The search for natural antimicrobial agents has gained increasing attention over the past few decades, driven by the rising resistance of microorganisms to synthetic antibiotics and growing concerns over chemical residues in food products and aquaculture systems (Amábile et al., 2024). Common chemical preservatives, such as sodium sulfite, sodium nitrite, sodium benzoate, tert-butylhydroquinone (tBHQ), and butylated hydroxyanisole (BHA), have raised public health concerns because of their potential carcinogenicity, genotoxicity, and allergenic efects when consumed over the long term (Kalairaj et al., 2024). One group of bioactive compounds with promising potential is bacteriocins, antimicrobial peptides naturally produced by bacteria, including lactic acid bacteria (LAB) (Hernández et al., 2021). Bacteriocins exhibit specific inhibitory activity against microorganisms, particularly food spoilage and pathogenic bacteria, without harming eukaryotic cells (Banerji et al., 2022). These properties make them ideal candidates for use as natural preservatives in the food industry.

Traditional fermented products, such as terasi (fermented shrimp paste made from Acetes spp.), are rich in complex microbial ecosystems that remain largely underexplored. Terasi is a fermented product of shrimp or fish with the addition of salt or other ingredients as a food flavoring due to its distinctive aroma and taste (Sumardianto et al., 2022; Jatmiko et al., 2025). Terasi, a product of spontaneous fermentation, may harbor bacterial isolates with high biosynthetic capabilities, including bacteriocin production. The raw material, rebon shrimp (Acetes spp.), has a high nutrient content that supports the growth of proteolytic microorganisms and LAB during fermentation (Herlina et al., 2024). Several studies have reported that fermented products, including shrimp paste, have the potential to produce bacteriocins with strong antibacterial activity. For example, bacteriocins produced by lactic acid bacteria (LAB) demonstrate inhibitory activity against pathogenic bacteria such as Staphylococcus aureus, Escherichia coli, and Salmonella typhimurium(Sukmawati et al., 2023). These bacteriocins were stable across a wide range of pH values, temperatures, and NaCl concentrations, indicating their robustness as biopreservatives.

Similarly, Herlina et al. (2024) reported that Indonesian traditional shrimp paste (terasi) produced LAB-derived bacteriocins that efectively inhibited E. coli, Vibrio parahaemolyticus, and S. aureus. Elayaraja et al. (2014) isolated Virgibacillus salexigens from fermented shrimp paste, which produced stable bacteriocins with strong antibacterial activity. Consistent with previous findings, Virgibacillus salexigens isolated from fermented shrimp paste (terasi) also produces heatand pH-stable bacteriocins with broadspectrum antibacterial activity, notably against Listeria monocytogenes(Kobayashi et al., 2016). These findings confirm that fermented seafood products, such as rebon shrimp paste, are rich sources of bacteriocin-producing bacteria. Therefore, bacteriocins derived from fermented products possess significant antibacterial potential and can be further developed as natural food preservatives to extend the shelf life and maintain the quality of food products. A nutrient-rich environment creates optimal conditions for the synthesis of various bioactive compounds (Chai et al., 2020).

In the context of seafood, particularly white shrimp (Litopenaeus vannamei), post-harvest quality degradation is a major concern, especially when handling and storage are inadequate (Berty, 2020). The primary factors contributing to spoilage include: a) endogenous enzyme activity (autolysis) – upon death, natural enzymes such as proteases begin breaking down muscle tissue, leading to softening and structural damage that accelerate spoilage (Das et al., 2023); b) microbial spoilage – spoilage bacteria such as Pseudomonas and other Gram-negative species proliferate rapidly under moist, room-temperature conditions, producing of-odors such as trimethylamine (TMA), ammonia, and sulfur compounds (Luqman et al., 2024); c) improper storage temperature – suboptimal temperature accelerates microbial growth and enzymatic reactions. Even under cool conditions without ice, shrimp quality declines significantly within 24–48 h (Gökoğlu, 2021). d) Lipid oxidation: Although the fat content in shrimp is low, existing lipids are prone to oxidation, generating rancid odors and degrading sensory quality (Feng et al., 2021); and e) cross-contamination: Poor hygiene during harvesting, processing, or transportation may introduce microbial contaminants that accelerate product spoilage (Akinsemolu et al., 2024).

