Microbial Community Structure and Diversity of Shrimp Paste at Different Fermentation Stages

High-throughput sequencing was used to reveal the highly diverse bacterial populations in shrimp paste at different fermentation stages. We studied three stages of fermentation and obtained 448,916 reads. Using this approach, we revealed the presence of 30 phyla, 55 classes, 86 orders, 206 families and 695 genera of bacteria in the shrimp paste. Shrimp paste in fermentation metaphase had a more diverse microbiota than that in fermentation prophase and fermentation anaphase. Diversity appeared greatest in fermentation anaphase. The four dominant phyla were Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes. The most common genera were Psychrobacter, Halomonas, Bacillus, Alteribacillus, and Lactococcus. Their content varied at different stages of fermentation. All the microbiome presented a variety of changes in the microbial diversity of shrimp paste. Importance Most research on the microbial diversity of shrimp paste has focused on the shrimp culture environment, or the chemical composition and sensory attributes of the paste. Little research has been conducted on the microbial diversity and composition of shrimp paste. The relationship between microbes and the flavor and quality of shrimp paste has thus been unknown. We therefore analyzed the microbial composition and variation of shrimp paste at different stages of fermentation. The dominant bacteria in fermentation prophase, metaphase, and anaphase were identified. Our preliminary findings give some insight into which microbes contribute to the flavor of shrimp paste and suggest how to improve its flavor. In addition, our findings are relevant to optimizing the production of shrimp paste and guaranteeing its quality and safety.


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Shrimp paste is widely consumed as a condiment and used as an ingredient 33 throughout China and the south-east Asian region (1-2) .It is normally produced by 34 fermenting small shrimp (Acetes vulgaris) with salt at a ratio of 5:1 (shrimp to salt, w/w). 35 The mixture is thoroughly blended or homogenized before being compacted in a 36 container. Shrimp paste is rich in protein, calcium, carotenoids, and chitin (3-4). It 37 exhibits anti-oxidant activity (5), lowers cholesterol and blood pressure, and enhances the 38 body's immune response and other biological activity (6). It thus has great potential as a 39 functional food. Shrimp paste produced using different fermentation technologies has 40 different fermentation cycles. Some pastes are fermented for 3-6 months (7), some for 2 41 months (8) and some for only 1 month, which is traditional in China (9). We took 1 42 month as a study period, and divided it into three fermentation stages: prophase, 43 metaphase, and anaphase, each of which was 10 days long.

