Bacterial diversity and community level physiological profiling of terasi (Indonesian shrimp paste) to ensure its food safety

Fermentation is a technology to increase the economic value and preserve of seasonal products such as tiny shrimps. This study aimed to ensure the safety and the length of production time of shrimp paste by determining the bacteria involved, during the shrimp paste fermentation process with 10% and 15% salt content. Identification of halophilic bacteria by 16S rRNA used primers 27F and 1492R. Identification of unculturable bacteria used bacterial amplification in the V3-V4 rRNA region. Bacterial activity was observed using the Community Level Physiology Profiling technique. The results showed that the dominant bacteria in shrimp paste with 10% and 15% salt were culturable, i.e., Staphylococcus nepalensis, Salinicoccus qingdaoensis, and Staphylococcus cochnii. In the unculturable identification, the dominant bacteria in shrimp paste with 10% salt were Alkalibacillus, Alkalibacterium, Tetragenococcus, whereas the shrimp paste with 15% salt was dominated by genus of Salimicrobium, Staphylococcus, Corticicoccus, Alkalibacterium and Lentibacillus. The shrimp paste with 10% salt used a high amount of carbohydrate and carboxylic acid substrate. Both 10% and 15% salt of terasi were safe to consume due to no pathogenic bacteria contained. The production of shrimp paste with 10% salt is more profitable due to the low production cost and faster production time.


Introduction
Shrimp paste is one of well-known fermented fish products.Shrimp paste is used as a food additive, flavor enhancer, or as a staple food and as a source of protein [1,2].These products are known as terasi in Indonesia.Shrimp paste is made from planktonic and tiny shrimp which less economic value.Processing krill, planktonic and tiny shrimps to be shrimp paste can increase the economic value and preserve the planktonic and tiny shrimps [1,3].Krill and tiny shrimps such as Acetes and Mesopopdosis was seasonal products [3].Several micro small and medium enterprise produce shrimp paste through ancient knowledge which is transferred from generation to the next generation.The fermentation technology of shrimp paste is easy to applied.Planktonic and tiny shrimps were dried, pound, fermented in closed container, second dried, second pound, packaged and sold [4].However, there is a little knowledge about how to produce good and safe quality of shrimp paste.The good quality of shrimp paste will influence the price of shrimp paste.Salt is one of factors that determines the safety and quality of shrimp paste.Salt affects diversity of bacteria and metabolites in shrimp paste [5], as well as the length of fermentation [6,7].Diversity of bacteria and their metabolites influence the safety, aroma, taste and consumer's preference.
There were differences in microbial dominance at the beginning of fermentation compared to the one in the middle and at the end of fermentation.At the beginning in shrimp sauce, the dominant bacteria are Vibrio, Photobacterium, Psychrobacter, Pseudoalteromonas and Enterovibriogenus [5].These microbes are pathogenic, but their abundance decreases along with the length of fermentation.Pathogenic and spoilage microbes in fermented food determine the unsafety of fermented food to consume.The bacteria found in Indonesia shrimp paste consist of Bacillus, Pseudomonas, Micrococcus, Kurthia, Sporolactobacillus [8], Tetragenococcus halophilius and T.muriaticus [9].These bacteria were culturable.However, only some bacteria can be cultured so that the data on microbial diversity in fermented foods are often biased and not reflecting actual circumstance [10].Hence, advanced approaches are needed to study bacterial diversity, including metagenomics [11,12].The diversity of shrimp paste bacteria obtained metagenomically by using high-throughput sequencing had been studied on shrimp paste made from Acetes chinensis in China [7], kapi in Thailand [6], Huanghua shrimp paste in China [2], and low salt Indonesian shrimp paste with 5% salt concentration [13].
One of the methods to determine the role of the bacterial community is through physiological analysis of bacteria on the use of substrates such as the BIOLOG Ecoplate [14,15].Substrate utilization can infer the substrate availability in fermented food.The aim of this study was to determine the bacterial diversity during the shrimp paste fermentation, both through culturable and unculturable method by High Throughput Sequencing.Moreover, the role of the bacterial community was observed through the substrate utilization on the BIOLOG Ecoplate.This research was expected to contribute to the respective industry on the safety and the quality of shrimp paste made with 10% and 15% salt concentration.

