Continuous Production of Chitin Oligosaccharides Utilizing an Optimized Enzyme Production-Adsorption-Enzymolysis-Product Separation (EAES) System

Abstract

Chitin oligosaccharide (CHOS) is a chitin derivative with excellent biological activities. Enzymatic hydrolysis of chitin-rich biomass into CHOS is a hot topic in research on the high-value utilization of chitin resources. The disadvantages of complex preparation and purification processes and the high cost of chitin-degrading enzymes limit large-scale enzymatic production and application of CHOS. In this study, the activity of chitinase increased from 1.8 U/mL to 3.52 U/mL by 94.4% after optimizing the carbon and nitrogen source of Chitiniphilus sp. LZ32 fermentation. An enzyme production-adsorption-enzymolysis-product separation (EAES) system was constructed using fermentation, an adsorption purification module, and a product ultrafiltration module of a chitin-degrading enzyme. CHOS production by continuous enzymatic hydrolysis was performed in an EAES system using housefly larval powder (HLP) as the substrate. After the C. sp. LZ32 fermentation broth was circulated in the adsorption module for 90 min, the adsorption rate of the chitin-degrading enzyme reached more than 90%. The ultrafiltration module effectively separated CHOS at an operating pressure of 2 bar. Four batches of CHOS were produced in the EAES system using repeated batch fermentation. The running time of a single batch decreased from 115 h in the first batch to 48 h, and the CHOS output of each batch gradually increased. The total CHOS output was 61 g, and the production efficiency was 0.24 g/h. The CHOS produced by the EAES system (ECHOS) has high antioxidant activity. In this study, the EAES system was used to simplify the purification and separation steps of CHOS preparation, and the continuous production of CHOS was realized, which has potential application prospects in the field of green CHOS production.

1. Introduction

Chitin is a non-toxic, water-insoluble, crystalline polymer consisting of N-acetylglucosamine monomers linked by β-1,4-glycosidic bonds. Chitin is widely found in the shells of crustaceans (e.g., shrimp, crab, and lobster), the cuticle of insects (e.g., beetles, ants, houseflies), and the cell walls of fungi [1]. As one of the most widely distributed biopolymers in nature, chitin is produced at 10¹¹–10¹⁴ tons per year and is the second most abundant organic compound after cellulose [2,3]. Although chitin has favorable properties, such as non-toxicity, biocompatibility, and biodegradability, it has far-reaching potential in applications such as drug delivery, tissue engineering, and cancer therapy. However, their high crystallinity and insolubility in most solvents limit the expansion of chitin applications [4]. In recent years, research on the utilization of chitin resources has attracted considerable attention because of its high-value-added derivative chitooligosaccharide (CHOS) [5]. CHOS is an oligosaccharide with a degree of polymerization less than 20 consisting of N-acetylglucosamine and a small amount of D-glucosamine, which is mainly obtained by chitin degradation [6]. CHOS not only has good water solubility but also exhibits many biological activities [7]. Chitooligosaccharides (COS), a partial deacetylation product of CHOS, and derivatives modified by its active group have been used as an antioxidant [8,9], antibacterial [10,11], antitumor [12,13], anti-inflammatory [14,15,16], and anti-obesity [17,18], for anti-Alzheimer’s diseases [19,20] and anti-human immunodeficiency viruses (HIV) [21,22], against plant pathogenic nematodes [23,24], and for stimulating plant defenses [25], showing excellent performance in many aspects. Therefore, CHOS shows broad application prospects in the fields of pharmaceuticals, food industry, and agriculture [26,27].

