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Effects of combined organic acid treatments during the cutting process on the natural microflora and quality of chicken drumsticks
Food Control 67 (2016) 1e8
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Effects of combined organic acid treatments during the cutting process on the natural microflora and quality of chicken drumsticks Yuanting Zhu a, Xiaolong Xia a, Aiping Liu a, Likou Zou b, Kang Zhou a, Xinfeng Han a, Guoquan Han 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 5 January 2016 Received in revised form 18 February 2016 Accepted 19 February 2016 Available online 23 February 2016
Reducing the microbial load on broiler chicken carcasses at each stage of poultry meat processing, is highly important for hygienic meat production. This study was conducted to assess the efficacy of various combined organic acid treatments during the cutting process on the microbial decontamination of chicken drumsticks. Changes in naturally occurring microflora before and after treatment were analyzed through microbiological counting and polymerase chain reactionedenaturing gradient gel electrophoresis (PCR-DGGE). Results revealed that the most effective treatments obtained through the combination of orthogonal design and sensory evaluation were as follows: 0.5% lactic acid (w/v), 1% citric acid (w/v), and spray-washing for 30 s. Microbiological counting results and PCR-DGGE analysis indicated that the microbial load on the chicken drumsticks decreased significantly after the treatment was administered. The treatment did not affect the physicochemical properties and sensory attributes of the quick-frozen chicken drumsticks during storage. Therefore, the proposed technique could be used to improve the microbial quality of chicken drumsticks. The technique could also be employed in poultry meat production chains. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Chicken drumsticks Organic acid Natural microflora Decontamination PCR-DGGE Sensory evaluation
1. Introduction A significant increase in global poultry meat consumption has been observed. This increase is likely to continue into the future (Henchion, McCarthy, Resconi, & Troy, 2014). During the conversion of chicken into meat for consumption, broiler carcasses are at a high risk of contamination from fecal soiling on feathers and skin during slaughtering, intestinal content leakage during evisceration, and processing equipment and general processing environments (Owens, Alvarado, & Sams, 2010). Therefore, poultry meat can become contaminated with various food-borne pathogens, including Salmonella, Campylobacter spp., Listeria monocytogenes, Clostridium perfringens, and Staphylococcus aureus (Bohaychuk € m & Molin, 1987). Various interventions et al., 2006; Ternstro have been implemented to reduce the number of food-borne pathogens on surfaces of broiler carcasses (Bolton, Meredith, Walsh, & McDowell, 2014; Koolman, Whyte, Meade, Lyng, &
* Corresponding author. E-mail address: [email protected] (S. Liu). http://dx.doi.org/10.1016/j.foodcont.2016.02.031 0956-7135/© 2016 Elsevier Ltd. All rights reserved.
Bolton, 2014a, 2014b; Musavian, Krebs, Nonboe, Corry, & Purnell, 2014). Furthermore, manufacturers should reduce or eliminate carcass contamination of psychrotrophic spoilage bacteria to ensure adequate shelf life of raw chilled products (Owens et al., 2010). Hazard analysis and critical control point (HACCP) systems have been widely used in poultry meat industries to minimize product contamination (Tompkin, 1994). HACCP includes critical control points (CCP) at which an intervention may be implemented to prevent, reduce, or eliminate microbial contamination. Decontamination is a bactericidal treatment applied to reduce pathogenic and spoilage organisms (Bolder, 1997). Chemical treatments can be used to eliminate the presence of pathogens and spoilage microorganisms and thus may provide a basis of an effective CCP intervention (Acuff, 2005). The United States Food and Drug Administration has approved the use of a number of chemicals as decontaminants of poultry meat. Several organic acids, such as lactic acid and acetic acid, have been generally recognized as safe substances for use in poultry processing plants. These acids could inhibit subsequent microbial growth and thus extend shelf life. The efficiency of organic acid solutions in reducing artificially
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inoculated pathogens has been extensively investigated (Koolman et al., 2014a, 2014b). Few have been evaluated against naturally occurring microflora on carcasses after treatment. Muyzer, De Waal, & Uitterlinden (1993) proposed denaturing gradient gel electrophoresis (DGGE) based on the separation of PCR amplicons with the same size but with different 16S rDNA sequences; this technique can be used to overcome the limitations of culturedependent techniques, to reveal microbial communities, and to analyze microbial diversity. As a well-established tool, this technique has also been applied to numerous fields. For instance, Li, Zhou, Xu, Li, and Zhu (2006) employed PCR-DGGE to investigate the bacterial diversity and the main flora in chilled pork. Cutting is the last step before broiler chicken products are quickly frozen. Therefore, the microbial decontamination efficacy in this process has an immediate influence on the quality of chicken products. This study aimed to evaluate the efficacy of microbial decontamination treatment during the cutting process in the slaughtering and processing chains of poultry meat. Combined organic acid treatment conditions for chicken drumsticks were optimized. Changes in naturally occurring bacterial community on chicken drumsticks before and after treatment were analyzed through PCR-DGGE. The effects of the optimum treatment on the physiochemical properties and sensory attributes of chicken drumsticks were also investigated. This study may provide a basis of chicken slaughtering and processing methods.
2. Materials and methods 2.1. Sampling 2.1.1. Sampling of chicken drumstick surface for microbial analyses Chicken drumstick samples were obtained in three stages, namely, earlier, middle, and later stages, of cutting process in the broiler cutting workshop at a local chicken slaughtering plant where about 30,000,000 broiler chickens are processed each year. The chicken drumstick surfaces were sampled in accordance with previously described methods (Gill, Badoni, Moza, Barbut, & Griffiths, 2005). Briefly, the surface covering 25 cm2 of each chicken drumstick sample was swabbed by using sterilized cotton swabs moistened with 0.1% peptone water. The swabs obtained after sampling were placed in stomacher bags and stored in an ice bath during transportation to our laboratory, and were determined within 3 h. The samples were collected for control and treated groups.
2.1.2. Sampling of quick-frozen chicken drumsticks for pathogen detection For each sample from control and treated groups, 25 g of thawed chicken drumsticks was incised, collected in a sterile plastic stomacher bag containing 225 mL of sterile saline water, and pummeled for 2 min. A 1-mL aliquot of each homogenate from control and treated drumsticks was subjected to enrichment for detection of S. aureus and Salmonella.
2.1.3. Sampling of quick-frozen chicken drumsticks for physicochemical and sensory indexes evaluation during storage The quick-frozen chicken drumstick samples, vacuum-packaged and stored at 18 C in a freezer, were collected on days 5, 10, 15, 20, 30, 40, 50, and 60, and then subjected to physicochemical index analysis and sensory evaluation. The samples were collected for both control and treated groups.
