# Exploring Bacillus velezensis in a biomedical context: a systematic review

Eduarda Guimarães Sousa, Gabriela Munis Campos, Ludmila Silva Quaresma, Thaís Fernandes Mendonça Mota, Nédia de Castilhos Ghisi, Gabriel Camargos Gomes, Rhayane Cristina Viegas Santos, Beatriz Gamarano Rocha de Souza, Éric Guédon, Siomar de Castro Soares, et al.

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Eduarda Guimarães Sousa, Gabriela Munis Campos, Ludmila Silva Quaresma, Thaís Fernandes Mendonça Mota, Nédia de Castilhos Ghisi, et al. Exploring Bacillus velezensis in a biomedical context: a systematic review. Academia Molecular Biology and Genomics, 2025, 2, 10.20935/acadmolbio-gen7598. hal-05015661

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# Exploring *Bacillus velezensis* in a biomedical context: a systematic review

Eduarda Guimarães Sousa <fnref n="1" />,*, Gabriela Munis Campos <fnref n="1" />, Ludmila Silva Quaresma <fnref n="1" />, Thaís Fernandes Mendonça Mota <fnref n="2" />,
Nédia de Castilhos Ghisi <fnref n="2" />, Gabriel Camargos Gomes <fnref n="1" />, Rhayane Cristina Viegas Santos <fnref n="1" />, Beatriz Gamarano Rocha de Souza <fnref n="1" />, Éric Guédon <fnref n="3" />,
Siomar de Castro Soares <fnref n="4" />, Joyce da Cruz Ferraz Dutra <fnref n="1" /> and Vasco Ariston de Carvalho Azevedo <fnref n="1" />,*

Academic Editor: Arturo Rojo-Domínguez

## Abstract

*Bacillus velezensis* is associated with producing secondary metabolites with antifungal, anti-inflammatory, antagonistic, and antimicrobial activities that have multiple applications in the food industry, agriculture, and farming. This study aims to review current knowledge to highlight the biomedical potential of *Bacillus velezensis* as a mammalian probiotic supported by in vivo and in vitro research and to compile existing knowledge. All information in this study was searched in the Dimensions, Scopus, PubMed, and Web of Science databases on 2023 Dec 10. The articles were selected according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) method and scientometric analysis. A total of 1585 records were identified after the screening process. Of these, 151 were used for scientometric analysis, and 13 were used for data analysis based on in vitro and in vivo studies. The literature review revealed the great biomedical potential of *B. velezensis* and its relevance in scientific research, especially in recent years, thanks to advances in genome sequencing and the search for alternative antibiotics. The production of secondary metabolites such as surfactin, bacilysin, and fengycin is of growing interest to industry and human health. It also has great probiotic potential due to its antagonistic activity, lack of antibiotic resistance, and immunomodulatory activity. However, the optimization of its use and a better understanding of cultivation methods for gene expression under variable environmental conditions are crucial. Future research should focus on refining cultivation methods and applying techniques to maximize the production of these bioactive compounds to exploit the full potential of *B. velezensis* in various applications.

**Keywords:** biomedical, probiotic, secondary metabolites, *Bacillus velezensis*

**Citation:** Sousa EG, Campos GM, Quaresma LS, Mota TFM, de Castilhos Ghisi N, Gomes GC, et al. Exploring *Bacillus velezensis* in a biomedical context: a systematic review. *Academia Molecular Biology and Genomics* 2025;2.
https://doi.org/10.20935/AcadMolBioGen7598

## 1. Introduction

The genus *Bacillus* comprises a diverse group of spore-forming bacteria with significant biotechnological relevance. Initially classified by Ferdinand Cohn in 1872 when *Vibrio subtilis* was renamed *Bacillus subtilis*, this genus belongs to the phylum Firmicutes, class Bacilli, order Bacillales, and family Bacillaceae. *Bacillus* species are characterized by their rod shape, Gram-positive nature, facultative aerobic metabolism, catalase positivity, and ability to form endospores [1]. These characteristics confer high resistance to extreme environmental conditions such as heat, desiccation, radiation, disinfectants, and pH fluctuations, allowing them to colonize diverse habitats [1]. Among them, *B. subtilis* and *B. velezensis* stand out due to their industrial applications, biotechnological properties, and generally recognized as safe (GRAS) status [2, 3].

*B. velezensis* and related species, such as *Bacillus amyloliquefaciens*, can produce secondary metabolites, including lipopeptides, polyketides, bacteriocins, and volatile compounds [4]. These bioactive molecules exhibit potent antimicrobial and antifungal properties, playing a key role in biocontrol. Notably, fengycins, surfactins, and bacillomycins are lipopeptides with antifungal activity, while surfactins also contribute to biofilm formation and environmental stress resistance. Additionally, bacteriocins such as bacilysin, polyketides like oxydifficidin, and volatile compounds such as acetoin and 2,3-butanediol have antimicrobial activity and act as plant growth stimulators [5-8].

<footnote n="1">¹Cellular and Molecular Genetics Laboratory, Institute of Biological Sciences, Department of General Biology, Federal University of Minas Gerais, Pampulha Campus, Belo Horizonte 31270-901, MG, Brazil.</footnote>

<footnote n="2">²Programa de Pós-Graduação em Biotecnologia (PPGBIOTEC), Universidade Tecnológica Federal do Paraná (UTFPR), Campus Dois Vizinhos, Estrada para Boa Esperança s/n, km 04, Comunidade São Cristóvão, P.O. Box 157, Dois Vizinhos 85660-000, PR, Brazil.</footnote>

<footnote n="3">³STLO, INRAE, Institut Agro, 4 allée Adolphe Bobierre, CS 61103, 35011 Rennes, France.</footnote>

<footnote n="4">⁴Laboratory of Bioinformatics, Institute of Biological and Natural Sciences, Department of Microbiology, Immunology and Parasitology, Federal University of Triângulo Mineiro, Educational Center Campus, Uberaba 38025-180, MG, Brazil.</footnote>

<footnote>*email: eduardaguimaraessousa@gmail.com (E.G.S.); vascoariston@gmail.com (V.A.)</footnote>
Given its ability to produce these bioactive metabolites, *B. velezensis* is widely used in agriculture as a biological control agent and plant growth promoter [9, 10]. Moreover, due to its high probiotic potential, this species has applications in the food and pharmaceutical industries, also genes associated with immunomodulation and the production of anti-inflammatory cytokines [11–13].

In aquaculture research, numerous studies have demonstrated the anti-inflammatory and antagonistic capabilities of *B. velezensis* in combating diseases caused by pathogens belonging to the *Vibrio* and *Aeromonas* genera [4, 14–16]. Such occurrences directly impact economic losses incurred by the aquaculture industry and indirectly affect the food industry. The bacterium has shown promising probiotic effects, stimulating the innate and humoral immune responses of aquatic organisms. Notably, *B. velezensis* enhances the expression of pro-inflammatory cytokines (e.g., TNF-α, IL-1β, IL-10, and IFN-γ) and promotes the production of immunoglobulins (IgM and IgD), playing a crucial role in immune modulation.

Beyond aquaculture, *B. velezensis* is widely distributed in various environments, including soil, fermented foods, and animal intestines [3]. Its ability to form spores, colonize plant surfaces, and produce antimicrobial compounds makes it a valuable organism for applications in agriculture, food production, and biomedical research [17–19]. The species' antimicrobial properties, biofilm formation capabilities, and antibiotic synthesis underscore its industrial importance [19–21].

In this context, *B. velezensis* has been the subject of extensive studies, particularly in agriculture and aquaculture; this is due to the high applicability of this bacterium in the production of secondary metabolites, which are related to immunomodulatory, antimicrobial, and antagonistic activities. In the context of human health and biomedical applications, a comprehensive review of this bacterium is essential to identify and consolidate existing knowledge.

The principal objective of this study is to demonstrate and review that *B. velezensis* has a high level of applicability in the biomedical field and significant potential for use as a probiotic in mammals. By analyzing in vivo and in vitro studies, the aim is to provide evidence of the effectiveness of this microorganism in stimulating the human host. In addition, this study employs scientometric and systematic analysis to comprehensively assess the existing research landscape. The scientometric approach offers an objective, quantitative evaluation of the literature, identifying key trends, influential publications, and collaborative networks, while the systematic review ensures a rigorous and reproducible selection of relevant studies. Together, these methodologies not only strengthen the validation of *B. velezensis'* probiotic potential but also underscore its broad applicability in biomedical research.

# 2. Methods

## 2.1. Selection and search strategy

This systematic review was based on the Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) method [22]. Studies published in the Dimensions, Scopus, PubMed, and Web of Science (Main Collection) databases were included. Documents

of all types were considered, with no publication date or language limitations. The specific question of the study guided the selection of keywords: to evaluate the potential of *B. velezensis* as a probiotic in biomedical applications. For this purpose, the database records were used, determined by the following search terms: ("Bacillus velezensis" OR "B. velezensis" OR "velezensis") AND ("Human" OR "Biomedicine" OR "Medicine" OR "Cell" OR "Microbiome" OR "Gastrointestinal tract" OR "Gut" OR "Brain" OR "Neurotransmitter" OR "Probiotic"). These search terms refer to the document's title, abstract, and keywords. The databases were last accessed on 2023 Dec 10.

The data processing of records identified in the Dimensions, Scopus, PubMed, and Web of Science databases followed the methodology described by [23]; this involved exporting records in CSV format, converting them to .xlx, identifying and removing duplicates, and handling records without DOIs using specific scripts. The articles were selected based on inclusion and exclusion criteria where two researchers independently assessed the downloaded documents, and any disagreements were resolved by a third reviewer. The manual review of titles and abstracts was followed by a thorough evaluation for systematic review eligibility (Figure 1).

## 2.2. Data extraction from included studies

The following information was extracted from the selected documents: (1) study characteristics, (2) bacterial culture conditions, and (3) experimental protocols. Two reviewers collected the data independently, with a third reviewer resolving any discrepancies.

## 2.3. Data analyses

The articles identified and screened were subsequently subjected to scientometric analysis. The WoS, Dimensions, and Scopus databases were selected as they index the articles in question. In particular, the "Analyze Results" and "Create Citation Report" tools were employed in the WoS database, and the H-index was investigated due to the availability of only this database. For all articles, data were generated and extracted for analysis using the CiteSpace software [23].

The scientometric analysis was conducted using CiteSpace, a software designed for visualizing and interpreting citation networks and research trends. The software processes the dataset obtained from each database and maps the relationships between different parameters, such as keywords, categories, countries, and journals (Supplementary Figure S1A). The analysis is represented through network visualizations, where each element is depicted as a "node" (colored circle). The size of each node corresponds to the frequency of occurrence of the evaluated parameter, while the thickness of the connecting lines indicates the strength of co-occurrence or correlation between nodes. Additionally, the outermost purple ring of a node is used to assess its centrality, which reflects the influence and prominence of a particular topic within the research field covered in the review (Supplementary Figure S1B). Centrality is a key scientometric metric, calculated by taking the sum of the links that pass through the node (i), divided by the total of the shortest links between circle j and k, as shown in the equation presented in the Supplementary Material, designated as "Equation 1" [23].
**Inclusion criteria**

1 - Studies testing *Bacillus velezensis* in vitro and in vivo

2 - English language

3 - Studies: Original documents with DOI

**Exclusion criteria**

1 - Articles that did not focus on *Bacillus velezensis* as the primary subject

2 - Book chaptes, posters and review articles

3 - Articles that were not in English

4 - Articles that adressed areas other than biomedical applications

5 - Articles that did not test *Bacillus velezensis* in vivo

**Figure 1** • Inclusion and exclusion criteria for selecting studies in the systematic review.

# 3. Results

## 3.1. General features and scientometric analysis

In this systematic review, 1585 records were identified in the Dimensions, Scopus, PubMed, and Web of Science databases. During the automated screening, 985 records were removed, which included files without DOI and duplicate files, resulting in 600 files. During the screening process, 449 articles were discarded for failing to meet the pre-established criteria, with 6 classified as book chapters, books, notes, letters, editorials, or reviews, and 443 were select hat did primarily focus on *B. velezensis* applied to biomedicine.

From the selected articles, 164 studies were selected, where 13 studies were used for data analysis, which included both in vivo and in vitro studies, and 151 were used for scientometric analysis. According to the results, 131 documents belonged to the main WoS database, 6 to Dimensions, and 14 to Scopus. This review underscores the focused and substantial interest in *B. velezensis* within the biomedical field and probiotic application, as evidenced by the comprehensive analysis of selected studies. These documents are shown in a flowchart in **Figure 2**.

### 3.1.1. Scientometric analysis of publication characteristics

The WoS dataset's H-index, which represents 86.7% of the publications, was 23. According to the number of publications per year on the subject, the first publication in the study set was in 2008. From 2016 onwards, the number of publications increased significantly (Figure 3), and 2021 and 2022 were the years with the highest publications (36 publications each year).

**Table 1** presents the most frequently cited articles, considered essential references due to their innovative contributions and relevance to biomedical research. The article "Probiotics isolated from yaks improve the growth performance, antioxidant activity, and cytokines related to immunity and inflammation in mice", published in 2019, was the most frequently cited article, with 80 citations. This article examines the impact of probiotics, specifically *B. subtilis* and *B. velezensis*, isolated from yaks, on their antioxidant capacity, immunological effect, and anti-inflammatory capabilities

in a mouse model. The work by Yuan and colleagues, entitled "Ultrafast Recovery of Uranium from Seawater by *Bacillus velezensis* Strain UUS-1 with Innate Anti-Biofouling Activity" and published in 2019, was the second most cited, with 72 citations. This article deals with the innate anti-biofouling activity of *B. velezensis*, specifically its ability to recover uranium. The remaining articles address other highly applicable biomedical, biotechnological, and probiotic topics.

### 3.1.2. Geographical pattern

China has the highest number of publications on the application of *B. velezensis* in biomedicine, exerting considerable influence in this area of research, and the highest centrality, followed by France, India, and the USA (Supplementary Figure S2).

### 3.1.3. Publishing journals

The journal with the highest number of publications was *Frontiers in Microbiology*, with 83 articles, followed by *PLoS One* with 60 and *Applied and Environmental Microbiology* from ASM Journals with 57. The highest centrality was observed for *Applied Microbiology and Biotechnology* from Springer (0.23) and *Applied and Environmental Microbiology* (0.18) (Supplementary Figure S3A). The *Canadian Journal of Microbiology* exhibited a notable surge in citations from 2019 to 2021, indicating a sudden shift in the citation pattern with a substantial increase (with a strength of 2.89), as illustrated in Supplementary Figure S3B.

### 3.1.4. Categories

"Biotechnology and Applied Microbiology" and "Microbiology" were the research categories with the highest frequency of published articles in the area (40 publications each), followed by "Food Science and Technology" (18 publications). "Biochemistry and Molecular Biology" had the highest centrality (0.34), followed by "Chemistry, Multidisciplinary" and "Biotechnology and Applied Microbiology" (0.30 and 0.29, respectively), as illustrated in **Figure 4**.
<figure><img src="image_2.png" /><figcaption>**Figure 2** • Prisma flowchart detailing all stages of document capture: identification, screening, and included.</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>**Figure 3** • Number of publications on biomedical applications of *Bacillus velezensis* over time.</figcaption></figure>

### 3.1.5. Keywords

Using keywords enables the synthesis and collation of content about a specific topic. The most prominent among the selected

publications are shown in **Figure 5A**: "*Bacillus velezensis*", "subtilis", "identification", "bacteria", "strain", "gene", and "apoptosis". The dimensions of the word and the node may be employed to indicate these keywords. Moreover, the interconnections between words, defined as co-occurrence, can be investigated using edges.
<table><caption>**Table 1** • The 10 most frequently cited articles on the use of *Bacillus velezensis* as a potential biomedical agent.</caption><thead><tr><td># Citation</td><td>Corresponding author country</td><td>Title</td><td>Source title</td><td>Total citations</td></tr></thead><tbody><tr><td>1</td><td>[24]<br>China</td><td>Probiotics isolated from yaks improves the growth performance, antioxidant activity, and cytokines related to immunity and inflammation in mice</td><td>BMC</td><td>80</td></tr><tr><td>2</td><td>[25]<br>China</td><td>Ultrafast Recovery of Uranium from Seawater by <i>Bacillus velezensis</i> Strain UUS-1 with Innate Anti-Biofouling Activity</td><td>Wiley Online Library</td><td>72</td></tr><tr><td>3</td><td>[26]<br>China</td><td>A novel fermented soybean, inoculated with selected Bacillus, Lactobacillus and Hansenula strains, showed strong antioxidant and anti-fatigue potential activity</td><td>Elsevier</td><td>62</td></tr><tr><td>4</td><td>[25]<br>China</td><td>Biological Degradation of Aflatoxin B1 by Cell-Free Extracts of <i>Bacillus velezensis</i> DY3108 with Broad PH Stability and Excellent Thermostability</td><td>MDPI</td><td>53</td></tr><tr><td>5</td><td>[27]<br>India</td><td>Azoreductase and dye detoxification activities of <i>Bacillus velezensis</i> strain AB</td><td>Springer</td><td>50</td></tr><tr><td>6</td><td>[28]<br>Iran</td><td>Microencapsulation of <i>Bacillus velezensis</i> Using Alginate-Gum Polymers Enriched with TiO2 and SiO2 Nanoparticles</td><td>MDPI</td><td>41</td></tr><tr><td>7</td><td>[7]<br>Denmark</td><td>Depiction of secondary metabolites and antifungal activity of <i>Bacillus velezensis</i> DTU001</td><td>Elsevier</td><td>40</td></tr><tr><td>8</td><td>[29]<br>India</td><td>Lipopeptide antibiotic production by <i>Bacillus velezensis</i> KLP2016</td><td>Journal of Applied Pharmaceutical Science</td><td>38</td></tr><tr><td>9</td><td>[30]<br>China</td><td>Purification, characterization and antitumor activity of an exopolysaccharide produced by <i>Bacillus velezensis</i> SN-1</td><td>Elsevier</td><td>37</td></tr><tr><td>10</td><td>[31]<br>China</td><td>In vivo assessment of the probiotic potentials of three host-associated Bacillus species on growth performance, health status and disease resistance of <i>Oreochromis niloticus</i> against <i>Streptococcus agalactiae</i></td><td>Elsevier</td><td>34</td></tr></tbody></table>

The term “Bacillus velezensis” was observed to have the highest frequency and centrality, with 64 publications and a centrality score of 0.61, as expected due to the main topic of the research. The second most frequent word was “subtilis” (30 publications), followed by “identification” and “bacteria” (20 and 14 publications, respectively). The second most central term was “apoptosis” (0.32), followed by “subtilis” (0.31). A more detailed visual representation can be found in **Figure 5B**.

Clusters are defined as groupings of similar publications. This analysis is based on the co-occurrence of keywords to identify search hotspots. Eight clusters were identified for this dataset, with silhouettes exceeding 0.7. The largest cluster was that comprising the term “antibiotics” (cluster #0), followed by cluster #1, which comprised the term “Bacillus velezensis sn-1”; cluster #2, which

comprised the term “Bacillus velezensis”; cluster #3, which comprised the term “autophagy”; cluster #4, which comprised the term “Bacillus”; cluster #5, which comprised the term “honeycombs”; cluster #6, which comprised the term “rifampicin resistance”; and cluster #7, which comprised the term “palm kernel shell” (Figure 6A). Cluster #0 first emerged in 2016 (Figure 6B), while the oldest cluster is #3, concerning autophagy, which commenced in 2008 and concluded in 2019. Cluster #7, which concerns palm kernel shells, is the most recent, having commenced in 2022.

## 3.2. Experimental features of studies

This review evaluated different strategies to explore the biomedical potential of *B. velezensis*. The selected studies included in vivo and in vitro experiments. **Table 2** presents the objectives of each
<figure><img src="image_4.png" /><figcaption>**Figure 4** • Research categories with the highest frequency in relation to the year of publication.</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>**Figure 5** • Map of the main keywords cited on the theme of this review. (A) illustrates the network of co-occurrences of keywords in *B. velezensis* research, together with the frequency and centrality of each keyword, which is also related to the year. (B) provides a more detailed, quantitative representation of centrality and frequency.</figcaption></figure>
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /><figcaption>**Figure 6** • Keyword-based knowledge clusters. (A) shows the main clusters formed according to keywords. (B) shows the clusters that emerged in relation to the years.</figcaption></figure>

study, the number of experiments conducted, and the methodology employed. Table S1 provides more detailed information about all the experiments carried out in each study. Most studies tested the ability of *B. velezensis* to inhibit the growth and activity of pathogens and assessed the probiotic potential of *B. velezensis* strains using animal models such as mice.

Additionally, three strategies were employed to evaluate the biomedical potential of this microorganism: in vivo, in vitro, and in silico experiments (69%). In the in silico experiments, 16S rRNA sequencing was observed in all studies, mainly for species and strain identification. However, three studies used techniques such

as metagenomics to observe microbiota colonization after using *B. velezensis* [32, 33].

Seven studies applied statistical analysis to ensure the reliability of their in vivo experiments. Zhang et al. 2022 [34] conducted the most extensive study, identifying, isolating, and purifying a potential antibacterial protein. Their methodology included purification techniques (Sephadex G-75, QAE-Sephadex A 25, C4 chromatography, and SDS-PAGE), protein identification (LC-MS/MS), and antibacterial activity assessments (MIC, time-kill kinetics, and stability testing). Additionally, antimicrobial capacity was evaluated through metal ion effects, ROS assays, ATP and AKP release,
<table><caption>**Table 2 • General features of the selected studies.**</caption><thead><tr><td>Article</td><td>Objective</td><td>General strategy</td><td>Reference</td></tr></thead><tbody><tr><td>An intestinal <i>Bacillus velezensis</i> isolate displays broad-spectrum antibacterial activity and prevents infection of both Gram-Positive and Gram-Negative pathogens in vivo</td><td><i>B. velezensis</i> with growth inhibitory activity against the pathogen and probiotic potential</td><td>In vitro, in vivo, and in silico studies</td><td>[21]</td></tr><tr><td>Structural characterization and hepatoprotective activity of exopolysaccharide from <i>Bacillus velezensis</i> SN-1</td><td>Hepatoprotective activity and prebiotic and probiotic potential of fractional released exopolysaccharide (rEPS) produced by <i>B. velezensis</i></td><td>In vitro, in vivo, and in silico studies</td><td>[32]</td></tr><tr><td>Whole genome sequence analysis and in vitro probiotic characterization of <i>Bacillus velezensis</i> FCW2 MCC4686 from spontaneously fermented coconut water</td><td>Probiotic potential</td><td>In vitro, in vivo, and in silico studies</td><td>[35]</td></tr><tr><td><i>Bacillus velezensis</i> DSM 33864 reduces <i>Clostridioides difficile</i> colonization without disturbing commensal gut microbiota composition</td><td><i>B. velezensis</i> with growth inhibitory activity against the pathogen and probiotic potential</td><td>In vitro, in vivo, and in silico studies</td><td>[33]</td></tr><tr><td>Indigenous <i>Bacillus</i> species isolated from <i>Aedes aegypti</i> larvae: Isolation, larvicidal toxicity screening, phenotypic characterization, and molecular identification</td><td><i>B. velezensis</i> with larvicidal toxicity</td><td>In vitro, in vivo, and in silico studies</td><td>[36]</td></tr><tr><td>Bioactive earthworm peptides produced by novel protease-producing <i>Bacillus velezensis</i> PM 35 and its bioactivities on liver cancer cell death via apoptosis, antioxidant activity, protection against oxidative stress, and immune cell activation</td><td>Bioactive peptides can promote liver cancer bioprophylactic activity, antioxidant, oxidative stress protection, and immune cell activation</td><td>In vitro and in vivo studies</td><td>[24]</td></tr><tr><td>In vivo efficacy of <i>Bacillus velezensis</i> isolated from Korean gochang bokbunja vinegar against carbapenem-resistant <i>Klebsiella pneumoniae</i> infections</td><td><i>B. velezensis</i> with growth inhibitory activity against the pathogen and probiotic potential</td><td>In vitro and in vivo studies</td><td>[37]</td></tr><tr><td>Discovery of a novel antibacterial protein CB6-C to target methicillin-resistant <i>Staphylococcus aureus</i></td><td><i>B. velezensis</i> with growth inhibitory activity against the pathogen</td><td>In vitro and in vivo studies</td><td>[34]</td></tr><tr><td>Purification and characterization of a fibrinolytic enzyme from marine <i>Bacillus velezensis</i> Z01 and assessment of its therapeutic efficacy in vivo</td><td>Fibrinolytic enzymes produced by <i>B. velezensis</i>, like thrombolytic agent</td><td>In vitro, in vivo, and in silico studies</td><td>[38]</td></tr><tr><td>Immune-enhancing effects of postbiotic produced by <i>Bacillus velezensis</i> Kh2-2 isolated from Korea Foods</td><td>Postbiotics with immune-enhancing effects</td><td>In vitro, in vivo, and in silico studies</td><td>[39]</td></tr><tr><td>Anti-staphylococcal activity of a cyclic lipopeptide, C15 -bacillomycin D, produced by <i>Bacillus velezensis</i> NST6</td><td><i>B. velezensis</i> with growth inhibitory activity against the pathogen</td><td>In vitro and in vivo studies</td><td>[40]</td></tr><tr><td>Probiotics isolated from animals in Northwest China improve the intestinal performance of mice</td><td>Probiotic potential</td><td>In vitro, in vivo, and in silico studies</td><td>[41]</td></tr><tr><td><i>Bacillus velezensis</i> AP183 inhibits <i>Staphylococcus aureus</i> biofilm formation and proliferation in murine and bovine disease models</td><td>Ability to inhibit biofilm formation in mouse and bovine models and <i>B. velezensis</i> with growth inhibitory activity against the pathogen</td><td>In vitro, in vivo, and in silico studies</td><td>[42]</td></tr></tbody></table>
<figure><img src="image_8.png" /><figcaption>**Figure 7** • Media used for the growth of *B. velezensis*.</figcaption></figure>

membrane permeability, intracellular morphological changes in MRSA, and histological experiments in mice.

### 3.3. Strategic media selection for optimal *Bacillus velezensis* growth

**Table 3** shows the media selection of *Bacillus velezensis* growth, based on strain, medium, shaking, growth temperature, and incubation time. Researchers primarily used the Luria-Bertani (LB) medium to grow four strains of *B. velezensis*: BVM7, DSM 33864, CB6, and PM35 (Figure 7). This medium, known for its versatility, effectively supports bacterial growth in various experiments. In contrast, researchers chose the de Man Rogosa Sharpe (MRS) medium to cultivate four other strains: *B. velezensis* FCW2, KCTC 13417, JT3-1, and Kh2-3. In addition to LB and MRS, researchers experimented with Tryptic Soy Broth (TSB), Nutrient Yeast Salt Medium (NYSM) and a custom-made medium. These choices highlight their efforts to optimize growth conditions and target specific experimental outcomes, such as enhancing the production of secondary metabolites.

Most studies incubated their cultures for 24 h (38%), finding this a sufficient time for the strains to reach the desired growth phase for further experiments. However, some strains required longer incubation time, such as 48 h for SN-1, CB6, and ZO1, and up to five days for AP143. These differences reflect the need to tailor conditions to each strain's growth requirements or experimental goals. Researchers employed shaking for strains with longer incubation times (48 h or more) to ensure adequate aeration and promote optimal growth in liquid media. On the other hand, they often chose static conditions for shorter incubation periods, like 24 h or overnight (approximately 12–16 h), which proved sufficient for certain studies or growth phases.

### 3.4. Antibiotic resistance in *Bacillus velezensis*

According to the study of Dhanya Raj et al., 2023 [35], *Bacillus velezensis* FCW2 harbors genes that confer resistance to

cephalosporins and tetracycline (Table 4). However, in vitro tests revealed that this strain remains susceptible to these antibiotics, suggesting that the resistance genes might be non-functional or not expressed under the conditions tested. The other studies did not show antibiotic resistance.

### 3.5. Inhibition of microorganisms by *Bacillus velezensis*

**Table 4** illustrates the inhibitory capacity of *B. velezensis*, which effectively inhibits a diverse range of microorganisms. *Staphylococcus aureus* was the most frequently inhibited, accounting for 38.46% of the strains tested. *Enterococcus faecalis* and *Klebsiella pneumoniae* were also significantly inhibited by 15.38% of the strains, highlighting *B. velezensis*'s broad-spectrum activity against these pathogens.

Other microorganisms, including *Escherichia coli*, *Clostridium difficile*, and *Pseudomonas aeruginosa*, exhibited inhibition by *B. velezensis*, though at comparatively lower levels than the primary targeted pathogens. This variation in inhibition is reflected in the data, where these species account for a smaller percentage of the total inhibition observed.

Similarly, *Candida gloeosporioides* KACC 40690, *Fusarium oxysporum* KACC 40031, and additional fungal species displayed inhibition rates that were significantly reduced compared to the most susceptible microorganisms. These findings highlight that while *B. velezensis* demonstrates broad-spectrum antimicrobial activity, its efficacy is highly dependent on the specific microorganism.

### 3.6. Production of secondary metabolites

**Table 5** shows 12 metabolites found among the 13 selected articles, of which surfactin is the most studied according to the literature. This lipopeptide, which is common for *Bacillus* subtilis and *B. velezensis* and other related bacteria [43], has a notable antagonistic effect, with antibacterial (*Ralstonia solanacearum* [44] and *S. aureus* [45]) and antifungal (*Fusarium moniliforme* [46] and *Zymoseptoria tritici* [47]) activity, showing its potential to be used
<table><caption>**Table 3** • Optimal *Bacillus velezensis* growth.</caption><thead><tr><th>Bacterial strain</th><th>Culture medium</th><th>Shaking</th><th>Growth temperature</th><th>Incubation time</th></tr></thead><tbody><tr><td>BVM7</td><td>Luria-Bertani (LB)</td><td>Yes</td><td>37 °C</td><td>24 h</td></tr><tr><td>SN-1</td><td>Custom-made: 2% KH<sub>2</sub>PO<sub>4</sub>, 16% K<sub>2</sub>HPO<sub>4</sub>, 2% MgSO<sub>4</sub>·7H<sub>2</sub>O, 1% CaSO<sub>4</sub>·2H<sub>2</sub>O, 2% FeCl<sub>3</sub>, 5% yeast extract, and 2% sucrose; pH 7</td><td>Yes</td><td>35 °C</td><td>48 h</td></tr><tr><td>FCW2</td><td>de Man Rogosa Sharpe (MRS)</td><td>No</td><td>-</td><td>24 h</td></tr><tr><td>DSM 33864</td><td>Luria-Bertani (LB)</td><td>No</td><td>-</td><td>-</td></tr><tr><td>LSD4.2</td><td>Nutrient Yeast Salt Medium (NYSM)</td><td>Yes</td><td>35 °C</td><td>48 h</td></tr><tr><td>PM35</td><td>NB Medium</td><td>No</td><td>37 °C</td><td>24 h</td></tr><tr><td>KCTC 13417</td><td>de Man Rogosa Sharpe (MRS)</td><td>No</td><td>37 °C</td><td>24 h</td></tr><tr><td>CB6</td><td>Luria-Bertani (LB)</td><td>Yes</td><td>37 °C</td><td>48 h</td></tr><tr><td>Zo1</td><td>-</td><td>-</td><td>37 °C</td><td>48 h</td></tr><tr><td>Kh2-3</td><td>de Man Rogosa Sharpe (MRS)</td><td>No</td><td>37 °C</td><td>overnight</td></tr><tr><td>NST6</td><td>Tryptic Soy Broth (TSB)</td><td>No</td><td>37 °C</td><td>overnight</td></tr><tr><td>JT3-1</td><td>de Man Rogosa Sharpe (MRS)</td><td>No</td><td>37 °C</td><td>24 h</td></tr><tr><td>AP143</td><td>Tryptic Soy Broth (TSB)</td><td>Yes</td><td>30 °C</td><td>5 days</td></tr></tbody></table>

as a natural antibiotic. Like the first lipopeptide, the compound bacilysin also shows antibacterial (*Xanthomonas oryzae* [48]) and antifungal (*Candida albicans* [49]) activity.

**Table 6** lists all the secondary metabolites mentioned in the selected articles. These metabolites have been classified according to their functions and potential biomedical applications, covering a variety of biological activities. Among the classifications, metabolites with antagonistic activity, which act by inhibiting or preventing the growth of pathogenic microorganisms, stand out. In addition, biosurfactants and metabolites with antioxidant properties have been identified, which are important in neutralizing free radicals and those with anti-inflammatory activities.

### 3.7. In vivo tests: experimental models, bacterial strains, and concentrations used

The articles included in this review describe the use of *B. velezensis* strains in a range of experimental models, including tests such as murine colonization, bacterial infection, toxicological assay, hepatoprotection, thrombosis model, immunosuppressive mouse model, general performance evaluation in mouse health, cutaneous wound model, and intramammary challenge. The preceding models were conducted in vivo, utilizing mice, including CD-1, C57BL/6, BALB/c, and Kunming mice, which were the most used animals (69%). Additionally, some of the experiments were performed in *C. elegans* (bacterial infection), *Aedes aegypti* larvae (larvicidal toxicity), zebrafish (toxicological assay), and dairy cows (intramammary challenge). The specific experimental model and its corresponding in vivo model are outlined in the following table (Table 7). The treatment was a viable bacterial formulation of *B. velezensis*, comprising cells or spores. The quantified dose of the formulation exhibited variability between different works, ranging

from 10⁵ to 10⁹ colony-forming units (CFU), as observed across various studies.

### 3.8. Applications of *Bacillus velezensis* in biomedical fields

Studies on *Bacillus velezensis* highlight its multiple therapeutic and biocontrol applications. The BVM7 strain, isolated from the human microbiome, demonstrated the production of potent antimicrobial peptides that inhibit *Vibrio cholerae* and *Klebsiella pneumoniae* [21]. The exopolysaccharide rEPS-2, extracted from *B. velezensis* SN-1, exhibited hepatoprotective effects in mice, alleviating liver damage and modulating the intestinal microbiota [32]. The DSM 33864 strain in murine models reduced *Clostridium difficile* colonization without negatively impacting the commensal microbiota [33]. Additionally, the fermentation of soybean meal with *B. velezensis* SCP024-S-4 increased the nutritional content and improved weight gain in mice [64]. Furthermore, *B. velezensis* PM35, isolated from an earthworm, produced bioactive peptides with antioxidant, anticancer, and immunomodulatory activities [24]. The CB6-C protein from *B. velezensis* successfully targeted methicillin-resistant *S. aureus* [34], while the enzyme *velefibrinase* proved effective in treating thrombotic diseases [38].

Regarding biocontrol, *B. velezensis* Kh2-2 produced nitric oxide and enhanced immune responses in animal models [39]. The NST6 strain exhibited strong antimicrobial activity against *S. aureus* and its resistant strains [40]. The JT3-1 strain reduced pro-inflammatory cytokines and improved intestinal health in mice [41]. Soybean meal fermented with *B. velezensis* GZ1 increased glycogen levels and reduced oxidative stress in mice [26]. *B. velezensis* AP183 inhibited *S. aureus* proliferation in murine and
<table><caption>**Table 4** • Antagonistic activity of *Bacillus velezensis*.</caption><thead><tr><th>Strain</th><th>Inhibited microorganism</th></tr></thead><tbody><tr><td>BVM7</td><td><em>Enterococcus faecalis</em><br><em>Vibrio cholerae</em></td></tr><tr><td>SN-1</td><td>N/A</td></tr><tr><td>FCW2</td><td><em>Staphylococcus aureus</em><br><em>Streptococcus</em> spp.<br><em>Escherichia coli</em><br><em>Enterococcus faecalis</em></td></tr><tr><td>DSM 33864</td><td><em>Clostridium difficile</em></td></tr><tr><td>LSD4.2</td><td>N/A</td></tr><tr><td>PM35</td><td>N/A</td></tr><tr><td>KCTC 13417</td><td><em>Klebsiella pneumoniae</em></td></tr><tr><td>CB6</td><td><em>Staphylococcus aureus</em></td></tr><tr><td>Zo1</td><td>N/A</td></tr><tr><td>Kh2-3</td><td>N/A</td></tr><tr><td>NST6</td><td><em>Escherichia coli</em><br><em>Klebsiella pneumonia</em><br><em>Salmonella typhimurium</em><br><em>Pseudomonas aeruginosa</em><br><em>Xanthomonas</em></td></tr><tr><td>NST7</td><td><em>Bacillus subtilis</em><br><em>Micrococcus leuteus</em><br><em>Enterococcus hirae</em><br><em>Staphylococcus aureus</em><br><em>S. epidiermidis</em><br><em>Staphylococcus aureus</em></td></tr><tr><td>NST8</td><td><em>Ascomycete</em> fungi<br><em>Fusarium oxysporum</em> (KACC 40031)<br><em>Colletotrichum gloeosporioides</em> (KACC 40690)</td></tr><tr><td>JT3-1</td><td>N/A</td></tr><tr><td>AP143</td><td><em>Staphylococcus aureus</em></td></tr></tbody></table>

<table><caption>**Table 5** • Secondary metabolites found in the selected studies.</caption><thead><tr><th>Metabolites observed</th><th>Reference</th></tr></thead><tbody><tr><td>Exopolysaccharides rEPS-1 and rEPS-2</td><td>Cao, C. et al., 2023 [32]</td></tr><tr><td>Macrolactin H, bacilysin, bacilibactin, surfactin, fengycin, plipastatin, butirosin</td><td>Dhanya Raj CT. et al., 2023 [35]</td></tr><tr><td>Amynthas arenulus earthworm peptides (AAEPs)</td><td>Wasunan P, Maneewong C. et al., 2022 [24]</td></tr><tr><td>Kitosanase</td><td>Zhang, H., Chen, J., Liu, Y. et al., 2022 [34]</td></tr><tr><td>Velefibrinase</td><td>Zhou Y. et al., 2022 [38]</td></tr></tbody></table>
<table><caption>**Table 6 • Metabolites by potential effect.**</caption><thead><tr><td>Metabolites</td><td>Antagonistic</td><td>Biosurfactant</td><td>Antioxidant</td><td>Anti-inflammatory</td><td>References</td></tr></thead><tbody><tr><td>Surfactin</td><td>X</td><td>X</td><td>X</td><td>X</td><td>[44, 50, 51]</td></tr><tr><td>Bacilysin</td><td>X</td><td>-</td><td>-</td><td>-</td><td>[48, 52]</td></tr><tr><td>Fengycin</td><td>X</td><td>X</td><td>-</td><td>-</td><td>[53, 54]</td></tr><tr><td>Macrolactin</td><td>X</td><td>-</td><td>-</td><td>X</td><td>[55, 56]</td></tr><tr><td>Bacillibactin</td><td>X</td><td>-</td><td>-</td><td>-</td><td>[57, 58]</td></tr><tr><td>Exopolysaccharides</td><td>X</td><td>X</td><td>X</td><td>X</td><td>[32, 59–61]</td></tr><tr><td>Plipastatin</td><td>X</td><td>-</td><td>X</td><td>-</td><td>[62, 63]</td></tr></tbody></table>

bovine models [42]. Finally, the study of *B. velezensis* NRRL B-23189 against *Penicillium roqueforti* s.l. showed promising in vitro results but limited effectiveness under silage conditions [65]. *B. velezensis* 71 and *Paenibacillus peoriae* T099 were identified as effective biocontrol agents against *Xanthomonas* pathogens, providing an alternative to copper-based pesticides [66]. These findings underscore the versatile potential of *B. velezensis* in various therapeutic and biocontrol applications.

# 4. Discussion

## 4.1. General characteristics of the studies

To obtain the main provisions and distributions of research on *B. velezensis* and its biomedical applications, a scientific analysis was carried out. The number of publications, countries, journals, areas of knowledge, and keywords were used as parameters to assess the applicability of this microorganism.

The H-index is a measure that was carried out using only the WoS database, which represents most publications. It quantitatively represents the productivity and impact of the scientific contribution of an area or researcher, combining the number of publications with the citation rate [67]. In this work, this index was 23, a good benchmark [68], reflecting the impact of articles in the biomedical field of *B. velezensis*, a relevant topic for discussion.

In this context, *B. velezensis* has a high degree of applicability in various areas, mainly due to the production of its secondary metabolites, which are involved in promoting antagonistic activity towards pathogens of interest to human health, have antimicrobial and anti-inflammatory activity, and are also being widely studied as potential probiotics [59, 69–71]. These factors may be associated with the number of publications having increased significantly in 2016, where 2021 and 2022 were the years with the highest number of publications (Figure 3). With the rise in new technologies and research in genomics, driven by decreasing costs of genome sequencing and advancements in next-generation sequencing (NGS) techniques [72], there is growing interest in this field. At the same time, increasing concern over high antibiotic resistance has spurred research into alternative solutions, such as probiotics and next-generation probiotics [73]. Given the high applicability of *Bacillus velezensis* both in probiotic studies and in agriculture and the food industry, there is a growing need for a deeper understanding of this bacterium, particularly for its use as a probiotic or a next-generation probiotic [74].

The value of studies on this subject can be seen in the objective of the ten most cited studies in Table 1, which primarily seek to evaluate the mechanisms of action using *B. velezensis* and its various applications. The year of these top ten articles correlates closely with the analysis of the growth in the number of publications discussed above, except for one article from 2008. These articles highlight the need to describe the various actions of this bacterium, including its potential as a probiotic, anti-biofouling activity, antioxidant and anti-fatigue potential, detoxification, secondary metabolites, antimicrobial, and antitumor activity, and other benefits that directly impact human health and well-being.

The countries with the greatest centrality and influence in this area of research are closely related to their high investment in scientific research and international collaborations. China, in particular, has shown the highest number of publications and collaborative networks, and it invests heavily in research involving alternatives for commercial probiotic products [75], as well as developing sustainable methods for using microorganisms in biocontrol and increasing agricultural productivity [76]. This country is also one of the largest aquaculture producers in the world, and there has been much research into the use of *B. velezensis* as a probiotic, as an alternative strategy to antibiotics [77, 78]. On the other hand, France, the USA, and India have many studies related to probiotics, which show the rapid expansion of clinical studies with them due to the developed markets for supplements and functional foods and the growth of the health and wellness market [79].

Research on *B. velezensis* has been published in several academic journals. Most publications are in *Frontiers in Microbiology*, followed by *PLoS One* and *Applied and Environmental Microbiology*. The latter is one of the main journals related to research into the potential of microbial life, medicine, and environmental microbiology, according to the respective scopes of the journal. The theme of this review is microorganisms, which are closely related to the fields of research. Furthermore, these journals have high impact factors, highlighting the relevance of this topic. In addition, the *Canadian Journal of Microbiology* had a relevant number of citations between the same years that an increase in publications in this area was observed. Also, it is a very influential journal in the microbiological field due to its scope and impact factor.

This review aims to examine the principal applications of *B. velezensis* in the biomedical field, focusing on its potential as a probiotic. The primary categories identified are aligned with the objective, including “Biotechnology and Applied Microbiology”, followed by “Food Science and Technology”. This bacterial species is frequently employed as a prospective probiotic in fermented foods
<table><caption>**Table 7** • The relationship between the in vivo model employed and its corresponding experimental model.</caption><thead><tr><td>Bacterial strain</td><td>In vivo model</td><td>Dose administered/treatment</td><td>Experimental model</td><td>Reference</td></tr></thead><tbody><tr><td>BVM7</td><td>CD-1 mice</td><td>5 × 10<sup>7</sup> CFU or 3 × 10<sup>8</sup> CFU spores</td><td>Murine colonization</td><td>[21]</td></tr><tr><td>DSM 33864</td><td>Female C57BL/6 mice</td><td>1 × 10<sup>9</sup> CFU spores</td><td>Murine colonization</td><td>[33]</td></tr><tr><td>CB6</td><td>Female BALB/c mice</td><td>Antibacterial protein CB6-C (10 mg/kg<sup>-1</sup>) or antibacterial protein CB6-C (20 mg/kg<sup>-1</sup>)</td><td>Bacterial infection in mice by methicillin-resistant Staphylococcus aureus (MRSA)</td><td>[34]</td></tr><tr><td>NST6</td><td>C. elegans</td><td>Chloroform extracts of NST6 mixed with the food bacteria at a final concentration of 50 µg/mL<sup>-1</sup></td><td>C. elegans infected with S. aureus</td><td>[40]</td></tr><tr><td>LSD4.2</td><td>Aedes aegypti larvae</td><td>13.8 × 10<sup>7</sup> CFU/mL</td><td>Larvicidal toxicity</td><td>[36]</td></tr><tr><td>FCW2</td><td>Zebrafish (Danio rerio)</td><td>10<sup>5</sup>, 10<sup>6</sup>, and 10<sup>7</sup> cells/mL were added to fish tanks</td><td>Toxicological assay</td><td>[35]</td></tr><tr><td>SN-1</td><td>Kunmig mice</td><td>N/A</td><td>Hepatoprotection</td><td>[32]</td></tr><tr><td>Z01</td><td>Male BALB/c mice</td><td>Purified fibrinolytic protease named Velefibrinase: low-dosage Velefibrinase (0.22 mg/kg), middle-dosage (0.44 mg/kg), and high-dosage (0.88 mg/kg)</td><td>Mouse tail thrombosis model</td><td>[38]</td></tr><tr><td>Kh2-2</td><td>Male ICR mice</td><td>1 × 10<sup>6</sup> CFU/mL</td><td>Immunosuppressive mouse model</td><td>[39]</td></tr><tr><td>JT3-1</td><td>Female Kunming mice</td><td>1 × 10<sup>8</sup> CFU</td><td>Model for evaluating general performance in growth, immune function, intestinal morphology, and microbiota of mice</td><td>[41]</td></tr><tr><td>AP143</td><td>Female C57BL/6J mice</td><td>1 × 10<sup>8</sup> CFU/mL spores</td><td>Cutaneous wound model</td><td>[42]</td></tr><tr><td>AP191</td><td>Female C57BL/6J mice</td><td>1 × 10<sup>8</sup> CFU/mL spores</td><td>Cutaneous wound model</td><td>[42]</td></tr><tr><td>AP218</td><td>Female C57BL/6J mice</td><td>1 × 10<sup>8</sup> CFU/mL spores</td><td>Cutaneous wound model</td><td>[42]</td></tr><tr><td>AB01</td><td>Female C57BL/6J mice</td><td>1 × 10<sup>8</sup> CFU/mL spores</td><td>Cutaneous wound model</td><td>[42]</td></tr><tr><td>AP183</td><td>Female C57BL/6J mice</td><td>1 × 10<sup>8</sup> CFU/mL spores or 1 × 10<sup>6</sup> CFU/mL spores; supernatant containing metabolites without spores or spores with supernatant (1.0 × 10<sup>6</sup> CFUs) co-administered with Staphylococcus aureus (1.0 × 10<sup>6</sup> CFUs)</td><td>Cutaneous wound model</td><td>[42]</td></tr><tr><td>AP183</td><td>Dairy cows</td><td>A high dose 10 mL of a 10<sup>8</sup> CFU/mL; a lower dose 10 mL of a 10<sup>2</sup> CFU/mL</td><td>Intramammary challenge</td><td>[42]</td></tr><tr><td>PM35</td><td>Liver cancer cells (HepG2) and normal murine fibroblast cells (L929)</td><td>N/A</td><td>MTT method test</td><td>[24]</td></tr><tr><td>KCTC 13417</td><td>Female BALB/c mice</td><td>1.5 × 10<sup>9</sup> CFU</td><td>Bacterial infection by Carbapenem-resistant K. pneumoniae (CRKP)</td><td>[37]</td></tr></tbody></table>
and animal feed, thus establishing a potential correlation [80]. The analysis of keywords also revealed the most representative terms related to *B. velezensis*, including the name of the bacterium itself, as well as “subtilis”, “identification”, “strain”, and others. The articles focused on confirming the identity of this species, which was previously classified as “subtilis” [81]. The beneficial activities are also strain-dependent, underscoring the importance of characterizing the specific strain and its genomic profile.

Based on the co-occurrence of keywords (Figure 5 and Figure 6), the primary clusters illustrate the extensive diversity of research on *B. velezensis*, encompassing a range of applications in agriculture, industry, and potential uses in human health. The largest identified cluster was cluster #0, entitled “antibiotics”. This cluster indicates research aimed at overcoming the significant challenge of antimicrobial resistance, a problem also observed in cluster #6. Additionally, this cluster reflects the search for alternatives in using microorganisms to address this challenge and to produce secondary metabolites with antimicrobial activities. This is complemented by investigating the cellular mechanisms underlying the bacterium’s activities, as evidenced by cluster #3. Finally, cluster #7 represents the most recent research, which is more specialized, and demonstrates the progress of research with *B. velezensis*, present in clusters #1 and #2. This new area of research concerns the use of agricultural waste to stimulate growth, or the production of bioactive compounds applied to biotechnology, one of the main categories represented in this review.

The selected studies adopted a comprehensive approach, encompassing not only in vivo and in vitro methods but also in silico techniques (Table 3 and Table S1), as well as Dhanya Raj et al. 2023 [35]. Furthermore, the papers that produced the most robust and reliable results—as indicated by Table 7 (in vivo experiments), Table 6 (potential for secondary metabolite production), Table 4 (antagonistic activity), the absence of resistance genes, which shows that it is a good probiotic, and Table 2 (choice of methodologies)—combined these three approaches and incorporated rigorous statistical analyses, which are essential for validating experimental findings, as well as Zhang et al., 2022 [34]. The integration of methods and utilization of statistical analyses permitted a comprehensive characterization of the *B. velezensis* strains, thereby underscoring their potential utility in the biomedical field and reinforcing the reliability of the presented results.

While this review compiles relevant findings on the biomedical applications of *Bacillus velezensis*, some limitations must be acknowledged. First, the methodological heterogeneity among the included studies (Table 2 and Table S1) poses a challenge for direct comparison. Differences in experimental models, bacterial strains, and analytical methods introduce variability in the reported outcomes, making direct comparisons difficult. Another important limitation is that most of the available data come from in vitro and preclinical studies, with limited clinical evidence. Although promising, the translational potential of *B. velezensis* in human applications remains to be fully validated. Further clinical studies are necessary to confirm its efficacy and safety in biomedical settings. Additionally, the optimization of cultivation conditions to enhance the production of bioactive metabolites varied across studies, with differences in culture media and incubation periods tailored to the specific requirements of each experiment. This variability highlights both the complexity and adaptability of *B. velezensis*, reinforcing its relevance in diverse biomedical applications.

Moreover, the number of studies available for analysis remains a limitation. While this review included 151 articles, this number remains well within the acceptable range for a meaningful scientific and qualitative review [82]. Although larger datasets allow for more robust analyses and comparative assessments, exceptions occur, particularly when integrating both quantitative and qualitative research evaluations in practical assessment processes. In this context, the selected dataset ensures a balance between comprehensiveness and analytical feasibility.

Furthermore, studies with institutional or field-specific constraints often rely on even smaller datasets. Given the methodological heterogeneity among the included studies, ranging from experimental models to bacterial strains and analytical techniques, the dataset used here is sufficiently representative to capture the key trends and biomedical applications of *B. velezensis*. Also, the systematic approach taken in this review ensures that the selected studies provide relevant and high-quality insights, mitigating the limitations of a smaller sample size. Ultimately, while additional research is needed to expand the available evidence base, the present dataset offers a robust foundation for evaluating the biomedical potential of *B. velezensis*.

## 4.2. Biomedical potential of *Bacillus velezensis*

In order to more fully comprehend the principal functions and applications of *B. velezensis* in the biomedical field, a comprehensive analysis was conducted, emphasizing its antimicrobial and probiotic activities, in addition to the production of secondary metabolites. The results demonstrate that *B. velezensis* has been extensively investigated for its capacity to impede a multitude of pathogenic microorganisms, including *Staphylococcus aureus* (Table 4), which represents a significant therapeutic target due to its resistance to conventional antibiotics such as methicillin (MRSA). The inhibition of *S. aureus* by 38.46% of the *B. velezensis* strains tested reflects the potential of this bacterium to offer an effective alternative in combating antibiotic-resistant pathogens, a topic increasingly discussed in the biomedical field.

Moreover, other studies have corroborated the efficacy of *B. velezensis* in inhibiting *S. aureus* in vivo using tests with mice and cattle, thereby substantiating its potential across diverse experimental models [42]. Furthermore, the iturins produced by *B. velezensis* have demonstrated efficacy in inhibiting *S. aureus*, particularly with regard to hemolytic activity [83]. Additionally, exopolysaccharides produced by this bacterium have exhibited the capacity to inhibit biofilm formation [84]. The *Bacillus* genus has been identified as a promising agent for the prevention and treatment of biofilms, with the potential to enhance the efficacy of antibiotics in clinical and veterinary settings [85]. In light of these findings, it seems reasonable to conclude that *Bacillus velezensis* strains have significant potential for combating infections and enhancing antimicrobial therapies.

Furthermore, research has demonstrated that *B. velezensis* exhibits considerable probiotic capabilities. In vivo experiments conducted with the DSM 33864 strain observed a reduction in *Clostridium difficile* colonization without causing dysbiosis, thereby highlighting the potential for this strain to be used in treatments to restore intestinal microbiota balance. In addition to the probiotic potential of this strain demonstrated in the biomedical field, other *B. velezensis* strains have been explored in different contexts.
For example, the FS26 strain displays probiotic characteristics in its genome and produces secondary metabolites that inhibit pathogens in agricultural settings. Additionally, it possesses genes that facilitate adhesion and confer tolerance to acids and bile salts [86]. Another study demonstrated that *B. velezensis* has robust antimicrobial capabilities against *V. cholerae* non-O1, can remove ammoniacal nitrogen and nitrite from culture water, and can stimulate the innate immune response of *Macrobrachium nipponense*. These findings highlight the potential of this strain as a probiotic in aquaculture [87].

The R-71003 strain has also undergone genome evaluation. A novel *B. velezensis* R-71003 strain exhibiting probiotic characteristics was isolated from the intestine of common carp. This strain met safety criteria based on in vivo and in vitro experiments. Furthermore, it was demonstrated that a low concentration of sodium gluconate can promote the growth of *B. velezensis* R-71003 [88].

Additionally, *B. velezensis* has been employed as a starter culture with probiotic potential in fermented foods. An in silico study demonstrated that the KMU01 strain, isolated from kimchi, exhibits survival characteristics under extreme conditions, such as gastric juice, as well as probiotic features, including adhesion, thiamine, and essential amino acid production, and the presence of enzymes such as gamma-glutamyltransferase, nattokinase, and bacteriocins [89]. This evidence suggests that *B. velezensis* has the potential to be explored as a probiotic in a range of fields, including biomedicine, agriculture, and aquaculture.

*B. velezensis*, as the results show (Table 5 and Table 6), can produce secondary metabolites such as surfactin, bacilysin, and fengycin, which have well-established antimicrobial and antifungal activities and are of great economic interest [66]. In addition to its antimicrobial function, surfactin stands out as a potent biosurfactant with applications in bioremediation. In addition, this molecule has shown an impact on the formation of biofilms, which facilitate host colonization and consequently facilitate probiotic activity [90]. This metabolite is also a highly effective biosurfactant, already being used in oil contamination remediation [50, 91]. It represents a renewable and natural alternative to artificial surfactants, especially in the cosmetic industry, showing low irritability to the skin, so it is potentially safe to use in gels and creams, for example [92]. However, the production of this peptide is costly. Therefore, many studies focus on production optimization by genetic engineering [93] and finding the optimal conditions for bacterial growth [94].

Studies on bacilysin have also shown the optimization of the production of this peptide, such as the characteristics of the medium [95] and gene editing [96], and the production of bacilysin is correlated to the process of sporulation [97], which is interesting in the industrial production of probiotic bacteria [98]. The third top metabolite, fengycin, is well known by its fungicidal activity against pathogens like *Fusarium graminearum* [53], *Rhizomucor variabilis* [99], and *Candida albicans* [100], and even antiviral effects, with *Cucumber mosaic virus* (CMV), showing a great potential for biological control, because of its biosafety and degradability. Strategies for improving fengycin production involve producing the ideal precursors for its biosynthesis, metabolic engineering, and genetic engineering [66].

Regarding antibiotic resistance, only two classes have been observed to exhibit resistance. Resistance to tetracycline has been observed in various microorganisms, including environmental,

commensal, human and animal species, and bacterial pathogens; this is due to the extensive use of tetracycline in recent decades, as it is a broad-spectrum antibiotic that has been widely used in agriculture, aquaculture, and the clinic [101]. Conversely, the emergence of resistance to cephalosporins, a β-lactam antibiotic, has prompted concerns regarding its impact on public health. Observing numerous resistant bacteria has led to an increased focus on heteroresistance to this drug class, which is widely used for more complex infections [102]. It was observed that the FCW2 strain, despite possessing resistance genes to cephalosporins and tetracyclines, did not express this resistance in in vitro tests; this raises questions about the expression of these genes under different environmental and physiological conditions, which still need to be investigated. The absence of antibiotic resistance observed in *B. velezensis* raises the question of whether these strains can be used as potential probiotics and whether they could transmit resistance genes through horizontal transfer to the intestinal microbiota, a topic that has been extensively debated [103].

The optimization of cultivation conditions to enhance the production of metabolites of interest has also been emphasized, with the utilization of diverse culture media and incubation periods to align with the requirements of each study. The variability in experimental approaches serves to illustrate the complexity and adaptability of *B. velezensis*, thereby underscoring its significance in a multitude of biomedical applications.

## 5. Conclusions

Our results show both a scientific analysis and an in-depth analysis of the applicability of *B. velezensis* in the biomedical field and its ability to be a promising probiotic candidate. In terms of general characteristics, the main dispositions and distributions of research on *B. velezensis* are systematically presented, demonstrating the great relevance of this topic in several publications, countries, journals, fields of knowledge, and keywords. The comprehensive analysis of *B. velezensis* revealed a significant biomedical potential, mainly related to producing secondary metabolites with antimicrobial and probiotic activity. However, optimizing its use and a better understanding of the gene expression under different cultivation methods and varying environmental conditions remain crucial. Future research should focus on refining and applying cultivation methods to maximize these bioactive compounds' yield, thereby realizing the full potential of *B. velezensis* in diverse applications.

## Acknowledgments

The authors gratefully acknowledge the Pro-Rectory of Research - Federal University of Minas Gerais, the Interuniversity Graduate Program in Bioinformatics and the Laboratory of Cellular and Molecular Genetics at UFMG, the Rede de Ciências Ómicas (RECOM), and INRAE and Institut Agro (Rennes, France) for their support. For the purpose of Open Access, a CC-BY public copyright license has been applied by the authors to the present document and will be applied to all subsequent versions up to the Author Accepted Manuscript arising from this submission.
## Funding

E.G.S. and G.C.G. were supported by the Research Support Foundation of the State of Minas Gerais (FAPEMIG), (5.18/2022). G.M.C. was supported by the International Cooperation Program CAPES/COFECUB Foundation at the Federal University of Minas Gerais funded by CAPES—the Brazilian Federal Agency for the Support and Evaluation of Graduate Education of the Brazilian Ministry of Education (88887.974576/2024-00). E.G. was supported by PHC CAPES/COFECUB (Me 1007/23).

## Author contributions

Conception and design of the study: E.G.S., G.M.C., S.d.C.S., J.d.C.F.D., and V.A.d.C.A.; Acquisition of data: E.G.S., G.M.C., L.S.Q., T.F.M.M., S.d.C.S., J.d.C.F.D., and V.A.d.C.A.; Analysis and interpretation of data: E.G.S., G.M.C., L.S.Q., T.F.M.M., G.C.G., R.C.V.S., B.G.R.S., E.G., S.d.C.S., J.d.C.F.D., and V.A.d.C.A.; Drafting of the article: E.G.S., G.M.C., E.G., S.d.C.S., J.d.C.F.D., and V.A.d.C.A.; Final approval of the version to be submitted: E.G., G.M.C, N.C.G, S.d.C.S., J.d.C.F.D., and V.A.d.C.A. All authors have read and agreed to the published version of the manuscript.

## Conflict of interest

The authors declare that they have no competing interests.

## Data availability statement

Data supporting these findings are available within the article, at https://doi.org/10.20935/AcadMolBioGen7598, or upon request.

## Institutional review board statement

Since the study was a retrospective analysis of laboratory data collected, and no additional investigations were performed with submitted specimens, no ethical approval was applied.

## Informed consent statement

Not applicable.

## Supplementary materials

The supplementary materials are available at https://doi.org/10.20935/AcadMolBioGen7598. Table S1: Detailed information about all the experiments and methodology carried out in each study.

## Additional information

Received: 2024-12-04

Accepted: 2025-03-07

Published: 2025-03-26

Academia Molecular Biology and Genomics papers should be cited as *Academia Molecular Biology and Genomics* 2025, ISSN 3064-9765, https://doi.org/10.20935/AcadMolBioGen7598. The journal's official abbreviation is *Acad. Mol. Biol. Genom*.

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## Copyright

© 2025 copyright by the authors. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

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