The large-scale preparation of CHOS is usually obtained by chemical and physical hydrolysis of chitin [28,29]. Chemical hydrolysis mostly uses strong acids such as hydrochloric acid, sulfuric acid, or phosphoric acid or sodium hydroxide and potassium hydroxide to hydrolyze chitin. Physical hydrolysis involves the use of methods such as high-pressure homogenization, ultrasonic treatment, and milling [30]. Chemical hydrolysis is dangerous, and the wastewater produced can damage the natural environment if it is not properly treated. Although physical hydrolysis is more environmentally friendly than chemical hydrolysis, the yield is lower, limiting large-scale production [26,31]. In contrast, enzymatic hydrolysis shows potential for industrial production owing to its advantages, such as high efficiency, environmental protection, and controllability [32]. However, most chitin-decomposing enzymes must be purified before use, and their hydrolysis substrates must be purified chitin or colloidal chitin [6,33]. The separation and purification of chitin-degrading enzymes and the treatment of chitin substrates not only complicate the production process but also increase the production cost, which has become the main limiting factor for large-scale production of CHOS [34]. Therefore, on the one hand, it is necessary to find chitin-degrading enzymes with crystalline chitin degradation ability and improve enzyme production and efficiency; on the other hand, there is a need to establish a simple and low-cost method for chitin-degrading enzyme purification and CHOS production.

Accumulation of research findings on chitinase has led to remarkable advancements in the enzymatic preparation of CHOS [35]. However, there are very few reports on the direct use of crystalline chitin or untreated chitin biomass as substrates to prepare CHOS. Only a few chitin enzymes and crude enzymes produced by strains can degrade crystalline chitin [36,37,38,39,40]. Many works of literature have reported chitin extraction methods and characterization analysis of various insects [41,42]. The content of the insect chitin is 25–60% of its dry weight, and the insects show yield values comparable to crustacean chitin, which is a good-quality alternative source in chitin [41,43]. Compared with shrimp and crab shell chitin, insect chitin has high biocompatibility and biodegradability and has superior anti-inflammatory activity, which makes it a potential for wide application in cosmetics and biomedical fields [43,44]. Furthermore, insect chitin is more susceptible to acid degradation than shrimp chitin and has a higher affinity for chitin enzymes [45,46]. The chitin extraction rate of Diptera insects exceeds 50%, and insect larvae are more suitable for extracting chitin [47]. Muscidea insects have the advantages of strong reproductive ability and short growth cycle and feeding cost. It has been widely cultivated commercially to provide livestock feed, protein, chitin, and chitosan [48,49,50].

In our previous study, we screened a strain of chitin-degrading bacteria Chitiniphilus sp. LZ32, which was able to degrade untreated housefly larval powder (HLP) to CHOS, showing better potential for crystalline chitin degradation [51]. Repeated batch fermentation is a process in which part of the fermentation culture is repeatedly replaced with fresh sterilized medium for uninterrupted culture. There is only one inoculation in the process of repeated batch fermentation, which is a fermentation strategy that effectively shortens the fermentation time and improves production efficiency. Repeated batch fermentation is widely used for metabolite production, biomass acquisition, biotransformation, and wastewater treatment [52,53,54,55,56]. Therefore, repeated batch fermentation may be a suitable method to improve chitin-degrading enzyme productivity.

The adsorption and immobilization of chitin decomposition enzymes are crucial for their separation of chitin enzymes, continuous catalysis, and product separation. This can not only realize the reuse of enzymes and help the downstream process but also improve the overall economic benefits. At present, a variety of immobilization materials have been applied to the immobilization of chitin enzymes, including magnetic nanoparticles, kaolin, charcoal, chitin, and chitosan [57,58,59,60,61,62]. Studies on the mechanism of action of chitin-degrading enzymes such as chitinase and lytic polysaccharide monoxygenase (LPMO) have shown that the carbohydrate-binding module (CBM) of chitin-degrading enzymes plays an important role in the degradation of chitin [63,64]. Zhou et al. developed CBM derived from the chitin enzyme of Chitinolyticbacter meiyuanensis SYBC-H1 as a tag for recombinant protein purification, achieving excellent adsorption and purification effects [65]. Based on the strong binding characteristics of ChiC8-1 to chitin, Zhao et al. developed a CHOS preparation process in which enzyme purification was performed simultaneously with chitin hydrolysis [66]. When studying the chitin-degrading enzymes of C. sp. LZ32, HLP cannot only efficiently adsorb chitin-degrading enzymes in large quantities of fermentation broth but can also be directly degraded into CHOS by chitin-degrading enzymes after adsorption [51]. The combination of C. sp. LZ32 and HLP shows great potential for the preparation of CHOS. In addition, membrane reactors have more advantages than traditional batch reactors in terms of improving production efficiency. For example, a more efficient continuous process can be used to recover and reuse the catalyst, and product separation can be achieved simultaneously [67].

