Changes in the bacterial communities of vacuum-packaged pork during chilled storage analyzed by PCR–DGGE

Changes in the bacterial communities of vacuum-packaged pork during chilled storage analyzed by PCR–DGGE

Meat Science 86 (2010) 889–895 Contents lists available at ScienceDirect Meat Science j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m /...

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Meat Science 86 (2010) 889–895

Contents lists available at ScienceDirect

Meat Science j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m e a t s c i

Changes in the bacterial communities of vacuum-packaged pork during chilled storage analyzed by PCR–DGGE Y. Jiang a,b, F. Gao a,c, X.L. Xu a, Y. Su c, K.P. Ye a, G.H. Zhou a,⁎ a b c

National Center of Meat Quality and Safety Control, Nanjing Agricultural University, Nanjing 210095, PR China Ginling College, Nanjing Normal University, Nanjing 210097, PR China College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, PR China

a r t i c l e

i n f o

Article history: Received 14 September 2009 Received in revised form 8 December 2009 Accepted 14 May 2010 Keywords: Vacuum-packaged pork Bacterial diversity Lactic acid bacteria PCR–DGGE

a b s t r a c t In this study, PCR-denaturing gradient gel electrophoresis (DGGE) was used to investigate the bacterial communities of vacuum-packaged pork during chilled storage. Eight kinds of lactic acid bacteria (LAB) were identified from the strains isolated from MRS plates by PCR–DGGE of the V3 region, and Lactobacillus sakei was the representative isolate at the end of the monitoring. By means of the direct meat analysis of PCR–DGGE, LAB increased gradually and Carnobacterium sp./Car. divergens, Lactobacillus sakei and Lactococcus sp./Lc. piscium, became the predominant bacteria at the end of the storage. The results of Lactobacillus-specific PCR and DGGE showed that different Lactobacillus populations were present at different storage periods and Lb. sakei became the predominant bacteria in the end. In conclusion, the PCR–DGGE technique as a culture-independent method is applicable to monitoring bacterial population dynamics in vacuum-packaged pork. © 2010 The American Meat Science Association. Published by Elsevier Ltd. All rights reserved.

1. Introduction Meat is easily contaminated to serve as substrate for various spoilage and pathogenic microorganisms due to its high water content and abundance of essential nutrients (Jackson, Acuff, & Dickson, 1997; Hilario, Buckley, & Young, 2004). Thus in practice, the proper storage method for raw meat needs to be developed to protect from this bacterial contamination. Vacuum packaging under chilled conditions has been proved to be very effective in extending the shelf life of perishable foods, such as fresh meat and meat products, and preventing the growth of food-borne pathogens (Church & Parsons, 1995). The composition of the bacterial flora of meat and the development of the spoilage flora under different storage conditions have been traditionally studied on the basis of cultivation, isolation and identification by phenotypic means (Nápravníková, Vorlová, & Malota, 2002; Blixt & Borch, 2002). Alternative molecular methods, independent of cultivation, have become a very important tool in the study of microbial communities because they are believed to overcome problems associated with selective cultivation and isolation of bacteria from natural samples. Denaturing gradient gel electrophoresis (DGGE) is perhaps the most commonly used among the culture-independent fingerprinting techniques (Ercolini, 2004). In the last decade, DGGE has been successfully applied to investigate the microbiology of fermented foods (Ampe, Omar, Moizan, Wacher, & Guyot, 1999; Cocolin,

Manzano, Aggio, Cantoni, & Comi, 2001; Cocolin, Manzano, Cantoni, & Comi, 2001; Temmerman, Masco, Vanhoutte, Huys, & Swings, 2003). Besides, several studies have exploited the DGGE/TGGE technique for identification of LAB isolated from beer and fermented sausages (Tsuchiya, Kano, & Koshino, 1994; Cocolin, Manzano, Cantoni, & Comi, 2000). As for fresh meat, there are several studies on beef (Ercolini, Russo, Torrieri, Masi, & Villani, 2006; Fontana, Cocconcelli, & Vignolo, 2006; Russo, Ercolini, Mauriello, & Villani, 2006), but the DGGE analysis on pork is still few. More recently, methods for qualitative analysis of specific populations with a complex ecosystem based on genus-specific PCR and DGGE have been developed (Satokari, Vaughan, Akkermans, Saarela, & de Vos, 2001; Heilig et al., 2002; Randazzo, Torriani, Akkermans, de Vos, & Vaughan, 2002). Until now, the changes of Lactobacillus-like communities in vacuum-packaged chill-stored pork analyzed by means of group-specific PCR and DGGE were rarely reported. Therefore, the objective of this study was using PCR–DGGE to evaluate the changes of bacterial communities, to identify LAB in vacuum-packaged pork during chilled storage. Meanwhile, Lactobacillus group-specific PCR combined with DGGE analysis were used to investigate the changes of Lactobacillus-like communities in vacuumpackaged pork during chilled storage. 2. Materials and methods 2.1. Sampling

