Title Indonesian Journal of Environmental Management and Sustainability e-ISSN:2598-6279 p-ISSN:2598-6260 Research Paper Potential Application of Consortium Microbe from Sea Cucumber Intestinal Symbiont as Preservatives for Vaname Shrimp Marwa Irfan Hanif1, Delianis Pringgenies1*, Gunawan Widi Santosa1, Ervia Yudiati1, Ali Djunaedi1, Koesoemadj1, 1Department of Marine Science, Faculty of Fisheries and Marine Science, Diponegoro University, Semarang, Indonesia *Corresponding author e-mail: pringgenies@yahoo.com Abstract Vaname shrimp is one of the most profitable export commodities in Indonesia. However, the shrimp meat undergoes rapid quality degradation when shipped without any preservatives. This is an issue since the preservatives commonly found on the market are formalin-based. This study aims to discover a natural preservative solution by utilizing microbes. The objective of the study is to discover natural preservatives made of symbiont microbe in sea cucumber’s intestinal organ by antimicrobial activity screening. Sea cucumber samples were collected from Bandengan waters of Jepara. There are 3 symbiotic microbe form intestinal cucumber that can inhibit the growth from Bacillus cereus and Pseudomonas aeruginosa. These three microbes are Listeria sp., Staphylococcus sp., and Rothia sp. Consequently, tested microbial samples were prepared into a consortium microbe and were tested further as a preservative agent for shrimp with a positive control parameter (cooling). The observations conducted in this study include organoleptic properties, acidity, total colony, proximate test (protein, water, ash, fat, and carbohydrate contents), and Total Volatile Base-Nitrogen (TVBN). The results found 3 active isolates are synergic one to each other as a bacterial consortium. Acidity test of sample extract treatment measured a pH of 7.44, compared to the non-treatment result of 7.14. Organoleptic test results of the shrimp indicated that the shrimp was acceptable for consumption. Proximate test of the treatment did not show a significant difference compared to the positive control treatment. Total colony and TVBN test on treated samples resulted in 48 x 105 CFU/ml and 39.62 mgN% respectively, whereas a similar test on non-treatment sample showed 119 x 105 CFU/ml and 45.31 mgN% respectively. It was concluded that the extract of sea cucumber symbiont microbe consortium showed potency in preventing meat quality degradation in shrimp, although treatment by freezing still produces a better result. Keywords Microbial Symbiont, Preservative, Sea Cucumber Received: 14 Agustus 2019, Accepted: 27 September 2019 https://doi.org/10.26554/ijems.2019.3.3.106-111 1. INTRODUCTION Food decay is known as food degradation in normal storage (Buckle et al., 2007). According toBarus et al. (2017), one factor in marine food decay was caused by bacteria with the growth of bacteria will cause physical and chemical changes of the food. Ma’ruf (2012), stated that microorganism such as P. aeruginosa dan B. cereus had cause fish and other marine food. In this case, one of the important export commodity in Indonesia with the risk of decay is a product of vanname shrimp (Wijayanto et al., 2017). Dealing with preservation in marine food in general such as frozen, smoking, radiation or food preservative additives. But this food preservation (radiation and frozen) will have a high operational cause. So that people will prefer to use low-cost synthetic preservative such as formalin (Goon, 2014). The use of formalin will have a very bad effect on a human. Formalin will cause irritation and inflammation in the mouth and esophagus, chest or stomach pain and in most savior condition is kidney failure. Therefore the option use of natural food preservation will be needed (Yulisa et al., 2014). The marine microbe is one of