Shrimp spoilage is commonly characterized by abnormal discoloration (e.g., grayish or reddish tints), strong ofensive odor, excessive surface slime, and soft, nonelastic texture (Whittle et al., 2002). Given the health risks associated with synthetic preservatives and the growing need to preserve seafood quality, exploring safer and more environmentally friendly alternatives, such as bacteriocins, is critical. Bacteriocins ofer several advantages: they are natural and safe for use in seafood products, do not alter the taste or aroma when used at optimal concentrations, and can be applied via spraying, immersion, or coating. Moreover, they are efective when combined with cold storage using a hurdle technology approach (Lahiri et al., 2022). Bacteriocins can extend shelf life, control foodborne pathogens, and contribute to public health (Parada et al., 2025). Their environmentally friendly nature and favorable safety profiles position them as promising candidates for future food preservation, with minimal adverse efects on humans or animals (Madoramae et al., 2025). These compounds also demonstrate significant thermal stability, maintain antimicrobial activity in the acidic to neutral pH range, and are efective against Salmonella typhimurium (Makhlouf et al., 2024).

Previous research successfully isolated and identified three bacteriocinproducing bacteria from shrimp paste: TRS1 (Bacillus tropicus strain MCCC 1A01406), TRS2 (Bacillus nitratireducens strain MCCC 1A00732), and TRS3 (Bacillus paramycoides strain MCCC 1A04098). These isolates produced bacteriocins with varying antimicrobial potencies, as reflected by their minimum inhibitory concentration (MIC) values, which ranged from 0.19% to 0.78% (Sukmawati et al., 2025). Based on this background, this study aimed to: (1) characterize bacteriocins produced by bacterial isolates from terasi udang rebon by determining their values to assess their relative toxicity against Artemia sp.; and (2) evaluate the eficacy of bacteriocins as a natural preservative on white shrimp based on findings

Artemia sp. (brine shrimp) is widely recognized as a reliable model organism for the preliminary toxicity testing of bioactive compounds, including bacteriocins and bacteriocin-like inhibitory substances. Its high sensitivity, short life cycle, low maintenance cost, and lack of ethical restrictions make it suitable for determining values in ecotoxicological assessments. Previous studies have demonstrated that Artemia salina exhibits consistent and quantifiable responses to both natural and synthetic toxicants, making it an eficient bioindicator for evaluating environmental and food safety (Chan et al., 2021). Recent studies have confirmed the validity of this method for assessing the toxicity of marine natural products and microbial metabolites (Chan et al., 2021; Mahmud et al., 2023).

The 24-hour LC₅₀ toxicity test of bacteriocins produced by TRS1 (Bacillus tropicus strain MCCC 1A01406), TRS2 (Bacillus nitratireducens strain MCCC 1A00732), and TRS3 (Bacillus paramycoides strain MCCC 1A04098) revealed distinct values, representing the lethal concentration of bacteriocins required to cause 50% mortality in the test organisms within a 24-hour period (Table 1). The bioassay was performed using Artemia sp. as the test organism, which is recognized as a reliable biological model for the preliminary toxicity evaluation of bioactive compounds. This organism was selected because of its high sensitivity and consistent response to a wide range of natural and synthetic toxic substances. This parameter is commonly used to assess the acute toxicity of bioactive compounds and provides a quantitative measure of their safety for potential applications in food and environmental systems.

Regression analysis indicated that the value of the bacteriocins was concentration-dependent and followed a linear relationship. The coeficient of determination (R²), which indicates the goodness of fit of the linear regression model between the logarithm of concentration and probit values, the higher the value, the more accurate and reliable the is (Figure 1; Figure 2; Figure 3).

