# Partial Purification and Characterization of Bacteriocin-Like Inhibitory Substances Produced byStreptomyces sp. Isolated from the Gut of Chanos chanos

Authors: Muhammad A. Kurnianto; Harsi D. Kusumaningrum; Hanifah N. Lioe; Ekowati Chasanah
Journal: BioMed Research International
 (2021)

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## Abstract

Bacteriocin-like inhibitory substances (BLIS) have sparked great interest because of their promising use in food as natural antimicrobial agents. In this work, sixStreptomyces isolates obtained from the gut of Chanos chanos demonstrated their ability to produce extracellular metabolites with inhibitory activity against Salmonella enterica serovar Typhimurium, Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus. Exposure of the extracellular metabolites to proteolytic enzymes (i.e., proteinase-K, trypsin, and pepsin) revealed high sensitivity and confirmed their proteinaceous nature. The metabolites were stable at high temperatures (up to 100°C for 30 min) and a wide range of pH (pH 2.0–7.0). Fractionation of the crude BLIS by filtration yielded three fractions based on molecular weight: <3 kDa, 3–10 kDa, and >10 kDa. Analysis of the antibacterial activity of these fractions showed increased specific activity, especially in the fraction with a molecular weight (MW) of <3 kDa, relative to the crude sample. The fraction with MW<3kDa had minimum inhibitory and bactericidal concentrations in ranges 0.04–0.62 mg·mL−1 and 0.08–1.25 mg·mL−1, respectively. This fraction also showed better temperature and pH stability compared with crude BLIS. Brine shrimp toxicity assay revealed that this fraction has moderate toxicity with a 50% lethal concentration of 226.975 μg·mL−1 (i.e., moderate toxicity) to Artemia salina. Identification of the peptide sequences of this fraction by liquid chromatography–tandem mass spectrometry yielded 130 proteins with retention times of 15.21–19.57 min. Eleven proteins with MWs of 1345.66–2908.35 Da and composed of less than 30 amino acid residues with high hydrophobicity (15.34–26.22 kcal·mol−1) appeared to be responsible for the antibacterial activity of the fraction. This study revealed the potential application of BLIS from Streptomyces, especially BLIS SCA-8, as antibacterial agents.


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## Body

## 1. Introduction

Increasing consumer awareness of the adverse health effects of chemical preservative in food has resulted in heightened interest in fresh and minimally processed food [1–3]. However, this trend presents a massive challenge because minimally processed food is closely related to various diseases caused by pathogenic bacteria [4, 5]. Hence, numerous explorations of potential natural preservatives, such as bacteriocins or bacteriocin-like inhibitory substances (BLIS), have been conducted in efforts to replace chemical preservatives to improve the shelf life and safety of food [6, 7].Bacteriocins are short-chain protein or polypeptide compounds synthesized ribosomally by microorganisms that could inhibit the growth of bacterial strains closely related to the bacteriocin-producing strains [7, 8]. The term BLIS describes a peptide or protein antimicrobial compound that is synthesized by ribosomes but not fully characterized by its amino acid sequence and biochemical properties [9]. Bacteriocins may be divided on the basis of their structure into three main classes: class I includes lantibiotics, class II includes small posttranslationally unmodified bacteriocins, and class III includes large antibacterial proteins (>10 kDa) and bacteriolysins [10]. Bacteriocins generally have a narrow antibacterial spectrum and are limited to closely related genera or species [7]. However, the growing demand for natural agents for food preservation (i.e., to replace chemical preservatives) or clinical applications renders the study and exploration of bacteriocins with broad spectrum antibacterial activity increasingly necessary [11, 12].Most bacteriocins are produced by lactic acid bacteria, such as those belonging toLactococcus, Lactobacillus, Streptococcus, and Pediococcus [7, 10]. Some bacteria from other genera, one of which is Streptomyces, are also capable of producing bacteriocins or BLIS with potential antibacterial activity and stability [13]. Several authors have examined the bacteriocins produced by Streptomyces pluripotens sp. nov. [14], Streptomyces scopuliridis sp. nov. [15], Streptomyces griseus (grisemycin) [16], and Streptomyces bottropensis (bottromycin A2) [17]. These studies show that Streptomyces bacteriocins have broad spectrum antibacterial activity against Gram-positive and negative bacteria and even resistant pathogenic bacteria, such as methicillin-resistant Staphylococcus aureus [14]. However, although the potential activity of some BLIS produced by Streptomyces has been reported, many other Streptomyces species producing these substances have not been described [13].Streptomyces is a Gram-positive bacterium widely known to produce various bioactive compounds [18]. These bacteria are widely abundant in various ecosystems and can form associations with eukaryotic hosts, such as fish [19, 20]. This ability confers the bacterium with the physiological and genetic ability to adapt and express metabolites of varying bioactivity [21]. Several studies have discovered metabolites with potential bioactivity produced by Streptomyces associated with fish [20, 22, 23]. Kurnianto et al. [23, 24], for instance, identified a suspected peptide-containing antibacterial metabolite with broad spectrum antibacterial activity produced by Streptomyces associated with Chanos chanos.The present study is aimed at analyzing the potential of sixStreptomyces isolates from the gut of C. chanos to produce BLIS, at analyzing the stability and antibacterial activity characteristics of BLIS, and at identifying the active fraction of BLIS using liquid chromatography-tandem mass spectrometry (LC-MS/MS).


## 2. Materials and Methods

### 2.1. Bacterial Strains and Culture Collection

SixStreptomyces (i.e., S. variabilis SCA-5, S. variabilis SCA-11, S. variabilis AIA-10, S. labedae SCA-8, S. globisporus AIA-12, and S. misionensis AIA-17) were obtained from the gut of milkfish (C. chanos) [23, 24]. The strains were identified through their morphological, physiological, biochemical, and molecular characteristics by 16S rRNA gene sequencing. The isolates were cultivated in ISP-2 medium (yeast extract, 4 g; malt extract, 10 g; dextrose broth, 4 g; bacteriological agar, 20 g; and distilled water, 1,000 mL) and incubated at 30°C. Target bacteria, including Salmonella enterica serovar Typhimurium ATCC 14028, Escherichia coli ATCC 25922, Listeria monocytogenes ATCC 35152, and S. aureus ATCC 25923, were grown on tryptic soy broth (Oxoid, UK) and incubated for 24 hours at 37°C.


### 2.2. Production and Preliminary Screening of BLIS-Producing Bacteria

The sixStreptomyces isolates were inoculated on ISP-2 and incubated at 30°C during the optimum antibacterial production time (9–11 days) with agitation in a shaker incubator (New Brunswick, Germany) [24]. Bacterial cultures were centrifuged (Hermle, Germany) at 7,000×g and 4°C for 15 min to separate the supernatant from the cell biomass. The supernatants were adjusted to pH 6.5–7.0 with 1 mol·L−1 NaOH (Merck, Germany) and passed through a 0.22 μm membrane filter (Sartorius, France) to obtain a neutralized cell-free supernatant (CFS). This CFS was analyzed for its inhibitory activity against two bacteria (i.e., E. coli ATCC 25922 and S. aureus ATCC 25922) via the microdilution method [25]. A total of 100 μL of the tested bacterial suspension (1×106CFU·mL−1), 80 μL of Mueller–Hinton broth medium (MHB) (Oxoid, UK), and 20 μL of the CFS were added to each well of a microplate. Test bacteria grown on MHB medium without exposure to the CFS were used as controls. The microplate was incubated for 24 h at 37°C, and the absorbance of each well of the microplate was measured at 600 nm using an ELISA reader (Bio-Rad, US). The growth inhibitory activity of the test bacteria was calculated from the absorbance of samples with and without treatment (control). An absorbance value lower than the control indicates inhibition of the growth of the test bacteria. CFS samples showing potential activity were concentrated using a freeze dryer (Christ, Germany) to obtain crude BLIS.


### 2.3. Confirmation of the Proteinaceous Nature of Crude BLIS

The proteinaceous nature of crude BLIS was assessed by exposure of the six concentrated CFS samples to various enzymes, including proteinase-K (Geneaid, Taiwan), trypsin (Sigma-Aldrich, USA), and pepsin (Sigma-Aldrich), at a final concentration of 10 mg·mL−1 and incubation for 2 h at 37°C. The samples were heated for 2 min to inactivate the enzyme, cooled to room temperature, and then subjected to antimicrobial activity testing [11] by using the agar well diffusion method.


### 2.4. Partial Purification via Ultrafiltration Membranes

The six crude BLIS samples were separated according to molecular weight by using 3 and 10 kDa ultracentrifuges (Amicon Ultra Centrifugal Unit, Regenerated Cellulose 3,000 and 10,000 MWCO, Merck Millipore Ltd., Germany) for 60 min at1,328.4×g and 4°C. This process separated the samples into three BLIS ultrafiltration fractions (BLIS-UF), i.e., <3 kDa, 3–10 kDa, and >10 kDa [26]. Each fraction was concentrated with a freeze dryer (Christ, Germany), adjusted to a final volume of 80 mL with deionized water, and then subjected to bacterial inhibitory activity tests to assess their bioactivity.


