Probiotics are defined as “live microorganisms” that, when ingested in sufficient qualities confer a health benefit on the host. Probiotics can maintain intestinal stability by regulating intestinal microbiota, repairing the intestinal mucosal barrier, and restricting the colonization of pathogenic bacteria in the intestinal [1]. In recent years, oral probiotic preparations have demonstrated promising outcomes in the prevention and alleviation of diseases such as colitis, irritable bowel syndrome, obesity, diabetes, and colon cancer. The prerequisite for their efficacy is that a sufficient number of viable cell stably reaches the colon and colonize for a long period [2]. However, the viability of most probiotics diminishes rapidly when exposed to food processing (light, heat, oxidation, moisture) and the harsh environment of gastrointestinal digestion (gastric acid, bile salts, digestive enzymes) [3]. Therefore, improving the storage stability and viability of probiotics before reaching the colon is crucial for the development of functional probiotic formulations [4].

Microencapsulation with natural polysaccharides represents an effective strategy for reducing probiotic viability loss. Alginate (ALG) is a natural anionic polysaccharide with the advantages of non-toxicity, hydrophilicity, biocompatibility and low cost. It is resistant to gastric acid, commonly used as a wall material for encapsulation, and can be degraded by intestinal microorganisms [5]. In addition, ALG exhibits excellent biomedical properties, including non-immunogenicity, mucosal adhesion, self-assembly with other nanomaterials to form nanoparticles, and prolonged release of candidate substances [6]. However, the high porosity of a single ALG gel network gives it a frail ability to resist harsh environments. Therefore, many studies have considered the introduction of nanomaterials (including silica, titanium dioxide, carbon dots, nanotubes, and nano clay) into polysaccharide-based encapsulation carriers to improve system design ability and structural strength [7]. Montmorillonite (MT) is a natural nanomaterial with silica-aluminate as the main component, which is non-toxic, has a high specific surface area, exhibits good adhesion, adsorption, and cation exchange capacity, and is often used as a carrier for controlled drug release [8]. However, nano clay is weakly dispersed, less stable in single polysaccharide matrices, and vulnerable to a variety of complex conditions [9]. Therefore, there is an urgent need to develop new processing means to replace the stabilizing effect of conventional surfactant addition. Cold plasma (CP) is an emerging non-thermal food processing technology with the potential for microbial purification, food functional modification, and carbohydrate modification [10]. The mixtures of excited state gas molecules and free radicals generated by CP under voltage applied are widely used for surface modification of polysaccharide-based and protein-based polymeric materials. For example, [11] successfully constructed a novel high internal phase Pickering emulsion with a complex formed by proanthocyanidin and CP-treated soy protein isolates, which possessed excellent rheological properties and better stability. However, CP for surface modification of polysaccharides and nanomaterials is rarely reported. Therefore, our study hypothesized that CP modification could improve the surface properties of alginate-montmorillonite nano gels, and the O3 released during CP ionization could provide a relatively sterile environment for probiotic encapsulation. With the updates of products and the increasing nutritional demands of consumers, people are paying more attention to the selection of strains and functional activities of ingredient components in newly developed products [12]. Kefir is a health drink that can prevent colon cancer, and Lactobacillus kefiranofaciens JKSP109 (LK) is a probiotic isolated from the Kefir strain [13]. Jingyang Fu brick tea is produced by solid-state fermentation of tea using aerobic and anaerobic microorganisms, especially the Aspergillus cristatus and Eurotium cristatum. The unique process resulted in FBTP content, abundance, structure, and function that are significantly different from other varieties of tea, including green, black, and oolong teas [14]. Therefore, FBTP can not only be used as a colloid to stabilize nano gels but also as a prebiotic and antioxidant to enhance the viability of probiotics.

The gastrointestinal tract (GIT) consists primarily of the stomach, small intestine, and large intestine, with major physiologic barriers including gastric acidity, enzymatic degradation, mucus, and epithelial barriers [15]. When probiotics are used in nutritional fortification, the temporary protection provided by encapsulating material often falls short of the expected health benefits of probiotic preparation [16]. Therefore, for this purpose, strategies for designing probiotic products include the enrichment of materials with antioxidant activity to repair the intestinal barrier at the site of intestinal mucosal injury, and mucus-adherent materials to prolong probiotic colonization in the intestine [17]. [18] incorporated polyphenols into synthetic materials and crosslinked them via transition metal ions to develop biofunctional materials for probiotic delivery. The strong mucosal adhesion of the materials prolonged the retention time of probiotics in the intestine without affecting the viability of probiotics. The synergistic effect of probiotics and bioactivity materials demonstrated excellent preventive and therapeutic efficacy against colitis. The non-covalent interactions between the molecules of polysaccharide-based materials can facilitate their application in probiotic delivery systems, which helps to address the balance of physicochemical interactions between the probiotic cell wall/membrane and the encapsulated materials [19]. In addition, the polysaccharide-based materials, nanomaterials, probiotics and targeting sites are biocompatible with each other. Based on gastric acid resistance and colonic release, it is particularly important to endow the delivery system with unique functionalities through the ingenious structure of the encapsulated material, including reactive oxygen species (ROS) scavenging, mucosal adhesion, and colonization enhancement [20].

In this study, ALG and nano-montmorillonite complexes were treated with CP and then interacted with FBTP to form microgels for probiotic encapsulation. This study aimed to improve the interfacial and antioxidant properties of the delivery system to enhance the viability of encapsulated probiotics. First, the effects of CP treatment and FBTP addition on the physicochemical properties, interfacial structure, and antioxidant activity of biopolymer solutions (BSs) were sequentially investigated by dynamic light scattering (DLS), rheological measurements, differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), and antioxidant experiments. The distribution of probiotics in BSs and the microstructure of LK-loaded microgels were characterized by fluorescence labeling and scanning electron microscopy. Then, the gastrointestinal destiny and storage stability of probiotics were evaluated, and the good colonic colonization of LK was observed by a laser scanning confocal microscope (CLSM). This newly developed polysaccharide-nanoparticle probiotic microgel by green facile CP technology can be used as a potential functional food additive.