(Litopenaeus vannamei) stored in acidic electrolyzed water ic

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Omics Technologies Applied to Agriculture and Food

New insights into the changes of proteome and microbiome of shrimp (Litopenaeus vannamei) stored in acidic electrolyzed water ice Li Zhao, Zhaohuan Zhang, Meng Wang, Jiangping Sun, Huan Li, Pradeep Kumar Malakar, Haiquan Liu, Yingjie Pan, and Yong Zhao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00498 • Publication Date (Web): 30 Apr 2018 Downloaded from http://pubs.acs.org on May 2, 2018

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Journal of Agricultural and Food Chemistry

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New insights into the changes of proteome and microbiome of shrimp

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(Litopenaeus vannamei) stored in acidic electrolyzed water ice

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Li Zhao1†, Zhaohuan Zhang1†, Meng Wang1†, Jiangping Sun1, Huan Li1, Pradeep K. Malakar1, Haiquan Liu1,2,3,4, Yingjie Pan1,2,3, Yong Zhao1,2,3*

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1

College of Food Science & Technology, Shanghai Ocean University, Shanghai

201306, China 2

Laboratory of Quality and Safety Risk Assessment for Aquatic Products on

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Storage and Preservation (Shanghai), Ministry of Agriculture, Shanghai 201306,

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China

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Shanghai Engineering Research Center of Aquatic-Product Processing &

Preservation, Shanghai 201306, China 4

Engineering Research Center of Food Thermal-processing Technology,

Shanghai Ocean University, Shanghai 201306, China

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†These authors have contributed equally to this work.

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* Corresponding author.

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Tel./fax: +86 21 6190 0503

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E-mail address: [email protected]

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ACS Paragon Plus Environment


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Abstract:

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Acidic electrolyzed water (AEW) ice is a novel technique for prolonging the

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shelf-life of foods, but there is limited knowledge of its preservation mechanism. A

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proteomics approach and 16S rRNA-based Illumina sequencing were employed to

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investigate the changes of key proteins and bacterial communities in shrimps stored in

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AEW ice and tap water ice (TW ice) for 7 days. Compared with TW ice, AEW ice

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markedly retards the degradation of myofibrillar proteins in shrimps, including

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myosin, actin and tropomyosin. Besides, sarcoplasmatic proteins which participate in

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carbohydrate catabolic process and amino acid metabolism were also influenced.

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Furthermore, the growth of spoilage bacteria which includes the genera

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Psychrobacter, Shewanella and Flavobacterium was significantly inhibited by AEW

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ice, and the inhibition rate at day 7 was 71.6%, 47.8% and 100% respectively

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(p 20.

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To facilitate taxonomic assignment, we adapted a pipeline to identify operational

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taxonomic units (OTUs) at the species level. Briefly, valid reads from the complete

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data set were binned into OTUs using a 97% identity threshold and employing CD8


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HIT33; this was followed by the selection of a representative sequence from each

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OTU31. Taxonomy was inferred by aligning the representative sequences to relevant

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sequences in several databases for each OTU. (RDP database, Greengenes, NCBI

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16SMicrobial database).

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Comparative and statistical analysis of diversity. To compare the relative

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abundance of OTUs, the numbers of valid reads were normalized. Read counts (sum

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of both technical replicates for each sample) were divided by a sample-specific

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scaling factor. Species richness and diversity statistics including, chao1, Simpson, and

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Shannon were calculated using QIIME. The Heatmap is plotted using the heatmap.2

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function in R package with the default parameters.

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Correlation analysis between microbiota changes and proteome changes

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during storage. A scatter diagram and regression line was performed using the

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Statistical Package for Social Sciences (SPSS Version 19.0 Chicago, Illinois, USA) to

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analyze the correlation between microbiota changes and proteome changes. As for

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microbiota, several bacteria related to food spoilage were applied to investigate the

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relationship with proteome. A R2 value more than 0.8 indicate good correlation

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between measurements.

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Results

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Quantitative image analysis of 2-DE gels and identify differently expressed

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spots. Figure 1 (a) shows coomassie brilliant blue-stained 2-DE gels of the muscle

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proteins obtained from AEW ice treatment group and TW ice control group.

