Description

Inspiration

Seafood spoilage is a pressing issue in the global food supply chain. Highly perishable after harvest, seafood undergoes rapid quality deterioration that not only shortens shelf life but also poses serious food safety risks, including diarrhea, vomiting, and even acute poisoning due to histamine accumulation and pathogenic bacteria. Although chemical preservatives and cold chain logistics are widely used to maintain freshness, they come with significant concerns. Additives like sulfites may pose carcinogenic risks, while fossil fuel-powered refrigeration contributes to carbon emissions and plastic pollution. As a result, the industry is caught in a dilemma between economic cost and environmental sustainability. In this context, developing safe and effective natural preservation technologies derived from marine biomass has emerged as a promising and necessary solution.

Meanwhile, shrimp and crab shell resources remain significantly underutilized. In the coastal region of Fujian, for instance, the Fujian Marine Environmental Protection Regulations ⁽¹⁾ mandate that marine aquaculture waste be transported to land for safe disposal. However, some aquaculture operators continue to illegally discard shrimp and crab shells into nearshore waters. These chitin-rich biomass residues are increasingly harming coastal ecosystems through acid leaching and other processes, turning a potentially renewable resource into an environmental burden. Therefore, it is imperative to develop more practical and sustainable approaches to shell waste utilization by establishing a closed-loop system of “recycling—processing—reuse”.

Chitosan, a degradation product of shrimp and crab shells, is the second most abundant natural biomass resource worldwide. While it exhibits broad-spectrum antimicrobial activity, its high degree of polymerization leads to poor water solubility, limiting its direct application in the food industry. However, by applying targeted degradation techniques, chitosan can be converted into low-molecular-weight chito-oligosaccharides (COS), thereby overcoming this limitation and offering a promising natural alternative to traditional chemical preservatives.

In response to the growing industry needs and recent technological advancements, our project focuses on establishing a screening system to identify novel chitosanases capable of efficiently degrading chitosan—derived from shrimp and crab shells—into glucosamine (GlcN) or chitobiose ((GlcN)₂). Building on these degradation products, we aim to develop an innovative seafood preservative that leverages the synergistic effects of COS derivatives and ε-polylysine. Through this approach, we seek to promote the recycling and high-value utilization of shrimp and crab shell waste, while advancing the development of safe, natural preservation technologies—ultimately fulfilling the vision of “taking from the sea and giving back to the sea.”

Background

As global seafood trade continues to grow, ensuring the freshness and safety of seafood has become a critical challenge. Due to their high protein and moisture content, seafood products are highly susceptible to microbial contamination during harvesting, processing, and transportation, leading to rapid spoilage and food safety risks. Traditional preservation methods—such as refrigeration, chemical additives, and modified atmosphere packaging—have their drawbacks: refrigeration is expensive to maintain, chemical additives raise health concerns, and advanced packaging technologies often require specialized equipment. With the growing consumer demand for natural, healthy food foods, the need for safe and effective natural preservatives has become more urgent. In this case, chitin-derived compounds stand out as promising alternatives to synthetic preservatives, owing to their renewability and diverse functional properties.

As a byproduct of marine fishery processing, shrimp and crab shells have undergone a transformation from waste to high-value products through the industrial chain of “shrimp and crab shells – chitosan – COS.” At the upstream stage, discarded shrimp and crab shells undergo deproteinization and decalcification to be converted into chitosan, effectively transforming waste into a valuable resource. In the midstream phase, enzymatic hydrolysis and physical processing are employed to modulate the molecular weight and functional groups of chitosan, which is then further degraded into COS. Due to its remarkable biological activity, COS is widely used in agriculture, medicine, and food industries ⁽²⁾ .

Fig.1 The “Shrimp and crab shells – chitosan – COS” industrial chain

Within this industrial chain, chitosan, as a midstream product, has become a research hotspot in the field of seafood preservation, owing to its natural antibacterial properties and biodegradability. Its preservation mechanism involves a combination of antibacterial, moisturizing, and antioxidant effects, forming an effective and safe barrier to prolong the freshness of seafood. However, the high molecular weight of chitosan limits its water solubility and bioavailability, and excessive intake may impose a burden on the digestive system. Therefore, it is essential to explore alternatives with improved performance and enhanced safety profiles compared to chitosan.

COS is the oligosaccharide form of chitosan. They not only overcome the limitations of poor water solubility, but also improve intestinal absorption, thereby reducing metabolic burden. In addition, COS exhibits stronger antibacterial activity than chitosan, highlighting its greater potential for applications in seafood preservation. As a result, increasing research efforts have focused on COS, the key degradation product of chitosan.

