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Evaluation of the spoilage potential of bacteria isolated from chilled chicken in vitro and in situ
Food Microbiology 63 (2017) 139e146
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Evaluation of the spoilage potential of bacteria isolated from chilled chicken in vitro and in situ Guang-yu Wang a, Hu-hu Wang a, Yi-wei Han b, Tong Xing b, Ke-ping Ye a, Xing-lian Xu a, *, Guang-hong Zhou a a
Key Laboratory of Meat Processing and Quality Control, Ministry of Education, Nanjing Agricultural University, Nanjing, Jiangsu 210095, PR China Collaborative Innovation Center of Food Safety and Nutrition, Ministry of Education and Finance, College of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, PR China
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 March 2016 Received in revised form 16 June 2016 Accepted 17 November 2016
Microorganisms play an important role in the spoilage of chilled chicken. In this study, a total of 53 isolates, belonging to 7 species of 3 genera, were isolated using a selective medium based on the capacity to spoil chicken juice. Four isolates, namely Aeromonas salmonicida 35, Pseudomonas fluorescens H5, Pseudomonas fragi H8 and Serratia liquefaciens 17, were further characterized to assess their proteolytic activities in vitro using meat protein extracts and to evaluate their spoilage potential in situ. The in vitro studies showed that A. salmonicida 35 displayed the strongest proteolytic activity against both sarcoplasmic and myofibrillar proteins. However, the major spoilage isolate in situ was P. fragi H8, which exhibited a fast growth rate, slime formation and increased pH and total volatile basic nitrogen (TVBN) on chicken breast fillets. The relative amounts of volatile organic compounds (VOCs) originating from the microorganisms, including alcohols, aldehydes, ketones and several sulfur compounds, increased during storage. In sum, this study demonstrated the characteristics of 4 potential spoilage bacteria on chilled yellow-feather chicken and provides a simple and convenient method to assess spoilage bacteria during quality management. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Chilled chicken Raw-chicken juice agar Spoilage bacteria Sensory analysis Volatile organic compounds
1. Introduction Meat is the most valuable livestock product. From a nutritional point of view, the importance of meat is derived from its highquality protein, which contains all essential amino acids, and its highly bioavailable minerals and vitamins. In particular, chicken is one of the most traded and consumed meats worldwide. The yellow-feather chicken, as a special species in Asia, has a more distinctive flavor than many other commercial broilers (Zhang et al., 2015). However, to reduce the ongoing outbreaks of animal influenza, particularly the H7N9 strain, live poultry markets have currently been restricted in the majority of cities in China. Consumers now buy fresh chilled chicken, which originates from slaughter plants, through shops and supermarkets. Hence, the demand for these products has increased markedly, and safety problems have become a public health concern. The safety issues associated with chilled chicken have been
* Corresponding author. E-mail address: [email protected] (X.-l. Xu). http://dx.doi.org/10.1016/j.fm.2016.11.015 0740-0020/© 2016 Elsevier Ltd. All rights reserved.
based mostly on the presence of toxicant and pathogenic bacteria in food, which may influence public health. It is noteworthy that according to Regulation 178/2002 of the European Parliament and Commission, a foodstuff is regarded as unsafe not only if it is harmful to consumer health but also if it is not fit for human consumption (Nowak et al., 2012). In this sense, spoiled food, which means food with an appearance, taste or flavor leading to its rejection, is also considered unsafe. Chicken meat is prone to deterioration in a short time, even under chilled conditions (Patsias et al., 2008). Microbiological contamination is one of the most important factors contributing to quality loss, resulting in slime, colony formation, compromised food texture, off-flavors and off-odors (Grama et al., 2002; Hyldgaard et al., 2015). Many studies have reported that the specific spoilage organisms (SSOs) in refrigerated poultry are Pseudomonas spp., Enterobacteriaceae, lactic acid bacteria, and Brochotrix thermosphacta (Chaillou et al., 2015; Grama et al., 2002; Meredith et al., 2014). However, few studies have examined microbial organisms with strong catabolic capacities on chicken meat at refrigeration temperatures. These bacteria may destroy the cell
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structure, promoting the outflow of nutrients that can be utilized by other spoilage bacteria. Therefore, it is necessary to characterize these organisms to better understand chilled yellow-feather chicken spoilage. The aims of the present work were to identify potential spoilage bacteria from chilled yellow-feather chicken using selective medium based on the capacity to spoil chicken juice and to assess the spoilage potential of these isolates both in vitro and in situ. Such information may eventually be used to better inform processing and preservation strategies to enhance the quality and shelf-life of chilled chicken. 2. Materials and methods 2.1. Preparation of the selective medium and samples The selective medium was prepared by mixing raw chicken juice (the fresh chicken breasts were homogenized with deionized water and then filtered through two layers of gauze) and 1% agar. Both ingredients were maintained at 45 C before mixing. This rawchicken juice agar (RJA) was subsequently sterilized by irradiation at a dose of 6 KGy (Kazanas, 1968) via the 60Co source at Hangyu (Hangyu Irradiation Technology Co., LTD, Nanjing, China). Yellow-feather chicken meat was collected from slaughterhouses and supermarkets. Immediately after collection, the samples were aseptically transferred to the laboratory in an ice box within 3 h. The samples were then stored at 8 C until spoilage before isolation was performed. 2.2. Isolation and identification of spoilage bacteria Fifty-four surface samples (25 g) from each muscle were aseptically weighed and homogenized in 225 mL sterilized 0.85% NaCl solution. Decimal dilutions were prepared in the same solution, and 0.1 mL of each of the appropriate dilutions was plated on RJA. The plates were then incubated aerobically at 25 C for 48 h. Colonies with a large decomposition zone were selected from each selective medium and subsequently streaked on the same medium twice to obtain a single colony. The isolates were identified based on 16S rRNA gene sequences, which were amplified using the universal primers 27F (50 -AGAGTTTGATCCTGGCTCAG-30 ) and 1492R (50 GGTTACCTTGTTACGACTT-30 ). The PCR products were purified and sequenced by Invitrogen (Invitrogen Biotechnology Co., Ltd, Shanghai, China). The sequences were compared with those in GenBank using the BLAST function, and the closest matches to each clone were determined based on specific probable identities and then confirmed using a VITEK2 automated system (BioMerieux, France). 2.3. Selection of spoilage bacteria via the RJA assay A total of 53 isolates that belonged to different genera or species were identified: Aeromonas spp. (Aeromonas salmonicida, Aeromonas hydrophila and Aeromonas media), Chryseobacterium shigense and Pseudomonas spp. (Pseudomonas fluorescens, Pseudomonas fragi and Pseudomonas putida). All strains were cultured in tryptone soy broth (TSB) for 24 h at 25 C. The cell density was adjusted to an optical density of 600 nm (OD600) of 0.4. Next, four replicate 2-mL aliquots of the isolates were spotted onto RJA, and decomposition was measured after 3 days of incubation at 25 C. The strains Pseudomonas fragi H8, Aeromonas salmonicida 35 and Pseudomonas fluorescens H5 were selected because they provided the three largest decomposition zone diameters (DZDs) among the species described above. The spoilage potentials of these strains were compared with that of a well-known spoilage
