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The effects of lactic acid-based spray washing on bacterial profile and quality of chicken carcasses
Food Control 60 (2016) 615e620
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Food Control journal homepage: www.elsevier.com/locate/foodcont
The effects of lactic acid-based spray washing on bacterial profile and quality of chicken carcasses Aiping Liu a, Zhen Peng a, Likou Zou b, Kang Zhou a, Xiaolin Ao a, Li He a, Shujuan Chen a, Shuliang Liu a, * a b
College of Food Science, Sichuan Agricultural University, Ya'an, Sichuan 625014, PR China The Laboratory of Microbiology, Dujiangyan Campus, Sichuan Agricultural University, Dujiangyan, Sichuan 611830, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 June 2015 Received in revised form 12 September 2015 Accepted 12 September 2015 Available online 14 September 2015
Chicken products with high water and nutrient contents provide essential nutrients for microbial growth. This study was designed to determine the effect of lactic acid-based spray washing. First, the reduction of microbial counts on chicken carcasses was evaluated through lactic acid-based spray washing. Second, bacterial diversity on chicken carcasses in different steps in a chicken-slaughtering chain was investigated through polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). The effect of spray washing on quick-frozen chicken quality was also examined. Results revealed that microbial counts decreased significantly after the chickens were spray-washed with 1.5% lactic acid at 50 C for 15s. PCR-DGGE revealed a decrease in microbial counts of several bacterial species, such as uncultured bacterium, uncultured Clostridiales sp., Pseudomonas fluorescens, Ruminococcus sp., and uncultured Ruminococcus sp. The spray washing performed did not remarkably affect the pH and total volatile basic nitrogen of quick-frozen chicken during storage. Therefore, the proposed technique may be applied to chicken-slaughtering chains and may be used to investigate bacterial diversity on chicken carcasses. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Chicken carcass Decontamination Food safety PCR-DGGE
1. Introduction Chicken industry has developed rapidly in China, however, chicken-slaughtering chains are challenged by several issues, such as microbial contamination (Lues, Theron, Venter, & Rasephei, 2007; Rosenquista, Sommerb, Nielsenc, & Christensena, 2006). Meat is easily contaminated and thus utilized as a substrate by microorganisms because meat is composed of high water and essential nutrient contents (Hilario, Buckley, & Young, 2004; Jackson, Acuff, & Dickson, 1997; Jiang et al., 2010). Therefore, microbial load on chicken carcasses should be reduced. Microbial count reductions of meat subjected to acid solution washing have been extensively investigated. Anderson and Marshall (1989) found that beef can be effectively treated by immersing meat in 3% lactic acid at 70 C for 15s. Ozdemir et al. (2006) utilized lactic acid and hot water treatments to reduce Salmonella typhimurium and Listeria monocytogenes. Carpenter, Smith,
* Corresponding author. E-mail address: [email protected] (S. Liu). http://dx.doi.org/10.1016/j.foodcont.2015.09.015 0956-7135/© 2015 Elsevier Ltd. All rights reserved.
and Broadbent (2011) compared spray washing with 2% levulinic acid at 55.4 C with spray washing with lactic or acetic acid to decontaminate pathogenic bacteria inoculated onto meat surfaces; only lactic acid washes exhibited a slightly higher decontaminating capacity than water. Burfoot and Mulvey (2011) developed a solution containing 4% lactic acid with pH 3.7 to reduce microbial counts on chicken and turkey carcasses. Although studies have investigated the efficiency of spray washing with acid solutions in reducing artificially inoculated pathogens, few publications have focused on the quality and microbial diversity of meat products through the whole process of meat-slaughtering chains. Molecular methods, which are independent of cultivation, have been commonly used to analyze microbial communities (Janssen, 2006). Among molecular methods, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) proposed by Muyzer, de Weal, and Uitterlinden (1993) is the most commonly employed method (Ercolini, 2004), which is a well-established tool for investigating microbial diversity in numerous laboratories (Chen et al., 2013; Cocolin, Manzano, Cantoni, & Comi, 2000; Heilig et al., 2002; Spano, Lonvaud-Funel, Claisse, & Massa, 2007).
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In the present study, samples were obtained from a chickenslaughtering enterprise where approximately 30 million chickens are processed each year. This study aimed to explore the appropriate conditions for lactic acid-based spray washing and the changes in bacterial diversity on chicken carcasses in the slaughtering chain through PCR-DGGE. This study also described the pH and total volatile basic nitrogen (TVBN) of quick-frozen chicken during storage and compared the obtained values between roomtemperature-water spray washing (control group, method utilized by the chicken-slaughtering enterprise) and lactic acid-based spray washing (treated group). This study may be used as a reference to investigate bacterial diversity on chicken carcasses. This study may also lay a better foundation for chicken slaughtering and processing. 2. Materials and methods 2.1. Materials Food-grade lactic acid was purchased from Jindan Company (Henan, China). Primers were synthesized by Weiketiantai Biotech Company (Sichuan, China). Gold view dye, DNA Marker DL2000, Premix Taq Version 2.0 and Vector pMD 19-T were purchased from Takara (Kyoto, Japan). Other reagents were of analytical grade. 2.2. Sampling 2.2.1. Sampling of chicken carcass surface for microbial count determination and PCR-DGGE Samples were collected with sterilized cotton swab, and 5 cm2 were rubbed in one spot with one cotton swab. The chicken carcass surface covering 50 cm2 (5 cm2 of head, 5 cm2 of anus, 10 cm2 of backside, 5 cm2 of wing, 5 cm2 of drumstick, 10 cm2 of chest and 10 cm2 of abdomen) was rubbed and then immersed in a 50 mL tube filled with sterile saline solution. The samples were transported to our laboratory in icebox within 4 h. 2.2.2. Sampling of quick-frozen chicken for PCR-DGGE The quick-frozen chicken was thawed and rubbed with sterilized cotton swab, and 50 cm2 of chicken carcass surface were rubbed as described in Section 2.2.1. Subsequently, the cotton swab was immersed in a 50 mL sterile homogeneous bag filled with sterile saline solution. After 30 min, 1 mL of the solution was collected for bacterial propagation. 2.2.3. Sampling of quick-frozen chicken for pH and TVBN evaluation In each determination, six chickens were placed in three different positions with two chickens per position in a freezer, and these six chickens were used from beginning to end. 2.3. Optimization of lactic acid-based spray washing to reduce microbial counts on chicken carcasses The optimal conditions for lactic acid-based spray washing were investigated by uniform design, and the effects of temperature, lactic acid concentration, and time were analyzed. Table 1 illustrated the factors and levels of the tests. After lactic acid-based spray washing was performed, the homogenized sample solution was serially diluted with 0.1 mol/L sterile phosphate-buffered saline (PBS, pH 7.0) and spread plated (0.1 mL) in duplicate on plate count agar. The plates were incubated aerobically at 37 C for 48 h, and the total CFU was recorded. Three independent replications of each group were performed, and the average was reported. The control group was decontaminated by room-temperature-water spray washing for 90 s.