potential resource for bioactive component commercialization. Microorganism with association with a marine organism such sea cucumber will be able to synthesize secondary metabolite in the host organism (Pringgenies, 2010). According to Ma’ruf et al. (2014), sea cucumber genus of Holothuria had been proven as potential antibacterial ability from its bioactive component. Antibacterial from Sea cucumber had been effectively had proven as an antibacterial compound for B. cereus and P. aeruginosa. Three symbiotic bacteria from intestine https://doi.org/10.26554/ijems.2019.3.3.106-111 Hanif et. al. Indonesian Journal of Environmental Management and Sustainability, 3 (2019) 106-111 of sea cucumber that are Listeria sp., Staphylococcus sp., and Rothia sp. had antibacterial activity against these two contaminant microbes Pringgenies et al. (2018). A consortium of bacteria is a community that had a more significant effect than a single isolate (Asri and Zulaika, 2016). Symbiotic bacteria consortium from the intestine of sea cucumber has the antibacterial capability for B. cereus and which will inhibit microbial contaminant which can be used as a natural preservative. 2. EXPERIMENTAL SECTION 2.1 Material The material used in the research was the three isolate of bacteria symbion, vanname shrimp (Liptopenaeus vannamei) for experiment test, Zobell 2216 E medium, and some other chemical reagent for proximate and TVBN test. equipment used for the research is oven, furnace, Kjeldahl flask, soxhlet, incubator, Conway disk, autoclave, and laminar airflow. 2.2 Synergistic test among symbiotic bacteria Listeria sp., Staphylococcus sp., and Rothia sp. from the intestine of sea cucumber tested for the synergistic test. One ose symbiotic bacteria isolate had been applied on top of each other in the Zobell 2216 E medium. The sample was incubated for 24 hours and observed. If there is no inhibition zone, the isolate was synergistic one to each other in the consortium (Asri and Zulaika, 2016). 2.3 Isolation of Secondary Metabolite Isolate of symbiotic bacteria consortium was grown as a starter on 10 ml liquid Zobell medium in 15 ml reaction tube and shook in 120 rpm speed for 3-5 days until its reach stationary phase. Isolate the moved into 40 ml liquid Zobell media the shook for 3-5 days with 120 rpm speed. Culture of bacteria move into 50 ml centrifuge tube and centrifuge with 2800 G-force speed for 15 minutes to produce supernatant (Rau et al., 2008). 2.4 Preservation test of Bacterial Consortium Su- pernatant in Vanname Shrimp The supernatant extract of the bacterial consortium was tested on vanname shrimp (L. vannamei ). Preservation test divides into five control that is cooling (ES), bacterial supernatant (3C), sterile seawater (AS), sterile medium (MS), and without treatment (N). Vanname shrimp shocked with all treatment for 10 hours (Putra and Mirdhayati, 2009). 2.4.1 Tested Parameters 1 pH (Santoso, 2017). 2 Total Plate Count (Sitakar et al., 2016). 3 Organoleptic (SNI 01-2728.1-2006) 4 Proximate: Water (SNI 01-2354.2-2006) Ash (SNI 23541-2010) Fat (SNI 01-2354.3-2006) Protein (SNI 01-2354.4-2006). Karbohidrat Atma (2018). 5 Total Volatile Base-Nitrogen (TVBN) (SNI 01-2354.8- 2009). 3. RESULTS AND DISCUSSION 3.1 Synergistic test Among Bacteria Result of the symbiotic bacterial synergistic test showed no inhibition zone formed. Meaning that the three symbiotic bacteria isolate were synergistic one to each other as a bacterial consortium. Synergic bacteria to form a consortium will give optimum result than a single isolate of bacteria. The factor of bacteria isolates to form synergism to one another still unknown. Some factor could effect of synergism are 1. synergism in the nutrition synthesis, 2. Synergism among bacteria in undegraded material by other bacteria, 3. One type of bacteria has the