bacterium, Serratia liquefaciens 17, which was isolated previously (data not shown). 2.4. In vitro proteolytic and lipolytic assays The proteolytic activities of the chicken sarcoplasmic and myofibrillar proteins were estimated based on SDS-PAGE analysis. In brief, each isolate was incubated in TSB. At the end of the exponential phase of growth, the cultures (1 mL) were centrifuged at 10,000 g for 5 min. The supernatant was used as the microbial extract for further assays. Sarcoplasmic and myofibrillar proteins were extracted as described by Mauriello et al. (2002) and then adjusted to a concentration of 1 mg/mL. The microbial extracts (200 mL) were incubated with sarcoplasmic and myofibrillar proteins (1 mL) for 20 h at 25 C. Next, SDS-PAGE was performed based on the method of Paramithiotis et al. (2000). Control samples containing sarcoplasmic or myofibrillar proteins were incubated for 20 h at 25 C as described above. The assessment of proteolytic activity was performed by comparing the protein profiles of control samples with those of the samples that had been incubated with the proteolytic strains. An overnight culture of each strain was inoculated onto nutrient agar plates supplemented with 10 g/L triolein (Drosinos et al., 2007), and the plates were incubated at 25 C for 10 days. The appearance of a clear zone surrounding the colonies indicated the occurrence of lipolytic activity. 2.5. In situ spoilage potential evaluation 2.5.1. Meat contamination A total of 620 samples sliced from chilled yellow-feather chicken breasts were prepared in two batches. The first batch, containing both raw and cooked chicken, was used for microbiological analysis. The second batch, containing only raw chicken, was used for biochemical analysis. All samples were sterilized via irradiation as described above. Each inoculation mixture of the four isolates was spread onto the surface of the chicken breast pieces at a concentration of 3 log CFU/g. Uninoculated breasts were included as a control. All samples were stored at 8 C for 7 days. The microbiological and biochemical analyses were performed after 2, 3, 4, 5, 6 and 7 days. 2.5.2. Microbiological analysis At each sampling date, samples were transferred aseptically to a stomacher bag and diluted 10 times in 0.85% NaCl solution. The mixture was homogenized using a stomacher and 1 mL of the homogenized solution was transferred from the stomacher bag for additional serial-dilution steps. Aliquots of the appropriate dilutions (0.1 mL each) were spread on TSA in duplicate, and the agar plates were incubated aerobically at 25 C for 24 h before counting. 2.5.3. Total volatile basic nitrogen (TVBN) and pH analysis TVBN was determined according to China National Food Safety Standard methods - Method for analysis of hygienic standard of meat and meat products (GB/T 5009.44e2003). TVBN contents were expressed as mg of TVBN per 100 g of chicken. The pH was determined according to the method described by Kang et al. (2014). Briefly, 10 g of chicken breast sample was homogenized (Ultra Turrax T25, IKA, Germany) with 40 mL of pre-cooled distilled water at 15,000 rpm for 10 s. The pH was then determined using a digital pH meter (Hanna, Italy). 2.5.4. Sensory analysis The samples were evaluated by five experienced panelists who participated in sensory tests from the National Center of Meat
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Quality and Safety Control of China and had been trained by professional trainers (Zhang et al., 2015). Free descriptors were chosen to define odors in preliminary sessions. Acceptability as a composite of overall acceptance, appearance, odor and surface slime was estimated using a scale ranging from 1 to 9. The scale points were defined as follows: excellent, 9; very good, 8; good, 7; acceptable, 6; between acceptable and unacceptable, 5; slightly unacceptable, 4; moderately unacceptable, 3; very much unacceptable, 2 and extremely unacceptable, 1. The samples were considered spoiled when the median of the grades given was 6 or less. 2.5.5. Volatile organic compound (VOC) analysis VOC analysis was performed after 7 days of storage at 8 C. The analysis was performed on an Agilent 7890A gas chromatograph coupled to an Agilent 5973C mass spectrometer. A slight modification of the method described by Jaffres et al. (2011) was used. Briefly, a 4-g portion was transferred into a 20-mL vial with a polypropylene screw-on cap and a PTFE/silicone septum to make it airtight. The vial was heated at 40 C for 40 min to equilibrate the system. The SPME fiber, an 85-mm carboxen/polydimethylsiloxane Stable Flex™ (Supelco), was inserted through the septum and exposed in the headspace of the vial for 30 min to allow absorption of the volatile compounds onto the fiber. The SPME was then introduced into the injector port of the gas chromatograph for 5 min in splitless mode, set at 250 C, to desorb the volatile compounds. The desorbed components were analyzed on an Agilent DB-WAX (30 m 0.25 mm 0.25 mm) capillary column. Helium was used as a carrier gas at a constant flow rate of 0.8 mL/min, and the oven temperature was programmed as follows: 40 C for 5 min, ramped at 3 C/min to 140 C, ramped at 10 C/min to 250 C, and then held for 5 min. The mass spectrometer was operated in electron impact mode, with an electron energy set at 70 eV and a scan range of 50e500 m/z. The NIST library and comparison with the spectra and retention times of the standards were used during the identification of volatile components. Because our aim was to monitor the detected compounds that were produced by specific isolates and that thus played important roles in the isolates’ sensory patterns, the results are reported as percentages based on the total peak area. 2.6. Statistical analysis All experiments were performed using 4 replicates, excluding the VOC measurements, which were conducted in triplicate. The results are presented as the mean ± standard deviation (SD). Statistical analyses were performed using the SPSS statistics program (Version 22, USA), and ANOVA was used to valuate differences between means. 3. Results and discussion 3.1. Isolation and identification of potential spoilage bacteria Over the past few decades, research attention has focused on microbial diversity and the SSOs in meat using culture-dependent (e.g., selective medium) or culture-independent methods (Doulgeraki et al., 2012). However, many bacteria would be overlooked if only species-specific selective media were used in meat quality research. In addition, not all bacterial groups present on meat are involved in spoilage (Dalgaard, 2000; Grama et al., 2002). Therefore, a screening approach that provides only the nutrients available in chicken as the energy and nutrient sources was needed. In this work, the isolates from chilled yellow-feather chicken were selectively isolated from RJA, and their spoilage potential was
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preliminarily evaluated. A total of 53 strains belonging to seven species of three genera were isolated from spoiled samples (Table 1). According to the RJA assay, different strains exhibited different DZD profiles. The DZDs of P. fragi H8 and A. salmonicida 35 were significantly larger (P < 0.05) than those of the other isolates, which indicated strong catabolic capacity on chicken meat. However, the DZD of S. liquefaciens 17 was rarely observed (Table 1, Fig. 1). Much of the published research has examined Pseudomonas spp. as the dominant spoilage bacteria on chilled meat under aerobic conditions. P. fragi is the most frequently identified species, followed by P. lundensis and P. fluorescens (Casaburi et al., 2015; Ercolini et al., 2010). Meanwhile, isolation of A. salmonicida from spoiled shrimp was reported by Mace et al. (2014), although A. salmonicida is not a well-known spoilage bacterium. Our metagenomic results for chilled yellow-feather chicken (data not shown) also revealed that the relative abundance of Aeromonas spp. was high at the beginning of storage. Of note, S. liquefaciens is the most common member of the Enterobacteriaceae detected in meat stored under different atmospheric conditions (Stanbridge and Davies, 1998).
3.2. Proteolytic and lipolytic activity levels The SDS-PAGE results are shown in Fig. 2. Comparison of the control and the treatment samples revealed no significant differences in the sarcoplasmic protein bands, except for A. salmonicida 35, for which several bands disappeared from 25 to 55 kDa and from 100 to 130 kDa (Fig. 2A). However, three important modifications could be observed for the myofibrillar proteins (Fig. 2B): one protein with a molecular weight of approximately 200 kDa disappeared in the A. salmonicida 35 isolate, whereas 2 proteins of approximately 43 and 35 kDa were hydrolyzed to different degrees in all four isolates. The 200- and 43-kDa proteins may represent myosin and actin, respectively. Myosin and actin are the two most abundant proteins in muscle myofibrils, and even small changes in myosin have the potential to influence protein solubility and fiber shear strength (Huff Lonergan et al., 2010). The 35-kDa protein may be troponin-T, which is part of the regulatory complex that mediates actinmyosin interactions. It is conceivable that troponin-T degradation may lead to changes that involve thick and thin filament interactions (Lehman et al., 2001). Deterioration of the meat nutritive value and sensory properties results first from the degradation of principal components, such as proteins and lipids (Nowak et al., 2012). Bacterial proteinases play important roles in the hydrolysis of meat proteins in particular. Many studies have reported the proteolytic activities of lactic acid bacteria (LAB) or Staphylococci in fermented meat products and Pseudomonas spp. in pork or beef (Drosinos et al., 2007; Ercolini et al., 2010; Mauriello et al., 2002; Zeng et al., 2014), but there is little information about the proteolytic activities of Aeromonas spp. and Serratia spp. In the in vitro study presented here, A. salmonicida 35 showed the highest proteolytic activity values for both sarcoplasmic and myofibrillar proteins, suggesting strong spoilage potential on chicken meat. In contrast, P. fluorescens H5, P. fragi H8 and S. liquefaciens 17 were proteolytic only against the myofibrillar protein fraction, implying a gradually decreasing spoilage potential on chicken meat. These findings are in agreement with the results of Tarrant et al. (1973), who studied the activity of a proteolytic enzyme from P. fragi on pig muscle and indicated that myofibrillar protein was more susceptible to hydrolysis than sarcoplasmic protein. All four strains displayed lipolytic activities, suggesting that they may have the capacity to degrade chicken skin.