The 24-hour toxicity assay of crude bacteriocins produced by TRS1 (Bacillus tropicus strain MCCC 1A01406), TRS2 (Bacillus nitratireducens strain MCCC 1A00732), and TRS3 (Bacillus paramycoides strain MCCC 1A04098) was conducted to evaluate their relative toxicity toward Artemia sp.. The value represents the lethal concentration of bacteriocins required to cause 50% mortality in the test organisms within a 24-hour exposure period, serving as an important parameter for assessing the safety of bioactive compounds. The results revealed distinct values among the three isolates, indicating diferences in their bioactivities and toxicological profiles. Bacteriocins from TRS1 exhibited the lowest value, suggesting stronger biological potency with relatively low acute toxicity, whereas TRS2 and TRS3 showed higher values, indicating lower toxicities. These variations are likely associated with diferences in the structural and functional characteristics of the bacteriocins produced by each Bacillus species. Following the assessment, the antibacterial efectiveness of

TRS1, TRS2, and TRS3 was further evaluated by testing their ability to inhibit spoilage bacteria in white shrimp (L. vannamei). Fresh shrimp samples were treated with bacteriocins based on their respective concentrations, ensuring safe yet efective exposure levels.

The results demonstrated that bacteriocin-treated shrimp exhibited a slower increase in total bacterial count than the untreated control, indicating that bacteriocins efectively suppressed the proliferation of spoilage-associated microorganisms. Among the treatments, TRS1 showed the most pronounced inhibitory efect, consistent with its stronger antimicrobial potency observed during the minimum inhibitory concentration (MIC) test. These findings confirmed that bacteriocins derived from Bacillus isolated from fermented shrimp paste could prolong the shelf life of white shrimp by inhibiting spoilage-associated microorganisms, thereby maintaining product freshness and safety (Sukmawati et al., 2025). The results showed significant bacterial suppression at various time points over a 24-hour observation period (Table 2).

The total bacterial count (TBC) analysis revealed distinct inhibitory efects of each bacteriocin treatment on microbial growth in white shrimp during 24 h of storage (Table 2). Among the treatments, the TRS1 bacteriocin exhibited the most pronounced antimicrobial activity, maintaining bacterial counts well below the SNI threshold CFU/g) throughout the observation period, with only a modest increase from at hour 3 to CFU/g at hour 24. Similarly, the TRS2 bacteriocin treatment demonstrated efective bacterial suppression, although slightly higher counts were recorded compared to TRS1, reaching CFU/g at hour 24. In contrast, the TRS3 bacteriocin-treated samples exhibited a steady increase in TBC from 7.9×10⁴ CFU/g at hour 3 to at 24 h. Although TRS3 initially inhibited microbial growth, maintaining bacterial levels below the SNI limit until hour 12-its overall antimicrobial performance was less efective than that of TRS1 and TRS2. Meanwhile, the untreated control group showed exponential microbial proliferation, with bacterial counts exceeding

CFU/g as early as hour 3 and reaching CFU/g by hour 24, far surpassing the acceptable microbial limit. Based on the Kruskal-Wallis test (Table 3), a significant diference (p<0.001) was observed among the bacteriocin treatment groups, indicating strong statistical evidence to reject the null hypothesis. These findings confirmed that the three bacteriocins (TRS1, TRS2, and TRS3) had significantly diferent efects on bacterial growth inhibition, with TRS1 showing the highest preservative eficacy in maintaining shrimp quality during storage. This finding is supported by the data patterns presented in Table 3.

Owing to the significant diference, a follow-up analysis using Dunn’s post hoc test was conducted. The results indicated that all bacteriocin treatments (TRS1, TRS2, and TRS3) showed significant diferences compared with the control group, confirming the efectiveness of bacteriocin application. However, no significant diferences were observed between TRS1 and TRS2 or between TRS2 and TRS3, suggesting that these treatments elicited relatively similar responses in the plants. Significant diferences were primarily evident between TRS1 and TRS3, as well as consistently across all treatments compared to the control, highlighting the variation in efects among treatments. These findings suggest that while all bacteriocin treatments efectively inhibited bacterial growth compared to the untreated control, the degree of inhibition varied depending on the bacterial strain that produced the bacteriocin. TRS1, derived from Bacillus tropicus, exhibited the strongest antimicrobial activity, likely due to its lower value and higher potency, as observed in previous toxicity and MIC assessments. In contrast, TRS3, produced by Bacillus paramycoides, showed weaker inhibition, possibly due to diferences in peptide structure, molecular weight, or stability under ambient conditions. The absence of significant diferences between TRS1 and TRS2 indicates that the bacteriocins produced by Bacillus tropicus and Bacillus nitratireducens may share similar functional characteristics or antimicrobial mechanisms. Overall, the statistical results support the hypothesis that bacteriocins can serve as efective natural preservatives by suppressing spoilage bacteria and maintaining shrimp quality during storage.