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 ⁽³⁾. As a novel cryoprotectant, COS forms hydrogen-bond networks with water molecules through its hydroxyl groups, which suppress ice crystal growth, protect the myofibrillar structure from mechanical damage, and enhance water-holding capacity by stabilizing protein secondary structures and reducing drip loss ⁽⁴⁾.. Meanwhile, the active functional groups of COS derivatives contribute to maintaining the elasticity and characteristic whiteness of fish fillets, highlighting their unique role in preserving seafood quality⁽⁵⁾.. This comprehensive cascade of actions—from molecular-level interactions to macroscopic preservation effects—underscores the technological advantages of COS as a natural preservative.

The Problem

Although the “shrimp and crab shells–chitosan–COS” industrial chain establishes a closed-loop system for the high-value utilization of marine resources and boasts many advantages, it still faces significant challenges in degradation processes and product applications.

At present, the conversion pathway from shrimp and crab shells to chitosan is relatively well-established, but the downstream production of COS from chitosan remains underdeveloped and lack of precision.

(1) Chemical Methods: COS can be produced by degrading chitosan using acids or oxidizing agents. For example, Ji et al. ⁽⁶⁾. employed hydrochloric acid to degrade chitosan. Jiao et al. ⁽⁷⁾. used hydrogen peroxide (H₂O₂) to react with chitosan and obtained COS with a molecular weight of approximately 2 kDa. However, due to their harsh reaction conditions, these methods often result in poorly controlled product structures and may lead to wastewater pollution or over-hydrolysis.
(2) Physical Methods: Physical degradation techniques such as microwave irradiation, ultrasonic disruption, and hydrodynamic cavitation have also been employed to depolymerize chitosan. For instance, Xing et al. ⁽⁸⁾. treated chitosan with 800W microwave radiation at 80°C for 25 minutes and obtained COS with a degree of polymerization (DP) ranging from 2 to 6. However, such methods are limited by low efficiency, high equipment requirements, and limited precision in controlling the extent of degradation.
(3) Enzymatic Hydrolysis: This method uses specific enzymes (e.g., chitinase, chitosanase) or non-specific enzymes (e.g., cellulase, papain) to hydrolyze chitosan. For example, Huang et al. ⁽⁹⁾. employed papain as a catalyst to hydrolyze chitosan, resulting in COS with a DP of 6 to 10 and a yield of 45.07%. Although enzymatic methods benefit from mild reaction conditions and high product specificity, they are constrained by a narrow range of enzyme types, poor catalytic stability, and low substrate specificity.

While the degradation process faces multi-faceted challenges, the downstream application of COS is also hindered by a “broad-but-shallow” development dilemma. Although COS has been widely applied in agriculture, medicine, and food industries, its functional development remains relatively superficial. In agriculture, COS is primarily used as a broad-spectrum fertilizer enhancer, yet lacks crop-specific and growth-stage-specific formulations. In the food industry, it is predominantly used as a general-purpose additive, with limited efforts to develop specialized functional products tailored to the specific characteristics of different food processing methods. ⁽¹⁰⁾. This undifferentiated application model has led to a shortage of high value-added COS products. Consequently, the industrial chain suffers from a persistent imbalance—marked by overcapacity at the low end and an unmet demand for high-end applications.

This imbalance is particularly pronounced in seafood preservation across coastal areas. Seafood, due to its protein and moisture content, is highly susceptible to microbial contamination, leading to rapid spoilage. In coastal regions like Fujian Province, the hot, humid climate and coastal pollution further exacerbate the risk of seafood spoilage. Traditional processing methods are often ineffective in preventing spoilage, and deficiencies in cold chain logistics frequently lead to temperature fluctuations during transportation, further aggravating the problem ⁽¹¹⁾. Moreover, traditional chemical preservatives and waste disposal practices may cause secondary pollution and pose health risks, conflicting with environmental protection and public health policies ⁽¹²⁾. Additionally, strict regulations on preservative residues in developed countries have heightened the challenges for the export of Chinese seafood products ⁽¹³⁾.

Although COS holds promise as an emerging technology for seafood preservation, its relatively weaker antibacterial and antioxidant activities compared to chemical preservatives have limited its large-scale industrial application ⁽¹⁴⁾. To address this technological bottleneck and satisfy the specific demands of seafood preservation, it is imperative to explore strategies that can enhance the functional efficacy of COS.