Preparation of low-salt shrimp paste (terasi)
Shrimps Acetes japonicus were sun dried for 4 hours.After cooled down, salt was added to the semidried shrimps.A total amount of 787.5 grams of shrimps was mixed with 87.5 grams of salt to obtain shrimp paste with a salt concentration of 10%.For the shrimp paste with a salt content of 15%, a total amount of 743.75 grams of shrimps was mixed with 131.25 grams of salt.Each shrimp paste was pounded and then fermented (as the early stage of the fermentation) for 48 hours at a temperature of 29.5ºC, before being sun dried again for 4 hours.After the drying process, the shrimp paste was remashed, packaged, and fermented at an advanced stage for 28 days.Sampling was carried out every week so that samples were obtained on day 0, 7, 14, 21, and 28.Ten grams each of shrimp paste from 3 replications were taken, mixed, and used as a test sample.

pH and salt concentration during fermentation
The pH testing was carried out by mixing 5g of crushed shrimp paste with 10 mL of distilled water and measured using a pH meter (Ohauss ST 2100-F, USA).The salt concentration test was carried out with the Mohr method [16].

Bacterial enumeration and identification during fermentation
A total amount of 1 gram of shrimp paste was weighed and dissolved with 9 mL of Phosphate Buffer Saline (PBS) solution.Serial dilutions were carried out, and each 100 µL of it was spread evenly using a spreading rod (glass rod L) on Salt Marine Agar medium.The culture was incubated at room temperature for 14 days [17].The colonies were counted in the range of 30-300 colonies.Distinguishable bacterial colonies were further observed for phenotypic characteristics (color, shape, elevation) and then purified for molecular testing with 16S rRNA.Molecular identification was carried out by identifying the sequences on 16S rRNA.DNA isolation was performed using a DNA Miniprep kit (ZymoResearch, USA).A total of 5 µL of DNA was used as a template for DNA amplification, polymerase enzyme using GoTaq PCR mix, and primers used for amplification were primers 27F and 1492R (Macrogen Lab., Korea).PCR conditions were carried out with predenaturation of 95ºC for 2 minutes, denaturation of 96ºC for 30 seconds, and elongation of 72ºC for 1 minute.These stages were carried out for 30 cycles.Final extension was done at on 72ºC for 5 min.Next, PCR product samples were sequenced using the sequencing services of Macrogen Inc. (Seoul, Korea).The obtained sequence results were compared to the database using Basic Local Alignment Search Tool (BLAST) on GenBank (National Centre for Biotechnology Information, NCBI), and the Ribosomal Database Project (RDP).

Unculturable bacteria
DNA extraction was carried out using DNA MiniPrep (Zymogen USA).The extracted metagenomic DNA was used as template for PCR amplification following the method described in Qubit 2.0 (Life USA).The primer set contained the appropriate Illumina adapters, with the reverse primer containing a unique error-correcting barcode for each sample.The PCR reaction included two rounds of amplification as described by [7].Before sequencing, the DNA concentration of each PCR product was determined using a Qubit 2.0 kit and its quality was controlled using a bioanalyzer (Agilent, USA) PCR product samples were sequenced using the sequencing services of Macrogen Inc. (Seoul, Korea).A sequencing library was prepared using Herculase II Fusion DNA Plymerase Nextera XT Index Kit V2 (Agilent Technologies, Inc), following the manufacturer's recommendations.Library quality was assessed by the Qubit 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system using a DNA 1000 chip.Prepared libraries were quantified using qPCR according to the Illumina qPCR Quantification Protocol Guide.The library was sequenced on an Illumina MiSeq platform (Illumina, San Diego, CA, USA) according to the manufacturer's instructions.The data analysis consisted of three steps: pre-processing and clustering, taxonomy assignment, and diversity statistics.Ambiguous reads were filtered out, and the extra-long tail was trimmed.Short reads were assembled using the FLASH program.Chimeras were identified and removed using rDNA tools PacBio and reference file (RDP) http://www.mothur.org/wiki/RDP_reference_file.QIIME-UCLUT/RDP and QIIME were used for taxonomy assignment (16S).

Community level physiology profilling
120 µL of shrimp paste suspension at a dilution of 10 -3 was added to the wells in the BIOLOG TM Ecoplate.Furthermore, the Ecoplate was incubated at a room temperature of 28ºC in the dark for 144 hours.Each Ecoplate was divided into 3 identical regions for replication.The substrates used in Ecoplate consisted of 7 carbohydrates, 8 carboxylic acids, 6 amino acids, 2 amines, 2 phenols, 4 polymers, and 2 carbon phosphates.The absorbance before and in every 12 hours of incubation was observed using a Microplate Reader at 590 and 750 nm wavelengths.The absorbance value of each carbon source was corrected by subtracting the absorbance value from the control well.The negative value was calculated as zero.The relative use of a carbon source (pi) was calculated as the ratio between the corrected absorbance value in each well and the total absorbance of one Ecoplate.

