Changes in the microbial communities of air-packaged and vacuum-packaged common carp (Cyprinus carpio) stored at 4 °C

Food Microbiology 52 (2015) 197e204

Contents lists available at ScienceDirect

Food Microbiology journal homepage: www.elsevier.com/locate/fm

Changes in the microbial communities of air-packaged and vacuumpackaged common carp (Cyprinus carpio) stored at 4
C Yuemei Zhang, Qian Li, Dongping Li, Xiaochang Liu, Yongkang Luo* College of Food Science and Nutritional Engineering, China Agricultural University, Beijing Higher Institution Engineering Research Center of Animal Product, Beijing 100083, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 1 April 2015 Received in revised form 3 August 2015 Accepted 5 August 2015 Available online 7 August 2015

The dominant microbiota of air-packaged (AP) and vacuum-packaged (VP) common carp fillets during storage were systematically identified. Culture-dependent methods were used for microbial enumeration and 16S rRNA genes of the isolated pure strains were sequenced and analyzed. Different packaging conditions affected the growth of microbiota and the shelf life of carp. Shelf-life of AP and VP fillets was 8 and 12 days, respectively. Vacuum packaging delayed the increase of biogenic amines levels compared to air packaging, especially for cadaverine and tyramine levels. In the present study, a total of 13 different genera comprised the microbial communities of fresh carp fillets and Acinetobacter dominated the indigenous flora of carp. However, variability in bacterial community composition was observed in these two packaging conditions. Pseudomonas were the only microbiota found in the spoiled AP carp, whereas Carnobacterium followed by Aeromonas were found mainly in VP samples. Other genera Shewanella, Lactococcus, and Pseudomonas were also found in low numbers at the end of the VP fillets’ shelf life. Additional microbial enumeration observed the highest Pseudomonas counts (8.77 log CFU/g on day 8) in AP samples and a relatively high level of lactic acid bacteria (7.74 log CFU/g on day 12) in VP samples. © 2015 Elsevier Ltd. All rights reserved.

Chemical compounds studied in this article: Phenethylamine (PubChem CID: 1001) Putrescine (PubChem CID: 1045) Cadaverine (PubChem CID: 273) Histamine (PubChem CID: 774) Tyramine (PubChem CID: 5610) Spermidine (PubChem CID: 1102) Spermine (PubChem CID: 1103) Keywords: Common carp Vacuum packaging Microbial communities Spoilage Biogenic amines

1. Introduction Common carp (Cyprinus carpio) is currently recognized as a freshwater fish species with high economic value and is often grown in aquaculture facilities in many countries (Winker et al., 2010). Total aquaculture production of common carp in China increased to approximately 3,022,494 tons in 2013 (Bureau of Fisheries of the Ministry of Agriculture, 2014). Due to its abundance, reasonable domestic price, and delicious taste, carp has become a popular species for many producers and consumers. It is usually distributed as the fresh whole fish in markets. However, there is a growing tendency among consumers who prefer fresh or thawed fillets because of their convenience. Given that the shelflife of air-packaged (AP) carp fillets is relatively short, research on

* Corresponding author. E-mail addresses: [email protected], [email protected] (Y. Luo). http://dx.doi.org/10.1016/j.fm.2015.08.003 0740-0020/© 2015 Elsevier Ltd. All rights reserved.

new packaging methods for fillets is required. Vacuum packaging is an effective packaging technology that offers a way of prolonging the shelf life of perishable fillets by excluding oxygen and inhibiting the growth of microorganisms (Noseda et al., 2012). Additionally, vacuum-packaged (VP) fillets have a small package volume, making international transport easier. Therefore, storage under vacuum packaging has been given increasing attention recently. The undesirable growth and metabolism of microorganisms are the primary causes for spoilage of fish (Gram and Huss, 1996). However, not all microorganisms are responsible for fish spoilage. In general, fish is always contaminated with a small quantity of microorganisms that are designated as specific spoilage organisms (SSOs) when they are considered inedible (Dalgaard, 1995). These SSOs are present in low numbers in fresh fish and can eventually become dominant in spoilage microorganisms (Pennacchia et al., 2011). Therefore, it is meaningful to identify the SSOs at the time of spoilage and to analyze the relationship between SSOs and the shelf-life of products that supply fresh products to consumers.

198

Y. Zhang et al. / Food Microbiology 52 (2015) 197e204

Under chilled, aerobic storage conditions, several Gram-negative genera, particularly Pseudomonas, Aeromonas, Shewanella, Enterobacteriaceae, dominate the spoilage microorganisms of freshwater and marine fish (Noseda et al., 2012; Wang et al., 2014). However, vacuum packaging can inhibit the growth of aerobic bacteria commonly present on fish, resulting in an increase in Gram-positive bacteria that can respire better than Gram-negative bacteria in this type of packaging (Lyhs et al., 2002; Mace et al., 2012; Noseda et al., 2012). With regard to common carp, Mahmoud et al. (2004) analyzed the microbiota that cause spoilage in skin, gills and intestines. Their analysis depended mostly on traditional microbiological methods, such as plate viable counts, isolation, and biochemical identification. However, the objective of this study was to characterize and monitor the changes in the microbial communities of AP and VP common carp fillets stored at 4
C using a combination of culturebased and 16S rRNA gene analysis methods. We also hoped to gain more knowledge on the dominant microorganisms at the species level so that we could develop suitable control methods. 2. Materials and methods 2.1. Sampling and packaging Twenty common carp (weight 1150 ± 180 g, length 43 ± 1 cm) were obtained from an aquatic products market in Beijing, China and were transported to the laboratory alive in September 2014. Subsequently, carp were stunned, deheaded, scaled, gutted, filleted, and washed immediately with cold sterile water. The fillets were then drained at 4
C for 3 min and prepared for packaging. The fillets (weight 220 ± 35 g, about 12  10  2 cm3) were randomly divided into two portions. Then they were packaged in air (n ¼ 18) in well-sealed polyvinyl chloride bags (about 250ⅹ200 mm) and under vacuum (n ¼ 21) in pouches of polyethylene/polyamide film (about 250  200 mm, having an oxygen permeability of 40e50 cm3/m2 per 24 h/atm at 85% relative humidity, 23
C), respectively. All pouches were stored at 4 ± 1
C. Samples of white dorsal muscle from three fillets were taken randomly for analyzing sensory scores, pH value, total volatile basic nitrogen (TVB-N), microbiological enumeration, and biogenic amines every 2 days. However, microbial communities were identified on days 0, 4, and 8 for AP samples and on days 6 and 12 for VP samples.

