Description

Inspiration

Despite record levels of global food production, the issue of hunger remains acute (1). In 2024, more than 295 million people worldwide still suffered from severe hunger. Food spoilage not only intensifies food waste but also presents significant health and safety risks. In 2021, the global loss and waste of edible aquatic products amounted to approximately 23.8 million tons, representing 14.8% of the total output (2). Furthermore, the World Health Statistics 2023 report indicates that approximately 600 million people globally suffer from foodborne diseases annually, with children under five constituting 40% of these cases, resulting in roughly 125,000 child deaths per year (3).

Currently, the spoilage and deterioration of aquatic products pose a significant challenge to global food safety. Shortly after harvest, aquatic products often experience a decline in quality, which not only decreases their shelf life but may also causes symptoms such as diarrhea, vomiting, and acute poisoning in consumers, primarily due to the histamine poisoning and the proliferation of pathogens. The dominant preservation methods in the industry—chemical additives and cold chain systems—present a significant dilemma. While ensuring product freshness, they force a trade-off between economic cost and environmental well-being: substances like sulfites cause the carcinogenic hazards and cold chains contribute to carbon emissions and plastic pollution for their reliance on fossil fuels. Hence, the development of safe and efficient natural preservation techniques from marine biomass resources is of paramount importance.

Extracted from shrimp and crab shells, chitosan is a degradation product and the second-largest global natural biomass resource. It exhibits substantial antimicrobial potential. However, its high degree of polymerization restricts its solubility in water, thus limiting its use in antimicrobial processes and within the food industry. By employing degradation techniques, chitosan can be transformed into chitooligosaccharides (COS). This conversion overcomes its solubility issues and opens up new avenues for replacing traditional preservatives and antimicrobial agents. COS not only enhances antimicrobial effectiveness but also extends the scope of applications beyond preservatives, serving as substrate of various high-value products and offering considerable research and market potential. Simultaneously, it has been observed that shrimp and crab shell resources are underutilized. In 2022, China produced over 50 million tons of marine crabs and shrimp, resulting in more than 10 million tons of related waste (4). Consequently, there is an urgent need to develop a more practical approach for utilizing these shells and to establish a comprehensive "recycle-process-reuse" closed-loop system.

In response to the identified industrial and technological demands, our project is dedicated to developing a screening system to identify novel chitosanases. These enzymes effectively degrade chitosan from shrimp and crab shells into COS, which possesses superior antimicrobial properties. Furthermore, we have formulated novel preservatives for aquatic products using these oligosaccharide derivatives. Our objective is to promote the recycling of shrimp and crab shell wastes through this approach, thereby advancing natural preservation technologies and embracing the concept of "sourced from aquatic products, used for aquatic products."

Background

The global aquatic products trade continues to expand, yet preserving the freshness of aquatic products presents significant challenges. Aquatic products, being rich in protein and moisture, are highly susceptible to microbial contamination during the fishing, processing, and transportation stages, which leads to quality degradation and increases food safety risks. Traditional preservation techniques primarily utilize cold storage, chemical additives, and modified atmosphere packaging (MAP). However, these methods are fraught with challenges: maintaining low temperatures is costly, chemical additives pose potential health risks, and physical packaging technologies require stringent equipment standards. Statistics reveal that up to 30%-35% of aquatic products are lost or wasted during transportation and processing (6). With a rising consumer demand for natural and healthy food options, there is a pressing need for the development of safe and effective natural preservatives within the industry. Chitosan derivatives, known for their renewable sources and versatility, show considerable promise as viable alternatives to chemical preservatives.

Derived from marine fishery processing by-products, shrimp and crab shells are transformed from waste into high-value products through the "Shrimp and Crab Shell - Chitosan - COS" industry chain. In this sequence, the waste shells are first converted into chitosan via deproteinization and decalcification for resource utilization. Subsequently, chitosan undergoes enzymatic and physical processing to control its molecular weight and functional groups, enabling its transformation into COS. As a result of its significant biological activity, COS is widely used in agriculture, medicine, and the food industry (7).