### 2.5. Quantification of Total Protein

The total protein concentration of each sample was determined on the basis of the Bradford method (Bradford 1976), which refers to Jamaluddin et al. [27]. A total of 160 μL of each sample was reacted with 40 μL of Bradford reagent in a 96-well microtiter plate and then incubated at 37°C in an orbital shaker for 10 min. Protein concentrations were determined by an ELISA reader (Bio-Rad) at 595 nm. Distilled water was used as a blank sample, and a protein standard curve was obtained using bovine serum albumin as a protein standard.


### 2.6. Determination of the Antibacterial Activity of BLIS

The antibacterial activity of BLIS was determined using the agar well diffusion method. A total of 25 mL of Mueller–Hinton agar (Oxoid, UK) containing the test bacteria (1%v/v; E. coli, S. Typhimurium, L. monocytogenes, and S. aureus) at a density of 1×106CFU·mL−1 was poured into the wells of a plate and allowed to solidify. A well (diameter, 6 mm) was made in the agar with a cork borer, and 100 μL samples of BLIS were placed into the wells. After incubation at 37°C for 24 h, the diameter of the inhibition zone (mm) was measured. Distilled water and ampicillin 10 μg·mL−1 were used as controls. The antibacterial activity of BLIS was expressed as activity (A) and specific activity (B) [28]. Purification fold (C) and yield activity (%) (D) of the samples were also calculated.BLIS activity (A) was calculated as shown in equation (1) and expressed in units of AU·mL−1, where 1 AU·mL−1 was defined as the unit area of the inhibition zone per unit volume (mm2·mL−1). In equation (1), Lz refers to the clear zone area (mm2), Ls refers to the well area (mm2), and V refers to the sample volume (mL).
(1)A=Lz−LsV.BLIS-specific activity (B) was defined as the ratio of the total BLIS activity of the sample to the total protein concentration (AU·μg−1) and calculated using equation (2), where A refers to the BLIS activity (AU·mL−1), Tp refers to the total protein concentration (μg) in the sample, and V refers to the sample volume (mL).
(2)B=A×VTp×V.Purification fold (C) was defined as the ratio of the specific activity of the fraction obtained at each purification step (Bp) to the specific activity of crude BLIS (B0) and calculated using equation (3), where Bp was the BLIS-specific activity of the fraction obtained at each steps of purification and B0 was the BLIS-specific activity of crude BLIS.
(3)C=BpB0.Yield activity (D) was defined as the ratio of the total BLIS activity (AU·mL−1) calculated at each step of purification to the total activity of crude BLIS and calculated using equation (4), where A was the BLIS activity (AU·mL−1) in each step, V was the sample volume (mL), and A0 was the BLIS activity of crude BLIS.
(4)D=A×VA0×100.


### 2.7. Analysis of Minimum Inhibitory and Bactericidal Concentrations

Determination of the MIC of BLIS was conducted by preparing BLIS samples at several concentrations (i.e., 10, 5.0, 2.5, 1.25, 0.625, 0.312, 0.156, 0.078, 0.04, 0.02, 0.009, and 0.004 mg·mL−1). Each BLIS sample was placed on a microplate that had previously been inoculated with 0.1 mL of test bacteria (density, 1×106) and 0.1 mL of MHB (HiMedia, India). The microplate was incubated for 24 h at 37°C. The growth of the test bacteria on the microplate was observed using a microplate reader (Bio-Rad) at 600 nm, and their viability was determined by the plate counting method [3]. The MIC was obtained from the lowest concentration of bacteriocin that could inhibit the growth of the test bacteria. The MBC of BLIS was evaluated as follows. After MIC determination, the liquid remaining on the microplate was inoculated with Mueller–Hinton agar (HiMedia) and then incubated for 24 h at 37°C. The MBC refers to the MIC of bacteriocin required to kill 99.9% of the tested bacteria.


### 2.8. Determination of the Temperature and pH Stability of BLIS

Evaluation of the temperature stability of BLIS was performed by dissolving all of the fractions in deionized water and then incubating the resulting solutions at 60, 80, or 100°C for 30 min or 121°C for 15 min. The solutions were cooled to room temperature and subjected to antibacterial activity tests [29]. The pH stability of BLIS was determined by adjusting the pH of the purified fractions to pH 2.0–10.0 by using 1 mol·L−1 HCl and 1 mol·L−1 NaOH as necessary and incubating for 2 h. Prior to the antibacterial activity test, the pH of the samples was set to pH 7.0. All antibacterial activity tests were performed by using the agar-well diffusion method, as described above.


### 2.9. Analysis of Toxicity by Using Brine Shrimp Lethality Assay

Artemia salina cysts were hatched in artificial seawater containing 27 g of NaCl in 3 L of distilled water and provided light and aeration. The larval hatching process was conducted for 48 h. Once hatched, the larval eggs were considered ready for analysis. The purified BLIS fraction showing the best specific activity was dissolved in artificial seawater to obtain test solutions with final BLIS concentrations of 2,000, 1,000, 200, and 20 μg·mL−1. A total of 1 mL of each test solution was transferred to a separate test tube and added with 10 two-day-old A. salina larvae by using a sterile syringe. The volume in the test tube was adjusted to 2 mL to achieve final BLIS concentrations of 1 mg·mL−1, 500 μg·mL−1, 100 μg·mL−1, and 10 μg·mL−1. The test tubes were incubated for 24 h, and the number of dead A. salina larvae was counted. The lethal concentration (LC50) of BLIS was determined via the probit analysis method with a 95% confidence interval using SPSS software. Each concentration was tested thrice, and artificial seawater was used as a negative control [30].


### 2.10. LC-MS/MS Analysis andIn Silico Study of the BLIS Fraction with the Greatest Activity

The BLIS fraction with the greatest antibacterial activity was dissolved in 1 mL of water, centrifuged for 1 min, and then injected into a NanoLC Ultimate 3000 Tandem Q Exactive Plus Orbitrap HRMS instrument equipped with a Thermo PepMap RSLC C18 capillary column (75μm×15cm, 3 μm, 100 Å) and a Thermo Scientific™ 164649 trap column (30 μm, 5 mm). The sample was eluted with H2O with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B) at a flow rate of 300 nL·min−1. The elution program was as follows: 2%–35% B for 30 min, 30%–90% B for 15 min, 90% B for 15 min, and 5% B for 30 min. Spectra were collected over the mass range of 200–2,000 m/z. The mass spectral results were analyzed using Proteome Discoverer 2.2 (Thermo Scientific), which uses the search engine SEQUEST HT, for database-based protein identification. The enzyme used was trypsin with a maximum miss cleavage of 2. The dynamic modifications applied were oxidation (for the amino acid methionine) and acetylation (for N-terminals). The percentages of hydrophobic and cationic amino acid residues were predicted using ProtParam (https://web.expasy.org/protparam/), and the physicochemical properties of the detected amino acid peptide sequences (i.e., sequence length, molecular weight (MW), hydrophobicity, and Boman index) were determined using PepDraw (https://www.pepdraw.com/).


### 2.11. Statistical Analysis

All experiments were performed as three independent replicates, and the results were expressed asmean±standard deviation. Data analysis was performed using SPSS 18.0 software. One-way analysis of variance (ANOVA) and Duncan’s multiple range test (95% confidence interval) were applied to detect significant differences between means.


## 2.1. Bacterial Strains and Culture Collection

SixStreptomyces (i.e., S. variabilis SCA-5, S. variabilis SCA-11, S. variabilis AIA-10, S. labedae SCA-8, S. globisporus AIA-12, and S. misionensis AIA-17) were obtained from the gut of milkfish (C. chanos) [23, 24]. The strains were identified through their morphological, physiological, biochemical, and molecular characteristics by 16S rRNA gene sequencing. The isolates were cultivated in ISP-2 medium (yeast extract, 4 g; malt extract, 10 g; dextrose broth, 4 g; bacteriological agar, 20 g; and distilled water, 1,000 mL) and incubated at 30°C. Target bacteria, including Salmonella enterica serovar Typhimurium ATCC 14028, Escherichia coli ATCC 25922, Listeria monocytogenes ATCC 35152, and S. aureus ATCC 25923, were grown on tryptic soy broth (Oxoid, UK) and incubated for 24 hours at 37°C.


