1. Introduction
Pacific white shrimp (
Litopenaeus vannamei) has been of increasing demand all over the world, commercially viable of the globe with high yield and high nutrition [
1,
2,
3]. In spite of their delicacy and popularity, this high value crustacean is highly perishable because of a limited shelf-life, mainly caused by the enzymatic autolysis, chemical oxidation and microbial deterioration [
4,
5]. The activity of microorganism is the main cause to affect the quality of fresh shrimp [
4]. Gram-negative psychrotrophic organisms are the most important bacteria in cold storage under aerobic conditions [
6], such as
Shewanella spp.,
Aeromonas spp.,
Pseudomonas spp. and genera of the
Enterobacteriaceae family [
7].
Shewanella spp. and
Aeromonas spp. are largely responsible for the specific spoilers in Pacific white shrimp, known as specific spoilage microorganism (SSO) [
8,
9]. The SSO of
Shewanella spp. and
Aeromonas spp. can produce amines, trimethylamine, protein degradation and off-odor in previous researches to decrease the quality of shrimp [
10,
11,
12,
13]. Hence, it is essential to control the activity of SSO in fresh shrimp during cold storage by innovative preservation techniques [
14]. Our previous research found that cell-free supernatant (CFS) from
Aeromonas sobria could inhibit the growth ability of
Shewanella putrefaciens, the ability of producing TVB-N and biogenic amines, the microbial decarboxylation of amino acids and the degradation of sarcoplasmic proteins of shrimp during cold storage at 4 °C. Because the role of metabolites of
A. sobria in CFS can influence the micro-environment and the regulate production of substance and controlling the activities of enzymes [
15,
16]. For example, the CFS from
Pseudomonas fluorescens had an inhibited effect on the activity of spoilage phenotypes in
Shewanella baltica [
12]. In the presence of
Carnobacterium maltaromaticum,
S. baltica imposed its characteristic to produce more volatile compounds, such as sulfur compounds like H
2S that can lead to typical cabbage/sulfur odors of spoiled samples [
17]. Hence, it is an innovative method to use the interaction between bacteria to inhibit microbial activity by extracting cell-free supernatant as an edible soaking solution.
During processing, transportation, retailing, and domestic storage, the temperature of food storage was not constant. Pacific white shrimp will be exposure to different temperatures and temperature fluctuation which have a negative effect on shrimps [
18], due to the high water and protein content [
19]. There were some related researches had been done about seafood storage during temperature fluctuation. For example, Shi et al. found a perfect method to predict the freshness of fillets stored from 0 °C to 10 °C based on feature viable by electronic nose and tongue in the cold chain [
19]. Zhang et al. studied the effect of frozen-then-chilled storage on free Ca
2+, proteolytic enzyme activity of calpains and the proteasome, water-holding capacity and sheer force of porcine (
longissimus thoracis) at lumborum muscle and found that frozen-then-chilled storage increased free Ca
2+ concentration, followed by a faster decrease of calpain-1 activity and activation of around 50% of calpain-2 and proteasome activity was reduced by around 40% following freezing-thawing [
20]. Therefore, the freshness and changes of quality on shrimp is a worthy subject to focus on stored during temperature fluctuation in the cold chain. Additionally, there is almost no research to study the effect of CFS from
A. sobria on the spoilage of
S. putrefaciens in Pacific white shrimp with temperature fluctuation.
The object of this work was to investigate the effect of CFS from A. sobria on the spoilage of S. putrefaciens in Pacific white shrimp during transportation, retailing, and domestic storage in which shrimp will be exposure to different temperatures and temperature fluctuation. The growth of S. putrefaciens was analyzed. Physical and chemical properties including sensory evaluation, total volatile basic nitrogen (TVB-N), amino acid and biogenic amines were analyzed. Myofibrillar proteins of shrimp were analyzed by poly-acrylamide gel electrophoresis (SDS-PAGE).
