Introduction

Perishable agricultural products are primarily animal and plant-based produce that are susceptible to quality issues, such as spoilage and decay, under natural environmental temperatures. Fruits, vegetables, meat, raw milk, and aquatic products are easily contaminated by spoilage bacteria during processing, storage, and marketing, leading to significant biochemical changes and accelerated deterioration, causing waste and severe economic losses (Usman et al., 2024; Aziz et al., 2023; Wang et al., 2019). Spoilage bacteria are microorganisms that induce food spoilage and pose food safety risks, including bacteria, molds, and yeasts. Once these microorganisms contaminate agricultural products, they emit distinct odors, make products soft and sticky, and increase volatile basic nitrogen by decomposing proteins, fats, and carbohydrates. According to the Food and Agriculture Organization of the United Nations (Koutsoumanis et al., 2021), global waste due to the spoilage of agricultural products accounts for about one-third of total output. Contamination and decay losses caused by spoilage bacteria are major challenges for the global agricultural processing industry and are of significant concern for governments in terms of agricultural product quality and supply security (Vanegas et al., 2017). With China’s “rural revitalization” and “big food concept” policies, along with the growing demand for safe, eco-friendly, low-carbon, high-quality, and commercialized fresh agricultural products, greener, safer, and higher-quality preservation and storage technologies have become the development trend for China’s agricultural cold chain storage and transportation industry. Ensuring that global agricultural products reach tables fresh and solving the preservation of perishable goods with advanced technology has drawn researchers’ attention. Currently, food and agricultural preservation methods include chemical, physical, and biological techniques to ensure food safety and sustainability (Aziz et al., 2024; Du et al., 2024; Shouket et al., 2024; Shouket et al., 2023; Khan et al., 2022).

Chemical preservation technology employs chemical agents through methods such as application, soaking, and spraying to treat the surface of agricultural products. This impacts the physiological and metabolic activities of organisms, thereby achieving storage and preservation (You et al., 2023). Predominantly, this technology utilizes chemical preservatives like various metal salts, chitosan, ethanol, and certain additives such as calcium salts. While chemical preservation is convenient, efficient, and easily promoted, prolonged use can lead to drug resistance in pathogenic bacteria and environmental pollution due to residual substances. Furthermore, excessive consumption of chemically preserved agricultural products may pose severe health risks, including cancer and teratogenic effects (Javanmardi et al., 2019). Physical preservation technology utilizes physical means to prevent agricultural products from deteriorating. These methods include low-temperature storage, controlled atmosphere storage, heat treatment, ozone storage, and irradiation storage. However, physical preservation has limitations, such as high energy consumption, significant investment, and nutrient loss. Moreover, its effectiveness is often influenced by various environmental factors. With the advancement of green industries, addressing the preservation challenges of perishable products through eco-friendly technologies has garnered increasing attention. There is an urgent need to find safe, non-toxic, and effective preservation methods (Ng G et al., 2020).

Biological preservation technology utilizes antibiotics, lysozyme, protease, and organic acids produced by antagonistic bacteria to maintain freshness. These substances inhibit harmful pathogens on the surface of agricultural products or prevent the loss of internal nutrients through competitive growth inhibition, heavy parasitism of pathogens, and induction of defense enzymes, thereby achieving preservation. Lee et al. discovered that the ethanol extract of chestnut inner shell exhibited antibacterial effects against Campylobacter jejuni in chicken at a concentration of 2 mg/mL (Lee et al., 2016). Khaleque et al. demonstrated that clove essential oil is more effective than cinnamon essential oil in inhibiting the growth of Listeria monocytogenes in beef surimi, enhancing the product’s safety (Khaleque et al., 2016). The decay of agricultural products is primarily caused by bacteria and fungi, and traditional methods of prevention and control have largely relied on chemical fungicides and physical preservation. However, these physical and chemical methods have significant drawbacks. In contrast, biological preservation offers advantages such as environmental friendliness, safety, high efficiency, and a reduced risk of inducing drug resistance in pathogens (Schillinger et al., 1996).

In recent years, the use of antagonistic bacteria for the biological preservation of agricultural products has garnered increasing attention from researchers worldwide. Numerous antibacterial substances have been extracted from these bacteria, including small molecular antibiotics, proteinaceous antibacterial substances, polypeptide antibiotics, and secondary metabolism-derived compounds. Studies have discovered that Bacillus subtilis can effectively control fruit diseases such as strawberry gray mold, anthracnose, grape gray mold, and apple anthracnose. Methylotrophic Bacillus has been shown to inhibit banana wilt, pear rot, and other diseases (Madhaiyan et al., 2010). Furthermore, various strains of Bacillus, Alternomonas, Pseudomonas, photosynthetic bacteria, Marinobacter bacteria, and Lactobacillus can effectively suppress Vibrio parahaemolyticus in aquatic products (Bhaskaran et al., 2023). Mannai et al. demonstrated that a 2:1 mixture of Trichoderma harzianum and Bacillus subtilis, applied three times consecutively, achieved an 80.4% control rate of strawberry gray mold. In another study (Mannai et al., 2023), Shi et al. inoculated whole milk with bacteriocin K7 produced by Leuconostoc mesenteroides K7, which exhibited a significant antagonistic effect on Listeria monocytogenes. This bacteriocin operates differently from nisin, indicating potential applications in milk preservation (Shi et al., 2016).

The application of microorganisms for biological control has achieved significant breakthroughs and is regarded as a promising alternative to chemical fungicides and physical preservation methods. When combined with packaging films, it can further inhibit microbial growth. However, even commercial biological control preparations are still less effective compared to chemical fungicides. Therefore, exploring new and efficient antagonistic bacteria and studying their effects and mechanisms in inhibiting the rot of agricultural products is of both theoretical and practical importance. This research can accelerate the commercialization of microbial control for agricultural products, aiding the rapid and healthy development of the agricultural industry and contributing to rural revitalization. This review outlines the current state and applications of antagonistic bacteria and their active preservation film technology in various perishable agricultural products. It aims to offer a theoretical basis for future research on this technology and its industrial application in the production, transportation, and storage of agricultural products.