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Table 1 Raw-chicken juice agar (RJA) assay of the isolates. Genera
Species
Number of isolates
Largest decomposition zone diameter (mm)
Aeromonas
Aeromonas salmonicida Aeromonas hydrophila Aeromonas media Chryseobacterium shigense Pseudomonas fragi Pseudomonas fluorescens Pseudomonas putida
35 2 1 1 4 8 2
19.3 17.1 17.1 14.7 21.8 17.3 15.1
Chryseobacterium Pseudomonas
± ± ± ± ± ± ±
0.89ab 0.38bc 0.23bc 2.52c 2.63a 1.12bc 0.31c
Values are expressed as the mean ± standard deviation (n ¼ 4). aec: Values with different lower case letters in superscript are significantly different (P < 0.05).
Fig. 1. The decomposition zone diameter (DZD) profiles of A. salmonicida 35, P. fluorescens H5, P. fragi H8 and S. liquefaciens 17.
Fig. 2. SDS-PAGE of sarcoplasmic (A) and myofibrillar (B) protein samples. Lane M, molecular weight markers; Lane C, control; Lane 35, A. salmonicida; Lane H5, P. fluorescens; Lane H8, P. fragi; Lane 17, S. liquefaciens. The arrows indicate protein bands that are altered in the samples.
3.3. Microbiological analysis The results for the total viable counts are shown in Fig. 3. In raw chicken samples, after two days of storage, the growth of the 4 strains reached between 5 and 6 log CFU/g; however, A. salmonicida 35 tended to exhibit slower growth over the next three days. At the end of storage, P. fragi H8 and S. liquefaciens 17 reached titers of 10.2 log CFU/g, whereas A. salmonicida 35 attained a titer of approximately 8.6 log CFU/g (Fig. 3A). In cooked chicken samples, the
growth patterns were similar to those in raw samples (Fig. 3B). P. fragi is present in nearly all the samples during storage under various conditions, including air, modified atmosphere packaging (MAP) and vacuum packaging (VP) (Ercolini et al., 2010; Pennacchia et al., 2011; Zhang et al., 2015). By contrast, P. fluorescens is more abundant than P. fragi on fresh meat, but the latter becomes dominant over time (Doulgeraki et al., 2012; Lebert et al., 1998). This finding is also in general agreement with a report demonstrating that S. liquefaciens represented the dominant isolate of
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S. liquefaciens 17 in fresh and cooked samples, chicken meat may be considered their ecological niche. Because the vast majority of chilled chicken sold in Chinese supermarkets and wet markets is stored aerobically, the environment may enforces a selection pressure on the bacterial community, so those groups of bacteria that are best adapted to the environment will outgrow the others, become dominant, and reach high numbers. 3.4. TVBN and pH changes The evolution of TVBN and pH values is depicted in Fig. 4. The initial concentration of TVBN was 8.2 mg/100 g. Subsequently, TVBN increased rapidly in P. fragi H8 and P. fluorescens H5 compared with S. liquefaciens 17 and A. salmonicida 35 (Fig. 4A). At the end of storage, the concentrations of TVBN in P. fragi H8 and P. fluorescens H5 were higher than 50 mg/100 g. Meanwhile, the pH value was
Fig. 3. Microbiological changes during storage of raw (A) and cooked (B) chicken samples inoculated with A. salmonicida 35, P. fluorescens H5, P. fragi H8 and S. liquefaciens 17 stored at 8 C. Each data point and the error bars show the mean ± standard deviation of 4 replicates.
Enterobacteriaceae during storage of minced beef and pork at a chilled temperature (Doulgeraki et al., 2012). Moreover, in the current study, A. salmonicida 35 showed a weak growth ability in fresh samples, consistent with the results of Pennacchia et al. (2011), who detected Aeromonas spp. in chilled beef at the beginning of storage but found that these species disappeared after 7 days. However, in the cooked samples in the present study, A. salmonicida 35 also quickly reached approximately 9.4 log CFU/g. This good growth status on cooked meat was identical to that in a previous study (Mace et al., 2014), in which cooked tropical shrimp samples inoculated with A. salmonicida were considered to be strongly spoiled after 8 days of storage. This result revealed that A. salmonicida may be associated with cooked meat spoilage. Therefore, due to their strong occurrence of P. fragi H8 and
Fig. 4. TVBN (A) and pH (B) changes of raw chicken samples inoculated with A. salmonicida 35, P. fluorescens H5, P. fragi H8 and S. liquefaciens 17 stored at 8 C. Each data point and the error bars show the mean ± standard deviation of 4 replicates.
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initially 5.8. During storage, the pH values in P. fragi H8 and P. fluorescens H5 increased substantially, reaching values of approximately 7.4 (Fig. 4B). Similar to what was observed for TVBN, the final pH values in P. fragi H8 and P. fluorescens H5were significantly higher than the initial values. TVBN is widely used as an indicator of meat spoilage because its components result from microbial degradation of protein and nonprotein nitrogenous compounds, such as amino acids and nucleotide catabolites (Liu et al., 2013). The limiting concentration of TVBN in meats for human consumption in China (GB 16869-2005) is 15 mg/100 g muscle. In the present study, on the 4th day of storage, P. fragi H8 and P. fluorescens H5 had exceeded this limit and became the fastest group. According to the proteolytic and microbiological analysis, strain 35 exhibited the strongest proteolytic activity in vitro but the lowest growth rate on meat, whereas P. fragi H8 and P. fluorescens H5 performed strongly in both analyses, which is likely the main reason for the difference in TVBN. Meanwhile, the pH value of normal chicken breast is 5.7e6.1 (Barbut et al., 2005). Changes in the pH value of meat have been attributed to many factors. In any case, a high pH in meat leads to a more rapid spoilage process due to more rapid bacterial growth and nutrient consumption (Iulietto et al., 2015). It is well established that LAB metabolism produces compounds such as lactic acid, resulting in a slight decrease in pH, whereas in non-lactic cultures, the pH increases slightly (Pogacic et al., 2015). Furthermore, the majority of Pseudomonas spp. produce only one type of proteinase, typically neutral zinc metallo proteinases with pH optima ranging from 6.5 to 8 (Fairbairn and Law, 1986; Tarrant et al., 1973). In the current study, the growth of P. fragi H8 and P. fluorescens H5 resulted in significant increases in pH, ranging from 5.8 to 7.4, thus achieving the most suitable pH values for proteinases. This finding may also explain why P. fragi H8 and P. fluorescens H5 showed the highest growth rate and TVBN values on chicken. 3.5. Sensory analysis As shown in Fig. 5, chicken fillet freshness was excellent over the first two days, and the freshness characteristics diminished
Fig. 5. Spoilage scores of raw chicken samples inoculated with A. salmonicida 35, P. fluorescens H5, P. fragi H8 and S. liquefaciens 17 stored at 8 C for 7 days, as determined in the sensory panel. Each data point and the error bars show the mean ± standard deviation of the 5 panelists’ scores.
