Subtilis Natto Research Articles

Bacillus subtilis natto Derivatives Inhibit Enterococcal Biofilm Formation via Restructuring of the Cell Envelope.

Enterococcus faecalis is considered a leading cause of hospital-acquired infections. Treatment of these infections has become a major challenge for clinicians because some E. faecalis strains are resistant to multiple clinically used antibiotics. Moreover, the presence of E. faecalis biofilms can make infections with E. faecalis more difficult to eradicate with current antibiotic therapies. Thus, our aim in this study was to investigate the effects of probiotic derivatives against E. faecalis biofilm formation. Bacillus subtilis natto is a probiotic strain isolated from Japanese fermented soybean foods, and its culture fluid potently inhibited adherence to Caco-2 cell monolayers, aggregation, and biofilm production without inhibiting the growth of E. faecalis. An apparent decrease in the thickness of E. faecalis biofilms was observed through confocal laser scanning microscopy. In addition, exopolysaccharide synthesis in E. faecalis biofilms was reduced by B. subtilis natto culture fluid treatment. Carbohydrate composition analysis also showed that carbohydrates in the E. faecalis cell envelope were restructured. Furthermore, transcriptome sequencing revealed that the culture fluid of B. subtilis natto downregulated the transcription of genes involved in the WalK/WalR two-component system, peptidoglycan biosynthesis and membrane glycolipid biosynthesis, which are all crucial for E. faecalis cell envelope synthesis and biofilm formation. Collectively, our work shows that some derivatives present in the culture fluid of B. subtilis natto may be useful for controlling E. faecalis biofilms.

A Novel Effective Formulation of Bioactive Compounds for Wound Healing: Preparation, In Vivo Characterization, and Comparison of Various Postbiotics Cold Creams in a Rat Model

The wound is a break in the integrity of the skin produced by injury, illness, or operation. Wound healing is an essential dynamic biological/physiological process that occurs in response to tissue damage. The huge health, economic, and social effects of wounds on patients and societies necessitate the research to find novel potential therapeutic agents in order to promote wound healing. Postbiotics, the newest member of the biotics family, are valuable functional bioactive substances produced by probiotics through their metabolic activity, which have several beneficial properties, including immunomodulatory, anti-inflammatory, antimicrobial, and angiogenesis characteristics, resulting in acceleration of wound healing. In the current study, three topical cold cream formulations containing postbiotics obtained from Lactobacillus fermentum, Lactobacillus reuteri, or Bacillus subtilis sp. natto probiotic strains were prepared. The effectiveness and wound healing activity of the developed postbiotics cold cream formulations were investigated compared to cold cream without postbiotics and no treatment via wound closure investigation, hydroxyproline content assay, and histological assessment in 25 Sprague Dawley rats divided into five groups. Interestingly, analysis of the results revealed that all three formulations containing postbiotics significantly accelerated the wound healing process. However, in general, the Bacillus subtilis natto cold cream manifested a better wound healing property. The pleasing wound healing characteristics of the topical postbiotics cold creams through the in vivo experiment suggest that formulations containing postbiotics can be considered as a promising nominee for wound healing approaches.

Preparation of pH-sensitive carboxymethyl cellulose/chitosan/alginate hydrogel beads with reticulated shell structure to deliver Bacillus subtilis natto

pH-sensitive hydrogels have been applied in delivering probiotics and drugs. However, pH sensitivity has been found to be contradictory with structural stability in hydrogel preparation. In this work, a novel strategy based on two systems of sodium carboxymethyl cellulose (CMC)/chitosan (CS) and sodium alginate (SA)/calcium chloride was designed to construct a reticulated shell structure stable for 3 h in simulated gastric fluid (pH 1.2) but began to break up at 2 h in simulated intestinal fluid (pH 6.8), exhibiting obvious pH sensitivity. The embedding rate of Bacillus subtilis natto reached to 67.3%, and the sustained release lasted for more than 10 h. It is implicated that the reticulated shell structure has harmoniously balanced the two incompatible properties of pH sensitivity and sustained release of CMC/CS/SA beads.

