packed silver carp - Wiley Online Library

Received: 21 January 2017

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Revised: 28 April 2017

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Accepted: 9 May 2017

DOI: 10.1111/jfpp.13389

ORIGINAL ARTICLE

Quality changes and microbiological spoilage analysis of air-packed and vacuum-packed silver carp (Hypophthalmichthys molitrix) fillets during chilled storage Dongping Li

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Qian Li | Yuemei Zhang | Xiaochang Liu | Hui Hong |

Yongkang Luo Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, 100083, People’s Republic of China Correspondence Yongkang Luo, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, People’s Republic of China. Email: [email protected] Funding information China Agriculture Research System (CARS-46); National Natural Science Foundation of China, Grant/Award Number: 31471683; Beijing Natural Science Foundation, Grant/Award Number: 6152017

Abstract The effects of air-packed (AP) and vacuum-packed (VP) on quality and microbial characteristics of silver carp (Hypophthalmichthys molitrix) fillets during chilled storage (4 6 1 8C) were investigated. The fillets were analyzed for sensory scores, total volatile basic nitrogen (TVB-N), ATP-related compounds (ATP, IMP, HxR, and Hx), K value, and biogenic amines (BAs). The results proved that VP inhibited the increase of microorganisms, TVB-N, Hx, and putrescine in silver carp, and slowed the reduction in sensory score. Therefore, VP can be applied for preservation of the silver carp fillets to improve its quality. For identification, 16S rRNA genes of the isolated pure strains were sequenced and analyzed. On the initial day of storage, Chryseobacterium was the dominant bacterial genus. At the end of shelf life, Pseudomonas was the most common group in AP fillets and Aeromonas followed by Yersinia were found mainly in VP samples.

Practical applications Silver carp (Hypophthalmichthys molitrix) are distributed widely in fresh water systems. The world aquaculture production of silver carp was 4,354,638 tons, and it ranked second highest among freshwater fish species in 2015, but they are perishable during storage because of microbial spoilage and biochemical reactions. Vacuum packaging (VP) has proved to be effective for extending the shelf-life of aquatic products by excluding oxygen that prevents the growth of spoilage bacterial. However, little information is available on the microbial succession of VP silver carp. Therefore, this work was to determine the differences of microbiological succession on chilled silver carp fillets under air-packed (AP) and VP conditions using a combination of culture-based and 16S rRNA gene analysis methods. Furthermore, this study will give valuable information about development and spoilage of VP silver carp fillets.

1 | INTRODUCTION

Agriculture Organization of the United Nations [FAO], 2016). However, they are perishable during storage because of microbial spoilage and

Fresh fish products have become more popular as the result of rich

biochemical reactions (Song et al., 2012). In the last few years, additional

nutrition, and their delicious and mild flavor. But the rich nutrients,

treatments have been taken to improve the quality of fish products and

high levels of moisture, and microbial activity in fish also make them

to extend shelf-life, such as sugar-salting packaging, active antimicrobial

more easily putrefactive (Fan, Luo, Yin, Bao, & Feng, 2014; Song, Luo,

packaging, vacuum packaging (VP), modified atmosphere packaging, and

You, Shen, & Hu, 2012). Therefore, we need to take additional meas-

so on (Fan et al., 2014; Remya et al., 2016). VP treatments have proved

ures to improve the quality of fish products and to extend shelf-life.

to be very effective for extending the shelf-life of these fish products by

Silver carp (Hypophthalmichthys molitrix) are distributed widely in

excluding oxygen that prevent the growth of spoilage bacterial (Mace

fresh water systems (Kasankala, Xiong, & Chen, 2012). The world aqua-

et al., 2012; Pennacchia, Ercolini, & Villani, 2011). In addition, VP fish

culture production of silver carp was 4,354,638 tons and it ranked sec-

transport easier due to the reduction in volume and therefore, VP has

ond highest among freshwater fish species in 2015 (Food and

attracted the attention of the food industry in recent years.

J Food Process Preserv. 2017;e13389. https://doi.org/10.1111/jfpp.13389

wileyonlinelibrary.com/journal/jfpp

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Pseudomonas, Aeromonas, Flavobacterium, Shewanella, Micrococcus,

sample (50 lL) was injected by an automatic sampler at a flow rate of

and Moraxella are the most common bacteria in fresh fish (Wang, Luo,

1.0 mL/min. The mobile phase was 0.05 M phosphate buffer (PBS, pH

Huang, & Xu, 2014). At the end of storage, fewer species dominate,

6.8) and the peak was detected at 254 nm. The amounts of ATP and

and they are referred to as specific spoilage organisms (SSOs), with a

related compounds were determined and calculated based on the

strong ability to corrupt the product (Gram & Huss, 1996). The rela-

standard of ATP (adenosine triphosphate), ADP (adenosine diphos-

tionship between microbiological spoilage and the shelf-life of silver

phate), AMP (adenosine monophosphate), IMP (inosine monophos-

carp is not well understood. The aim of this work was to determine the

phate), HxR (hypoxanthine ribonucleoside), and Hx (hypoxanthine). The

differences of microbiological succession on chilled silver carp fillets

K value was calculated as follows (Saito, Arai, & Matsuyoshi, 1959):

under air-packed (AP) and VP conditions using a combination of culture-based and 16S rRNA gene analysis methods. The SSOs of AP

K value ð%Þ5

ð½HxR1½HxÞ3100 ð½ATP1½ADP1½AMP1½IMP1½HxR1½HxÞ

and VP silver carp fillets were discussed also.

2 | MATERIALS AND METHODS

2.5 | Determination of BAs The determination of BAs followed Shi, Cui, Lu, Shen, and Luo (2012).