Result and discussion
The pH of shrimp pastes with a salt content of 10% and 15% tended to be neutral with a range of 7.67-7.76for shrimp paste with a salt content of 10% and 7.77-7.85for shrimp paste with a salt content of  1).The pH of shrimp paste with a salt content of 10% and 15% tended to be neutral due to the formation of alkaline compounds such as ammonia or other protein degradation products [18,19,20].The shrimp paste with 10% salt contained higher organic acids, causing a lower pH.According to [20], decreased pH in shrimp paste caused by accumulation of organic acids due to metabolism of lactic acid bacteria.The salt concentration of the shrimp paste tended to decrease until 14 days of fermentation with both 10% and 15% salt and increased again on the 21st day (Table 1).This decrease could be due to the dissociation of NaCl into Na + and Cl -ions.Both of these ions will be absorbed and used for bacterial growth.The increase in salt concentration can also be caused by reduced water concentration due to evaporation [19].
The fermentation of shrimp paste involved a group of halophilic bacteria [21].Both shrimp paste with 10% and 15% salt had no different total number of halophilic bacteria (Table 1).Halophilic bacteria of the shrimp paste on the same salt concentration tended to show the decrease of the number of the microbes along with the length of fermentation time.The growth of bacteria in a fermentation without the addition of new substrates caused the exhaustion of nutrients and toxic metabolites were produced so that the total bacteria number decreased at the end of the shrimp paste fermentation process.
Both of the shrimp paste with 10% and 15% salt in this study were dominated by bacteria from the Staphylococcus and Salinicoccus (Figure 1a).[17] reported on the fish fermentation process that at 14 to 28 days, the fermentation was dominated by the Staphylococcus.There were two species in the Staphylococcus genus in shrimp paste with 10% and 15% salt, i.e., S. nepalensis and S. cochnii.Staphylococcus is a bacterium found in fish sauce, as reported by [22] who stated that groups of S. saprophyticus and S. nepalensis have been isolated from several fish sauces.The bacteria Salinicoccus siamiensis found in shrimp paste is also found in Kapi [23].Salinococcus qingdaonensis is a moderately halophilic, non-motile, heterotrophic bacterium that does not form spores [24].The Salinicoccus genus is usually found in salty environments such as salt mines, salted fish, and fermented seafood [23].Bacillus is also a bacterium found in shrimp paste [25].Population dynamics of culturable bacteria occurred in shrimp paste with 10 and 15% salt (Figure 1a).In this study, Salinicoccus siamensis grew optimally in 10% salt (Figure 1a).In line with [23] stated that Salinicoccus siamensis grew optimally in 10% salt kapi (fermented shrimp paste from Thailand).
The differences in bacteria between the culturable and unculturable methods can be caused by the growth conditions including the media that is not suitable for certain bacteria as most bacteria cannot be cultivated in specific nutrient media [20].The shrimp paste with 15% salt was dominated by the order Bacilalles during fermentation (Figure 1b).The shrimp paste with 10% salt was dominated by the order Bacillales at 0,7 and day 21 of fermentation.However, it was dominated by the order Lactobacillales at day 14 and 28 of fermentation (figure 1b).In line with the dynamic of pH in 14 days of fermentation decreased due to the activity of Lactobacillales in liberation of organic acid.In shrimp paste with 10% salt, Tetragenococcus and Alkalibacterium had higher relative abundance compared to 15% salt shrimp paste (Figure 1c).Both of these bacteria that were included in the category of lactic acid bacteria caused the pH of the shrimp paste with 10% salt to be lower than 15%.Some studies of fermented food showed that lactic acid bacteria decrease while salt concentration increase [26].Due to lactic acid bacteria abundance in shrimp paste with 10% salt concentration, it is considered that it is better to produce shrimp paste with 10% salt concentration.Lactic acid bacteria is well known as Generally Recognized As Safe (GRAS) [27,28].The shrimp paste with 15% salt had higher Salimicrobium and Corticicoccus compared to 10% salt (Figure 1c).The shrimp paste with 15% salt was dominated by genus of Salimicrobium, Staphylococcus, and Corticicoccus at the beginning of fermentation.The bacteria at the end of the fermentation were dominated by Alkalibacterium and Lentibacillus.