Heterologous expression of nattokinase from B. subtilis natto using Pichia pastoris GS115 and assessment of its thrombolytic activity

BackgroundNattokinase is a fibrinolytic enzyme that has huge market value as a nutritional supplement for health promotion. In order to increase nattokinase yields, fermentation conditions, strains, cultivation media, and feeding strategies have been optimized. Nattokinase has been expressed using several heterologous expression systems. Pichia pastoris heterologous expression system was the alternative.ResultsThis report aimed to express high levels of nattokinase from B. subtilis natto (NK-Bs) using a Pichia pastoris heterologous expression system and assess its fibrinolytic activity in vivo. Multicopy expression strains bearing 1–7 copies of the aprN gene were constructed. The expression level of the target protein reached a maximum at five copies of the target gene. However, multicopy expression strains were not stable in shake-flask or high-density fermentation, causing significant differences in the yield of the target protein among batches. Therefore, P. pastoris bearing a single copy of aprN was used in shake-flask and high-density fermentation. Target protein yield was 320 mg/L in shake-flask fermentation and approximately 9.5 g/L in high-density fermentation. The recombinant nattokinase showed high thermo- and pH-stability. The present study also demonstrated that recombinant NK-Bs had obvious thrombolytic activity.ConclusionsThis study suggests that the P. pastoris expression system is an ideal platform for the large-scale, low-cost preparation of nattokinase.

The expression of β-glucosidase during natto fermentation increased the active isoflavone content

Natto is a traditional food fermented by Bacillus subtilis natto with soybean as raw material and contains a variety of active substances, especially flavonoid. Flavonoids in soybean usually exist in the form of inactive glycosides, which can only play biological activities when being decomposed into free isoflavone aglycone by β-glucosidase. In this study, the relationship between the activity of β-glucosidase, the expression of β-glucosidase coding genes and the content and structure of active isoflavones in Bacillus natto was systematically elucidated for the first time. In this research, more than 70 kinds of flavonoids have been identified and the active isoflavones in natto increased by 2.2 times compared with soybeans. Furthermore, the activity of β-glucosidase and the expression level of its coding genes were both increased, which laid a theoretical foundation for further understanding the active substances and physiological functions of natto and the application of β-glucosidase in food industry.

Comparative transcriptome analysis reveals the underlying mechanism for over-accumulation of menaquinone-7 in Bacillus subtilis natto mutant

This study combined whole-genome sequencing, compound mutagenesis, and comparative transcriptome analysis methods to investigate the menaquinone-7 (MK-7) metabolism and regulation in Bacillus subtilis natto. Whole genome sequencing identified that the wild-type MK-7 producing strain BN0 contains one single circular chromosome in size of 4,119,230 bp, harboring 4603 protein-coding genes, and two plasmids in size of 64,309 and 5820 bp respectively. In addition, UV-ARTP (Ultraviolet -atmospheric and room temperature plasm) compound mutagenesis was performed in BN0, resulting in 50.6% improvement of MK-7 production in the mutant BN19-A. Further comparative transcriptome analysis revealed that: (1) the key genes (e.g glpA, dxs, ispF and ispH) involved in the glycerol metabolism pathway and MEP pathway were significantly up-regulated; (2) most genes related to the formation of spore were down-regulated; and (3) many genes (e.g tatA, bmr and bcr) involved in Tat secretion pathway, ABC transporters and MFS transporters were up-regulated. These results help us get insights into the underlying metabolic mechanism of MK-7 production in B. subtilis natto, and provide more potential molecular targets to be engineered for further strain improvement.

Live Bacillus subtilis natto Promotes Rumen Fermentation by Modulating Rumen Microbiota In Vitro.

Simple SummaryAlthough there is much research on the applications of Bacillus subtilis natto in dairy cows, the regulation of it on rumen microorganisms and the mechanisms of microbiota that affect rumen fermentation is still unclear, such as the mechanism of improving ruminal ammonia nitrogen concentration and the pathway of increasing propionic acid production. In this study, we explored the effects of live and autoclaved B. subtilis natto on rumen microbiota in vitro by 16S rRNA gene sequencing to clarify the ruminal microbial composition and diversity and their underlying mechanisms.Previous studies have shown that Bacillus subtilis natto affects rumen fermentation and rumen microbial community structure, which are limited to detect a few microbial abundances using traditional methods. However, the regulation of B. subtilis natto on rumen microorganisms and the mechanisms of microbiota that affect rumen fermentation is still unclear. This study explored the effects of live and autoclaved B. subtilis natto on ruminal microbial composition and diversity in vitro using 16S rRNA gene sequencing and the underlying mechanisms. Rumen fluid was collected, allocated to thirty-six bottles, and divided into three treatments: CTR, blank control group without B. subtilis natto; LBS, CTR with 109 cfu of live B. subtilis natto; and ABS, CTR with 109 cfu of autoclaved B. subtilis natto. The rumen fluid was collected after 0, 6, 12, and 24 h of fermentation, and pH, ammonia nitrogen (NH3-N), microbial protein (MCP), and volatile fatty acids (VFAs) were determined. The diversity and composition of rumen microbiota were assessed by 16S rRNA gene sequencing. The results revealed LBS affected the concentrations of NH3-N, MCP, and VFAs (p < 0.05), especially after 12 h, which might be attributed to changes in 18 genera. Whereas ABS only enhanced pH and NH3-N concentration compared with the CTR group (p < 0.05), which might be associated with changes in six genera. Supplementation with live B. subtilis natto improved ruminal NH3-N and propionate concentrations, indicating that live bacteria were better than autoclaved ones. This study advances our understanding of B. subtilis natto in promoting ruminal fermentation, providing a new perspective for the precise utilization of B. subtilis natto in dairy rations.