ability to inhibit toxic compound to other bacteria (Asri and Zulaika, 2016). Figure 1. Synergistic test Result 3.2 pH Test on Shrimp Preservation Value of some pH test in the shrimp preservation is on table 1. Based on pH test in shrimp preservation range from 6,46 – 7,77 where according to Purwa et al. (2012), pH ranged from 6,5 – 7,5 as a suitable pH for contaminant bacteria. pH value on Treatment on shrimp with extract was 7,44 while on positive control is 7,42. Table 1. pH Test Result No. Treatment pH 1 ES 7,42 ± 0,01 2 3C 7,44 ± 0,01 3 AS 7,10 ± 0,01 4 MS 6,46 ± 0.00 5 N 7,14 ± 0,01 According to Harsojo (2008), good pH value for food should be lower than 5,3 where the possibility of bacteria contaminant will be minimum. In reality value of pH in the shrimp product in the market range from 6,6 – 7,5. © 2019 The Authors. Page 107 of 111 Hanif et. al. Indonesian Journal of Environmental Management and Sustainability, 3 (2019) 106-111 Meaning that is neutral and nonacid for human, but it is a good condition for bacteria contaminant. 3.3 Total Colony Test of Contaminant Bacteria (TPC) Result of total colony count in shrimp preservation as in table 2. Table 2. Total Bacteria Count No. Treatment Bacteria Colony Value (CFU/ml) 1 ES 19 x 105 2 3C 48 x 105 3 AS 114 x 105 4 MS 82 x 105 5 N 119 x 105 Result of TPC in positive control (cooling) has value 19 x 105 CFU/ml, while in the supernatant value is 48 x 105 CFU/ml. This two value is are much lower than shrimp without any treatment that is 119 x 105 CFU/ml. Result of TPC of all treatment is above the standard of 5 x 105 CFU/ml. But, according to Purwa et al. (2012) based on Directorate General of Food and Drug. Value of TPC in food product should be about 50 x 105 CFU/ml and above this number is not suitable for consumption. Based on result shrimp with treatment in ES and 3C is still suitable for consumption. Value of TPC is affected by the initial bacteria amount (Sukmawati and Hardianti, 2018). According to Harsojo (2008), the value of bacterial contaminant in the shrimp product in the market is 10 – 58 x 105. Value of negative control indicates that sterile media and sterile seawater will not effect on bacteria supernatant extract. 3.4 Organoleptic Test on Shrimp Preservation Result of organoleptic test based on 10 respondent as in table 3. Organoleptic tests were carried out by 10 respondents with a range of values 1-9. The evaluation points observed were shrimp appearance, shrimp smell, and shrimp texture. The highest organoleptic value was obtained by ES shrimp and the lowest was by shrimp N (without treatment). Based on SNI 01-2728.1-2006 if the average organoleptic value is must be lower than 7, if the organoleptic value of the shrimp under that value, the shrimp is not suitable for consumption. Untreated shrimp (N) has a value of 5.70. So, based on the quality standards the shrimp is not suitable for consumption. While the organoleptic value of shrimp with other treatments is more than 7. 3.5 Water and Protein Content Test The results of the water and protein content in the shrimp product shows as in Table 4. The highest value of water con- tent is found in N shrimp with a value of 78.73% where this value is directly proportional to bacterial colonies number in Table 3. Organoleptic Test Result No. Treatment Organoleptic Value 1 ES 8,53 2 3C 7,77 3 AS 7,37 4 MS 7,43 5 N 5,70 shrimp N which is as much as 119 x 105 CFU / ml. The ex- cess water content in the material will cause the material to be easily overgrown by contaminant bacteria. This is similar to Bawinto et al. (2015) the content of excess water content in a material can cause food to become rapidly damaged because it is used as a microorganisms growth’s medium. Shrimp with extracts or 3C shrimp have an average wa- ter content value so that bacterial growth