2. Materials and Methods
2.1. Bacterial Strains
Strains of S. putrefaciens QY38 and A. sobria QY32 isolated from spoiled Pacific white shrimp previously were identified by 16SrRNA and VITEK®2 CompactA system (BIOMÉRIEUX, Lyon, France). They were stored at −80 °C in sterilized Tryptone soy broth (TSB; Qingdao Hope Bio-Technology Co., Ltd., Qingdao, China) containing 25% glycerine. The isolates were pre-cultured individually in brain heart infusion broth (BHI; Qingdao Hope Bio-Technology Co., Ltd., Qingdao, China) for 18 h and then in TSB for 8 h at 27 °C, before use. The cell concentration after preculture was about 7~9 log CFU/mL.
2.2. Preparation of CFS
A. sobria was cultured overnight in BHI at 30 °C. Cultures were then centrifuged at 10,000
g for 15 min at 4 °C for removing the cells from the growth medium. For obtaining CFS of
A. sobria, the supernatant was filtered through a 0.22-μm-pore-size filter (Whatman, Inc., Cilfton, NJ, USA) and then stored at −20 °C [
21]. The initial pH was 7.19.
2.3. Sample Preparation
Pacific white shrimp about 6 ± 2 g for each one were transported to the laboratory alive from the local market (Shanghai, China). All the shrimps were sacrificed and washed with ice slurry. Then the shrimps were sterilized by dipping in 75% ethanol for 120 s and washed twice with sterilized distilled water [
22]. Finally, the shrimps were sterilized by ultraviolet (UV) again for 20 min. The shrimps were randomly divided into 6 groups. Overnight cultures of
S. putrefaciens were inoculated into sterilized distilled water and then three group shrimps were dipping in the solutions. These three groups were used as control samples (CK). The other three groups were dipping in the above prepared
S. putrefaciens solution containing a final concentration of 100% (
v/
v) CFS of
A. sobria for 1 min. These three groups were used as test samples (SA). After draining in the relatively sterile environment, six shrimp groups were stored separately according to the cold chain logistics process in
Figure 1, recorded as CK1, SA1; CK2, SA2; CK3, SA3.
According to the basic requirements of food technology during cold chain logistics and food sold in supermarket, the temperature of food stored in cold storage is 0 °C, the temperature of the refrigerated transport box is 4 °C, the temperature of the ice station for retailing is 0 °C and the temperature of domestic storage is 4 °C. According to the temperature of the above simulated cold chain logistics process, the prepared shrimp samples were stored in a refrigerator with the corresponding temperature, respectively.
2.4. Microbiological Analysis
The bacteriological analysis in this study carried out including total H
2S producer. Twenty-five grams of shrimps were homogenized with 225 mL sterilized saline water (NaCl, 0.85%,
w/
v, Qingdao Hope Biol-Technology Co., Ltd., Qingdao, China). Then, for bacteriological analysis [
23], serial decimal dilutions of each group were carried out with sterilized saline water. 1.0 mL of the dilutions were spread plated into Iron Agar (IA, Qingdao Hope Biol-Technology Co., Ltd., Qingdao, China) for the enumeration of total H
2S producer. The IA plates were inoculated at 30 °C for 72 h and black colonies were enumerated.
2.5. Sensory Evaluation
The whole shrimp samples for sensory analysis were displayed on a cleaned white porcelain plate by thirsty untrained panelists from the College of Food Science and Technology, Shanghai Ocean University, using a 9-point hedonic scales (9 = like extremely to 1 = dislike extremely). Sensory characteristics include odors, texture, color and other general appearance [
24,
25]. The mean of the scores represented the overall sensory quality of shrimps. The panelists should be familiar with the rating scales beforehand.
2.6. Determination of Total Volatile Basic Nitrogen (TVB-N)
TVB-N was determined according to the method of Dabadé et al. with the application of an Automatic Kjeldahl Apparatus (KjeltecTM8400; FOSS Quality Assurance Co., Ltd., Copenhagen, Denmark) [
6]. TVB-N contents were expressed as mg N/100 g shrimp flesh.