## 2.2. Production and Preliminary Screening of BLIS-Producing Bacteria

The sixStreptomyces isolates were inoculated on ISP-2 and incubated at 30°C during the optimum antibacterial production time (9–11 days) with agitation in a shaker incubator (New Brunswick, Germany) [24]. Bacterial cultures were centrifuged (Hermle, Germany) at 7,000×g and 4°C for 15 min to separate the supernatant from the cell biomass. The supernatants were adjusted to pH 6.5–7.0 with 1 mol·L−1 NaOH (Merck, Germany) and passed through a 0.22 μm membrane filter (Sartorius, France) to obtain a neutralized cell-free supernatant (CFS). This CFS was analyzed for its inhibitory activity against two bacteria (i.e., E. coli ATCC 25922 and S. aureus ATCC 25922) via the microdilution method [25]. A total of 100 μL of the tested bacterial suspension (1×106CFU·mL−1), 80 μL of Mueller–Hinton broth medium (MHB) (Oxoid, UK), and 20 μL of the CFS were added to each well of a microplate. Test bacteria grown on MHB medium without exposure to the CFS were used as controls. The microplate was incubated for 24 h at 37°C, and the absorbance of each well of the microplate was measured at 600 nm using an ELISA reader (Bio-Rad, US). The growth inhibitory activity of the test bacteria was calculated from the absorbance of samples with and without treatment (control). An absorbance value lower than the control indicates inhibition of the growth of the test bacteria. CFS samples showing potential activity were concentrated using a freeze dryer (Christ, Germany) to obtain crude BLIS.


## 2.3. Confirmation of the Proteinaceous Nature of Crude BLIS

The proteinaceous nature of crude BLIS was assessed by exposure of the six concentrated CFS samples to various enzymes, including proteinase-K (Geneaid, Taiwan), trypsin (Sigma-Aldrich, USA), and pepsin (Sigma-Aldrich), at a final concentration of 10 mg·mL−1 and incubation for 2 h at 37°C. The samples were heated for 2 min to inactivate the enzyme, cooled to room temperature, and then subjected to antimicrobial activity testing [11] by using the agar well diffusion method.


## 2.4. Partial Purification via Ultrafiltration Membranes

The six crude BLIS samples were separated according to molecular weight by using 3 and 10 kDa ultracentrifuges (Amicon Ultra Centrifugal Unit, Regenerated Cellulose 3,000 and 10,000 MWCO, Merck Millipore Ltd., Germany) for 60 min at1,328.4×g and 4°C. This process separated the samples into three BLIS ultrafiltration fractions (BLIS-UF), i.e., <3 kDa, 3–10 kDa, and >10 kDa [26]. Each fraction was concentrated with a freeze dryer (Christ, Germany), adjusted to a final volume of 80 mL with deionized water, and then subjected to bacterial inhibitory activity tests to assess their bioactivity.


## 2.5. Quantification of Total Protein

The total protein concentration of each sample was determined on the basis of the Bradford method (Bradford 1976), which refers to Jamaluddin et al. [27]. A total of 160 μL of each sample was reacted with 40 μL of Bradford reagent in a 96-well microtiter plate and then incubated at 37°C in an orbital shaker for 10 min. Protein concentrations were determined by an ELISA reader (Bio-Rad) at 595 nm. Distilled water was used as a blank sample, and a protein standard curve was obtained using bovine serum albumin as a protein standard.


## 2.6. Determination of the Antibacterial Activity of BLIS

The antibacterial activity of BLIS was determined using the agar well diffusion method. A total of 25 mL of Mueller–Hinton agar (Oxoid, UK) containing the test bacteria (1%v/v; E. coli, S. Typhimurium, L. monocytogenes, and S. aureus) at a density of 1×106CFU·mL−1 was poured into the wells of a plate and allowed to solidify. A well (diameter, 6 mm) was made in the agar with a cork borer, and 100 μL samples of BLIS were placed into the wells. After incubation at 37°C for 24 h, the diameter of the inhibition zone (mm) was measured. Distilled water and ampicillin 10 μg·mL−1 were used as controls. The antibacterial activity of BLIS was expressed as activity (A) and specific activity (B) [28]. Purification fold (C) and yield activity (%) (D) of the samples were also calculated.BLIS activity (A) was calculated as shown in equation (1) and expressed in units of AU·mL−1, where 1 AU·mL−1 was defined as the unit area of the inhibition zone per unit volume (mm2·mL−1). In equation (1), Lz refers to the clear zone area (mm2), Ls refers to the well area (mm2), and V refers to the sample volume (mL).
(1)A=Lz−LsV.BLIS-specific activity (B) was defined as the ratio of the total BLIS activity of the sample to the total protein concentration (AU·μg−1) and calculated using equation (2), where A refers to the BLIS activity (AU·mL−1), Tp refers to the total protein concentration (μg) in the sample, and V refers to the sample volume (mL).
(2)B=A×VTp×V.Purification fold (C) was defined as the ratio of the specific activity of the fraction obtained at each purification step (Bp) to the specific activity of crude BLIS (B0) and calculated using equation (3), where Bp was the BLIS-specific activity of the fraction obtained at each steps of purification and B0 was the BLIS-specific activity of crude BLIS.
(3)C=BpB0.Yield activity (D) was defined as the ratio of the total BLIS activity (AU·mL−1) calculated at each step of purification to the total activity of crude BLIS and calculated using equation (4), where A was the BLIS activity (AU·mL−1) in each step, V was the sample volume (mL), and A0 was the BLIS activity of crude BLIS.
(4)D=A×VA0×100.


## 2.7. Analysis of Minimum Inhibitory and Bactericidal Concentrations

Determination of the MIC of BLIS was conducted by preparing BLIS samples at several concentrations (i.e., 10, 5.0, 2.5, 1.25, 0.625, 0.312, 0.156, 0.078, 0.04, 0.02, 0.009, and 0.004 mg·mL−1). Each BLIS sample was placed on a microplate that had previously been inoculated with 0.1 mL of test bacteria (density, 1×106) and 0.1 mL of MHB (HiMedia, India). The microplate was incubated for 24 h at 37°C. The growth of the test bacteria on the microplate was observed using a microplate reader (Bio-Rad) at 600 nm, and their viability was determined by the plate counting method [3]. The MIC was obtained from the lowest concentration of bacteriocin that could inhibit the growth of the test bacteria. The MBC of BLIS was evaluated as follows. After MIC determination, the liquid remaining on the microplate was inoculated with Mueller–Hinton agar (HiMedia) and then incubated for 24 h at 37°C. The MBC refers to the MIC of bacteriocin required to kill 99.9% of the tested bacteria.


## 2.8. Determination of the Temperature and pH Stability of BLIS

Evaluation of the temperature stability of BLIS was performed by dissolving all of the fractions in deionized water and then incubating the resulting solutions at 60, 80, or 100°C for 30 min or 121°C for 15 min. The solutions were cooled to room temperature and subjected to antibacterial activity tests [29]. The pH stability of BLIS was determined by adjusting the pH of the purified fractions to pH 2.0–10.0 by using 1 mol·L−1 HCl and 1 mol·L−1 NaOH as necessary and incubating for 2 h. Prior to the antibacterial activity test, the pH of the samples was set to pH 7.0. All antibacterial activity tests were performed by using the agar-well diffusion method, as described above.


## 2.9. Analysis of Toxicity by Using Brine Shrimp Lethality Assay

Artemia salina cysts were hatched in artificial seawater containing 27 g of NaCl in 3 L of distilled water and provided light and aeration. The larval hatching process was conducted for 48 h. Once hatched, the larval eggs were considered ready for analysis. The purified BLIS fraction showing the best specific activity was dissolved in artificial seawater to obtain test solutions with final BLIS concentrations of 2,000, 1,000, 200, and 20 μg·mL−1. A total of 1 mL of each test solution was transferred to a separate test tube and added with 10 two-day-old A. salina larvae by using a sterile syringe. The volume in the test tube was adjusted to 2 mL to achieve final BLIS concentrations of 1 mg·mL−1, 500 μg·mL−1, 100 μg·mL−1, and 10 μg·mL−1. The test tubes were incubated for 24 h, and the number of dead A. salina larvae was counted. The lethal concentration (LC50) of BLIS was determined via the probit analysis method with a 95% confidence interval using SPSS software. Each concentration was tested thrice, and artificial seawater was used as a negative control [30].


## 2.10. LC-MS/MS Analysis andIn Silico Study of the BLIS Fraction with the Greatest Activity

The BLIS fraction with the greatest antibacterial activity was dissolved in 1 mL of water, centrifuged for 1 min, and then injected into a NanoLC Ultimate 3000 Tandem Q Exactive Plus Orbitrap HRMS instrument equipped with a Thermo PepMap RSLC C18 capillary column (75μm×15cm, 3 μm, 100 Å) and a Thermo Scientific™ 164649 trap column (30 μm, 5 mm). The sample was eluted with H2O with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B) at a flow rate of 300 nL·min−1. The elution program was as follows: 2%–35% B for 30 min, 30%–90% B for 15 min, 90% B for 15 min, and 5% B for 30 min. Spectra were collected over the mass range of 200–2,000 m/z. The mass spectral results were analyzed using Proteome Discoverer 2.2 (Thermo Scientific), which uses the search engine SEQUEST HT, for database-based protein identification. The enzyme used was trypsin with a maximum miss cleavage of 2. The dynamic modifications applied were oxidation (for the amino acid methionine) and acetylation (for N-terminals). The percentages of hydrophobic and cationic amino acid residues were predicted using ProtParam (https://web.expasy.org/protparam/), and the physicochemical properties of the detected amino acid peptide sequences (i.e., sequence length, molecular weight (MW), hydrophobicity, and Boman index) were determined using PepDraw (https://www.pepdraw.com/).


