Fibrinolytic Protease-Producing Bacteria with Varied Hemolysis Pattern Associated with Marine Algae Dictyota sp.

The main death factor of cardiovascular disease (CVD) is the formation of a blood clot (thrombus). Thrombus is formed by the action of fibrin, playing a role as a blood coagulation agent. Administration of fibrinolytic enzymes can degrade fibrin through the fibrinolysis process. Therefore, searching for new sources of fibrinolytic enzymes becomes critical in eradicating diseases by fibrinolysis of thrombus. This study aims to isolate fibrinolytic protease-producing bacteria associated with fermented brown algae products Dictyota sp, of Awur Bay, Jepara, Indonesia, and to observe their hemolysis pattern. As many as 14 unique bacterial colonies previously isolated from fermented Dictyota sp. were sub-cultured using Zobell Agar (ZA) medium. Skim Milk Agar (SMA) and Fibrin Agar (FA) were then used as selective media to detect the presence of fibrinolytic protease-producing bacteria, which was indicated by their ability to form a clear proteolytic and fibrinolytic zone simultaneously around bacterial colonies. Hemolysis characteristics of fibrinolytic bacteria were determined using Blood Agar Plate (BAP) to test their ability to produce hemolysin toxin. As a result, of these 14 isolates, 3 of them, namely FD-09, FD-13, and FD-14 (FD= Fermented Dictyota), could produce both proteolytic and fibrinolytic zone with a fibrinolytic index range of 2.0–2.9. Isolate FD-09 is the least pathogenic (g-hemolytic) compared to other fibrinolytic isolates, FD-13 (b-hemolytic) and FD-14 (a-hemolytic), in terms of hemolysin toxicity. In conclusion, fermented Dictyota sp. is a potential source of bacteria-producing fibrin-degrading protease with varied hemolysis patterns. It is necessary to identify bacteria-producing fibrinolytic protease isolates Dictyota sp. and further characterization regarding the specificity and activity of the resulting protease to develop its potential as an antithrombotic agent

AKTIVITAS ANTI-BIOFILM BAKTERI DARI PRODUK ALGA COKLAT Dictyota sp.

Infeksi bakteri dapat memperlambat penyembuhan, menyebabkan deformitas dan kematian sel. Hal ini disebabkan bakteri menghasilkan biofilm yang memberikan sifat resistensi terhadap antibiotik yang diberikan pada luka yang terinfeksi bakteri itu. Infeksi yang terkait dengan biofilm merupakan mayoritas dari semua infeksi bakteri yang kronis atau berulang dalam tubuh manusia. Alginat liase.merupakan enzim yang mampu mendegradasi alginat, yang merupakan komponen utama biofilm bakteri. Oleh karena itu, pencarian sumber baru alginat liase menjadi penting dalam pemberantasan penyakit infeksi terutama yang terkait dengan pembentukan biofilm. Penelitian ini bertujuan untuk mengetahui kemampuan simbion dari produk fermentasi alga coklat Dictyota sp. yang diperoleh dari Teluk Awur, Jepara, Indonesia dalam mendegradasi biofilm bakteri. Sampel makroalga segar difermentasi terlebih dahulu selama 7 hari untuk merangsang aktivitas degradasi oleh bakteri simbion secara umum. Data yang diperoleh dari hasil kultur dan resistensi dapat dijadikan sebagai dasar dilakukan terapi empiris. Bakteri simbion Dictyota sp ditumbuhkan pada media Zobell Agar (ZA) dan kemudian masing-masing koloni spesifik yang tumbuh dimurnikan menggunakan media Nutrient Agar (NA). Agar minimal alginat kemudian digunakan sebagai media selektif untuk mendeteksi keberadaan bakteri alginolitik yang ditunjukkan dengan kemampuannya membentuk zona alginolitik yang jernih disekitar koloni bakteri. Dari 14 isolat bakteri simbion Dictyota sp, 3 yaitu FD-01, FD-03, dan FD-04, dapat menghasilkan zona alginoliti dengan nilai indek alginolitik berkisar antara 0,5 – 1,0. Kesimpulannya, Dictyota sp. merupakan sumber potensial bakteri penghasil enzim antibiofilm, alginat lias

PRODUCTION OPTIMIZATION, PARTIAL PURIFICATION, AND THROMBOLYTIC ACTIVITY EVALUATION OF PROTEASE OF Bacillus cereus HSFI-10