Current Solution

To overcome limitations in degradation pathways of chitosan, recent studies focus on improving enzymatic methods.

In terms of enzymatic performance, Zhao et al. ⁽¹⁵⁾ identified a novel chitosanase, ChiC8–1, whose unique molecular structure confers both high catalytic efficiency and strong substrate affinity. The enzyme integrates purification and hydrolysis processes; however, it exhibits peak activity only under specific environmental conditions, which significantly limits its applicability in diverse real-world scenarios.

Regarding enzymatic hydrolysis technology, Jia et al. ⁽¹⁶⁾ introduced an ultrasound–microwave pretreatment method combined with a multi-enzyme system, significantly enhancing COS yield and endowing the product with antioxidant properties. Nevertheless, this physically assisted enzymatic approach demands precise control over multiple parameters, which not only raises operational costs but also reduces overall production efficiency.

Meanwhile, modern seafood preservation strategies are evolving toward greater diversification and technological sophistication. Among traditional methods, low-temperature preservation remains the most widely used approach. It works by maintaining a subzero environment to suppress microbial growth and enzymatic activity, thereby effectively slowing down seafood spoilage. Chemical preservation, on the other hand, involves the addition of specific substances such as preservatives and antioxidants to inhibit deterioration. While this method is simple and effective, it may pose risks to human health, necessitating strict regulation of the types and concentrations of chemical additives used. Modified atmosphere packaging (MAP) technology works by adjusting the composition of surrounding gases to suppress cellular respiration and slow metabolic activity in stored seafood. Although MAP can preserve seafood quality over extended periods, it demands advanced equipment and precise control of gas composition ⁽¹⁷⁾.

In addition, several innovative preservation technologies continue to emerge. For instance, the team led by Xie Jing overcame technical bottlenecks in integrating marine and land-based cold chain systems by developing an energy-efficient refrigeration system for fishing vessels, significantly improving the capabilities of marine freezing and ultra-low-temperature preservation equipment ⁽¹⁸⁾.

To address the relatively weak antibacterial activity of COS, recent studies have focused on various site-specific modifications. Studies have shown that COS-nisin conjugates formed via the Maillard reaction exhibit significantly enhanced antimicrobial activity, possibly due to the formation of new chemical bonds and functional groups during the reaction, which improve the antibacterial properties of the conjugate. Additionally, other studies have introduced various functional groups—such as quaternary ammonium and acyl thiourea groups—onto COS molecules via chemical modification, resulting in modified COS with significantly stronger antimicrobial activity than its natural counterpart ⁽⁵⁾.

Project

To enhance the efficiency of the chitosan-to-COS conversion and address the challenges in the seafood preservation industry, we ultimately chose to focus on the targeted screening of highly efficient chitosan-degrading enzymes, along with the development of high-value product chains based on COS.

First, we constructed mutant libraries through directed mutations of chitosanases (McChoA, BamCsn, SaCsn46A, GlmTk). These mutant enzymes were secreted by E. coli BL21(DE3) to break down chitosan into COS. We then developed a screening system regulated by ChsR.

The ChsR protein is a key LacI family transcriptional regulator in Vibrio cholerae, playing a vital role in metabolic adaptation. It directly binds to the promoter regions of the tcpP and chsB genes, forming a regulatory hub that upregulates chsB expression. The chsB gene encodes a GlcN transporter involved in the chitin metabolic pathway, providing carbon sources for bacterial growth. Studies have shown that COS molecules impair ChsR's regulatory function through competitive inhibition ⁽¹⁹⁾. Both GlcN and (GlcN)2 specifically bind ChsR, blocking its interaction with DNA promoters, thereby simultaneously suppressing virulence gene expression and chitin metabolism.

We designed the ChsR binding site as an operator and inserted the sfGFP reporter gene to construct a genetic circuit. This system was then introduced into E. coli BL21(DE3) for expression. By taking advantage of ChsR’s specific binding to GlcN and (GlcN)2, we achieved semi-quantitative detection through fluorescence intensity measurement.

Through this screening system, we successfully identified novel chitosanases capable of efficiently degrading chitosan into GlcN and (GlcN)2. To achieve high-value utilization of the resulting COS, we further developed a potent antibacterial preservative based on COS derivatives.