Table 1 Factorelevel list for uniform design. Groups
Control 1 2 3 4 5 6
Treatment Temperature ( C)
Concentration (%)
Time (s)
e 30 35 40 45 50 55
e 1 2 3 0.5 1.5 2.5
e 45 90 30 75 15 60
2.4. PCR-DGGE analysis 2.4.1. DNA extraction The solution samples (50 mL) from each step (dehairing, chamber cutting, carcass washing, pre-cooling, meat cutting, and quick freezing) in the chicken-slaughtering chain (control group and treated group) were centrifuged at 3000 g for 3 min to remove debris; the supernatant was centrifuged for 3 min at 12,000 g. The pellet obtained was washed thrice with 0.1 mol/L PBS (pH 7.0) and stored at 20 C for further analysis. Bacterial DNA was extracted using column bacterial DNA OUT™ (Tiandz, Beijing, China) in accordance with the manufacturer's instructions and then suspended in 100 mL of TE buffer. DNA solution was estimated by 1.0% agarose gel electrophoresis. 2.4.2. PCR reaction Primers U968-GC (50 -CGCCCGGGGCGCGCCCCGGGCGGGGCGGGGGCA. CGGGGGGAACGCGAAGAACCTTAC-30 ), which contained GC clamp, and L1401 (50 -GCGTGTGTA CAAGACCC-30 ) (Nubel et al., 1996) were applied to amplify the V6eV8 regions of the bacterial 16S rRNA gene. The PCR system (50 mL) included 25 mL of Premix Taq Version 2.0, 1 mL of DNA temperlate (approximately 10 ng), 1 mL of primers (0.5 mL for each, 10 mmol), and 23 mL of deionized water. The PCR was programmed as (1) 94 C for 5 min; (2) 35 cycles of 94 C for 30 s, 64 C for 30 s, and 72 C for 40 s; (3) 72 C for 7 min. The result was analyzed through 1.0% agarose gel electrophoresis. Afterwards, the PCR products were purified and a second PCR was conducted by using the initially purified PCR products as a DNA template. 2.4.3. DGGE protocol A temporal temperature gradient gel electrophoresis system (CBS Scientific, CA, USA) was applied for DGGE analysis, and DGGE was performed in accordance with previously described methods (Nubel et al., 1996) with slight modifications. In brief, PCR products were loaded in 8% polyacrylamide gel (acrylamide: bisacrylamide ¼ 38: 2) containing a denaturing gradient of 45%e 55% urea-formamide, which was electrophoresed at 200 V for 30 min and then at 120 V for 16 h. DGGE gel was stained with AgNO3. After the color was developed, the gel was scanned using a gel documentation system (GelDoc-XR, Bio-Rad, CA, USA). 2.4.4. DNA reclaim and sequence analysis The evident bands on the DGGE gel were incised and reclaimed using a universal DNA purification kit (Tiangen Biotech, Beijing, China) with reference to the manufacturer's protocol. The reclaimed DNA was amplified through PCR with the primers U968 without a GC clamp and L1401 as described in Section 2.5. The PCR products were purified using an agarose gel DNA purifcation kit (Ver. 2.0; Takara, Hilden, Germany) in accordance with the manufacturer's protocol; the products were electrophoresed in 1.0% agarose gel and then analyzed. The purified products were ligated
A. Liu et al. / Food Control 60 (2016) 615e620
to T-vector and transformed into Escherichia coli DH5a. Monoclonal colonies were collected, sent to Invitrogen (Shanghai, China), and sequenced. These sequences (350e500 bp) were identified with the Advanced Basic Local Alignment Search Tool similarity search option in the GenBank DNA database (Altschul et al., 1997). 2.5. Effect of lactic acid-based spray washing on pH and TVBN of the quick-frozen chicken products during storage 2.5.1. Determination of pH value The pH of quick-frozen chicken was measured after the chicken was subjected to room temperature. Before pH was determined, the pH meter (Cyberscan PC 510, Eutech, Shanghai, China) was standardized against standard buffers (pH 4.0, 6.86 and 9.18). A 10 g sample was homogenized in 100 mL of distilled water (pH 7.0) for 20 min, and the mixtures were filtered using Whatman filter paper. The filtrate was used subsequently for analysis. Analysis was performed in triplicate for each chicken, and the average result was reported. 2.5.2. Determination of TVBN In brief, 10 g of sample, free of fat, tendon, and bone, was homogenized and immersed in 100 mL of deionized water for 30 min. The mixtures were filtered using Whatman filter paper, and 5 mL of the filtrate was mixed with 5 mL of magnesium oxide (10 g/L) in a sample tube of a distillation unit. The distillate was collected in a receiving flask that contained 10 mL of 2% boric acid and 5e6 drops of 0.2% methyl red and 0.1% bromocresol green indicators. The distillate solution was finally titrated with standardized 0.01 mol/L hydrochloric acid. The results were expressed as mg/ 100 g sample. Each chicken was analyzed in triplicate, and the average result was reported. 3. Results and discussion 3.1. Effect of lactic acid-based spray washing on microbial counts The reduction of microbial counts on chicken carcasses using lactic acid was investigated through uniform design optimization; this technique could remarkably reduce the amount of the required test points and ensure that results reflecting the major characteristics of the experimental system were obtained (Liang, Fang, & Xu, 2001; Wu et al., 2008). Table 2 summarized the microbial counts in the treated and control groups. Compared with the microbial counts in the control group, the microbial counts in the treated group were reduced by 1.88, 1.98, 2.03, 1.92, 2.03 and 1.98lg cfu/g. The statistical significance of data was analyzed; the effect of reduction in each treatment group was not evident (P > 0.05). However, the reduction effect that occurred after each treatment