The Kruskal-Wallis test on the TRS1 of bacteriocin efectiveness based on observation time (every 3 h until 24 h) showed a significant diference (p = 0.042) (Table 4).

The results of the analysis indicated that bacteriocin significantly afected bacterial colony counts across diferent time points. That is, TRS1 was efective in inhibiting bacterial growth at 3, 6, 9, 12, 15, 18, 21, and 24 h. However, no diferences were observed among the time points of observation, as shown by Dunn’s post hoc test.

The results of Dunn’s post hoc test on the efectiveness of bacteriocin TRS1 in Pacificwhite shrimp across diferent observation times showed that most pairwise comparisons were not statistically significant. Although some diferences were observed at the early observation points, particularly between 3 and 6, 9, 21, and 24 h, as well as between 15 and 21 and 24 h , these results did not remain significant after adjustment (Adj. Sig. > 0.05). These findings indicate that the efectiveness of bacteriocin TRS1 remained relatively stable throughout the observation period, with variations across time points not strong enough to be considered statistically significant.

The Kruskal-Wallis test results showed that the efectiveness of the TRS2 bacteriocin was similar across the time points of observation (every 3 h until 24 h) (Table 4). Meanwhile, TRS3 bacteriocin showed diferences in its efectiveness across the time points of observation (Table 4). However, the diferences existed only between hours 3 and 23 and hours 3 and 24, as indicated by Dunn’s post hoc test.

The results of Dunn’s post hoc test indicated that the diferences in the efectiveness of TRS3 bacteriocin across observation times were largely not significant after adjustment (Adj. Sig. >0.05). However, significant diferences were observed at certain time comparisons, particularly between 3 and 21 h and between 3 and 24 h . This finding suggests that the efectiveness of TRS3 undergoes a notable change during the early observation period (3 h) compared to later periods (21–24 h).

Overall, although some variation in efectiveness was observed across diferent times, most diferences were not statistically significant after correction, suggesting that the activity of TRS3 bacteriocin remained relatively stable throughout the observation period, with significant changes only evident between the earliest and latest time points.

TRS1 bacteriocin showed an value of 0.22 µL/mL against Artemia, indicating a very high level of toxicity. Meanwhile, TRS2 and TRS3 had values of 4.40 and 2.48 µL/ mL, respectively, which also fall into the highly toxic category, although they are less toxic than TRS1. These values are classified as highly toxic based on toxicity categories, where compounds with values <100 µL/ mL are considered highly toxic; 100–500 µL/ mL toxic; 500–1,000 µL/mL moderately toxic; 1,000–2,500 µL/mL slightly toxic; and >2,500 µL/mL are regarded as practically non-toxic (Meyer et al., 1982; USEPA, 2022; Pohan et al., 2023).

Among the tested bacteriocins, TRS1 (produced by B. tropicus) exhibited the lowest concentration (0.22 µL/mL), indicating the highest toxicity to Artemia sp. In contrast, the bacteriocins of TRS2 and TRS3 had much higher LC ₈₀ values (4.40 and 2.48 µL/mL, respectively). These findings are critical for the development of bacteriocins as natural preservatives in fishery products, particularly shrimp products. Ideally, bacteriocins used for preservation should exhibit high toxicity against microorganisms to enable the use of very low concentrations but with high efectiveness. The three bacteriocins isolated from the shrimp paste in this study are promising candidates for further applications, either as antimicrobial coatings or as preservatives/additives in shrimp processing.