Shimadzu, Kyoto, Japan) equipped with a COSMOSIL 5C18-PAQ (4.6  250 mm) column and an SPD-10A (V) detector. Ammonium acetate (0.1 M; solvent A) and acetonitrile (solvent B) were used as mobile phases. The gradient elution program was as follows: 0 min, 50% B; 25 min, 90% B; 35 min, 90% B; 45 min, 50% B. The samples were detected at 254 nm with a flow rate of 0.8 mL/min and an injection volume of 50 mL. The column temperature was 30
C. 2.5. Enumeration of microbial communities Plate dilution gradient methods were carried out on samples to enumerate different microbial communities using the method of Wang et al. (2014). For all microbial enumeration, samples of serial dilutions (100 mL) were spread on the surface of dry media. Total viable counts (TVC) were determined in plate count agar (PCA) and they were incubated at 30 ± 1
C for 72 h. Lactic acid bacteria (LAB) were enumerated in overlaid pour-plates of MRS agar and incubated at 30
C for 48 h. Pseudomonas sp. were determined on Pseudomonas CFC Selective Agar (CFC) at 20
C for 48 h. H2S-producing bacteria were evaluated on iron agar medium (IA); black colonies produced on IA were enumerated after incubating at 20
C for 4 days. All colony forming units were recorded as log CFU/g. All of the culture media were supplied by Hai Bo Biological Technology Co., Ltd. (Qingdao, China). 2.6. Isolation and identification of microorganisms 2.6.1. Isolation and purification Bacterial DNA were isolated and purified in accordance with the method of Wang et al. (2014). After TVC enumeration, bacteria were isolated on PCA for identification of the dominant microbiota from fresh carp fillets (53 isolates), AP fillets stored on days 4 and 8 (46 and 55 isolates, respectively), and from VP fillets stored on days 6 and 12 (40 and 48 isolates, respectively), with the goal of purifying these strains. All of the colonies were selected from the highest dilution PCA spread plates which usually contained 30e100 isolates, using an inoculation loop, and sub-cultured at 30 ± 1
C for 24e48 h in 5 mL of tryptic soy broth (TSB) (Aoboxing Universeen Bio-Tech Co., Ltd., Beijing, China). After being cultured, by repeated plate streaking, Gram staining and microscopy, the isolates from each sample were purified for identification by 16S rRNA gene sequence analysis. A single purified colony was sub-cultured in 5 mL of TSB at 30 ± 1
C for 24e48 h.

2.2. Sensory analyses The sensory characteristic of each fillet (fresh and cooked) was evaluated using the method of Hong et al. (2012). Cooked carp fillets were prepared by steaming for 10 min at 100
C. Seven members were trained to evaluate the color, odor, elasticity, and the morphology of raw fish muscle, as well as the flavor, odor, and broth turbidity of cooked carp fillets, scoring each on a scale from 1 to 5 points. A total score of 35.0 points was considered fresh, while 15.0 was regarded as the lowest acceptable limit. 2.3. Determination of TVB-N and pH TVB-N value was measured by the semi-micro steam distillation method (Hong et al., 2012). The pH value was detected using a digital pH meter (Mettler Toledo FE20/EL20, Shanghai, China). 2.4. Determination of biogenic amines Extraction and derivatization of biogenic amines (BAs) were carried out as described by Shi et al. (2012). Subsequently, BAs were determined and quantified by using HPLC (Shimadzu LC-10A;

2.6.2. Extraction and identification of DNA by 16S rRNA gene analysis The 2 mL sample of TSB culture was transferred, each containing a single purified colony, to a centrifuge tube, where cells were collected by centrifugation. DNA was extracted from the centrifugal sediment using the bacterial DNA extraction kit (Bomad Biological Technology Co., Ltd., Beijing, China) according to the manufacturer's instructions. 1.0% agarose gel electrophoresis was used to determine quality of these DNA extracts. These extracts, which showed the clear, bright ladder in the agarose gel, were picked as the DNA templates for PCR (TC-512, Techne, UK). Bacterial 16S rRNA was amplified by PCR using the universal bacterial forward primer 27f (50 -GAGATTTGATCCTGGCTCAG-30 ) and the reverse primer 1495r (50 -CTACGGCTACCTTGTTACGA-30 ) (Wang et al., 2014). All the oligonucleotide PCR primers used in this study were received from Bomad Biological Technology Co., Ltd. (Beijing, China; BBT). The PCR system was comprised of 12.5 mL 2  Taq PCR Master Mix (containing thermostable DNA polymerase, MgCl2, dNTPs, buffer), 1 mL of template DNA, 10.5 mL double distilled water, and 0.5 mL of each primer at a concentration of 10 mM with a total reaction volume of 25 mL. Amplification was performed under