2.2. Optimization of combined organic acid-based spray washing during cutting to reduce microbial load on chicken drumsticks The optimal conditions for combined organic acid-based spray washing were investigated by using an orthogonal experimental design [L16(45)], and the effects of lactic - citric acid concentrations [food-grade, Jindan Company (Henan, China)], and spray-washing time were analyzed. Table 1 lists the factors and levels of the tests. After spray washing was administered, total viable counts (TVC) of chicken drumsticks in treated groups and control groups were performed. The control group received no treatment. In addition, the sensory attributes of chicken drumsticks in the treated groups were evaluated. The most promising treatments were obtained on the basis of both decontamination effects and sensory evaluation. 2.3. Microbiological analysis The cotton swabs in stomacher bags as described in Section 2.1.1 were immersed for 30 min with sterile saline solution. Serial tenfold dilutions were then prepared and plated onto the appropriate media. All media used in the microbiological experiments were purchased from Hangzhou Microbial Reagent Co., Ltd. (Hangzhou, China). For enumeration, the results were reported as the logarithm of colony forming units (cfu) per cm2 (lg cfu/cm2). TVC were performed using the pour-plate method with a plate count agar (PCA) referring to the National Standard of China (GB 4789.2-2010). Coliform bacteria were enumerated via a violet red bile agar (VRBA) method, and a minimum of 10 representative colonies from VRBA plates were inoculated into brilliant green lactose bile (BGLB) and incubated to confirm the coliforms (GB 4789.3-2010). Intestinal enterococci were cultivated at 35 ± 2 C for 24 h (SN/T, 1933.12007). Enumeration of Pseudomonas spp. was performed on cetrimide fucidin cephaloridine (CFC) agar plate and incubated at 25 C for 48 h in accordance with the ISO 13720: Meat and meat product enumeration of Pseudomonas spp (Talon et al., 2007). The prevalence of Salmonella was performed according to the National Standard of China (GB 4789.4-2010). 1 mL of homogenate in Section 2.1.2 was inoculated into BPW solution (buffered peptone water, 2%), and cultured at 37 C for 24 h and the presence of Salmonella was confirmed through selective enrichment and further PCR assays of presumptive colonies based on specific virulence genes (inv A and hut gene) of Salmonella. Occurrence of S. aureus was detected by surface plating on BairdeParke Agar plate and incubated at 37 C for 45e48 h in accordance with the methods described in Chinese standards (GB 4789.10-2010). The prevalence of Salmonella and S. aureus in quick-frozen drumsticks after the treatment were calculated by dividing the total number of samples by the number of positive samples. 2.4. PCR-DGGE analysis 2.4.1. DNA extraction The cotton swabs after sampling as described in Section 2.1.1
Table 1 Factors and levels of orthogonal test [L16 (45)]. Factors
A Lactic acid concentration (w/v, %) B Citric acid concentration (w/v, %) C Spray washing time (s)
Levels 1
2
3
4
0.5 0.5 15
1.0 1.0 30
1.5 1.5 45
2.0 2.0 60
Y. Zhu et al. / Food Control 67 (2016) 1e8
(control groups and treated groups) were immersed in 50-mL volume of sterile saline solution for 30 min to obtain the sample solutions. The sample solution was centrifuged for 10 min at 10,000 rpm, and the sediment was washed for three times with 0.1 mol/L PBS (pH 7.0) and then stored at 20 C for further analysis. Bacterial DNA was extracted using the TIANamp Bacteria DNA Kit (Tiangen, Beijing, China) according to manufacturer instructions and then suspended in 90 mL of TE buffer. DNA solution was analyzed through 1.0% agarose gel electrophoresis and then stored at 20 C for the subsequent experiments. 2.4.2. PCR The universal bacterial primers targeting the 16S rDNA V3 variable region, F338-GC(5ʹ-CGCCCGCCGCGCGCGGCGGGC GGGGCGGGGGCACGGGGGGACTCCTACGGGAGGCAGCAG-3ʹ) and R518 (5ʹ-GTATTACCGCGGCTGCTGG-3ʹ) were used to amplify fragments with a size of approximately 200 bp (Muyzer et al., 1993). A GC clamp was added to the F338-GC primer to enable DGGE. These primers were synthesized by Shanghai Invitrogen and Biotechnology Co., Ltd. (Shanghai, China). A PCR mixture with a total volume of 50 mL contained 1 mL of template (approximately 10 ng), 0.5 mL of each of the primers (10 mmol), 25 mL of Taq PCR Master Mix, and 23 mL of dd H2O. PCR was performed under the following conditions: 94 C for 5 min; amplification for 30 cycles (94 C for 1 min, 66 C for 1 min and 72 C for 20 s); and 72 C for 10 min. An aliquot of 5 mL of the PCR product was analyzed through electrophoresis on an agarose gel (1.0%) to check for amplicon size and concentration. Gels were visualized and digitized with the Gel Doc™ 2000 Gel Documentation System (Bio-Rad, CA, USA).
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sequence alignment by using CLUSTAL X version 1.81. The DGGE profiles were subjected to cluster analysis in NYSYS 2.1 software. 2.5. Effects of combined organic acid-based spray washing on physico-chemical indices and sensory attributes of the quick-frozen drumstick products during storage 2.5.1. pH and total volatile base nitrogen (TVB-N) measurements The pH of quick-frozen chicken drumsticks during storage was measured after the samples were thawed. A pH meter (Cyberscan PC 510, Eutech, Shanghai, China) was standardized with a threepoint method before each determination against standard buffers (pH 4.00, 6.86, and 9.18). The drumstick samples were homogenized in 100 mL of dd H2O for 20 min, and the slurry was filtered using a Whatman filter paper. The filtrate was subsequently analyzed. TVB-N was determined, as described previously (Liu et al., 2016). The TVB-N content was expressed as mg of TVB-N per 100 g of sample. 2.5.2. Sensory evaluation Sensory evaluation was conducted within 2 months after organic acid treatment and storage in a freezer at 18 C in accordance with the methods of National Standard of China (GB 16869-2005). Seven experienced panelists were asked to evaluate each batch of samples before and after treatment were administered. The samples were presented in a randomized order. Scores were assigned for color, texture, odor, and broth on the basis of a structured four-point hedonic scale (9e10, 8e9, 7e8, and 6). 2.6. Statistical analysis
2.4.3. DGGE protocol DGGE was conducted using a temporal temperature gradient gel electrophoresis system (CBS Scientific Company, CA, USA) in accordance with the previous methods with slight modifications (Muyzer et al., 1993). In brief, the PCR products were loaded in 8% polyacrylamide gel (acrylamide:bisacrylamide ¼ 38:2) with a denaturing gradient of 40%e50% ureaeformamide and electrophoresed at 200 V for 30 min and then at 120 V for 17 h at 60 C. Afterward, the gels were washed with a fixing solution (10% v/v ethanol and 0.5% v/v acetic acid) for 30 min, stained with a staining solution (0.2% w/v AgNO3 and 0.08% formaldehyde) for 20 min, washed twice in double-distilled (dd) H2O, and developed in a precooling developing solution (1.5% w/v sodium hydroxide and 0.2% v/v formaldehyde) for 10 min. 2.4.4. Similarity analysis The DGGE gel was scanned using a Gel Doc™ 2000 gel documentation system. The electrophoretic profiles were subjected to cluster analyses via an unweighted pair group method by using arithmetic averages (UPGMA) and Dice coefficients with Quantity One in a Bio-Rad gel documentation system (Rademaker, Louws, Rossbach, Vinuesa, & De Bruijn, 1999). The similarity indices of the DGGE profiles were calculated (Arakere et al., 2005). 2.4.5. DNA reclaimed from the DGGE gels and subjected to sequence analysis All detectable bands on the DGGE gel were incised, reclaimed, and amplified by PCR, and sent to Shanghai Sangon Biotechnology Company (Shanghai, China) for sequencing to further analyze the microbial community structure and diversity in the three stages of cutting process before and after the treatment. The obtained DNA sequences were analyzed to determine their homology to those in the available databases at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) by using the BLAST-N software. On the basis of these sequences, we performed
Three independent replications of each sample were performed, and mean and standard deviation were calculated and subjected to ANOVA. Statistical significance was determined by using SPSS. In all cases, P < 0.05 indicated significant differences. 3. Results and discussion 3.1. Optimization of combined organic acid-based spray washing during cutting Organic acid concentration and treatment time are considered as important factors that influence the decontamination effects on chicken drumsticks. Table 2 lists the microbial counting data of orthogonal test design [L16 (45)]. Compared with the control group, total viable counts in the treated groups were significantly reduced (P < 0.05), with a decline of 1.21e2.64 log10 cfu/cm2, which indicated that the spray washing of combined lactic and citric acids was effective. The result of the orthogonal test presented the optimum conditions (A3B3C4): 1.5% lactic acid, 1.5% citric acid, and spray-washing for 60 s. However, the color of the chicken drumsticks changed significantly under these conditions. Therefore, based on the results of both orthogonal design and sensory evaluation, group 2 with 0.5% lactic acid, 1% citric acid and spray-washing time 30 s was selected as the preferable treatment. 3.2. Decontamination effect of combined organic acid-based spraywashing on the chicken drumsticks from the cutting process The microbial counts on the chicken drumsticks spray-washed with a combination of lactic acid (0.5%) and citric acid (1%) for 30 s were significantly lower than those on the samples in the control group (Fig. 1). The decontamination treatment brought about a TVC reduction of 1.68 log10 cfu/cm2 as compared to the
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Table 2 L16 (45) orthogonal test results. Test number
Factors
Control 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 K1 K2 K3 K4 R Optimal level Optimal combination Order
(A) Lactic acid concentration (%)
(B) Citric acid concentration (%)
(C) Spray washing time (s)
e A1 A1 A1 A1 A2 A2 A2 A2 A3 A3 A3 A3 A4 A4 A4 A4 8.36 7.02 8.65 6.37 2.28 A3 A3B3C4 C>A>B
e B1 B2 B3 B4 B1 B2 B3 B4 B1 B2 B3 B4 B1 B2 B3 B4 7.22 7.29 8.35 7.54 1.13 B3
e C1 C2 C3 C4 C2 C1 C4 C3 C3 C4 C1 C2 C4 C3 C2 C1 6.63 6.98 8.89 9.00 2.37 C4
Microbial counts (log10 cfu/cm2)
Significancec
4.17 ± 0.26a 1.41b 1.97b 2.41b 2.57b 1.87b 1.30b 2.09b 1.76b 1.84b 2.24b 2.64b 1.93b 2.10b 1.78b 1.21b 1.28b
a d h k l f c hi d ef j l g i e b bc
Note:. a Mean value of TVC (log10 cfu/cm2) of the control group. b Log10 cfu/cm2 reduction ¼ (log10 cfu/cm2 before treatment) (log10 cfu/cm2 after treatment). c Values followed by the same letters in the column ‘Significance’ were not significantly different (P > 0.05), as indicated by the ANOVA test. R refers to the result of extreme analysis. Ki (i ¼ 1, 2, 3, 4) refers to the sum of four values at the same level for each factor in each column.
5
Control group Treated group
lg (cfu/cm2)
4
3
2
1
0
TVC
Enterococcus Coliform bacteria Pseudomonas
Fig. 1. Conditions of the specific microbial contamination in chicken drumsticks before and after treatment (spray washing conditions: a combination of 0.5% lactic acid and 1% citric acid for 30 s; temperature, 10 C).
untreated control. Previously, it has been reported that spray washing with 0.25% lactic acid at different stages of processing lowered the mean TVC by 0.76 lg cfu/cm2 after scalding, by 0.60 lg cfu/cm2 after defeathering and by 0.78 lg cfu/cm2 after evisceration (Sakhare, Sachindra, Yashoda, & Rao, 1999). Also, TVC reductions of 0.59e2.48 log10 cfu/cm2 were observed, with the greater reductions achieved at the higher concentrations (up to 5% citric acid) (Bolton et al., 2014). Accordingly, the findings here were satisfactory. The initial mean enterococcus populations of 1.13 log10 cfu/cm2 were reduced to undetectable level by enumeration after the treatment. In addition, a significant reduction of coliform bacteria (1.31 log10 cfu/cm2) and Pseudomonas (1.85 log10 cfu/cm2) was observed, respectively. Organic acids are considered to inhibit microbial activity by two
primary mechanisms: by cytoplasmic acidification with subsequent uncoupling of energy production and regulation, and by accumulation of the dissociated acid anion to toxic levels (Mani-Lopez, pez-Malo, 2012). Lactic acid is lipophilic acid, which García, & Lo can easily penetrate across the microbial plasma membrane in the undissociated form; an alkaline environment is encountered, favoring the dissociation of the acid into the acid anion and free proton, thus reducing the internal pH of the cell and disrupting important cellular processes (Smulders, Barendsen, Van Logtestijn, Mossel, & Van Der Marel, 1986). Citric acid is known to efficiently inhibit pathogens by destabilizing the outer membrane through metal chelation or intercalation, in some instances resulting in enhanced pathogen inhibition versus the monocarboxylic like lactic acid, but its usage is potentially limited by possible negative sensory impact and the need for an appropriate pH for optimum bactericidal properties (Mller, Call, & Whitin, 1993). Therefore, above results indicated that lactic acid and citric acid can be administered in combination to treat chicken drumsticks during the cutting process in slaughtering and processing chains.
3.3. Reduction in S. aureus and Salmonella populations of quickfrozen chicken drumsticks after the treatment Salmonella and S. aureus are two kinds of common foodborne pathogens in retail poultry meat. These pathogens pose potential threats to consumers. In this study, the population of S. aureus and Salmonella was 46.67% (56/120) and 14.15% (17/120) frozen drumstick products in the control group, respectively, while the population of treated products was 14.17% (17/120) and 3.33% (4/120), respectively. The results indicated that the combined organic acidbased spray washing induced a significant reduction (P < 0.05) in the prevalence of the two typical pathogenic bacteria found in the quick-frozen chicken drumsticks.
Y. Zhu et al. / Food Control 67 (2016) 1e8
3.4. PCR-DGGE analysis 3.4.1. DNA extraction and PCR amplification of V3 region of 16S rDNA The results of agarose gel electrophoresis analysis revealed that the length of the DNA extracted from each sample exceeded 2000 bp (Fig. 2a). The V3 region of 16S rDNA was amplified by using the extracted DNA as a template in PCR. In Fig. 2b, a distinct target band (approximately 250 bp) was detected. In addition, in order to ensure a successful DGGE analysis, adequate PCR products were obtained through a secondary PCR. 3.4.2. DGGE analysis The bands in the DGGE gel represented microbial species or operational taxonomic units in the microbial community. The change in the band number in the DGGE lanes indicated the complexity and variability of the bacterial community on the chicken drumsticks (Ercolini, 2004). The results (Fig. 3) revealed that the DGGE profile varied among the different stages of the cutting process. The species and quantities of the bacteria on the surface of the chicken drumsticks in each stage decreased significantly after the chicken drumsticks were subjected to combined organic acid treatments. Thirteen distinct bands in Fig. 3 were successfully reclaimed to further analyze the naturally occurring microflora in the three different stages of cutting process. With reference to NCBI BLAST, bands b, e, and g were identified as uncultured Pseudomonas sp. Bands d, f, l, and m were found as uncultured bacteria. Band c corresponded to the uncultured Flavobacteria. These microorganisms were viable but non-culturable (VBNC) bacterial cells. These cells could not form colonies on solid media but could maintain metabolic activities and elongation abilities after the administration of nutrients (Gauthier, 2000). Therefore, these VBNC cells are of particular concern because they may remain undetected via routine bacteriological procedures. Bands h and i identified respectively as Enterobacter cloacae and Gamma proteobacterium could be found on the surface of the chicken drumsticks in each stage of the cutting process. Band j corresponded to Pseudomonas moraviensis sp., appeared in the middle stage of the cutting process. Band k identified as Pseudomonas putida was just detected in the earlier stage. Fig. 4 presents the similarity indices of the DGGE profiles. The highest similarities (71%) were found in the samples obtained in the earlier and middle stages before the treatment. Before the treatment, the similarity indices in the different stages of cutting
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process ranged from 62% to 71%. The results indicated that the naturally occurring microflora in the different stages of cutting were similar. After the treatment was administered, the similarity index decreased significantly. The similarity indices between 1 and 1ʹ, 2 and 2ʹ, and 3 and 3ʹ, which corresponded to the same stages during cutting before and after the treatment, were 37%, 44%, and 37%, respectively. These findings suggested that the microbial counts were significantly reduced by the combined organic acid treatment, and the microbial communities were strongly affected simultaneously.
3.5. pH change in quick-frozen chicken drumsticks during storage pH has been used as an important criterion to evaluate meat quality (Chouliara et al., 2006). Fig. 5A illustrates the change in pH of the quick-frozen chicken drumsticks during storage. The pH of the quick-frozen chicken drumsticks ranged from approximately 6.0 to 7.0. Similar pH values were observed in both untreated and treated samples throughout storage. In addition, pH showed no statistically significant changes (P > 0.05) for all of the eight treated chicken drumstick samples during the 2-month storage. As a result, organic acid-based spray washing did not elicit considerable effects on the pH of chicken drumsticks.
3.6. TVB-N determination of quick-frozen chicken drumsticks during storage As an important reference index, the TVB-N content of meat produced by the microbial degradation of nitrogenous tissues is used to reveal microbiological spoilage and to evaluate the quality of meat and other meat products (Chan et al., 2006). For instance, it has been reported that the levels of TVB-N as an indicator of skinless chicken breast spoilage were approximately 18 and 17 mg N per 100 g sample when stored at 4 and 10 C (Rukchon, Nopwinyuwong, Trevanich, Jinkarn, & Suppakul, 2014). In our study, as can be seen in Fig. 5B, the TVB-N content of the quickfrozen chicken drumsticks before and after the treatment presented a similar trend in the 2-month storage. Although the difference between the control group and treated group was not significant (P > 0.05), the TVB-N content of quick-frozen chicken drumsticks increased more slowly in the treated group, because microbial population was significantly reduced after the treatment.
Fig. 2. Agarose gel of DNA from each sample (A) and agarose gel of the PCR products of the V3 regions of DNA for DGGE (B). M represents Marker. Lanes 1, 2, and 3 represent chicken drumstick samples from earlier, middle, and later stages of cutting process in the control groups, respectively. Lanes 1ʹ, 2ʹ, and 3ʹ represent the chicken drumstick samples from earlier, middle, and later stages of cutting process after the treatment was administered.
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Fig. 3. DGGE profiles of the PCR products of the DNA samples from the chicken drumsticks in different stages of the cutting workshop before and after decontamination treatment (A); Schematic of the DGGE profile (B). Each lane corresponds to samples in Fig. 2. Bands a, b, c, d, e, f, h, i, j, k, l, and m are the detectable bands on the DGGE gel of different lanes.
Fig. 4. Similarity index of the DGGE profiles. 1, 2, 3, 1ʹ, 2ʹ, and 3ʹ correspond to Fig. 2.
3.7. Sensory properties of quick-frozen chicken drumsticks during storage Panelists provided similar preference scores for the control group and treated group during freezing storage, which indicated
that the treated samples were highly acceptable. No significant difference (P > 0.01) was found between the control and treated samples for all characteristics evaluated. Therefore, it could be concluded that the treatment based on organic acid spray-washing did not affect the organoleptic quality of chicken drumsticks.
Y. Zhu et al. / Food Control 67 (2016) 1e8
7
10
7.0
A
6.8
B
9
6.6
8
TVBN(mg/100g)
6.4
pH
6.2 6.0 5.8
Control group Treated group
5.6
7 6 5
Control group Treated group
4
5.4 5.2
3
5.0 0
10
20
30
40
50
60
Time (d)
0
10
20
30
40
50
60
Time (d)
Fig. 5. pH change (A) and TVB-N content (B) of the quick-frozen chicken drumsticks during storage before and after treatment.
4. Conclusion This work demonstrated the effectiveness of the antimicrobial effect of acid treatments in different stages of the cutting process. The most effective treatment conditions were 0.5% lactic acid, 1.0% citric acid, and spray-washing for 30 s. The changes in naturally occurring microflora were analyzed through microbiological counting and PCR-DGGE analysis. Physicochemical indices, including pH and TVB-N, and the sensory attributes of the quickfrozen chicken drumsticks during storage before and after the treatment were also investigated. On the basis of these comprehensive profiling, we can conclude that decontamination during the cutting process by spray washing with water containing a combination of lactic acid and citric acid possibly improved the microbiological quality of chicken drumsticks without significant sensory changes. The improvement in the microbiological quality of the treatment further enhanced the shelf life of poultry meat. Acknowledgments The authors are grateful for the financial support of the National Natural Science Foundation of China (Grant No. 31400066) and the Science & Technology Foundation of Sichuan Province (Grant No. 14NZ0012) to this research. References Acuff, G. (2005). Chemical decontamination strategies for meat. Improving the safety of fresh meat. Boca Raton: CRC Press. Arakere, G., Nadig, S., Swedberg, G., Macaden, R., Amarnath, S. K., & Raghunath, D. (2005). Genotyping of methicillin-resistant Staphylococcus aureus strains from two hospitals in Bangalore, South India. Journal of Clinical Microbiology, 43(7), 3198e3202. Bohaychuk, V., Gensler, G., King, R., Manninen, K., Sorensen, O., Wu, J., et al. (2006). Occurrence of pathogens in raw and ready-to-eat meat and poultry products collected from the retail marketplace in Edmonton, Alberta, Canada. Journal of Food Protection®, 69(9), 2176e2182. Bolder, N. (1997). Decontamination of meat and poultry carcasses. Trends in Food Science & Technology, 8(7), 221e227. Bolton, D., Meredith, H., Walsh, D., & McDowell, D. (2014). The effect of chemical treatments in laboratory and broiler plant studies on the microbial status and shelf-life of poultry. Food Control, 36(1), 230e237. Chan, S. T., Yao, M. W., Wong, Y., Wong, T., Mok, C., & Sin, D. W. (2006). Evaluation of chemical indicators for monitoring freshness of food and determination of volatile amines in fish by headspace solid-phase microextraction and gas chromatography-mass spectrometry. European Food Research and Technology, 224(1), 67e74. Chouliara, I., Samelis, J., Kakouri, A., Badeka, A., Savvaidis, I., Riganakos, K., et al. (2006). Effect of irradiation of frozen meat/fat trimmings on microbiological and physicochemical quality attributes of dry fermented sausages. Meat Science,
74(2), 303e311. Ercolini, D. (2004). PCR-DGGE fingerprinting: novel strategies for detection of microbes in food. Journal of Microbiological Methods, 56(3), 297e314. Gauthier, M. J. (2000). Environmental parameters associated with the viable but nonculturable state. In R. R. Colwell, & D. J. Grimes (Eds.), Nonculturable microorganisms in the environment (pp. 87e112). Washington, D.C.: Springer. Gill, C., Badoni, M., Moza, L., Barbut, S., & Griffiths, M. (2005). Microbiological sampling of poultry carcass portions by excision, rinsing, or swabbing. Journal of Food Protection®, 68(12), 2718e2720. Henchion, M., McCarthy, M., Resconi, V. C., & Troy, D. (2014). Meat consumption: trends and quality matters. Meat Science, 98(3), 561e568. Koolman, L., Whyte, P., Meade, J., Lyng, J., & Bolton, D. (2014a). A combination of chemical and ultrasonication treatments to reduce Campylobacter jejuni on raw poultry. Food and Bioprocess Technology, 7(12), 3602e3607. Koolman, L., Whyte, P., Meade, J., Lyng, J., & Bolton, D. (2014b). Use of chemical treatments applied alone and in combination to reduce Campylobacter on raw poultry. Food Control, 46, 299e303. Liu, A., Peng, Z., Zou, L., Zhou, K., Ao, X., He, L., et al. (2016). The effects of lactic acidbased spray washing on bacterial profile and quality of chicken carcasses. Food Control, 60, 615e620. Li, M., Zhou, G., Xu, X., Li, C., & Zhu, W. (2006). Changes of bacterial diversity and main flora in chilled pork during storage using PCR-DGGE. Food microbiology, 23(7), 607e611. pez-Malo, A. (2012). Organic acids as antimicroMani-Lopez, E., García, H. S., & Lo bials to control Salmonella in meat and poultry products. Food Research International, 45(2), 713e721. Mller, A. J., Call, J. E., & Whitin, R. C. (1993). Comparison of organic acid salts for Clostridium botulinum control in an uncured turkey product. Journal of Food Protection®, 56(11), 958e962. Musavian, H. S., Krebs, N. H., Nonboe, U., Corry, J. E., & Purnell, G. (2014). Combined steam and ultrasound treatment of broilers at slaughter: a promising intervention to significantly reduce numbers of naturally occurring Campylobacters on carcasses. International Journal of Food Microbiology, 176, 23e28. Muyzer, G., De Waal, E. C., & Uitterlinden, A. G. (1993). Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Applied and Environmental Microbiology, 59(3), 695e700. Owens, C. M., Alvarado, C., & Sams, A. R. (2010). Poultry meat processing (2nd ed.). Boca Raton: CRC Press. GB 4789.2-2010. National food safety Standard. Food microbiological examination: Aerobic plate count.. GB 4789.3-2010. National food safety Standard. Food microbiological examination: Enumeration of coliforms.. GB 4789.4-2010. National food safety Standard. Food microbiological examination: Salmonella.. GB 4789.10-2010. National food safety Standard. Food microbiological examination: Staphylococcus aureus.. Rademaker, J., Louws, F., Rossbach, U., Vinuesa, P., & De Bruijn, F. (1999). Computerassisted pattern analysis of molecular fingerprints and database construction. Molecular Microbial Ecology Manual, 7(3), 1e33. Rukchon, C., Nopwinyuwong, A., Trevanich, S., Jinkarn, T., & Suppakul, P. (2014). Development of a food spoilage indicator for monitoring freshness of skinless chicken breast. Talanta, 130, 547e554. Sakhare, P., Sachindra, N., Yashoda, K., & Rao, D. N. (1999). Efficacy of intermittent decontamination treatments during processing in reducing the microbial load on broiler chicken carcass. Food Control, 10(3), 189e194. Smulders, F., Barendsen, P., Van Logtestijn, J., Mossel, D., & Van Der Marel, G. (1986). Review: lactic acid: considerations in favour of its acceptance as a meat
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decontamininant. International Journal of Food Science & Technology, 21(4), 419e436. SN/T 1933.1e2007. Exit inspection and quarantine standards of the People's Republic of China. Detection of Enterococci in food and water e Part 1: Method for plate count and MPN. Talon, R., Lebert, I., Lebert, A., Leroy, S., Garriga, M., Aymerich, T., et al. (2007). Traditional dry fermented sausages produced in small-scale processing units in Mediterranean countries and Slovakia. 1: microbial ecosystems of processing
environments. Meat Science, 77(4), 570e579. €m, A., & Molin, G. (1987). Incidence of potential pathogens on raw pork, Ternstro beef and chicken in Sweden, with special reference to Erysipelothrix rhusiopathiae. Journal of Food Protection®, 50(2), 141e149. Tompkin, R. (1994). HACCP in the meat and poultry industry. Food Control, 5(3), 153e161. GB 16869-2005. National food safety Standard. Fresh and frozen poultry product.
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Effects of combined organic acid treatments during the cutting process on the natural microflora and quality of chicken drumsticks Yuanting Zhu a, Xiaolong Xia a, Aiping Liu a, Likou Zou b, Kang Zhou a, Xinfeng Han a, Guoquan Han 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 5 January 2016 Received in revised form 18 February 2016 Accepted 19 February 2016 Available online 23 February 2016
Reducing the microbial load on broiler chicken carcasses at each stage of poultry meat processing, is highly important for hygienic meat production. This study was conducted to assess the efficacy of various combined organic acid treatments during the cutting process on the microbial decontamination of chicken drumsticks. Changes in naturally occurring microflora before and after treatment were analyzed through microbiological counting and polymerase chain reactionedenaturing gradient gel electrophoresis (PCR-DGGE). Results revealed that the most effective treatments obtained through the combination of orthogonal design and sensory evaluation were as follows: 0.5% lactic acid (w/v), 1% citric acid (w/v), and spray-washing for 30 s. Microbiological counting results and PCR-DGGE analysis indicated that the microbial load on the chicken drumsticks decreased significantly after the treatment was administered. The treatment did not affect the physicochemical properties and sensory attributes of the quick-frozen chicken drumsticks during storage. Therefore, the proposed technique could be used to improve the microbial quality of chicken drumsticks. The technique could also be employed in poultry meat production chains. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Chicken drumsticks Organic acid Natural microflora Decontamination PCR-DGGE Sensory evaluation
1. Introduction A significant increase in global poultry meat consumption has been observed. This increase is likely to continue into the future (Henchion, McCarthy, Resconi, & Troy, 2014). During the conversion of chicken into meat for consumption, broiler carcasses are at a high risk of contamination from fecal soiling on feathers and skin during slaughtering, intestinal content leakage during evisceration, and processing equipment and general processing environments (Owens, Alvarado, & Sams, 2010). Therefore, poultry meat can become contaminated with various food-borne pathogens, including Salmonella, Campylobacter spp., Listeria monocytogenes, Clostridium perfringens, and Staphylococcus aureus (Bohaychuk € m & Molin, 1987). Various interventions et al., 2006; Ternstro have been implemented to reduce the number of food-borne pathogens on surfaces of broiler carcasses (Bolton, Meredith, Walsh, & McDowell, 2014; Koolman, Whyte, Meade, Lyng, &
* Corresponding author. E-mail address: [email protected] (S. Liu). http://dx.doi.org/10.1016/j.foodcont.2016.02.031 0956-7135/© 2016 Elsevier Ltd. All rights reserved.
Bolton, 2014a, 2014b; Musavian, Krebs, Nonboe, Corry, & Purnell, 2014). Furthermore, manufacturers should reduce or eliminate carcass contamination of psychrotrophic spoilage bacteria to ensure adequate shelf life of raw chilled products (Owens et al., 2010). Hazard analysis and critical control point (HACCP) systems have been widely used in poultry meat industries to minimize product contamination (Tompkin, 1994). HACCP includes critical control points (CCP) at which an intervention may be implemented to prevent, reduce, or eliminate microbial contamination. Decontamination is a bactericidal treatment applied to reduce pathogenic and spoilage organisms (Bolder, 1997). Chemical treatments can be used to eliminate the presence of pathogens and spoilage microorganisms and thus may provide a basis of an effective CCP intervention (Acuff, 2005). The United States Food and Drug Administration has approved the use of a number of chemicals as decontaminants of poultry meat. Several organic acids, such as lactic acid and acetic acid, have been generally recognized as safe substances for use in poultry processing plants. These acids could inhibit subsequent microbial growth and thus extend shelf life. The efficiency of organic acid solutions in reducing artificially
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inoculated pathogens has been extensively investigated (Koolman et al., 2014a, 2014b). Few have been evaluated against naturally occurring microflora on carcasses after treatment. Muyzer, De Waal, & Uitterlinden (1993) proposed denaturing gradient gel electrophoresis (DGGE) based on the separation of PCR amplicons with the same size but with different 16S rDNA sequences; this technique can be used to overcome the limitations of culturedependent techniques, to reveal microbial communities, and to analyze microbial diversity. As a well-established tool, this technique has also been applied to numerous fields. For instance, Li, Zhou, Xu, Li, and Zhu (2006) employed PCR-DGGE to investigate the bacterial diversity and the main flora in chilled pork. Cutting is the last step before broiler chicken products are quickly frozen. Therefore, the microbial decontamination efficacy in this process has an immediate influence on the quality of chicken products. This study aimed to evaluate the efficacy of microbial decontamination treatment during the cutting process in the slaughtering and processing chains of poultry meat. Combined organic acid treatment conditions for chicken drumsticks were optimized. Changes in naturally occurring bacterial community on chicken drumsticks before and after treatment were analyzed through PCR-DGGE. The effects of the optimum treatment on the physiochemical properties and sensory attributes of chicken drumsticks were also investigated. This study may provide a basis of chicken slaughtering and processing methods.
2. Materials and methods 2.1. Sampling 2.1.1. Sampling of chicken drumstick surface for microbial analyses Chicken drumstick samples were obtained in three stages, namely, earlier, middle, and later stages, of cutting process in the broiler cutting workshop at a local chicken slaughtering plant where about 30,000,000 broiler chickens are processed each year. The chicken drumstick surfaces were sampled in accordance with previously described methods (Gill, Badoni, Moza, Barbut, & Griffiths, 2005). Briefly, the surface covering 25 cm2 of each chicken drumstick sample was swabbed by using sterilized cotton swabs moistened with 0.1% peptone water. The swabs obtained after sampling were placed in stomacher bags and stored in an ice bath during transportation to our laboratory, and were determined within 3 h. The samples were collected for control and treated groups.
2.1.2. Sampling of quick-frozen chicken drumsticks for pathogen detection For each sample from control and treated groups, 25 g of thawed chicken drumsticks was incised, collected in a sterile plastic stomacher bag containing 225 mL of sterile saline water, and pummeled for 2 min. A 1-mL aliquot of each homogenate from control and treated drumsticks was subjected to enrichment for detection of S. aureus and Salmonella.
2.1.3. Sampling of quick-frozen chicken drumsticks for physicochemical and sensory indexes evaluation during storage The quick-frozen chicken drumstick samples, vacuum-packaged and stored at 18 C in a freezer, were collected on days 5, 10, 15, 20, 30, 40, 50, and 60, and then subjected to physicochemical index analysis and sensory evaluation. The samples were collected for both control and treated groups.
2.2. Optimization of combined organic acid-based spray washing during cutting to reduce microbial load on chicken drumsticks The optimal conditions for combined organic acid-based spray washing were investigated by using an orthogonal experimental design [L16(45)], and the effects of lactic - citric acid concentrations [food-grade, Jindan Company (Henan, China)], and spray-washing time were analyzed. Table 1 lists the factors and levels of the tests. After spray washing was administered, total viable counts (TVC) of chicken drumsticks in treated groups and control groups were performed. The control group received no treatment. In addition, the sensory attributes of chicken drumsticks in the treated groups were evaluated. The most promising treatments were obtained on the basis of both decontamination effects and sensory evaluation. 2.3. Microbiological analysis The cotton swabs in stomacher bags as described in Section 2.1.1 were immersed for 30 min with sterile saline solution. Serial tenfold dilutions were then prepared and plated onto the appropriate media. All media used in the microbiological experiments were purchased from Hangzhou Microbial Reagent Co., Ltd. (Hangzhou, China). For enumeration, the results were reported as the logarithm of colony forming units (cfu) per cm2 (lg cfu/cm2). TVC were performed using the pour-plate method with a plate count agar (PCA) referring to the National Standard of China (GB 4789.2-2010). Coliform bacteria were enumerated via a violet red bile agar (VRBA) method, and a minimum of 10 representative colonies from VRBA plates were inoculated into brilliant green lactose bile (BGLB) and incubated to confirm the coliforms (GB 4789.3-2010). Intestinal enterococci were cultivated at 35 ± 2 C for 24 h (SN/T, 1933.12007). Enumeration of Pseudomonas spp. was performed on cetrimide fucidin cephaloridine (CFC) agar plate and incubated at 25 C for 48 h in accordance with the ISO 13720: Meat and meat product enumeration of Pseudomonas spp (Talon et al., 2007). The prevalence of Salmonella was performed according to the National Standard of China (GB 4789.4-2010). 1 mL of homogenate in Section 2.1.2 was inoculated into BPW solution (buffered peptone water, 2%), and cultured at 37 C for 24 h and the presence of Salmonella was confirmed through selective enrichment and further PCR assays of presumptive colonies based on specific virulence genes (inv A and hut gene) of Salmonella. Occurrence of S. aureus was detected by surface plating on BairdeParke Agar plate and incubated at 37 C for 45e48 h in accordance with the methods described in Chinese standards (GB 4789.10-2010). The prevalence of Salmonella and S. aureus in quick-frozen drumsticks after the treatment were calculated by dividing the total number of samples by the number of positive samples. 2.4. PCR-DGGE analysis 2.4.1. DNA extraction The cotton swabs after sampling as described in Section 2.1.1
Table 1 Factors and levels of orthogonal test [L16 (45)]. Factors
A Lactic acid concentration (w/v, %) B Citric acid concentration (w/v, %) C Spray washing time (s)
Levels 1
2
3
4
0.5 0.5 15
1.0 1.0 30
1.5 1.5 45
2.0 2.0 60
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(control groups and treated groups) were immersed in 50-mL volume of sterile saline solution for 30 min to obtain the sample solutions. The sample solution was centrifuged for 10 min at 10,000 rpm, and the sediment was washed for three times with 0.1 mol/L PBS (pH 7.0) and then stored at 20 C for further analysis. Bacterial DNA was extracted using the TIANamp Bacteria DNA Kit (Tiangen, Beijing, China) according to manufacturer instructions and then suspended in 90 mL of TE buffer. DNA solution was analyzed through 1.0% agarose gel electrophoresis and then stored at 20 C for the subsequent experiments. 2.4.2. PCR The universal bacterial primers targeting the 16S rDNA V3 variable region, F338-GC(5ʹ-CGCCCGCCGCGCGCGGCGGGC GGGGCGGGGGCACGGGGGGACTCCTACGGGAGGCAGCAG-3ʹ) and R518 (5ʹ-GTATTACCGCGGCTGCTGG-3ʹ) were used to amplify fragments with a size of approximately 200 bp (Muyzer et al., 1993). A GC clamp was added to the F338-GC primer to enable DGGE. These primers were synthesized by Shanghai Invitrogen and Biotechnology Co., Ltd. (Shanghai, China). A PCR mixture with a total volume of 50 mL contained 1 mL of template (approximately 10 ng), 0.5 mL of each of the primers (10 mmol), 25 mL of Taq PCR Master Mix, and 23 mL of dd H2O. PCR was performed under the following conditions: 94 C for 5 min; amplification for 30 cycles (94 C for 1 min, 66 C for 1 min and 72 C for 20 s); and 72 C for 10 min. An aliquot of 5 mL of the PCR product was analyzed through electrophoresis on an agarose gel (1.0%) to check for amplicon size and concentration. Gels were visualized and digitized with the Gel Doc™ 2000 Gel Documentation System (Bio-Rad, CA, USA).