gradually over time. The differences in appearance and odor among the four isolates became apparent from the 4th day of storage. For example, samples inoculated with P. fragi H8 and P. fluorescens H5 exhibited slight surface slime, and A. salmonicida 35 displayed special off-odors, whereas S. liquefaciens 17 did not produce any typical characteristic slime or intense odors. After 6 days of storage, all samples were considered spoiled (Table 2, Fig. 5). Samples inoculated with P. fragi H8 and P. fluorescens H5 were considered grossly spoiled as they exhibited strong slime formation and emitted unpleasant odors that were described as “sour”, “fruity” and “cheesy”. In contrast, “grassy” and “sour” descriptors were cited for A. salmonicida 35. Slime production gives bacteria an advantage because slime constitutes a protective layer that keeps bacteria moist and allows €rkroth and microorganisms to grow at a lower temperature (Bjo Korkeala, 1997). This protection allows the bacteria to survive and to compete with other bacteria in meat. Consequently, the use of a low temperature for meat storage does not prevent the formation of slime, although refrigeration does result in longer product shelflife. In the present study, there were certain correlations between the sensory rejection time and the quantity of TVBN, in contrast to the conclusions of Joffraud et al. (2006) and Jaffres et al. (2011), who suggested that TVBN was not likely to be a good indicator of sensory detection of seafood spoilage. 3.6. VOC analysis Over the last few years, several studies have demonstrated a correlation between the release of spoilage VOCs and the growth of specific microbial species during meat storage (Argyri et al., 2015; Ercolini et al., 2009; Joffraud et al., 2001). Numerous VOCs detected in the present study have previously been reported in the literature as bacterial metabolites and are involved in the undesirable odor characteristics of the spoilage process. Without taking into account compounds that were present in trace amounts, a total of 25 VOCs were considered. Metabolites from aromatic amino acids and unsaturated fatty acids, such as alcohols (e.g., 1-hexanol, 1-heptanol and 1-octen-3-ol), aldehydes (e.g., nonanal, octanal and hexanal) and ketones (e.g., 2-pentanone, 2-heptanone, 2-nonanone), were detected in this study (Table 3). Specifically, in the headspace of A. salmonicida 35, the concentrations of 1-hexanol, hexanal, indole, 1-octen-3-ol and 2,3-butanediol were significantly higher (P < 0.05) than the concentrations of other compounds. These VOC profiles of A. salmonicida 35 were similar to those in the control group. A higher 1-hexanol level may et al., 2001). Meanwhile, associated with a “green” flavor (Falque indoles are produced by the bacterial decomposition of tryptophan and have been used as an indicator of spoilage in seafood (Sarnoski et al., 2010; Snellings et al., 2003). The formation of 2,3-butanediol is also associated with microbial activity during storage (Josephson et al., 1983; Ui et al., 1986). When samples were spoiled by P. fluorescens H5, the most significant compounds (P < 0.05) detected were 2-nonanone, 2-heptanone, 2-undecanone and acetic acid. This group also produced 2-octenyl acetate, which was not produced by the other groups. For P. fragi H8, 2-heptanone, 2nonanone, 2-heptanol, 3-methyl-1-butanol, 2-nonanol, 1undecene and acetic acid were detected. P. fluorescens H5 and P. fragi H8 exhibited similar increased concentrations of volatile compound profiles. In this study, 2-heptanone and 2-nonanone were the two most abundant compounds found in both samples. Casaburi et al. (2015) found that the presence of ketones in stored fresh meat is mainly associated with the presence of Pseudomonas spp., Carnobacterium spp. and Enterobacteriaceae. 2-Heptanone and 2-nonanone were also detected in meat contaminated with Pseudomonas spp., as reported by Joffraud et al. (2001) and Leroy et al.
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Table 2 Sensory evaluation and odors assigned to samples of chicken breast fillets on the 6th day following inoculation with the bacterial isolates. Bacterial isolate
Sensory evaluation
Off-odor characteristics
P. fragi H8 P. fluorescens H5 A. salmonicida 35 S. liquefaciens 17
Extremely unacceptable: juice loss, off-odor, slime Very much unacceptable: juice loss, off-odor, slime Moderately unacceptable: juice loss, off-odor Moderately unacceptable: juice loss, slightly off-odor
Sour/fruity/cheesy Sour/fruity/cheesy Sour/grassy Garlicky/cabbage-like
Table 3 Volatile organic compounds (VOCs) identified in inoculated samples after 7 days of storage at 8 C. Volatile compound
Alcohols 3-Methyl-1-butanol 1-Hexanol 1-Octen-3-ol 2-Nonanol 2,3-Butanediol 2-Heptanol 1-Heptanol 1-Pentanol Aldehydes Nonanal Hexanal Octadecanal Octanal Ketones 2-Pentanone 2-Heptanone 2-Nonanone Acetoin 2-Undecanone 2,3-Octanedione Other 1-Undecene Acetic acid Dimethyl disulfide 2,5-Dimethylpyrazine Dimethyl trisulfide 2-Octenyl acetate Indole
Control
Meat inoculated with A. salmonicida 35
P. fluorescens H5
P. fragi H8
S. liquefaciens 17
<1 26.42 ± 0.83a 8.75 ± 1.64c <1 2.50 ± 1.05ef <1 1.65 ± 0.19f 1.33 ± 0.16f
<1 31.84 ± 2.15a 10.48 ± 0.84d <1 6.20 ± 1.23e <1 1.19 ± 0.46g 2.20 ± 0.37fg
2.62 ± 0.78c <1 <1 1.96 ± 0.24c <1 <1 <1 <1
2.95 ± 0.67e <1 <1 7.04 ± 0.36d <1 16.75 ± 0.82c <1 <1
10.93 ± 1.66a 1.58 ± 0.58d 4.43 ± 2.2c 1.03 ± 0.41d 1.31 ± 0.06d <1 <1 1.25 ± 0.21d
4.34 ± 0.83d 19.06 ± 1.56b <1 1.68 ± 0.39f
2.19 ± 0.65fg 18.56 ± 1.88b 4.47 ± 1.06ef 1 ± 0.26g
<1 <1 <1 <1
<1 <1 <1 <1
1.95 ± 0.14cd <1 <1 <1
<1 <1 <1 3.71 ± 0.48de <1 2.09 ± 0.52ef
<1 <1 <1 1.74 ± 1.19fg <1 2.17 ± 0.36fg
<1 7.48 ± 0.52b 38.59 ± 2.24a <1 7.32 ± 1.27b <1
<1 31.83 ± 2.34a 19.94 ± 1.44b <1 <1 <1
3.03 7.99 1.69 4.53 <1 <1
<1 <1 <1 <1 <1 <1 <1
<1 4.31 ± 0.29ef <1 <1 <1 <1 14.46 ± 2.23c
<1 7.11 ± 2.55b <1 <1 <1 7.38 ± 0.85b <1
3.71 ± 1.98e 4.49 ± 1.23e <1 <1 <1 <1 <1
<1 <1 12.84 ± 1.18a 12.93 ± 2.77a 1.37 ± 0.23d <1 <1
± ± ± ±
1.28cd 1.31b 0.48d 1.94c
Values are expressed as the mean ± standard deviation of the area ratio (%) of the volatile compounds identified in each inoculated sample group (n ¼ 3). aeg: Values with different lower case letters in superscript, within a column, are significantly different (P < 0.05).
(2009). However, acetoin, the ketone most often found in fresh meat, was not found in this study. Alterations by S. liquefaciens 17 led to the production of 3-methyl-1-butanol, 2-nonanol, 2pentanone, 2-heptanone and 2-nonanone. This strain also produced dimethyl disulfide, 2,5-dimethylpyrazine and dimethyl trisulfide which were not produced by the other groups. 3-Methyl1-butanol is one of the most common alcohols found in spoiled raw meat and is produced from leucine catabolism (Smit et al., 2005). This alcohol can be associated with a whiskey-like odor (Larrouture-Thiveyrat et al., 2003), and the presence of its corresponding ketones may enhance the odor intensity because the odor intensities of compounds with similar odor qualities has been reported to be additive (Meilgaard and Peppard, 1986). The formation of sulfur compounds and 3-methyl-1-butanol during spoilage has also been reported in beef samples containing S. liquefaciens, indicating that these VOCs may be associated with Serratia metabolism (Hernandez-Macedo et al., 2012). In this work, the spoilage potential of 4 isolates, as assessed based on proteolytic and lipolytic activities in vitro and growth kinetics, biochemical changes, sensory alterations and VOC patterns in situ, exhibited different profiles than the same species reported in previous studies because these tests were individually based, and the test strains were preliminarily screened in an RJA
assay. Our results indicate that the spoilage potential of strains was species, strain and food matrix dependent. 4. Conclusion These findings have contributed to the characterization of the spoilage potential of bacterial species isolated from chilled yellowfeather chicken meat using RJA. In vitro, A. salmonicida 35 presented the strongest proteolytic capacity against myofibrillar protein, whereas P. fragi H8 led to a strong occurrence, rapid increases in TVBN and pH, off-odors and serious slime formation in situ. Therefore, it is likely that P. fragi H8 plays a significant role in the spoilage of chilled chicken. Furthermore, the availability of RJA may be useful for effective analyses in studies aimed at identifying the potential spoilage bacteria during the natural spoilage of meat and meat products. Nevertheless, because the present study was performed under pure culture conditions, further investigations are needed to determine spoilage potential in mixed cultures. Acknowledgments This study was supported by China Agriculture Research System (CARS-42) funded by the China Ministry of Agriculture.