In this study, the enzyme-producing medium of C. sp. LZ32 was optimized to improve the yield of chitin-degrading enzymes because of its excellent HLP degradation performance. Repeated batch fermentation was used to improve the enzyme production efficiency of C. sp. LZ32. An enzyme production-adsorption-enzymolysis-product separation (EAES) system for CHOS production was constructed. The core of the EAES system consists of a fermentation enzyme production module, adsorption of chitin degradation enzymes, an HLP enzymatic hydrolysis module, and a CHOS separation module. By optimizing the process parameters, the EAES system enables repeated continuous batch production of CHOS using HLP as a substrate. This study offers a reference framework for the direct preparation of CHOS from natural crystalline chitin.

2. Materials and Methods

2.1. Materials, Bacterial Strains

Sterile dry housefly larvae were purchased from a local market in Liaocheng city (China). HLP was prepared by grinding and sieving through a mesh sieve (10 mesh). Chitin was prepared from housefly larvae according to a previously described method [68]. CHOS with a degree of polymerization from 2 to 6 was purchased from Qingdao BZ Oligo Biotech CO., LTD (Qingdao, China). All reagents were of analytical or chromatographic grade and purchased from local suppliers. C. sp. LZ32 was isolated from soil samples, maintained on agar slants, stored at 4 °C, and renewed periodically for further use [51].

2.2. Optimization of Carbon and Nitrogen Source in Culture Medium

The culture medium was optimized to improve chitinase activity during fermentation by C. sp. LZ32. The basal culture medium contained HLC, 5 g; peptone, 2 g; glucose, 10 g; K₂HPO₄, 0.7 g; KH₂PO₄, 0.3 g; and MgSO₄, 0.5 g; pH 7.0, and distilled water up to 1 L. The effects of different carbon and nitrogen sources were evaluated by replacing glucose and peptone in basal culture medium. The carbon sources included glucose, galactose, fructose, arabinose, lactose (as monosaccharide), cellobiose, sucrose, maltose, starch, and cellulose at 10 g/L. Beef extract, yeast extract, corn syrup, urea, sodium nitrate, ammonium sulfate, and ammonium chloride were tested as nitrogen sources at concentrations of 3 g/L. The initial pH of the medium was adjusted to 7.0 with 1 N NaOH. The biomass and chitinase activities were also investigated. The effects of carbon and nitrogen concentrations on chitinase activity were investigated at different concentrations.

2.3. Batch and Repeated Batch Fermentation

One loopful of C. sp. LZ32 seed culture was inoculated into a 500 mL shake flask containing 80 mL of seed medium (15 g/L fructose, 5 g/L yeast extract, K₂HPO₄, 0.7 g/L; KH₂PO₄, 0.3 g/L, MgSO₄, 0.5 g/L; pH 7.0) and then cultured for 18 h at 30 °C and 150 rpm. Then, the seed culture was inoculated into a 5 L fermenter containing 3 L of fermentation medium at an inoculum concentration of 2%. Batch fermentation was performed in 5 L fermenter at 300 rpm, while aeration was maintained at 2 vvm. The fermentation temperature was maintained at 30 °C using a recirculating water bath. The pH was automatically controlled at 7.0 by adding 1 N NaOH. For repeated batch fermentation, 90% of the culture was removed after specific fermentation time of single-batch fermentation and supplemented with an equivalent amount of sterilized fermentation medium [24]. The medium was changed at 48 h intervals during repeat batch operations. Four repeated batches were performed.

