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Marine Drugs 22 (10): 445 (2024)
Pohling, Julia
Ramakrishnan, Vegneshwaran Vasudevan
Hossain, Abul
Trenholm, Sheila
Dave, Deepika
Shrimp shells are a key source of chitin, commonly extracted through chemical methods, which may cause minor molecular damage. Nowadays, there is great interest in achieving close to zero protein content in crude chitin in order to use it for high-end markets. Therefore, this study optimized the enzymatic deproteination using two commercial proteases (SEB Pro FL100 and Sea-B Zyme L200) for effective and fast removal of residual protein from Northern shrimp (Pandalus borealis) shell chitin for the first time. The protein content was determined using both the Kjeldahl method and amino acid analysis using gas chromatography–mass spectrometry (GC-MS). The performance of papain (Sea B Zyme L200) was superior to fungal protease (SEB Pro FL100) for this application, and it achieved residual protein content of 2.01%, while the calculated optimum for the latter enzyme was 6.18%. A model was developed using 24 factorial design, and it was predicted that the lowest residual protein content using fungal protease and papain could be achieved at the following conditions: a pH of 4.2 and 7, and an enzyme concentration of 4 and 1.5%, respectively. Thus, the low-protein content obtained using enzymatic deproteination could be an alternative approach to the traditional methods, indicating their potential to produce premium-quality chitin.
Citation: Pohling, J.; Ramakrishnan,V.V.; Hossain, A.; Trenholm, S.; Dave,D. Optimization of EnzymaticDeproteination of Northern Shrimp(Pandalus borealis) Shell Chitin UsingCommercial Proteases. Mar. Drugs2024, 22, 445. https://doi.org/10.3390/md22100445Academic Editor: Paola LaurienzoReceived: 7 September 2024Revised: 24 September 2024Accepted: 26 September 2024Published: 28 September 2024Copyright: © 2024 by the authors.Licensee MDPI, Basel, Switzerland.This article is an open access articledistributed under the terms andconditions of the Creative CommonsAttribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).marine drugs ArticleOptimization of Enzymatic Deproteination of Northern Shrimp(Pandalus borealis) Shell Chitin Using Commercial ProteasesJulia Pohling 1 , Vegneshwaran Vasudevan Ramakrishnan 1,2, Abul Hossain 2,3 , Sheila Trenholm 1and Deepika Dave 1,*1 Marine Institute, Center for Aquaculture and Seafood Development, Memorial University of Newfoundland,St. John’s, NL A1C5R3, Canada; julia.pohling@mi.mun.ca (J.P.); vvasudevanra@mun.ca (V.V.R.);sheila.trenholm@mi.mun.ca (S.T.)2 Department of Biochemistry, Memorial University of Newfoundland, St. John’s, NL A1C 5S7, Canada;abulh@mun.ca3 Faculty of Land and Food Systems, The University of British Columbia, Vancouver, BC V6T 1Z4, Canada* Correspondence: deepika.dave@mi.mun.caAbstract: Shrimp shells are a key source of chitin, commonly extracted through chemical methods,which may cause minor molecular damage. Nowadays, there is great interest in achieving close tozero protein content in crude chitin in order to use it for high-end markets. Therefore, this studyoptimized the enzymatic deproteination using two commercial proteases (SEB Pro FL100 and Sea-BZyme L200) for effective and fast removal of residual protein from Northern shrimp (Pandalus borealis)shell chitin for the first time. The protein content was determined using both the Kjeldahl method andamino acid analysis using gas chromatography–mass spectrometry (GC-MS). The performance ofpapain (Sea B Zyme L200) was superior to fungal protease (SEB Pro FL100) for this application, and itachieved residual protein content of 2.01%, while the calculated optimum for the latter enzyme was6.18%. A model was developed using 24 factorial design, and it was predicted that the lowest residualprotein content using fungal protease and papain could be achieved at the following conditions: apH of 4.2 and 7, and an enzyme concentration of 4 and 1.5%, respectively. Thus, the low-proteincontent obtained using enzymatic deproteination could be an alternative approach to the traditionalmethods, indicating their potential to produce premium-quality chitin.Keywords: high-quality chitin; commercial proteases; Atlantic shrimp; optimization; waste utilization1. IntroductionChitin is