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Sequencing and bioinformatic analysis. 64 DNA was extracted from shrimp paste at 11 stages of fermentation. Following total 65 Applied and Environmental Microbiology 4 genomic DNA extraction, amplicons of V3-V4 16S rRNA genes were generated, and 66 448,916 reads were obtained through high-throughput sequencing, corresponding to 67 153,766 reads from fermentation prophase, 169,915 reads from fermentation metaphase 68 and 125,235 reads from fermentation anaphase. Species diversity and richness were 69 calculated for each time point (Table 1) Bacteroidetes were present at the lowest levels (3.09% and 4.89%, respectively). 82 The proportion of Proteobacteria in the fermentation prophase was higher than in 83 metaphase and anaphase. Firmicutes was most abundant in the metaphase stage, and 84 present at the lowest levels in the prophase stage. Bacteroidetes showed a gradually 85 decreasing trend across the three stages. The content of Actinobacteria and 86 Applied and Environmental Microbiology 5 Verrucomicrobia differed little across the three stages. Planctomycetes and Fusobacteria 87 were more abundant in the fermentation prophase than in metaphase or anaphase, and 88 were present at extremely low levels in late fermentation (Table 2). 89 At the genus level, Psychrobacer was the dominant microorganism, accounting for 90 23.78%, followed by Halomonas at 9.46% (Fig. 1). During the whole fermentation 91 process, levels of Psychrobacer decreased (Fig.2), and the proportion of Psychrobacer 92 was greater in fermentation prophase than in metaphase or anaphase (Table 2). 93 Halomonas was not found in Stage 1 to Stage 4, but began to appear in Stage5, and 94 gradually increased and stabilized (Fig. 2). This growth pattern may be related to the salt 95 concentration of the shrimp paste during fermentation. Levels of Bacillus slowly 96 increased from Stage 1 to Stage 3, began to decrease at Stage 4 and then remained 97 essentially unchanged (Fig. 2). In general, Bacillus was most abundant in the 98 pre-fermentation period (Table 2). Alteribacillus began to appear at a low level at Stage 2, 99 gradually increased to Stage 8, then gradually reduced (Fig. 2). Its content was highest in 100 fermentation metaphase than in the other two periods (Table 2). Lactococcus began to 101 reduce after Stage 3 and then remained unchanged. After Stage 9 it decreased 102 significantly (Fig. 2). The proportion of Lactococcus declined across the three 103 fermentation phases (Table 2). Carnobacterium had the highest content mid-fermentation. 104 Marinobacter levels were extremely low in the early stage of fermentation, and increased 105 over time. Salinicoccus,Chromohalobacter,Salimicrobium,Allobacillus,and 106 Tetragenococcus were almost nonexistent at the early stage and began to appear in the 107 Applied and Environmental Microbiology 6 middle and late stage. There was little change in the content of Oceanisphaera, Kocuria,108 Pseudomonas, Pseudoalteromonas, and Aliivibrio in the three stages. The proportions of 109 Tissierella, Photobacterium,Gelidibacter,Pseudorhodobacterm,Moritella,Vibrio,110 Roseovarius, Aequorivita, and Flavobacterium were higher in the early stage of 111 fermentation than in the other stages. Staphylococcus content was highest in fermentation 112 metaphase, followed by anaphase, and lowest in prophase.

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In this study, high-throughput sequencing provided detailed insights into the 120 complex microbiota of shrimp paste at different fermentation stages. We found that there 121 were 30 phyla, 55 classes, 86 orders, 206 families, and 695 genera in the shrimp paste.

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Proteobacteria was dominant, reaching 55.59%, and it was high at the 123 pre-fermentation stage. Proteobacteria is a major group of gram-negative bacteria, which 124 is widespread in humans (24), shrimp (25) and crabs (26)(27). It is the dominant group in 125 coastal areas and aquaculture ponds (28-32). The shrimp used in this study came from the 126 Yellow Sea, where the major bacterial group is Proteobacteria, and therefore the 127 dominance of Proteobacteria in the shrimp paste may be related to its source materials.
128 Applied and Environmental Microbiology 7 The Proteobacteria is the largest group of bacteria, and it includes many pathogenic 129 bacteria, such as E. coli, Salmonella, and Helicobacter pylori (33). Proteobacteria levels 130 in food are mainly related to its freshness, and transport and storage hygiene conditions. 131 Therefore, Proteobacteria may greatly influence the quality of shrimp paste, and it is 132 necessary to strictly control their growth during its fermentation, production, and storage 133 to standardize production.    The main phyla and genera in three fermentation stages of shrimp paste Figure 1 Phylogenetic tree of shrimp paste The pivot points in the graph represent the corresponding Taxonomy records in the NCBI database, the English name is spelled near the pivot point. The larger the abundance of a species is, the larger the circle of the fulcrum is. When a number of samples are plotted simultaneously, the relative abundance of different samples can be expressed in different colors by means of a small pie chart at the branches or nodes.

Figure 2 Assignment of shrimp paste at the genus level
The horizontal axis is the number of each sample, and the longitudinal axis is the relative abundance ratio. The color corresponds to the species name under the taxonomic level, and the width of different color blocks indicates the relative abundance ratio of differential species.

Figure 3 Principal coordinate analysis graphs of shrimp paste
Different colors represent different samples or different group samples in the graph, the higher the similarity between samples, the more likely to be aggregated in the graph.