Figure 1 The "Shrimp and crab shells - chitosan - COS" industrial chain.

In this context, chitosan emerges as a research hotspot in aquatic products preservation, attributed to its natural antimicrobial properties and biodegradability. It acts through antimicrobial, moisturizing, and antioxidant activities to form a safe, effective protective coating that maintains freshness. Nonetheless, the water solubility and biological utilization of chitosan are restricted by its molecular weight, and excessive consumption can strain the digestive system, which underscore the need for developing enhanced alternatives.

COS, a derivative of chitosan, effectively addresses the water solubility limitations of its predecessor. This compound not only promotes the growth of beneficial gut bacteria, functioning as a prebiotic, but also aids in maintaining intestinal flora equilibrium. Additionally, COS displays enhanced antibacterial properties compared to chitosan, thereby revealing significant potential for preservation applications. Furthermore, COS has a broader range of applications in agriculture, food, and medicine, showing significant developmental potential. Consequently, it has garnered considerable attention from the scientific community.

Specifically, the antibacterial activity of COS primarily relies on its ability to disrupt bacterial cell membrane integrity, causing leakage of intracellular components and ultimately leading to cell death through physical damage. Additionally, COS can penetrate bacteria cells, interfere with the electron transport chain, and inhibit key enzymatic steps in the tricarboxylic acid (TCA) cycle, thereby establishing a dual-mode bactericidal mechanism (8). As a novel cryoprotectant, COS forms hydrogen-bond networks with water molecules through its hydroxyl groups, which suppresses ice crystal growth, protects the myofibrillar structure from mechanical damage, and enhances water-holding capacity by stabilizing protein secondary structures and reducing drip loss (9). This comprehensive cascade of actions—from molecular-level interactions to macroscopic preservation effects—underscores the technological advantages of COS as a natural preservative.

The principal antibacterial mechanism of COS involves disrupting cell membrane integrity, which results in bacterial death through physical damage. Moreover, the molecule can penetrate bacteria, disrupting electron transport chain activity and obstructing key processes in the TCA cycle (8). Additionally, COS serves as an innovative freezing protective agent, forming a hydrogen bond network that inhibits ice crystal formation, stabilizes protein secondary structure, and minimizes thawing drip loss, thus enhancing the water-holding capacity (9). This comprehensive action chain from molecular effects to macroscopic preservation demonstrates COS's technical superiority as a natural preservative.

The Problem

The "shrimp and crab shell-chitosan-COS" industrial chain has successfully established a high-value utilization cycle for marine resources and presents several advantages. However, it encounters substantial challenges in practical applications, especially in the degradation efficiency and product performance.

➤ The chemical method degrades chitosan into COS using acids or oxidants such as hydrochloric acid (10) and hydrogen peroxide (11). While rapid, this approach may result in uncontrollable product structures and produce problematic waste, including acidic or alkaline wastewater or induce excessive hydrolysis.
➤ The physical method involves degrading chitosan using techniques like microwave radiation (12), ultrasonic fragmentation, and turbo cavitation. Although it can yield COS with specific degree of polymerization (DP), this method demands sophisticated equipment and lacks precise control over the degradation level.
➤ The enzymatic method utilizes enzymes such as chitinase, chitosanase, or nonspecific enzymes like cellulase or papain to hydrolyze chitosan (13). This method is gentle and yields highly specific products but is constrained by the enzymes' selectivity, stability, and substrate specificity.

A significant gap persists between the current industrialization level and the preservation sector's needs. Aquatic products, being rich in protein and moisture, are highly perishable due to rapid microbial growth. This issue is exacerbated in coastal areas with high temperatures and humidity, such as Fujian, where the risk of spoilage escalates. Traditional preservation methods often fail to effectively prevent spoilage, while long-term freezing significantly damages the texture and flavor of aquatic products (14). Furthermore, the excessive use of traditional chemical preservatives and their residues adversely affects human health, contravening the objectives of environmental sustainability and public health (15). Additionally, stringent international regulations on preservative residues complicate the global trade of aquatic products (16). Given these challenges, COS, a novel aquatic products preservation technology, is poised to become increasingly vital.