Influence of the Medium Composition and the Culture Conditions on Surfactin Biosynthesis by a Native Bacillus subtilis natto BS19 Strain.

An effective microbial synthesis of surfactin depends on the composition of the culture medium, the culture conditions and the genetic potential of the producer strain. The aim of this study was to evaluate the suitability of various medium components for the surfactin producing strain and to determine the impact of the culture conditions on the biosynthesis of surfactin isoforms by the newly isolated native strain Bacillus subtilis natto BS19. The efficiency of surfactin biosynthesis was determined by measuring the surface tension of the medium before and after submerged culture (SmF) and by qualitative and quantitative analysis of the obtained compound by high performance liquid chromatography. The highest efficiency of surfactin biosynthesis was achieved using starch as the carbon source and yeast extract as the nitrogen source at pH 7.0 and 37 °C. Potato peelings were selected as an effective waste substrate. It was shown that the increase in the percentage of peel extract in the culture medium enhanced the biosynthesis of surfactin (mg/L) (2–30.9%; 4–46.0% and 6–58.2%), while reducing surface tension of the medium by about 50%. The obtained results constitute a promising basis for further research on biosynthesis of surfactin using potato peelings as a cheap alternative to synthetic medium components.

Organoleptic and Proximate Analysis of Natto (Yellow Soy Fermented Food by The Bacterium Bacillus subtilis natto)

This research aims to conduct organoleptic and proximate analysis of yellow soybean natto which is a food fermented from soybeans by Basillus subtillus natto. Natto is a traditional food originating from Japan that is classified as a probiotic food because it is consumed in fresh condition with the bacteria Basillus subtillus natto in it. Organoleptic analysis of natto in this study consisted of tests of texture, taste, color and aroma, while proximate analysis consisted of analysis of water content, protein content, fat content, carbohydrate content, and ash content. The results of the yellow soybean natto analysis show that the natto originating from Vedca Cianjur has a slimy sticky texture, brown color, a slightly sour and musty aroma like semangit tempeh and the savory taste of soybeans which is the distinctive aroma of natto. Proximate analysis shows that natto contains an average water level of 58.4± 0.50 %wb, average protein content of 20,1 ± 0.65 %wb, average fat content of 9.4 ± 0.42% wb, average carbohydrate content of 7.6 ± 0.47% wb, and the average ash content of 2.3 ± 0.19% wb. Keywords: physical, proximate, natto, Basillus subtillus natto

Bifunctional mechanisms of autophagy and apoptosis regulations in melanoma from Bacillus subtilis natto fermentation extract

Melanoma is one of the most dangerous malignant epidermal cancers. Natto freeze-drying extract (NFDE) and natto water extract (NWE) were isolated from natto, soybeans fermented by Bacillus subtilis natto, which were assessed as potential anti-melanoma agents. Cell cytotoxicity assays revealed significant anti-melanoma effects of NFDE and NWE in a dose-dependent manner, and exhibited low influences on normal skin cells, including Hs68, HaCaT and adipose tissue-derived stem cells (ADSCs), respectively. Through a flow cytometer assay and autophagy acridine orange staining, the cellular death phenomenon shifted from autophagy to apoptosis with the increased dosages. Reactive oxygen species (ROS) were enhanced using DCFDA (2,7-dichlorodihydrofluorescein diacetate) staining when melanoma cells were treated with the extract. NFDE and NWE treatments increase the oxidative stress of cancer cells and cause apoptosis by inhibiting AMP-activated protein kinase (AMPK). NFDE and NWE were considered to play a critical role in cell death through ROS adjustment, autophagy regulation and apoptosis promotion.