Overview of Antagonistic Bacteria

Definition of antagonistic bacteria

Antagonistic bacteria produce specific metabolites or alter environmental conditions during their life processes, thereby inhibiting the growth and reproduction of other microorganisms, or even killing them. Due to their unique advantages, such as environmental friendliness, good preservation effects, strong antibacterial properties, and high tolerance (Wang et al., 2022), they align well with the development needs of the agricultural product industries worldwide (Ferreira et al., 2021). Consequently, antagonistic bacteria have become a popular biopreservation technology in the field of agricultural product preservation.

Antibacterial mechanism of antagonistic bacteria

Understanding the preservation mechanism of antagonistic bacteria is crucial for their effective application in preserving agricultural products and advancing new preservation technologies (Madhaiyan et al., 2010). Generally, these mechanisms can be categorized into four types: competition for resources, hyperparasitism, secretion of antimicrobial substances, and induced host resistance (Dianpe et al., 2010).

Competition for trophic space

Nutrient and space competition is a crucial biocontrol mechanism employed by antagonistic bacteria. These bacteria swiftly dominate fruit surfaces by competing with pathogens for essential nutrients such as carbohydrates and nitrogen sources. Yu et al. discovered that Candida guillermondensis rapidly proliferates at peach fruit wounds through nutritional competition, effectively preventing soft rot (Yu et al., 2009). Similarly, Wang et al. found that B. cepacia B-1 inhibits the growth of Botrytis cinerea and Penicillium expansum in strawberry wounds by competing with these pathogens for nutrients and space (Wang et al., 2018).

Heavy parasitic

Hyperparasitism, a phenomenon where fungi parasitize other fungi, is a well-documented natural occurrence and a crucial mechanism through which biocontrol bacteria eliminate plant pathogens. This process includes identification, contact, entanglement, penetration, and ultimately parasitism (Ahmed et al., 2000). It has been observed that certain yeasts can attach to pathogenic bacteria and directly parasitize them to exert antibacterial effects. Arras et al. discovered that Candida namibiae can colonize fungal mycelium and promote its lysis, thereby inhibiting the development of citrus penicillium (Giovanni et al., 1996). Claudia et al. found that Pseudozyma aphidis can parasitize the hyphae of powdery mildew and Botrytis cinerea, competing for nutrients and thereby controlling strawberry powdery mildew and gray mold (Calder et al., 2019). Presently, our understanding of hyperparasitism mechanisms remains limited, with many aspects still to be further explored.

Secretion of antimicrobial substances

Research indicates that when antagonistic bacteria interact with pathogenic bacteria, significant biomass accumulates around yeast cells at the wound sites of fruits. This biomass includes antibiotics, hydrolytic enzymes, organic acids, and toxic proteins. These extracellular active substances likely play a role in the interactions between antagonistic and pathogenic bacteria. Rabbee et al. noted that Bacillus subtilis secretes antibacterial substances that inhibit sweet cherry rot (Muhammad, 2019). Similarly, Sharma et al. highlighted that Pseudomonas cepacia Burkh can secrete antibacterial compounds that suppress the growth of Botrytis cinerea and Penicillium expansum on apple surfaces, as well as Penicillium digitatum in lemons (Sharma et al., 2009). Current research underscores that the secretion of antibacterial substances is a critical mechanism through which antagonistic bacteria inhibit pathogens. However, it is important to consider that some antibacterial substances, like antibiotics, may induce resistance in pathogenic bacteria, diminishing the effectiveness of disease control over time. Additionally, the antibiotics produced by antagonistic bacteria might pose risks to consumers (Rodríguez et al., 2007).

Inducing Host Resistance

In recent years, the induction of resistance in postharvest fruits by antagonistic bacteria has emerged as a research hotspot in biological control. Wang et al. discovered that Hansenispora uvarum (H. uvarum) in grape juice could activate the SA signaling pathway in strawberries by upregulating the expression of resistance genes and enhancing the activity of defense-related enzymes, thereby preventing postharvest gray mold (Wang et al., 2019). Tang et al. observed that Clauretii, at an appropriate concentration, could elevate the expression of resistance genes and defense-related enzymes in cherry tomatoes, significantly bolstering their resistance to Botrytis cinerea and Alternaria alternata (Tang et al., 2021). Charles et al. reported that Pichia membranefaciens could heighten the disease resistance of citrus fruits by inducing increased activity of defense enzymes or phytoprotectin synthesis, thereby averting the onset of citrus green mold and blue mold (Charles, 1992).

Synergistic effect

The preservation mechanisms for different antagonistic bacteria are often multifaceted, working synergistically to prevent the decay of agricultural products. Research has demonstrated that a mixed culture of Debaryomyces hansenii and Stenotrophomonas rhizophila significantly inhibits the proliferation and lesion size of Fusarium on melon fruits. This effect is attributed to the competition for nutrients among antagonistic bacteria, as well as the production of hydrolytic enzymes and secondary metabolites (Rivas-Garcia et al., 2019).

Evaluation of antagonistic bacteria in fresh keeping of agricultural products

As the study of antagonistic bacteria mechanisms progresses, our understanding of their preservation capabilities continues to deepen. An increasing number of researchers are examining the preservation effects of various antagonistic bacteria on different agricultural products. This method, which involves using beneficial microorganisms or the antibacterial substances they produce to combat pathogenic or spoilage microorganisms, is known as biological preservation. Currently, yeast, lactic acid bacteria, and actinomycetes are among the most effective antagonistic bacteria used for preserving agricultural products (Lahmamsi et al., 2024).