## 2.11. Statistical Analysis

All experiments were performed as three independent replicates, and the results were expressed asmean±standard deviation. Data analysis was performed using SPSS 18.0 software. One-way analysis of variance (ANOVA) and Duncan’s multiple range test (95% confidence interval) were applied to detect significant differences between means.


## 3. Results and Discussion

### 3.1. Antibacterial Activity of Crude BLIS

The neutralized CFS of sixStreptomyces isolates showed potential inhibitory activity (Figure 1). The growth inhibition of E. coli and S. aureus was highest in the CFS of S. globisporus AIA12 and S. misionensis AIA17. Meanwhile, the lowest inhibition of growth of E. coli and S. aureus was found in CFS produced by S. labedae SCA-11 and S. variabilis AIA-10, respectively. ANOVA followed by Duncan’s multiple range test indicated that the growth absorbance of test bacteria exposed to CFS was significantly lower than that of test bacteria not exposed to CFS. This result indicates that the presence of extracellular metabolites produced by Streptomyces in CFS could inhibit the growth of the test bacteria and supports previous findings demonstrating the potential antibacterial properties of metabolites produced by Streptomyces isolated from the gut of C. chanos [23, 24]. The difference in antibacterial activity produced is due to differences in the concentration of extracellular metabolites produced [13]. Analysis of antibacterial activity using the agar well diffusion method confirmed these findings. Specifically, the concentrated CFS formed inhibition zones against all tested bacteria (Figure 2). These results show that crude BLIS has broad spectrum antibacterial activity.Figure 1
The growth absorbance (in 600 nm) of test bacteria in Mueller Hinton broth exposed to cell-free supernatant (CFS); white square:E. coli; light-gray square: S. aureus; C (−): control negative using Mueller Hinton broth inoculated with test bacteria without being exposed to CFS; C (+): control positive using ampicillin 10 μg·mL−1.Figure 2
Inhibition zone (mm) of concentrated cell-free supernatant (crude BLIS) against test bacteria determined using the agar well diffusion method; white square:E. coli; dark-gray square: S. aureus; light-gray square: L. monocytogenes; black square: S. Typhimurium; C (−): control negative using distilled water; C (+): control positive using ampicillin 10 μg·mL−1.The zones of inhibition determined in this study indicated that the six crude BLIS samples have better inhibitory activity against Gram-negative bacteria than against Gram-positive bacteria. This finding contradicts most studies reporting that bacteriocins are only active against Gram-positive bacteria [31, 32]. However, recent studies have reported the ability of several bacteriocins to inhibit Gram-negative bacteria. For instance, the bacteriocins synthesized by Streptomyces nigrescens inhibit Vibrio parahaemolyticus, bacteriocin SLG10 inhibits E. coli, and bacteriocin JLA-9 inhibits E. coli, S. Typhimurium, Pseudomonas fluorescens, and Shigella flexneri [3, 13, 33]. According to Pei et al. [3], the inhibitory activity of bacteriocins against Gram-negative bacteria is due to the ability of these substances to form interactions with nucleic acids and bacterial intracellular enzymes and damage the integrity of bacterial cell membranes. Choi and Lee [34] added that the inhibitory activity of bacteriocins against Gram-negative bacteria may also be due to their low MW. These bacteriocins can penetrate cell membranes through porins and directly attack the intracellular components of cells.


### 3.2. Proteinaceous Nature Properties of Crude BLIS

Since bacteriocins and BLIS are considered peptides, confirmation of the proteinaceous nature of these compounds is necessary. Analysis of the proteinaceous nature of the crude BLIS revealed that the antimicrobial activity of the six extracts againstE. coli was completely lost after treatment with proteinase-K and trypsin (Table 1). Moreover, whereas other crude BLIS fractions showed a significant decrease in activity, only the crude BLIS of S. variabilis SCA-11 and SCA-5 completely lost their activity following pepsin treatment. These findings are consistent with those of Lasik-Kurdyś and Sip [35], who reported that BLIS produced by Oenococcus oeni loses its activity after exposure to pronase E, proteinase-K, trypsin, pepsin, and α-chymotrypsin. Moračanin et al. [36] also found that the bacteriocin produced by Leuconostoc mesenteroides loses its antilisteria activity after exposure to pepsin, papain, and proteinase-K. These results confirm the proteinaceous nature of active metabolites in crude BLIS, which may be responsible for the latter’s antibacterial activity.Table 1
Inhibitory zone (mm) of crude BLIS after proteolytic enzyme treatment againstEscherichia coli.
TreatmentInhibition zone (mm)SCA-5SCA-8SCA-11AIA-10AIA-12AIA-17Proteinase-K000000Pepsin07.0±0.3207.3±0.036.4±0.077.4±0.10Trypsin000000Untreated control19.2±0.1016.5±0.1324.1±0.1222.6±0.0521.7±0.1820.4±0.19


### 3.3. Stability of Crude BLIS

Knowledge of the temperature and pH stability of BLIS is important because good stability is necessary to realize its applications in food processing. Among the crude samples assayed in this work, the crude BLIS ofS. variabilis SCA-5 and S. labedae SCA-8 were the most stable (Figure 3). Both extracts could maintain over 50% of their antibacterial activity after heating up to 100°C. Even in crude BLIS of S. variabilis SCA-5, the activity was maintained up to 121°C. Besides, both extracts were also able to maintain their antibacterial activity at exposure to a wide range of pH 2.0–7.0. However, significant decreases in activity occurred when the temperature and pH were increased beyond these ranges. This finding is consistent with the results of Hernández-Saldaña et al. [13], who reported that the bacteriocin produced by S. griseus and S. nigrescens shows activity loss of up to 50% at 100°C and stable at pH 3–7. Meanwhile, in S. violaceoruber and S. bottropensis, the activity decreased up to 70% at the same temperature. Du et al. [31] reported that plantaricin GZ1-27 maintains its activity at temperatures of up to 80°C for 30 min and is stable at pH 2.0–6.0.Figure 3
Inhibitory zone (mm) of crude BLIS after heat (a) and pH (b) treatment againstE. coli; ∎: SCA-5; ●: SCA-8; ⬦: SCA-11; ▲: AIA-10; ○: AIA-12; ⬥: AIA-17.
(a)(b)