2.4. EAES System Construction

A schematic representation of the EAES system is shown in Figure 1. It mainly consists of a 5 L stirred fermenter, an enzyme adsorption column (ID: 4 cm; height: 30 cm), an ultrafiltration membrane module with a molecular weight cut-off (MWCO) of 3 kDa, a replenishment tank, and a storage tank. All modules were connected to a peristaltic pump. When the EAES system was operating, the HLP suspension was pumped into an adsorption column pre-filled with glass beads (D: 0.5 cm) using a replenishment tank. After the adsorption column was filled with HLP, the HLP suspension was replaced with pH 7.0 phosphate buffer. Both ends of the adsorption column were equipped with a stainless-steel wire mesh and a screen plate to avoid particle leakage. The adsorption column and ultrafiltration membrane module were maintained at the required temperature using an ultra-constant-temperature water bath.

2.5. COS Production in EAES System

Inoculate 30 mL of seed into a fermenter containing 3 L of the fermentation medium. Fermentation was incubated for 72 h under conditions identical to those used for batch fermentation. A peristaltic pump was used to circulate the fermentation broth in the enzyme adsorption column at various flow rates. When the chitinase concentration in the fermentation broth decreased below 0.3 U/mL, the circulation between the fermenter and enzyme adsorption column was terminated, and the temperature of the enzyme adsorption column was elevated to 40 °C. Following a 30 min incubation period, circulation between the enzyme adsorption column and the ultrafiltration membrane assembly was initiated. The filtrate was collected, and an equivalent volume of buffer (pH 7.0) was introduced from the refill bottle into the supplement storage tank to maintain a constant feed volume. Samples were collected at regular intervals using various operating parameters for product analysis. The EAES system employed for COS production was also operated in repeat batch mode. Repeat batches were fermented for 48 h, with the exception of the initial batch. The first batch of the EAES system consists of two batches of enzyme-producing fermentation. The cycle between the enzyme adsorption column and ultrafiltration membrane module was discontinued at the conclusion of each batch, and the processes of enzyme adsorption, enzymatic digestion, and product separation were repeated for CHOS production.

2.6. Analysis of Antioxidant Activity of CHOS Produced by EAES System

The antioxidant activity of CHOS (ECHOS) produced by the EAES system was evaluated using the hydroxyl-radical scavenging, DPPH-radical scavenging, and reducing power methods and compared with commercially available CHOS and Vitamin C. The hydroxyl-radical scavenging ability of ECHOS was determined with reference to the method of Guo et al. [69]. The volume of the total reaction mixture was controlled to 4.5 mL, and 1 mL of ECHOS solution of different mass concentrations, 1 mL of phosphate buffer solution (150 mM, pH 7.4), 0.5 mL of EDTA-Fe²⁺ solution (220 μM), 1 mL of safranine O solution (0.23 μM), and 1 mL of H₂O₂ solution (60 μM) were sequentially added to the test tube. After homogeneous mixing, the reaction mixture was heated in an environment of 37 °C for 30 min, and its absorbance value was measured at a wavelength of 520 nm. Scavenging effect (%) = [(A_{sample 520nm} − A_{blank 520nm})/(A_{control 520nm} − A_{blank 520nm})] × 100. The superoxide radical scavenging ability and reducing ability of ECHOS were examined according to the method established by Zhang et al. [70]. ECHOS was dissolved in deionized water to prepare solutions of different concentrations. Different concentrations of ECHOS solution and 2 mL of 1,1-diphenyl-2-picrylhydrazyl (DPPH) ethanol solution (180 μmol/L) were sequentially added to the test tubes. A total of 1 mL of deionized water was used to replace the sample as the blank group, and 2 mL of absolute ethanol was used to replace the DPPH ethanol solution as the control group. The absorbance was measured at a wavelength of 517 nm after 20 min of incubation at room temperature in the dark after mixing the reaction systems. The DPPH radical scavenging rate was calculated as follows: scavenging effect (%) = [(A_{sample 517nm} − A_{blank 517nm})/(A_{control 517nm} − A_{blank 517nm})] × 100. The measurement of reducing power is to mix 1 mL of ECHOS solution of different concentrations with 1 mL of 1% potassium ferricyanide solution, heat it at 50 °C for 20 min, cool to room temperature, add 1 mL of trichloroacetic acid (10%), centrifuge at 3000 rpm for 5 min, and take 1.5 mL of supernatant and mix it with 1.2 mL of distilled water and 0.3 mL of ferric chloride solution (0.1%). The absorbance value of the reaction system was measured at 700 nm after standing at room temperature for 30 min.