Saccharomycetes

Antagonistic yeast is characterized by its wide availability, strong adaptability, robust reproductive ability, non-toxic metabolites, and lack of sensitization (Tahir, 2022). Currently, research on the preservation of agricultural products using antagonistic yeasts primarily targets fruit and vegetable preservation. These yeasts predominantly originate from fruit surfaces, leaves, roots, soil, and seawater, with the majority isolated from fruit surfaces (Li et al., 2017). For instance, Zhang et al. identified Meyerozyma guilliermondii from healthy broccoli, discovering that this strain enhances the synthesis of phenolic, terpenoid, and alkaloid substances, thereby improving broccoli’s disease resistance (Zhang et al., 2022). Lin et al. isolated Hannaella sinensis from orchard soil, demonstrating its significant efficacy in controlling postharvest penicillium disease in apples and reducing natural decay (Lin et al., 2022). Wang et al. isolated Yarrowia lipolytica from grape pericarp, which effectively controls Penicillium rubens post-harvest (Wang et al., 2018).

Figure 1. Antibacterial mechanism of antagonistic bacteria.

Lactic acid bacteria

Lactic acid bacteria are recognized as safe microorganisms in agriculture. They have promising applications due to their abilities to antagonize pathogenic bacteria, enhance immune function, strengthen the intestinal barrier, and balance intestinal flora. These features make them potential candidates as the next generation of safe, stable, and economical biological antibiotics, with the possibility of reducing or even replacing traditional antibiotics (Wang et al., 2018). Currently, research on the use of lactic acid bacteria in meat preservation primarily focuses on specific strains like Lactococcus, Streptococcus, Enterococcus, and Pediococcus. These bacteria produce bacteriostatic or bactericidal bacteriocins to achieve preservation. For instance, Nisin produced by Lactococcus lactis can improve meat taste, yet it is costly and unstable in acidic environments (Anumudu et al., 2021); Enterocin from Enterococcus faecium exhibits strong bactericidal properties but requires stringent environmental conditions to be effective (Wei et al., 2024); Pediocin from Pediococcus offers good bacteriostasis without affecting the taste and flavor of meat (Rodríguez et al., 2007).

Actinomycetes

Actinomyces is a prokaryotic microorganism with characteristics between fungi and bacteria. Research indicates that actinomycetes exhibit antagonistic properties against specific microorganisms. For instance, they show antagonism towards pathogens responsible for common fruit and vegetable diseases, such as tomato gray mold, cucumber downy mildew (Jijakli et al., 1998), Botryosphaeria dothidea, Fusarium oxysporum f. sp. niveum, Phytophthora capsici, and Sclerotinia sclerotiorum (Wu et al., 2018). Hao et al. identified a strain of Streptomyces albiflaviniger from soil, whose fermentation filtrate effectively inhibits poplar fusarium wilt, watermelon anthracnose, soybean bacterial spots, and rice bacterial spots (Hao et al., 2019). Similarly, Jain et al. isolated a strain of Streptomyces sampsonii from tomatoes. This strain was found to inhibit spore germination and hyphal growth of Botryosphaeria dothidea, enhance tomato resistance enzyme activity, and provide an excellent preservative effect on stored tomatoes (Jain et al., 2007). It is notable that despite the numerous research findings highlighting the potential of antagonistic bacteria as biological preservatives, there are currently few commercial examples available. This is primarily due to the inherent characteristics of antagonistic bacteria, such as low solubility, weak desorption ability, rapid release, and easy degradation, which restrict their application in agricultural products. Additionally, environmental factors such as pH, storage temperature and duration, oxygen levels, and light exposure can also impact the effectiveness of natural preservatives. Therefore, to enhance their biological efficacy and applicability, they are typically used in conjunction with other preservation methods, leveraging synergistic effects to achieve preservation and anti-corrosion goals. Packaging is one effective strategy to broaden the applicability of natural preservatives in agricultural products, as it can enhance stability while maintaining antibacterial properties, thus increasing versatility in agricultural product processing. Furthermore, with the growing societal focus on the safety of agricultural products, there is a rising demand for improved packaging materials (Yu et al., 2021). Consequently, the development of antibacterial biological cling films has emerged as a significant research interest (Min et al., 2010).

Application of Antibacterial Activity Preservative Film in Agricultural Products

Classification of packaging films suitable for perishable products

Agricultural product packaging is crucial for preventing microbial contamination and is an essential component of processing and storage. Given the diverse characteristics of packaging materials, selecting the appropriate material is particularly important. Currently, common packaging film materials include polyethylene (PE), high-density polyethylene (HDPE), low-density polyethylene (LDPE), polyamide (PA), polyvinyl ether (PVE), and polyvinyl alcohol (PVA). Common types of packaging films include microporous, antibacterial, anti-fog, and nanocomposite films. This article discusses the properties of these films, categorizing them into polymer chemically synthesized films, degradable films, and edible films.

Degradable film

Natural polymers such as starch, cellulose, and chitosan are extensively utilized for postharvest fruit preservation due to their excellent reproducibility, biodegradability, biocompatibility, and environmental friendliness (Mohammad et al., 2013). Polylactic acid (PLA) is a degradable semi-crystalline polyester produced by fermenting sugars, corn, and other substances into lactic acid, which is then polymerized. However, the varying degrees of PLA crystallization and its low impact toughness limit its application in the packaging industry (Lizundia et al., 2016). Geng et al. developed a degradable Cur-ZIF-8/CS/Zein composite membrane by chemically crosslinking the zinc ion of imidazole framework zeolite-8, containing curcumin, with hydrophilic chitosan (CS) and corn flavone. This approach addressed the interfacial incompatibility issues between hydrophobic and hydrophilic polymers. While chitosan membranes exhibit poor barrier properties against water vapor, oxygen, and carbon dioxide and tend to dissolve easily, zein films suffer from limited flexibility and brittleness, restricting their use in fruit preservation. The Cur-ZIF-8/CS/Zein composite film demonstrated superior barrier properties, mechanical strength, and antibacterial efficacy compared to traditional polymer composites, ultimately extending the shelf life of lychee by 8 days (Geng et al., 2023).