d.
All dominant bacterial genera found in this study have been reported to be present in shrimp paste or shrimp sauce.Tetragenococcus, Staphylococcus were reported as the predominant bacteria of shrimp paste in China [2][7] [20].On the other hand, Lentibacillus, Salinococcus, Salimicrobium, Alkalibacterium, and Staphylococcus are the dominant bacteria in kapi [6].The Alkalibacillus genus is also found in shrimp sauce (saeu-jeout) and fish sauce (myeokchi-aeukjot) [29,20].Alloiococcus has been reported as the dominant bacterium in kapi from the Rayong and Ranong areas in Thailand [6].Atopostipes was found in shrimp paste in China [19].Corticicoccus is a genus that has not been reported in previous studies on shrimp paste or shrimp sauce.However, this genus is closely related to Salinicoccus [2].Salinicoccus is one of the dominant genera of kapi in Thailand [6].Several studies on shrimp paste or shrimp sauce revealed that there were differences in bacteria that affect the metabolites or characters of shrimp paste, shrimp sauce or other fermented food produced [31] [32].The bacterial community at 10% salt concentration used more and higher substrates than shrimp paste with 15% salt concentration (Figure 1d).The shrimp paste with 10% salt concentration had the ability to use higher polymers, carbohydrates, and carboxylic acids.The catabolic activity on these substrates caused shrimp paste with 10% salt to have a more acidic pH than shrimp paste with 15% salt.
In shrimp paste with 10% salt, the bacterial community used a polymer substrate which was higher at the beginning of the fermentation (Figure 2).At 7,14,21 days of fermentation, the bacterial community used more carbohydrates and at the end of the 21 days fermentation, it expressed the higher the use of carboxylic acid.It showed that shrimp paste with 10% salt contained higher carbohydrate.Carbohydrate degraded to be carboxylic acid, so the use of carboxylic acid increased in the end of fermentation.Carboxylic acids are known as preservative compound in food.
In the shrimp paste with 15% salt, at 0,7,14 days, bacterial communities used carbohydrate substrates, and at the end of fermentation (21 days and 28 days), they used higher amino acids.It means that, in the shrimp paste with 15% salt, protein degradation occurred at the end fermentation and synthesize amino acids.The amino acid such as glutamic acid is known as umami flavor enhancer [33].The fermentation process changed the substrate usage profile by the bacterial community.The type and availability of carbon are one of the factors in the environment that has been promoted to contribute to the diversity of bacterial communities, with only certain bacteria using certain carbon sources [34].
The use of substrates by bacteria is closely related to the availability of substrates in their natural environment.Shrimp paste with 10% salt contained bacteria consumed amino acids higher than 15% (Figure 2).It showed that shrimp paste with 10% salt contained higher amino acids.The highest of glycyl-L glutamic acid utilization of bacteria in shrimp paste with 15% salt concentration (Figure 2) showed that glutamic acid was abundance in this time.As an agricultural product, the production of shrimp paste with salt 10% is more profitable.Apart from using less salt, shrimp paste with 10% salt also produces products that can be sold more quickly, while shrimp paste with 15% salt requires 21 to 28 days of fermentation to produce delicious shrimp paste.Shrimp paste is consumed all over the world, especially in Asia, so it is necessary to determine the quality of shrimp paste which is good for international needs.Nowadays, public awareness of healthy and low salt food is increasing to avoid health risks such as hypertension, so the use of products with low salt is preferred.Small and medium businesses in Indonesia can use 10% salt with a fermentation to get safe, sweeter, and rich amino acid of shrimp paste.

Conclusions
Salt concentration and fermentation time affect the diversity of bacteria.Shrimp paste with 10% salt contained higher lactic acid bacteria and was dominated by Tetragenococcus and Alkalibacterium genus, while shrimp paste with 15% salt was dominated by Salimicrobium and Corticicoccus genus at the beginning of fermentation and ended by Lentibacillus and Alkalibacillus.This difference also caused differences in the use of the affected substrate profile.The bacterial community in shrimp paste with 10% salt used many and higher substrates.The bacterial composition in shrimp paste with 10% salt correlated with polymer substrates, carbohydrates, and carboxylic acids.Since both 10% and 15% salt concentration did not contain pathogenic bacteria, these shrimp pastes were safe to consume.Since 10% salt concentration contained more lactic acid bacteria, it is considered that it is safer to produce shrimp paste with 10% salt concentration.Shrimp paste with 15% salt concentration contained high glutamic acid at the end of fermentation.It is better to ferment shrimp paste with 15% salt concentration until 21 or 28 days of fermentation due to the umami taste.The production of shrimp paste with 10% salt is more profitable because it reduces production costs due to the use of less salt and faster production time.

Figure 2 .
Figure 2. Heatmap the difference substrate's utilization of shrimp paste with 10% salt concentration and 15% salt concentration

Table 1 .
pH, salinity and total halophilic bacteria of shrimp paste with 10% and 15% salt content Data were represented in the table average of three replicates