in shrimp 3C is minimum. However, the water content in the ES positive control was noted to be high, this was probably due to the ice melting and seeping into the shrimp meat. The lowest value of water content is found in AS shrimp where it is estimated that immersion in sterile seawater will cause the liquid in the shrimp to come out to a higher concentration. The value of protein content will also affect the number of contaminant bacteria that grow and cause the decay path faster. The highest value of protein content is found in AS shrimp. High levels of protein result in reduced water con- tent in shrimp, this may also be a factor why the AS water content is the lowest. According to Yuarni et al. (2015), the high protein content will result in increased bacterial growth because protein is an important nutrient for bacteria. So this factor is what causes the total value of colonies in AS shrimp to be very high. The value of protein content in 3C shrimp is not much different from the value in the positive control ES shrimp where the value of both includes the average value. Overall the value of shrimp protein levels still meets the quality standard of more than 15%. Table 4. Add caption No. Treatment Water (%) Protein (%) 1 ES 78,15 18,69 2 3C 77,77 18,45 3 AS 76,57 19,41 4 MS 78,27 17,26 5 N 78,73 17,78 3.6 Fat and Ash Content Test The value of fat content and an ash content of shrimp in each treatment as in Table 5. Fat content in shrimp samples from three samples of ES, 3C, and AS shrimp still showed values that were suitable with existing quality standards. © 2019 The Authors. Page 108 of 111 Hanif et. al. Indonesian Journal of Environmental Management and Sustainability, 3 (2019) 106-111 According to Fendjalang (2018), the fat content in fishery products ranges from 1.00 - 1.40%. The value of fat content in MS and N shrimp is less than 1.00%, this is probably due to the high water content in these two shrimp. Khasrad and Arfal (2016) mention that the low level of fat in food caused by high water content. Contaminant bacteria will also play a role in changing the conditions of fat in a substance. The value of ash content obtained in this study shows that the ash content in MS shrimp samples exceeds the existing quality standard. The value of ash content in fishery products must be lower than 2%. According to Erni et al. (2018), high ash content can also cause by the high temperature and duration of drying in a test material. Table 5. Fat and Ash Content Test Result No. Treatment Fat (%) Ash (%) 1 ES 1,05 0,91 2 3C 1,15 1,49 3 AS 1,10 1,67 4 MS 0,86 2,44 5 N 0,94 1,16 Table 6. Carbohydrate Content Calculation Result No. Treatment Carbohydrate (%) 1 ES 1,20 2 3C 1,17 3 AS 1,25 4 MS 1,17 5 N 1,39 3.7 Carbohydrate Content Calculation Carbohydrate content was calculated using the by differ- ence method as in Table 6. Calculation by difference is a gross calculation that subtracts 100% with the value of other chemical content so that crude fiber will be included in the counting (Suptijah, 2012). The highest carbohydrate value is in the N sample then AS, while the ES, 3C, and MS samples are lower than that number. High carbohydrate values can be an indicator of the high growth of contaminant bacteria. According to Andarti and Wardani (2015), carbo- hydrates are a source of energy for the body and a simple form of carbohydrates can be used as an energy source for contaminant bacteria growth. 