Enhancement of natural killer cell activity by oral administration of a fermented soybean product in dogs.

Background:Probiotics are known for their ability to enhance cellular immunity, including the activation of macrophages, natural killer (NK) cells, and cytotoxic T lymphocytes. Natto is a Japanese traditional probiotic food made by fermenting soybean with bacteria Bacillus subtilis var. natto. Components of natto include spores of B. subtilis natto, poly-γ-glutamic acid, and levan, which have demonstrated their immunoadjuvant and anti-allergic effects through various in vitro and in vivo studies. However, it remains unclear whether oral administration of natto can modulate the immune activity in animals. Aim:This study aimed to investigate the effects of oral administration of natto on the immune system of dogs.Methods:Eight dogs were randomly divided into two groups: a natto-treated group and an untreated group. The dogs in the natto-treated group were fed with 10 g/head/day of a freeze-dried natto product in addition to a usual amount of regular dry food for 14 days, whereas the dogs in the untreated group were fed with the regular dry food alone. To determine cellular immune activity, the cell surface antigen analysis of peripheral blood lymphocytes and cytotoxicity analysis of peripheral blood mononuclear cells were carried out before and after the natto administration period. Additionally, a relative expression of inflammatory cytokines in peripheral blood monocytes after the introduction of antigen-stimulation was also examined.Results:At the end of the administration period, a proportion of NK cells (CD3− CD5− CD21− cells and CD3+ CD5dim CD8+ cells) in peripheral blood lymphocytes were found to be significantly increased, and the cytotoxic effect of the peripheral blood mononuclear cells on canine tumor cells were greatly enhanced in the natto-treated group, but not in the untreated group. The expression of TNF-α in peripheral blood mononuclear cells following an antigen-stimulation was increased considerably in the dogs after administration of natto.Conclusion:We conclude that oral administration of natto activated the cytotoxic activity of peripheral NK cells in dogs, and a daily intake of natto might be helpful in augmenting cellular immune activity.

In vitro and in vivo antioxidant activities of soy protein isolate fermented with Bacillus subtilis natto.

The Bacillus subtilis natto fermented soy protein isolate (FSPI) exhibited concentration-dependent scavenging activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS+·) and hydroxyl (·OH) free-radicals. In addition, FSPI administration significantly increased superoxide dismutase activity in mice liver and serum by 20.2% and 86.2%, and suppressed the production of malondialdehyde by 51.3% and 35.1%, respectively, compared to high-fat control (HFC) group. Notably, the movement of mice treated with FSPI was livelier and more active, and its weight gain was significantly lower than that of both NC and HFC groups. The production and accumulation of perirenal fat was also significantly inhibited by FSPI, however, no significant difference in TG and TC levels were observed between FSPI and HFC groups. The results revealed the great potential of FSPI applying in the development of health food or sports food.

Co-fermentation of lentils using lactic acid bacteria and Bacillus subtilis natto increases functional and antioxidant components.

We identified lentil products with both nutritional value and antioxidant capacity by studying the changes of probiotics and functional substances during single fermentation with lactic acid bacteria (LAB) or co-fermentation using LAB and Bacillus subtilis natto. After fermentation, the best growth of LAB was observed in anaerobic solid-state co-fermentation, whereby the viable counts of Lactobacillus plantarum TK9 and Lactobacillus paracasei TK1501 reached 2.77 × 109 and 2.78 × 109 CFU/g, respectively. Furthermore, the total phenol and genistin content produced by the two mixed groups, respectively, increased by 0.52- and 0.66-fold, as well as 0.63- and 0.64-fold, compared with unfermented samples. Similarly, the free amino acid content increased by 0.53- and 0.49-fold, respectively. The 50% inhibitory concentrations for the radical-scavenging against 1,1-diphenyl-2-picrylhydrazyl and α-glucosidase inhibitory activity were lower following anaerobic co-fermentation. Consistently, products of anaerobic mixed solid-state fermentation had higher oxygen radical absorbance capacity. Therefore, anaerobic solid-state co-fermentation of lentils using B. subtilis natto may promote the multiplication of LAB and enhance the antioxidant activity of fermented lentil products. PRACTICAL APPLICATION: Simple and efficient food handling is more suitable for industrial production. Co-fermentation is a good method to optimize the fermentation process. Co-culture technology has high potential in terms of functionality and antioxidant capacity.

