Drug-resistant bacterium is one of the biggest threats to global public health and development [[1], [2], [3]]. The World Health Organization reported that bacterial antimicrobial resistance caused approximately 1.27 million direct deaths in 2019 and contributed to a total of 4.95 million deaths when considering both direct and indirect effects [4]. By 2050, bacterial drug resistance is projected to have a substantial global impact, with an estimated 1.91 million deaths directly due to drug-resistant infections and an additional 8.22 million deaths associated with complications arising from this resistance (Fig. 1A) [5]. To address this pressing problem, bacteriophages (also known as phages), have been found to exert antimicrobial effects through mechanisms distinctly different from antibiotics, making them a promising solution to the drug-resistant crisis (Fig. 1B-D) [[6], [7], [8], [9], [10]]. Phages are natural predators of bacteria, which infect bacteria in a specific manner [6,11]. They attach to bacterial surfaces, inject their genomes into the bacteria, and replicate within them. This infection process is highly targeted, as phages typically infect specific bacterial hosts [6,12,13]. The extensive biodiversity of phages in nature allows for “bioprospecting”: the discovery and development of naturally evolving phages with properties suitable for bactericidal applications, such as phage therapy, environmental disinfection, and food decontamination [[14], [15], [16]].

In this context, phage therapy has attracted a growing amount of interest. It is regarded as one of the most promising applications against bacterial infections, involving administering virulent phages directly to patients to lyse pathogenic bacteria. This approach has gained increasing traction since its initial development in 1919 [[24], [25], [26]]. Its biological characteristics such as high specificity, no adverse allergic reactions, short incubation period, large outbreak volume, and wide host range. The main methods of phage therapy include topical dressings [27], intravenous injection [28,29], oral or nebulized inhalation [30], and local instillation [31]. Furthermore, the implementation methods of phage therapy mainly include monotherapy therapy [32,33], polyphase therapy [34,35], combination therapy of phages and antibiotics [36,37], and phage-derived proteins therapy [38]. Phage therapy is primarily conducted using the three main life cycle types of phages: lytic cycle, lysogenic cycle, and chronic cycle [11]. Lytic phages infect and rapidly lyse their bacterial host cells, while lysogenic phages display environment-dependent dual behavior: they can either integrate stably into the host's genome or enter the lytic cycle in response to specific inducers. These inducers may include physical or chemical agents such as ultraviolet radiation, mitomycin C, and hydrogen peroxide. (Fig. 1E) [[39], [40], [41]]. Lysogenic phages can protect their host from reinfection by other phages and alter the bacterial phenotype through the expression of viral genes, a process known as lysogenic conversion. (Fig. 1F) [42]. Last but not least, the chronic cycle refers to non-bactericidal phage infections, typically occurring in filamentous phages, where progeny phages are synthesized without causing significant damage to the bacterial cell walls. (Fig. 1G) [11]. The different life cycles provide these phages with distinct advantages for various applications.

In addition to phage therapy, the antimicrobial function of phages can be also used for environmental [43,44] and food decontamination [45]. Phages selectively target and lyse specific bacterial strains, making them ideal for reducing harmful pathogens in food products, surfaces (e.g. hospital equipment [[46], [47], [48]] and food processing factory [45,49]), and water systems [50,51] without disrupting beneficial microorganisms. This method has the potential to enhance food safety by reducing the risk of foodborne illnesses and spoilage [52,53], while also promoting sustainability by minimizing the use of chemical disinfectants and antibiotics in agricultural and industrial practices [[54], [55], [56]]. The United States Food and Drug Administration has approved several bactericidal phage products for food and animal husbandry [57,58].

Among the three types of phages, lytic phages kill a significant proportion of the bacterial cells they infect, making them suitable for therapeutic applications aimed at treating bacterial infections [[59], [60], [61]]. Lysogenic phages can be engineered to become obligatorily lytic, transforming them into potential therapeutic candidates for treating bacterial infections [62,63]. For instance, a technology called phage recombineering of electroporated DNA can achieve unmarked deletions or precise insertion of genes, making it simple and effective to construct targeted phage mutants, thereby transforming lysogenic phages into therapeutic candidates [64]. Unlike the other two, chronic phages, in spite of being poor candidates for phage therapy, can achieve a high concentration for constructing materials which will be illustrated later.

Besides natural bacterial disinfection, phages can also be engineered to achieve other functions using techniques such as constructing phage-built biomaterials [[65], [66], [67], [68], [69], [70]] and phage display [71,72]. Phage-built biomaterial technology mainly employs filamentous phages, such as M13, as building blocks. Their elongated, linear morphology allows highly ordered alignment and hierarchical assembly into fibers, films, or scaffolds, which would not be feasible with tailed or icosahedral phages [73]. In addition, the dense and repetitive arrangement of capsid proteins on their surface provides programmable chemical and biological functionalities, enabling precise molecular modification and large-scale material construction through self-assembly (Fig. 2, Fig. 3) and/or crosslinking [[74], [75], [76], [77]]. These materials have been reported in various forms, including hydrogels [78], aerogels [79], microspheres [66], fibers [[80], [81], [82]], and films (Fig. 4) [65,83,84]. They have been widely used in scaffolds [[84], [85], [86], [87]], biosensing [83,[88], [89], [90]], optical devices [65], piezoelectric materials [91], and cell culture substrate (Fig. 5) [66].