2.1 | Samples and treatment

After the extraction and derivatization of BAs, the sample (50 lL) was

Silver carp (weight 1,390 6 81 g, length 44 6 1 cm) was purchased

injected by an automatic sampler and analyzed by HPLC (Shimadzu,

from an aquatic products market (Beijing, China) and transferred to the laboratory alive in March 2015. Immediately, the fresh silver carp were stunned, scaled, gutted, sectioned and washed with sterile water. After draining in the incubator at 4 6 1 8C for 5 min, 36 of the fillets (AP) were packaged in polyethylene bags (about 250 mm 3 200 mm) and 44 of

LC-10A Tseries). The eight standard BAs, tryptamine (TRY), phenylethylamine (PHE), putrescine (PUT), cadaverine (CAD), histamine (HIM), tyramine (TYM), spermidine (SPD), and spermine (SPM) were purchased from Sigma-Aldrich (Shanghai, China).

the fillets (VP) were packaged and sealed in pouches of polyethylene/

2.6 | Microbial enumeration

polyamide film (about 250 mm 3 200 mm, with an oxygen permeability

The total number of different genera was determined according to the

of 40–50 cm3/m2 per 24 h/atm at 85% relative humidity, 23 8C). All of

plate count method. The treatment of the samples followed Hong, Luo,

the samples were stored in the refrigerator at 4 6 1 8C for temperature

Zhou, and Shen (2012). The samples (100 lL) of appropriate decimal

equalization. Every 2 days, three random fillets were taken for analysis

dilution were spread on the surface of the culture medium. Total viable

to determine sensory scores, TVB-N, ATP-related compounds, K value,

counts (TVC), lactic acid bacteria (LAB), H2S-producing bacteria, Pseu-

BAs, and bacterial counts. The microbial communities of AP were identi-

domonas sp., and Aeromonas sp. were determined in plate count agar

fied at 0, 6, and 12 days and of VP at 0, 12, and 18 days.

(PCA), MRS agar, iron agar medium, CM0559 pseudomonas agar base (PAB), and CM0833 Aeromonas medium base (AMB), respectively.

2.2 | Sensory analysis

Then, they were incubated at 30 6 1 8C for 72 h, 30 6 1 8C for 48 h,

The sensory evaluation of the raw fish fillets was based on the meth-

20 6 1 8C for 96 h, 25 6 1 8C for 48 h, and 30 6 1 8C for 24 h, respec-

ods of Ojagh, Rezaei, Razavi, and Hosseini (2010). Nine trained panel-

tively. All counts were performed in duplicate and expressed as log10

ists evaluated the color, odor, texture, and tonicity of the fish samples. Maximum score for each of the four indices was 5, and a total score of 20 represented fish that were absolutely fresh.

2.3 | Determination of TVB-N TVB-N value was measured by the semi-micro steam distillation method following Zhang, Li, Lu, Shen, and Luo (2011).

(colony forming units [CFU]/g). The culture media were bought from Beijing Land Bridge Technology Company, except for PAB and AMB (OXOID, Basingstoke, UK).

2.7 | Isolation and identification of microorganisms The separation and purification of microorganisms were described by Wang et al. (2014). The bacteria from PCA were taken for separation and identification at 0, 6, and 12 days for AP fillets and at 0, 12, and

2.4 | Determination of ATP-related compounds and K value

18 days for VP fillets. The isolated bacteria were identified by using the 16S rRNA gene sequence. DNA from bacterial isolates was extracted following the method

We determined ATP-related compounds as described by Fan, Chi, and

of Wang et al. (2014). The DNA extracts with clear ladder on the 1.0%

Zhang (2008) with some modification. ATP-related compounds were

agarose electrophoretic gel were chosen for further analysis.

extracted from 1 g samples of fillet muscle. Subsequently, the extrac-

The amplification of bacterial 16S rRNA gene was performed using

tion was filtered by a 0.22 lm drainage membrane and then analyzed

a PCR (TC-512; Techne, UK) with universal bacterial primers: forward

by high performance liquid chromatography (HPLC) (Shimadzu, LC-10A

primer 27f (50 -GAGATTTGATCCTGGCTCAG-30 ) and reverse primer

Tseries, Japan) equipped with a COSMOSIL 5C18-PAQ (4.6 mm id 3

1495r (50 -CTACGGCTACCTTGTTACGA-30 ). The PCR system was

250 mm) column and a UV detector (Shimadzu, LC-10A Tseries). The

formed with 12.5 lL 23 Taq PCR Master Mix (containing thermostable

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DNA polymerase, MgCl2, dNTPs, buffer), 1 lL template DNA, 10.5 lL

were considered to be unacceptable when the sensory score was

double distilled water, and 0.5 lL of each primer at a concentration of

below 10. Therefore, the AP samples were unacceptable after storing

10 lM. The reaction was run at a temperature of 94 8C for 5 min, fol-

for 8 days and the VP fillets were inedible after storing for 12 days.

lowed by 35 cycles (denaturation at 94 8C, 30 s; primer annealing at

The difference in both cases may be attributed to the variance in

54 8C, 30 s; primer extension at 72 8C, 1 min), and then kept at a tem-

atmosphere and microbial growth. Similar findings were also observed

perature of 72 8C for 10 min. Thereafter, PCR amplification products

et al. (2012). Mace et al. reported that the sensory score by Mace

were examined using 1.0% agarose gel electrophoresis. The PCR rea-

showed a significant decline in raw salmon fillets packaged under vac-

gent was supplied from Bomad Biological Technology (BBT) Co., Ltd.

uum, and the samples were strongly spoiled after storing for 10 days.

(Beijing, China) (Wang et al., 2014) The sequencing of the samples was performed by the BBT Co., Ltd., and the identification was completed using EzTaxon (Chun et al., 2007) (http://www.eztaxon.org/). Sequences with over 97% similarity were considered to be the same species.

experiments

were

The increase in TVB-N during storage was attributed to the microbiological and autolytic activities (Sallam, Ahmed, Elgazzar, & Eldaly, 2007). Fillets are considered acceptable when the TVB-N value 20 mg/ 100 g (Ojagh et al., 2010). The initial TVB-N value of raw silver carp fil-

2.8 | Statistical analysis All

3.2 | TVB-N

lets was 10.27 mg/100 g (Table 1). During storage, the TVB-N values

replicated

three

times.

The

mean

under AP and VP conditions increased with a slight fluctuation from

values 6 standard deviations of the measurements were reported for

the 2nd to the 8th day. A significant difference was observed between

each case. The least significant difference (LSD) procedure was used to

the two treatments on the 10th day. AP fillets reached 22.87 mg/

test for differences between means (significance was defined at

100 g at the end of storage (12th day), exceeding the highest accepta-

p < .05) using SPSS 21.0 (SPSS Inc., Chicago, IL) software.

ble level of 20 mg/100 g, but the VP fillets were still below this maximal permissible level at the end of storage (18th day). This may be attributed to production of organic acids in VP, which might be derived

3 | RESULTS AND DISCUSSION

from glycolysis by acid-forming bacteria. Another possible reason is

3.1 | Sensory score

that vacuum condition controlled the reproduction of aerobic microor-

Changes in sensory score of silver carp treated with AP and VP stored at 4 8C are shown in Table 1. The initial sensory score of silver carp fillets was 19.67. The sensory scores of all samples showed a significant decline (p < .05) with the increase of storage time. There were no significant differences (p > .05) between the sensory scores of AP and VP

ganisms and, thus, inhibited the microbial degradation of proteins. Similar findings have been also observed in grass carp (Ctenopharyngodon idellus) fillets (Zhang et al., 2011).

3.3 | ATP-related compounds and K value

fillets within 2 days. After being stored for 8 days, the sensory scores

The changes in the content of ATP and its related compounds are usu-

of AP and VP fillets were 10.33 and 12.67, respectively. The fillets

ally an effective index during storage of fish samples. IMP, HxR, and Hx

Changes in sensory score, TVB-N, ATP-related compounds (ATP, IMP, HxR, and Hx) and K value of silver carp treated with airpacked (AP) and vacuum-packed (VP) stored at 4 8C

T A B LE 1

Storage time (days)

Sensory score

AP

0 2 4 6 8 10 12

19.67 17.33 14.33 11.67 10.33 9.00 8.33

6 6 6 6 6 6 6

0.58j 1.15hi 0.58g 0.58de 0.58cd 1.00bc 1.15ab

10.27 9.40 9.06 11.76 13.63 18.67 22.87

6 6 6 6 6 6 6

0.32ab 0.65a 0.86a 2.57abc 2.76cd 1.29e 2.65f

3.19 0.56 0.74 0.59 0.47 0.79 0.35

6 6 6 6 6 6 6

1.02b 0.48a 0.27a 0.18a 0.12a 0.21a 0.14a

4.88 6.04 5.85 4.15 3.16 2.50 1.70

6 6 6 6 6 6 6

0.02g 0.68h 0.70h 0.10f 0.55e 0.30d 0.40c

0.69 1.62 2.00 1.93 2.42 2.94 2.63

6 6 6 6 6 6 6

0.13a 0.33b 0.18bcde 0.05bcd 0.38ef 0.41gh 0.26fg

0.05 0.13 0.19 0.22 0.56 0.86 1.96

6 6 6 6 6 6 6

0.01a 0.04a 0.03ab 0.03ab 0.19cd 0.23ef 0.14g

8.69 17.71 24.78 34.66 50.22 54.14 56.94

6 6 6 6 6 6 6

1.19a 0.17b 3.59c 5.86d 1.28e 6.53ef 4.70f

VP

0 2 4 6 8 10 12 14 16 18

19.67 18.67 16.00 13.67 12.67 11.00 10.67 8.67 7.67 7.00

6 6 6 6 6 6 6 6 6 6

0.58j 0.58ij 1.00h 0.58fg 0/58ef 1.00d 1.53d 0.58b 1.15ab 1.00a

10.27 10.64 10.08 10.46 12.70 10.36 10.92 13.72 14.19 14.93

6 6 6 6 6 6 6 6 6 6

0.32ab 0.56ab 1.19ab .32ab 0.32bcd 1.19ab 1.12ab 1.48cd 1.71cd 2.05d

3.19 0.20 0.21 0.23 0.24 0.24 0.20 0.23 0.23 0.23

6 6 6 6 6 6 6 6 6 6

1.02b 0.04a 0.01a 0.02a 0.01a 0.01a 0.04a 0.05a 0.05a 0.05a

4.88 6.96 6.03 5.25 4.80 4.19 3.87 1.00 0.14 0.13

6 6 6 6 6 6 6 6 6 6

0.02g 0.53i 0.36h 0.24g 0.20g 0.18f 0.34f 0.05b 0.06a 0.04a

0.69 1.98 2.17 3.59 4.35 3.97 3.25 3.26 2.28 1.72

6 6 6 6 6 6 6 6 6 6

0.13a 0.08bcde 0.22cde 0.39ij 0.39k 0.08jk 0.10hi 0.24hi 0.24def 0.22bc

0.05 0.16 0.18 0.28 0.46 0.68 0.98 2.38 2.99 3.66

6 6 6 6 6 6 6 6 6 6

0.01a 0.07ab 0.01ab 0.03abc 0.10bcd 0.31de 0.28f 0.40h 0.10i 0.17j

8.69 17.59 25.71 36.28 48.16 48.37 53.59 89.41 89.45 89.72

6 6 6 6 6 6 6 6 6 6

1.19a 1.56b 1.54c 3.63d 2.70e 0.61e 5.99ef 0.86g 0.98g 2.16g

Treatment

TVB-N (mg/100 g)

ATP (lmol/g)

Same lowercase letters in a column indicate no significant differences (p > .05).

