Protease are class of degradative enzymes that hydrolyze proteins, breaking its peptide bond, into smaller peptides and amino acids (Dos Santos Aguilar & Sato, 2018). Proteases represent one of the most economically significant groups of industrial enzymes, contributing to over 60% of total enzyme sales worldwide (Singh & Panesar, 2023). Microbial proteases have attracted considerable interest because they offer advantages such as lower production costs, higher yields, shorter and more sustainable production cycles, reduced space requirements, and greater ease of genetic manipulation compared with proteases derived from plant and animal sources (Barzkar et al., 2022). Moreover, microbial proteases can be cultured on a large scale using inexpensive substrates, including agricultural waste, and their extracellular nature simplifies downstream processes (Solanki et al., 2021).

Interest of halotolerant and halophilic protease from halotolerant microbes has been raising, as these enzymes retain stability and activity under high-salt conditions. These properties support their application in industrial processes involving high-salt environment (Kochhar et al., 2022). Halotolerant and halophilic proteases have been used in detergent industries (Mokashe et al., 2017), leather industry for dehairing (Li et al., 2022), and food processing (Naveed et al., 2021).

Previous research have explored halotolerant and halophilic protease-producing bacteria from various food source such as fish sauce (Abd Samad et al., 2017), several Thai fermented fish (Taprig, 2013), anchovy paste (OPTIMASI WAKTU INKUBASI PRODUKSI PROTEASE DAN AMILASE ISOLAT BAKTERI ASAL TERASI IKAN TERI Stolephorus sp, 2018), Chinese fermented catfish paste (Yuan et al., 2022), and fermented shrimp paste (Yao et al., 2025). Beyond food products, environmental samples are being explored as sources of proteolytic bacteria, including seagrasses (Asar et al., 2016), lakes ((Benmebarek & Kharroub, 2023); (Ibrahim et al., 2019); (Ruginescu et al., 2020)), hypersaline water (Proca et al., 2020), mud volcano (Rohman et al., 2012), and salt mines (Ali et al., 2016).

Terasi, fermented shrimp paste from Indonesia, traditionally produced through spontaneous fermentation of shrimp with salt addition, generating habitat dominated by halotolerant and halophilic bacteria (Kobayashi et al., 2003). These bacteria secrete extracellular enzymes, including protease to hydrolyze protein in shrimp as raw material (Prihanto et al., 2021). This condition makes terasi a potential source for isolating halotolerant, protease-producing bacteria.

Lampung is one of Indonesia's terasi producers, with exports reaching US$50,000 per year (Lampung, XXXX). Lampung's naturally fermented terasi represents promising source of novel halotolerant proteolytic bacterial isolates. However, studies on halotolerant bacteria from Lampung's terasi with proteolytic activities remain limited, indicating the need for further exploration to optimize their potential applications. The potential of halotolerant bacterial enzymes underscores the importance of exploring protease-producing bacteria from fermented foods, one of which is Lampung's shrimp paste.

Proteolytic bacteria were isolated from wet terasi collected in Labuhan Maringgai using two media types: Nutrient Agar supplemented with 5% NaCl (w/v) (NA+5% NaCl) and Nutrient Agar supplemented with 10% NaCl (w/v) (NA+10% NaCl). As shown in Table 1 and Table 2, a total of 20 and 15 bacterial isolates exhibiting distinct colony morphologies were obtained from NA+5% NaCl and NA+10% NaCl, respectively. Macroscopic morphological characteristics of the isolates, including colony color, shape, elevation, and margin, were recorded according to standard methods (Cappuccino & Sherman, 2008).

All isolates that grew were then purified and inoculated on Skim Milk Agar (SMA) and incubated for 24 hours to observe the protease enzyme activity of each isolate. Skim Milk Agar is a differential medium used to observe the qualitative activity of protease because it contains casein as the main substrate of the protease enzyme. Bacteria that are able to hydrolyse and have proteolytic activity against casein will have a clear zone around the isolate (Figure 1) (Chung et al., 2021)(Decimo et al., 2014). Based on the screening results, 85% of the bacterial isolates obtained from NA + 5% NaCl exhibited proteolytic activity, whereas 67% of the isolates derived from NA + 10% NaCl showed similar activity.

Qualitative activity of protease enzymes was assessed by measuring the diameter of the clear zone formed around the bacterial colonies grown on SM agar media. Casein, the major protein component of skim milk, consists of phosphoproteins that bind calcium and form calcium calceinate salts, resulting white appearance of the medium (Amin, 2018). Protease enzyme produced by bacteria hydrolize casein to soluble amino acids, causing local decolorization of the medium and producing transparent and clear zone surrounding the colony (Mamangkey & Suryanto, 2021). The clear zone indicates the protease activity produced by the bacterial isolate. The larger the clear zone indicates higher extracellular protease enzyme activity (Cahyaningrum et al., 2021).

Based on the clear zone measurements depicts in Figure 1 and Figure 2, the bacterial isolates exhibited varying levels of protease activity. The formation of these clear zones confirms that the isolates release protease into the growth medium and are capable of degrading the casein in SM agar as their primary protein substrate (Artha et al., 2019)(Ramadhan et al., 2021). All isolates showed the ability to produce protease enzyme with varying activity. Based on the proteolytic index category, index value less than 1.5 is classified as low, IP 1.5 to 3.5 is classified as medium, and IP values above 3.5 are classified as high (Ayuningrum et al., 2022). Majority of the bacterial isolates isolated from two media showed a low index.

As shown in Figure 1 and Figure 2, isolates obtained from NA + 5% NaCl exhibited a proteolytic index of 0.3 – 3.0, while those isolated with NA + 10% NaCl showed an activity of 0.18 - 3.06. In NA+ 5% NaCl and NA+10% NaCl media, the highest proteolytic index was shown by isolates BTCL5-07 and BTCL10-05 with values ​​of 3.0 and 3.06, respectively. Based on the proteolytic index classification, both isolates fall within the medium-activity category. Previous studies on proteolytic bacteria from shrimp paste have also reported diverse levels of activity. For example, (Handayani et al., 2025) identified 41 isolates from seven shrimp paste samples had proteolytic activity with a clear zone greater than 2 cm. Another study by Susanti et al., (2021) found 16 proteolytic bacteria with proteolytic index ranging from 1.60-3.32. These findings indicate that halotolerant bacteria from Terasi Lampung represent a promising source of potential proteolytic enzymes. Further research is required to optimize enzyme production.

Isolates BTCL5-07 and BTCL10-05 from Lampung fermented shrimp paste (terasi) exhibited the highest proteolytic activity, with proteolytic indices of 3.0 and 3.06, respectively. These findings indicate that halotolerant bacteria from Lampung terasi represent a promising source of potential proteolytic enzymes. Further research is required to optimize enzyme production.