Additionally, phages can be utilized in drug delivery applications, including gene therapy, targeted drug delivery, and targeted imaging, where they transport therapeutic agents or imaging probes to specific tissues or target organs [103]. In gene therapy, phages can effectively deliver genetic material to particular tissues or cell types by displaying targeting peptide sequences on their capsids, enabling the in situ production of therapeutic proteins [104]. The tissue and cell-type specificity of phages also facilitates targeted drug delivery, allowing therapies to precisely act on diseased tissues while minimizing toxic effects on healthy cells [105]. Finally, phages can be employed in targeted imaging. For instance, Li et al. chemically modified three reactive groups on the M13 phage capsid and conjugated fluorescent particles to two of them. When these phages were further functionalized with folic acid, they exhibited high-affinity binding to KB cancer cells, thereby enabling effective cell visualization [106].

Beyond their role as building blocks for phage-built biomaterials and drug carriers, phages have also been engineered as versatile platforms for ligand discovery through phage display technology. Phage display is a selection method used to identify amino acid sequences based on their affinity for specific substrates, making it a powerful technology for screening and isolating target-specific peptides [107]. It has been widely used for investigating protein-protein interactions [108,109] investigating enzyme specificity [110,111], carrying out immunotherapy [112], developing vaccine [113], screening drug [114], and developing biosensors (Fig. 6) [[115], [116], [117], [118]].

All the aforementioned applications, ranging from phage therapy to phage display technology, share a fundamental prerequisite: the availability of high-titer phage stocks that can be diluted to various concentrations appropriate for specific applications. Phage therapy, in particular, has the most stringent standards for phage purity and dosage, as therapeutic efficacy is critically dependent on the administered titer [123,124]. A substantial body of research indicates that, within a defined therapeutic window, higher phage titers are generally correlated with superior treatment outcomes [[125], [126], [127], [128]]. For instance, in a chicken model of E. coli septicemia, intramuscular injection of a phage dose as low as 102 PFU provided only partial protection, whereas 104 PFU conferred significant protection and 106 PFU completely prevented mortality [129]. Similarly, in a murine model of bacteremia induced by drug-resistant Enterococcus, a single intraperitoneal dose of 3 × 108 PFU rescued 100 % of the animals, whereas lower doses (3 × 107 PFU and 3 × 106 PFU) yielded rescue rates of 60 % and 40 %, respectively [33]. In addition to therapeutic use, phage-based biomaterials often require concentrations far exceeding those needed for phage therapy. The fabrication of structural materials, such as films, fibers, hydrogels, or scaffolds, relies on dense packing and ordered assembly of phage particles, which typically requires titers on the order of 1013–1014 PFU/mL to provide sufficient building blocks for supramolecular organization and/or crosslinking [73,94]. This clear dose-response relationship underscores the paramount importance of producing high-concentration, high-purity phage preparations for diverse application. However, phage purification is challenging due to the presence of numerous impurities and by-products in the process of obtaining, isolating, and preparing phages. These include bacteria, lysed bacterial debris, endotoxins, peptidoglycan, exotoxins, flagella, nucleic acids, residual precipitates, culture medium and dust [130,131]. Among all these impurities and by-products, endotoxins released from lysed bacteria pose the greatest safety concern and are the most challenging to isolate from phages [27,[132], [133], [134]]. It has been extensively reported that endotoxins have high immunogenicity. If present in large quantities, they may cause sepsis (endotoxin) shock through cytokine signaling, leading to intravascular coagulation, multiple organ failure, and even death [135]. In addition to endangering people's lives [[136], [137], [138]], these impurities can also pollute the environment [139,140], interfere the structure of phage-built materials [131], and affect the function and stability of phage [123,[141], [142], [143], [144]]. Therefore, establishing methods for phage purification is critical in the field of phage research.

While phage purification is essential for the practical applications of phages, the purification process itself could cause the detriment of phage bioactivity [145,146]. In this process, the activity of phages is not only influenced by the regular factors (pH, temperature, ions), but also affected by the purification steps, such as the usage of precipitation initiator (e.g. polyethylene glycol), centrifugation, and filtration [147]. The sophisticated control of these influencing factors presented additional challenges for designing phage purification procedures.

To address these issues, we summarized the reported methods of producing purified phages, covering the three main stages of phage production: acquisition and identification of phages, preparation of crude phage suspension, and purification and concentrating of phages (Fig. 7). For the purification and concentrating of phages, we detailed various techniques (e.g. polyethylene glycol precipitation, ultracentrifugation, chromatography, ultrafiltration, and dialysis), as well as the comprehensive procedures that selectively combine these techniques in specific sequences tailored to various phages. We also discussed the relationships and differences among these methods to provide a thorough guide for the practical preparation of purified phages.