⁎ Corresponding author. Tel.: +86 25 84395376. E-mail address: [email protected] (G.H. Zhou).

Three pork loins, labeled as group A, B and C, were removed from pork carcasses at 24 h post-mortem in a commercial meat plant. Each

0309-1740/$ – see front matter © 2010 The American Meat Science Association. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2010.05.021

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loin was divided into six steaks, and steaks originating from the same loin were rubbed together in order to spread the naturally acquired contamination evenly. They were transported to the laboratory on ice in cooler boxes within 30 min. Each steak was individually placed in a sterile plastic bag (Hongbaoli Package Company, China; O2 transmission rate, 30 cm3 m− 2 atm− 1 24 h− 1 at 23 °C) and vacuum-packaged using a vacuum-packaging machine (DZQ500/2SB, Zhejing Baochun Package Equipment Plant, China). The vacuum-packaged steaks were stored at 4 °C. One pack from each group was analyzed at zero time and days 3, 6, 14, 18 and 23 of storage, respectively. 2.2. Enumeration and isolation of microorganisms Twenty five grams of each sample was homogenized in 225 ml of sterile peptone saline (8 g/l NaCl, 1 g/l bacteriological peptone, LuQiao Company, Beijing, China). After shaking at 230 rpm for 10 min with a stomacher, further decimal dilutions were made, and the following analyses were carried out on duplicate agar plates: (a) mesophilic aerobic bacterial counts on Plate Count Agar (PCA, LuQiao Company, Beijing, China) incubated at 30 °C for 48 h; (b) LAB on de Man Rogosa Sharpe agar (MRSA, LuQiao Company, Beijing, China) incubated with a double layer at 30 °C for 48 h; (c) Enterobacteriaceae on Violet Red Bile Glucose agar (VRBGA, LuQiao Company, Beijing, China) incubated at 37 °C for 24 h. After counting, means and standard deviations were calculated. Five to ten LAB strains from MRS plates for each sample were randomly selected, streaked on MRS agar three times, and stored at − 20 °C in MRS broth containing 30% glycerol. All isolates were examined by performing Gram staining and catalase tests. Grampositive and catalase-negatives strains were considered LAB. 2.3. DNA extraction of pure LAB cultures Two milliliters of a 24 h culture were centrifuged at 12,000 ×g for 10 min at 4 °C to pellet the cells, which were subjected to DNA extraction using Axyprep Bacterial Genomic DNA Miniprep kit (Axygen, USA) according to the manufacturer's instructions. 2.4. DNA extraction from pork From each sampling point, 10 g pork samples of each group were homogenized in a stomacher tube with 100 ml of saline peptone water, followed by agitation for 30 min at room temperature. The tubes were centrifuged for 5 min at 200 ×g in order to separate the debris. The supernatant (30 ml) was transferred to a 50 ml sterile centrifuge tube, and a second centrifugation was carried out at 12,000 ×g for 10 min. The pellet was resuspended in 150 μl of Buffer S of Axyprep Bacterial Genomic DNA Miniprep kit (Axygen, USA) and transferred to a sterile 2 ml tube. Twenty microlitres of lysozyme (50 mg/ml) (Sigma) was added and incubated for 1 h at 37 °C. Thirty microlitres of proteinase K (10 mg/ml) (Tiangen, China) was added and incubated for 2.5 h at 55 °C. The preparations were subjected to DNA extraction using Axyprep Bacterial Genomic DNA Miniprep kit (Axygen, USA) according to the manufacturer's instructions. 2.5. Polymerase chain reaction All primers used in this study are listed in Table 1. DNA of LAB was amplified with primers GC338f and 518r (Table 1) spanning the V3 region of the 16S rRNA gene (Muyzer, De Wall, & Uitterlinden, 1993; Ampe et al., 1999; Ampe & Miambi, 2000). GoTaq Green Master Mix (Promega, USA) was used in the PCR reaction. The amplification were carried out with a Mastercycler ep (Eppendorf, Germany) by using a final volume of 25 μl containing GoTaq Green Master Mix (2×) 12.5 μl, each primer 1 μl (0.4 μM), DNA template 1 μl, and ddH2O 9.5 μl. The template DNA was denatured for 5 min at 94 °C. To increase the specificity of the amplification and reduce the