### 3.4. Antibacterial Activity of BLIS Ultrafiltered Fractions

Antibacterial activity analysis using the agar well diffusion method showed that the BLIS ultrafiltered (BLIS-UF) fraction withMW>10kDa could produce the largest inhibition zone (Figure 4). This trend is similar to the trend of BLIS activity observed. However, BLIS-specific activity calculations indicated that this fraction has the lowest activity among the fractions obtained. BLIS-specific activity describes the ratio of BLIS activity to the total protein concentration, where this value is the activity per one μg of protein contained in the fraction. The greater the BLIS-specific activity of a fraction, the more active it is [28]. Compared with the BLIS-UF fraction with MW>10kDa, those with MW<3kDa and 3–10 kDa had higher BLIS-specific activity. These results indicate that the active fraction of BLIS Streptomyces has a low MW, consistent with previous studies reporting that BLIS produced by Streptomyces have MW<3kDa, such as Streptomyces griseus IFO 1330 (1,833 kDa), Streptomyces griseus XylebKG-1 ADFC02 (~2 kDa), and Streptomyces nigrescens ATCC 23941 (3 kDa). BLIS shows inhibitory activity against Bacillus cereus, Vibrio parahaemolyticus, Enterococcus casseliflavus, L. monocytogenes, and Micrococcus luteus [13, 16].Figure 4
Inhibition zone (mm) of BLIS ultrafiltration fraction based on molecular weight (<3 kDa, 3–10 kDa, and >10 kDa) againstE. coli (a), S. aureus (b), L. monocytogenes (c), and S. Typhimurium (d) using the agar well diffusion method; light-gray square: >10 kDa; dark-gray square: 3–10 kDa; white square: <3 kDa.
(a)(b)(c)(d)Among the fractions obtained, BLIS-UF SCA-8 withMW<3kDa showed the highest BLIS-specific activity. This fraction revealed broad spectrum antibacterial effects against E. coli, S. aureus, L. monocytogenes, and S. Typhimurium with specific activities of 635.6, 167.9, 664.5, and 615.5 AU·μg−1, respectively (Table 2). These results reflect an increase in BLIS-specific activity of up to 396.9 AU·μg−1 compared with that of crude BLIS. Such results may be attributed to the purification level of crude BLIS, which was increased by up to 2.5 times. Application of ultrafiltration membranes appeared to be quite effective in separating crude BLIS into purified fractions based on MW. Yield recoveries ranged from 15.7% to 99.6%. Among the fractions obtained, that with MW<3kDa revealed the lowest yield. The results of the present study are similar to those of Zacharof et al. [37], who found a decrease in yield recovery from 68% to 36% after filtration using 1 kDa MWCO nanofilters.Table 2
Total protein (μg), total BLIS activity (AU), BLIS-specific activity (AU·μg−1), purification level, and yield (%) during fractionation steps of BLIS produced by Streptomyces sp.
IsolatesPurification stepVolume (mL)Total protein (μg)Total BLIS activity (AU)aBLIS-specific activity (AU·μg−1)bPurification levelcYield (%)dECSALMSTECSALMSTECSALMSTECSALMSTSCA-5CFS1,20015,029NDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDCrude BLIS1002,914424,094131,187765,890492,080145.545.0262.8168.91.01.01.01.0100100100100BUF (kDa)>10802,741264,296131,152616,528453,04896.447.8224.9165.30.71.10.91.062.3100.080.592.13–10801,113224,456118,144627,040340,208201.6106.1563.2305.61.42.22.11.852.990.181.969.1<380845157,79293,152387,312397,928186.8110.3458.6471.21.31.01.72.837.271.050.680.9SCA-8CFS1,20014,044NDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDCrude BLIS1002,305850,141322,553616,900861,330368.8139.9267.6373.61.01.01.01.0100100100100BUF (kDa)>10801,793672,344244,136614,088785,864375.0136.2342.5438.31.01.01.31.279.175.799.591.23–1080858469,152242,688567,784533,010546.7266.2622.9584.71.51.92.31.655.275.292.061.9<380912579,334144,112570,245528,174635.6167.9664.5615.51.71.22.51.668.144.792.461.3SCA-11CFS1,20021,790NDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDCrude BLIS1002,332722,526253,013602,200664,730309.9108.5258.3285.11.01.01.01.0100100100100BUF (kDa)>10802,287664,184239,928598,320661,792290.4104.9261.6289.40.91.01.01.091.994.899.499.63–10801,098569,024232,968565,294575,300518.2212.2514.8523.91.72.02.01.878.892.193.986.5<380899385,736130,016421,728585,505429.2144.7469.2651.51.41.31.82.353.451.470.088.1AIA-10CFS1,20020,997NDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDCrude BLIS1002,237631,981137,861414,090308,081282.661.6185.2137.81.01.01.01.0100100100100BUF (kDa)>10802,228550,840116,624330,354297,728247.252.3148.3133.60.90.80.81.087.284.679.896.63–10801,288429,63995,344226,938185,438333.574.0176.2144.01.21.21.01.068.069.254.860.2<3801,059264,72833,76864,914305,260250.031.961.3288.30.90.50.32.141.924.515.799.1AIA-12CFS1,20019,219NDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDCrude BLIS1002,327564,889115,696497,410399,570242.849.7213.8171.71.01.01.01.0100100100100BUF (kDa)>10802,141538,42493,624432,776397,912251.543.7202.2185.91.00.90.91.195.380.987.099.63–1080910477,71560,080487,506275,671525.066.0535.8303.02.21.32.51.884.651.998.069.0<380794326,77618,200151,688307,120411.422.9191.0386.71.70.50.92.357.815.730.576.9AIA-17CFS1,20017,060NDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDCrude BLIS1002,250495,133269,670355,920312,520220.1119.9158.2138.91.01.01.01.0100100100100BUF (kDa)>10801,946214,480145,008254,319288,400110.274.5130.7148.20.50.60.81.143.353.871.592.33–10801,332388,558129,688193,157305,264291.897.4145.0229.21.30.80.91.778.548.154.397.7<380833295,27298,00021,457271,976354.5117.625.8326.51.61.00.22.459.636.36.087.0∗CFS: cell-free supernatant; BUF: BLIS ultrafiltration fraction; ND: not determined; tested bacteria: E. coli (EC), S. aureus (SA), L. monocytogenes (LM), and S. Typhimurium (ST). aTotal BLIS activity was determined by agar well diffusion, considering the total sample volume. bBLIS-specific activity was determined by the ratio between total BLIS activity and total protein content. cPurification level was determined by the ratio between specific activity in each step and the specific activity of crude BLIS. dYield was determined by the ratio between total BLIS activity in each step and the total activity in crude BLIS.


## 3.1. Antibacterial Activity of Crude BLIS

The neutralized CFS of sixStreptomyces isolates showed potential inhibitory activity (Figure 1). The growth inhibition of E. coli and S. aureus was highest in the CFS of S. globisporus AIA12 and S. misionensis AIA17. Meanwhile, the lowest inhibition of growth of E. coli and S. aureus was found in CFS produced by S. labedae SCA-11 and S. variabilis AIA-10, respectively. ANOVA followed by Duncan’s multiple range test indicated that the growth absorbance of test bacteria exposed to CFS was significantly lower than that of test bacteria not exposed to CFS. This result indicates that the presence of extracellular metabolites produced by Streptomyces in CFS could inhibit the growth of the test bacteria and supports previous findings demonstrating the potential antibacterial properties of metabolites produced by Streptomyces isolated from the gut of C. chanos [23, 24]. The difference in antibacterial activity produced is due to differences in the concentration of extracellular metabolites produced [13]. Analysis of antibacterial activity using the agar well diffusion method confirmed these findings. Specifically, the concentrated CFS formed inhibition zones against all tested bacteria (Figure 2). These results show that crude BLIS has broad spectrum antibacterial activity.Figure 1
The growth absorbance (in 600 nm) of test bacteria in Mueller Hinton broth exposed to cell-free supernatant (CFS); white square:E. coli; light-gray square: S. aureus; C (−): control negative using Mueller Hinton broth inoculated with test bacteria without being exposed to CFS; C (+): control positive using ampicillin 10 μg·mL−1.Figure 2
Inhibition zone (mm) of concentrated cell-free supernatant (crude BLIS) against test bacteria determined using the agar well diffusion method; white square:E. coli; dark-gray square: S. aureus; light-gray square: L. monocytogenes; black square: S. Typhimurium; C (−): control negative using distilled water; C (+): control positive using ampicillin 10 μg·mL−1.The zones of inhibition determined in this study indicated that the six crude BLIS samples have better inhibitory activity against Gram-negative bacteria than against Gram-positive bacteria. This finding contradicts most studies reporting that bacteriocins are only active against Gram-positive bacteria [31, 32]. However, recent studies have reported the ability of several bacteriocins to inhibit Gram-negative bacteria. For instance, the bacteriocins synthesized by Streptomyces nigrescens inhibit Vibrio parahaemolyticus, bacteriocin SLG10 inhibits E. coli, and bacteriocin JLA-9 inhibits E. coli, S. Typhimurium, Pseudomonas fluorescens, and Shigella flexneri [3, 13, 33]. According to Pei et al. [3], the inhibitory activity of bacteriocins against Gram-negative bacteria is due to the ability of these substances to form interactions with nucleic acids and bacterial intracellular enzymes and damage the integrity of bacterial cell membranes. Choi and Lee [34] added that the inhibitory activity of bacteriocins against Gram-negative bacteria may also be due to their low MW. These bacteriocins can penetrate cell membranes through porins and directly attack the intracellular components of cells.


## 3.2. Proteinaceous Nature Properties of Crude BLIS

Since bacteriocins and BLIS are considered peptides, confirmation of the proteinaceous nature of these compounds is necessary. Analysis of the proteinaceous nature of the crude BLIS revealed that the antimicrobial activity of the six extracts againstE. coli was completely lost after treatment with proteinase-K and trypsin (Table 1). Moreover, whereas other crude BLIS fractions showed a significant decrease in activity, only the crude BLIS of S. variabilis SCA-11 and SCA-5 completely lost their activity following pepsin treatment. These findings are consistent with those of Lasik-Kurdyś and Sip [35], who reported that BLIS produced by Oenococcus oeni loses its activity after exposure to pronase E, proteinase-K, trypsin, pepsin, and α-chymotrypsin. Moračanin et al. [36] also found that the bacteriocin produced by Leuconostoc mesenteroides loses its antilisteria activity after exposure to pepsin, papain, and proteinase-K. These results confirm the proteinaceous nature of active metabolites in crude BLIS, which may be responsible for the latter’s antibacterial activity.Table 1
Inhibitory zone (mm) of crude BLIS after proteolytic enzyme treatment againstEscherichia coli.
TreatmentInhibition zone (mm)SCA-5SCA-8SCA-11AIA-10AIA-12AIA-17Proteinase-K000000Pepsin07.0±0.3207.3±0.036.4±0.077.4±0.10Trypsin000000Untreated control19.2±0.1016.5±0.1324.1±0.1222.6±0.0521.7±0.1820.4±0.19