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Contents lists available at ScienceDirect
Food Microbiology journal homepage: www.elsevier.com/locate/fm
Evaluation of the spoilage potential of bacteria isolated from chilled chicken in vitro and in situ Guang-yu Wang a, Hu-hu Wang a, Yi-wei Han b, Tong Xing b, Ke-ping Ye a, Xing-lian Xu a, *, Guang-hong Zhou a a
Key Laboratory of Meat Processing and Quality Control, Ministry of Education, Nanjing Agricultural University, Nanjing, Jiangsu 210095, PR China Collaborative Innovation Center of Food Safety and Nutrition, Ministry of Education and Finance, College of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, PR China
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 March 2016 Received in revised form 16 June 2016 Accepted 17 November 2016
Microorganisms play an important role in the spoilage of chilled chicken. In this study, a total of 53 isolates, belonging to 7 species of 3 genera, were isolated using a selective medium based on the capacity to spoil chicken juice. Four isolates, namely Aeromonas salmonicida 35, Pseudomonas fluorescens H5, Pseudomonas fragi H8 and Serratia liquefaciens 17, were further characterized to assess their proteolytic activities in vitro using meat protein extracts and to evaluate their spoilage potential in situ. The in vitro studies showed that A. salmonicida 35 displayed the strongest proteolytic activity against both sarcoplasmic and myofibrillar proteins. However, the major spoilage isolate in situ was P. fragi H8, which exhibited a fast growth rate, slime formation and increased pH and total volatile basic nitrogen (TVBN) on chicken breast fillets. The relative amounts of volatile organic compounds (VOCs) originating from the microorganisms, including alcohols, aldehydes, ketones and several sulfur compounds, increased during storage. In sum, this study demonstrated the characteristics of 4 potential spoilage bacteria on chilled yellow-feather chicken and provides a simple and convenient method to assess spoilage bacteria during quality management. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Chilled chicken Raw-chicken juice agar Spoilage bacteria Sensory analysis Volatile organic compounds
1. Introduction Meat is the most valuable livestock product. From a nutritional point of view, the importance of meat is derived from its highquality protein, which contains all essential amino acids, and its highly bioavailable minerals and vitamins. In particular, chicken is one of the most traded and consumed meats worldwide. The yellow-feather chicken, as a special species in Asia, has a more distinctive flavor than many other commercial broilers (Zhang et al., 2015). However, to reduce the ongoing outbreaks of animal influenza, particularly the H7N9 strain, live poultry markets have currently been restricted in the majority of cities in China. Consumers now buy fresh chilled chicken, which originates from slaughter plants, through shops and supermarkets. Hence, the demand for these products has increased markedly, and safety problems have become a public health concern. The safety issues associated with chilled chicken have been
* Corresponding author. E-mail address: [email protected] (X.-l. Xu). http://dx.doi.org/10.1016/j.fm.2016.11.015 0740-0020/© 2016 Elsevier Ltd. All rights reserved.
based mostly on the presence of toxicant and pathogenic bacteria in food, which may influence public health. It is noteworthy that according to Regulation 178/2002 of the European Parliament and Commission, a foodstuff is regarded as unsafe not only if it is harmful to consumer health but also if it is not fit for human consumption (Nowak et al., 2012). In this sense, spoiled food, which means food with an appearance, taste or flavor leading to its rejection, is also considered unsafe. Chicken meat is prone to deterioration in a short time, even under chilled conditions (Patsias et al., 2008). Microbiological contamination is one of the most important factors contributing to quality loss, resulting in slime, colony formation, compromised food texture, off-flavors and off-odors (Grama et al., 2002; Hyldgaard et al., 2015). Many studies have reported that the specific spoilage organisms (SSOs) in refrigerated poultry are Pseudomonas spp., Enterobacteriaceae, lactic acid bacteria, and Brochotrix thermosphacta (Chaillou et al., 2015; Grama et al., 2002; Meredith et al., 2014). However, few studies have examined microbial organisms with strong catabolic capacities on chicken meat at refrigeration temperatures. These bacteria may destroy the cell
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structure, promoting the outflow of nutrients that can be utilized by other spoilage bacteria. Therefore, it is necessary to characterize these organisms to better understand chilled yellow-feather chicken spoilage. The aims of the present work were to identify potential spoilage bacteria from chilled yellow-feather chicken using selective medium based on the capacity to spoil chicken juice and to assess the spoilage potential of these isolates both in vitro and in situ. Such information may eventually be used to better inform processing and preservation strategies to enhance the quality and shelf-life of chilled chicken. 2. Materials and methods 2.1. Preparation of the selective medium and samples The selective medium was prepared by mixing raw chicken juice (the fresh chicken breasts were homogenized with deionized water and then filtered through two layers of gauze) and 1% agar. Both ingredients were maintained at 45 C before mixing. This rawchicken juice agar (RJA) was subsequently sterilized by irradiation at a dose of 6 KGy (Kazanas, 1968) via the 60Co source at Hangyu (Hangyu Irradiation Technology Co., LTD, Nanjing, China). Yellow-feather chicken meat was collected from slaughterhouses and supermarkets. Immediately after collection, the samples were aseptically transferred to the laboratory in an ice box within 3 h. The samples were then stored at 8 C until spoilage before isolation was performed. 2.2. Isolation and identification of spoilage bacteria Fifty-four surface samples (25 g) from each muscle were aseptically weighed and homogenized in 225 mL sterilized 0.85% NaCl solution. Decimal dilutions were prepared in the same solution, and 0.1 mL of each of the appropriate dilutions was plated on RJA. The plates were then incubated aerobically at 25 C for 48 h. Colonies with a large decomposition zone were selected from each selective medium and subsequently streaked on the same medium twice to obtain a single colony. The isolates were identified based on 16S rRNA gene sequences, which were amplified using the universal primers 27F (50 -AGAGTTTGATCCTGGCTCAG-30 ) and 1492R (50 GGTTACCTTGTTACGACTT-30 ). The PCR products were purified and sequenced by Invitrogen (Invitrogen Biotechnology Co., Ltd, Shanghai, China). The sequences were compared with those in GenBank using the BLAST function, and the closest matches to each clone were determined based on specific probable identities and then confirmed using a VITEK2 automated system (BioMerieux, France). 2.3. Selection of spoilage bacteria via the RJA assay A total of 53 isolates that belonged to different genera or species were identified: Aeromonas spp. (Aeromonas salmonicida, Aeromonas hydrophila and Aeromonas media), Chryseobacterium shigense and Pseudomonas spp. (Pseudomonas fluorescens, Pseudomonas fragi and Pseudomonas putida). All strains were cultured in tryptone soy broth (TSB) for 24 h at 25 C. The cell density was adjusted to an optical density of 600 nm (OD600) of 0.4. Next, four replicate 2-mL aliquots of the isolates were spotted onto RJA, and decomposition was measured after 3 days of incubation at 25 C. The strains Pseudomonas fragi H8, Aeromonas salmonicida 35 and Pseudomonas fluorescens H5 were selected because they provided the three largest decomposition zone diameters (DZDs) among the species described above. The spoilage potentials of these strains were compared with that of a well-known spoilage