2.7. Biogenic Amines Analysis
The extraction of biogenic amines was carried out according to the method of Ikonic et al. and Tasic et al. [
26,
27]. Then the accumulation of biogenic amines was analyzed by HPLC system (SHIMADZU, LC-2010C HT, Kyoto, Japan) after filtered through a 0.45-μm membrane filter. The parameters of HPLC analysis were settled according to the method of Qian et al. [
28].
2.8. Amino Acid Analysis
The amino acid contents of shrimp samples were determined using an automatic amino acid analyzer (Hitachi Global Co., Ltd., Tokyo, Japan) [
29]. Briefly, after 10min of nitrogen blowing, 100 mg shrimp samples were hydrolyzed in 6 M HCl solution (Sinopharm Chemical Reagent Co., Ltd., Beijing, China) including drops of phenol (Sinopharm Chemical Reagent Co., Ltd., Beijing, China) for 24 h at 110 °C. The residual was dissolved in 1.0 mL of citrate buffer (pH 2.2,Sinopharm Chemical Reagent Co., Ltd., Beijing, China) and then passed through a 0.22-μm membrane filter for injection into the analyzer.
2.9. Extraction of Muscle Proteins and Electrophoresis
The myofibrillar proteins were extracted using the method of Qian et al. [
2]. The shrimp muscle (5 g) were m inced and stirred in phosphate buffer A (15.6 mmol/L Na
2HPO
4, 3.5 mmol/L KH
2PO
4,
I = 0.05, pH 7.5, 4 °C; Sinopharm Chemical Reagent Co., Ltd., Beijing, China). The precipitate was collected after centrifugation at 1000
g and then centrifuged with phosphate buffer B (15.6 mmol/L Na
2HPO
4, 3.5 mmol/L KH
2PO
4, 0.45 mol/L KCl,
I = 0.5, pH 7.5, 4 °C) at 1000
g, 4 °C. The Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, GenScript Co., Ltd., Nanjing, China) was conducted referring to the method of Qian et al. [
28].
2.10. Statistical Analysis
All the experiments were repeated at least three times and the results are reported as averages. F test (LSD and Duncan’s test) was performed to evaluate the mean differences between measurements at the 5% confidence level (p < 0.05) using the SPSS software program (SPSS Inc., Chicago, IL, USA).
4. Discussions
This study obtained an insight into the effect of CFS from
A. sobria on the spoilage of
S. putrefaciens in Pacific white shrimp in different cold chain logistics during transportation, retailing, and domestic storage, which have a negative effect on shrimp [
18,
19]. Our previous research also indicated that
Shewanella and
Aeromonas species were identified the specific spoilage bacteria in the spoiled Pacific white shrimp and that CFS from
A. sobria could inhibit the growth ability and the spoilage abilities of
S. putrefaciens during the refrigerated condition. Therefore, the effect of CFS from
A. sobria on the spoilage of
S. putrefaciens in Pacific white shrimp in different cold chain logistics is a worthy subject to be studied. Until now, the related results have been largely unknown. Our data indicated that the initial bacterial counts of the fresh shrimp samples inoculated by
S. putrefaciens were initially around 4.0 log CFU/g. The inhibitory effect on bacterial growth was temperature-related.
S. putrefaciens counts in group CK1 and SA1 grew slowly, indicating that 0 °C is not suitable for
S. putrefaciens, while CK2 and SA2 grew much faster. Similar studies were also reported that the total counts (TAC) in tilapia fillets increased faster at 10 °C than stored at 0, 4 and 7 °C [
19].