Table 1 Primers used in this study. Primer

a

8f 798r 338f b 518r P1 P4 Bact0011f Lab0677r Lab0159f Uni0515r b GC clamp a b

Primer sequence (5′ to 3′)

Reference

GGA GAG TTT GAT C(A/C)T GGC T CCA GGG TAT CTA ATC CTG TT ACT CCT ACG GGA GGC AGC AG ATT ACC GCG GCT GCT GG GCG GCG TGC CTA ATA CAT GC ATC TAC GCA TTT CAC CGC TAC AGA GTT TGA T(C/T) (A/C) TGG CTC AG CAC CGC TAC ACA TGG AG GGA AAC AG(A/G) TGC TAA TAC CG ATC GTA TTA CCG CGG CTG CTG GCA CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG G

Li et al., 2006 Li et al., 2006 Ampe et al., 1999 Muyzer et al., 1993 Klijn et al., 1991 Klijn et al., 1991 Lane, 1991 Heilig et al., 2002 Heilig et al., 2002 Lane, 1991 Muyzer et al., 1993

f, forward; r, reverse. The GC clamp was attached to the 5′ end of the primer.

formation of spurious byproducts, a “touchdown” PCR was carried out. The initial annealing temperature of 65 °C was 10 °C above the expected annealing temperature and decreased 0.5 °C every second cycle until the touchdown temperature of 55 °C was reached, 15 additional cycles were then carried out at 55 °C. A denaturation step of 94 °C for 1 min was used and extension was carried out at 72 °C for 3 min with a final extension at 72 °C for 10 min. The reaction mixture was then cooled to 4 °C. Nested PCR and touchdown PCR were used to amplify the V3 region of 16S rRNA gene of DNA extracted directly from pork. In the first round of PCR, the primers 8f and 798r (Table 1) were designed to amplify the approximate 800 bp 16S rRNA gene fragments (Li, He, Wu, & Jiang, 2006). The amplification reactions were carried out in a 25 μl reaction volume containing GoTaq Green Master Mix (2×) 12.5 μl, each primer 2.5 μl (1 μM), DNA template 1 μl, and ddH2O 6.5 μl. The following PCR program was used: initial denaturation at 95 °C for 2 min, 25 cycles of denaturation at 94 °C for 1 min, annealing at 58 °C for 1 min, extension at 72 °C for 2 min, and final extension at 72 °C for 2 min and cooling to 4 °C. The presence of PCR products was verified on a 1.2% agarose gel. In order to eliminate the remaining oligonucleotides and original template DNA, the purification of the amplicons was performed using the Wizard SV Gel and PCR Clean-up System (Promega, USA) according to the manufacturer's instructions. Subsequently, a second PCR was performed, using the purified amplicons of the first PCR as template DNA (Temmerman et al., 2003). The second round of touchdown PCR was carried out using primers GC338f and 518r spanning the V3 region of the 16S rRNA gene according to the amplification protocols for DNA of LAB, as described above. The Lactobacillus group-specific primer Lab0677r and Bact0011f (Table 1) were used to amplify the V1–V3 region of 16S rRNA gene of DNA extracted directly from pork, and the resulting 700 bp amplicons were used as templates in nested PCR with primers Lab0159f and Uni0515-GCr (Heilig et al., 2002). Amplification reactions were carried out in a 25 μl reaction volume containing GoTaq Green Master Mix (2×) 12.5 μl, each primer 2.5 μl (1 μM), DNA template 1 μl, and ddH2O 6.5 μl. The PCR program was performed according to the specification of Heilig et al. (2002). Primer Lab0677r is specific for members of the Lactobacillus group (Heilig et al., 2002) and was used to investigate the changes of Lactobacillus-like community in vacuumpackaged pork during refrigerated storage. DNA extracted from pure LAB strains isolated in this study was amplified with the primers Lab0159f and Uni0515-GCr using the corresponding conditions described as above. The PCR product (5 μl) was analyzed by electrophoresis in a 1.2% agarose gel. 2.6. DGGE analysis The Dcode universal mutation detection system (Bio-Rad, Richmond, California) was used for DGGE analysis of the PCR products