bacterium, Serratia liquefaciens 17, which was isolated previously (data not shown). 2.4. In vitro proteolytic and lipolytic assays The proteolytic activities of the chicken sarcoplasmic and myofibrillar proteins were estimated based on SDS-PAGE analysis. In brief, each isolate was incubated in TSB. At the end of the exponential phase of growth, the cultures (1 mL) were centrifuged at 10,000 g for 5 min. The supernatant was used as the microbial extract for further assays. Sarcoplasmic and myofibrillar proteins were extracted as described by Mauriello et al. (2002) and then adjusted to a concentration of 1 mg/mL. The microbial extracts (200 mL) were incubated with sarcoplasmic and myofibrillar proteins (1 mL) for 20 h at 25 C. Next, SDS-PAGE was performed based on the method of Paramithiotis et al. (2000). Control samples containing sarcoplasmic or myofibrillar proteins were incubated for 20 h at 25 C as described above. The assessment of proteolytic activity was performed by comparing the protein profiles of control samples with those of the samples that had been incubated with the proteolytic strains. An overnight culture of each strain was inoculated onto nutrient agar plates supplemented with 10 g/L triolein (Drosinos et al., 2007), and the plates were incubated at 25 C for 10 days. The appearance of a clear zone surrounding the colonies indicated the occurrence of lipolytic activity. 2.5. In situ spoilage potential evaluation 2.5.1. Meat contamination A total of 620 samples sliced from chilled yellow-feather chicken breasts were prepared in two batches. The first batch, containing both raw and cooked chicken, was used for microbiological analysis. The second batch, containing only raw chicken, was used for biochemical analysis. All samples were sterilized via irradiation as described above. Each inoculation mixture of the four isolates was spread onto the surface of the chicken breast pieces at a concentration of 3 log CFU/g. Uninoculated breasts were included as a control. All samples were stored at 8 C for 7 days. The microbiological and biochemical analyses were performed after 2, 3, 4, 5, 6 and 7 days. 2.5.2. Microbiological analysis At each sampling date, samples were transferred aseptically to a stomacher bag and diluted 10 times in 0.85% NaCl solution. The mixture was homogenized using a stomacher and 1 mL of the homogenized solution was transferred from the stomacher bag for additional serial-dilution steps. Aliquots of the appropriate dilutions (0.1 mL each) were spread on TSA in duplicate, and the agar plates were incubated aerobically at 25 C for 24 h before counting. 2.5.3. Total volatile basic nitrogen (TVBN) and pH analysis TVBN was determined according to China National Food Safety Standard methods - Method for analysis of hygienic standard of meat and meat products (GB/T 5009.44e2003). TVBN contents were expressed as mg of TVBN per 100 g of chicken. The pH was determined according to the method described by Kang et al. (2014). Briefly, 10 g of chicken breast sample was homogenized (Ultra Turrax T25, IKA, Germany) with 40 mL of pre-cooled distilled water at 15,000 rpm for 10 s. The pH was then determined using a digital pH meter (Hanna, Italy). 2.5.4. Sensory analysis The samples were evaluated by five experienced panelists who participated in sensory tests from the National Center of Meat
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Quality and Safety Control of China and had been trained by professional trainers (Zhang et al., 2015). Free descriptors were chosen to define odors in preliminary sessions. Acceptability as a composite of overall acceptance, appearance, odor and surface slime was estimated using a scale ranging from 1 to 9. The scale points were defined as follows: excellent, 9; very good, 8; good, 7; acceptable, 6; between acceptable and unacceptable, 5; slightly unacceptable, 4; moderately unacceptable, 3; very much unacceptable, 2 and extremely unacceptable, 1. The samples were considered spoiled when the median of the grades given was 6 or less. 2.5.5. Volatile organic compound (VOC) analysis VOC analysis was performed after 7 days of storage at 8 C. The analysis was performed on an Agilent 7890A gas chromatograph coupled to an Agilent 5973C mass spectrometer. A slight modification of the method described by Jaffres et al. (2011) was used. Briefly, a 4-g portion was transferred into a 20-mL vial with a polypropylene screw-on cap and a PTFE/silicone septum to make it airtight. The vial was heated at 40 C for 40 min to equilibrate the system. The SPME fiber, an 85-mm carboxen/polydimethylsiloxane Stable Flex™ (Supelco), was inserted through the septum and exposed in the headspace of the vial for 30 min to allow absorption of the volatile compounds onto the fiber. The SPME was then introduced into the injector port of the gas chromatograph for 5 min in splitless mode, set at 250 C, to desorb the volatile compounds. The desorbed components were analyzed on an Agilent DB-WAX (30 m 0.25 mm 0.25 mm) capillary column. Helium was used as a carrier gas at a constant flow rate of 0.8 mL/min, and the oven temperature was programmed as follows: 40 C for 5 min, ramped at 3 C/min to 140 C, ramped at 10 C/min to 250 C, and then held for 5 min. The mass spectrometer was operated in electron impact mode, with an electron energy set at 70 eV and a scan range of 50e500 m/z. The NIST library and comparison with the spectra and retention times of the standards were used during the identification of volatile components. Because our aim was to monitor the detected compounds that were produced by specific isolates and that thus played important roles in the isolates’ sensory patterns, the results are reported as percentages based on the total peak area. 2.6. Statistical analysis All experiments were performed using 4 replicates, excluding the VOC measurements, which were conducted in triplicate. The results are presented as the mean ± standard deviation (SD). Statistical analyses were performed using the SPSS statistics program (Version 22, USA), and ANOVA was used to valuate differences between means. 3. Results and discussion 3.1. Isolation and identification of potential spoilage bacteria Over the past few decades, research attention has focused on microbial diversity and the SSOs in meat using culture-dependent (e.g., selective medium) or culture-independent methods (Doulgeraki et al., 2012). However, many bacteria would be overlooked if only species-specific selective media were used in meat quality research. In addition, not all bacterial groups present on meat are involved in spoilage (Dalgaard, 2000; Grama et al., 2002). Therefore, a screening approach that provides only the nutrients available in chicken as the energy and nutrient sources was needed. In this work, the isolates from chilled yellow-feather chicken were selectively isolated from RJA, and their spoilage potential was
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preliminarily evaluated. A total of 53 strains belonging to seven species of three genera were isolated from spoiled samples (Table 1). According to the RJA assay, different strains exhibited different DZD profiles. The DZDs of P. fragi H8 and A. salmonicida 35 were significantly larger (P < 0.05) than those of the other isolates, which indicated strong catabolic capacity on chicken meat. However, the DZD of S. liquefaciens 17 was rarely observed (Table 1, Fig. 1). Much of the published research has examined Pseudomonas spp. as the dominant spoilage bacteria on chilled meat under aerobic conditions. P. fragi is the most frequently identified species, followed by P. lundensis and P. fluorescens (Casaburi et al., 2015; Ercolini et al., 2010). Meanwhile, isolation of A. salmonicida from spoiled shrimp was reported by Mace et al. (2014), although A. salmonicida is not a well-known spoilage bacterium. Our metagenomic results for chilled yellow-feather chicken (data not shown) also revealed that the relative abundance of Aeromonas spp. was high at the beginning of storage. Of note, S. liquefaciens is the most common member of the Enterobacteriaceae detected in meat stored under different atmospheric conditions (Stanbridge and Davies, 1998).
3.2. Proteolytic and lipolytic activity levels The SDS-PAGE results are shown in Fig. 2. Comparison of the control and the treatment samples revealed no significant differences in the sarcoplasmic protein bands, except for A. salmonicida 35, for which several bands disappeared from 25 to 55 kDa and from 100 to 130 kDa (Fig. 2A). However, three important modifications could be observed for the myofibrillar proteins (Fig. 2B): one protein with a molecular weight of approximately 200 kDa disappeared in the A. salmonicida 35 isolate, whereas 2 proteins of approximately 43 and 35 kDa were hydrolyzed to different degrees in all four isolates. The 200- and 43-kDa proteins may represent myosin and actin, respectively. Myosin and actin are the two most abundant proteins in muscle myofibrils, and even small changes in myosin have the potential to influence protein solubility and fiber shear strength (Huff Lonergan et al., 2010). The 35-kDa protein may be troponin-T, which is part of the regulatory complex that mediates actinmyosin interactions. It is conceivable that troponin-T degradation may lead to changes that involve thick and thin filament interactions (Lehman et al., 2001). Deterioration of the meat nutritive value and sensory properties results first from the degradation of principal components, such as proteins and lipids (Nowak et al., 2012). Bacterial proteinases play important roles in the hydrolysis of meat proteins in particular. Many studies have reported the proteolytic activities of lactic acid bacteria (LAB) or Staphylococci in fermented meat products and Pseudomonas spp. in pork or beef (Drosinos et al., 2007; Ercolini et al., 2010; Mauriello et al., 2002; Zeng et al., 2014), but there is little information about the proteolytic activities of Aeromonas spp. and Serratia spp. In the in vitro study presented here, A. salmonicida 35 showed the highest proteolytic activity values for both sarcoplasmic and myofibrillar proteins, suggesting strong spoilage potential on chicken meat. In contrast, P. fluorescens H5, P. fragi H8 and S. liquefaciens 17 were proteolytic only against the myofibrillar protein fraction, implying a gradually decreasing spoilage potential on chicken meat. These findings are in agreement with the results of Tarrant et al. (1973), who studied the activity of a proteolytic enzyme from P. fragi on pig muscle and indicated that myofibrillar protein was more susceptible to hydrolysis than sarcoplasmic protein. All four strains displayed lipolytic activities, suggesting that they may have the capacity to degrade chicken skin.