2.7. Analytical Methods and Statistical Analysis

Biomass was determined by drying the cells to a constant weight in an oven at 65 °C. Chitin enzyme activity was measured according to a previously described method [6]. The enzymatic hydrolysis products were analyzed using HPLC. The enzymatic reaction was terminated by combining the sample with 70% acetonitrile in a 1:1 ratio. Subsequently, the sample was centrifuged at 12,000 rpm for 10 min and filtered through a 0.22 μm membrane for HPLC analysis. A Prevail Carbohydrate ES column (4.6 × 250 mm) was used for chromatographic separation. The mobile phase consisted of acetonitrile and water at a 70:30 ratio, with a flow rate of 1 mL/min and an injection volume of 10 µL. The ultraviolet detector was set at a wavelength of 205 nm, and the column temperature was maintained at 30 °C. The gradient elution program was as follows: 0 min, 75% acetonitrile; 7 min, 75% acetonitrile; 8 min, 65% acetonitrile; 15 min, 65% acetonitrile; 16 min, 75% acetonitrile; 22 min, 75% acetonitrile. The total duration of the assay was 30 min. The data obtained are expressed as the mean ± standard deviation of three determinations and were statistically analyzed using ANOVA.

3. Results and Discussion

3.1. Optimization of Carbon and Nitrogen Sources in Culture Medium

To identify a suitable medium for cell growth and chitin enzyme production by C. sp. LZ32, fermentation was carried out using different carbon and nitrogen sources in the basal medium (Figure 2). The results show that C. sp. LZ32 can utilize a wide variety of carbon and nitrogen sources and can produce chitin enzyme under all tested carbon and nitrogen sources. The highest chitin enzyme activity and biomass were obtained using fructose as the carbon source, followed by glucose. The enzyme activity of arabinose was only 32% of that of fructose (Figure 2a). Chitin-degrading enzymes are typical exocrine enzymes, and the secretion and production of most enzymes is induced and regulated by chitin and its derivatives. The relationship between the induced expression of chitin-degrading enzymes and carbon source selection is a complex process, and glucose, as an easy-to-use carbon source, usually exerts an inhibitory effect on chitin-degrading enzymes [71,72]. Ni and Westpheling’s studies suggest that this inhibitory effect may be achieved through direct repeats in the promoter region, which are involved in both chitin induction and glucose inhibition [73]. In the culture environment where glucose and chitin exist at the same time, glucose inhibits the production of chitin-degrading enzymes, resulting in low enzyme activity of chitin. As an isomer of glucose, fructose may not inhibit the secretion of chitin-degrading enzymes, but it can be easily absorbed and utilized by bacteria. In addition, chitosan and N-acetyl-β-D-glucosamine (GlcNAc) produced by chitin degradation can be used as carbon and nitrogen sources for the growth of bacterial cells [74,75]. Therefore, fructose is more conducive to the growth of bacteria and the secretion of chitin-degrading enzymes than glucose in the presence of chitin. The maximum chitin enzyme activity in fermentation broth with 15 g/L fructose as the carbon source was 2.14 U/mL. The biomass obtained at different fructose concentrations did not differ much (Figure 2b). To evaluate nitrogen sources, various inorganic and organic nitrogen sources were independently added to chitin enzyme production. As shown in Figure 2c, ten nitrogen sources were examined, and yeast extract was beneficial for cell growth and chitin enzyme production. When the yeast extract concentration was increased from 3 g/L to 5 g/L, the biomass and chitin enzyme activities increased, and the maximum chitin enzyme activity (3.08 U/mL) and biomass (5.58 g/L) were obtained with the addition of yeast extract at 5 g/L (Figure 2d). The optimal medium contains 5 g/L HLC; 15 g/L fructose, 5 g/L yeast extract, 0.7 g/L K₂HPO₄; 0.3 g/L KH₂PO₄; And 0.5 g/L MgSO₄, greatly improving the chitin enzyme production of C. sp. LZ32. In addition, the experimental results also showed that enzyme yield was positively correlated with biomass.

