2-enriched MAP (TMAP: 50% O2/40% CO2/10% N2) rendered steaks with desirable appearance and odor freshness during storage through inhibiting bacterial growth and reducing the accumulation of acetoin, 2-ethyl-1-hexanol, 1,3-butanediol, diacetyl and 2-heptanone (P < 0.05). 1-Octen-3-ol and 2-ethyl-1-hexanol were main VOCs in TMAP steaks and were positively related to Brochothrix thermosphacta levels. Acetoin and 2-ethyl-1-hexanol could be used as potential spoilage indicators in CMAP steaks, which were positively related to B. thermosphacta and Pseudomonas levels. Overall, the desirable odor freshness of TMAP steaks was mainly attributed to the CO2-inhibitory effect on the off-odor VOCs production correlated with B. thermosphacta, Pseudomonas and Serratia growth.