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Table 1 Raw-chicken juice agar (RJA) assay of the isolates. Genera
Species
Number of isolates
Largest decomposition zone diameter (mm)
Aeromonas
Aeromonas salmonicida Aeromonas hydrophila Aeromonas media Chryseobacterium shigense Pseudomonas fragi Pseudomonas fluorescens Pseudomonas putida
35 2 1 1 4 8 2
19.3 17.1 17.1 14.7 21.8 17.3 15.1
Chryseobacterium Pseudomonas
± ± ± ± ± ± ±
0.89ab 0.38bc 0.23bc 2.52c 2.63a 1.12bc 0.31c
Values are expressed as the mean ± standard deviation (n ¼ 4). aec: Values with different lower case letters in superscript are significantly different (P < 0.05).
Fig. 1. The decomposition zone diameter (DZD) profiles of A. salmonicida 35, P. fluorescens H5, P. fragi H8 and S. liquefaciens 17.
Fig. 2. SDS-PAGE of sarcoplasmic (A) and myofibrillar (B) protein samples. Lane M, molecular weight markers; Lane C, control; Lane 35, A. salmonicida; Lane H5, P. fluorescens; Lane H8, P. fragi; Lane 17, S. liquefaciens. The arrows indicate protein bands that are altered in the samples.
3.3. Microbiological analysis The results for the total viable counts are shown in Fig. 3. In raw chicken samples, after two days of storage, the growth of the 4 strains reached between 5 and 6 log CFU/g; however, A. salmonicida 35 tended to exhibit slower growth over the next three days. At the end of storage, P. fragi H8 and S. liquefaciens 17 reached titers of 10.2 log CFU/g, whereas A. salmonicida 35 attained a titer of approximately 8.6 log CFU/g (Fig. 3A). In cooked chicken samples, the
growth patterns were similar to those in raw samples (Fig. 3B). P. fragi is present in nearly all the samples during storage under various conditions, including air, modified atmosphere packaging (MAP) and vacuum packaging (VP) (Ercolini et al., 2010; Pennacchia et al., 2011; Zhang et al., 2015). By contrast, P. fluorescens is more abundant than P. fragi on fresh meat, but the latter becomes dominant over time (Doulgeraki et al., 2012; Lebert et al., 1998). This finding is also in general agreement with a report demonstrating that S. liquefaciens represented the dominant isolate of
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S. liquefaciens 17 in fresh and cooked samples, chicken meat may be considered their ecological niche. Because the vast majority of chilled chicken sold in Chinese supermarkets and wet markets is stored aerobically, the environment may enforces a selection pressure on the bacterial community, so those groups of bacteria that are best adapted to the environment will outgrow the others, become dominant, and reach high numbers. 3.4. TVBN and pH changes The evolution of TVBN and pH values is depicted in Fig. 4. The initial concentration of TVBN was 8.2 mg/100 g. Subsequently, TVBN increased rapidly in P. fragi H8 and P. fluorescens H5 compared with S. liquefaciens 17 and A. salmonicida 35 (Fig. 4A). At the end of storage, the concentrations of TVBN in P. fragi H8 and P. fluorescens H5 were higher than 50 mg/100 g. Meanwhile, the pH value was
Fig. 3. Microbiological changes during storage of raw (A) and cooked (B) chicken samples inoculated with A. salmonicida 35, P. fluorescens H5, P. fragi H8 and S. liquefaciens 17 stored at 8 C. Each data point and the error bars show the mean ± standard deviation of 4 replicates.
Enterobacteriaceae during storage of minced beef and pork at a chilled temperature (Doulgeraki et al., 2012). Moreover, in the current study, A. salmonicida 35 showed a weak growth ability in fresh samples, consistent with the results of Pennacchia et al. (2011), who detected Aeromonas spp. in chilled beef at the beginning of storage but found that these species disappeared after 7 days. However, in the cooked samples in the present study, A. salmonicida 35 also quickly reached approximately 9.4 log CFU/g. This good growth status on cooked meat was identical to that in a previous study (Mace et al., 2014), in which cooked tropical shrimp samples inoculated with A. salmonicida were considered to be strongly spoiled after 8 days of storage. This result revealed that A. salmonicida may be associated with cooked meat spoilage. Therefore, due to their strong occurrence of P. fragi H8 and
Fig. 4. TVBN (A) and pH (B) changes of raw chicken samples inoculated with A. salmonicida 35, P. fluorescens H5, P. fragi H8 and S. liquefaciens 17 stored at 8 C. Each data point and the error bars show the mean ± standard deviation of 4 replicates.
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initially 5.8. During storage, the pH values in P. fragi H8 and P. fluorescens H5 increased substantially, reaching values of approximately 7.4 (Fig. 4B). Similar to what was observed for TVBN, the final pH values in P. fragi H8 and P. fluorescens H5were significantly higher than the initial values. TVBN is widely used as an indicator of meat spoilage because its components result from microbial degradation of protein and nonprotein nitrogenous compounds, such as amino acids and nucleotide catabolites (Liu et al., 2013). The limiting concentration of TVBN in meats for human consumption in China (GB 16869-2005) is 15 mg/100 g muscle. In the present study, on the 4th day of storage, P. fragi H8 and P. fluorescens H5 had exceeded this limit and became the fastest group. According to the proteolytic and microbiological analysis, strain 35 exhibited the strongest proteolytic activity in vitro but the lowest growth rate on meat, whereas P. fragi H8 and P. fluorescens H5 performed strongly in both analyses, which is likely the main reason for the difference in TVBN. Meanwhile, the pH value of normal chicken breast is 5.7e6.1 (Barbut et al., 2005). Changes in the pH value of meat have been attributed to many factors. In any case, a high pH in meat leads to a more rapid spoilage process due to more rapid bacterial growth and nutrient consumption (Iulietto et al., 2015). It is well established that LAB metabolism produces compounds such as lactic acid, resulting in a slight decrease in pH, whereas in non-lactic cultures, the pH increases slightly (Pogacic et al., 2015). Furthermore, the majority of Pseudomonas spp. produce only one type of proteinase, typically neutral zinc metallo proteinases with pH optima ranging from 6.5 to 8 (Fairbairn and Law, 1986; Tarrant et al., 1973). In the current study, the growth of P. fragi H8 and P. fluorescens H5 resulted in significant increases in pH, ranging from 5.8 to 7.4, thus achieving the most suitable pH values for proteinases. This finding may also explain why P. fragi H8 and P. fluorescens H5 showed the highest growth rate and TVBN values on chicken. 3.5. Sensory analysis As shown in Fig. 5, chicken fillet freshness was excellent over the first two days, and the freshness characteristics diminished
Fig. 5. Spoilage scores of raw chicken samples inoculated with A. salmonicida 35, P. fluorescens H5, P. fragi H8 and S. liquefaciens 17 stored at 8 C for 7 days, as determined in the sensory panel. Each data point and the error bars show the mean ± standard deviation of the 5 panelists’ scores.