3.2. Production of Chitin Enzymes by Batch and Repeat Batch Processes

The results of single-batch fermentation enzyme production of C. sp. LZ32 show that the production of chitin enzyme occurred only after the growth of bacteria, and the two were in a semi-coupled relationship (Figure 3a). C. sp. LZ32 reached its maximum biomass (7.26 g/L) at 56 h of culture, followed by a significant decrease. Chitin enzyme production started at 16 h and reached a peak of 3.52 U/mL at 64 h, which was 1.96 times higher than before unoptimized media (1.8 U/mL) [51]. To further optimize the chitin production process, repeated batch fermentation was carried out. As shown in Figure 3b, four batches of fermentation were performed for 208 h. Except for the first batch, the duration of fermentation was 64 h. The fermentation time for the other three batches was 48 h. Bacterial growth was delayed in the first batch. Little lag was observed in subsequent batches, and the highest biomass of 7.42 g/L was achieved in the second batch. The biomass of the third and fourth batches was 7.12 g/L and 6.76 g/L, respectively. Although the biomass of the third and fourth batches decreased compared to the second batch, it was still higher than the biomass (5.53 g/L) obtained from a single-batch fermentation of C. sp. LZ32 for 40 h (Figure 3a). Similarly, the highest value of chitin enzyme activity (3.58 U/mL) also appeared in the second batch, followed by a downward trend. However, after 48 h of fermentation in the fourth batch, the enzyme activity was 2.71 U/mL, which was still higher than the enzyme activity produced by single-batch fermentation for 48 h (2.32 U/mL). The total amount of enzyme produced in the four batches was 12.88 U/mL, and the production efficiency was 0.062 U/mL/h. Compared with C. sp. LZ32 single-batch fermentation (0.042 U/mL/h), the production efficiency of chitin enzyme in repeated-batch fermentation was improved by 47.62%. This is consistent with the results of repeated-batch fermentation for the fermentative production of other products [54,61,76,77]. Repeated-batch fermentation can shorten the fermentation time without reducing the enzyme production of C. sp. LZ32, which is an effective strategy to improve the production efficiency of chitin-degrading enzymes. In addition, chitin-degrading enzymes are mostly inducible enzymes, and the secretion of chitin-degrading enzymes increases significantly when microorganisms are induced by chitin and its degradation products [78,79]. In repeated-batch operation, after fresh culture medium is injected, the bacteria will be induced by the residual products of the previous batch during the growth process, which is beneficial to the increase in enzyme yield.

3.3. Optimization of Operating Conditions for HLP Adsorption of Chitin Enzyme

Previous studies have shown that HLP has excellent adsorption performance for chitin-degrading enzymes in C. sp. LZ32 fermentation broth. Using HLP as an adsorption material for chitin enzyme purification can effectively simplify the purification of chitin-degrading enzymes and reduce production costs [51]. HLP was selected as the adsorbent material for chitin-degrading enzyme purification in the EAES system, and the effect of the flow rate on adsorption was studied. As shown in Figure 4, with an increase in the circulation time of the fermentation broth between the fermenter and the enzyme adsorption column, both enzyme activity and biomass in the fermenter decreased. This shows that in addition to chitin-degrading enzymes being adsorbed on HLP, some bacteria are also intercepted by the adsorption column. At a high flow rate (60 mL/s), the enzyme activity in the fermentation broth decreased to 0.1 U/mL and the biomass decreased from 7.28 g/L to 3.57 g/L after 150 min of circulation. Therefore, when adsorbing chitin-degrading enzymes, many bacteria are trapped in the adsorption column, which has a significant impact on subsequent enzymatic hydrolysis and ultrafiltration processes. Although a low operating flow rate (40 mL/min) can effectively reduce the retention of bacteria, it prolongs the operating time, which is not conducive to efficient operation of the EAES system. At a flow rate of 50 mL/min, the enzyme activity in the fermentation broth was less than 0.3 U/mL after 90 min of cycling, and the biomass was only reduced by 14.4%. Therefore, a flow rate of 50 mL/min was selected for subsequent studies.