a natural polysaccharide that is abundantly found in the exoskeleton ofarthropods (crustaceans and insects) and fungi. Chitin and its deacetylated derivative,chitosan, have a versatile range of applications in the medical, environmental, cosmetic, andfood/nutrition industries [1,2]. Typical for a natural polymer, chitin’s molecular structureand macrostructural arrangement in the cuticle vary widely between sources, seasons, bodyparts, animal health, and extraction methods [3]. The most critical descriptors of chitinare the degree of deacetylation (DDA) and the molecular weight, which render chitin andchitosan distinct properties [4–6]. Inconsistencies between chitosan products are consideredone of the main reasons why chitosan is not yet approved as a medical ingredient anda generally recognized as safe (GRAS) substance, although it is often claimed to be aGRAS [7,8]. In contrast to synthetic polymers, parameters concerning chitin synthesis arecompletely out of our control, while parameters during shell handling and chitin extractionare somewhat controllable.Northern shrimp (Pandalus borealis) is a small, soft-shelled species, and plays it avital role in the Newfoundland and Labrador (NL) fishing industry [9]. According toFisheries and Oceans Canada (DFO), in 2021, the total landing volume of shrimp in NL was35,998 t (29.7% of the total shellfish landing), with a landing value of $140 million [10]. InMar. Drugs 2024, 22, 445. https://doi.org/10.3390/md22100445 https://www.mdpi.com/journal/marinedrugsMar. Drugs 2024, 22, 445 2 of 18NL, Pandalus borealis is machine-peeled in fully automated processing facilities. The shell,amounting up to 75% of the harvested biomass, is landfilled, composted, disposed at sea, orused in low-value applications, such as fertilizer. The shell is composed of approximately17% chitin, 41.9% protein, 34.2% minerals, and 5.2% lipids [11]. While the value of chitinin the shells has been recognized in Canada for years, industries have been strugglingto establish economically viable businesses. Factors such as production cost, health andsafety regulations around the transportation, storage, and handling of large volumes ofconcentrated acids and bases, as well as wastewater treatment, are the major challenges torecovering chitin from shrimp.Chitin can be extracted by several chemical, enzymatic, or biotechnological methodsto remove mineral deposits, fats, proteins, and pigments [4,6,7]. The majority of theworld’s chitin is produced using a traditional chemical approach. Minerals (mainly calciumcarbonate) are dissolved by reaction with hydrochloric acid. Proteins are denatured andwashed off using a dilute sodium hydroxide solution at elevated temperatures (belowboiling). While this process is very reliable and consistently produces chitin of highpurity, some minor molecular damage, mainly in the form of depolymerization, occurs [5].In recent decades, the focus of the research has shifted away from chemically-intensiveprocesses, and towards developing gentle extraction methods that minimize changes tothe native molecular structure [7,8]. For example, demineralization has been achievedsuccessfully using citric and acetic acids [12–14], as well as microwave-assisted deepeutectic solvent (DES) extraction [15]. The green approach for removing proteins from chitinreplaces sodium hydroxide with proteolytic enzymes [4,6,11]. Despite the research efforts,caustic deproteination using sodium hydroxide is still the method of choice for proteinremoval because the process is simple and removes protein reliably and completely [4–6,15].However, the agreement on which alternative process is best is still ambiguous. Theadvantage of enzymatic protein removal is that it allows for the recovery of the shrimpprotein hydrolysate as a secondary product. Shrimp protein is particularly attractive asit contains all of the essential amino acids required by the body. The residual proteincontent in enzymatically deproteinated chitin has been a major issue in preventing thewidespread commercialization of green processes. Achieving a high-purity chitin is vitalfor several reasons. Because natural polymers like chitin or cellulose compete with highlypure synthetic polymers for many applications, it is inherently essential to remove allother contaminants in order to achieve