Y. Zhang et al. / Food Microbiology 52 (2015) 197e204

the following conditions: 94
C for 5 min and then 35 cycles (denaturation at 94
C, 30 s; primer annealing at 54
C, 30 s; primer extension at 72
C, 1 min) followed by a final elongation step at 72
C for 10 min. 1.0% agarose gel electrophoresis was used to examine the quality and quantity of these PCR amplification products (5 mL). Sequencing was performed in the BBT Co., Ltd. (Beijing, China). The 16S partial sequences were mostly approximately 1400 bp. The identification of the colonies was completed by using the EZTaxon database, which contains 16S rRNA gene sequences of type strains with validly published prokaryotic names (Chun et al., 2007) (http://www.eztaxon.org/). Minimum values of 97% similarity were used to determine whether the sequences belonged to the same species (Noseda et al., 2012). 2.7. Statistical analysis Each analysis was performed in triplicate (except microbiological analyses, which were performed in duplicate). The least significant difference (LSD) procedure was used to test for differences between means (significance was defined at P < 0.05) using SPSS 17.0 (SPSS Inc., Chicago, IL) software. 3. Results and discussion 3.1. Sensory analysis Fig. 1a shows the changes of sensory scores in air-packaged (AP) and vacuum-packaged (VP) carp fillets. Sensory scores of each group significantly decreased (P < 0.05) as storage time increased for both AP and VP fillets. After storing for 6 days, the sensory scores

(a)

40

for AP and VP samples declined from initial values of 34.0 to 19.5 and 23.0, respectively. Adopting a total sensory score of 15.0 or less as the sensory rejection point, as determined by our panelists, AP and VP fillets were inedible after being stored for 8 and 12 days, respectively. 3.2. Changes in biochemical quality (TVB-N, pH and biogenic amines) Variations in TVB-N values of AP and VP common carp fillets are presented in Fig. 1b. The initial TVB-N value of carp was 12.43 mg/ 100 g. Significant differences (P < 0.05) between TVB-N values for AP and VP conditions were observed on the 8th day. The TVB-N values in AP fillets suddenly increased to 26.41 mg/100 g on that day, whereas those in VP samples remained at a low level until they increased to 22.91 mg/100 g on the 12th day. Genç et al. (2013) reported similar results. Connell (1995) proposed that a level of 25 mg/100 g could be regarded as the limit of acceptability for fish quality. Therefore, we could consider that AP fillets had a shelf life of 8 days, whereas VP samples had a shelf life of about 12 days. As shown in Fig. 1c, a fluctuation of pH was observed for each packaging condition in which pH varied between 6.51 and 6.83 in AP fillets, between 6.55 and 6.83 in VP samples. The pH value in AP samples decreased to a minimum (6.51) on day 6, whereas the pH value in VP fillets reached the minimum (6.55) on day 10. The later increase in pH was possibly related to the formation of volatile basic components, amines, and bacterial metabolites (Atrea et al., 2009). The pH values in AP and VP fillets increased to the values (6.73 and 6.63) on days 8 and 12, respectively, which is probably because vacuum packaging inhibited the growth of aerobic bacteria and prolonged the shelf life of carp fillets.

AP

35

Sensory scores

199

VP

30 25 20 15

10 5 0 0

(b)

2

4

6

8

Storage time/days

10

12

(c)

35

7.20

AP

7.00

VP

AP VP

25

6.80

20

pH

TVB-N (mg/100g)

30

6.60

15 6.40

10

6.20

5

6.00

0 0

2

4

6

8

Storage time/days

10

12

0

2

4

6

8

10

12

Storage time/days

Fig. 1. Changes in (a) sensory scores, (b) TVB-N contents and (c) pH of air-packaged (AP) and vacuum-packaged (VP) common carp fillets during chilled storage.

200

Y. Zhang et al. / Food Microbiology 52 (2015) 197e204

Table 1 Changes in biogenic amine contents of air-packaged (AP) and vacuum-packaged (VP) common carp fillets during chilled storage. Storage time/days

Biogenic amine (mg/kg) PHE

AP

VP

0 2 4 6 8 0 2 4 6 8 10 12

2.84 5.66 10.16 5.84 2.12 2.84 3.45 9.24 3.29 5.14 5.20 2.74

PUT ± ± ± ± ± ± ± ± ± ± ± ±

ab

0.13 0.65 0.97 0.29 1.24 0.13 0.42 0.39 1.05 0.90 0.95 1.98

0.00 1.70c 3.30d 0.81c 1.26a 0.00ab 1.10ab 1.45c 0.47ab 0.00ab 3.83ab 0.45ab

CAD ± ± ± ± ± ± ± ± ± ± ± ±

a

0.00 0.24bc 0.58cd 0.13ab 0.08d 0.00a 0.03ab 0.10ab 0.21b 0.23ab 0.00ab 0.66c

HIM

ND 0.57 ± 0.12a 1.19 ± 0.00b 0.94 ± 0.00b 9.11 ± 0.18c ND 0.92 ± 0.37a 0.54 ± 0.00a ND 0.95 ± 0.00a 0.37 ± 0.07a 15.08 ± 0.40b