## 3.3. Stability of Crude BLIS

Knowledge of the temperature and pH stability of BLIS is important because good stability is necessary to realize its applications in food processing. Among the crude samples assayed in this work, the crude BLIS ofS. variabilis SCA-5 and S. labedae SCA-8 were the most stable (Figure 3). Both extracts could maintain over 50% of their antibacterial activity after heating up to 100°C. Even in crude BLIS of S. variabilis SCA-5, the activity was maintained up to 121°C. Besides, both extracts were also able to maintain their antibacterial activity at exposure to a wide range of pH 2.0–7.0. However, significant decreases in activity occurred when the temperature and pH were increased beyond these ranges. This finding is consistent with the results of Hernández-Saldaña et al. [13], who reported that the bacteriocin produced by S. griseus and S. nigrescens shows activity loss of up to 50% at 100°C and stable at pH 3–7. Meanwhile, in S. violaceoruber and S. bottropensis, the activity decreased up to 70% at the same temperature. Du et al. [31] reported that plantaricin GZ1-27 maintains its activity at temperatures of up to 80°C for 30 min and is stable at pH 2.0–6.0.Figure 3
Inhibitory zone (mm) of crude BLIS after heat (a) and pH (b) treatment againstE. coli; ∎: SCA-5; ●: SCA-8; ⬦: SCA-11; ▲: AIA-10; ○: AIA-12; ⬥: AIA-17.
(a)(b)


## 3.4. Antibacterial Activity of BLIS Ultrafiltered Fractions

Antibacterial activity analysis using the agar well diffusion method showed that the BLIS ultrafiltered (BLIS-UF) fraction withMW>10kDa could produce the largest inhibition zone (Figure 4). This trend is similar to the trend of BLIS activity observed. However, BLIS-specific activity calculations indicated that this fraction has the lowest activity among the fractions obtained. BLIS-specific activity describes the ratio of BLIS activity to the total protein concentration, where this value is the activity per one μg of protein contained in the fraction. The greater the BLIS-specific activity of a fraction, the more active it is [28]. Compared with the BLIS-UF fraction with MW>10kDa, those with MW<3kDa and 3–10 kDa had higher BLIS-specific activity. These results indicate that the active fraction of BLIS Streptomyces has a low MW, consistent with previous studies reporting that BLIS produced by Streptomyces have MW<3kDa, such as Streptomyces griseus IFO 1330 (1,833 kDa), Streptomyces griseus XylebKG-1 ADFC02 (~2 kDa), and Streptomyces nigrescens ATCC 23941 (3 kDa). BLIS shows inhibitory activity against Bacillus cereus, Vibrio parahaemolyticus, Enterococcus casseliflavus, L. monocytogenes, and Micrococcus luteus [13, 16].Figure 4
Inhibition zone (mm) of BLIS ultrafiltration fraction based on molecular weight (<3 kDa, 3–10 kDa, and >10 kDa) againstE. coli (a), S. aureus (b), L. monocytogenes (c), and S. Typhimurium (d) using the agar well diffusion method; light-gray square: >10 kDa; dark-gray square: 3–10 kDa; white square: <3 kDa.
(a)(b)(c)(d)Among the fractions obtained, BLIS-UF SCA-8 withMW<3kDa showed the highest BLIS-specific activity. This fraction revealed broad spectrum antibacterial effects against E. coli, S. aureus, L. monocytogenes, and S. Typhimurium with specific activities of 635.6, 167.9, 664.5, and 615.5 AU·μg−1, respectively (Table 2). These results reflect an increase in BLIS-specific activity of up to 396.9 AU·μg−1 compared with that of crude BLIS. Such results may be attributed to the purification level of crude BLIS, which was increased by up to 2.5 times. Application of ultrafiltration membranes appeared to be quite effective in separating crude BLIS into purified fractions based on MW. Yield recoveries ranged from 15.7% to 99.6%. Among the fractions obtained, that with MW<3kDa revealed the lowest yield. The results of the present study are similar to those of Zacharof et al. [37], who found a decrease in yield recovery from 68% to 36% after filtration using 1 kDa MWCO nanofilters.Table 2
Total protein (μg), total BLIS activity (AU), BLIS-specific activity (AU·μg−1), purification level, and yield (%) during fractionation steps of BLIS produced by Streptomyces sp.
IsolatesPurification stepVolume (mL)Total protein (μg)Total BLIS activity (AU)aBLIS-specific activity (AU·μg−1)bPurification levelcYield (%)dECSALMSTECSALMSTECSALMSTECSALMSTSCA-5CFS1,20015,029NDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDCrude BLIS1002,914424,094131,187765,890492,080145.545.0262.8168.91.01.01.01.0100100100100BUF (kDa)>10802,741264,296131,152616,528453,04896.447.8224.9165.30.71.10.91.062.3100.080.592.13–10801,113224,456118,144627,040340,208201.6106.1563.2305.61.42.22.11.852.990.181.969.1<380845157,79293,152387,312397,928186.8110.3458.6471.21.31.01.72.837.271.050.680.9SCA-8CFS1,20014,044NDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDCrude BLIS1002,305850,141322,553616,900861,330368.8139.9267.6373.61.01.01.01.0100100100100BUF (kDa)>10801,793672,344244,136614,088785,864375.0136.2342.5438.31.01.01.31.279.175.799.591.23–1080858469,152242,688567,784533,010546.7266.2622.9584.71.51.92.31.655.275.292.061.9<380912579,334144,112570,245528,174635.6167.9664.5615.51.71.22.51.668.144.792.461.3SCA-11CFS1,20021,790NDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDCrude BLIS1002,332722,526253,013602,200664,730309.9108.5258.3285.11.01.01.01.0100100100100BUF (kDa)>10802,287664,184239,928598,320661,792290.4104.9261.6289.40.91.01.01.091.994.899.499.63–10801,098569,024232,968565,294575,300518.2212.2514.8523.91.72.02.01.878.892.193.986.5<380899385,736130,016421,728585,505429.2144.7469.2651.51.41.31.82.353.451.470.088.1AIA-10CFS1,20020,997NDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDCrude BLIS1002,237631,981137,861414,090308,081282.661.6185.2137.81.01.01.01.0100100100100BUF (kDa)>10802,228550,840116,624330,354297,728247.252.3148.3133.60.90.80.81.087.284.679.896.63–10801,288429,63995,344226,938185,438333.574.0176.2144.01.21.21.01.068.069.254.860.2<3801,059264,72833,76864,914305,260250.031.961.3288.30.90.50.32.141.924.515.799.1AIA-12CFS1,20019,219NDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDCrude BLIS1002,327564,889115,696497,410399,570242.849.7213.8171.71.01.01.01.0100100100100BUF (kDa)>10802,141538,42493,624432,776397,912251.543.7202.2185.91.00.90.91.195.380.987.099.63–1080910477,71560,080487,506275,671525.066.0535.8303.02.21.32.51.884.651.998.069.0<380794326,77618,200151,688307,120411.422.9191.0386.71.70.50.92.357.815.730.576.9AIA-17CFS1,20017,060NDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDCrude BLIS1002,250495,133269,670355,920312,520220.1119.9158.2138.91.01.01.01.0100100100100BUF (kDa)>10801,946214,480145,008254,319288,400110.274.5130.7148.20.50.60.81.143.353.871.592.33–10801,332388,558129,688193,157305,264291.897.4145.0229.21.30.80.91.778.548.154.397.7<380833295,27298,00021,457271,976354.5117.625.8326.51.61.00.22.459.636.36.087.0∗CFS: cell-free supernatant; BUF: BLIS ultrafiltration fraction; ND: not determined; tested bacteria: E. coli (EC), S. aureus (SA), L. monocytogenes (LM), and S. Typhimurium (ST). aTotal BLIS activity was determined by agar well diffusion, considering the total sample volume. bBLIS-specific activity was determined by the ratio between total BLIS activity and total protein content. cPurification level was determined by the ratio between specific activity in each step and the specific activity of crude BLIS. dYield was determined by the ratio between total BLIS activity in each step and the total activity in crude BLIS.