The observed toxicity was much higher than that reported in previous studies. For example, bacteriocin from Lactobacillus plantarum exhibited inhibitory activity against Listeria monocytogenes, with a minimum inhibitory concentration (MIC) ranging from 2–4 µL/mL when applied to processed meat products (Sobrino-Lopez & Martin-Belloso, 2008). Meanwhile, a liquid filtrate containing bacteriocin-like substances from Bacillus subtilis inhibited the growth of Staphylococcus aureus and Escherichia coli at concentrations of 1–3 µL/mL (Wahyudi et al., 2010). Enterocin, a bacteriocin produced by Enterococcus faecium, showed an value against Artemia salina in the range of mL (Delgado et al., 2007). Bacteriocins from Streptomyces isolated from the gut of milkfish (Chanos chanos) demonstrated extracellular metabolite activity against Salmonella enterica serovar Typhimurium, Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus, with an value of 0.227 µL/mL (Kurnianto et al., 2021). Bacteriocin P7 from Bacillus velezensis, isolated from a mangrove endophyte, has an MIC value of 30.35 µL/mL (Li et al., 2025). Another study found that a crude extract from Bacillus velezensis strain PD9 was efective against MRSA, with an MIC value of 125 µL/mL (Baharuddin et al., 2021). Bacteriocins produced by Lactococcus spp.. HY449 inhibited the growth of S. epidermidis ATCC 12228, S. aureus ATCC 65389, Streptococcus. pyogenes ATCC 21059, and P. acnes ATCC 6919, with an estimated of approximately 50 mg/mL (Oh et al., 2006).

Following the assessment, the efectiveness of bacteriocins isolated from TRS1, TRS2, and TRS3 in inhibiting spoilage bacteria in white shrimp was further evaluated. This involved applying bacteriocins to shrimp and monitoring the total bacterial count (TBC) at regular intervals. The results showed significant efectiveness in bacterial suppression at various time points over a 24-hour observation period (Table 2). TRS1 demonstrated the highest efectiveness in inhibiting bacterial growth. Even at the 24- hour mark, the TBC remained relatively low , far below both the control and the Indonesian National Standard (SNI) threshold Bacteriocins from TRS2 and TRS3 also exhibited inhibitory efects. At 24 h, the TBC of TRS2 bacteriocin reached CFU/g, whereas that of TRS3 exceeded the SNI limit with a TBC of CFU/g. In contrast, the control group showed a sharp increase in TBC from at 3 h to at 24 h, illustrating rapid microbial growth in the absence of bacteriocins. Only TRS1 and TRS2 bacteriocins maintained TBC values below the SNI threshold throughout the 24- hour period. The TRS3 was efective only up to 12 h, after which the TBC values surpassed the permitted maximum limit.

During the 24-hour observation period, the TRS1 and TRS2 bacteriocintreated shrimps showed fluctuations in their total bacterial counts. Initially, there was an increase in TBC from and , respectively, at hour 3 to and respectively, at hour 6. This early increase may be attributed to the adaptive phase of microorganisms adjusting to the bacteriocin environment (Negash & Tsehai, 2020), during which not all microbes are immediately inhibited (Todorov et al., 2022). The difusion and penetration times required for bacteriocins to act within shrimp tissues may also influence their initial efectiveness (Silva et al., 2018). From hours 6 to 15, a gradual decline in TBC was observed, indicating optimal antimicrobial activity. Bacteriocins likely disrupt bacterial cell membranes, interfere with metabolic processes, and ultimately lead to cell death (Sharma et al., 2021; Soltani et al., 2021). Interestingly, a rebound in bacterial growth occurred from 18 to 24 h, possibly due to the development of microbial resistance or regrowth from previously suppressed or dormant populations (Telhig et al., 2020).

In contrast, the TRS3 bacteriocintreated samples exhibited a steady increase in TBC from at hour 3 to CFU/g at hour 24. Although TRS3 showed some early inhibition (with TBC under the SNI threshold at hour 12), its overall antimicrobial performance was less efective than that of TRS1 and TRS2. The untreated control group showed exponential microbial growth, confirming the necessity of antimicrobial interventions for shrimp preservation.

This study concluded that crude bacteriocins TRS1, TRS2, and TRS3 exhibited 24-hour values of 0.22, 4.40, and 2.48 µL/mL, respectively, indicating that all three were highly toxic to Artemia sp. larvae based on standard toxicity categories. In terms of application, crude bacteriocins of TRS1 and TRS2 efectively inhibited microbial growth in fresh Pacific white shrimp over a 24-hour storage period, with total bacterial counts (TBC) consistently remaining below the SNI threshold. In contrast, the TRS3 crude bacteriocin efectively suppressed microbial growth only up to 12 h, after which the TPC exceeded the acceptable limit. Therefore, TRS1 and TRS2 are the most promising candidates for use as natural preservatives in white shrimp, ofering superior microbial inhibition during storage. On the other hand, the TRS3 crude bacteriocin might require a higher concentration to sustain long-term eficacy, which requires further analysis.