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obtained from single cultures and directly from vacuum-packaged pork according to the specifications of Muyzer et al. (1993) with the following modifications. The PCR products were loaded onto 8% (wt/ vol) polyacrylamide gels (acrylamide/bisacrylamide = 37.5:1) in 0.5× TAE with a 35–55% gradient of urea and formamide (UF) increasing in the direction of parallel electrophoresis. Gradients of 30–60% were used for the separation of the Lactobacillus-specific amplicons, as described previously (Heilig et al., 2002). A 100% denaturant acrylamide was defined as 7 M urea and 40% formamide. Electrophoresis was performed for 10 min at 200 V and then for 16 h at 85 V at a constant temperature of 60 °C. Gels were stained with ethidium bromide (0.5 mg/L) for 10 min, then rinsed for 20 min in milli-Q water and photographed with UV transillumination using the GelDoc 2000 system (Bio-Rad). The DGGE profiles obtained were analyzed by clustering via the unweighted pair group method with arithmetic mean (UPGMA; Dice coefficient of similarity), using the Quantity One software package (Bio-Rad).

2.7. Sequencing of LAB strains and DGGE bands LAB strains with the same DGGE profiles were grouped and representatives of each group were amplified using primers P1 and P4 as described by Klijn, Weerkamp, & de Vos (1991), targeting a 700 bp region of the V1–V3 16S rRNA gene. After purification, products were sent to a commercial sequencing facility (Invitrogen, Shanghai, China). Small pieces of selected DGGE bands were excised with a sterile scalpel. The pieces were then each transferred into 20 μl of sterile water and incubated overnight at 4 °C to allow diffusion of the DNA. Two microlitres of the eluted DNA was re-amplified using the conditions described above. The PCR products were analyzed by DGGE to confirm that the expected products had been isolated. Those samples yielding a single band co-migrating with that of the original sample were then excised and amplified with the primer without the GC clamp, purified, and sent to Invitrogen Company (Shanghai, China) for sequencing. Sequences were aligned with those in GenBank with the Blast program (Altschul et al., 1997) to determine the closest known relatives of the partial 16Sr RNA gene sequences obtained.

2.8. Nucleotide sequence accession numbers The nucleotide sequences determined in this study have been deposited in the GenBank database. The accession numbers for the 16S rRNA gene partial sequences of the eight LAB isolates are EU621982 through EU621989. The accession numbers for the nucleotide sequences obtained from the DGGE bands are: EU621990, EU621991, EU621992, EU621993, EU621996, EU621997, EU621998, EU621999 and GQ161949.

3. Results 3.1. Bacterial enumeration Bacterial counts of vacuum-packaged pork during chilled storage are shown in Table 2. Total aerobic counts increased their initial value with one log within the first 6 days, while LAB counts were one log lower approximately at time zero and also grew to 4 log cfu g− 1 at day 6. After 6 days both of them increased gradually and reached to over 7 log cfu g− 1 at the end of the monitoring. Enterobacteriaceae increased slowly, and the maximum number was just 5 log cfu g− 1. The numbers of Enterobacteriaceae varied between replicates, giving a high standard deviation.

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3.2. Differentiation of LAB by DGGE analysis A total of 130 isolates showed gram-positive staining and were catalase negative. After DNA extraction and amplification with primers GC338f and 518r targeting the V3 region of the 16S rRNA gene, all of the strains gave the PCR products of the expected size (230 bp). Analysis of amplicons on a DGGE gel with a denaturing gradient from 35% to 55% showed eight different profiles (Fig. 1). By means of sequencing and alignment of partial 16S rRNA gene generated with primer pair P1–P4, the representative strains with different DGGE profiles were identified as Lactococcus lactis subsp. lactis (band A), Carnobacterium divergens (band B), Lactobacillus curvatus (band C), Lb. sakei (band D), Car. sp. (band E), Leuconostoc mesenteroides (band F), Weissella cibaria (band G), and Wei. viridescens (band H), respectively. The relative identification obtained by alignment in GenBank and accession numbers for the submitted sequences are reported in Table 3. Total LAB isolates were grouped on the basis of the migration profiles (Table 4). At day 0 and 3, Wei. viridescens were most frequently encountered and the percentage was 47% and 80%, respectively. The dominating species were Car. divergens from day 6 to 18. Lb. sakei increased gradually from day 6, and became the most frequently found species (71.4%) at the end of the monitoring. 3.3. Direct analysis of pork by DGGE A low product yield was observed for the amplification of DNA extracted directly from pork samples of day 0 and 3, by using a single step PCR with the primers GC338f and 518r, and therefore we opted for the use of a nested PCR approach. The DGGE results of the microbial community which had developed in vacuum-packaged pork were reported in Fig. 2. The bands marked with letters in Fig. 2 were sequenced after re-amplification, and the relative identification obtained by alignment in GenBank is reported in Table 3. Other weak bands were not labeled. As shown in Fig. 2, when a denaturing gradient of 35–55% was applied to a 230 bp PCR product, a fluctuation in the population was observed. At day 0, a high microbial diversity was noticed but only bands a, b and c were with high intensity. Bands a, b and c were identified as Serratia marcescens/Serratia sp./Pseudomonas fluorescens, Ser. marcescens/Pseudomonas sp., and Achromobacter xylosoxidans/Achromobacter sp., respectively, and their intensity began to decrease after 6 days of storage. At day 6, the intensity of some other bands increased and became the dominant ones in the latter period of storage. Band d of group B, identified as Brochothrix sp./B. thermosphacta, was increased from day 6 to day 18. Band h of group B, identified as Lactobacillus algidus, increased intensity from day 14 till day 18. Bands e, f and g of group B, identified as Carnobacterium sp./ Car. divergens, Lb. sakei and Lactococcus sp./Lc. piscium, respectively, became the dominant bands at the end of the monitoring. The DGGE profiles of three groups were not exactly the same, while there was similar trend in the changes of bands among three groups during the whole storage. The high similarities were revealed by the cluster analysis among three samples from group A, B and C at day 0, and the similarity indices were above 80% (Fig. 3). All samples but the sixth day sample of the group B in the former period of the storage performed a coherent cluster with similarity indices above 76%. The low similarities were obtained between the former phase and the latter phase of storage in samples with similarity indices of 21% (Fig. 3). 3.4. Population dynamics and diversity of the Lactobacillus community The diversity and population dynamics of the Lactobacillus community were investigated with primer Lab0677r by using nested PCR and DGGE on DNA extracted directly from the vacuum-packaged

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Table 2 Microbial growth (log cfu g− 1 ± SD) of the vacuum-packaged pork during chilled storage. Microorganisms

Day 0

Day 3

Day 6

Day 14

Day 18

Day 23

Aerobic bacteria Lactic acid bacteria Enterobacteriaceae

3.77 ± 0.11 2.85 ± 0.10 2.98 ± 0.23

4.28 ± 0.07 3.16 ± 0.14 3.11 ± 0.37

4.64 ± 0.11 4.58 ± 0.16 3.98 ± 0.44

6.28 ± 0.03 6.11 ± 0.01 5.01 ± 0.21

6.83 ± 0.18 6.69 ± 0.11 5.58 ± 0.12

7.56 ± 0.09 7.51 ± 0.04 5.23 ± 0.24

pork during chilled storage. At days 0 and 3, some bands with very low intensity were observed, including band L. Several bands with high intensity (band I, J and K) were detected at day 6. After 6 days of storage, band K became more intense while other bands turned to fainter. Band K became the most intense band (band M) at the end of the monitoring (Fig. 4). The DGGE profiles of the three groups were similar, and the result of group B was shown in Fig. 4. To identify the origin of the bands in the Lactobacillus DGGE patterns of vacuum-packaged pork, DNA extracted from eight pure LAB strains isolated in this study were amplified with primers Lab0159f and Uni0515-GCr. The PCR products were performed DGGE together with the amplicons of vacuum-packaged pork (Fig. 4). The PCR of eight pure isolates and their amplicans was successful performed and subsequently separated by DGGE. There were several bands for the strain of Weissella cibaria (band G). Bands I, J, K and L were identified by comparing their relative position of migration in the acrylamide gel with the DGGE profiles of the control strains such as Wei. viridescens (band H), Wei. cibaria (band G), Lb. sakei (band D) and Car. sp. (band E), respectively. Other bands with low intensity were not labeled. In order to verify the correspondence of bands, band M was excised from the acrylamide gel, re-amplified with primers Lab0159f and Uni0515r for sequence. The band was subsequently identified as Lb. sakei, and its sequence exhibited 99.7% identity with the sequence in the GenBank databases (Table 3). 4. Discussion In the last decades, using molecular approaches and direct sampling of the DNA in complex microbial systems is a fast-moving research area. In this study, PCR–DGGE was used to study the changes of bacterial communities in vacuum-packaged pork during chilled storage.

plating method (Egan, 1983; Boers, Dijkmann, & Wijngaards, 1994; Jeremiah, Gibson, & Argnosa, 1995; Borch, Kant-Muermans & Blixt, 1996; Nápravníková et al., 2002), the current study confirmed that LAB increased gradually and became the dominant bacteria later. 4.2. DGGE analysis of LAB isolates At present, several studies have exploited the DGGE technique for identification of LAB isolated from food such as beer, fermented sausages, the food environment, and so on (Tsuchiya et al., 1994; Cocolin et al., 2000; Ercolini, Moschetti, Blaiotta & Coppola, 2001). In the current study, LAB isolated from pork were identified by amplification of the V3 region of the 16S rRNA gene and showed eight different DGGE profiles, allowing them to be immediately distinguished. The genera of LAB most frequently encountered on vacuum or modified atmosphere packaged (MAP) meat are Lactobacillus, Leuconostoc and Carnobacterium.(Dainty & Mackey, 1992; McMullen & Stiles, 1993). Lb. sakei was considered as representative isolate of psychrotropic microbial flora on vacuum-packed meat and meat product (Gill, 1983; Hugas, 1998; Samelis, Kakouri, & Rementzis, 2000), on the basis of traditional cultivation and isolation methods. Yost & Nattress (2000) used multiplex PCR to identify LAB strains isolated from vacuum-packed pork stored at 2 °C and Lb. sakei and Leuconostoc spp. was considered as representatives. By means of PCR– DGGE analysis of bulk colonies from selective media, an alternative to isolation (Ercolini, 2004), Weissella hellenica was found among the dominant LAB population in the last spoilage phase of MAP beef stored at 5 °C (Ercolini et al., 2006). Strains of Lactococcus lactis, an organism traditionally associated with dairy and vegetable products, were also isolated from raw pork (Garver & Muriana, 1993), cooked poultry meat (Barakat, Griffiths, & Harris, 2000), fermented sausages (Rodriguez et al., 1995) and vacuum-packed seafood (Mauguin &

4.1. Enumeration of microorganisms In agreement with previous studies on the shelf life of vacuumpackaged chilled stored (≤4 °C) pork conducted by the traditional

Table 3 Strains and DGGE bands identified in this study by means of partial 16S rRNA gene sequencing. Bands A B C D E F G H a b c d e

Fig. 1. DGGE fingerprints of PCR products originated with gc338f-518r primer set from the strains isolated in this study. A, Lactococcus lactis subsp. lactis; B, Carnobacterium divergens; C, Lactobacillus curvatus; D, Lactobacillus sakei; E, Carnobacterium sp.; F, Leuconostoc mesenteroides; G, Weissella cibaria; H, Weissella viridescens. The positions of the bands are indicated on the right.

f g h M a

a

Closest relative

Length (bp)

Identity (%)

Accession no.

Lactococcus lactis subsp. lactis Carnobacterium divergens Lactobacillus curvatus Lactobacillus sakei Carnobacterium sp. Leuconostoc mesenteroides Weissella cibaria Weissella viridescens Serratia marcescens/Serratia sp./ Pseudomonas fluorescens Serratia marcescens/Pseudomonas sp. Achromobacter xylosoxidans/ Achromobacter sp. Brochothrix sp./Brochothrix thermosphacta Carnobacterium sp./Carnobacterium divergens Lactobacillus sakei Lactococcus sp./Lactococcus piscium Lactobacillus algidus Lactobacillus sakei

674 683 688 700 631 677 709 707 197

100 99.0 100 100 100 99.7 95.3 99.7 100

EU621982 EU621983 EU621984 EU621985 EU621986 EU621987 EU621988 EU621989 EU621990

198 197

100 100

EU621991 EU621992

200

100

EU621993

192

100

EU621996

199 199 166 394

100 100 100 99.7

EU621997 EU621998 EU621999 GQ161949

Bands are lettered as indicated on the DGGE gels shown in Figs. 1, 2 and 4.