## 4. MIC and MBC of BLIS Ultrafiltration SCA-8 withMW<3kDa Fraction

The MIC and MBC of BLIS-UF SCA-8 withMW<3kDa are shown in Table 3. This fraction revealed MICs ranging from 0.04 mg·mL−1 to 0.62 mg·mL−1 and MBCs ranging from 0.08 mg·mL−1 to 1.25 mg·mL−1. The lowest MIC of this fraction (0.04 mg·mL−1) was obtained in S. aureus and L. monocytogenes, and its lowest MBC (0.08 mg·mL−1) was observed in S. aureus. The MIC and MBC obtained in this study are higher than those of the bacteriocins SLG10 and JLA-9. The bacteriocin SLG10 produced by Lactobacillus plantarum shows MICs in the range of 0.016–0.032 mg·mL−1 and MBCs in the range of 0.016–0.064 mg·mL−1 and is most lethal to L. monocytogenes CICC 21529 [3]. Bacteriocin JLA-9 has MBCs in the range of 0.016–0.032 mg·mL−1 [33]. Differences in MICs and MBCs obtained in this study relative to those in previous reports may be related to the purity of the bacteriocins. Specifically, bacteriocins SLG10 and JLA-9 were tested in their pure form, whereas BLIS-UF SCA-8 with MW<3kDa is only semipurified at best. Sharma et al. [38] indicated that further purification may reduce MIC, which has implications for smaller MIC doses in certain microorganisms.Table 3
The MIC and MBC of BLIS-UF SCA-8 with molecular weight less than 3 kDa.
Bacterial testMIC (mg·mL−1)MBC (mg·mL−1)Escherichia coli ATCC 259220.310.62Salmonella Typhimurium ATCC 140280.621.25Staphylococcus aureus ATCC 259230.040.08Listeria monocytogenes ATCC 351520.040.16


### 4.1. Stability of BLIS Ultrafiltration SCA-8 withMW<3kDa Fraction

Among the fractions obtained, BLIS-UF SCA-8 withMW<3kDa revealed the greatest BLIS-specific activity. Stability analysis of this fraction showed better stability at high temperatures and pH, as well as improved sensitivity to enzymes, compared with crude BLIS. BLIS-UF SCA-8 with MW<3kDa retained over 70% of its antibacterial activity despite exposure to temperatures of 60–100°C for 30 min and solution pH of 2.0–7.0. It also showed enhanced sensitivity to pepsin (Table 4). Considering that food materials comprise various matrices and are subjected to various processing methods, such as heating or acidification [39, 40], the excellent stability of BLIS-UF SCA-8 with MW<3kDa reveals its promising potential use as a food biopreservative.
(5)∗Residueactivity=inhibitionzoneofthreatedsample−6inhibitionzoneofunthreatedcontrol−6×100.Table 4
SCA-8 withMW<3kDa against Escherichia coli.
Stability testInhibition zone (mm)Residue activity (%)∗Heat60°C23.78±0.2597.1380°C23.50±0.0795.63100°C18.90±0.4970.49121°C12.23±0.3234.02pH222.80±0.2191.80422.00±0.2887.43623.53±0.1195.77721.85±0.4286.61814.83±0.2548.221000EnzymeProteinase-K00Pepsin6.35±0.141.93Trypsin00Untreated control24.3±0.14100


### 4.2. Median Lethal Concentration of BLIS Ultrafiltration SCA-8 withMW<3kDa Fraction

The most promising BLIS fraction, BLIS-UF SCA-8 withMW<3kDa, was subjected to the brine shrimp lethality assay to determine its LC50 [41]. The test results showed that the mortality rate of A. salina increased with increasing BLIS concentration, in which a probit test showed that this fraction has LC50 of 226.975 μg·mL−1 (moderate toxicity) (Table 5). Hamidi et al. [42] classified toxicity based on LC50 as follows: highly toxic, 0–100 μg·mL−1; moderately toxic, 100–500 μg·mL−1; weakly toxic, 500–1,000 μg·mL−1; and nontoxic, >1,000 μg·mL−1. The LC50 of BLIS-UF SCA-8 with MW<3kDa was higher than that of the purified bacteriocin from Lactobacillus lactis, which revealed an LC50 of 21.54 μg·mL−1 [43]. The difference in LC50 between BLIS samples may be attributed to differences in the purity of the samples.Table 5
LC50 by the brine shrimp lethality test of most potential BLIS SCA-8 with MW<3kDa fraction.
SampleConcentration (μg·mL−1)Mortality (%)LC50 (μg·mL−1)BLIS SCA-8 withMW<3100096.6226.97550066.6100401013.3LC50 in A. salina and LD50 in animal models has a positive correlation, in which LC50<10μg·mL−1 has an LD50 of 100–1,000 mg·kg−1, LC50<20μg·mL−1 has an LD50 of 1,000–2,500 mg·kg−1, and LC50>25μg·mL−1 has an LD50 of 2,500–8,000 mg·kg−1 [44]. On the basis of these studies, BLIS-UF SCA-8 with MW<3kDa may be assumed to have an LD50 of over 2500 mg·kg−1 (weakly toxic) because its LC50 to A. salina is 226.975 μg·mL−1. Erhirhie et al. [45] classified LD50 according to the dose range as follows: extremely toxic, <5 mg·kg−1; highly toxic, 5–50 mg·kg−1; moderately toxic, 50–500 mg·kg−1; weakly toxic, 500–5,000 mg·kg−1; nontoxic, 5000–15,000 mg·kg−1, and relatively harmless, >15,000 mg·kg−1. These findings demonstrate that BLIS-UF SCA-8 with MW<3kDa may be developed as a promising preservative agent.


### 4.3. Identification of the Peptide Sequence of the BLIS Ultrafiltration SCA-8 withMW<3kDa Fraction

Peptide identification by LC-MS/MS can provide information on the molecular mass of the amino acid sequence of the peptide constituents of a sample [42]. LC-MS/MS of BLIS-UF SCA-8 with MW<3kDa followed by database-based protein analysis using Proteome Discoverer 2.2 succeeded in identifying 130 proteins (retention times of 15.21–19.98 min) (Figure 5; Table 6). Eleven peptides with over 60% relative abundance and MW ranging from 1.34 kDa to 2.90 kDa appeared to be responsible for the antibacterial activity of this fraction (Table 6). In silico analysis of physicochemical properties using ProtParam (https://web.expasy.org/protparam/) and PepDraw (http://www.tulane.edu/~biochem/WW/PepDraw/) revealed that each peptide having hydrophobic amino acids ranging from 38.5 to 75%,and high hydrophobicity (15.34–26.5 kcal·mol−1) (Table 6). Gadde et al. [46] found that antimicrobial peptides are generally hydrophobic and cationic. Cationic properties may help promote interactions between peptides and bacterial cell membranes or walls [47]. Hydrophobic properties could destroy cell walls or membranes by forming pores, resulting in cell lysis [48]. Protein-binding identification (Boman index) was conducted using an APD-based prediction program (http://aps.unmc.edu/AP/main.php). The detected proteins revealed high protein binding potential, especially those detected at 16.77 min (Table 6). Boman [49] stated that if a peptide or protein has a positive Boman index and is over 2.48 kcal·mol−1, the protein or peptide is highly likely to be able to bind to bacterial cell membranes.Figure 5
LC-MS/MS chromatogram profile of BLIS UF-SCA-8 withMW<3kDa fraction.Table 6
Identified peptide sequences (relative abundance > 60%) by Proteome Discoverer 2.2 (Thermo Scientific) in BLIS-UF SCA-8 withMW<3kDa fraction.
RT (min)Relative abundance (%)AccessionSequenceMolecular weight (Da)Number of amino acidHydrophobicity (kcal·mol−1)Boman index16.1161.67F2IY40TAVTEWTVAHAGGER1584.771520.841.5616.5261.37A0A2A4MKN2VTMNKEIDGLER1420.711222.161.1616.7769.94A0A087LGR3EQLSAFLQSEEGK1465.711321.492.916.9182A0A1C9VDC5SAGPMERIFASGMR1345.661415.342.1716.9386.02K2NT65AGGAYVPIDPGYPSER1493.691619.311.8117.0898.53A0A087LFK0EVGASSKADMGK1195.561223.861.9217.46100A0A2G7E6K9TAAGLEAGETVR1174.601219.561.1718.0777.3A0A217EF20QAAPYPLPADPDAR1481.731418.361.79518.898.53A0A1U9ZU28AFDPPPTTHPPATDAR1690.811620.561.4519.4471.08A0A397RL80DLGAWPALPGEDTSTSSSGPTAAPSISAHQ2908.353026.221.8419.9880.64A0A1B1AP88RDAPGASPGGTAGRGGPR1636.821826.52.27Accession: the master protein accession as found in the database (UniProt).Bacteriocins may be divided into three classes. Class I includes bacteriocins with posttranslational modified amino acid, which heat stable, have a low MW (<5 kDa), and are only able to inhibit Gram-positive bacteria. Class II includes bacteriocins with low MW (<10 kDa), heat stable, which do not contain a posttranslational modified amino acid and can inhibit both Gram-positive and negative bacteria. Finally, class III includes bacteriocins with large MW (>30 kDa) and is generally heat labile [50]. According to this classification and the data obtained in the present study, BLIS-UF SCA-8 with MW<3kDa may be a class II bacteriocin. This assumption is based on the MW of BLIS-UF SCA-8 (<3 kDa), its heat stable characteristics, its ability to inhibit Gram-positive and negative bacteria, and the absence of a posttranslational modified amino acid in its peptide sequence.