Current Solution

To overcome limitations in chitosan degradation pathways, recent studies have concentrated on enhancing enzymatic methods. Zhao et al. (17) identified a novel chitosanase, ChiC8-1, characterized by its unique molecular structure that enables high catalytic efficiency and strong substrate affinity. Although this enzyme integrates purification and hydrolysis processes, it only reaches optimal activity under specific conditions, considerably limiting its practical applications. Jia et al. (18) pioneered an ultrasound-microwave pretreatment technique paired with a multi-enzyme system, which markedly improves the yield of COS and imparts antioxidant properties to the product. However, this method reliant on physical processes requires meticulous control of several parameters, leading to increased operational costs and decreased production efficiency.

Modern aquatic products preservation strategies are embracing diversification and advanced technology. Low-temperature preservation is predominant, utilizing subzero conditions to inhibit microbial and enzymatic activities, thus effectively retarding spoilage. Conversely, chemical preservation entails adding preservatives and antioxidants to prevent degeneration, a straightforward yet potent method that necessitates stringent regulation due to potential health risks. Modified atmosphere packaging technology adjusts atmospheric gas components to curb cellular respiration and slow metabolism, maintaining the quality of aquatic products over longer durations. Though effective, MAP requires sophisticated equipment and precise gas control (19). Additionally, breakthroughs in preservation technology continue to emerge. For instance, the team led by Xie Jing has developed an energy-efficient refrigeration system for fishing vessels, addressing the challenges of integrating marine and terrestrial cold chain technologies and enhancing the standard of freezing and ultra-low-temperature storage (20).

Project

1. Preliminary Research

Early Stage Research: At the initial stage of our project, we conducted an in-depth investigation into aquatic products preservation and the preparation of COS. Our team engaged with staff from the Xiamen Municipal Marine Bureau and individual merchants in the aquatic products market. They reported significant losses during the storage and transportation process of aquatic products and highlighted that traditional preservation methods are notably deficient in terms of cost, safety, and environmental protection. Moreover , during our visit to Xiamen Bluebay Science & Technology Co.,Ltd., we learned that the resource utilization of by-products from shrimp and crab shells is extremely low, and the lack of recycling efforts not only results in resource wastage but also exacerbates environmental pressures.

Technical Research: In the technical domain, we held discussions with numerous experts, scholars, and corporate technicians. There was a general consensus that, although enzyme degradation of chitosan possesses environmental potential, the chitosanases currently in use still face limitations in activity and stability. These discussions have provided critical reference points for our subsequent enzyme engineering and screening system design.

2. New Solution

To address the issue of the low efficiency of chitosanases, we conducted random mutations on various chitosanases (CsnCA, CsnMY002, McchoA, BamCsn, SaCsn46A, Glm_Tk) to construct a mutation library. These mutated enzymes were expressed using Escherichia coli JM109(DE3). For the origins of the six enzymes, please refer to the Design.

To address the issue that the antimicrobial capacity of chitosan enzymatic degradation products failed to meet standards , we established a screening system based on toxicity characterization. We used various mutated enzymes to degrade chitosan into COS and incubated these degradation products with wild-type Escherichia coli BL21(DE3). By monitoring the decrease in the OD₆₀₀ value of the bacterial cultures, we were able to efficiently identify enzymes that produce COS with high antimicrobial activity.

Figure 2 Screening workflow for chitosanases and its application.

To mitigate the high production costs associated with enzymes, we enhanced enzyme secretion by semi-rational design of the excretion signal peptide LMT (BBa_25LUSMUT), thus circumventing costly protein purification processes. Furthermore, we improved the expression efficiency of the enzymes and performed comprehensive characterization of the products using analytical techniques including HPLC and TLC.

We have successfully developed several new enzymes capable of efficiently degrading chitosan, and based on these enzymes, we have further developed an efficient antibacterial preservative made from COS. After approved by the Safety and Security Committee (Safety Form), we applied this preservative successfully in the preservation of fish and fruits, proving its significant antibacterial and preservation effects. Compared to traditional chemical preservatives, our formula contains no chemical additives, achieving a true breakthrough in green preservation technology.