Chemical synthesis of polymer films

Polymer films exhibit superior strength, heat resistance, and chemical stability, with the ability to adjust the CO₂/O₂ transmission ratio based on the characteristics of agricultural products. Despite their numerous benefits, polymer flexible packaging materials contribute significantly to environmental pollution. Mannan et al. developed PLLA-PEG-PLLA (PLGLxGy) and PLLA-PCL-PLLA (PLCLxCy) copolymers using polyethylene glycol (PEG) and poly (L-lactic acid) (PLLA), which were applied in the packaging of cherry tomatoes and strawberries. The study revealed that both polymer films could maintain suitable high CO₂ and low O₂ concentrations. This environment effectively inhibits fruit respiration and water loss, suppresses microbial growth and enzyme reactions, delays ripening and aging, and significantly extends the shelf life of cherry tomatoes and strawberries (Mannan et al., 2013).

Edible film

Edible films are utilized as packaging materials and components of agricultural products. They are designed to be efficient, safe, and environmentally friendly. These films can be composed of proteins, carbohydrates, lipids, and other compounds. Among these, chitosan is a popular active packaging material (Gomes et al., 2019). Various agricultural products have unique packaging requirements, necessitating the selection of appropriate films based on their specific characteristics. Due to their environmental benefits and high efficiency, edible films have become a focal point in agricultural preservation. Sharma et al. used chitosan films containing Candida utilis to inhibit rot in tomatoes caused by Alternaria alternata and Geotrichum candidum. The results indicated that the lesion diameters with 0.25% and 0.5% chitosan were 16±3mm and 15±1mm, respectively, while another study showed a lesion diameter of 6mm and a significantly reduced decay rate with the same chitosan concentrations (Sharma et al., 2006). De Lacey et al. prepared edible films from gelatin solutions enriched with probiotics (Bifidobacterium bifidum and Lactobacillus acidophilus), sorbitol, and glycerol as plasticizers, evaluating the survival rate and functionality of the probiotics. The results demonstrated that the probiotic count in the coating remained stable for ten days. To avoid impacting the sensory properties of fish, the number of probiotics stabilized after high-pressure treatment to remove gram-negative bacteria (Guo et al., 2025). The findings showed that lactic acid bacteria and bifidobacteria were unaffected by high pressure when combined with the film (De Lacey et al., 2012). Soukoulis et al. incorporated Lactobacillus rhamnosus GG into films made of starch, soy protein concentrate, gelatin, and sodium caseinate, using glycerol as a plasticizer. During the drying process, the Lactobacillus rhamnosus count decreased significantly across the four film solutions, ranging from 0.81 to 1.87 log CFU/g. The inactivation rate was highest in starch films without protein. The activity of Lactobacillus rhamnosus increased by 3-4 times and 5-7 times when proteins were added to starch films made from corn and rice (Soukoulis et al., 2016).

While edible films are widely used in preservation, they still have some drawbacks. For instance, their water solubility reduces the environmental resistance of the films, which can easily impact the appearance and quality of agricultural products when exposed to moisture. However, edible films combined with other technologies or additives can not only maximize the benefits of these films but also mitigate certain defects.

Combination mode and effect of antagonistic bacteria and fresh-keeping film

The primary function of antibacterial cling film is to enhance the quality and safety of agricultural products while extending their shelf life. This is achieved by incorporating active substances into the polymer matrix, which are then slowly and continuously released from the packaging to provide long-term antibacterial effects (Patrícia et al., 2010), as illustrated in Figure 2.

Figure 2. Mechanism of action of antibacterial plastic film.

The primary techniques for combining antagonistic bacteria with packaging films to achieve antibacterial and preservative effects include casting, blowing, dipping, and spraying (Zhang et al., 2017).

Casting method

The casting method involves thoroughly mixing the antagonist solution with the preservative film matrix solution, pouring the mixture into a mold, and drying it to create a uniform preservative film with antagonistic properties. This fresh-keeping film contains antagonistic bacteria both within and on the surface, enabling a slow and even release onto perishable products, thereby ensuring long-term preservation. Moreover, when perishable products are placed in environments where antagonistic bacteria struggle to survive, the plastic wrap acts as a protective barrier, aiding in the preservation of these bacteria (Cheng et al., 2016). A. M. López de Lacey et al. incorporated Lactobacillus acidophilus and Bifidobacterium bifidum directly into the film-forming solution, adjusting the bacterial concentration to 10⁹ CFU. The solution was then poured into a square plastic dish and dried in an oven at 45°C for 15 hours, resulting in a uniform dry film containing viable bacteria. The number of viable bacteria in the film remained stable for six days. Tapia et al. developed edible alginate and gellan coatings containing Bifidobacterium lactis BB-12, applying them to apples and papayas. The researchers dissolved alginate or gellan gum and heated the solutions to form the film. Bifidobacteria were then added, and the mixture was poured into dishes to dry, forming a film. Although the concentration of viable bacteria in the film solution was two logarithmic cycles lower compared to the film on the fruit, the results showed that the bacteria on the fruit film remained viable and stable in number over ten days at 2°C (Tapia et al., 2007).

Blown film method

The blown film method involves using a circular machine head to inflate a thin film material into a tube, which is then cooled and shaped by a wind ring. This thin film, now double-layered and half the circumference of the film tube, is gradually stacked by a zigzag plate and finally rolled up by a curling device to form a semi-finished film. This method is both economical and straightforward for thin film formation and processing (Benjamín et al., 2024). Barbiroli et al. developed a paper infused with lysozyme and lactoferrin, metabolites of antagonistic bacteria. Their tests on the Gram-positive bacterium Listeria monocytogenes revealed that the combined antibacterial agents significantly extended the lag period from 1.86 hours to 6.5 hours, whereas lysozyme alone extended it only to 5.81 hours. For Gram-negative bacteria such as Escherichia coli, lysozyme alone extended the lag period from 1.08 hours to 1.79 hours, while the paper containing lactoferrin extended it from 1.80 hours to 3.25 hours (Barbiroli et al., 2012). Wang et al. found that integrating Nisin, chitosan, and perilla essential oil into a composite film via an open-loop reaction exhibited effective antibacterial activity against Staphylococcus aureus, Escherichia coli, Salmonella enterica, and Pseudomonas aeruginosa. Additionally, Nisin enhanced the film’s mechanical properties, water vapor barrier properties, and optical attributes (Wang et al., 2021).