## 4.1. Stability of BLIS Ultrafiltration SCA-8 withMW<3kDa Fraction

Among the fractions obtained, BLIS-UF SCA-8 withMW<3kDa revealed the greatest BLIS-specific activity. Stability analysis of this fraction showed better stability at high temperatures and pH, as well as improved sensitivity to enzymes, compared with crude BLIS. BLIS-UF SCA-8 with MW<3kDa retained over 70% of its antibacterial activity despite exposure to temperatures of 60–100°C for 30 min and solution pH of 2.0–7.0. It also showed enhanced sensitivity to pepsin (Table 4). Considering that food materials comprise various matrices and are subjected to various processing methods, such as heating or acidification [39, 40], the excellent stability of BLIS-UF SCA-8 with MW<3kDa reveals its promising potential use as a food biopreservative.
(5)∗Residueactivity=inhibitionzoneofthreatedsample−6inhibitionzoneofunthreatedcontrol−6×100.Table 4
SCA-8 withMW<3kDa against Escherichia coli.
Stability testInhibition zone (mm)Residue activity (%)∗Heat60°C23.78±0.2597.1380°C23.50±0.0795.63100°C18.90±0.4970.49121°C12.23±0.3234.02pH222.80±0.2191.80422.00±0.2887.43623.53±0.1195.77721.85±0.4286.61814.83±0.2548.221000EnzymeProteinase-K00Pepsin6.35±0.141.93Trypsin00Untreated control24.3±0.14100


## 4.2. Median Lethal Concentration of BLIS Ultrafiltration SCA-8 withMW<3kDa Fraction

The most promising BLIS fraction, BLIS-UF SCA-8 withMW<3kDa, was subjected to the brine shrimp lethality assay to determine its LC50 [41]. The test results showed that the mortality rate of A. salina increased with increasing BLIS concentration, in which a probit test showed that this fraction has LC50 of 226.975 μg·mL−1 (moderate toxicity) (Table 5). Hamidi et al. [42] classified toxicity based on LC50 as follows: highly toxic, 0–100 μg·mL−1; moderately toxic, 100–500 μg·mL−1; weakly toxic, 500–1,000 μg·mL−1; and nontoxic, >1,000 μg·mL−1. The LC50 of BLIS-UF SCA-8 with MW<3kDa was higher than that of the purified bacteriocin from Lactobacillus lactis, which revealed an LC50 of 21.54 μg·mL−1 [43]. The difference in LC50 between BLIS samples may be attributed to differences in the purity of the samples.Table 5
LC50 by the brine shrimp lethality test of most potential BLIS SCA-8 with MW<3kDa fraction.
SampleConcentration (μg·mL−1)Mortality (%)LC50 (μg·mL−1)BLIS SCA-8 withMW<3100096.6226.97550066.6100401013.3LC50 in A. salina and LD50 in animal models has a positive correlation, in which LC50<10μg·mL−1 has an LD50 of 100–1,000 mg·kg−1, LC50<20μg·mL−1 has an LD50 of 1,000–2,500 mg·kg−1, and LC50>25μg·mL−1 has an LD50 of 2,500–8,000 mg·kg−1 [44]. On the basis of these studies, BLIS-UF SCA-8 with MW<3kDa may be assumed to have an LD50 of over 2500 mg·kg−1 (weakly toxic) because its LC50 to A. salina is 226.975 μg·mL−1. Erhirhie et al. [45] classified LD50 according to the dose range as follows: extremely toxic, <5 mg·kg−1; highly toxic, 5–50 mg·kg−1; moderately toxic, 50–500 mg·kg−1; weakly toxic, 500–5,000 mg·kg−1; nontoxic, 5000–15,000 mg·kg−1, and relatively harmless, >15,000 mg·kg−1. These findings demonstrate that BLIS-UF SCA-8 with MW<3kDa may be developed as a promising preservative agent.


## 4.3. Identification of the Peptide Sequence of the BLIS Ultrafiltration SCA-8 withMW<3kDa Fraction

Peptide identification by LC-MS/MS can provide information on the molecular mass of the amino acid sequence of the peptide constituents of a sample [42]. LC-MS/MS of BLIS-UF SCA-8 with MW<3kDa followed by database-based protein analysis using Proteome Discoverer 2.2 succeeded in identifying 130 proteins (retention times of 15.21–19.98 min) (Figure 5; Table 6). Eleven peptides with over 60% relative abundance and MW ranging from 1.34 kDa to 2.90 kDa appeared to be responsible for the antibacterial activity of this fraction (Table 6). In silico analysis of physicochemical properties using ProtParam (https://web.expasy.org/protparam/) and PepDraw (http://www.tulane.edu/~biochem/WW/PepDraw/) revealed that each peptide having hydrophobic amino acids ranging from 38.5 to 75%,and high hydrophobicity (15.34–26.5 kcal·mol−1) (Table 6). Gadde et al. [46] found that antimicrobial peptides are generally hydrophobic and cationic. Cationic properties may help promote interactions between peptides and bacterial cell membranes or walls [47]. Hydrophobic properties could destroy cell walls or membranes by forming pores, resulting in cell lysis [48]. Protein-binding identification (Boman index) was conducted using an APD-based prediction program (http://aps.unmc.edu/AP/main.php). The detected proteins revealed high protein binding potential, especially those detected at 16.77 min (Table 6). Boman [49] stated that if a peptide or protein has a positive Boman index and is over 2.48 kcal·mol−1, the protein or peptide is highly likely to be able to bind to bacterial cell membranes.Figure 5
LC-MS/MS chromatogram profile of BLIS UF-SCA-8 withMW<3kDa fraction.Table 6
Identified peptide sequences (relative abundance > 60%) by Proteome Discoverer 2.2 (Thermo Scientific) in BLIS-UF SCA-8 withMW<3kDa fraction.
RT (min)Relative abundance (%)AccessionSequenceMolecular weight (Da)Number of amino acidHydrophobicity (kcal·mol−1)Boman index16.1161.67F2IY40TAVTEWTVAHAGGER1584.771520.841.5616.5261.37A0A2A4MKN2VTMNKEIDGLER1420.711222.161.1616.7769.94A0A087LGR3EQLSAFLQSEEGK1465.711321.492.916.9182A0A1C9VDC5SAGPMERIFASGMR1345.661415.342.1716.9386.02K2NT65AGGAYVPIDPGYPSER1493.691619.311.8117.0898.53A0A087LFK0EVGASSKADMGK1195.561223.861.9217.46100A0A2G7E6K9TAAGLEAGETVR1174.601219.561.1718.0777.3A0A217EF20QAAPYPLPADPDAR1481.731418.361.79518.898.53A0A1U9ZU28AFDPPPTTHPPATDAR1690.811620.561.4519.4471.08A0A397RL80DLGAWPALPGEDTSTSSSGPTAAPSISAHQ2908.353026.221.8419.9880.64A0A1B1AP88RDAPGASPGGTAGRGGPR1636.821826.52.27Accession: the master protein accession as found in the database (UniProt).Bacteriocins may be divided into three classes. Class I includes bacteriocins with posttranslational modified amino acid, which heat stable, have a low MW (<5 kDa), and are only able to inhibit Gram-positive bacteria. Class II includes bacteriocins with low MW (<10 kDa), heat stable, which do not contain a posttranslational modified amino acid and can inhibit both Gram-positive and negative bacteria. Finally, class III includes bacteriocins with large MW (>30 kDa) and is generally heat labile [50]. According to this classification and the data obtained in the present study, BLIS-UF SCA-8 with MW<3kDa may be a class II bacteriocin. This assumption is based on the MW of BLIS-UF SCA-8 (<3 kDa), its heat stable characteristics, its ability to inhibit Gram-positive and negative bacteria, and the absence of a posttranslational modified amino acid in its peptide sequence.


## 5. Conclusions

Streptomyces is a promising producer of bioactive metabolites. The present study showed that six isolates of Streptomyces isolated from the gut of C. chanos could produce BLIS. The proteinaceous properties of BLIS were confirmed with assays involving several proteolytic enzymes. Crude BLIS showed broad spectrum antibacterial activity and high temperature and pH stability. Fractionation with ultrafiltration membranes revealed that BLIS fractions with MWs of <3 kDa and 3–10 kDa present the greatest activity. Calculation of the specific activities of these fractions demonstrated that BLIS-UF SCA-8 with MW<3kDa was more active than the fraction with MW of 3–10 kDa. BLIS-UF SCA-8 with MW<3kDa showed an increase in purity of up to 2.5 times, good pH and heat stability, and moderate toxicity to A. salina. Identification of the peptide sequence of this fraction through LC-MS/MS revealed that the peptides of this fraction are mostly composed of hydrophobic amino acids with low MW. Overall, the results of this study demonstrate the promising potential of BLIS Streptomyces as a candidate antibacterial agent for food applications.



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*Source: 7190152-2021-12-14.xml*