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group was statistically higher (P < 0.05) than that of the control group; this result indicated that lactic acid-based spray washing was effective. As can be seen, Group 3 and 5 resulted in the same reduction, but the lactic acid concentration of group 3 was higher than that of group 5; as a result, money and water resources were put to waste. Besides, the possibility of appearance changes decreased (Burfoot & Mulvey, 2011). Therefore, the preferable treatment was 1.5% lactic acid at 50 C for 15s. 3.2. PCR-DGGE analysis 3.2.1. DNA extraction and PCR amplification of V6eV8 regions of 16S rDNA DNA was extracted from each sample, as described in Section 2.4.1; agarose gel analysis results were shown in Fig. 1. The length of the DNA extracted from each sample was considerably more than 2000 bp (Fig. 1). Each band was evident and used for further application. The V6eV8 regions of 16S rDNA were amplified using the extracted DNA as s template through two rounds of PCR, and the PCR products were analyzed through agarose gel electrophoresis (Fig. 2). Herewith, a secondary PCR was performed to obtain adequate PCR products to ensure a successful DGGE analysis. The obvious target band (~450 bp) was obtained (Fig. 2), and this finding was consistent with that of Li, Zhou, Xu, Li, and Zhu (2006). 3.2.2. DGGE analysis Each band in DGGE represented a dominant microorganism or operational taxonomic unit in the microbial community. The change in the band number in DGGE lanes indicated the complexity and variability of bacterial flora on chicken carcasses. The DGGE profile of the six steps, namely, dehairing, chamber cutting, carcass washing, pre-cooling, meat cutting, and quick freezing, in the chicken-slaughtering chain was investigated. The results (Fig. 3) revealed that the profile varied among different steps, and the highest and brightest bands were obtained from carcass washing. This result indicated that the contamination in carcass washing process was the worst. The species and quantities of bacteria on the chicken carcass surface of the last four steps decreased after the chickens were subjected to lactic acid-based spray washing. Eleven evident bands from Fig. 3 were reclaimed to further analyze the microbial community structure and diversity in the six steps of the chicken-slaughtering chain. The reclaimed DNA was amplified through PCR with the primers U968 without the GC clamp and L1401; the PCR products were then purified using an agarose gel DNA purification kit (Ver.2.0; Takara, Hilden, Germany) in accordance with the manufacturer's protocol before these
Table 2 Mean lg reductionb of microbial counts as affected by different lactic acid-based spray washing (lg cfu/g). Groups
Control 1 2 3 4 5 6 a b
Treatment
Microbial counts
Temperature ( C)
Concentration (%)
Time (s)
e 30 35 40 45 50 55
e 1 2 3 0.5 1.5 2.5
e 45 90 30 75 15 60
5.74 ± 0.04a 1.88b 1.98b 2.03b 1.92b 2.03b 1.98b
Mean S. typhimurium counts (lg cfu/g) for control group. Lg reduction ¼ (lg cfu/g before treatment) e (lg cfu/g after treatment).
Fig. 1. Agarose gel of total DNA extracted from each sample. M, Marker; 1, 2, 3, 4, 5, and 6 represent sample from dehairing, chamber cutting, carcass washing, pre-cooling, meat cutting, and quick freezing (control group, room-temperature-water spray washing), respectively; 30 , 40 , 50 , and 60 represent sample from carcass washing, precooling, meat cutting, and quick freezing (treated group, lactic acid-based spray washing), respectively.
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Fig. 2. Agarose gel of secondary PCR amplification products of V6eV8 regions of 16S rDNA. Each lane corresponds to samples in Fig. 1.
in the pre-cooling pool. Band c, which corresponded to Enterobacteriaceae bacterium, could be found in the six steps in the control group. However, the band brightness weakened after lactic acid-based spray washing was performed; this result indicated the effectiveness of the technique in decontaminating chicken carcasses containing Enterobacteriaceae bacterium. Bands d and f corresponded to uncultured Clostridiales bacterium and Ruminococcus sp., respectively, and the brightest part was visible in carcass washing; these results were consistent with those of Gong et al. (2007). However, the brightness of these two bands decreased sharply after lactic acid-based spray washing was conducted, and these bands almost disappeared in quick freezing. Bands e and g
Fig. 3. DGGE profile of V6eV8 regions of 16S rDNA from each sample. (A) Scanned image of a silver-stained DGGE gel; (B) Schematic diagram of DGGE profile. Each lane corresponds to samples in Fig. 1.
products were analyzed in agarose gel (Fig. 4). The purified products were ligated to T-vector and transformed into E. coli DH5a. Monoclonal colonies were collected and sent for sequencing. Table 3 lists the types of bacteria identified on the basis of the detectable bands in Figs. 3 and 4. Band a, which was identified as Lactobacillus sp., could be found on the chicken carcass surface in each step (Fig. 3). Band b, which was identified as uncultured bacterium, disappeared after lactic acid-based spray washing was conducted. However, band b was visible in pre-cooling possibly because of the cross contamination
corresponded to uncultured bacterium, which severely contaminated the dehairing, carcass washing, and meat cutting processes. Band h, which corresponded to Pseudomonas sp., disappeared but appeared in quick freezing. The cross contamination in the storage environment might induce this appearance. Bands i and j indicated Pseudomonas fluorescens and uncultured Ruminococcus sp., respectively. These bands were reduced remarkably compared with those of the samples from the control group. Band k was uncultured Pseudomonas sp., which severely contaminated the washing and quick freezing steps. Fig. 5 presented the similarity indices of the DGGE profiles. The
Table 3 Type of bacterial communities corresponding to the bands in Fig. 3
Fig. 4. Purified products of reclaimed DNA from DGGE profile after PCR amplification. M, Marker; a to k represent DNA marked in Fig. 3.
