Biosafety Assessment Research Articles

Exploration of risk analysis and elimination methods for a Cr(VI)-removal recombinant strain through a biosafety assessment in mice

Though recombinant strains are increasingly recognized for their potential in heavy metal remediation, few studies have evaluated their safety. Moreover, biosafety assessments of fecal-oral pathway exposure at country as well as global level have seldom analyzed the health risks of exposure to microorganisms from a microscopic perspective. The present study aimed to predict the long-term toxic effects of recombinant strains by conducting a subacute toxicity test on the chromium-removal recombinant strain 3458 and analyzing the gut microbiome. The available disinfection methods were also evaluated. The results showed that strain 3458 induced liver damage and affected renal function and lipid metabolism at 1.0 × 1011 CFU/mL, which may be induced by its carrier strain, pET-28a. Strain 3458 poses the risk of increasing the number of pathogenic bacteria under prolonged exposure. When 500 mg L−1 chlorine-containing disinfectant or 250 mg L−1 chlorine dioxide disinfectant was added for 30 min, the sterilization rate exceeded 99.9 %. These findings suggest that existing wastewater disinfection methods can effectively sterilize strain 3458, ensuring its application value. The present study can serve a reference for the biosafety evaluation of the recombinant strain through exposure to the digestive tract and its feasibility for application in environmental pollution remediation.

A standard procedure for rapid toxicity evaluation of carbon dots both in vitro and in vivo

Carbon dots (CDs) are an emerging class of fluorescent quantum dot nanomaterials that have attracted considerable scientific attention for biomedical or bioimaging applications due to their physicochemical and biochemical properties. With the emergence of massive novel synthetic CDs applying to biomedical fields of science, evaluating their biosafety before any biological application is essential. However, there is no universal protocol or routine procedures for toxicity detection and biosafety assessment of CDs in general biological environments. Herein, we provide an ideal and fast operating system to detect the biotoxicity of CDs, which has been preliminary practiced. Briefly, the obtained CDs will be evaluated by in vitro cytotoxicity assay using cell counting kit-8, lactate dehydrogenase assay kit, and flow cytometry. Meanwhile, the model creature zebrafish is employed to perform in vivo evaluation by measuring body length, hatching rate, heart rate, and morphological observation. Our operating procedure condenses previous scattered biosafety detection methods into a rapid standard evaluation protocol that can be applied to early biotoxicity screening of CDs. This protocol will accelerate CDs biological exploitation and guide future industrialized biosafety assessment in large-scale applications.

Green Synthesis and Biosafety Assessment of MXene.

The rise of MXene-based materials with fascinating physical and chemical properties has attracted wide attention in the field of biomedicine, because it can be exploited to regulate a variety of biological processes. The biomedical applications of MXene are still in its infancy, nevertheless, the comprehensive evaluation of MXene's biosafety is desperately needed. In this review, the composition and the synthetic methods of MXene materials are first introduced from the view of biosafety. The evaluation of the interaction between MXene and cells, as well as the safety of different forms of MXene applied in vivo are then discussed. This review provides a basic understanding of MXene biosafety and may bring new inspirations to the future applications of MXene-based materials in biomedicine.

Simulating ozone degradation of deoxynivalenol and its bio-safety assessment by mouse model.

Deoxynivalenol (DON), a trichothecene mycotoxin, is one of the most prevalent mycotoxins globally, primarily produced by Fusarium species. DON exposure could cause a range of symptoms, including nausea, vomiting, gastroenteritis, growth retardation, immunosuppression, and intestinal flora disorders in both humans and animals. Recently, ozone degradation technology has been applied for DON control. However, the safety of the contaminated grain after degradation was often ignored. Therefore, the implementation technology for assessing the safety of DON-contaminated grain degradation is of great significance for food safety. In this study, based on previous degradation result of DON, we further studied and assessed the toxicity of corn contaminated with ozone-degrading DON by animal experiments in mice. We simulated feed made from corn contaminated with DON produced by inoculated Fusarium graminearum, which was treated with an ozone aqueous solution. DON treated by ozone could effectively increase the expression of total protein in mice and improve the immune system efficacy. Meanwhile, compared with DON directly-exposed mice, the corn with degrading DON could effectively maintain the level of liver and kidney immune function, and improved growth performance, enterohepatic circulation, and energy metabolism. Our study indicated that the toxicity of fed corn contaminated with degrading-DON decreased significantly after ozone degradation, resulting in a much lower toxicity compared to the DON group, or nontoxicity to some extent. Therefore, we hope that this mouse model could be used as a promising approach for assessing the risk of fungal toxins on metabolism, immunity, and intestinal health.

Coumarins from carcinogenic phenol: synthesis, characterization, in silico, biosafety, anticancer, antioxidant, and anti-inflammatory assessments

Coumarins from carcinogenic phenol: synthesis, characterization, in silico, biosafety, anticancer, antioxidant, and anti-inflammatory assessments

Laboratory evaluation of the effects of Portunus pelagicus extracts against Culex pipiens larvae and aquatic non-target organisms

Culex pipiens (Diptera: Culicidae) is a pathogen-bearing mosquito found worldwide. Marine organisms are a promising source to search for mosquito-killing substances. The potential of the different solvent extracts of Portunus pelagicus was examined using Cx. pipiens 3rd instar. The bio-safety evaluation was carried out using Danio rerio (zebrafish) and Artemia nauplii (brine shrimp). The larvicidal activity of Portunus pelagicus (blue crab) was found in chloroform extracts against Cx. pipiens 3rd instars, with LC50 value of 109.11 and LC90 value of 88.44 ppm after 48 h of treatment. A Biosafety assessment of P. pelagicus methanol extract on D. rerio and A. nauplii revealed that the extract had toxic effects on A. nauplii but was safe for D. rerio. However, the non-toxicity of the extract towards D. rerio may be misleading; further investigation on other aquatic organisms is required. Thus, the extract should be used with caution.

Imidazole based nanofungicides: antifungal efficacy against Rhizoctonia solani and biosafety assessment

Various nanotechnology advancements are used to protect crops and the safety concerns of nanopesticides for soil quality need to be addressed. Soil enzymes and certain beneficial bacteria play a vital role in preserving soil health by carrying out required biochemical reactions. In the present study, clotrimazole, econazole nitrate, and miconazole nitrate based nano-fungicides were investigated for their antifungal efficacy followed by their biosafety assessment. Antifungal studies of all the test nano-fungicides demonstrated their effectiveness against Rhizoctonia solani. The relative lesion heights were 29–38% under field conditions and they ranged from 17% to 33% in pot experiments. Impact of these nano-fungicides on urease activity in soil was comparable with the conventional fungicide hexaconazole, as evident from the presence of 0.5–2.7 mg/kg urea-nitrogen after 24 h of 200 mg/kg urea-nitrogen application. In all the treatments, the activity of soil nitrifiers was not adversely affected after application of these nano-fungicides. After 21 days of incubation, the nitrate-N contents were in the range of 150–180 mg/kg in nano-fungicide treated soils as compared to 180 mg/kg of nitrate-N in urea control. Virtual risk assessment using docking of these imidazole derivatives on soil enzymes revealed their behavior similar to that of hexaconazole.

Optimization of ultrasound extraction technology of DTD from Ageratina adenophora, and the preparation and characterization of nanoemulsions: Insecticidal activity and safety evaluation

Due to the imperfect extraction process of 4,7-dimethyl-1-(propan-2-ylidene)− 1,4,4a,8a-tetrahydronaphthalene-2,6(1 H,7 H)-dione (DTD) and the nonconstruction of delivery systems, their practical application in the field of botanical pesticides is limited. To overcome these problems, ultrasound-assisted extraction technology was optimized using BoxBehnken design (BBD). The optimal ultrasound extraction conditions of DTD were as follows: time (39 min), solid to liquid ratio (1:31), and temperature (68 ℃). The extraction yield was 0.4528 ± 0.0182 %. Nanoemulsions (NEs) loaded with DTD were prepared and characterized, and the optimal formulation of DTD NEs was determined as follows: 1 wt% DTD, 1.5 wt% S-200, 1 wt% TW-80, and 96.5 wt% deionized water. The NEs prepared by the optimal formula had small contact angles and tension levels, indicating that they had good wetting properties. The larvicidal activity of DTD NEs is similar to that of azadirachtin EC. In the biosafety assessment, the DTD NE treatment group had low levels of cell apoptosis, the toxicity of the DTD NEs to earthworms was low, and the DTD NEs had no adverse effect on maize growth. Therefore, DTD NEs offer a high degree of biosafety. DTD NEs have the potential to serve as a safe, green alternative pesticide for IPM projects to control important agricultural pests.

TGFβ Inhibitor A83-01 Enhances Murine HSPC Expansion for Gene Therapy.

Murine hematopoietic stem and progenitor cells (HSPCs) are commonly used as model systems during gene therapeutic retroviral vector development and preclinical biosafety assessment. Here, we developed cell culture conditions to maintain stemness and prevent differentiation during HSPC culture. We used the small compounds A83-01, pomalidomide, and UM171 (APU). Highly purified LSK SLAM cells expanded in medium containing SCF, IL-3, FLT3-L, and IL-11 but rapidly differentiated to myeloid progenitors and mast cells. The supplementation of APU attenuated the differentiation and preserved the stemness of HSPCs. The TGFβ inhibitor A83-01 was identified as the major effector. It significantly inhibited the mast-cell-associated expression of FcεR1α and the transcription of genes regulating the formation of granules and promoted a 3800-fold expansion of LSK cells. As a functional readout, we used expanded HSPCs in state-of-the-art genotoxicity assays. Like fresh cells, APU-expanded HSPCs transduced with a mutagenic retroviral vector developed a myeloid differentiation block with clonal restriction and dysregulated oncogenic transcriptomic signatures due to vector integration near the high-risk locus Mecom. Thus, expanded HSPCs might serve as a novel cell source for retroviral vector testing and genotoxicity studies.

Improvement of Methods for Safety Control of Microbial Producers and Food Produced Therewith.

The study substantiates the necessity to implement the algorithm of molecular-genetic assessment of biosafety of the genetically modified microorganisms (GMM) and to develop standardized methods to test the genetically modified strains producing enzymes, bioactive substances, and other products of microbial synthesis prior to their use in food industry. Analysis of microbial producers and related food products for the presence of GMM-associated DNA revealed high incidence of the marker genes amp and lacZ in enzyme preparations and in mycelium of industrial genetically modified producer of Aspergillus genus. The procedure of extraction of DNA from mycelium of mold fungi is optimized by including the stage of additional purification of the extracts, assessment of their purity by PCR with universal ITS primers, and determination of effective DNA concentration in the samples prior to conduction of the molecular genetic assay. For identification and genotyping of mold fungi (the biotechnological producers of enzyme preparations), the Sanger sequencing method was adapted. Using this modified method, we determined the species of five equivocally identified strains of Aspergillus genus. To identify the closely-related micromycetes of Ascomycota division, a genotyping algorithm was developed based on amplification of total DNA with expanded panel of primers and DNA sequencing by capillary electrophoresis.

Hemolysis and hypersensitive tests ease culture collection management of antagonistic bacteria

A biosafety assessment is a crucial first step in the management of microbial culture collection to screen and determine unexpected potential plant and human pathogenic bacteria. It is common to collect and store as many fresh culture collections from natural resources before being further evaluated for antagonist bacteria. As a result, a bulk of isolates must be preserved which required more effort and budget. Safety evaluations based on the hemolysis and hypersensitive reactions offer simple tests to ease culture collection management of antagonist bacteria. The study aimed to evaluate the safety of bacterial culture collection for their hemolysis and hypersensitive reactions. Ninety-five isolates of rhizosphere and endophytic bacterial isolates from the culture collections of the Department of Plant Protection-IPB University, Indonesian Industrial and Beverages Crops Research Institute, and Indonesian Spice and Medicinal Crops Research Institute, were evaluated their safety using the hemolysis and hypersensitive tests. The hemolysis test was conducted using blood agar media, from which isolates with a negative (?) reaction were then tested for the hypersensitivity reaction on tobacco leaves. Bacterial isolates passed from both hemolysis and hypersensitivity tests were then preserved by the lyophilization method for long-term storage of culture collection. Based on the hemolysis test, 68 out of 95 bacterial isolates (71.57%) were found to be positive (? or ?) reactions. The hypersensitive test showed that 22 of 27 negative hemolysis isolates did not trigger hypersensitivity reactions in tobacco leaves, therefore, they were preserved by lyophilization. The study indicated that a high number of bacterial isolates in the present collection, 68 positive hemolysis, and 5 hypersensitive, need to be re-evaluated due to their safety concerns. The present study highlights the importance of biosafety tests performed in an early stage before the to permanent collection of antagonist isolates.

Biosafety Assessment of a Selenium-Containing Nanocomposite Based on a Polysaccharide Matrix of Starch

Biosafety Assessment of a Selenium-Containing Nanocomposite Based on a Polysaccharide Matrix of Starch

Improved salt-tolerance of transgenic soybean by stable over-expression of AhBADH gene from Atriplex hortensis.

The salt-tolerance of transgenic soybean cleared for environmental release was improved by stable over-expression of AhBADH gene from Atriplex hortensis, which was demonstrated through molecular analysis and field experiments. An effective strategy for increasing the productivity of major crops under salt stress conditions is the development of transgenics that harbor genes responsible for salinity tolerance. Betaine aldehyde dehydrogenase (BADH) is a key enzyme involved in the biosynthesis of the osmoprotectant, glycine betaine (GB), and osmotic balance in plants, and several plants transformed with BADH gene have shown significant improvements in salt tolerance. However, very few field-tested transgenic cultivars have been reported, as most of the transgenic studies are limited to laboratory or green house experiments. In this study, we demonstrated through field experiments that AhBADH from Atriplex hortensis confers salt tolerance when transformed into soybean (Glycine max L.). AhBADH was successfully introduced into soybean by Agrobacterium mediated transformation. A total of 256 transgenic plants were obtained, out of which 47 lines showed significant enhancement of salt tolerance compared to non-transgenic control plants. Molecular analyses of the transgenic line TL2 and TL7 with the highest salt tolerance exhibited stable inheritance and expression of AhBADH in progenies with a single copy insertion. TL1, TL2 and TL7 exhibited stable enhanced salt tolerance and improved agronomic traits when subjected to 300mM NaCl treatment. Currently, the transgenic line TL2 and TL7 with stable enhanced salt tolerance, which have been cleared for environmental release, are under biosafety assessment. TL 2 and TL7 stably expressing AhBADH could then be applied in commercial breeding experiments to genetically improve salt tolerance in soybean.

In vitro biosafety and bioactivity assessment of the goat milk protein derived hydrolysates peptides

AbstractGoat milk and protein hydrolysate peptides have generated significant interest due to their diverse range of biological functions. To produce these peptides, casein (CP), and whey proteins (WP) were partially purified from goat milk and subsequently hydrolyzed with gastrointestinal endopeptidases such as pepsin, trypsin, and α‐chymotrypsin based on in silico selection to achieve gastrointestinal stable peptides. The hydrolysates were characterized using various techniques, including zeta sizer, polydispersity index (PDI), zeta potential, and reverse‐phase high‐performance liquid chromatography (RP‐HPLC). The resulting peptides were then purified using RP‐HPLC. To assess cytotoxicity, an MTT (3‐[4, 5‐dimethylthiazol‐2‐yl]‐2, 5‐diphenyl tetrazolium bromide) assay was performed with fluorescence microscopy. Finally, cytokine levels were determined using enzyme‐linked immunosorbent assay (ELISA). In comparison to WP and CP, the casein protein hydrolysates (CPH) and whey protein hydrolysates (WPH) peptides average particle size and PDI were decreased. The zeta potential of casein and whey peptides hydrolysates there were no differences. The RP‐HPLC analysis revealed the production of peptides through the hydrolysis of CP and WP. These hydrolysates peptides were found to possess diverse biological activities, as evidenced by their significant inhibition of α‐amylase, pancreatic lipase (PL), and angiotensin‐converting enzyme (ACE), as well as their antibacterial properties against selected pathogens. Further investigations were carried out to understand the mode of action of these peptides using fluorescence and scanning electron microscope (SEM) microscopy. This study provides the first evidence of gastrointestinal stable peptides derived from goat milk with inhibitory activity against α‐amylase, PL, and ACE, as well as antibacterial properties against specific pathogens. Additionally, the peptides demonstrated significant cytokine inhibition and low toxicity toward the HT‐29 cell line. This study strongly suggests that the biologically active peptides responsible for the health‐related bioactive properties described here, which are considered functional and nutraceutical ingredients, should be identified and validated in subsequent research, particularly the most effective hydrolysate peptides derived from goat milk protein. Goat milk is widely recognized as an excellent source of milk protein. In this study, bioactive peptides were produced using casein and whey proteins from goat milk, which exhibited inhibitory activity against angiotensin converting enzyme, α‐amylase, and pancreatic lipase, as well as antibacterial properties. Goat milk protein is a promising source for developing high‐quality protein products with excellent safety standards that have potential applications in the pharmaceutical and food industries.

Low-dose graphene oxide promotes tumor cells proliferation by activating PI3K-AKT-mTOR signaling via cellular membrane protein integrin αV

Along with the increasing production and application of graphene oxide (GO), its environmental health and safety (EHS) risks have become a global concern. Numerous studies have investigated the biosafety and toxicity mechanisms associated with GO, however, the majority of previous studies were based on its direct toxic dose, which could not reflect the realistic state of environmental exposure of GO with an indirect toxic dose (low dose). Meanwhile, the effects of low-dose GO on the progression of tumors are still unclearly. Herein, we found that GO can promote multiple types of tumor cell proliferation under its low-dose treatment. Moreover, the lateral size of GO has no obvious distinction on its promoting effect on tumor proliferation. The mechanistic investigation revealed that low-dose GO treatment increased the expression level of integrin αV protein, a cell membrane receptor, and further lead to the constitutively activated PI3K/AKT/mTOR signaling pathway and promoted mitotic progression. Collectively, these findings increased our understanding of the detrimental effects of GO in promoting tumor proliferation, as well as improved our biosafety assessment at its realistic exposure doses.

A Chemical Characterization Workflow for Nontargeted Analysis of Complex Extracts from Polymer Based Medical Device Using High Resolution LC/MS.

The chemical characterization of extractables and leachables (E&Ls) is an important aspect of biosafety and biocompatibility assessment in medical device industry. The advent of the body-contact use of medical devices in patient treatment has introduced a potential source for extractables and leachables as these medical devices are comprised of various polymeric materials. Several industry working groups, the FDA and USP, have recognized the guidance for chemical characterizations and nontargeted analysis of medical device extracts, such as ISO 10993-18:2020. The MS application of nontargeted analysis has played a critical role in understanding the E&Ls from medical device extracts. However, there have been very few reports about the MS based workflow with nontargeted analysis for medical device extracts and there is little guidance about the exact methodologies which should be used, even though there is an urgent need for a clearly defined process for the identification of medical device extracts. In this study, we demonstrated an analytical LC/MS (liquid chromatography/mass spectrometry) workflow using high resolution Exploris120 Orbitrap instrument for data acquisition and Compound Discoverer 3.3 intelligent software for data processing to profile the polymer related E&Ls from a balloon dilation catheter device extracted with 40% ethanol. An E&L ID workflow combining LC separation, data-informed MS acquisition strategy, MS information mining (including adduct ions, MS information from both electrospray ionization (ESI) (+) and ESI (-), in-source fragmentation, common fragment ions (CFIs), common neutral losses (CNLs), and in silico MS simulation was described with intelligent software processing and manual data interpretation. The workflow developed in this study was proven to be effective to provide a comprehensive profile of polymer related degradation products, polymer impurities and additives including surfactants, UV curing agent, antioxidants, and plasticizers for the device analyzed. The classification of E&L compounds using CFIs and CNLs was very effective to facilitate the identification of polymer related impurities and extract the polymer related impurities with common structures in a large data result set.

Probing the deep brain: Enhanced multi-photon imaging by aggregation-induced emission luminogens via nanocrystallization

The latest requirement of optical imaging is to visualize vital, thick, and complex biological tissues in real-time and minimum-invasive mode. Among diverse available diagnostic tools, multi-photon imaging (MPI) with long wavelength excitation and exceptional lateral resolution has been a perfect option. However, the existing palette of MPI contrast agents either suffers from sub-optimal imaging performance or non-negligible risk in biosafety, exploration of suitable probes is thus of great significance. To address these concerns, in this work, pure organic nanocrystals (NCs) were developed based on aggregation-induced emission luminogens (AIEgens) (DPTA-BT) with an ultrasound-assisted method. Compared to the amorphous DPTA-BT nanoparticles (NPs), NCs with orderly arranged molecules exhibited highly enhanced nonlinear optical signals, including up to 15.35 folds of two-photon action cross-section (2PACS) and up to 4.11 folds of three-photon action cross-section (3PACS). The combination of MPI and DPTA-BT NCs has contributed to a 1900 μm imaging depth of vasculature after craniotomy and a 765 μm imaging depth through the intact skull. The safe use of DPTA-BT NCs was verified by the in vitro and in vivo biosafety assessment. This work is thus expected to encourage more innovation in the development of organic contrast agents for MPI.

Specifics of Organizational and Design Solutions for Buildings of the Bureau of Forensic Medical Examination

Introduction: Nowadays, biosafety of medical personnel and visitors of the Bureau of Forensic Medical Examination (BFME) is of special importance. One of the most significant issues in terms of infection prevention and control is the design solution for premises of the BFME and departments of anatomic pathology. Up-to-date forensic medical examination institutions shall combine functionality, good technical equipment, and engineering design, all ensuring human comfort and safety. Objective: To evaluate sanitary and hygienic measures, especially organizational and design solutions for BFME buildings ensuring occupational safety and health of the personnel and proper infection control, based on the analysis of scientific publications and valid regulatory documents. Materials and methods: We have reviewed recent Russian and English-language scientific papers on current approaches to infection prevention and control in bureaus of forensic medical examination published in 2018–2022. They were selected using a logical search on such platforms as eLIBRARY.ru, PubMed, CyberLeninka, and Google Scholar. The criteria for inclusion of publications in the review were a non-zero Hirsch index (h-index) or the Russian Science Citation Index (RSCI) and the presence of the following phrases in the title, abstract, and/or keywords: autopsy biosafety, infection of medical staff, infection prevention and control, design of forensic medical examination premises, and occupational safety in forensic medical services. Of 143 sources selected, 47 articles contained a proven risk assessment of biosafety of medical personnel and description of preventive measures and were thus found eligible for inclusion in the review. Results: We established problems in the design of construction or reconstruction of BFME premises in terms of creating a safe working environment. We have noted that the workflow, functionality of the premises, and the level of sanitary measures aimed at prevention of infection in staff and visitors is often disregarded. The procedure of instrument sterilization is not regulated by documents of the Russian Federation. Organization of forensic activities in areas remote from large cities and in towns of 25,000 residents or less remains challenging. It is essential to revise and supplement current regulations to ensure biosafety of forensic medical services. Conclusion: The article describes the shortcomings of design solutions that must be taken into account in construction and/or reconstruction of BFME premises. The design of such buildings in terms of infection control shall be specified in regulatory documents.

Quantum Dots Meet Enzymes: Hydrophobicity of Surface Ligands and Size Do Matter.

Colloidal quantum dots (QDs) are a class of representative fluorescent nanomaterials with tunable, bright, and sharp fluorescent emission, with promising biomedical applications. However, their effects on biological systems are not fully elucidated. In this work, we investigated the interactions between QDs with different surface ligands and different particle sizes and α-chymotrypsin (ChT) from the thermodynamic and kinetic perspectives. Enzymatic activity experiments demonstrated that the catalytic activity of ChT was strongly inhibited by QDs coated with dihydrolipoic acid (DHLA-QDs) with noncompetitive inhibitions, whereas the QDs coated with glutathione (GSH-QDs) had weak effects. Furthermore, kinetics studies showed that different particle sizes of DHLA-QDs all had high suppressive effects on the catalytic activity of ChT. It was found that DHLA-QDs with larger particle sizes had stronger inhibition effects because more ChT molecules were bound onto the surface of QDs. This work highlights the importance of hydrophobic ligands and particle sizes of QDs, which should be considered as the primary influencing factors in the assessment of biosafety. Meanwhile, the results herein can also inspire the design of nano inhibitors.

Techno-functional characterization of fecal lactobacilli isolates of Bos indicus calves for probiotic properties.

In this study, 105 bacterial colonies were isolated from the feces of newborn healthy Bos indicus calves and 37 isolates were confirmed using morphological, biochemical tests, and genus-specific PCR as lactobacilli. 11 isolates were then short-listed for in vitro probiotic testing based on their ability to dwell under acid and bile stress. Species-level identification using 16S rRNA gene sequencing revealed that they were Ligilactobacillus salivarius. These isolates flourished in 0.4% phenol, depicting resistance in adverse conditions encountered in the gastrointestinal tract. The results of cell surface hydrophobicity were found to be 74.50% for RBL12 and 62.62% for RBL09 in hexadecane and xylene, respectively, and that of auto-aggregation was highest in RBL26 (58.92%). These isolates also produced digestive enzymes like amylase, protease, and β-galactosidase. Further assays reiterated their antimicrobial and coaggregation potential against diarrhea-causing pathogens like Escherichia coli ATCC-25922 and Salmonella arizonae ATCC-13314. Biosafety assessment revealed that none of the tested isolates were hemolytic and mucinolytic in nature. Furthermore, the antioxidant potential of the isolates was also confirmed using 1,1‑diphenyl‑2‑picrylhydrazyl (DPPH) and ferric ion-reducing antioxidant power (FRAP) assay. Along with efficient utilization of inulin, isolates showed promising adhesion ability to the HT-29 cell line. The current findings hence conclude that these Lactobacillus isolates can be exploited as animal probiotics for potentialapplication in young calves to foster gut health and immunity.