3.8 TVBN (Total Volatile Base-Nitrogen) Test Result of TVBN in each treatment shows as in table 7. The results of TVBN values in shrimp are directly proportional to the total value of colonies of contaminant bacteria in shrimp as in figure 2. TVBN or Total Volatile Base-Nitrogen is a measurement of volatile material in a substance (Tapotubun Figure 2. Comparison Between TVBN and Total Colony Table 7. TVBN Test Result No. Treatment Nilai TVB-N (mgN%) 1 ES 36,30 2 3C 39,32 3 AS 44,49 4 MS 43,95 5 N 45,31 and Laouhenapessy, 2008). As the graph in figure 2, the more total contaminant bacterial colonies will increase the value of TVBN in the sample. This was since contaminant bacteria will accelerate the formation of enzymes that func- tion as protein degradation. Farahita et al. (2012) opine that the process of TVB occurs due to the work of prote- olytic enzymes that break protein bonds and the result in this degradation will produce a number of volatile bases such as ammonia, H2S, and foul-smelling trimethylamine. Enzymes roles in degradation down proteins are produced by bacterial activity. 4. CONCLUSIONS Three isolates of symbiotic bacteria from sea cucumber Lister sp., Staphylococcus sp., and Rothia sp. have synergic one to another. Supernatant Extract of the three bacterial consortia has a significant potential for shrimp product preservation compared to nontreatment. Result of positive control using cooling treatment still give a better result. REFERENCES Andarti, I. Y. and A. K. Wardani (2015). Pengaruh Lama Fermentasi Terhadap Karakteristik Kimia, Mikrobiologi, dan Organoleptik Miso Kedelai Hitam (Glycine max (L)). Jurnal Pangan dan Agroindustri, 3(3); 889–898 Asri, A. C. and E. Zulaika (2016). Sinergisme Antar Isolat Azobacter yang Dikonsorsiumkan. Jurnal Sains dan Seni, 5(2); 57–59 © 2019 The Authors. Page 109 of 111 Hanif et. al. Indonesian Journal of Environmental Management and Sustainability, 3 (2019) 106-111 Atma, Y. (2018). Prinsip Analisis Komponen Pangan Makro & Mikro Nutrien. Deepublish. Sleman; 126 Barus, J. G., P. E. Santosa, and D. Septinova (2017). Pen- garuh Lama Perendaman dengan Menggunakan Larutan Daun Salam (Szygium Polyanthum) sebagai Pengawet Ter- hadap Total Plate Count dan Salmonella Daging Broiler. Jurnal Riset dan Inovasi Peternakan, 1(3); 42–47 Bawinto, A. S., E. Mongli, and B. E. Kaseger (2015). Anal- isa Kadar Air, pH, Organoleptik, dan Kapang pada Pro- duk Ikan Tuna (Thunnus sp.) Asap, di Kelurahan Girian Bawah, Kota Bitung, Sulawesi Utara. Jurnal Media Teknologi Hasil Perikanan, 3(2); 55–65 BSN (2006a). Cara Uji Kimia – Bagian 2: Penentuan Kadar Air pada Produk Perikanan (SNI 01-2345.2-2006) BSN (2006b). Cara Uji Kimia – Bagian 3: Penentuan Kadar Lemak Total pada Produk Perikanan (SNI 01-2354.3- 2006) BSN (2006c). Cara Uji Kimia – Bagian 4: Penentuan Kadar Protein dengan Metode Total Nitrogen pada Pro- duk Perikanan (SNI 01-2354.4-2006) BSN (2006d). Udang Segar – Bagian 1: Spesifikasi (SNI 01-2728.1-2006) BSN (2009). Cara Uji Kimia – Bagian 1: Penentuan Kadar Abu dan Abu tak Larut dalam Asam pada Produk Perikanan (SNI 01-2354.1-2010) BSN (2010). Penentuan Kadar Total Volatil Base Nitrogen (TVB-N) dan Trimetil Amin Nitrogen (SNI 01-2354.8- 2009) Buckle, K. A., R. E. Edwards, G. H. Fleet, and W. M (2007). Ilmu Pangan. UI Press. Jakarta Erni, N., Kadirman, and R. Fadilah (2018). Pengaruh Suhu dan Lama Pengeringan Terhadap Sifat Kimia dan Organoleptik Tepung Umbi Talas (Colocasia esculenta). Jurnal Pendidikan Teknologi Pertanian, 4; 95–105 Farahita, Y., Junianto, and N. Kurniawati (2012). Karakter- istik Kimia Caviar Nilem dalam Perendaman Campuran Larutan Asam Asetat dengan Larutan Garam Selama Penyimpanan Suhu Dingin. Jurnal Perikanan dan Kelau- tan, 3(4); 165–170 Fendjalang, S. N. M. (2018). Analisis Kimia Ikan Tuna Asap pada Beberapa Pasar Tradisional di Tobelo, Kabupaten Halmahera Utara. In Prosiding Seminar Nasional KSP2K, volume 1. pages 174–178 Goon, B. M. S. I. M. S., S. (2014). Fish Marketing Status with Formalin Treatment in Bangladesh. International Journal of Public Health Science, 3(2); 95–100 Harsojo (2008). Kualitas Udang yang Dijual di Pasar Jakarta Selatan dari Aspek Mikrobiologi. Berkas Penelitian Hay- ati