Band of DGGE
Identity (%)
Closest relative
a b c d e f g h i j k
99 99 99 97 96 94 99 99 99 98 97
Lactobacillus sp. Uncultured bacterium Enterobacteriaceae bacterium Uncultured Clostridiales bacterium Uncultured bacterium Ruminococcus sp. Uncultured bacterium Pseudomonas sp. Pseudomonas fluorescens Uncultured Ruminococcus sp. Uncultured Pseudomonas sp.
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Fig. 5. Cluster analysis of PCR-DGGE profile. 1, 2, 3, 30 , 4, 40 , 5, 50 , 6, 60 correspond to Fig. 1.
highest similarities were discovered among the samples from 30 , 50 and 6. After lactic acid-based spray washing was completed, the bacterial diversity of the two most severely contaminated steps were similar to that of the quick freezing step in the control group. Therefore, the lactic acid-based spray washing reduced contamination. The lowest similarities were found between 60 and other samples probably because of the significant reduction of microbial counts. 3.3. pH change in quick-frozen chicken during storage pH was an important index for meat quality evaluation. Fig. 6 presented the pH change in quick-frozen chicken during storage. As can be seen, the pH was 6.0e7.0. Statistical analysis results revealed that the pH of quick-frozen chicken did not significantly differ between room-temperature-water spray washing and lactic acid-based spray washing (P > 0.05). Low lactic acid concentration was used in bacteria reduction, and lactic acid content further decreased after other processes were performed in slaughtering chains. As a result, lactic acid-based spray washing did not elicit considerable effects on the pH of chicken.
two months, the TVBN content of quick-frozen chicken with roomtemperature-water spray washing and lactic acid-based spray washing indicated an upward trend (Fig. 7). Protein was decomposed to produce alkaline nitrogenous substances via enzymes and bacteria. Although the difference between room-temperaturewater spray washing and lactic acid-based spray washing was not significant (P > 0.05), the TVBN of quick-frozen chicken increased slowly after lactic acid-based spray washing was performed because few bacteria remained after the treatment was administered.
4. Conclusion
TVBN, as a measure of volatile nitrogen-containing compounds present in samples through steam distillation, was used to reveal microbiological spoilage (Loughran & Diamond, 2000). In the first
Lactic acid-based spray washing was applied to reduce microbial counts on chicken carcasses in this study. The optimal treatment was 1.5% lactic acid at 50 C for 15s. Subsequently, the changes in the bacterial communities of chicken carcasses in the six steps, namely dehairing, chamber cutting, carcass washing, pre-cooling, meat cutting, and quick freezing, in a chickenslaughtering chain were analyzed through PCR-DGGE. The pH and TVBN of chicken carcasses with room-temperature-water spray washing and lactic acid-based spray washing were evaluated. The lactic acid-based spray washing did not cause significant changes in pH and TVBN. Therefore, this study may be beneficial for studies on bacterial diversity on chicken carcasses. This study may also provide a basis for shelf life and food safety improvement of meat products.
Fig. 6. pH change of quick-frozen chicken during storage.
Fig. 7. TVBN determination of quick-frozen chicken during storage.
3.4. TVBN determination of quick-frozen chicken during storage
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Acknowledgments The authors extend their gratitude to the National Natural Science Foundation of China (No. 31400066) for the financial support. References Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W., et al. (1997). Gapped BLAST and PSIBLAST: a new generation of protein database search programs. Nucleic Acids Research, 25, 3389e3402. Anderson, M. E., & Marshall, R. T. (1989). Reducing microbial populations on beef tissues: concentration and temperature of lactic acid. Journal of Food Safety, 10(3), 181e190. Burfoot, D., & Mulvey, E. (2011). Reducing microbial counts on chicken and turkey carcasses using lactic acid. Food Control, 22(11), 1729e1735. Carpenter, C. E., Smith, J. V., & Broadbent, J. R. (2011). Efficacy of washing meat surfaces with 2% levulinic, acetic, or lactic acid for pathogen decontamination and residual growth inhibition. Meat Science, 88, 256e260. Chen, F., Wang, M., Zheng, Y., Li, S. J., Wang, H. Z., Han, D. D., et al. (2013). The effect of biocontrol bacteria on rhizosphere bacterial communities analyzed by plating and PCR-DGGE. Current Microbiology, 67(2), 177e182. Cocolin, L., Manzano, M., Cantoni, C., & Comi, G. (2000). Development of a rapid method for the identification of Lactobacillus spp. isolated from fermented Italian sausages using a polymerase chain reaction-temperature gradient gel electrophoresis. Letters in Applied Microbiology, 30, 126e129. Ercolini, D. (2004). PCR-DGGE fingerprinting: novel strategies for detection of microbes in food. Journal of Microbiological Methods, 56(3), 297e314. Gong, J. H., Si, W., Forster, R. J., Huang, R., Yu, H., Yin, Y., et al. (2007). 16S rRNA genebased analysis of mucosa-associated bacterial community and phylogeny in the chicken gastrointestinal tracts: from crops to ceca. FEMS Microbiology Ecology, 59(1), 147e157. Heilig, G. H. J., Zoetendal, E. G., Vaughan, E. E., Marteau, P., Akkermans, A. D. L., & de Vos, W. M. (2002). Molecular diversity of Lactobacillus spp., and other lactic acid bacteria in the human intestine as determined by specific amplification of 16S ribosomal DNA. Applied and Environmental Microbiology, 68, 114e123. Hilario, E., Buckley, T. R., & Young, J. M. (2004). Improved resolution of the phylogenetic relationships among Pseudomonas by the combined analysis of atpD, carA, recA and 16S rDNA. Antonie van Leeuwenhoek, 86, 51e64. Jackson, T. C., Acuff, G. R., & Dickson, J. S. (1997). Meat, poultry, and seafood. In