0.70 0.51 0.57 0.94 0.56 0.70 0.50 0.66 0.95 0.77 0.71 0.49

TYM ± ± ± ± ± ± ± ± ± ± ± ±

a

0.27 0.05a 0.10a 0.02b 0.00a 0.27ab 0.04a 0.22ab 0.01b 0.01ab 0.34ab 0.04a

35.30 28.31 37.18 44.84 63.03 35.30 14.70 19.05 6.62 24.15 27.49 25.10

SPD ± ± ± ± ± ± ± ± ± ± ± ±

a

0.00 1.11a 2.23a 0.00a 24.91b 0.00a 3.98b 4.21bc 0.09d 3.19ce 3.27e 8.30ce

2.13 3.41 3.23 3.08 2.84 2.13 4.74 1.96 2.00 1.90 2.33 2.22

SPM ± ± ± ± ± ± ± ± ± ± ± ±

a

0.03 0.79a 1.00a 0.72a 1.28a 0.03a 0.56b 0.57a 0.01a 0.10a 0.56a 0.93a

11.61 12.67 14.36 8.97 12.46 11.61 14.52 12.89 8.54 8.76 11.49 10.49

± ± ± ± ± ± ± ± ± ± ± ±

5.46a 3.87a 1.16a 3.55a 2.03a 5.46ab 1.90b 2.76ab 1.10a 0.83ab 2.79ab 3.75ab

Same lowercase letters in a column indicate no significant differences (P > 0.05) (ND: not determined; PHE: phenylethylamine; PUT: putrescine; CAD: cadaverine; HIM: histamine; TYM: tyramine; SPD: spermidine; SPM: spermine).

TVC of carp fillets was 3.53 log CFU/g. Taking the 7 log CFU/g as an upper acceptable level for freshwater fish (ICMSF, 1986), TVC in AP samples exceeded this count on day 6, whereas those in VP samples exceeded 7 log CFU/g on day 8. Similarly, Noseda et al. (2012) reported that VP extended a microbiological shelf life of Pangasius hypophthalmus fillets by 3 days. Pseudomonas in AP samples exhibited curves similar to the TVC, reaching 8.77 log CFU/g on day 8, whereas VP samples showed significantly lower (P < 0.05) Pseudomonas counts than the AP samples throughout the storage period. Atrea et al. (2009) also found that vacuum packaging could reduce the counts of Pseudomonas. Unlike Pseudomonas, the H2Sproducing bacteria and LAB counts increased slightly, not exceeding the 7 log CFU/g after 8 days of storage under air packaging conditions. However, under vacuum packaging conditions, H2S-producing bacteria counts proliferated to 7.49 log CFU/g on the final day 12th of storage. In addition, the initial count of LAB was 2.08 log CFU/g, which showed a stepwise trend, and their counts reached 7.74 log CFU/g by day 12. LAB might comprise an important part of the main bacterial loads towards the end of the shelf life of VP carp fillets. Similar results have also been reported in VP rainbow trout (Oncorhynchus mykiss) (Lyhs et al., 2001) and Pangasius hypophthalmus fillets (Noseda et al., 2012).

Biogenic amines, such as phenylethylamine (PHE), putrescine (PUT), cadaverine (CAD), histamine (HIM), tyramine (TYM), spermidine (SPD), and spermine (SPM), are low molecular weight nitrogenous compounds. In the present study, seven biogenic amines were determined in fresh carp (Table 1). The initial values of PUT and HIM were approximately 0.13 mg/kg and 0.70 mg/kg, respectively. SPD and SPM, which are naturally found in foods (Hong et al., 2013), showed no significant difference (P > 0.05) between these two treatments. CAD levels in AP and VP samples were fairly constant until days 6 and 10 of storage. Thereafter, CAD levels tended to increase significantly (P < 0.05) to 9.11 mg/kg for AP samples on day 8 and 15.08 mg/kg for VP samples on day 12. The increase of CAD levels coincided with a sharp decrease of sensory scores in VP fillets. Earlier studies showed that the contents of CAD were directly correlated with the decomposition of fish flesh (Shi et al., 2012; Wang et al., 2014). Therefore, the increase of CAD levels may be a result of accumulated microbial communities, especially for Carnobacterium (Table 4), which might reveal a strong ability to produce CAD. K
rı
zek et al. (2004) also reported that vacuum packaging could delay the growth of PUT and CAD in carp fillets stored at 3
C. In the present study, PUT levels increased slightly throughout the storage process, reaching 1.24 mg/kg for AP samples on day 8 and 1.98 mg/kg for VP samples on day 12. PHE and HIM levels did not differ significantly between AP and VP samples. However, VP treatments obviously reduced the accumulation of TYM compared to the control throughout the storage period.

3.4. Diversity in microbial communities The results of 16S rRNA sequencing for each packaging condition are shown in Tables 3 and 4. The indigenous microbiota of fish from temperate waters typically consist of several Gram-negative genera including Acinetobacter, Flavobacterium, Pseudomonas, Moraxella (ICMSF, 2005). A high bacterial diversity was generally observed before storage, when the initial TVC of carp fillets was very low

3.3. Enumeration of microbial communities The changes in microbial communities (log CFU/g) during chilled storage in AP and VP carp fillets are given in Table 2. The initial

Table 2 Changes in microbial communities (log CFU/g) during chilled storage of carp fillets packed in air (AP) and under vacuum (VP). Storage time/days

Microbial communities (log CFU/g)

0 AP

VP

2 4 6 8 2 4 6 8 10 12

́

Total viable counts

Pseudomonas

H2S-producing bacteria

Lactic acid bacteria

3.53 ± 0.17a

2.96 ± 0.24a

2.53 ± 0.21a

2.08 ± 0.15a

3.88 6.43 8.67 8.79 3.89 6.01 6.38 7.32 7.65 8.47

± ± ± ± ± ± ± ± ± ±

0.18a 0.19b 0.09c 0.13c 0.08a 0.04b 0.54b 0.15c 0.19c 0.16d

Same lowercase letters in a column indicate no significant differences (P > 0.05).