3
sequence alignment by using CLUSTAL X version 1.81. The DGGE profiles were subjected to cluster analysis in NYSYS 2.1 software. 2.5. Effects of combined organic acid-based spray washing on physico-chemical indices and sensory attributes of the quick-frozen drumstick products during storage 2.5.1. pH and total volatile base nitrogen (TVB-N) measurements The pH of quick-frozen chicken drumsticks during storage was measured after the samples were thawed. A pH meter (Cyberscan PC 510, Eutech, Shanghai, China) was standardized with a threepoint method before each determination against standard buffers (pH 4.00, 6.86, and 9.18). The drumstick samples were homogenized in 100 mL of dd H2O for 20 min, and the slurry was filtered using a Whatman filter paper. The filtrate was subsequently analyzed. TVB-N was determined, as described previously (Liu et al., 2016). The TVB-N content was expressed as mg of TVB-N per 100 g of sample. 2.5.2. Sensory evaluation Sensory evaluation was conducted within 2 months after organic acid treatment and storage in a freezer at 18 C in accordance with the methods of National Standard of China (GB 16869-2005). Seven experienced panelists were asked to evaluate each batch of samples before and after treatment were administered. The samples were presented in a randomized order. Scores were assigned for color, texture, odor, and broth on the basis of a structured four-point hedonic scale (9e10, 8e9, 7e8, and 6). 2.6. Statistical analysis
2.4.3. DGGE protocol DGGE was conducted using a temporal temperature gradient gel electrophoresis system (CBS Scientific Company, CA, USA) in accordance with the previous methods with slight modifications (Muyzer et al., 1993). In brief, the PCR products were loaded in 8% polyacrylamide gel (acrylamide:bisacrylamide ¼ 38:2) with a denaturing gradient of 40%e50% ureaeformamide and electrophoresed at 200 V for 30 min and then at 120 V for 17 h at 60 C. Afterward, the gels were washed with a fixing solution (10% v/v ethanol and 0.5% v/v acetic acid) for 30 min, stained with a staining solution (0.2% w/v AgNO3 and 0.08% formaldehyde) for 20 min, washed twice in double-distilled (dd) H2O, and developed in a precooling developing solution (1.5% w/v sodium hydroxide and 0.2% v/v formaldehyde) for 10 min. 2.4.4. Similarity analysis The DGGE gel was scanned using a Gel Doc™ 2000 gel documentation system. The electrophoretic profiles were subjected to cluster analyses via an unweighted pair group method by using arithmetic averages (UPGMA) and Dice coefficients with Quantity One in a Bio-Rad gel documentation system (Rademaker, Louws, Rossbach, Vinuesa, & De Bruijn, 1999). The similarity indices of the DGGE profiles were calculated (Arakere et al., 2005). 2.4.5. DNA reclaimed from the DGGE gels and subjected to sequence analysis All detectable bands on the DGGE gel were incised, reclaimed, and amplified by PCR, and sent to Shanghai Sangon Biotechnology Company (Shanghai, China) for sequencing to further analyze the microbial community structure and diversity in the three stages of cutting process before and after the treatment. The obtained DNA sequences were analyzed to determine their homology to those in the available databases at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) by using the BLAST-N software. On the basis of these sequences, we performed
Three independent replications of each sample were performed, and mean and standard deviation were calculated and subjected to ANOVA. Statistical significance was determined by using SPSS. In all cases, P < 0.05 indicated significant differences. 3. Results and discussion 3.1. Optimization of combined organic acid-based spray washing during cutting Organic acid concentration and treatment time are considered as important factors that influence the decontamination effects on chicken drumsticks. Table 2 lists the microbial counting data of orthogonal test design [L16 (45)]. Compared with the control group, total viable counts in the treated groups were significantly reduced (P < 0.05), with a decline of 1.21e2.64 log10 cfu/cm2, which indicated that the spray washing of combined lactic and citric acids was effective. The result of the orthogonal test presented the optimum conditions (A3B3C4): 1.5% lactic acid, 1.5% citric acid, and spray-washing for 60 s. However, the color of the chicken drumsticks changed significantly under these conditions. Therefore, based on the results of both orthogonal design and sensory evaluation, group 2 with 0.5% lactic acid, 1% citric acid and spray-washing time 30 s was selected as the preferable treatment. 3.2. Decontamination effect of combined organic acid-based spraywashing on the chicken drumsticks from the cutting process The microbial counts on the chicken drumsticks spray-washed with a combination of lactic acid (0.5%) and citric acid (1%) for 30 s were significantly lower than those on the samples in the control group (Fig. 1). The decontamination treatment brought about a TVC reduction of 1.68 log10 cfu/cm2 as compared to the
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Table 2 L16 (45) orthogonal test results. Test number
Factors
Control 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 K1 K2 K3 K4 R Optimal level Optimal combination Order
(A) Lactic acid concentration (%)
(B) Citric acid concentration (%)
(C) Spray washing time (s)
e A1 A1 A1 A1 A2 A2 A2 A2 A3 A3 A3 A3 A4 A4 A4 A4 8.36 7.02 8.65 6.37 2.28 A3 A3B3C4 C>A>B
e B1 B2 B3 B4 B1 B2 B3 B4 B1 B2 B3 B4 B1 B2 B3 B4 7.22 7.29 8.35 7.54 1.13 B3
e C1 C2 C3 C4 C2 C1 C4 C3 C3 C4 C1 C2 C4 C3 C2 C1 6.63 6.98 8.89 9.00 2.37 C4
Microbial counts (log10 cfu/cm2)
Significancec
4.17 ± 0.26a 1.41b 1.97b 2.41b 2.57b 1.87b 1.30b 2.09b 1.76b 1.84b 2.24b 2.64b 1.93b 2.10b 1.78b 1.21b 1.28b
a d h k l f c hi d ef j l g i e b bc
Note:. a Mean value of TVC (log10 cfu/cm2) of the control group. b Log10 cfu/cm2 reduction ¼ (log10 cfu/cm2 before treatment) (log10 cfu/cm2 after treatment). c Values followed by the same letters in the column ‘Significance’ were not significantly different (P > 0.05), as indicated by the ANOVA test. R refers to the result of extreme analysis. Ki (i ¼ 1, 2, 3, 4) refers to the sum of four values at the same level for each factor in each column.
5
Control group Treated group
lg (cfu/cm2)
4
3
2
1
0
TVC
Enterococcus Coliform bacteria Pseudomonas
Fig. 1. Conditions of the specific microbial contamination in chicken drumsticks before and after treatment (spray washing conditions: a combination of 0.5% lactic acid and 1% citric acid for 30 s; temperature, 10 C).
untreated control. Previously, it has been reported that spray washing with 0.25% lactic acid at different stages of processing lowered the mean TVC by 0.76 lg cfu/cm2 after scalding, by 0.60 lg cfu/cm2 after defeathering and by 0.78 lg cfu/cm2 after evisceration (Sakhare, Sachindra, Yashoda, & Rao, 1999). Also, TVC reductions of 0.59e2.48 log10 cfu/cm2 were observed, with the greater reductions achieved at the higher concentrations (up to 5% citric acid) (Bolton et al., 2014). Accordingly, the findings here were satisfactory. The initial mean enterococcus populations of 1.13 log10 cfu/cm2 were reduced to undetectable level by enumeration after the treatment. In addition, a significant reduction of coliform bacteria (1.31 log10 cfu/cm2) and Pseudomonas (1.85 log10 cfu/cm2) was observed, respectively. Organic acids are considered to inhibit microbial activity by two
primary mechanisms: by cytoplasmic acidification with subsequent uncoupling of energy production and regulation, and by accumulation of the dissociated acid anion to toxic levels (Mani-Lopez, pez-Malo, 2012). Lactic acid is lipophilic acid, which García, & Lo can easily penetrate across the microbial plasma membrane in the undissociated form; an alkaline environment is encountered, favoring the dissociation of the acid into the acid anion and free proton, thus reducing the internal pH of the cell and disrupting important cellular processes (Smulders, Barendsen, Van Logtestijn, Mossel, & Van Der Marel, 1986). Citric acid is known to efficiently inhibit pathogens by destabilizing the outer membrane through metal chelation or intercalation, in some instances resulting in enhanced pathogen inhibition versus the monocarboxylic like lactic acid, but its usage is potentially limited by possible negative sensory impact and the need for an appropriate pH for optimum bactericidal properties (Mller, Call, & Whitin, 1993). Therefore, above results indicated that lactic acid and citric acid can be administered in combination to treat chicken drumsticks during the cutting process in slaughtering and processing chains.
3.3. Reduction in S. aureus and Salmonella populations of quickfrozen chicken drumsticks after the treatment Salmonella and S. aureus are two kinds of common foodborne pathogens in retail poultry meat. These pathogens pose potential threats to consumers. In this study, the population of S. aureus and Salmonella was 46.67% (56/120) and 14.15% (17/120) frozen drumstick products in the control group, respectively, while the population of treated products was 14.17% (17/120) and 3.33% (4/120), respectively. The results indicated that the combined organic acidbased spray washing induced a significant reduction (P < 0.05) in the prevalence of the two typical pathogenic bacteria found in the quick-frozen chicken drumsticks.