M. P. Doyle, & T. J. Beuchat (Eds.), Food microbiology: Fundamentals and frontiers (pp. 83e100). Washington, D.C: ASM Press. Janssen, P. H. (2006). Identifying the dominant soil bacterial Taxa in libraries of 16S rRNA and 16S rRNA genes. Applied and Environmental Microbiology, 72(3), 1719e1728. Jiang, Y., Gao, F., Xu, X. L., Su, Y., Ye, K. P., & Zhou, G. H. (2010). Changes in the bacterial communities of vacuum-packaged pork during chilled storage analyzed by PCR-DGGE. Meat Science, 86, 889e895. Liang, Y. Z., Fang, K. T., & Xu, Q. S. (2001). Uniform design and its applications in chemistry and chemical engineering. Chemometrics and Intelligent Laboratory Systems, 58, 43e57. Li, M. Y., Zhou, G. H., Xu, X. L., Li, C. B., & Zhu, W. Y. (2006). Changes of bacterial diversity and main flora in chilled pork during storage using PCR-DGGE. Food Microbiology, 23, 607e611. Loughran, M., & Diamond, D. (2000). Monitoring of volatile bases in fish sample headspace using an acid chromic dye. Food Chemistry, 69, 97e103. Lues, J. F. R., Theron, M. M., Venter, P., & Rasephei, M. H. R. (2007). Microbial Composition in bioaerosols of a high-throughput chicken-slaughtering facility. Poultry Science, 86, 142e149. Muyzer, G., de Weal, E. C., & Uitterlinden, A. (1993). Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16SrRNA. Applied and Environmental Microbiology, 59, 695e700. Nubel, U., Engelen, B., Felske, A., Snaidr, J., Wieshuber, A., Amann, R. I., et al. (1996). Sequence heterogeneities of genes encoding 16S rRNAs in Paenibacilluspolymyxa detected by temperature gradient gel electrophoresis. Journal of Bacteriology, 178, 5636e5643. Ozdemir, H., Yildirim, Y., Kuplulu, O., Koluman, A., Goncuoglu, M., & Inat, G. (2006). Effects of lactic acid and hot water treatments on Salmonella typhimurium and Listeria monocytogenes on beef. Food Control, 17, 299e303. Rosenquista, H., Sommerb, H. M., Nielsenc, N. L., & Christensena, B. B. (2006). The effect of slaughter operations on the contamination of chicken carcasses with thermotolerant Campylobacter. International Journal of Food Microbiology, 108(2), 226e232. Spano, G., Lonvaud-Funel, A., Claisse, O., & Massa, S. (2007). In Vivo PCR-DGGE analysis of lactobacillus plantarum and Oenococcus oeni populations in red wine. Current Microbiology, 54(1), 9e13. Wu, M., Li, D., Wang, L. J., Zhou, Y. G., Brooks, M. S. L., Chen, X. D., et al. (2008). Extrusion detoxification technique on flaxseed by uniform design. Optimization Separation and Purification Technology, 61, 51e59.
Contents lists available at ScienceDirect
Food Control journal homepage: www.elsevier.com/locate/foodcont
The effects of lactic acid-based spray washing on bacterial profile and quality of chicken carcasses Aiping Liu a, Zhen Peng a, Likou Zou b, Kang Zhou a, Xiaolin Ao a, Li He a, Shujuan Chen a, Shuliang Liu a, * a b
College of Food Science, Sichuan Agricultural University, Ya'an, Sichuan 625014, PR China The Laboratory of Microbiology, Dujiangyan Campus, Sichuan Agricultural University, Dujiangyan, Sichuan 611830, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 June 2015 Received in revised form 12 September 2015 Accepted 12 September 2015 Available online 14 September 2015
Chicken products with high water and nutrient contents provide essential nutrients for microbial growth. This study was designed to determine the effect of lactic acid-based spray washing. First, the reduction of microbial counts on chicken carcasses was evaluated through lactic acid-based spray washing. Second, bacterial diversity on chicken carcasses in different steps in a chicken-slaughtering chain was investigated through polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). The effect of spray washing on quick-frozen chicken quality was also examined. Results revealed that microbial counts decreased significantly after the chickens were spray-washed with 1.5% lactic acid at 50 C for 15s. PCR-DGGE revealed a decrease in microbial counts of several bacterial species, such as uncultured bacterium, uncultured Clostridiales sp., Pseudomonas fluorescens, Ruminococcus sp., and uncultured Ruminococcus sp. The spray washing performed did not remarkably affect the pH and total volatile basic nitrogen of quick-frozen chicken during storage. Therefore, the proposed technique may be applied to chicken-slaughtering chains and may be used to investigate bacterial diversity on chicken carcasses. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Chicken carcass Decontamination Food safety PCR-DGGE
1. Introduction Chicken industry has developed rapidly in China, however, chicken-slaughtering chains are challenged by several issues, such as microbial contamination (Lues, Theron, Venter, & Rasephei, 2007; Rosenquista, Sommerb, Nielsenc, & Christensena, 2006). Meat is easily contaminated and thus utilized as a substrate by microorganisms because meat is composed of high water and essential nutrient contents (Hilario, Buckley, & Young, 2004; Jackson, Acuff, & Dickson, 1997; Jiang et al., 2010). Therefore, microbial load on chicken carcasses should be reduced. Microbial count reductions of meat subjected to acid solution washing have been extensively investigated. Anderson and Marshall (1989) found that beef can be effectively treated by immersing meat in 3% lactic acid at 70 C for 15s. Ozdemir et al. (2006) utilized lactic acid and hot water treatments to reduce Salmonella typhimurium and Listeria monocytogenes. Carpenter, Smith,
* Corresponding author. E-mail address: [email protected] (S. Liu). http://dx.doi.org/10.1016/j.foodcont.2015.09.015 0956-7135/© 2015 Elsevier Ltd. All rights reserved.
and Broadbent (2011) compared spray washing with 2% levulinic acid at 55.4 C with spray washing with lactic or acetic acid to decontaminate pathogenic bacteria inoculated onto meat surfaces; only lactic acid washes exhibited a slightly higher decontaminating capacity than water. Burfoot and Mulvey (2011) developed a solution containing 4% lactic acid with pH 3.7 to reduce microbial counts on chicken and turkey carcasses. Although studies have investigated the efficiency of spray washing with acid solutions in reducing artificially inoculated pathogens, few publications have focused on the quality and microbial diversity of meat products through the whole process of meat-slaughtering chains. Molecular methods, which are independent of cultivation, have been commonly used to analyze microbial communities (Janssen, 2006). Among molecular methods, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) proposed by Muyzer, de Weal, and Uitterlinden (1993) is the most commonly employed method (Ercolini, 2004), which is a well-established tool for investigating microbial diversity in numerous laboratories (Chen et al., 2013; Cocolin, Manzano, Cantoni, & Comi, 2000; Heilig et al., 2002; Spano, Lonvaud-Funel, Claisse, & Massa, 2007).