food-grade or biomedical-grade products, andto ensure that the polymer can be further processed without the presence of interferingsubstances [7,8].While protein and ash contents of <1% are acceptable, the lower the residual con-taminants, the higher the potential suitability for high-end markets [16]. The desire is toachieve a protein content as close to zero as possible. From the medical point of view,protein content should be minimized to avoid allergic reactions, which can occur as animmunological response to protein-based allergens. The presence of allergens compro-mises their clinical usage [16]. In particular, the protein tropomyosin has been identifiedas a potent allergen that should be removed completely [17]. From the processing pointof view, a low residual protein content in chitin improves the conversion process to chi-tosan. In this process, called deacetylation, chitin is converted to chitosan under harshalkaline conditions (>50% NaOH) at high temperatures (>100 ◦C). The reaction produceschitosan with a DDA of around 80–90%. To achieve a higher DDA, the process must berepeated several times. The higher-purity chitin achieves a higher DDA in the first stepof conversion, shortening the deacetylation process and the number of times the productis exposed to harsh denaturing conditions. Because each deacetylation step causes somedepolymerization, high purity of chitin is critical in producing high molecular weightchitosan [18]. Therefore, this study focused on optimizing enzymatic deproteination usingtwo commercial proteases for effective and fast removal of residual proteins from Northernshrimp (Pandalus borealis) shells to produce chitin for the first time. This approach couldMar. Drugs 2024, 22, 445 3 of 18address the full utilization of Northern shrimp by utilizing processing waste and producinghigh-quality and low-protein chitin.2. Results and Discussions2.1. Enzymatic DeproteinationOptimization of enzymatic deproteination was performed for two industrial enzymes,SEB Pro FL100 and Sea B Zyme L200. The performance of Sea B Zyme L200 was superiorto SEB Pro FL100 for this application, and it achieved a residual protein content of 2.01%,while the calculated optimum for SEB Pro FL100 was 6.18%. In the following two sections,the detailed results of optimization experiments are presented.2.2. SEB Pro FL100Table 1 shows the residual protein content for the 24 factorial design. The modelpredicts the theoretical residual protein content of 3.75% at the following conditions: pH 3;temperature, 55 ◦C; enzyme concentration, 2.58%; and reaction time, 2 h. The deproteinationreaction appears to be completed within the first 2 h. When the conditions were favourable,the enzyme was able to produce a residual protein content between 5–8% within 2 h.However, when they were unfavourable for deproteination, the residual protein contentranged from 11–16%, even after a 12 h reaction. None of the parameter combinations testedwithin the matrix were able to achieve protein contents below 5%.Table 1. 24 Factorial design for SEB Pro FL100.ID pH Temp (◦C) Conc (%) Time (h) Mean TotalN (%) SD Total N Protein N (%)Residual Protein(%)1 6.00 55.0 0.10 12 8.95 0.30 2.23 13.922 6.00 40.0 4.00 2 8.83 0.00 2.11 13.213 6.00 55.0 4.00 12 8.93 0.16 2.21 13.794 6.00 40.0 0.10 12 9.26 0.06 2.54 15.875 3.00 55.0 0.10 2 8.47 0.09 1.75 10.946 3.00 40.0 4.00 12 7.81 0.05 1.09 6.827 3.00 55.0 4.00 2 7.74 0.05 1.02 6.378 3.00 55.0 4.00 12 7.52 0.16 0.80 5.039 6.00 40.0 0.10 2 9.31 0.02 2.59 16.2210 6.00 55.0 0.10 2 9.10 0.00 2.38 14.8511 4.50 47.5 2.05 7 7.89 0.09 1.17 7.3112 3.00 55.0 0.10 12 8.58 0.04 1.86 11.6213 3.00 40.0 0.10 12 8.52 0.06 1.80 11.2814 6.00 40.0 4.00 12 8.77 0.11 2.05 12.8315 3.00 40.0 4.00 2 8.08 0.02 1.36 8.4916 4.50 47.5 2.05 7 7.87 0.03 1.15 7.1717 6.00 55.0 4.00 2 8.82 0.11 2.10 13.1418 4.50 47.5 2.05 7 7.82 0.01 1.10 6.8719 3.00 40.0 0.10 2 8.83 0.08 2.11 13.18As shown in the Pareto chart (Figure 1), the most important individual factors are thepH, enzyme concentration, and temperature. The pH × enzyme concentration was theonly significant two-way interaction identified with a p-value of 0.013. The reaction timehad no significant influence on the response factor.Figure 2 shows that the enzyme works best at the lower end of the pH range anddrastically decreases as the pH increases. