Salvia hispanica L. seeds are a well-consumed food in different countries worldwide due to their numerous benefits to human health . Its seeds are rich in nutraceutical compounds, resulting in high nutritional and health/medical values [13, 32]. Today, their incorporation - whole or flour - into yogurt, salads, bread, cakes, and others is the main form of consumption. Overall, chia seeds provide a new opportunity for their consumption as a functional food and a source of safe bioactive compounds with benefits to human health . Indeed, the in vitro biological activities of its digested protein fractions have been reported [16–19]. However, studies with isolated proteins from chia seeds are still scarce, despite their biotechnological applications. Among these compounds, protease inhibitors represent a large group of molecules in plant seeds with high agronomical, biotechnological, and biomedical added value [10–12], which has already been found in chia seeds but is still unexplored .
Many biological activities have been reported for PPIs, such as antitumoral, antioxidant, anticoagulant, and insecticidal . They have also been tested against osteoporosis, cardiovascular and inflammatory diseases, neurological disorders, and antimicrobial agents . Therefore, PPIs offer a remarkable opportunity for drug discovery and development. In this sense, considering their diversity already identified (> 6700 plant-derived proteinaceous PIs) and those still unexplored, determining the potential effects of each novel PPI on human health is of great interest [11, 33].
The initial ShTI purification steps involved heat treatment and affinity chromatography. These combined methods have been successfully employed for PPI purification [31, 34, 35]. In fact, due to the high physicochemical stability commonly associated with PPIs, heat treatment is carried out at the initial purification steps with minimal loss of inhibitory activity. Such a procedure allows the elimination of thermolabile proteins while concentrating the thermostable protein of interest [31, 34, 35]. Affinity chromatography was also an efficient and convenient step for ShTI purification that allowed us to remove the most contaminants present in HCE, as observed in the tricine-SDS–PAGE profile. Nevertheless, a reversed-phase C18 column was used in the purification process as a final step to obtain ShTI, showing a homogeneous single protein band after tricine-SDS–PAGE.
Previously, Souza et al.  isolated a trypsin inhibitor from commercial chia seed and flour in a protocol involving protein extraction, ammonium sulfate fractionation, and affinity chromatography. This protocol led to a low yield, and the soluble protein content of the retained peak from trypsin Sepharose CNBr 4B was detected in a discrete amount (0.001 mg/mL) by the Bradford method. Because of this, it was not possible to determine the specific activity. In contrast, the purification process applied in this work proved to be more efficient, achieving a 0.19% ShTI protein yield based on the protein content present in the crude extract. This value is similar to those found in RcTI from Ricinus communis L. cake (0.1%)  and LzaBBI, a Bowman-Birk protease inhibitor from Luetzelburgia auriculata seeds (0.2%) .
After purification of ShTI, its physicochemical characterization was performed. ShTI showed an apparent molecular mass of approximately 11 kDa, as evidenced by tricine-SDS–PAGE under nonreducing conditions and confirmed by mass spectrometry analysis (11.558 kDa). This molecular mass is close to those reported for other trypsin inhibitors, such as JcTI from Jatropha curcas seed cake (10.252 kDa)  and RcTI (14 kDa) . Although ShTI was characterized as a glycoprotein, the presence of carbohydrate moieties covalently bound to trypsin inhibitor structures is not a consensus. While JcTI  and AvTI from Acacia victoriae seeds  were also classified as glycosylated proteins, RcTI  and ClTI from Cassia leiandra seeds  did not show covalently linked carbohydrates.
PPIs are remarkably stable to temperature, pH, chemical agents (detergents, reductants, and oxidants), ionic strength, and proteolysis. Such characteristics are attributed to their compact tridimensional structure linked by disulfide bonds. Noncovalent interactions (e.g., hydrogen bonds and electrostatic interactions) also contribute to protein folding and overall stability [12, 38]. Considering the importance of PPI physical and chemical stability for biotechnological applications, ShTI stability was studied under prolonged exposure to extreme temperatures (100°C; 120 min), a wide range of pH values, and in the presence of a reducing agent (DTT).
ShTI proved to be thermostable and resistant to a wide range of pH values. Due to the rigidity of the trypsin inhibitor scaffold, in the face of high-temperature treatment and an extensive range of pH values, small changes in the tridimensional structure can be observed [12, 38]. Molecules with physicochemical stability are of great interest to agricultural, industrial, and pharmaceutical applications. These compounds do not demand special storage conditions (e.g., cold or warm temperatures), formulations with stabilizers, preservation in buffers, and a nonoxidative environment. Furthermore, they can resist the adverse conditions of the gastrointestinal tract, which is essential for the development of orally administered drugs . When exposed to 0.1 M DTT treatments (30–120 min.), ShTI trypsin inhibitory activity was partially reduced (23–38% loss), reinforcing the involvement and importance of disulfide bridges in its native structure.
PPIs can act reversibly or irreversibly regarding the inhibition mechanism to target proteases . Stoichiometric studies with ShTI and trypsin indicated an inhibitor:enzyme molar ratio of 1:1, showing an IC50 of 1.74 x 10− 8 M. The result suggests that ShTI presents a single reactive site trypsin complex formation, as also observed for IvTI (Inga vera trypsin inhibitor)  and EaTI (Entada acaciifolia trypsin inhibitor) . Kinetic studies of trypsin by ShTI described a competitive inhibition mode of action with a low Ki value of 1.79 x 10− 8 M, indicating high affinity toward trypsin. Different degrees of interaction have been determined for the trypsin and PPI relationship. For instance, a similar affinity toward trypsin was described for RcTI (1.90 ×10− 8 M) , while EaTI (1.75 x 10− 9 M) showed a more potent degree of interaction . Notably, the low Ki value found in PPIs has prompted their use in preclinical studies, highlighting their potential as therapeutic agents in drug development . In this sense, ShTI may also be a promising candidate to be exploited.
In the biomedical field, extensive studies have revealed the antibacterial activity of PPIs alone [25, 31, 39, 41]. However, strategies involving their effects in drug combination therapies have not been well explored. Therefore, we investigated the antibacterial properties of ShTI independently and in simultaneous treatment with oxacillin, a conventional antibiotic in clinical practice.
This study found that ShTI showed selective activity against S. aureus, including MRSA strains (MICs range from 15.83 to 19.03 µM). In contrast, no significant inhibitory effect was observed against Gram-negative bacteria. Generally, the antibacterial properties of PPIs are attributed to their ability to inhibit the activity of proteases from microorganisms, resulting in antinutritional outcomes [8, 25, 41]. In addition, perturbations of the membrane permeability of pathogens have also been reported [8, 31, 41].
Staphylococcus aureus represents an important Gram-positive human pathogen involved in most nosocomial and community-acquired infections worldwide. In the past, β-lactam antibiotics have been the first line for S. aureus infection treatment, including oxacillin and second-generation penicillin. However, the emergence of MRSA made early-generation β-lactam antibiotics no longer be used clinically [4, 42]. In this context, the World Health Organization (WHO) has issued a global priority pathogens list, including MRSA as a high priority group for research and development of new therapeutics . Here, ShTI interacted synergistically with oxacillin against sensitive and MRSA strains. Barros et al.  also observed a positive interaction between antibiotics (ciprofloxacin and vancomycin) and EpTI (a trypsin inhibitor from Erythrina poeppigiana seeds) against Klebsiella pneumoniae and S. aureus. Several advantages have been attributed to combination therapies over monotherapy, such as broadening the microorganism spectrum susceptible to available drugs, decreasing the risk of resistance development, diminishing therapeutic doses and treatment durations, and minimizing adverse effects .
To evaluate the antibacterial action mode of ShTI, MRSA-treated cells were analyzed under fluorescence and scanning electron microscopy. Its ability to disrupt cell membranes was demonstrated as indicated by intracellular propidium iodide detection. Propidium iodide has a strong affinity to interact with DNA in a complex that emits red fluorescence. However, as a membrane-impermeable fluorescent dye, it can only internalize cells with compromised plasma membranes. Disbalance into membrane permeabilization can lead to cytoplasmic content loss, resulting in cell death . The ShTI pore-forming capacity was confirmed by SEM.
Martins et al.  estimated a protein size of 3.62 nm in diameter of LzaBBI (17.3 kDa) by using a theoretical tool (http://www.calctool.org/CALC/prof/bio/protein_size). At the same platform, ShTI showed a theoretical diameter of 3.15 nm. Thus, considering the porous structure of Gram-positive bacterial cell wall dimensions of 40–80 nm, it is reasonable to assume that ShTI can reach and interact with the S. aureus plasma membrane, disturbing its surface, as suggested by Martins et al. . Notably, the cell wall of Gram-negative bacteria possesses an outer membrane that prevents the passage of some antibacterial compounds that are not present in Gram-positive microorganisms . This intrinsic resistance attributed to the cell wall structure might explain the particular activity found to ShTI against those pathogens.
ShTI also induced the production of reactive oxygen species (ROS) in S. aureus. Overproduction of ROS can lead to a toxic environment for microorganisms, in which damage to proteins, DNA, and lipids may occur, finally resulting in cell death. Although other PPIs have also been involved in inducing ROS accumulation in bacterial and fungal cells [9, 31], their mechanisms are not well understood. It has been suggested that ROS overproduction results from the activation of mitochondria to assure the increased energetic metabolism necessary to repair and maintain membrane cell physiological function, which was previously damaged .
Taken together, considering the mode of action of oxacillin and ShTI, it is reasonable to assume that oxacillin may perturb cell wall structure, which in turn improves the entry and interactions between ShTI and the plasma membrane, thereby generating the observed synergistic effects. Thus, considering the potential therapeutic application of ShTI in treating infections caused by S. aureus alone or in drug combination therapy, its toxicological assessment and in vivo responses deserve further study.