Impregnation method

The dipping method involves submerging a prepared film in a solution containing antagonistic bacteria to aid in future fruit preservation. The soaked film, with bacteria on both surfaces, can effectively contact the antagonistic bacteria with the fruit by wrapping it, thus facilitating bacterial colonization on the fruit’s surface. Dawson et al. discovered that soybean film with lauric acid (8%) and nisin (2.5%) significantly reduced the number of Listeria monocytogenes in turkey intestines within the first 8 hours, although its bacteriostatic effect was notably lower compared to the film in a liquid medium (Dawson et al., 2002). Xie et al. soaked PVCD film in bacteriocin plantaricin BM-1 solution, applied it to Listeria-inoculated pork, and observed a distinct bacteriostatic effect. The bacteriostatic zone measured approximately 16.28 ± 0.37 mm, indicating a positive effect on extending pork’s cold storage period (Xie et al., 2018).

Spray method

The spraying method involves evenly applying antagonistic bacteria on the surface of a preservative film, integrating it with the common preservation of agricultural products. Zhang et al. combined PE, low-density PE (LDPE), and high-density PE (HDPE) films with the bacteriocin plantaricin BM-1 using the spraying method. They discovered that the inhibitory effect of active films incorporating bacteriocin on Listeria at 25°C remained stable for at least 120 days, indicating significant potential for these active preservative films in controlling Listeria infection (Zhang et al., 2017.).

The integration of antagonistic bacteria with active films benefits the preservation of agricultural products. However, the preservation methods involving these bacteria and films have drawbacks. For instance, starch and pullulan exhibit excellent physicochemical and mechanical properties as films. Paulraj Kanmani et al. developed new edible films based on pullulan and starch by incorporating various probiotic strains. The inclusion of probiotics was found to decrease the viscosity, pH, transparency, and water vapor permeability of the film-forming solution, while the addition of starch reduced the probiotics’ activity. Moreover, issues such as poor film stability and inadequate adhesion between the film and antagonistic bacteria cannot be overlooked. To enhance long-term preservation effects, antimicrobial active preservative films can be combined with other preservation methods to counteract the limitations of using these films alone. Kanmani et al. have explored the combination of antimicrobial active cling films with controlled atmosphere technology, refrigeration, and high-pressure techniques, demonstrating that such combinations more effectively preserve perishable products (Kanmani et al., 2013).

Application of Antimicrobial Active Biofilm and Other Technologies in Agricultural Products

Fruits, vegetables, aquatic products, fresh meat, and dairy products are highly perishable. Without proper preservation, these agricultural products can rot and deteriorate, leading to significant economic losses and posing health risks if consumed unknowingly.

Application in fruit preservation

According to statistics, China experiences an annual post-harvest fruit loss of approximately 80 million tons, which accounts for about 25%-30% of the total yield (Neegam et al., 2021). In developed countries, the loss rate of fruits and vegetables is controlled to below 5%, while the loss rate in the logistics link is only 1%-2%, highlighting a significant gap. Due to their high moisture and sugar content, fruits are highly susceptible to infection by pathogenic bacteria and damage after harvest. For instance, citrus fruits suffer from postharvest rot primarily caused by Penicillium digitatum, Penicillium italicum, brown rot, and black rot. Pears are mainly affected by Penicillium expansum and Botrytis cinerea, while peaches are predominantly infected by Penicillium expansum, Botrytis cinerea, and Rhizopus stuccoides. Strawberries, on the other hand, are primarily infected by Penicillium expansum, Botrytis cinerea, and Rhizopus stuccoides (Gianfranco et al., 2001). Infection by these pathogens leads to a reduction in hardness, color changes, and decreases in ascorbic acid, titratable acid, reducing sugars, and soluble solids, thereby diminishing the sensory characteristics and commercial value of the fruits.

Active preservative films have been developed as natural, pollution-free alternatives to common waxes. These films extend the shelf life of fruits, enhance their appearance, and reduce water loss. Numerous antagonistic bacteria, particularly antagonistic yeasts, are now employed in fruit disease treatment due to their high inhibitory capabilities, survival rates, rapid colonization, production of volatile organic compounds, and low nutritional requirements. Fan et al. tested the addition of Cryptococcus laurenti to an alginate coating to extend the shelf life of strawberries. After five days of storage, the mildew rate in the active preservative film group was reduced by 25% compared to the control group. The edible alginate biofilm had no significant impact on the strawberries’ external color or anthocyanin content but did significantly decrease decay and weight loss rates, maintain fruit hardness, and improve overall quality and storage performance. The inhibition mechanisms may include: 1) the film sealing the fruit surface, and 2) the antagonistic yeast inhibiting pathogenic bacteria infection. Additionally, plasticizers and hydrophilic compounds can enhance the film’s flexibility, plasticity, and hydrophilicity. Glycerol, for example, reduces the film’s brittleness, and these additives optimize its performance, prolonging the activity and distribution of microorganisms within the film (Fan et al., 2009). Ren et al. studied the preservation effects of carboxymethyl cellulose and alginate-based coatings, which included Brewer’s Yeast, on grapes. After 13 days at room temperature, coatings with 1.5×10⁷ ∼ 1.5×10^- cfu/mL Brewer’s Yeast reduced decay compared to the control. By the 13th day, peroxidase activity in grapes treated with the coating plus yeast increased by 48.4% and 27.5% compared to the control and coating-only treatments, respectively. The yeast-enhanced coating also reduced weight loss, maintained soluble solids content, and elevated the activities of superoxide dismutase, peroxidase, and catalase, which together neutralize free radicals and minimize fruit cell damage. Moreover, this coating protected ascorbic acid from degradation, delaying senescence and preventing fruit damage (Ren et al., 2013). Aloui et al. compared abnormal Wickerhamomyces anomalus in sodium alginate and locust bean gum base films. The locust bean gum film demonstrated superior flexibility and barrier properties. After 14 days, the yeast population decreased by 0.75 log CFU/cm² in the locust bean gum film and by 0.95 log CFU/cm² in the alginate film, likely due to nutrient depletion and reduced moisture content. Overall, after 21 days of storage at 25°C, microbial viability did not significantly drop; both types of films retained 85% of the initial yeast population, suggesting that both films could provide nutrients to Wickerhamomyces anomalus (Aloui et al., 2015). Parafati et al. applied locust bean gum-based coatings, supplemented with strains of Wickerhamomyces anomalus, Metschnikovia pulcherrima, and Aureobasidium pullulans, as biocontrol agents on citrus surfaces. All yeast strains bound to the film reduced the incidence of citrus diseases and significantly improved yeast viability. The antagonistic bacteria enhanced citrus disease resistance by stimulating POD and SOD enzyme production, with SOD activity levels exceeding control values. High concentrations of antagonistic bacteria can boost pathogen inhibition capabilities, and immersion experiments demonstrated that membraned bacteria had increased survival rates, aiding their antibacterial efficacy (Parafati et al., 2016).