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Approximately 600 spots in each gel were detected and most of these spots were of

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molecular weight of 6.5-200 kDa and distributed between pH 4 to 7. Quantitative

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image analysis of three biological replicates of each sample revealed that 90 protein

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spots displayed > 2-fold change (p < 0.05) in expression at day 1 between the two 9



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groups, in which 86 spots were up-regulated with AEW ice treatment. At day 3, there

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were 81 spots were significantly expressed (p < 0.05), all these spots were up

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regulated with AEW ice treatment. At day 5, 59 significantly (p < 0.05) different

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protein spots were detected, where 25 spots were up regulated with AEW ice

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treatment. At day 7, only 6 spots expressed differently (p < 0.05), 4 spots were up

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regulated when shrimp were stored in AEW ice. (Table 2).

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As shown in Figure 1(b), a total of 60 spots showed more than 2.0-fold

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difference in abundance in at least two storage time point in AEW ice when compared

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to TW ice. These spots were excised from 2-DE gels and subjected to in-gel trypsin

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digestion and subsequent MALDI-TOF/TOF identification. Results showed 52 spots

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could be identified, while 8 protein spots were unidentified (identification results and

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quantitative analysis were shown at Table S2).

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Changes in concentration of identified proteins. Among these 52 identified

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spots, 45 spots were mainly classified into three major categories: myosin, actin and

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tropomyosin, and the identification results and GO analysis were shown in Table S1.

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Figure 2 showed the variation of intensity for these proteins from shrimp muscle at

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day 1, 3, 5 and 7, and the fold changes between AEW ice and TW ice treatments of

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these proteins were also compared. We analyzed the protein concentrations using the

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average of intensities of these spots, and the fold change of each spot was shown in

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Table S2.

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Results showed that intensities of three proteins decreased during the period of

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storage. However, these proteins were much more abundant in AEW ice treatment

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group compared with TW ice. More Specifically, for the myosin, the intensity of

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myosin heavy chain type 1 of shrimp stored in AEW ice was 4.83, 3.25 and 2.59 fold

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than in TW ice at day 1, 3 and 5 respectively (p < 0.05). And myosin heavy chain type 10


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2 of shrimp stored in AEW ice was also larger than that treated with TW ice. For the

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actins, both actin 2 and β actin of shrimps was at least 1.5 fold change between AEW

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ice and TW ice treatment (p < 0.05). Additionally, the intensity of tropomyosin which

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was related to cytoskeletal binding were also larger than that treated with TW ice with

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the 3.28, 3.45, 1.29 and 1.41 fold changes at day 1, 3, 5 and 7 (p < 0.05).

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Moreover, sarcoplasmatic proteins which participate in carbohydrate catabolic

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process and amino acid metabolism were also influenced by AEW ice treatment. The

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Triose-phosphate

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biphosphate-aldolase and pyruvate kinase were involved in carbohydrate catabolic

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process (Table S1). As shown in Figure 3, Triose-phosphate isomerase was down-

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regulated when shrimps stored in AEW ice during the first 5 days and increased

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sharply at day 7, while it showed a opposite trend in TW ice. The 2-phosphoglycerate

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dehydratase of shrimp stored in AEW ice showed a rapid decrease in concentration

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from day 1 to day 7, but it remained steady when shrimps stored in TW ice. Besides,

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the abundance of fructose 1,6-biphosphate-aldolase A and pyruvate kinase revealed a

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trend of fluctuations both in AEW ice and TW ice treatment group. Additionally,

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mitochondrial cysteine desulfurase, which was involved in cellular amino acid

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metabolic process (Table S1), showed a reduction in abundance in AEW ice

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treatment group, while its trend was smooth and lightly decrease in TW ice.

isomerase,

2-phosphoglycerate

dehydratase,

fructose

1,6-

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Protein-protein interaction network of the differentially expressed proteins.