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Table 4 Identification results obtained from the LAB isolated from the vacuum-packaged pork during different storage. Identification

No. of isolates (percentage) Day 0

Lactococcus lactis subsp. lactis Carnobacterium divergens Lactobacillus curvatus Lactobacillus sakei Carnobacterium sp. Leuconostoc mesenteroides Weissella cibaria Weissella viridescens Total

Day 3

4 (23.5)

Day 6 1 (5.3) 9 (47.4) 1 (5.3)

1 (5.9) 2 (11.8) 2 (11.8) 8 (47.0) 17 (100)

4 (20.0) 16 (80.0) 20 (100)

Novel, 1994). In the current study, the LAB strains isolated from pork with different DGGE profiles were identified as Lc. lactis subsp. lactis, Car. divergens, Lb. curvatus, Lb. sakei, Car. maltaromaticum, Leu. mesenteroides, Wei. cibaria and Wei. Viridescens, respectively. The composition of the LAB community varied at different stages of storage, and Lb. sakei was the dominant species at the end of storage.

4.3. DGGE analysis of the 16S V3 amplicons from microbial DNA directly extracted from meat samples To increase sensitivity, nested PCR has been used in many studies of low abundance organisms (Gibbons & Awad-El-Kariem, 1999; Verdin, Saillard, Labble, Bove, & Kobisch, 2000). In this study, the PCR product yield of DNA extracted directly from pork at early stages of storage was low, may be due to the lower bacterial concentration in pork samples, and the nested PCR yielded enough product for the further DGGE analysis. The microflora of vacuum-packaged chill-stored pork has been extensively studied and it is generally accepted that LAB are the predominant bacteria in the spoilage microflora (Boers et al., 1994; Jeremiah et al., 1995; Blixt & Borch, 2002; Holley, Peirson, Lam, & Tan, 2004). Other bacteria on vacuum-packaged chill-stored pork grow limitedly or slowly Dainty & Mackey (1992). In this study, the direct meat analysis by PCR–DGGE also indicated that LAB, such as

2 (10.5) 2 (10.5) 4 (21.0) 19 (100)

Day 14

Day 18

18 (69.3)

11 (55.0) 3 (15.0) 6 (30.0)

20 (71.4)

20 (100)

28 (100)

6 (23.1) 1 (3.8)

Day 23 8 (28.6)

1 (3.8) 26 (100)

Carnobacterium sp./Car. divergens (band e), Lb. sakei (band f) and Lactococcus sp./Lc. piscium (band g), increased rapidly in the later phases of storage and became the predominant bacteria (Fig. 2). Lc. piscium was consistently detected in vacuum packed, refrigerated beef in Japan when incubation of the plated samples was a 7 °C for 10– 14 days (Sakala, Hayashidani, Kato, Hirata, & Makino, 2002). In addition, Lb. algidus was detected in chilled vacuum-packaged pork only in the middle phase of storage in this study although it was reported as one of the predominant species in chilled vacuumpackaged beef (Sakala et al., 2002). Serratia, belonging to Enterobacteriaceae, was higher at early stages of storage but decreased after 6 days, while Brochothrix sp./B. thermosphacta was detectable in the middle of the storage but was difficult to be observed at the last sampling point, which confirmed that these spoilage bacteria were unable to compete against lactic acid bacteria in chill-stored meat under anaerobic conditions (Collins-Thompson, & Lopez, 1980; Sakala et al., 2002; Holley et al., 2004; Russo et al., 2006).

4.4. Lactobacillus group-specific PCR–DGGE The diversity and population dynamics of the Lactobacillus community were further investigated by using group-specific PCR and DGGE. Eight pure LAB strains isolated in this study were performed with DGGE simultaneously. Eight different LAB isolates, amplified with primers Lab0159f and Uni0515-GCr, could be

Fig. 2. DGGE profiles of the bacterial community from DNA directly extracted from vacuum-packaged pork samples of three groups at storage time 0, 3, 6, 14, 18 and 23 days. Bands indicated by letters a to h were excised and, after re-amplification, subjected to sequencing. The bands are discussed in the text, and the positions of the bands are indicated on the right.