3.75 6.23 8.49 8.77 3.70 5.79 5.64 5.69 6.90 7.69

± ± ± ± ± ± ± ± ± ±

0.15b 0.06c 0.01d 0.07d 0.00b 0.57c 0.12c 0.12c 0.00d 0.80e

3.34 5.06 6.06 6.50 3.58 5.14 6.40 6.95 7.04 7.49

± ± ± ± ± ± ± ± ± ±

0.19b 0.20c 0.05d 0.71d 0.00b 0.01c 0.22c 0.00d 0.27d 0.40d

2.57 4.44 5.79 6.46 3.43 4.94 5.57 6.73 7.11 7.74

± ± ± ± ± ± ± ± ± ±

0.29a 0.12b 0.51c 0.15d 0.12b 0.07c 0.21c 0.11d 0.17d 0.32e

Table 3 Diversity in microbial communities of air-packaged (AP) common carp fillets stored at 4
C based on 16S rRNA gene sequencing of pure isolates. Storage time (days) 0

4

Species identification Closest relative (Accession no.) Acinetobacter Acinetobacter johnsonii Acinetobacter lwoffii Xanthomonas

Identity No. of isolates (%) (N ¼ 53)

52.8 APON01000005 27

98.28e99.71

AIEL01000120

99.85

1

1.9 Y10760

1

97.89

Chryseobacterium haifense Chryseobacterium taihuense Microbacterium

EF204450

1

99.09

JQ283114

1

97.43

3.8

3.8 AB234028

2

98.92e98.94

Y16264

2

99.17e99.19

3.8

Pseudomonas Pseudomonas alcaligenes Arthrobacter Arthrobacter oxydans Sphingomonas Sphingomonas panni Brevundimonas Brevundimonas aurantiaca Brevundimonas diminuta Moraxella Moraxella osloensis Moraxella osloensis Enhydrobacter Enhydrobacter aerosaccus Vogesella Vogesella perlucida Dermacoccus Dermacoccus profundi

3.8 BATI01000076

2

99.54e100

X83408

1

98.9

AJ575818

2

99.4e99.74

AJ227787

1

98.69

GL883089

1

99.76

X74897 EU499677

5 2

99.58e99.84 99.45e99.61

AJ550856

2

99.93e100

EF626691

1

99.27

AY894329

1

99.92

1.9

3.8

Shewanella Shewanella xiamenensis Macrococcus

Closest relative (Accession no.)

Identity No. of isolates (%) (N ¼ 46)

Distribution Species (%) identification 4.3

FJ589031

2

99.51 4.3

Macrococcus Y15711 caseolyticus Acinetobacter

2

99.24e99.37

Acinetobacter APON01000005 johnsonii Pseudomonas

1

Pseudomonas deceptionensis Pseudomonas fragi Pseudomonas psychrophila Aeromonas Aeromonas veronii Aeromonas media Aeromonas jandaei

GU936597

6

98.88e99.58

AF094733

8

99.09e100

AB041885

17

Identity Closest relative No. of (Accession no.) isolates (%) (N ¼ 55)

Pseudomonas Pseudomonas GU936597 deceptionensis Pseudomonas AF094733 fragi Pseudomonas AB041885 psychrophila

Distribution (%)

17

99.57e99.84 100

10

99.55e99.93

28

99.78e100

2.2 99.01 67.4

100 21.7

X60414

7

99.58e99.85

X74679

2

99.58e99.72

X60413

1

99.65

Y. Zhang et al. / Food Microbiology 52 (2015) 197e204

Xanthomonas cucurbitae Chryseobacterium

Microbacterium aoyamense Kocuria Kocuria rhizophila

Distribution Species (%) identification

8

3.8

13.2

3.8

1.9 1.9

201

202

Table 4 Diversity in microbial communities of vacuum-packaged (VP) common carp fillets stored at 4
C based on 16S rRNA gene sequencing of pure isolates. Storage time (days) 0 Species identification Acinetobacter Acinetobacter johnsonii Acinetobacter lwoffii Xanthomonas

6 Closest relative No. Isolates Identity (Accession no.) (N ¼ 53) (%)

52.8 APON01000005 27

98.28e99.71

AIEL01000120

99.85

1

1.9 1

Chryseobacterium haifense Chryseobacterium taihuense Microbacterium

EF204450

1

99.09

JQ283114

1

97.43

Microbacterium aoyamense Kocuria

AB234028

Kocuria rhizophila

Y16264

2

99.17e99.19

BATI01000076

2

99.54e100

X83408

1

98.9

AJ575818

2

99.4e99.74

AB041885

3.8

Pseudomonas psychrophila Brochothrix

AODI01000055

3.8

Brochothrix thermosphacta Enterobacter Enterobacter xiangfangensis Lactococcus Lactococcus raffinolactis Lysinibacillus Lysinibacillus macroides Aeromonas Aeromonas veronii Aeromonas hydrophila subsp. ranae Aeromonas hydrophila subsp. hydrophila Aeromonas allosaccharophila Carnobacterium Carnobacterium divergens Carnobacterium maltaromaticum