IMP (lmol/g)

HxR (lmol/g)

Hx (lmol/g)

K value (%)

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Changes in the content of biogenic amines (BAs) in silver carp treated with air-packed (AP) and vacuum-packed (VP) stored at 4 8C BAs (mg/kg)

Treatment

Storage time Tryptamine (days) (TRY)

Phenylethylamine (PHE)

Putrescine (PUT)

Cadaverine (CAD)

Histamine (HIM)

Tyramine (TYM)

Spermidine (SPD)

Spermine (SPM)

AP

0 2 4 6 8 10 12

0.88 1.74 4.58 6.24 9.34 11.50 12.00

6 6 6 6 6 6 6

0.09bc 0.14ef 0.12i 0.83j 0.10k 0.11l 0.12m

1.45 0.62 22.46 16.66 11.60 10.27 6.63

6 6 6 6 6 6 6

0.11a 0.09a 0.60h 3.32f 2.53e 0.50de 0.80bc

0.44 0.16 0.96 1.27 11.01 17.79 35.86

6 6 6 6 6 6 6

0.10a 0.04a 0.33ab 0.48ab 0.50f 3.06g 1.00i

6 6 6 6 6 6 6

0.02a 0.06ab 0.04a 0.04a 0.08a 0.69b 0.80c

1.98 0.07 5.83 7.21 2.58 0.55 0.78

6 6 6 6 6 6 6

0.40bc 0.10a 0.96f 0.50g 0.15cd 0.10a 0.10a

34.83 10.13 39.46 27.95 31.65 24.47 35.00

6 6 6 6 6 6 6

3.06fg 1.20a 3.00h 3.37c 0.07ef 1.00c 1.00fg

18.88 5.91 21.67 14.81 16.91 9.66 12.78

6 6 6 6 6 6 6

2.29f 0.58a 1.33g 1.69d 1.22e 0.80b 0.80c

3.82 1.81 5.00 3.19 4.85 5.14 5.35

6 6 6 6 6 6 6

0.30de 0.03a 0.19gh 0.16bc 0.93gh 0.33gh 0.50h

VP

0 2 4 6 8 10 12 14 16 18

0.88 1.47 1.24 2.76 2.17 0.71 0.36 0.90 2.22 2.54

6 6 6 6 6 6 6 6 6 6

0.09bc 0.25de 0.47cd 0.20h 0.77fg 0.10ab 0.19a 0.20bc 0.42g 0.20gh

1.45 6.14 20.43 7.97 20.77 16.85 10.35 8.04 9.60 7.90

6 6 6 6 6 6 6 6 6 6

0.11a 0.50b 0.80g 1.50c 0.29g 1.20f 0.90de 0.33c 0.10d 0.57c

0.44 0.45 1.64 2.38 3.78 5.47 9.00 12.04 16.63 31.52

6 6 6 6 6 6 6 6 6 6

0.10a 0.87 6 0.10a 1.14 6 0.21ab 1.01 6 0.45bc 1.53 6 0.64c 1.05 6 0.50d 2.23 6 0.80e 3.61 6 0.53f 4.41 6 2.54g 10.02 6 1.00h 23.18 6

0.02a 0.47a 0.11a 0.31ab 0.20a 0.18b 0.20c 1.75c 1.06d 1.00e

1.98 0.30 3.26 1.87 3.38 0.28 0.36 1.50 3.74 2.09

6 6 6 6 6 6 6 6 6 6

0.40bc 0.27a 0.10de 0.75bc 0.61e 0.03a 0.22a 0.94b 0.50e 0.16bc

34.83 17.94 32.37 37.47 40.06 28.03 19.89 30.22 31.16 32.27

6 6 6 6 6 6 6 6 6 6

3.06fg 1.24b 1.08ef 1.00gh 1.21h 3.01c 1.00b 5.44de 4.69de 1.00ef

18.88 9.81 18.45 6.70 21.20 13.41 11.73 14.95 12.51 16.98

6 6 6 6 6 6 6 6 6 6

2.29f 0.39b 0.74ef 0.09a 0.29g 1.27cd 0.80c 2.42d 0.52c 0.90e

3.82 2.77 4.61 4.68 6.37 4.13 2.80 3.38 3.68 3.33

6 6 6 6 6 6 6 6 6 6

0.30de 0.27b 0.16fg 0.44g 0.38i 0.20ef 0.50b 0.28cd 0.00cde 0.14bcd

0.87 1.40 0.58 1.01 0.65 2.31 4.33


are strongly related with acceptable levels of fish freshness (Howgate,

(Mylopharyngodon piceus) (Fan et al., 2014) and grass carp (Wang et al.,

2006). The K value is the index for the degradation of ATP. In this study,

2014). The content of TYM fluctuated from 10.13 to 40.06 mg/kg dur-

changes in ATP, IMP, HxR, and Hx compounds of silver carp during

ing storage. HIM levels in the AP and VP fillets were relatively lower,

chilled storage were investigated (Table 1). The initial ATP, IMP, HxR,

and fluctuated from 0.07 to 7.21 mg/kg. TRY levels in AP fillets

and Hx concentration in the muscle of silver carp were 3.19, 4.88, 0.69,

increased significantly from 0.88 to 12.00 mg/kg, but TRY levels were

and 0.05 lmol/g, respectively. The ATP concentration of AP and VP fil-

fairly constant in VP samples during storage. CAD in AP samples

lets fell sharply during the first 2 days. Similarly, Nde, Paredi, and Crup-

showed no significant difference (p > .05) compared to the VP samples

kin (2001) reported that the ATP content of Scallop decreased rapidly

during the first 12 days of storage, but CAD had increased significantly

initially. The concentration of IMP increased during the first 2 days and

on the 16th and 18th days in VP samples. At the end of VP storage

then decreased gradually until the end of storage. IMP concentration of

(18th day), the content of CAD was 23.18 mg/kg. The PUT content of

VP was higher than that of AP after storage for 4 days. The content of

20 mg/100 g has been proposed as the maximum acceptable level by

Hx of AP and VP fillets showed no significant differences (p > .05) until

Krízek, Pavlíček, and Vacha (2002). During the first 6 days of storage,

day 6 of storage. Significant differences (p < .05) between Hx concen-

PUT increased very little. Thereafter, PUT levels increased significantly

trations for AP and VP samples were observed on the 12th day. The K

(p < .05) to 35.86 mg/kg for AP samples on the 12th day and

value showed a significant increasing trend with storage time under

31.52 mg/kg for VP samples on the 18th day. Furthermore, the PUT

both packaging conditions, and the initial K value of silver carp fillets

content in AP samples was much higher than in VP samples. Some

was 8.69% (Table 1). At the end of the AP storage (12th day), the K

reports have found that the occurrence of PUT and CAD in meat prod-

value of fillets had increased to 56.94%, and the K value of fillets was

ucts was related to ornithine-decarboxylase and lysine-decarboxylase

89.72% at the end of the VP storage (18th day). The K value of the fillets

activity of Enterobacteriaceae (Bover-Cid, Izquierdo-Pulido, & Vidal-

under two conditions showed little difference.

Carou, 2001). The PUT and CAD contents of VP fillets increased significantly after the 8th and 10th days, respectively, probably due to the

3.4 | BAs Table 2 shows the concentrations of eight BAs in both AP and VP silver carp fillets at 4 8C. In the present study, eight BAs were detected in silver carp fillets on the first day of storage. The initial values of TRY, PHE, PUT, CAD, HIM, TYM, SPD, and SPM were 0.88, 1.45, 0.44,

occurrence of Yersinia, which is also in the family Enterobacteriaceae. Similar results have been observed also in common carp (Cyprinus carpio) (Krízek et al., 2002).

3.5 | Enumeration of the different bacterial groups

0.87, 1.98, 34.83, 18.88, and 3.82 mg/kg, respectively. The total BAs

Table 3 shows the changes in the microbial groups in silver carp fillets

of AP samples were higher than the VP samples. PHE, HIM, TYM, SPD,

during chilled storage under the AP and VP conditions. The maximum

and SPM fluctuated during storage and showed no significant differ-

acceptable level of microbial enumeration for freshwater and marine

ence between AP and VP. During storage of the fillets, PHE, HIM,

fish is 7.00 log10 CFU/g (ICMSF, 1986). The TVC of the silver carp fil-

TYM, SPD, and SPM fluctuated which was reported also in black carp

lets was 3.51 log10 CFU/g at the beginning of storage, and then

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Enumeration of different groups of microorganisms in silver carp fillets during air-packed (AP) and vacuum-packed (VP) at 4 8C Microbial groups (log10 CFU/g)

Treatment

Storage time (days)

Total viable counts

Pseudomonas

Aeromonas

H2S-producing bacteria

AP

0 2 4 6 8 10 12

3.51 3.52 5.71 6.37 7.44 8.28 8.51

6 6 6 6 6 6 6

0.10a 0.11a 0.05c 0.08d 0.21g 0.07h 0.24h

2.50 3.06 5.18 6.35 8.23 8.43 8.45

6 6 6 6 6 6 6

0.17a 0.06b 0.23cd 0.06g 0.08h 0.08h 0.21h

2.19 2.24 5.18 6.41 6.34 7.47 7.42

6 6 6 6 6 6 6

0.26a 0.33a 0.22d 0.06fg 0.16f 0.12h 0.06h

2.26 3.30 4.95 6.02 6.28 7.69 7.85

6 6 6 6 6 6 6

0.28a 0.79b 0.06d 0.02ef 0.03fg 0.13i 0.07i

2.00 2.67 4.55 4.63 5.56 5.88 6.46

6 6 6 6 6 6 6

0.00a 0.05b 0.18d 0.21d 0.10ef 0.10fg 0.10ij

VP

0 2 4 6 8 10 12 14 16 18

3.51 4.38 4.49 5.52 5.73 6.15 6.73 7.04 7.29 7.54

6 6 6 6 6 6 6 6 6 6

0.10a 0.20b 0.17b 0.21c 0.24c 0.14d 0.16e 0.01f 0.21fg 0.23g

2.50 2.59 4.89 5.48 5.63 5.84 6.66 6.43 6.63 6.55

6 6 6 6 6 6 6 6 6 6

0.17a 0.15a 0.23c 0.02de 0.30ef 0.40f 0.14g 0.14g 0.14g 0.14g

2.19 2.99 4.48 5.26 5.83 6.55 6.50 6.65 6.76 6.75

6 6 6 6 6 6 6 6 6 6

0.26a 0.07b 0.40c 0.26d 0.34e 0.06fg 0.09fg 0.05fg 0.20g 0.21g

2.26 3.63 4.16 4.64 5.76 5.83 6.12 6.60 6.26 6.70

6 6 6 6 6 6 6 6 6 6

0.28a 0.45b 0.37c 0.39d 0.12e 0.36e 0.05ef 0.22gh 0.12fg 0.12h

2.00 3.78 4.35 5.27 5.95 6.29 6.41 6.83 6.60 6.38

6 6 6 6 6 6 6 6 6 6

0.00a 0.23c 0.04d 0.39e 0.16gh 0.36hi 0.16i 0.18j 0.19ij 0.41i

Lactic acid bacteria


increased significantly under both treatments with increasing storage

lates were identified by 16S rRNA gene sequencing. Sequences are

time. The microbial population was exceeded 7.00 log10 CFU/g after 8

considered to be of the same species if they group at greater than 97%

and 14 days of chilled storage under AP and VP, respectively. The

similarity in GenBank (Madigan et al., 2014). Of the 271 isolates, 55

growth rate of TVC under the VP treatment was significantly lower

isolates were identified, which were collected from fresh silver carp fil-

than under the AP treatment (p < .05). These findings are in agreement

lets. At the beginning of chilled storage, the bacterial diversity was

with Noseda et al. (2012), who reported a shelf life for Pangasianodon

higher than at the end of storage, whereas the TVC of the fresh fillets

hypophthalmus fillets of 7, 10, 12, and 14 days stored at 4 8C under air,

was low (3.51 log10 CFU/g). Similar findings have been observed also

under vacuum, in 50%CO2–50%N2 and 50%CO2–50%O2, respectively.

et al., 2012). The initial microbial communities in raw salmon (Mace

All of the bacterial groups increased during lag phase, exponential

were distributed across 16 different genera. Of the 55 isolates, 11 of

phase, and stationary phase under AP and VP conditions. Until the end

them were identified at the genus level as Chryseobacterium, nine of

of storage, microbial levels remained quite constant. In the samples

them as Kocuria, six of them as Pseudoclavibacter, and five of them as

packaged in air, the number of Pseudomonas sp. reached 8.45 log10

Brevundimonas; they represented 20, 16, 11, and 9% of the total iso-

CFU/g at day 12, which almost equaled the TVC. The counts of Pseu-

lated microbial strains, respectively. Chryseobacterium is the most com-

domonas sp. under the VP condition were significantly lower (p < .05)

mon bacterial genus in fresh fish, and 10 of the 11 isolates from this

than under the AP condition during the entire storage. The growth of

genus were identified at the species level as Chryseobacterium haifense.

Pseudomonas was also inhibited under VP in salmon (Salmo salar)

Acinetobacter has been reported as the most common bacterial genus in

(Paludan-Muller, Dalgaard, Huss, & Gram, 1998). LAB, H2S-producing

grass carp under chilled storage (Wang et al., 2014). All of the Kocuria

bacteria, and Aeromonas sp. counts were lower than the Pseudomonas

and Pseudoclavibacter were identified at the species level as Kocuria rhi-

sp. counts during air storage at 4 8C. Especially for LAB, the counts of

zophila and Pseudoclavibacter helvolus, respectively. Other microorgan-

LAB did not exceed 7.00 log10 CFU/g until the end of both AP and VP

isms in fresh fillets, such as Acinetobacter, Microbacterium, Psychrobacter,

storage. However, LAB counts under VP conditions were significantly

Pseudoxanthomonas, Arthrobacter, Stenotrophomonas, Rhodococcus, Mor-

higher than under AP conditions, which may be attributed to the anaer-

axella, Devosia, Flavobacterium, Nocardioides, and Methylobacterium, only

obic properties of LAB. Under VP at 4 8C, Aeromonas sp. reached 6.75

contained 1–3 isolates.

log10 CFU/g by the 18th day, but the other microbes were not signifi-

With the storage duration, microbial communities of AP samples

cant. We conclude that Pseudomonas was the predominant genus at

changed greatly, including the genus and the species (Table 4). Late in

the end of shelf life for AP fillets and Aeromonas was found mainly in

storage, Chryseobacterium became uncommon and many other bacterial

VP samples.

genera appeared, such as Shewanella, Pseudomonas, Carnobacterium, Lactococcus and Janthinobacterium. A total of 65 isolates from six dif-

3.6 | Identification of bacterial isolates

ferent genera were identified from AP fillets on the 6th day. At the end of the AP chilled storage (12th day), 67 isolates from three different

Tables 4 and 5 showed the microbial multiformity of silver carp fillets

genera were identified (Table 4). During the first 6 days of AP storage,

under AP and VP conditions at 4 8C, respectively. A total of 271 iso-

Pseudomonas increased rapidly and represented 37% of the total of 65

Y17227

Microbacterium oxydans

AJ421528

Psychrobacter faecalis

Brevundimonas

D12785

AY553293

Chryseobacterium antarcticum

Brevundimonas

EF204450

Chryseobacterium haifense

Chryseobacterium

AJ437696

Psychrobacter pulmonis

Psychrobacter

Y17231

Microbacterium esteraromaticum

Microbacterium

1

1

10

1

1

2

1

APON01000005 2

Acinetobacter johnsonii

1

100.00

99.09

97.68–98.02

99.78

99.71

99.85–100.00

99.86

99.48–99.63

98.17

Number of isolates Identity (N 5 55) (%)

KC843488

Closest relative (accession no)

Acinetobacter harbinensis

Acinetobacter

Species identification

0

9.09

20.00

3.64

5.45

5.45

Distribution (%)

Pseudomonas

Pseudomonas deceptionensis

Pseudomonas psychrophila

Pseudomonas helmanticensis

Pseudomonas

Shewanella hafniensis

Shewanella baltica

Shewanella xiamenensis

Shewanella putrefaciens

Shewanella

Acinetobacter parvus

Acinetobacter haemolyticus


Acinetobacter


6

18

AF074384

GU936597

AB041885

HG940537

AB205566

AJ000214

FJ589031

X81623

AIEB01000124

1

11

9

3

2

1

1

12

1

99.93

99.55–99.56

99.61–99.70

99.41–99.48

99.40–99.41

98.89

99.02

99.27–99.62

100.00

98.53

97.50–99.84

Number of Isolates Identity (N 5 65) (%)

APQQ01000002 1

KC843488


36.92

24.62

30.77

Distribution (%)

Bacterial diversity of air-packed (AP) silver carp fillets during storage at 4 8C based on 16S rRNA gene of pure isolates

Storage time (days)

T A B LE 4

Pseudomonas

Pseudomonas brenneri

Pseudomonas kilonensis

Pseudomonas prosekii

Pseudomonas gessardii

Pseudomonas deceptionensis

Pseudomonas migulae

Pseudomonas psychrophila

Pseudomonas costantinii

Pseudomonas fragi

Pseudomonas


Shewanella baltica

Shewanella


12

8

HG940537 1

AF268968 8

AJ292426 2

JN814372 1

AF074384 1

GU936597 7

AF074383 2

AB041885 12

AF374472 1

AF094733 18

X81623

99.47

99.78–99.93

99.53–99.55

99.56

99.92

99.04–99.70

99.62

99.85–100.00

99.70

99.28–99.77

99.27–99.47

99.16–99.19


AJ000214 3


(Continues)

79.10

16.42

Distribution (%)

6 of 12

| LI ET AL.

Y16264

X77440

X79186

FR714842

Rhodococcus

CP007597

D88211

Rhodococcus fascians

Rhodococcus

Stenotrophomonas rhizophila

Stenotrophomonas

Arthrobacter creatinolyticus

Arthrobacter

Pseudoxanthomonas AB008507 japonensis

Pseudoxanthomonas

Kocuria rhizophila

Kocuria

Pseudoclavibacter helvolus

Pseudoclavibacter

1

1

3

1

1

9

6

99.69

100.00

100.00

99.85

99.56

99.04–99.93

99.85–99.92

100.00

5.45

5.45

1.82

1.82

16.36

10.91

Psychrobacter maritimus

Psychrobacter

Lactococcus raffinolactis

Lactococcus

AJ609272

EF694030

Carnobacterium AF184247 maltaromaticum

1

2

2

99.85

99.78

100.00

1.54

3.08

3.08


Janthinobacterium DQ355146 1 svalbardensis

2

100.00

99.56–99.84

99.92


FN645213 1


Janthinobacterium Y08846 lividum

Janthinobacterium

Pseudomonas arsenicoxydans

4

Distribution (%)

Carnobacterium

FJ544245

Number of Isolates Identity (N 5 65) (%)

Brevundimonas naejangsanensis


Closest relative (accession no) helmanticensis

Distribution (%)

12

gessardii


6

bullata


0


(Continued)

Storage time (days)

T A B LE 4

(Continues)

4.48

Distribution (%)

LI ET AL.

| 7 of 12

Methylobacterium pseudosasae

Methylobacterium

Nocardioides dubius

Nocardioides

Flavobacterium granuli

Flavobacterium

Devosia subaequoris

Devosia

Moraxella osloensis

Moraxella

Rhodococcus qingshengii

cerastii


0

EU912442

AY928902

AB180738

AM293857

X74897

DQ090961


(Continued)

Storage time (days)

T A B LE 4

1

2

1

1

3

1

100.00

99.85

98.86

97.75

99.56–99.85

100.00


1.82

3.64

1.82

1.82

5.45

Distribution (%) Species identification

6 Closest relative (accession no) Number of Isolates Identity (N 5 65) (%) Distribution (%) Species identification

12 Closest relative (accession no)


Distribution (%)

8 of 12

| LI ET AL.

Y17227

Microbacterium oxydans

AJ421528

Psychrobacter faecalis

AY553293

Chryseobacterium antarcticum

6

4

1

1

10

1

1

2

1

99.85–99.92

100.00

100.00

99.09

97.68–98.02

99.78

99.71

99.85–100.00

99.86

99.48–99.63

98.17

10.91

9.09

20.00

3.64

5.45

5.45

1

26

X81623

X81623

HM031078

M58816

AF184247

DQ343754

Janthinobacterium DQ355146 svalbardensis

Janthinobacterium


Shewanella

Iodobacter limnosediminis

Iodobacter

Carnobacterium divergens

Carnobacterium maltaromaticum

Carnobacterium

Lactococcus piscium

1

2

6

1

3

2

99.78

99.41–99.70

98.90–99.63

100.00

99.85–99.92

100.00

2.33

4.65

13.95

9.30

Yersinia ruckeri

Yersinia

Aeromonas salmonicida subsp. pectinolytica

Aeromonas salmonicida subsp. salmonicida

Aeromonas punctata subsp. caviae

Aeromonas bestiarum

Aeromonas sobria

Aeromonas eucrenophila

Aeromonas

2

1

3

10

1

2

1

6

X75275

14

ARYZ01000167 1

X74681

X74674

X60406

X74683

X60411

Serratia AJ233434 proteamaculans


Shewanella

Carnobacterium AF184247 maltaromaticum

Carnobacterium


99.56–99.78

100.00

99.78–99.93

99.34

100.00

99.69–100.00

99.55

99.18–99.19

99.47

99.93–100.00

Number Closest of relative isolates Identity (accession no) (N 5 41) (%)

Lactococcus 4.65

65.12

Distribution (%)

18

Serratia

100.00

100.00

99.69–100.00

No. of isolates Identity (N 5 43) (%)

ARYZ01000167 1

X60406

X74683


pectinolytica

Aeromonas salmonicida subsp

Aeromonas bestiarum

Aeromonas sobria

Aeromonas

Distribution Species (%) identification

12

(Continues)

35.00

42.50

5.00

2.50

15.00

Distribution (%)

ET AL.

|

Pseudoclavibacter

X77440

FJ544245

Brevundimonas naejangsanensis

Pseudoclavibacter

D12785

Brevundimonas bullata

Brevundimonas

EF204450

Chryseobacterium haifense

Chryseobacterium

AJ437696

Psychrobacter pulmonis

Psychrobacter

Y17231

Microbacterium esteraromaticum

Microbacterium

APON01000005 2

Acinetobacter johnsonii

1

KC843488



Acinetobacter


0


Bacterial diversity of vacuum-packed (VP) silver carp fillets during storage at 4 8C based on 16S rRNA gene of pure isolates

Storage time (days)

T A B LE 5

LI 9 of 12

DQ090961

Rhodococcus qingshengii

AM293857

EU912442

AY928902

1

2

1

1

3

1

1

1

3

1

1

9

100.00

99.85

98.86

97.75

99.56–99.85

100.00

99.69

100.00

100.00

99.85

99.56

99.04–99.93


1.82

3.64

1.82

1.82

5.45

5.45

5.45

1.82

1.82

16.36

Distribution Species (%) identification

12 Closest relative (accession no) No. of isolates Identity (N 5 43) (%) Distribution (%) Species identification

18 Number Closest of relative isolates Identity (accession no) (N 5 41) (%)

Distribution (%)

|

Methylobacterium pseudosasae

Methylobacterium

Nocardioides dubius

Nocardioides

Flavobacterium granuli AB180738

Flavobacterium

Devosia subaequoris

Devosia

Moraxella osloensis

X74897

FR714842

Rhodococcus cerastii

Moraxella

X79186

CP007597

D88211

AB008507

Y16264

Rhodococcus fascians

Rhodococcus

Stenotrophomonas rhizophila

Stenotrophomonas

Arthrobacter creatinolyticus

Arthrobacter

Pseudoxanthomonas japonensis

Pseudoxanthomonas

Kocuria rhizophila

Kocuria

helvolus


0


(Continued)

Storage time (days)

T A B LE 5

10 of 12 LI ET AL.

LI

|

ET AL.

11 of 12

isolates. Acinetobacter, which increased from 5 to 31% of all isolates

detected. Chryseobacterium dominated the bacterial genera in fresh

during the first 6 days of AP storage, became the second most com-

fish. In AP silver carp fillets, Pseudomonas followed by Acinetobacter

mon bacterium. Shewanella accounted for 25%. The remaining isolates

was predominant at day 6, and it became the largest group of microbial

were identified as Carnobacterium, Lactococcus, and Psychrobacter,

genera of silver carp at day 12. Based on the identification using 16S

which represented 3, 3, and 2% of all isolates, respectively. At the end

rRNA gene sequencing, Pseudomonas isolates were comprised mostly

of the shelf life of AP samples, the microbiota was dominated by

of the species Pseudomonas fragi and P. psychrophila. While under VP

Gram-negative fermentative bacteria, including Pseudomonas, Shewa-

storage, Aeromonas was the predominant genus on the 12th day. At

nella, and Janthinobacterium. Pseudomonas species became the largest

the end of shelf life of VP silver carp fillets, Aeromonas followed by Yer-

group of microbes in silver carp at day 12, accounting for 79% (53 iso-

sinia was the predominant microbiota. In addition, VP inhibited the

lates) of the 67 isolates. Pseudomonas isolates were comprised of 11

increase in BAs, especially PUT. Furthermore, the spoilage ability of the

different species, which were dominated by Pseudomonas fragi (18 iso-

SSOs and the relation between the production of BAs and the capacity

lates) and Pseudomonas psychrophila (12 isolates). Shewanella was the

of different bacteria need to be researched.

second major microbe, and represented 16% of the total number of isolates. These results were consistent with plate count results. Pseudo-

ACKNOWLEDGMENTS

monas is common species of aquatic products under AP chilled storage

This study was supported by the earmarked fund for China Agricul-

(Parlapani, Kormas, & Boziaris, 2015; Tryfinopoulou, Tsakalidou, &

ture Research System (CARS-46), National Natural Science Founda-

Nychas, 2002). Otherwise, Aeromonas did not exist in AP fillets based

tion of China (award no. 31471683), and Beijing Natural Science

on our identification using 16S rRNA gene sequencing. This result did

Foundation (award no. 6152017). I would like to thank Thomas A.

not confirm with the plate count results. This may be attributed to the

Gavin, Professor Emeritus, Cornell University, for help with editing

growth of Rahnella and Yersinia in AMB, which we verified.

the English in this paper.

In the microbial composition, there were obvious differences between AP and VP samples. A total of 84 isolates from VP fillets were identified (Table 5): 43 of them were from the day close to spoilage (12th day), and the others were from the end of storage (18th day). Compared to AP fillets, the microbial composition at day 12, when close to spoilage, changed completely in VP fillets under chilled storage. Pseudomonas, as the predominant bacteria in AP samples, did not appear in VP samples. However in the plate count results, the counts of Pseudomonas increased to 6.55 log10 CFU/g on the 18th day. Tryfinopoulou, Drosinos, and Nychas (2001) found that Aeromonas, Enterobacteriaceae, and Shewanella can grow on PAB, which may be the reason for this phenomenon. At day 12, Aeromonas isolates in VP samples were at a higher proportion (65%) than those in AP samples. At day 18, Aeromonas was also represented by the highest proportion. The predominant species of Aeromonas was Aeromonas sobria, both on the 12th and 18th days. Aeromonas has been reported as the major spoilage bacteria of many freshwater fish species (Gui et al., 2014). Chryseobacterium was one of the major bacteria detected at the beginning of storage, but was no longer detected on the 12th and 18th days under the VP chilled storage. At the end of shelf life of VP silver carp fillets, Aeromonas followed by Yersinia was the predominant bacteria. In the present study, all of the Yersinia species were identified by 16S rRNA gene sequencing as Yersinia ruckeri.

4 | CONCLUSION In the present study, the succession of microbial communities and the changes in chemical indices in AP and VP silver carp fillets stored at

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How to cite this article: Li D, Li Q, Zhang Y, Liu X, Hong H, Luo Y. Quality changes and microbiological spoilage analysis of airpacked and vacuum-packed silver carp (Hypophthalmichthys molitrix) fillets during chilled storage. J Food Process Preserv. 2017;e13389. https://doi.org/10.1111/jfpp.13389