gradually over time. The differences in appearance and odor among the four isolates became apparent from the 4th day of storage. For example, samples inoculated with P. fragi H8 and P. fluorescens H5 exhibited slight surface slime, and A. salmonicida 35 displayed special off-odors, whereas S. liquefaciens 17 did not produce any typical characteristic slime or intense odors. After 6 days of storage, all samples were considered spoiled (Table 2, Fig. 5). Samples inoculated with P. fragi H8 and P. fluorescens H5 were considered grossly spoiled as they exhibited strong slime formation and emitted unpleasant odors that were described as “sour”, “fruity” and “cheesy”. In contrast, “grassy” and “sour” descriptors were cited for A. salmonicida 35. Slime production gives bacteria an advantage because slime constitutes a protective layer that keeps bacteria moist and allows €rkroth and microorganisms to grow at a lower temperature (Bjo Korkeala, 1997). This protection allows the bacteria to survive and to compete with other bacteria in meat. Consequently, the use of a low temperature for meat storage does not prevent the formation of slime, although refrigeration does result in longer product shelflife. In the present study, there were certain correlations between the sensory rejection time and the quantity of TVBN, in contrast to the conclusions of Joffraud et al. (2006) and Jaffres et al. (2011), who suggested that TVBN was not likely to be a good indicator of sensory detection of seafood spoilage. 3.6. VOC analysis Over the last few years, several studies have demonstrated a correlation between the release of spoilage VOCs and the growth of specific microbial species during meat storage (Argyri et al., 2015; Ercolini et al., 2009; Joffraud et al., 2001). Numerous VOCs detected in the present study have previously been reported in the literature as bacterial metabolites and are involved in the undesirable odor characteristics of the spoilage process. Without taking into account compounds that were present in trace amounts, a total of 25 VOCs were considered. Metabolites from aromatic amino acids and unsaturated fatty acids, such as alcohols (e.g., 1-hexanol, 1-heptanol and 1-octen-3-ol), aldehydes (e.g., nonanal, octanal and hexanal) and ketones (e.g., 2-pentanone, 2-heptanone, 2-nonanone), were detected in this study (Table 3). Specifically, in the headspace of A. salmonicida 35, the concentrations of 1-hexanol, hexanal, indole, 1-octen-3-ol and 2,3-butanediol were significantly higher (P < 0.05) than the concentrations of other compounds. These VOC profiles of A. salmonicida 35 were similar to those in the control group. A higher 1-hexanol level may et al., 2001). Meanwhile, associated with a “green” flavor (Falque indoles are produced by the bacterial decomposition of tryptophan and have been used as an indicator of spoilage in seafood (Sarnoski et al., 2010; Snellings et al., 2003). The formation of 2,3-butanediol is also associated with microbial activity during storage (Josephson et al., 1983; Ui et al., 1986). When samples were spoiled by P. fluorescens H5, the most significant compounds (P < 0.05) detected were 2-nonanone, 2-heptanone, 2-undecanone and acetic acid. This group also produced 2-octenyl acetate, which was not produced by the other groups. For P. fragi H8, 2-heptanone, 2nonanone, 2-heptanol, 3-methyl-1-butanol, 2-nonanol, 1undecene and acetic acid were detected. P. fluorescens H5 and P. fragi H8 exhibited similar increased concentrations of volatile compound profiles. In this study, 2-heptanone and 2-nonanone were the two most abundant compounds found in both samples. Casaburi et al. (2015) found that the presence of ketones in stored fresh meat is mainly associated with the presence of Pseudomonas spp., Carnobacterium spp. and Enterobacteriaceae. 2-Heptanone and 2-nonanone were also detected in meat contaminated with Pseudomonas spp., as reported by Joffraud et al. (2001) and Leroy et al.
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Table 2 Sensory evaluation and odors assigned to samples of chicken breast fillets on the 6th day following inoculation with the bacterial isolates. Bacterial isolate
Sensory evaluation
Off-odor characteristics
P. fragi H8 P. fluorescens H5 A. salmonicida 35 S. liquefaciens 17
Extremely unacceptable: juice loss, off-odor, slime Very much unacceptable: juice loss, off-odor, slime Moderately unacceptable: juice loss, off-odor Moderately unacceptable: juice loss, slightly off-odor
Sour/fruity/cheesy Sour/fruity/cheesy Sour/grassy Garlicky/cabbage-like
Table 3 Volatile organic compounds (VOCs) identified in inoculated samples after 7 days of storage at 8 C. Volatile compound
Alcohols 3-Methyl-1-butanol 1-Hexanol 1-Octen-3-ol 2-Nonanol 2,3-Butanediol 2-Heptanol 1-Heptanol 1-Pentanol Aldehydes Nonanal Hexanal Octadecanal Octanal Ketones 2-Pentanone 2-Heptanone 2-Nonanone Acetoin 2-Undecanone 2,3-Octanedione Other 1-Undecene Acetic acid Dimethyl disulfide 2,5-Dimethylpyrazine Dimethyl trisulfide 2-Octenyl acetate Indole
Control
Meat inoculated with A. salmonicida 35
P. fluorescens H5
P. fragi H8
S. liquefaciens 17
<1 26.42 ± 0.83a 8.75 ± 1.64c <1 2.50 ± 1.05ef <1 1.65 ± 0.19f 1.33 ± 0.16f
<1 31.84 ± 2.15a 10.48 ± 0.84d <1 6.20 ± 1.23e <1 1.19 ± 0.46g 2.20 ± 0.37fg
2.62 ± 0.78c <1 <1 1.96 ± 0.24c <1 <1 <1 <1
2.95 ± 0.67e <1 <1 7.04 ± 0.36d <1 16.75 ± 0.82c <1 <1
10.93 ± 1.66a 1.58 ± 0.58d 4.43 ± 2.2c 1.03 ± 0.41d 1.31 ± 0.06d <1 <1 1.25 ± 0.21d
4.34 ± 0.83d 19.06 ± 1.56b <1 1.68 ± 0.39f
2.19 ± 0.65fg 18.56 ± 1.88b 4.47 ± 1.06ef 1 ± 0.26g
<1 <1 <1 <1
<1 <1 <1 <1
1.95 ± 0.14cd <1 <1 <1
<1 <1 <1 3.71 ± 0.48de <1 2.09 ± 0.52ef
<1 <1 <1 1.74 ± 1.19fg <1 2.17 ± 0.36fg
<1 7.48 ± 0.52b 38.59 ± 2.24a <1 7.32 ± 1.27b <1
<1 31.83 ± 2.34a 19.94 ± 1.44b <1 <1 <1
3.03 7.99 1.69 4.53 <1 <1
<1 <1 <1 <1 <1 <1 <1
<1 4.31 ± 0.29ef <1 <1 <1 <1 14.46 ± 2.23c
<1 7.11 ± 2.55b <1 <1 <1 7.38 ± 0.85b <1
3.71 ± 1.98e 4.49 ± 1.23e <1 <1 <1 <1 <1
<1 <1 12.84 ± 1.18a 12.93 ± 2.77a 1.37 ± 0.23d <1 <1
± ± ± ±
1.28cd 1.31b 0.48d 1.94c
Values are expressed as the mean ± standard deviation of the area ratio (%) of the volatile compounds identified in each inoculated sample group (n ¼ 3). aeg: Values with different lower case letters in superscript, within a column, are significantly different (P < 0.05).
(2009). However, acetoin, the ketone most often found in fresh meat, was not found in this study. Alterations by S. liquefaciens 17 led to the production of 3-methyl-1-butanol, 2-nonanol, 2pentanone, 2-heptanone and 2-nonanone. This strain also produced dimethyl disulfide, 2,5-dimethylpyrazine and dimethyl trisulfide which were not produced by the other groups. 3-Methyl1-butanol is one of the most common alcohols found in spoiled raw meat and is produced from leucine catabolism (Smit et al., 2005). This alcohol can be associated with a whiskey-like odor (Larrouture-Thiveyrat et al., 2003), and the presence of its corresponding ketones may enhance the odor intensity because the odor intensities of compounds with similar odor qualities has been reported to be additive (Meilgaard and Peppard, 1986). The formation of sulfur compounds and 3-methyl-1-butanol during spoilage has also been reported in beef samples containing S. liquefaciens, indicating that these VOCs may be associated with Serratia metabolism (Hernandez-Macedo et al., 2012). In this work, the spoilage potential of 4 isolates, as assessed based on proteolytic and lipolytic activities in vitro and growth kinetics, biochemical changes, sensory alterations and VOC patterns in situ, exhibited different profiles than the same species reported in previous studies because these tests were individually based, and the test strains were preliminarily screened in an RJA
assay. Our results indicate that the spoilage potential of strains was species, strain and food matrix dependent. 4. Conclusion These findings have contributed to the characterization of the spoilage potential of bacterial species isolated from chilled yellowfeather chicken meat using RJA. In vitro, A. salmonicida 35 presented the strongest proteolytic capacity against myofibrillar protein, whereas P. fragi H8 led to a strong occurrence, rapid increases in TVBN and pH, off-odors and serious slime formation in situ. Therefore, it is likely that P. fragi H8 plays a significant role in the spoilage of chilled chicken. Furthermore, the availability of RJA may be useful for effective analyses in studies aimed at identifying the potential spoilage bacteria during the natural spoilage of meat and meat products. Nevertheless, because the present study was performed under pure culture conditions, further investigations are needed to determine spoilage potential in mixed cultures. Acknowledgments This study was supported by China Agriculture Research System (CARS-42) funded by the China Ministry of Agriculture.
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