HF679035

1

99.55

EF694030

2

99.78

AJ628749

1

99.71

1.9

3.8

3.8

1

98.69

Brevundimonas diminuta Moraxella Moraxella osloensis Moraxella osloensis Enhydrobacter Enhydrobacter aerosaccus

GL883089

1

99.76

X74897

5

99.58e99.84

EU499677

2

99.45e99.61

AJ550856

2

99.93e100

15

JN175353

3.8

AJ227787

99.57

Prolinoborus fasciculus Pseudomonas

98.92e98.94

Brevundimonas aurantiaca

1

Y15711

97.89

13.2

3.8

Distribution Species (%) identification 2.5

FJ589031

Macrococcus caseolyticus Prolinoborus

3.8

2

Shewanella Shewanella xiamenensis Macrococcus

12 Identity Closest relative No. of (Accession no.) isolates (%) (N ¼ 40)

6

1

1

100 2.5

EF694030

2.5

Lactococcus raffinolactis Aeromonas

100

100 2.5

5

2.5

50 X60414

16

99.58e100

AJ508766

2

99.64e99.65

CP000462

1

100

S39232

1

99.48

M58816

4

99.7e99.78

AF184247

2

99.93

15

1

99.71 2.1

FJ589031

100

Distribution (%) 2.1

GU936597

Shewanella xiamenensis Lactococcus

2.5 1

Pseudomonas Pseudomonas deceptionensis Shewanella

Identity Closest relative No. of (Accession no.) isolates (%) (N ¼ 48)

1

99.57 2.1

Aeromonas X60414 veronii Aeromonas S39232 allosaccharophila Carnobacterium Carnobacterium AF184247 maltaromaticum

1

99.86 18.7

5

99.79e99.93

4

99.57e99.64 75

36

99.52e100

Y. Zhang et al. / Food Microbiology 52 (2015) 197e204

Xanthomonas Y10760 cucurbitae Chryseobacterium

Pseudomonas Pseudomonas alcaligenes Arthrobacter Arthrobacter oxydans Sphingomonas Sphingomonas panni Brevundimonas

Distribution Species (%) identification

1.9 1 AY894329

99.92

1

Vogesella Vogesella perlucida Dermacoccus Dermacoccus profundi

EF626691

99.27

1.9

Y. Zhang et al. / Food Microbiology 52 (2015) 197e204

203

(3.53 log CFU/g). In the present study, a total of 13 different genera comprised the microbial communities of fresh carp fillets in which Acinetobacter were considered as a major microbiota, representing 52.8% of the total isolates (Table 3). Some researchers also reported Acinetobacter dominated the indigenous microbiota of farmed sea bream (Sparus aurata) and grass carp (Ctenopharyngodon idellus) (Parlapani et al., 2013; Wang et al., 2014). Using 16S rRNA gene sequencing, 27 of the 28 Acinetobacter isolates were confirmed at the species level as Acinetobacter johnsonii. Additionally, 13.2% of the total isolates belonged to the genus Moraxella. The remaining isolates present in the indigenous microbiota of carp fillets were identified as Xanthomonas, Chryseobacterium, Microbacterium, Kocuria, Pseudomonas, Arthrobacter, Sphingomonas, Brevundimonas, Enhydrobacter, Vogesella, and Dermacoccus. However, each of these genera only contained 1e2 isolates. We observed a large shift in the microbial communities during spoilage, in which the proportion of Acinetobacter became less common and several bacterial groups appeared and gradually proliferated (Tables 3 and 4). There were obvious differences in the microbial composition of AP and VP fillets. A total of 86 isolates were identified from each packaged fillet that was close to spoilage, including 46 sequences originated from AP fillets stored on day 4 (Table 3) and 40 isolates from VP samples stored on day 6 (Table 4). In AP fillets, Pseudomonas increased during the first 4 days after storage, increased very rapidly, and eventually represented 67.4% of the 46 sequences. This was consistent with plate count results (Table 2). Aeromonas were the second most common microbiota, accounting for 21.7% of the total isolates. However, the microbial composition on day 6 changed completely in VP fillets, compared to AP fillets. Aeromonas isolates in VP samples constituted a higher proportion (50%) than those in AP samples; they were the largest group in VP samples. Furthermore, Gram-positive bacteria in the genera Macrococcus and Carnobacterium emerged and accounted for 15% of the total isolates in VP samples. Noseda et al. (2012) reported that Carnobacterium maltaromaticum was also recovered in VP Pangasius hypophthalmus fillets. In addition, unlike the abundance of Pseudomonas species in AP fillets, there was only 1 strain identified as Pseudomonas in all of the genera isolated from the 6th day of storage in VP fillets. Based on the results of the sensory analysis, AP and VP fillets were considered spoiled and rejected on days 8 and 12, respectively, at the same time that TVC proliferated to 8.79 and 8.47 log CFU/g, respectively. At the end of the shelf life, we collected 55 isolates from AP samples and 48 isolates from VP samples on PCA spread plates of carp fillets (Tables 3 and 4). In AP fillets, Pseudomonas were in low numbers on day 0 (3.8%), but it increased rapidly under aerobic conditions, reached 67.4% on day 4 and became the only microbiota at the end of storage (Table 3). This was also reflected in the plate count results in which Pseudomonas were 100 times more common (2 log difference) than H2S-producing bacteria and LAB (Table 2). Pseudomonas were also found to be the predominant microbiota in other aquatic products under aerobic, chilled conditions (Parlapani et al., 2013, 2015; Tryfinopoulou et al., 2002; Hozbor et al., 2006). However, more bacterial diversity was observed at the end of the shelf life in VP fillets (Table 4). After 12 days of storage, a turnover was observed in the spoiled VP fillets in favor of LAB, although Aeromonas constituted the first largest group (50%) and LAB represented only 20% on day 6. Of the 48 isolates collected from spoiled VP fillets, LAB in particular Carnobacterium became the major predominant microbiota and Carnobacterium constituted 75% of the total isolates. All of them were identified at the species level as C. maltaromaticum (Table 4). Plate counts however indicated that the bacterial numbers on MRS were approximately 0.7 log CFU/g lower than those on PCA, and they were similar to the counts on CFC and IA (Table 2). This is probably because Carnobacterium cannot grow well