Khasrad, S., Anwar and R. Arfal (2016). Perbandingan Kualitas Kimia (Kadar Air, Kadar Protein, Kadar Lemak) Otot Biceps Femoris pada Beberapa. Bangsa Sapi. In Sem- inar Nasional Hasil Penelitian dan Pengabdian Kepada Masyarakat di Bali. pages 1–6 Ma’ruf, N. S. T. A., W. F. (2012). Uji Bioaktifitas Ek- strak Teripang Pasir (Holothuria scabra) Terhadap Bak- teri Pseudomonas aeruginosa dan Bacillus cereus. Jurnal Perikanan, 1(2); 1–9 Ma’ruf, W. F., E. M. Sari, and Sumardianto (2014). Kajian Senyawa Bioaktif Ekstrak Teripang Hitam (Holothuria edulis) Basah dan Kering sebagai Antibakteri Alami. Ju- rnal Pengolahan dan Bioteknologi Hasil Perikanan, 3(4); 16–24 Pringgenies, D. (2010). Karakteristik Senyawa Bioaktif Bakteri Simbion Moluska dengan GC-MS. Jurnal Ilmu dan Teknologi Kelautan, 2(2); 34–40 Pringgenies, D., Y. E, D. A, S. G. W, and Koesoemadji (2018). Penelusuran Bakteri Simbion dari Pencernaan Teripang sebagai Antibakteri Strain MDR (Multi Drug Resistant) sebagai Bahan Produksi Antiseptik Purwa, N., T. Junianto, and Herawati (2012). Karakteris- tik Bakteri Caviar Nilem dalam Perendaman Campuran Larutan Asam Asetat dengan Larutan Garam pada Peny- impanan Suhu Rendah. Jurnal Perikanan dan Kelautan, 3(4); 171–175 Putra, I. S. and I. Mirdhayati (2009). Penggunaan Madu Lebah (Genus Apis) sebagai Bahan Pengawet Alami Dag- ing Sapi. Jurnal Peternakan, 6(1); 14–20 Rau, C. H., A. Yudistira, and H. E. I. Simbala (2008). Iso- lasi, Identifikasi secara Molekular Menggunakan Gen 16S rRNA, Uji Aktivitas Antibakteri Bakteri Simbion Endofit yang Diisolasi dari Alga Halimeda opuntia. Pharmacon Jurnal Ilmiah Farmasi, 7(2); 53–61 Santoso, L. E. A. E., M. A. R. (2017). Efektivitas Ekstrak Daun Mangga sebagai Pengawet Alami Terhadap Masa Simpan Fillet Nila pada Suhu Rendah. Jurnal Perikanan dan Kelautan, 8(2); 57–67 Sitakar, N., M. Nurliana, J. F, M. Akbar, Z. H. Manaf, and Sugito (2016). Pengaruh Suhu Pemeliharaan dan Masa Simpan Daging Ikan Nilai (Oreochromis niloticus) pada Penyimpanan Suhu -20oC Terhadap Jumlah Total Bakteri. Jurnal Medika Veterinaria, 10(2); 162–165 Sukmawati and F. Hardianti (2018). Analisis Total Plate Count (TPC) Mikroba pada Ikan Asin Kakap di Kota Sorong Papua Barat. Jurnal Biodjati, 3(1); 72–78 Suptijah, J. A. M. D. N., P. (2012). Karakteristik dan Bioavailabilitas Nanokalsium Cangkang Udang Vannamei (Litopenaeus vannamei). Jurnal Akuatika, 3(1); 63–73 Tapotubun, N. and Laouhenapessy (2008). Efek Waktu Pemanasan Terhadap Mutu Presto Beberapa Jenis Ikan. Ichityos, 7(2); 65–70 Wijayanto, D., D. B. Nursanto, F. Kurohman, and R. A. Nugroho (2017). Profit Maximization of Whitleg Shrimp (Litopenaeus vannamei) intensive culture in Situbondo Regency, Indonesia. AACL Bioflux, 10(6); 1436–1444 Yuarni, D., Kadirman, and Jamaluddin. (2015). Laju Pe- rubahan Kadar Air, Kadar Protein dan Uji Organoleptik Ikan Lele Asin Menggunakan Alat Pengering Kabinet (Cabinet Dryer) dengan Suhu Terkontrol. Jurnal Pen- didikan Teknologi Pertanian, 1; 12–21 © 2019 The Authors. Page 110 of 111 Hanif et. al. Indonesian Journal of Environmental Management and Sustainability, 3 (2019) 106-111 Yulisa, N., E. Asni, and M. Azrin (2014). Uji Formalin pada Ikan Asin Gurami di Pasar Tradisional Pekanbaru. Jurnal Online Mahasiswa Fakultas Kedokteran, 1(2); 1–12 © 2019 The Authors. Page 111 of 111 INTRODUCTION EXPERIMENTAL SECTION Material Synergistic test among symbiotic bacteria Isolation of Secondary Metabolite Preservation test of Bacterial Consortium Supernatant in Vanname Shrimp Tested Parameters RESULTS AND DISCUSSION Synergistic test Among Bacteria pH Test on Shrimp Preservation Total Colony Test of Contaminant Bacteria (TPC) Organoleptic Test on Shrimp Preservation Water and Protein Content Test Fat and Ash Content Test Carbohydrate Content Calculation TVBN (Total Volatile Base-Nitrogen) Test CONCLUSIONS