The use of antagonistic activity cling film in the preservation of fruits has become widespread, demonstrating significant effectiveness. The primary mechanisms through which this film preserves fruits are hypothesized as follows: the film isolates fruits from direct contact with the external environment, regulates the permeability of carbon dioxide, oxygen, and water vapor, providing a low-oxygen and low-humidity environment. This slows down pathogenic infections, reduces the respiration rate and other biochemical reactions of the fruits, thereby maintaining their quality. The antagonistic bacteria within the film enhance the fruits’ disease resistance by inhibiting pathogenic bacteria and competing for nutritional space. This includes increasing the activity of disease-resistant enzymes and delaying fruit aging. The protein-based membrane may also nourish the antagonistic bacteria, facilitating their growth and rapid colonization. Introducing high concentrations of antagonistic bacteria into the film ensures they dominate the fruit surface, thereby enhancing their pathogen-inhibiting capabilities. The combination with the membrane boosts the survival rate of directly inoculated antagonistic bacteria, aiding in rapid colonization. Additives in the film improve its mechanical properties and contribute to fruit preservation. Optimizing the film’s formula further prolongs the activity and distribution of the antagonistic bacteria, enhancing the inhibition rate of pathogens.

The future research direction primarily focuses on exploring the synergistic combination of substances that can enhance the biocontrol efficacy of antagonistic bacteria, along with substances that sustain the probiotic population. Additionally, investigating the integration of antagonistic bioactive cling film with other preservation methods is crucial (Guimaraes et al., 2018). Considering the unique characteristics of different agricultural products, it is also essential to develop film formulations tailored to each specific product type.

In vegetable preservation

Fresh vegetables are highly fragile and moist, which makes them susceptible to mechanical damage during the picking process. This damage intensifies respiration and metabolic reactions, leading to a series of physiological and biochemical changes such as discoloration, taste alteration, aging, and water loss. The infection of exogenous microorganisms like Pseudomonas spp. (Pinto et al., 2015), aerobic thermophilic bacteria, yeast, and gray mold (Mansur et al., 2015) further diminishes their freshness before sale, significantly impacting their commercial value. According to statistics, approximately 35% to 40% of vegetables rot and deteriorate post-harvest annually, resulting in a total loss of 80 million tons and economic losses amounting to 80 billion yuan (Peng et al., 2022). Thus, there is an urgent need to develop efficient and cost-effective vegetable preservation technologies. Antagonistic bacteria preservative film technology is a safe, low-cost, and user-friendly method. It has been increasingly applied in packaging materials and remains a current research focus. Guo et al. discovered that a composite membrane made of nisin, gellan gum, and guar gum can inhibit bacterial growth in water chestnut, thereby extending its shelf life. This composite membrane forms a thin film on the surface of the water chestnut, reducing its exposure to external microorganisms (Guo et al., 2020). Song found that combining nisin, ε-polylysine, and chitosan effectively maintains the quality of fresh-cut carrots, suppresses microbial growth and reproduction, and prolongs shelf life. This is achieved by forming a film on the carrot surface, creating an internal environment of high CO2 and low O2, which enhances enzyme activity and increases the resistance of the fresh-cut carrots (Song et al., 2017). Sami et al. observed that treatment with 2.0 g•L^–1 chitosan, 0.4 g•kg^–1 nisin, and 1.5% acetic acid extends the shelf life of postharvest Pleurotus eryngii to 7 days. This may be due to chitosan and nisin reducing polyphenol oxidase activity by slowing the respiratory rate of Pleurotus eryngii (Sami et al., 2021).

Figure 3. Bacteriostasis mechanism of biological preservative film in fruits.

By examining the application of antagonistic bacterial preservative films in vegetable preservation, we can identify three primary mechanisms through which these films function: Formation of a semi-permeable barrier: These films typically create a semi-permeable layer on the vegetable surface when applied by spraying or soaking, serving as a protective barrier against mechanical damage. Regulation of postharvest physiology: The semi-permeable layer maintains a gas composition of low O₂ and high CO₂ around the vegetables, thereby regulating various physiological and metabolic activities of plant tissues and enhancing postharvest quality. Natural bacteriostatic agents: The films often contain natural bacteriostatic substances, which work synergistically to inhibit the growth and reproduction of microorganisms.

The combination of antagonistic bacteria and film is characterized by safety and health benefits. More importantly, it can be used as an additive to enhance the functionality of the film. The incorporation of various agricultural preservatives has improved the film’s forming characteristics, rendering its antibacterial properties more stable and effective in extending the shelf life of fresh vegetables. Nonetheless, the variety of known film-forming materials remains limited, and certain agricultural preservatives pose safety risks. Therefore, future efforts should focus on discovering new film-forming materials and natural additives to ensure zero health risks and achieve better product efficacy.

Application in cold fresh meat preservation

Meat is a food with high nutritional value, but its high protein content makes it susceptible to contamination by Bacillus subtilis, Pseudomonas fluorescens, and Proteus vulgaris, leading to spoilage. The corruption of meat products has caused significant economic losses to agricultural processing enterprises. According to WWF’s report, 18 percent of the meat is wasted per person. In China, the decay rate of meat products is as high as 12%, resulting in losses worth hundreds of billions of yuan due to microbial contamination. Therefore, finding effective preservation methods for meat is urgent. Compared to other preservation methods, antagonistic bacterial biofilm technology not only retains moisture and prevents the migration of oxygen and solutes but also effectively reduces juice loss and dehydration. It prevents contact between meat products and spoilage microorganisms, thereby delaying spoilage. This technology has emerged as a highly promising method for meat preservation (Shi et al., 2025; Adame et al., 2024a; Shi et al., 2024; Adame et al., 2024b; Lin et al., 2024; Cui et al., 2024a; Sji et al., 2024b; Cui et al., 2024b; Ayhan et al., 2019). Currently, numerous studies have explored the application of antagonistic bacterial biofilm technology in meat preservation. For instance, Rajabian et al. discovered that a potato starch composite film containing Lactiplantibacillus plantarum 7-1 can maintain the quality of chicken and extend its storage period. This may be due to Lactiplantibacillus plantarum 7-1 metabolizing and producing lactic acid and bacteriocins, which lower the environmental pH, as well as producing hydrogen peroxide, which exerts antioxidant effects and inhibits bacterial growth (Rajabian et al., 2019). Gialamas et al. prepared edible films by inoculating Saccharomyces cerevisiae into a sodium caseinate solution, demonstrating a significant inhibitory effect on Listeria monocytogenes in fresh beef. This is likely because Saccharomyces cerevisiae secretes lactic acid and competes for space and nutrients with Listeria monocytogenes (Haralampos et al., 2010). Silva et al. (2023) found that sodium alginate carboxymethyl cellulose edible films containing Lactococcus lactis 23609 can extend the shelf life of beef. This might be attributed to Nisin, a metabolic product of Lactococcus lactis, which diffuses to the beef surface to kill microorganisms. Additionally, the film isolates oxygen and inhibits the growth of aerobic (Silva et al., 2023).

Figure 4. Bacteriostasis mechanism of biological preservative film of antagonistic bacteria in vegetables.

Through the analysis of the aforementioned application examples, we can summarize the preservation mechanisms of antagonistic bacteria biological preservative films as follows: The coating materials partially isolate oxygen and moisture; the films prevent contamination from external microorganisms on chilled meat; antagonistic bacteria within the films inhibit spoilage bacteria by disrupting the structure, permeability, and integrity of cell membranes, thereby extending the storage period of chilled meat. Additionally, these coating materials exhibit excellent barrier properties. Applying the preservation coating on the surface of chilled meat helps prevent moisture migration caused by external environmental factors and reduces juice loss in the meat.

Meat and meat products are rich in nutrients and play a crucial role in people’s daily diet. However, current preservation technologies are often limited, and the overall state of preservation is not encouraging (Sánchez-Ortega et al., 2014). Antagonistic bacteria preservative films offer unique advantages such as natural composition, high efficiency, and environmental friendliness, presenting promising commercial prospects. Currently, the use of single antagonistic bacteria or their metabolites combined with film results in unstable preservation effects. Therefore, future research should focus on developing technologies that combine multiple antagonistic bacteria or integrate them with other natural preservatives.

Figure 5. Bacteriostasis mechanism of biological preservative film of antagonistic bacteria in chilled meat.

Application in preservation of aquatic products

China is a major producer and consumer of aquatic products. In 2021, the country produced 66.9029 million tons of aquatic products, marking a year-on-year increase of 2.16%. In the same year, the global apparent consumption of aquatic animal food was 162.5 million tons. According to a report by the Ministry of Agriculture and Rural Affairs, aquatic products offer a more effective source of protein compared to meat, with additional dietary advantages (Çiçek et al., 2023). Preservation methods for aquatic products primarily include salting and icing. However, due to their high moisture content and rich protein levels, these products are susceptible to oxidation, spoilage, and microbial contamination by pathogens like Salmonella, Listeria, and Staphylococcus aureus. These factors compromise the quality and reduce the commercial value of aquatic products. Statistics reveal that about 30% of aquatic products are wasted due to microbial spoilage, while chemical degradation accounts for an additional 25% of total losses in primary agricultural and fishery products annually, leading to significant economic losses for the country and society (Xie et al., 2024). Research indicates that the loss rate due to spoilage after catching fish can reach 15% in China, compared to less than 5% in developed countries (Jinjin et al., 2023). Thus, developing effective packaging films with active antagonistic bacteria to inhibit oxidation and microbial contamination is crucial.

Lu et al. coated northern Chinese black fish fillets with calcium alginate films containing cinnamon oil and Nisin. Compared to the control group, these edible films provided antioxidant and antibacterial protection, significantly preserving the quality of the fish (Lu et al., 2010). De Lacey et al. incorporated Lactobacillus acidophilus and Bifidobacterium into gelatin edible films. The results demonstrated that Bifidobacterium significantly inhibited H2S-producing microorganisms in cod. Additionally, the combination of edible film, bacteria, and high pressure exhibited a synergistic effect, effectively reducing volatile alkali content and lowering pH by over 1 unit, resulting in notable preservation effects (De Lacey et al., 2012). Shi et al. added lysozyme produced by antagonistic bacteria to chitosan to create an antagonistic bacteria film applied to pomfret fish. This treatment effectively slowed the growth of volatile basic nitrogen (TVB-N), trimethylamine (TMA), and thiobarbituric acid (TBA) values, delaying the spoilage of pomfret and extending the shelf life to 12 days compared to 5-6 days under refrigeration alone (Shi et al., 2013). Hadis et al. explored the preparation of Piscidin 1 PG/polylactic acid (Pis-1 PG/PLA) nanofiber membranes via electrospinning to treat sea bass meat. The total colony count in treated fish was reduced by 2.02-3.23 lg(cfu/mL) compared to the control group, effectively inhibiting increases in pH and volatile amino nitrogen content, and extending the cold storage period of sea bass by more than 3 days (Hadis et al., 2020).

Through a review of the literature, the mechanisms behind the antibacterial activity of fresh-keeping films for aquatic products can be summarized as follows:

Antagonistic bacteria can replace and compete for nutrients, as well as produce antibacterial metabolites, which can kill or inhibit the growth of closely related competitor bacteria and pathogenic bacteria in aquatic products. When aquatic products are treated with antagonistic antibacterial film, microbial growth is effectively inhibited, and the production and accumulation of alkaline substances are slowed, stabilizing pH levels and enhancing the preservation of freshness. Additionally, due to the slow-release effect of the film, antagonistic bacteria are continuously released, providing long-term inhibition of microorganisms in fish. The combination of bacterial films also broadens the bacteriostatic range of single organisms, improving bacteriostatic efficacy and extending the shelf life of aquatic products.

To examine its interaction with various packaging methods, such as vacuum packaging, and its retention in different polymers, the goal is to elucidate possible mechanisms of action for additives and optimize the use of antagonists and derived metabolites in aquatic products.

Application in raw milk preservation

Raw milk is rich in fat, protein, lactose, vitamins, and minerals, making it an ideal dietary food due to its unique nutritional value. However, its high nutritional content not only benefits human health but also serves as an ideal culture medium for the growth of spoilage bacteria and pathogenic microorganisms. Raw milk is highly susceptible to environmental influences, leading to microbial contamination and spoilage. The quality of raw milk is crucial for dairy products, and it typically has a short shelf life. Statistics show that approximately one-quarter of raw milk is lost at the production level or wasted at retail and consumption annually (Martin et al., 2021), second only to meat (Buzby et al., 2014). Therefore, developing new preservation technologies is essential. For instance, adding antagonistic bacteria can extend the shelf life of raw milk without compromising its sensory and quality attributes, thus meeting consumer demands. Jin et al. observed the efficacy of a mixture coating in inactivating Listeria monocytogenes when skim milk inoculated with Listeria monocytogenes Scott A was stored in glass jars coated with a mixture of polylactic acid (PLA) polymer and nisin. They found that PLA and nisin coating treatments effectively inactivated Listeria monocytogenes cells in these foods at both 4°C and 10°C (Jin, 2010). Cui et al. used a coating film of nisin and sodium lactate to study milk preservation. After five days of low-temperature storage, the inhibitory effect of PE film without coating, coating film only containing carrier (chitosan), coating film containing sodium lactate, coating film containing nisin, and coating film containing nisin and sodium lactate on microorganisms in milk increased sequentially (Cui et al., 2017). Kim et al. studied the migration differences of nisin in various coating films. When nisin was infiltrated into a vinyl acetate-ethylene copolymer coating film, the migration speed was higher and faster, showing significant inhibition of microbial growth in milk (Kim et al., 2002). Lee et al. prepared antimicrobial paperboard by co-coating nisin and chitosan, providing a broad antimicrobial spectrum that effectively inhibited Gram-positive bacteria and Listeria monocytogenes, thus improving the microbial stability of milk at 10°C (Lee et al., 2003). Wan et al. advocated for the use of an alginate and nisin complex membrane to preserve skim milk. This combination, which integrates alginate with nisin at a rate of 87%-93%, maintains 100% activity against Lactobacillus curvatus in skim milk (Wan et al., 1997).

Figure 6. Bacteriostasis mechanism of antagonistic bacteria biological preservative film in aquatic products.

The primary mechanisms of the antibacterial active fresh-keeping film for raw milk preservation include: (1) The compound film prevents nutrients from reaching bacteria, disrupting their normal metabolic activities, thereby inhibiting microbial growth and the decomposition of proteins and fats in raw milk; (2) The film increases the water activity of raw milk, slightly alters the protein structure, reduces fat oxidation, and preserves the flavor and texture of raw milk; (3) It inhibits lactic acid bacteria and other contaminants, resulting in lower lactic acid production and slower pH changes; (4) The compound film serves as a barrier to isolate air, delaying the dehydration and oxidation of raw milk; (5) The combined packaging effectively inhibits protein degradation, reduces oil precipitation, and extends the shelf life of raw milk.

The composition of raw milk is both complex and nutritious, with variations in milk sources, processing methods, and production equipment across different products. Consequently, it is challenging to uniformly apply a single antibacterial approach. Therefore, multiple preservation technologies must be explored to achieve synergistic effects that inhibit the growth of harmful microorganisms in raw milk. Further investigation into the metabolic pathways of antagonistic bacteria at the molecular level can lay a solid foundation for practical applications (Wu et al., 2024; Liu et al., 2023a; Liu et al., 2023b)

Conclusion and Future Perspective

Although significant progress has been made in the research, development, and application of antagonistic bacteria biological preservative films, their commercial production remains limited, particularly in agricultural applications, where they are still largely experimental. The understanding of the bacteriostatic mechanisms of these films is still at a fundamental theoretical stage, with insufficient research at the molecular level. Additionally, films created by simply combining antagonistic bacteria with film-forming materials have certain shortcomings. These include unstable coating effects, susceptibility to environmental factors, extended drying times, and a higher risk of microbial contamination, all of which hinder the industrial production of such preservation methods. Therefore, future development should focus on creating compound antagonistic bacteria preservative films with enriched components and incorporating natural additives that enhance film-forming characteristics. This approach would facilitate the commercialization of these films for agricultural products, significantly contributing to the rapid and healthy development of the agricultural industry and rural revitalization.

Figure 7. Bacteriostasis mechanism of antagonistic bacteria biological preservative film in dairy products.

Figure 8. Treatment changes of antibiofilm in perishable agricultural products.