204

Y. Zhang et al. / Food Microbiology 52 (2015) 197e204

on MRS (pH 6), in contrast to PCA (pH 7). Similar results were reported in sea bream fillets and Atlantic salmon (Salmo salar) packed under modified atmospheric packaging conditions (Parlapani et al., 2015; Powell and Tamplin, 2012). Carnobacterium also has contributed to the spoilage of VP cold-smoked salmon (Paludan-Müller et al., 1998). Other genera were also observed on day 12 including Lactococcus, Aeromonas, Pseudomonas and Shewanella. Aeromonas comprised 18.7% of the total strains from VP fillets. The fact that the proportion of Aeromonas decreased compared to those on PCA from VP samples stored on day 6 might be associated with the selective action of storage conditions and the inability of the microbiota to compete with Carnobacterium, which have the ability to grow well under anaerobic conditions (Leisner et al., 2007; Madigan et al., 2014). The spoilage potential of Aeromonas strains needs to be further evaluated. In the present study, only 1 isolate of Pseudomonas deceptionensis and 1 isolate of Shewanella xiamenensis were identified in the spoiled VP fillets, which suggested that they may contribute little to the spoilage of VP fillets. Plate counts however indicated that Pseudomonas and Shewanella were both found in high numbers (7.69 and 7.49 log CFU/g on CFC and IA, respectively). Tryfinopoulou et al. (2001) reported that the isolates on CFC might be identified as Aeromonas and Shewanella. Additionally, LAB groups had the ability to grow on IA (Mace et al., 2012). Macrococcus, Prolinoborus, Brochothrix, Enterobacter, Lysinibacillus observed on day 6 were not to be found at the end of storage (Table 4). 4. Conclusion This study has systematically identified the dominant microbiota of AP and VP common carp fillets during storage. More diversity under both packaging conditions was generally observed during the early storage period. A total of 13 different genera comprised the microbial communities of fresh carp fillets and Acinetobacter dominated the indigenous flora of carp. However, a large shift in the bacterial communities occurred during spoilage. In AP carp fillets, Pseudomonas became the only dominant microorganism at the end of the shelf life. Vacuum packaging extended the shelf life of carp fillets to 12 days compared to 8 days observed for fillets packed in air. Additionally, vacuum packaging changed the microbial composition in the spoiled carp fillets (5 different genera isolated). The 16S rRNA gene sequencing analysis showed that Carnobacterium followed by Aeromonas were the predominant microbiota at the end of the VP carp fillets’ shelf life. In addition, vacuum packaging delayed the increase of biogenic amines compared to air packaging, especially for CAD and TYM levels. Therefore, further work needs to be done to ascertain the spoilage potential and activity of Carnobacterium and Aeromonas in VP carp fillets and to understand the capacity of different bacteria to produce biogenic amines in the future. Acknowledgment This study was supported by the earmarked fund for China Agriculture Research System (CARS-46), National Natural Science Foundation of China (award no. 31471683) and Beijing Natural Science Foundation (award no. 6152017). References Atrea, I., Papavergou, A., Amvrosiadis, I., Savvaidis, I.N., 2009. Combined effect of vacuum-packaging and oregano essential oil on the shelf-life of Mediterranean octopus (Octopus vulgaris) from the Aegean Sea stored at 4
C. Food Microbiol. 26, 166e172. Bureau of Fisheries of the Ministry of Agriculture, 2014. China Fishery Statistical Yearbook. China, Beijing.