3.4. Optimization of Ultrafiltration Conditions for CHOS Separation

In the EAES system, an ultrafiltration membrane module was used to separate CHOS and recycle chitin-degrading enzymes. The variation in the membrane flux with ultrafiltration time was studied at different operating pressures. As shown in Figure 5a, the membrane flux increased significantly with an increasing operating pressure. When the operating pressure was increased from 1 to 2 bar, the initial membrane flux doubled. However, the amplitude of the membrane flux decreased significantly when the operating pressure exceeded 3 bar. Furthermore, although a maximum permeate flux of 4.3 (L/(m²h)) was reached at an operating pressure of 4 bar, this value decreased by nearly 50% after 50 min of operation and to 0.92 (L/(m²h)) after 120 min, which was only 21.40% of the initial flux. This may be because the accumulation of proteins and bacteria on the membrane surface thickens the filter cake layer on the membrane surface, which reduces the effective radius of the fluid passing through the membrane pores, resulting in attenuation of the membrane flux. However, the membrane flux always showed a slow downward trend at 1 bar. This shows that a lower operating pressure is beneficial for alleviating the permeate flux decay. However, a lower membrane flux is not a suitable separation condition for CHOS. In contrast, the maximum membrane flux (1.38 (L/(m²h)) was obtained by ultrafiltration for 120 min at 2 bar, indicating that 2 bar may be the optimal operating pressure for the ultrafiltration membrane module.

The composition of CHOS in the permeate at different ultrafiltration times was analyzed using HPLC at 2 bar. Statistical analysis was conducted on the proportion (mass percentage) of monomers with different degrees of polymerization in CHOS. The analysis revealed that as the duration of ultrafiltration increased, the proportion of these monomers in the permeate changed. Specifically, the proportions of monomers and dimers gradually decreased with extended operation time, whereas the proportions of pentamers and hexamers in CHOS progressively increased. As illustrated in Figure 5b, after 30 min of ultrafiltration, the proportions of trimers, tetramers, pentamers, and hexamers were 6%, 8%, 24%, and 19%, respectively. The sum of these proportions, totaling 57%, exceeded 50% of the total CHOS content. After 50 min of ultrafiltration, the proportions of pentamers and hexamers were 33% and 27%, respectively. The proportions of the various monomers stabilized after 70 min of ultrafiltration. Compared to the initial stage of ultrafiltration (10 min), the proportion of pentamers increased from 16% to 34%, while the proportion of hexamers increased from 14% to 30%. The ratio of CHOS at each degree of polymerization tends to be stable after 70 min of ultrafiltration. Compared with the initial stage of ultrafiltration (10 min), the proportions of pentamers and hexamers increased from 14% and 16% to 34% and 30%, respectively. Compared with other technologies, membrane separation technology has the advantages of high separation efficiency, low energy consumption, and simple process and can realize the purification and concentration of fermentation products simultaneously. The combination of the bioreactor and membrane separation device has been applied to fermentation production, biocatalysis, and sewage treatment. It has the advantages of increasing cell concentration, reducing product inhibition, and simplifying downstream processes [80,81]. The ultrafiltration membrane module in the EAES system could effectively separate the CHOS generated in the adsorption column module. Timely separation of CHOS also effectively prevented its continued degradation by the chitin-degrading enzymes. This may be the reason why the proportion of CHOS with a high degree of polymerization increased with the extension of the operation time.