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3.4. PCR-DGGE analysis 3.4.1. DNA extraction and PCR amplification of V3 region of 16S rDNA The results of agarose gel electrophoresis analysis revealed that the length of the DNA extracted from each sample exceeded 2000 bp (Fig. 2a). The V3 region of 16S rDNA was amplified by using the extracted DNA as a template in PCR. In Fig. 2b, a distinct target band (approximately 250 bp) was detected. In addition, in order to ensure a successful DGGE analysis, adequate PCR products were obtained through a secondary PCR. 3.4.2. DGGE analysis The bands in the DGGE gel represented microbial species or operational taxonomic units in the microbial community. The change in the band number in the DGGE lanes indicated the complexity and variability of the bacterial community on the chicken drumsticks (Ercolini, 2004). The results (Fig. 3) revealed that the DGGE profile varied among the different stages of the cutting process. The species and quantities of the bacteria on the surface of the chicken drumsticks in each stage decreased significantly after the chicken drumsticks were subjected to combined organic acid treatments. Thirteen distinct bands in Fig. 3 were successfully reclaimed to further analyze the naturally occurring microflora in the three different stages of cutting process. With reference to NCBI BLAST, bands b, e, and g were identified as uncultured Pseudomonas sp. Bands d, f, l, and m were found as uncultured bacteria. Band c corresponded to the uncultured Flavobacteria. These microorganisms were viable but non-culturable (VBNC) bacterial cells. These cells could not form colonies on solid media but could maintain metabolic activities and elongation abilities after the administration of nutrients (Gauthier, 2000). Therefore, these VBNC cells are of particular concern because they may remain undetected via routine bacteriological procedures. Bands h and i identified respectively as Enterobacter cloacae and Gamma proteobacterium could be found on the surface of the chicken drumsticks in each stage of the cutting process. Band j corresponded to Pseudomonas moraviensis sp., appeared in the middle stage of the cutting process. Band k identified as Pseudomonas putida was just detected in the earlier stage. Fig. 4 presents the similarity indices of the DGGE profiles. The highest similarities (71%) were found in the samples obtained in the earlier and middle stages before the treatment. Before the treatment, the similarity indices in the different stages of cutting
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process ranged from 62% to 71%. The results indicated that the naturally occurring microflora in the different stages of cutting were similar. After the treatment was administered, the similarity index decreased significantly. The similarity indices between 1 and 1ʹ, 2 and 2ʹ, and 3 and 3ʹ, which corresponded to the same stages during cutting before and after the treatment, were 37%, 44%, and 37%, respectively. These findings suggested that the microbial counts were significantly reduced by the combined organic acid treatment, and the microbial communities were strongly affected simultaneously.
3.5. pH change in quick-frozen chicken drumsticks during storage pH has been used as an important criterion to evaluate meat quality (Chouliara et al., 2006). Fig. 5A illustrates the change in pH of the quick-frozen chicken drumsticks during storage. The pH of the quick-frozen chicken drumsticks ranged from approximately 6.0 to 7.0. Similar pH values were observed in both untreated and treated samples throughout storage. In addition, pH showed no statistically significant changes (P > 0.05) for all of the eight treated chicken drumstick samples during the 2-month storage. As a result, organic acid-based spray washing did not elicit considerable effects on the pH of chicken drumsticks.
3.6. TVB-N determination of quick-frozen chicken drumsticks during storage As an important reference index, the TVB-N content of meat produced by the microbial degradation of nitrogenous tissues is used to reveal microbiological spoilage and to evaluate the quality of meat and other meat products (Chan et al., 2006). For instance, it has been reported that the levels of TVB-N as an indicator of skinless chicken breast spoilage were approximately 18 and 17 mg N per 100 g sample when stored at 4 and 10 C (Rukchon, Nopwinyuwong, Trevanich, Jinkarn, & Suppakul, 2014). In our study, as can be seen in Fig. 5B, the TVB-N content of the quickfrozen chicken drumsticks before and after the treatment presented a similar trend in the 2-month storage. Although the difference between the control group and treated group was not significant (P > 0.05), the TVB-N content of quick-frozen chicken drumsticks increased more slowly in the treated group, because microbial population was significantly reduced after the treatment.
Fig. 2. Agarose gel of DNA from each sample (A) and agarose gel of the PCR products of the V3 regions of DNA for DGGE (B). M represents Marker. Lanes 1, 2, and 3 represent chicken drumstick samples from earlier, middle, and later stages of cutting process in the control groups, respectively. Lanes 1ʹ, 2ʹ, and 3ʹ represent the chicken drumstick samples from earlier, middle, and later stages of cutting process after the treatment was administered.
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Fig. 3. DGGE profiles of the PCR products of the DNA samples from the chicken drumsticks in different stages of the cutting workshop before and after decontamination treatment (A); Schematic of the DGGE profile (B). Each lane corresponds to samples in Fig. 2. Bands a, b, c, d, e, f, h, i, j, k, l, and m are the detectable bands on the DGGE gel of different lanes.
Fig. 4. Similarity index of the DGGE profiles. 1, 2, 3, 1ʹ, 2ʹ, and 3ʹ correspond to Fig. 2.
3.7. Sensory properties of quick-frozen chicken drumsticks during storage Panelists provided similar preference scores for the control group and treated group during freezing storage, which indicated
that the treated samples were highly acceptable. No significant difference (P > 0.01) was found between the control and treated samples for all characteristics evaluated. Therefore, it could be concluded that the treatment based on organic acid spray-washing did not affect the organoleptic quality of chicken drumsticks.
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Fig. 5. pH change (A) and TVB-N content (B) of the quick-frozen chicken drumsticks during storage before and after treatment.
4. Conclusion This work demonstrated the effectiveness of the antimicrobial effect of acid treatments in different stages of the cutting process. The most effective treatment conditions were 0.5% lactic acid, 1.0% citric acid, and spray-washing for 30 s. The changes in naturally occurring microflora were analyzed through microbiological counting and PCR-DGGE analysis. Physicochemical indices, including pH and TVB-N, and the sensory attributes of the quickfrozen chicken drumsticks during storage before and after the treatment were also investigated. On the basis of these comprehensive profiling, we can conclude that decontamination during the cutting process by spray washing with water containing a combination of lactic acid and citric acid possibly improved the microbiological quality of chicken drumsticks without significant sensory changes. The improvement in the microbiological quality of the treatment further enhanced the shelf life of poultry meat. Acknowledgments The authors are grateful for the financial support of the National Natural Science Foundation of China (Grant No. 31400066) and the Science & Technology Foundation of Sichuan Province (Grant No. 14NZ0012) to this research. References Acuff, G. (2005). Chemical decontamination strategies for meat. Improving the safety of fresh meat. Boca Raton: CRC Press. Arakere, G., Nadig, S., Swedberg, G., Macaden, R., Amarnath, S. K., & Raghunath, D. (2005). Genotyping of methicillin-resistant Staphylococcus aureus strains from two hospitals in Bangalore, South India. Journal of Clinical Microbiology, 43(7), 3198e3202. Bohaychuk, V., Gensler, G., King, R., Manninen, K., Sorensen, O., Wu, J., et al. (2006). Occurrence of pathogens in raw and ready-to-eat meat and poultry products collected from the retail marketplace in Edmonton, Alberta, Canada. Journal of Food Protection®, 69(9), 2176e2182. Bolder, N. (1997). Decontamination of meat and poultry carcasses. Trends in Food Science & Technology, 8(7), 221e227. Bolton, D., Meredith, H., Walsh, D., & McDowell, D. (2014). The effect of chemical treatments in laboratory and broiler plant studies on the microbial status and shelf-life of poultry. Food Control, 36(1), 230e237. Chan, S. T., Yao, M. W., Wong, Y., Wong, T., Mok, C., & Sin, D. W. (2006). Evaluation of chemical indicators for monitoring freshness of food and determination of volatile amines in fish by headspace solid-phase microextraction and gas chromatography-mass spectrometry. European Food Research and Technology, 224(1), 67e74. Chouliara, I., Samelis, J., Kakouri, A., Badeka, A., Savvaidis, I., Riganakos, K., et al. (2006). Effect of irradiation of frozen meat/fat trimmings on microbiological and physicochemical quality attributes of dry fermented sausages. Meat Science,
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