3. Human Practices

In terms of human practices, we conducted public awareness campaigns and collected feedback. We designed and distributed surveys aiming to understand consumer awareness and acceptance of natural preservatives. At the same time, we utilized channels such as public lectures and social media articles to promote concepts of reducing food waste and encouraging the recycling of marine resources. Additionally, our team organized educational activities at locations like Yanwu Primary School, residential communities, and science museums. Through experimental demonstrations and interactive workshops, we introduced the role of synthetic biology in food preservation and environmental protection to youths, thereby enhancing the project's social impact and educational value (see Human Practices for details).

Biosafety

To address the environmental risk of potential escapes of engineered Escherichia coli, we have designed a dual suicide switch system aimed at preventing accidental bacterial release and ensuring biosafety. It consists of two independent and complementary genetic circuits, with each capable of inducing cell death under specific conditions, thereby providing a strong safety guarantee.

The first kill circuit employs the ccdB gene, encoding a toxin that inhibits DNA gyrase (topoisomerase II) and consequently induces cell death. The expression of ccdB is regulated by the pCymR promoter and is induced by cuminic acid. To reduce cellular burden caused by leaky toxin expression, the antitoxin gene ccdA is expressed at a low level via a weak constitutive promoter. When cuminic acid is added, the expression of CcdB surpasses the neutralizing capacity of CcdA, effectively triggering cell death.

The second kill circuit is based on the toxic overexpression of an essential gene. Specifically, the tyrS gene, encoding tyrosyl-tRNA synthetase, is regulated by a T5 phage promoter paired with a lacO operator. In the absence of IPTG, tyrS expression is repressed; however, IPTG addition triggers the overexpression of TyrS, leading to mistranslation and resulting in cell death due to proteotoxic stress.

We integrated two independent suicide circuits into a plasmid to ensure functionality even if one is inactivated due to mutation. These systems operate independently and provide mutual backup, ensuring rapid cell death and reducing the risk of strain escape.

Figure 3 Cuminic acid-inducible and IPTG-inducible kill circuit.

Future Prospect

The combination of technological innovation and sustainable practices demonstrates great potential for utilizing shrimp and crab shell resources in the field of food preservation. In downstream applications, the industrial production and circular economic model of chitosan will become crucial factors. Our project, through a 'recycle-process-reuse' closed-loop system, not only integrates the collection network and biotransformation factories for shrimp and crab shells but also transforms low-value raw materials into high-value biomanufacturing products, achieving natural processing techniques for food preservation.

Furthermore, considering the broad-spectrum antibacterial and antioxidant properties of this preservative, we plan to further test its application in the post-harvest preservation of various fruits, such as blueberries, peaches, strawberries, and tomatoes. By systematically assessing its potential and effectiveness in the actual preservation process, we aim to expand the application scope of chitosan as a natural preservative.

Due to the limitations of the season, we were unable to conduct an in-depth study of the antibacterial mechanism of chitosan. In the future, we plan to thoroughly investigate the inhibitory effects of COS on various harmful microorganisms. Furthermore, we will conduct quantitative studies to analyze the degree of polymerization and proportions of COS produced by different enzyme mutants degrading chitosan, in order to gain a deeper understanding of the catalytic mechanisms at key mutation sites.

Conclusion

Aquatic products are essential source of various vital nutrients, providing unparalleled nutritional benefits to our diets. Being rich in protein and moisture, they are also highly susceptible to microbial contamination during the fishing, processing, and transportation stages, which leads to quality degradation and increased food safety risks. We are trying to find critical breakthroughs from marine biomass resources, focusing on screening efficient chitosan degrading enzymes and exploring the transformation of high-value COS. Through our project, we can essentially establish a closed-loop system of "recycling-processing-reuse" for shrimp and crab shell resources, thereby promoting the development of natural preservative technologies.

References

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20. https://www.shou.edu.cn/2018/0110/c7082a218133/page.psp