3.5. EAES System Continuously Produces CHOS

A continuous production study of CHOS was performed using the EAES system at an adsorption flow rate of 50 mL/min and an operating pressure of 2 bar, as described in Section 2.5, for a total of four continuous batches, with membrane flux and CHOS yield detected at the end of each batch (Figure 6). The first batch was terminated after 115 h of operation, and the subsequent three batches were all run for 48 h, with a total operation time of 259 h. The first batch consisted of two batches of fermentative enzyme production processes to ensure that sufficient enzyme remained on the adsorption column for subsequent enzymatic hydrolysis. The results of membrane flux examination at the end of each batch showed that at the end of the first batch, the membrane flux was 2.02 (L/(m²h)), while by the end of the fourth batch, the membrane flux decreased to 0.87 (L/(m²h)). This may be caused by the accumulation of insoluble particles in the bacteria and enzymatic hydrolysates on the ultrafiltration membrane. From the perspective of the output of CHOS, it shows an upward trend with an increase in batches. The output of the fourth batch is 23.2 g, which is 3.74 times, 1.86 times, and 1.21 times that of the first three batches. The total yield of the four batches of CHOS was 61 g of COS, and the production efficiency was 0.24 g/h. The EAES system combines enzyme production, enzyme separation, enzymatic hydrolysis, and product separation processes for CHOS production, providing a new approach for the continuous production of CHOS.

3.6. Antioxidant Activity of Enzymolysis Products

Reactive oxygen species (ROS) are oxygen-active molecules formed during metabolism. Excessive ROS and its oxidative stress can damage the body and induce cancer, cardiovascular disease, and inflammatory disease [70,82]. Antioxidants can delay or prevent the oxidation process to reduce the damage caused by oxidative stress. Existing research showed that COS has certain antioxidant activities, such as free-radical scavenging capacity and reducing capacity [83,84,85]. The antioxidant activity of ECHOS was evaluated by hydroxyl-radical scavenging, DPPH-radical scavenging, and reducing power methods and compared with commercially available CHOS and Vitamin C. The hydroxyl-radical scavenging activity, DPPH-radical scavenging activity, and reducing power of ECHOS increased with increasing concentration (Figure 7a–c). At the same concentration, the three antioxidant activities of ECHOS were higher than CHOS. In addition, when the concentration of ECHOS was higher than 0.6 mg/mL, the hydroxyl-radical scavenging activity was significantly improved. At the concentration of 1 mg/mL, the scavenging capacity of ECHOS reached 55.29%, which was 1.67 times that of CHOS (Figure 7a). ECHOS had high DPPH-radical scavenging activity. At the concentration of 0.2 mg/mL, the clearance rates of ECHOS and CHOS were 57.38% and 33.88%, respectively. The clearance rate at 1 mg/mL was 92.71%, which was close to the clearance rate of Vitamin C (Figure 7b). The reduction force of ECHOS is shown in Figure 7c. Compared with Vitamin C, ECHOS and CHOS had weak reduction ability. The absorbance values at a concentration of 0.1 mg/mL were 0.168 and 0.082 A, representing 17.91% and 8.74% of the absorbance of Vitamin C, respectively. With increasing concentration, ECHOS showed strong reducing power. At 1 mg/mL, the absorption value of ECHOS was 0.872, which was 43.02% of Vitamin C and 1.57 times of CHOS. According to the above analysis, compared with CHOS at the same concentration, ECHOS had a higher scavenging rate of hydroxyl and DPPH radicals and a stronger reduction ability. Therefore, the effective degradation of HLP by the EAES system and the antioxidant performance of degradation products indicate that it has great potential in high-value utilization of waste shrimp and crab shells.

4. Conclusions

In this study, HLP-degrading strain C. sp. LZ32 was studied. By screening for the optimal carbon and nitrogen sources in the enzyme-producing medium and optimizing the addition amount, the yield of chitin-degrading enzymes can be effectively increased. Repeated batch fermentation improves the production efficiency of chitin-degrading enzymes. By constructing an EAES system and optimizing the operating parameters, continuous production of CHOS was achieved using a repeated batch process. The results demonstrate the feasibility of continuous production of CHOS in the EAES system. This provides a new pathway for enzymatic hydrolysis of crystalline chitin substrates to produce HCOS.