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Considering that LAB strains were randomly selected from traditional MRS plates and Lb. algidus strain failed to grow at temperatures N30 °C in MRS agar (Sakala et al., 2002), it is not surprising that Lb. algidus strain was only observed in Fig. 2 (band h) while was not found in Fig. 1. Interestingly, some LAB obtained in Fig. 1, such as Leu. mesenteroides (band F), Wei. cibaria (band G), and Wei. viridescens (band H), were not observed in Fig. 2. This may be either due to the fact that these bacteria concentrations are too low to detect by the direct PCR–DGGE analysis from pork or to that these bacteria produce weak bands (Fig. 2), which were not analyzed in this study. It is noticed that the population of Lactobacillus species detected by DGGE in Fig. 2 was not the same as that in Fig. 4. Lb. algidus was identified easily in the middle of the stage in Fig. 2 (band h) while there was only one band with high intensity corresponding to Lb. sakei observed in Fig. 4. The exact reason is not known but the selective amplification of 16S rRNA gene may contribute to this discrepancy.

5. Conclusions Fig. 3. Cluster analysis of molecular banding patterns generated by PCR–DGGE of Fig. 2. The dendrogram was obtained with the UPGMA clustering algorithm. Percentage similarity between different samples is shown on the scale above the dendrogram.

differentiated well. It is noteworthy that several bands for a species in a DGGE profile do not necessarily represent different strains of that species due to the presence of multiple copies of the ribosomal genes and the fact that the gene copies have evolved differently (Ueda, Seki, Kudo, Yoshida, & Kataoka, 1999). Heilig et al. (2002) found that a reverse primer, Lab0677r, targeting E. coli positions 677 to 693, would specifically encompass Lactobacillus, Leuconostoc, Weissella, Pediococcus, and Aerococcus. An interesting Lactobacillus DGGE patterns of vacuum-packaged pork was obtained in the present study. The Lactobacillus community was limited in the early phase, but increased rapidly in the later phases, especially Lb. sakei, which became the predominant bacterium at the end of the monitoring. The results obtained from this study are in agreement with previous studies, which underline how Lb. sakei association largely dominates fresh beef, pork, and meat products under vacuum or modified atmospheres (Hugas, 1998; Samelis, Tsakalidou, Metaxopoulos, & Kalantzopoulos, 1995; Samelis et al., 2000; Yost & Nattress, 2000; Lyhs, Björkroth, & Korkeala, 2002; Ercolini et al., 2006).

In this study, the changes in the bacterial communities of vacuumpackaged pork during chilled storage were analyzed by PCR–DGGE. Eight kinds of LAB were identified from MRSA isolates of chilled vacuum-packaged pork, and Lb. sakei was the representative isolate at the end of the monitoring. By means of the direct meat analysis of PCR–DGGE, the predominant bacteria of vacuum-packaged pork at the end of the storage were Carnobacterium sp./Car. divergens, Lb. sakei and Lactococcus sp./Lc. piscium. Analysis of the Lactobacillus group-specific PCR and DGGE also found that Lb. sakei become the predominant bacterium in the end, which is consistent with the LAB isolates identification results obtained from this study. It is applicable for the PCR–DGGE technique to use as a culture-independent method to monitor bacterial population dynamics in vacuum-packaged pork.

Acknowledgements This research was funded by the Ministry of Science and Technology of China (grant no: 2008AA100804) and the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (grant no: 08KJB550005). We are grateful to Dr Yumin Bao of the Alltech Asia-Pacific Biosciences Centre, USA for preliminary language improvement of the original manuscript.

Fig. 4. DGGE profiles of Lactobacillus-group PCR products from the strains isolated in this study (lanes 1 to 8) and from DNA directly extracted from vacuum-packaged pork samples at storage time 0, 3, 6, 14, 18 and 23 days (lanes 9 to 14). Bands A to H correspond to the same bands in Fig. 1. The bands are discussed in the text, and the positions of the bands are indicated on the right. The band indicated by letter M was excised and, after re-amplification, subjected to sequencing.

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