A functional dairy product rich in Menaquinone-7 and FeOOH nanoparticles

Menaquinone-7 (MK-7) has been identified as an essential nutrient in enhancing bone mineral density, bone health, and reducing the risk of osteoporosis. Although there have been worthy achievements in MK-7 fermentation process using Bacillus subtilis natto, the low yield of fermentation, costly downstream extraction and purification processes remain the main challenges. In this research, a new approach was taken to study the feasibility of the production of a functional fermented dairy-based product rich in MK-7 by the use of iron oxide hydroxide (FeOOH) nanoparticles (NPs) to increase the concentration of MK-7. In this regard, milk a rich source of calcium, a necessary nutrient for bone health was chosen as the fermentation medium for MK-7 biosynthesis using B. subtilis natto. Two different types of FeOOH NPs, namely naked and biocompatible xanthan gum (XG)-coated NPs, were synthesised (with 10 nm average particle size) and used to evaluate their effect on developing a fermented dairy-based product rich in MK-7 and iron. According to the results, in the presence of 3 mg/L of XG-coated and 12 mg/L of naked FeOOH NPs, the concentration of MK-7 significantly enhanced, and approximately 27% and 35% more MK-7 biosynthesis were observed respectively as compared with control samples with only 1.9 mg/L MK-7 production.

The effect of aeration and mixing in developing a dairy-based functional food rich in menaquinone-7.

Vitamin menaquinone-7 (MK-7) supplementation improves bone health and reduces the incidence of osteoporosis. Despite the recent developments in MK-7 fermentation using Bacillus subtilis natto, low fermentation yields, as well as complicated downstream processing steps, are still the main reasons for the expensive final product. To overcome these issues, developing a fermented dairy-based product rich in MK-7 by avoiding costly downstream steps and optimising the fermentation operating conditions to enhance the MK-7 concentration would be an alternative approach. The present study, therefore, aims to evaluate the role of agitation and aeration as the key operating conditions on MK-7 production by Bacillus subtilis natto using a milk media. The agitation and aeration rates of 525 RPM and 5 VVM were found to be the optimum levels leading to the production of 3.54mg/L of MK-7. Further, the sensory evaluation was performed to compare the sensory properties of the freeze-dried fermented samples with non-fermented milk samples. The results illustrated that the fermented samples had a significant saltiness with intense aroma resulting in the less acceptability of them by the panellists.

Exploring the Potential for Fungal Antagonism and Cell Wall Attack by Bacillus subtilis natto.

To develop more ecologically sustainable agricultural practices requires that we reduce our reliance on synthetic chemical pesticides for crop protection. This will likely involve optimized biocontrol approaches – the use of beneficial soil microbes to attack potential plant pathogens to protect plants from diseases. Many bacterial species, including strains of Bacillus subtilis, have been explored for their biocontrol properties, as they can control the growth of harmful fungi, often by disrupting the fungal cell wall. A strain that is not often considered for this particular application is Bacillus subtilis natto, primarily known for fermenting soybeans via cell wall degradation in the Japanese probiotic dish “natto.” Because deconstruction of the fungal cell wall is considered an important biocontrol trait, we were motivated to explore the possible anti-fungal properties of the B. subtilis natto strain. We show that B. subtilis natto can use complex fungal material as a carbon source for growth, and can effectively deconstruct fungal cell walls. We found degradation of fungal cell wall proteins, and showed that growth on a mix of peptides was very strong. We also found that intact fungal cell walls can induce the secretion of chitinases and proteases. Surprisingly, we could show that chitin, the bulk component of the fungal cell wall, does not permit successful growth of the natto strain or induce the secretion of chitinolytic enzymes, although these were produced during exposure to proteins or to complex fungal material. We have further shown that protease secretion is likely a constitutively enabled mechanism for nutrient scavenging by B. subtilis natto, as well as a potent tool for the degradation of fungal cell walls. Overall, our data highlight B. subtilis natto as a promising candidate for biocontrol products, with relevant behaviors that can be optimized by altering growth conditions. Whereas it is common for bacterial biocontrol products to be supplied with chitin or chitosan as a priming polysaccharide, our data indicate that this is not a useful approach with this particular bacterium, which should instead be supplied with either glucose or attenuated fungal material.

Isolation, Identification and Antimicrobial Evaluation of Bactericides Secreting Bacillus subtilis Natto as a Biocontrol Agent

Herein, a bactericide-secreting Bacillus strain, potentially useful as a biocontrol agent, was isolated from the commercial Yanjing Natto food. Following the biochemical and physiological evaluation, the molecular identification was performed using 16S rDNA sequencing of polymerase chain reaction-amplified DNA that confirmed the natto isolate as Bacillus subtilis natto (B. subtilis natto). The biocontrol (microbial inhibitory) capability of B. subtilis natto was investigated against Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, and yeast (Yarrowia lipolytica) and recorded. The antimicrobial activity of B. subtilis natto was further enhanced by optimizing the growth medium for optimal bactericides secretion. Under optimized conditions, B. subtilis natto exhibited much higher inhibitory activity against S. aureus with a zone of inhibition diameter up to 27 mm. After 48 h incubation, the optimally yielded B. subtilis natto broth was used to extract and purify the responsible bactericides by silica gel column chromatography, gel column chromatography, and semi-preparative high-performance liquid chromatography. Structural identification of purified bactericides (designated as NT-5, NT-6, and NT-7) from B. subtilis natto was performed by 13C-nuclear magnetic resonance (NMR) and mass spectral analyses. The NMR comparison also revealed that NT-5, NT-6, and NT-7 had identical structures, except for the fatty chain. In summary, the present study suggests the improved biocontrol and/or microbial inhibitory potential of newly isolated bactericides secreting B. subtilis natto.

Production of fructooligosaccharides by Bacillus subtilis natto CCT7712 and their antiproliferative potential.

Fructooligosaccharides (FOSs) known for their health properties and β-(2→6)-levan-type FOSs have shown prebiotic and immunomodulatory activities that overcome those of commercial β-(2→1)-FOSs, but costs do not favour their use. Moreover, FOSs can reach the bloodstream through the diet, and little is known about their direct effect on cells. The aim of this work was to produce high-content FOSs by Bacillus subtilis natto CCT7712 in a bioreactor using commercial sucrose and to evaluate their antiproliferative effects in OVCAR-3 cells. FOS production reached 173·60gl-1 , 0·2 vvm aeration and uncontrolled pH. Levan-type FOSs, composed of β-(2→6) links and mainly GF3 (6-nystose), were identified using RMN spectroscopy, FT-IR and ESI-MS. FOSs decreased the viability and proliferation of OVCAR-3 cells, and the effects were associated with an increased pro-inflammatory response by the induction of IL-8 and TNF-α, and the repression of ER-β genes. The metabolic profiles showed disruption of cellular homeostasis that can be associated with a decrease in proliferation. The high production of levan-type FOSs from B. subtilis natto CCT7712 in a bioreactor was achieved, and they showed antiproliferative potential in OVCAR-3 cells. FOS could be a good target for future therapeutic studies and commercial use.

A Study on Biomineralization using &lt;i&gt;Bacillus Subtilis&lt;/i&gt; Natto for Repeatability of Self-Healing Concrete and Strength Improvement

A Study on Biomineralization using &lt;i&gt;Bacillus Subtilis&lt;/i&gt; Natto for Repeatability of Self-Healing Concrete and Strength Improvement

Evaluation of various methods of selection of B. subtilis strains capable of secreting surface-active compounds.

The aim of the study was the evaluation of a three-step method for the selection of bacterial strains capable of producing surfactin. The procedure consisted of the following steps: 1.blood agar test, 2. measurement of the surface tension (ST) of the medium using the du Nouy method before and after submerged culture, 3. qualitative and quantitative assessment of surfactin by HPLC. Forty five Bacillus subtilis natto strains producing haemolysis zones (≥3mm) were selected. Nineten of them reduced ST of the medium to ≤ 40 mN/m; in six cases, the reduction was as much as 50%. All indicated strains produced surfactin. Positive correlations (p <0.5) between the percentage reduction of ST of the medium and surfactin concentration (r = 0.44), indicate that this parameter is determinant of the ability to synthesize this compound. The blood agar test has been shown to be useful only as a pre-selection criterion for surfactin producers (18 strains selected by this method reduced ST by only ≤30%). The proposed selection strategy proved effective and made it possible to select the BS15 strain that reduced the ST of the medium to 30.56 ± 0.15 mN/m and simultaneously provided a high concentration of surfactin compared to other strains.