Chun, J., Lee, J.H., Jung, Y., Kim, M., Kim, S., Kim, B.K., Lim, Y.W., 2007. EzTaxon: a web-based tool for the identification of prokaryotes based on 16S ribosomal RNA gene sequences. Int. J. Syst. Evol. Microbiol. 57, 2259e2261. Connell, J.J., 1995. Control of Fish Quality, fourth ed. London, U.K. Dalgaard, P., 1995. Qualitative and quantitative characterization of spoilage bacteria from packed fish. Int. J. Food Microbiol. 26, 319e333. _ Genç, I.Y., Esteves, E., Aníbal, J., Diler, A., 2013. Effects of chilled storage on quality of vacuum packed meagre fillets. J. Food Eng. 115, 486e494. Gram, L., Huss, H.H., 1996. Microbiological spoilage of fish and fish products. Int. J. Food Microbiol. 33, 121e137. Hong, H., Luo, Y., Zhou, Z., Bao, Y., Lu, H., Shen, H., 2013. Effects of different freezing treatments on the biogenic amine and quality changes of bighead carp (Aristichthys nobilis) heads during ice storage. Food Chem. 138, 1476e1482. Hong, H., Luo, Y., Zhou, Z., Shen, H., 2012. Effects of low concentration of salt and sucrose on the quality of bighead carp (Aristichthys nobilis) fillets stored at 4
C. Food Chem. 133, 102e107. Hozbor, M.C., Saiz, A.I., Yeannes, M.I., Fritz, R., 2006. Microbiological changes and its correlation with quality indices during aerobic iced storage of sea salmon (Pseudopercis semifasciata). LWT e Food Sci. Technol. 39, 99e104. ICMSF, 1986. Sampling plans for fish and shellfish. In: Microorganisms in Foods 2. Sampling for Microbiological Analysis: Principles and Specific Applications. University of Toronto Press, Toronto, pp. 181e196. ICMSF, 2005. Micro-organisms in Foods 6: Microbial Ecology of Food Commodities, second ed. Kluwer Academic, Plenum Publishers, New York.
2004. Biogenic amines in , L., Luk kova , S., K
rı
zek, M., V acha, F., Vorlova a
sov a, J., Cupa vacuum-packed and non-vacuum-packed flesh of carp (Cyprinus carpio) stored at different temperatures. Food Chem. 88, 185e191. vost, H., Drider, D., Dalgaard, P., 2007. Carnobacterium: Leisner, J.J., Laursen, B.G., Pre positive and negative effects in the environment and in foods. FEMS Microbiol. Rev. 31, 592e613. € rkroth, J., 2002. Identification of lactic acid bacteria from Lyhs, U., Korkeala, H., Bjo spoiled, vacuum-packaged ‘gravad’ rainbow trout using ribotyping. Int. J. Food Microbiol. 72, 147e153. Lyhs, U., Lahtinen, J., Fredriksson-Ahomaa, M., Hyyti€ a-Trees, E., Elfing, K., Korkeala, H., 2001. Microbiological quality and shelf-life of vacuum-packaged ‘gravad’ rainbow trout stored at 3 and 8
C. Int. J. Food Microbiol. 70, 221e230. Mace, S., Cornet, J., Chevalier, F., Cardinal, M., Pilet, M.F., Dousset, X., Joffraud, J.J., 2012. Characterisation of the spoilage microbiota in raw salmon (Salmo salar) steaks stored under vacuum or modified atmosphere packaging combining conventional methods and PCR-TTGE. Food Microbiol. 30, 164e172. Madigan, T.L., Bott, N.J., Torok, V.A., Percy, N.J., Carragher, J.F., de Barros Lopes, M.A., Kiermeier, A., 2014. A microbial spoilage profile of half shell Pacific oysters (Crassostrea gigas) and Sydney rock oysters (Saccostrea glomerata). Food Microbiol. 38, 219e227. Mahmoud, B.S.M., Yamazaki, K., Miyashita, K., Il-Shik, S., Dong-Suk, C., Suzuki, T., 2004. Bacterial microflora of carp (Cyprinus carpio) and its shelf-life extension by essential oil compounds. Food Microbiol. 21, 657e666. Noseda, B., Islam, M.T., Eriksson, M., Heyndrickx, M., De Reu, K., Van Langenhove, H., Devlieghere, F., 2012. Microbiological spoilage of vacuum and modified atmosphere packaged Vietnamese Pangasius hypophthalmus fillets. Food Microbiol. ́ 30, 408e419. Paludan-Müller, Christine, Dalgaard, P., Huss, H.H., Gram, L., 1998. Evaluation of the role of Carnobacterium piscicola in spoilage of vacuum- and modifiedatmosphere-packed cold-smoked salmon stored at 5
C. Int. J. Food Microbiol. 39, 155e166. Parlapani, F.F., Kormas, K.A., Boziaris, I.S., 2015. Microbiological changes, shelf life and identification of initial and spoilage microbiota of sea bream fillets stored under various conditions using 16S rRNA gene analysis. J. Sci. Food Agric. 95, 2386e2394. Parlapani, F.F., Meziti, A., Kormas, K.A., Boziaris, I.S., 2013. Indigenous and spoilage microbiota of farmed sea bream stored in ice identified by phenotypic and 16S rRNA gene analysis. Food Microbiol. 33, 85e89. Pennacchia, C., Ercolini, D., Villani, F., 2011. Spoilage-related microbiota associated with chilled beef stored in air or vacuum pack. Food Microbiol. 28, 84e93. Powell, S.M., Tamplin, M.L., 2012. Microbial communities on Australian modified atmosphere packaged Atlantic salmon. Food Microbiol. 30, 226e232. Shi, C., Cui, J., Lu, H., Shen, H., Luo, Y., 2012. Changes in biogenic amines of silver carp (Hypophthalmichthys molitrix) fillets stored at different temperatures and their relation to total volatile base nitrogen, microbiological and sensory score. J. Sci. Food Agric. 92, 3079e3084. Tryfinopoulou, P., Drosinos, E.H., Nychas, G.J.E., 2001. Performance of Pseudomonas CFC-selective medium in the fish storage ecosystems. J. Microbiol. Meth. 47, 243e247. Tryfinopoulou, P., Tsakalidou, E., Nychas, G.J.E., 2002. Characterization of Pseudomonas spp. associated with spoilage of Gilt-Head sea bream stored under various conditions. Appl. Environ. Microbiol. 68, 65e72. Wang, H., Luo, Y., Huang, H., Xu, Q., 2014. Microbial succession of grass carp (Ctenopharyngodon idellus) filets during storage at 4
C and its contribution to biogenic amines' formation. Int. J. Food Microbiol. 190, 66e71. Winker, H., Weyl, O.L., Booth, A.J., Ellender, B.R., 2010. Validating and corroborating the deposition of two annual growth zones in asteriscus otoliths of common carp Cyprinus carpio from South Africa's largest impoundment. J. Fish. Biol. 77, 2210e2228.