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Figure 4 shows the interaction network of differentially expressed proteins. Dots in

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the network represent proteins. The dots in the left part of figure 4 represent

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sarcoplasmatic proteins including various enzyme proteins, and the right dots were

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myofibrillar proteins including myosin heavy chains, actin, tropomyosin etc. Rounded

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rectangles represent biological process or metabolic pathway. The lines between dots 11



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represent different protein-protein interaction modes, and the lines between dots and

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rounded rectangle represent metabolic processes which proteins dot might participate

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in.

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As shown in Figure 4, sarcoplasmatic proteins including fructose 1,6-

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diphosphate aldolase, triose-phosphate isomerase, pyruvate kinase and enolase mainly

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involved in energy metabolism pathway, and myofibrillar proteins including myosin,

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actin and tropomyosin mainly involved in cellular organic system regulation pathway.

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These two categories of pathways were also interacted mainly through the interaction

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between the enolase (abbreviated as eno1b) and beta actin (abbreviated as acta 2).

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That means the changes of sarcoplasmatic proteins and myofibrillar proteins can

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influence each other.

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Microbial community diversity analysis of shrimp stored in AEW ice and

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TW ice. A total of 104644 high quality bacterial V3-V4 Illumina sequences with a

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median value of 10968 ± 3324 per sample were obtained from the two groups after 0,

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1, 3, 5 and 7 days treatment with AEW ice and TW ice. The total number of OTUs at

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97% similarity level was 5027 and significant overlap was found among the

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microbiota of the different samples. The α-diversity, which is measured by the

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Shannon index and Simpson index was generally lower in shrimp under TW ice

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treatment than those under AEW ice treatment. This observation was confirmed by

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the chao1 and observed species index (Table 3).

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Microbial community composition of shrimp stored in AEW ice and TW ice.

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All sequences were classified from the phylum to species using sequence alignment in

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the RDP database, Greengenes and NCBI 16SMicrobial database. At the phylum level

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(shown at Figure 5), Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes

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were abundant in both the AEW ice and TW ice group, but their proportions were 12


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different. Abundance of phylum Bacteroidetes, Actinobacteria and Firmicutes were

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decreased with TW ice treatment while the major phylum Proteobacteria was

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increased. The initial ratio of Proteobacteria in untreated shrimp at day zero was

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54.71%, during the subsequent storage in TW ice, the percentage increased to 95%. In

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AEW ice the ratio of Proteobacteria remained within 55.71%-68.38%. The second

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major phylum Bacteroidetes didn’t show a decrease when shrimp were treated with

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AEW ice and the ratio during 1, 3, 5 and 7 day storage was 17.76%, 23.76%, 31.01%

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and 14.28%. Additionally, Actinobacteria and Verrucomicrobia were significantly

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more abundant in shrimps stored in AEW ice than those of TW ice ( p < 0.01).

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Figure 6 showed the hierarchical heatmap at bacterial genus level. Information

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of sample UNIFRAC clustering and OTU prevalence were easy to be found in this

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figure. Results indicated that 9 samples could be organized into two main branches.

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The first branch was composed of the 6 treatment samples in 2 clusters where 0 day,

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AEW 1 day and TW 1 day formed the 1st cluster and AEW 3 day, AEW 5 day and

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AEW 7 day formed the 2nd cluster. The second branch was composed of three

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treatment samples, TW 3 day, TW 5 day and TW 7 day. Furthermore, there were

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several major genus were found in these samples.

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Table 4 detailed the abundance of these core geniuses in the different sample.

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The microbiota of shrimp stored in AEW ice mainly consisted of Psychrobacter,

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Octadecabacter, Haliea, Tamlana and the unclassified genera belongs to

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Rhodobacteraceae and Flavobacteriaceae. The most abundant genera in TW ice

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treated shrimp included Psychrobacter, Shewanella and the unclassified genus

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belongs to Rhodobacteraceae and Flavobacteriaceae. It’s easy to see that genera

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Psychrobacter (increased from 32.3% to 82.4%) and Shewanella (increased from

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0.1% to 9.2%) growing fast and become dominant bacteria when shrimps were stored 13



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with TW ice. After statistical analysis, the relative abundance of Psychrobacter and

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Shewanella were found to be significantly lower in AEW ice treated shrimps (p

(Litopenaeus vannamei) stored in acidic electrolyzed water ic

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