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In the present study, samples were obtained from a chickenslaughtering enterprise where approximately 30 million chickens are processed each year. This study aimed to explore the appropriate conditions for lactic acid-based spray washing and the changes in bacterial diversity on chicken carcasses in the slaughtering chain through PCR-DGGE. This study also described the pH and total volatile basic nitrogen (TVBN) of quick-frozen chicken during storage and compared the obtained values between roomtemperature-water spray washing (control group, method utilized by the chicken-slaughtering enterprise) and lactic acid-based spray washing (treated group). This study may be used as a reference to investigate bacterial diversity on chicken carcasses. This study may also lay a better foundation for chicken slaughtering and processing. 2. Materials and methods 2.1. Materials Food-grade lactic acid was purchased from Jindan Company (Henan, China). Primers were synthesized by Weiketiantai Biotech Company (Sichuan, China). Gold view dye, DNA Marker DL2000, Premix Taq Version 2.0 and Vector pMD 19-T were purchased from Takara (Kyoto, Japan). Other reagents were of analytical grade. 2.2. Sampling 2.2.1. Sampling of chicken carcass surface for microbial count determination and PCR-DGGE Samples were collected with sterilized cotton swab, and 5 cm2 were rubbed in one spot with one cotton swab. The chicken carcass surface covering 50 cm2 (5 cm2 of head, 5 cm2 of anus, 10 cm2 of backside, 5 cm2 of wing, 5 cm2 of drumstick, 10 cm2 of chest and 10 cm2 of abdomen) was rubbed and then immersed in a 50 mL tube filled with sterile saline solution. The samples were transported to our laboratory in icebox within 4 h. 2.2.2. Sampling of quick-frozen chicken for PCR-DGGE The quick-frozen chicken was thawed and rubbed with sterilized cotton swab, and 50 cm2 of chicken carcass surface were rubbed as described in Section 2.2.1. Subsequently, the cotton swab was immersed in a 50 mL sterile homogeneous bag filled with sterile saline solution. After 30 min, 1 mL of the solution was collected for bacterial propagation. 2.2.3. Sampling of quick-frozen chicken for pH and TVBN evaluation In each determination, six chickens were placed in three different positions with two chickens per position in a freezer, and these six chickens were used from beginning to end. 2.3. Optimization of lactic acid-based spray washing to reduce microbial counts on chicken carcasses The optimal conditions for lactic acid-based spray washing were investigated by uniform design, and the effects of temperature, lactic acid concentration, and time were analyzed. Table 1 illustrated the factors and levels of the tests. After lactic acid-based spray washing was performed, the homogenized sample solution was serially diluted with 0.1 mol/L sterile phosphate-buffered saline (PBS, pH 7.0) and spread plated (0.1 mL) in duplicate on plate count agar. The plates were incubated aerobically at 37 C for 48 h, and the total CFU was recorded. Three independent replications of each group were performed, and the average was reported. The control group was decontaminated by room-temperature-water spray washing for 90 s.
Table 1 Factorelevel list for uniform design. Groups
Control 1 2 3 4 5 6
Treatment Temperature ( C)
Concentration (%)
Time (s)
e 30 35 40 45 50 55
e 1 2 3 0.5 1.5 2.5
e 45 90 30 75 15 60
2.4. PCR-DGGE analysis 2.4.1. DNA extraction The solution samples (50 mL) from each step (dehairing, chamber cutting, carcass washing, pre-cooling, meat cutting, and quick freezing) in the chicken-slaughtering chain (control group and treated group) were centrifuged at 3000 g for 3 min to remove debris; the supernatant was centrifuged for 3 min at 12,000 g. The pellet obtained was washed thrice with 0.1 mol/L PBS (pH 7.0) and stored at 20 C for further analysis. Bacterial DNA was extracted using column bacterial DNA OUT™ (Tiandz, Beijing, China) in accordance with the manufacturer's instructions and then suspended in 100 mL of TE buffer. DNA solution was estimated by 1.0% agarose gel electrophoresis. 2.4.2. PCR reaction Primers U968-GC (50 -CGCCCGGGGCGCGCCCCGGGCGGGGCGGGGGCA. CGGGGGGAACGCGAAGAACCTTAC-30 ), which contained GC clamp, and L1401 (50 -GCGTGTGTA CAAGACCC-30 ) (Nubel et al., 1996) were applied to amplify the V6eV8 regions of the bacterial 16S rRNA gene. The PCR system (50 mL) included 25 mL of Premix Taq Version 2.0, 1 mL of DNA temperlate (approximately 10 ng), 1 mL of primers (0.5 mL for each, 10 mmol), and 23 mL of deionized water. The PCR was programmed as (1) 94 C for 5 min; (2) 35 cycles of 94 C for 30 s, 64 C for 30 s, and 72 C for 40 s; (3) 72 C for 7 min. The result was analyzed through 1.0% agarose gel electrophoresis. Afterwards, the PCR products were purified and a second PCR was conducted by using the initially purified PCR products as a DNA template. 2.4.3. DGGE protocol A temporal temperature gradient gel electrophoresis system (CBS Scientific, CA, USA) was applied for DGGE analysis, and DGGE was performed in accordance with previously described methods (Nubel et al., 1996) with slight modifications. In brief, PCR products were loaded in 8% polyacrylamide gel (acrylamide: bisacrylamide ¼ 38: 2) containing a denaturing gradient of 45%e 55% urea-formamide, which was electrophoresed at 200 V for 30 min and then at 120 V for 16 h. DGGE gel was stained with AgNO3. After the color was developed, the gel was scanned using a gel documentation system (GelDoc-XR, Bio-Rad, CA, USA). 2.4.4. DNA reclaim and sequence analysis The evident bands on the DGGE gel were incised and reclaimed using a universal DNA purification kit (Tiangen Biotech, Beijing, China) with reference to the manufacturer's protocol. The reclaimed DNA was amplified through PCR with the primers U968 without a GC clamp and L1401 as described in Section 2.5. The PCR products were purified using an agarose gel DNA purifcation kit (Ver. 2.0; Takara, Hilden, Germany) in accordance with the manufacturer's protocol; the products were electrophoresed in 1.0% agarose gel and then analyzed. The purified products were ligated