Cardiovascular disease is the primary cause of mortality in the world due to the formation of blood clots or thrombi in blood vessels. Bacterial proteases commonly function as thrombus dissolver agents in the pharmaceutical industry. Bacterial isolate HSFI-10 (Holothuria scabra Fermented Intestine-10) previously isolated from Rusip fermented sea cucumber had demonstrated thrombolytic activity. This study aimed to produce crude protease of HSFI-10 strain at an optimized incubation time and determine the thrombolytic activity of crude and dialysate proteases on A, B, AB, and O blood types. Isolate HSFI-10 was first molecularly identified and found to be Bacillus cereus with a homology level of 99.80% with Bacillus cereus strain ST06. The optimum crude enzyme was obtained after 48-h incubation with an activity of 222.52 U/mL, which increased to 438.84 U/mL after ammonium sulfate precipitation and dialysis. Clot lysis activity of crude enzymes was measured based on the gravimetry method on blood in the ABO system, showing results that ranged from 68.99% to 69.76%, while the dialysate ranged from 81.16% to 82.52%. In conclusion, partial purification of bacterial protease could increase both its specific and thrombolytic activities on human blood in the ABO system, with only 1% activity variability between A, B, AB, and O blood types

Characteristics and Substrate Specificity of Semi-Purified Bacterial Protease of Bacillus thuringiensis HSFI-12 with Potential as Antithrombotic Agent

Commercial proteases, such as Nattokinase (NK), Staphylokinase (SAK), and Streptokinase (SK) play an important role in the destruction of thrombus, the main cause of death in cardiovascular disease. The latest technology combining enzymes with certain drugs is the target of new research in the thrombolytic area. The first step is to develop protease from Bacillus thuringiensis HSFI-12 bacteria as an antithrombotic agent, characterization of the bacterial enzyme is necessary. This study aims to determine the specificity of protease from Bacillus thuringiensis HSFI-12 to explore its potential as an antithrombotic agent in terms of anticoagulant and fibrinolytic activities. The molecular weight and specificity of bacterial protease were determined with a zymographic method with casein as substrate. Bacillus thuringiensis HSFI-12 was first cultured on Nutrient Agar (NA) media and then on Skim Milk Agar (SMA) media. The obtained crude protease from Skim Milk Broth (SMB) was then concentrated as dialysate. Both crude and dialysate proteases were tested for their specific activity, as well as anticoagulant and fibrinolytic activities. Next, the dialysate’s molecular weight and specificity on the casein substrate were investigated using the zymographic method. As result, protease activity in crude form is lower than that in dialysate, which was 0.5570 ± 0,004 U/mL and 2.1767 ± 0,005 U/mL, respectively. The molecular weight of the obtained bacterial protease was between 117 – 133 kDa and the enzyme is capable of degrading casein as shown on the zymogram. Overall, both crude and dialysate proteases of Bacillus thuringiensis HSFI-12 show potential as an antithrombotic agent for exhibiting anticoagulant and antiplatelet activities. Yet, it could not exhibit direct fibrinolytic activity implying the possibility that the enzyme plays a role as a plasminogen activator, which can dissolve fibrin by activating plasmin

DETECTION AND PATHOGENICITY LEVEL SCREENING OF HEMOLYTIC BACTERIA ISOLATED FROM UNPACKED CHICKEN NUGGET : Received: 04th October 2023 Revised: 13th October 2023, 02nd November 2023, 06th November 2023 Accepted: 08th November 2023

Food poisoning is a severe threat that can lead to death. According to the WHO, cases of food poisoning have been on the rise in recent years. Pathogenic bacteria are responsible for food poisoning, often originating from contaminated food. Chicken nuggets, a popular ready-to-eat food, are susceptible to bacterial growth due to the risk of microbial contamination during storage. This study aims to assess the pathogenicity levels of bacteria in chicken nuggets when stored at room temperature for seven days. Samples from chicken nuggets left at room temperature were diluted and cultured on Nutrient Agar (NA) media. Subsequently, bacterial pathogenicity screening was conducted using MacConkey Agar (MC), Blood Agar Plate (BAP), and Chocolate Agar Plate (CAP). The results revealed six bacterial isolates labeled FMCN-1 to FMCN-6 (FMCN denoting Fatmawati Market chicken nugget). Most isolates exhibited a moderate level of pathogenicity (FMCN-2, FMCN-3, FMCN-4, FMCN-5, and FMCN-6), with only one isolate demonstrating low pathogenicity (FMCN-1). In conclusion, storing chicken nuggets at room temperature for seven days can lead to bacterial contamination with mostly moderate pathogenicity. Further molecular analysis is required to identify the potentially harmful bacteria