To address the limited antibacterial efficacy of native COS, we adopted the enhancement strategy proposed by Xia et al. ⁽¹⁴⁾ for structural modification. From a molecular perspective, the reactivity of functional groups at different positions within the COS molecule varies significantly. The amino group at the C-2 site exhibits the highest reactivity, followed by the primary hydroxyl group at the C-6 site, while the secondary hydroxyl group at C-3 displays relatively low reactivity due to steric hindrance. This variation in reactivity provides a theoretical foundation for rational COS design—specifically, by selectively introducing novel functional groups to enhance its antibacterial and antioxidant properties.

Furthermore, according to follow-up studies by Xia et al., COS derivatives mixed with ε-polylysine can achieve complete (100%) antibacterial activity at certain concentrations ⁽²⁰⁾. ε-Polylysine is a natural biopolymer produced by Streptomyces albulus through deep-liquid aerobic fermentation. It consists of 25–35 L-lysine residues linked via amide bonds between the α-carboxyl and ε-amino groups. Its linear structure and cationic nature confer broad-spectrum antimicrobial activity, earning it the reputation of a “natural biological preservative.”

Specifically, we utilized a thiol–ene click reaction to achieve precise modification of COS, sequentially introducing an allyl groups (Bro) at the C-6 hydroxyl site and a cysteine (Cys) at the C-2 amino site. This yielded an N,O-bis-substituted thioether derivative (COS-Bro-Cys) with significantly enhanced antibacterial activity. Subsequently, ε-polylysine was added to act synergistically with COS-Bro-Cys, resulting in a near-complete (∼100%) antibacterial effect.

The novel seafood preservative we developed establishes a dual antibacterial defense system by integrating targeted site-specific modification with the synergistic action of ε-polylysine. Compared with conventional chemical preservatives, our formulation not only achieves efficient antibacterial performance but also effectively preserves the natural color and moisture content of seafood. Throughout the entire process, no chemical additives are involved, representing a genuine technological breakthrough in green preservation.

Fig. 2 Technical route

Biosafety

To prevent our engineered E. coli from escaping the screening system, we designed a suicide switch compatible with our system. The tyrS gene, an essential enzyme for bacterial survival, encodes tyrosyl-tRNA synthetase. And either deficiency or overexpression of tyrS lead to bacterial death. We developed a tyrS-deficient E. coli strain and introduced plasmids with three coordinated regulatory circuits to achieve the suicide function.

Fig. 3 Schematic diagram of suicide switch

(1) Light-Control Switch: LexRO is a photosensitive protein that binds to the pColE408 in the absence of blue light when bacterial escape occurs, suppressing ChsR-LacI or LacI expression. This leads to tyrS overexpression and cell death. When exposed to blue light, LexRO dissociates and activates downstream gene expression. As a result, ChsR-LacI and LacI suppress tyrS overexpression, ensuring bacterial survival and normal function.
(2) ChsR-LacI-COS System: The ChsR-LacI fusion protein combines the environmental sensing module (ESM) of ChsR with the DNA recognition module (DRM) of LacI. It binds to the LacO operator to suppress tyrS overexpression. During screening, GlcN and (GlcN)2 relieves this suppression and triggers tyrS overexpression. However, LacI's repression remains, the bacteria survive and work.
(3) LacI-IPTG System: LacI binds to the LacO operator to inhibit gene expression. After screening, adding IPTG can remove this inhibition, activating tyrS overexpression and causing bacterial death.

Future Prospect

The integration of technological innovation with sustainable practices unlocks the vast potential of shrimp and crab shell resource utilization in the field of food preservation. Through advanced research in genetic engineering and synthetic biology, it is possible to develop highly efficient and substrate-selective chitosanase systems capable of precisely degrading chitosan into COS with a defined degree of polymerization, enhancing its application value in the food industry.

In downstream applications, the industrialization of COS production and the adoption of a circular economy model will be critical. The “recycling—processing—reuse” closed-loop system proposed in our project integrates shell waste collection networks with bioconversion facilities. It not only upgrades low-value raw materials into high value-added biomanufacturing products, but also enables natural food preservation processes.

Conclusion

There are several technological bottlenecks in the field of seafood preservation, prompting us to seek breakthroughs through the utilization of marine biomass resources. But the resource utilization of shrimp and crab shells faces two major challenges: the midstream degradation pathways for chitosan are numerous but lack precision, while downstream applications of COS widespread but insufficiently refined. These issues have driven our focus toward the screening of highly efficient chitosan-degrading enzymes and the high-value transformation of COS.

Through our project, we aim to establish a closed-loop system of “recycling—processing—reuse” for shrimp and crab shell resources, thereby advancing the vigorous development of natural preservation technologies.

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