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to T-vector and transformed into Escherichia coli DH5a. Monoclonal colonies were collected, sent to Invitrogen (Shanghai, China), and sequenced. These sequences (350e500 bp) were identified with the Advanced Basic Local Alignment Search Tool similarity search option in the GenBank DNA database (Altschul et al., 1997). 2.5. Effect of lactic acid-based spray washing on pH and TVBN of the quick-frozen chicken products during storage 2.5.1. Determination of pH value The pH of quick-frozen chicken was measured after the chicken was subjected to room temperature. Before pH was determined, the pH meter (Cyberscan PC 510, Eutech, Shanghai, China) was standardized against standard buffers (pH 4.0, 6.86 and 9.18). A 10 g sample was homogenized in 100 mL of distilled water (pH 7.0) for 20 min, and the mixtures were filtered using Whatman filter paper. The filtrate was used subsequently for analysis. Analysis was performed in triplicate for each chicken, and the average result was reported. 2.5.2. Determination of TVBN In brief, 10 g of sample, free of fat, tendon, and bone, was homogenized and immersed in 100 mL of deionized water for 30 min. The mixtures were filtered using Whatman filter paper, and 5 mL of the filtrate was mixed with 5 mL of magnesium oxide (10 g/L) in a sample tube of a distillation unit. The distillate was collected in a receiving flask that contained 10 mL of 2% boric acid and 5e6 drops of 0.2% methyl red and 0.1% bromocresol green indicators. The distillate solution was finally titrated with standardized 0.01 mol/L hydrochloric acid. The results were expressed as mg/ 100 g sample. Each chicken was analyzed in triplicate, and the average result was reported. 3. Results and discussion 3.1. Effect of lactic acid-based spray washing on microbial counts The reduction of microbial counts on chicken carcasses using lactic acid was investigated through uniform design optimization; this technique could remarkably reduce the amount of the required test points and ensure that results reflecting the major characteristics of the experimental system were obtained (Liang, Fang, & Xu, 2001; Wu et al., 2008). Table 2 summarized the microbial counts in the treated and control groups. Compared with the microbial counts in the control group, the microbial counts in the treated group were reduced by 1.88, 1.98, 2.03, 1.92, 2.03 and 1.98lg cfu/g. The statistical significance of data was analyzed; the effect of reduction in each treatment group was not evident (P > 0.05). However, the reduction effect that occurred after each treatment
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group was statistically higher (P < 0.05) than that of the control group; this result indicated that lactic acid-based spray washing was effective. As can be seen, Group 3 and 5 resulted in the same reduction, but the lactic acid concentration of group 3 was higher than that of group 5; as a result, money and water resources were put to waste. Besides, the possibility of appearance changes decreased (Burfoot & Mulvey, 2011). Therefore, the preferable treatment was 1.5% lactic acid at 50 C for 15s. 3.2. PCR-DGGE analysis 3.2.1. DNA extraction and PCR amplification of V6eV8 regions of 16S rDNA DNA was extracted from each sample, as described in Section 2.4.1; agarose gel analysis results were shown in Fig. 1. The length of the DNA extracted from each sample was considerably more than 2000 bp (Fig. 1). Each band was evident and used for further application. The V6eV8 regions of 16S rDNA were amplified using the extracted DNA as s template through two rounds of PCR, and the PCR products were analyzed through agarose gel electrophoresis (Fig. 2). Herewith, a secondary PCR was performed to obtain adequate PCR products to ensure a successful DGGE analysis. The obvious target band (~450 bp) was obtained (Fig. 2), and this finding was consistent with that of Li, Zhou, Xu, Li, and Zhu (2006). 3.2.2. DGGE analysis Each band in DGGE represented a dominant microorganism or operational taxonomic unit in the microbial community. The change in the band number in DGGE lanes indicated the complexity and variability of bacterial flora on chicken carcasses. The DGGE profile of the six steps, namely, dehairing, chamber cutting, carcass washing, pre-cooling, meat cutting, and quick freezing, in the chicken-slaughtering chain was investigated. The results (Fig. 3) revealed that the profile varied among different steps, and the highest and brightest bands were obtained from carcass washing. This result indicated that the contamination in carcass washing process was the worst. The species and quantities of bacteria on the chicken carcass surface of the last four steps decreased after the chickens were subjected to lactic acid-based spray washing. Eleven evident bands from Fig. 3 were reclaimed to further analyze the microbial community structure and diversity in the six steps of the chicken-slaughtering chain. The reclaimed DNA was amplified through PCR with the primers U968 without the GC clamp and L1401; the PCR products were then purified using an agarose gel DNA purification kit (Ver.2.0; Takara, Hilden, Germany) in accordance with the manufacturer's protocol before these
Table 2 Mean lg reductionb of microbial counts as affected by different lactic acid-based spray washing (lg cfu/g). Groups
Control 1 2 3 4 5 6 a b
Treatment
Microbial counts
Temperature ( C)
Concentration (%)
Time (s)
e 30 35 40 45 50 55
e 1 2 3 0.5 1.5 2.5
e 45 90 30 75 15 60
5.74 ± 0.04a 1.88b 1.98b 2.03b 1.92b 2.03b 1.98b
Mean S. typhimurium counts (lg cfu/g) for control group. Lg reduction ¼ (lg cfu/g before treatment) e (lg cfu/g after treatment).