Table 1 shows that
S. putrefaciens counts in group CK3 and SA3 were higher than group CK2 and SA2, respectively (
p < 0.05), which indicated that temperature fluctuation during transportation and retailing was beneficial for the growth of
S. putrefaciens [
18]. The CFS from
A. sobria results in the decline on the growth of
S. putrefaciens after the early stationary phase at refrigerated storage consistent with our previous research results. It was noticeable that bacteria counts were lower in group SA3 treated with CFS from
A. sobria in comparison with that in group CK3, which showed that the CFS also has a role in the condition of temperature fluctuation during storage. Similar studies were also reported that the addition of CFS from
P. fluorescens to
S. baltica culture has caused cell decline during the stationary phase, while had no effect on the growth rate during the lag and exponential phase [
12]. It could be hypothesized that plenty of metabolites secreted by
A. sobria may influence the micro-environment and regulate the production of substance, which might inhibit the growth of
S. putrefaciens.
The acceptability of shrimp to customer is closely related to their sensory evaluation, including color, odor, texture and elasticity of shrimp. The fresh shrimps were fresh sea-weedy odor and firm texture, while it became strong ammonia odor, sulfur odor and soft texture by the end of storage [
30]. The sensory scores for the evaluated attributes of shrimp samples (
Figure 3) all decreased with increasing storage time at different cold chain logistics. These results were consistent with Özogul et al. who found that the odor and texture of cooked fillets reduced significantly during refrigerated storage (
p < 0.05) [
31]. The differences between CK2 and CK3 were not significant, and CK1 seemed to be much better than them. The results indicated that the temperature fluctuation can decrease the sensory quality of shrimp. The sensory quality of group SA3 was significantly different from that of group CK3 (
Table 2). Therefore, CFS from
A. sobria could offset the quality deterioration of shrimp exposed to temperature fluctuation during cold chain logistics. TVB-N is composed of basic nitrogenous substances, such as ammonia and amines, due to the activity of spoilage bacteria and endogenous enzymes. The spoilage activity of
S. putrefaciens can be quantified by determining the accumulation of TVB-N [
22]. Highest TVB-N value after 6 days of storage were found in CK3 and CK2, and lowest value were found in SA1. Therefore, constant temperature and CFS from
A. sobria could inhibit the accumulation of TVB-N. The differences of TVB-N between CK3 and SA3 were less significant than that between CK1 and SA1.
The spoilage potential of
S. putrefaciens also relates to the activity of amino acid decarboxylase, which catalyzes the production of biogenic amine [
11].
S. putrefaciens is chiefly involved in the production of putrescine and cadaverine. Application of CFS from
A. sobria in culturing of
S. putrefaciens produced a reduction in the formation of putrescine and cadaverine during the experimental period. In addition, there was not a significant difference in the contents of cadaverine in group CK2 and SA2 (
Table 5), indicated that the CFS had few effects on the accumulation of cadaverine in shrimps stored at 4 °C. The contents of two biogenic amines in CK3 were higher than CK1 and CK2 and group CK3 was obviously increased on 6 days, due to temperature fluctuation stimulating the growth of bacteria and then accelerating the amino acid decarboxylase. The contents of two biogenic amines in group SA3 were lower than that of group CK3 at the end of the storage. It is concluded that the application of CFS could inhibit the accumulation of biogenic amines in shrimps exposed to temperature fluctuation during cold chain logistics. Arginine and ornithine are the main precursors of putrescine, and lysine is for cadaverine [
13,
32]. The changes between amino acids and biogenic amines showed a strong relationship, as the content of arginine and lysine also decreased significantly during the experimental period. It was assumed that the metabolites of
A. sobria influenced the activities of enzymes in
S. putrefaciens and related to the content of metabolites in cold environment [
7]. The decreasing in the contents of serine, glutamic acid and glycine of Pacific white shrimp may also lead to the reduction of umami characteristics and then affecting its flavor and taste [
33]. The results of the SDS-PAGE profile of myofibrillar proteins of shrimp were consistent with that of TVB-N and biogenic amine. It showed that application of CFS from
A. sobria lead to less protein hydrolysis.