Fig. 1. Agarose gel of total DNA extracted from each sample. M, Marker; 1, 2, 3, 4, 5, and 6 represent sample from dehairing, chamber cutting, carcass washing, pre-cooling, meat cutting, and quick freezing (control group, room-temperature-water spray washing), respectively; 30 , 40 , 50 , and 60 represent sample from carcass washing, precooling, meat cutting, and quick freezing (treated group, lactic acid-based spray washing), respectively.
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Fig. 2. Agarose gel of secondary PCR amplification products of V6eV8 regions of 16S rDNA. Each lane corresponds to samples in Fig. 1.
in the pre-cooling pool. Band c, which corresponded to Enterobacteriaceae bacterium, could be found in the six steps in the control group. However, the band brightness weakened after lactic acid-based spray washing was performed; this result indicated the effectiveness of the technique in decontaminating chicken carcasses containing Enterobacteriaceae bacterium. Bands d and f corresponded to uncultured Clostridiales bacterium and Ruminococcus sp., respectively, and the brightest part was visible in carcass washing; these results were consistent with those of Gong et al. (2007). However, the brightness of these two bands decreased sharply after lactic acid-based spray washing was conducted, and these bands almost disappeared in quick freezing. Bands e and g
Fig. 3. DGGE profile of V6eV8 regions of 16S rDNA from each sample. (A) Scanned image of a silver-stained DGGE gel; (B) Schematic diagram of DGGE profile. Each lane corresponds to samples in Fig. 1.
products were analyzed in agarose gel (Fig. 4). The purified products were ligated to T-vector and transformed into E. coli DH5a. Monoclonal colonies were collected and sent for sequencing. Table 3 lists the types of bacteria identified on the basis of the detectable bands in Figs. 3 and 4. Band a, which was identified as Lactobacillus sp., could be found on the chicken carcass surface in each step (Fig. 3). Band b, which was identified as uncultured bacterium, disappeared after lactic acid-based spray washing was conducted. However, band b was visible in pre-cooling possibly because of the cross contamination
corresponded to uncultured bacterium, which severely contaminated the dehairing, carcass washing, and meat cutting processes. Band h, which corresponded to Pseudomonas sp., disappeared but appeared in quick freezing. The cross contamination in the storage environment might induce this appearance. Bands i and j indicated Pseudomonas fluorescens and uncultured Ruminococcus sp., respectively. These bands were reduced remarkably compared with those of the samples from the control group. Band k was uncultured Pseudomonas sp., which severely contaminated the washing and quick freezing steps. Fig. 5 presented the similarity indices of the DGGE profiles. The
Table 3 Type of bacterial communities corresponding to the bands in Fig. 3
Fig. 4. Purified products of reclaimed DNA from DGGE profile after PCR amplification. M, Marker; a to k represent DNA marked in Fig. 3.
Band of DGGE
Identity (%)
Closest relative
a b c d e f g h i j k
99 99 99 97 96 94 99 99 99 98 97
Lactobacillus sp. Uncultured bacterium Enterobacteriaceae bacterium Uncultured Clostridiales bacterium Uncultured bacterium Ruminococcus sp. Uncultured bacterium Pseudomonas sp. Pseudomonas fluorescens Uncultured Ruminococcus sp. Uncultured Pseudomonas sp.
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Fig. 5. Cluster analysis of PCR-DGGE profile. 1, 2, 3, 30 , 4, 40 , 5, 50 , 6, 60 correspond to Fig. 1.
highest similarities were discovered among the samples from 30 , 50 and 6. After lactic acid-based spray washing was completed, the bacterial diversity of the two most severely contaminated steps were similar to that of the quick freezing step in the control group. Therefore, the lactic acid-based spray washing reduced contamination. The lowest similarities were found between 60 and other samples probably because of the significant reduction of microbial counts. 3.3. pH change in quick-frozen chicken during storage pH was an important index for meat quality evaluation. Fig. 6 presented the pH change in quick-frozen chicken during storage. As can be seen, the pH was 6.0e7.0. Statistical analysis results revealed that the pH of quick-frozen chicken did not significantly differ between room-temperature-water spray washing and lactic acid-based spray washing (P > 0.05). Low lactic acid concentration was used in bacteria reduction, and lactic acid content further decreased after other processes were performed in slaughtering chains. As a result, lactic acid-based spray washing did not elicit considerable effects on the pH of chicken.
two months, the TVBN content of quick-frozen chicken with roomtemperature-water spray washing and lactic acid-based spray washing indicated an upward trend (Fig. 7). Protein was decomposed to produce alkaline nitrogenous substances via enzymes and bacteria. Although the difference between room-temperaturewater spray washing and lactic acid-based spray washing was not significant (P > 0.05), the TVBN of quick-frozen chicken increased slowly after lactic acid-based spray washing was performed because few bacteria remained after the treatment was administered.
4. Conclusion
TVBN, as a measure of volatile nitrogen-containing compounds present in samples through steam distillation, was used to reveal microbiological spoilage (Loughran & Diamond, 2000). In the first
Lactic acid-based spray washing was applied to reduce microbial counts on chicken carcasses in this study. The optimal treatment was 1.5% lactic acid at 50 C for 15s. Subsequently, the changes in the bacterial communities of chicken carcasses in the six steps, namely dehairing, chamber cutting, carcass washing, pre-cooling, meat cutting, and quick freezing, in a chickenslaughtering chain were analyzed through PCR-DGGE. The pH and TVBN of chicken carcasses with room-temperature-water spray washing and lactic acid-based spray washing were evaluated. The lactic acid-based spray washing did not cause significant changes in pH and TVBN. Therefore, this study may be beneficial for studies on bacterial diversity on chicken carcasses. This study may also provide a basis for shelf life and food safety improvement of meat products.
Fig. 6. pH change of quick-frozen chicken during storage.
Fig. 7. TVBN determination of quick-frozen chicken during storage.
3.4. TVBN determination of quick-frozen chicken during storage
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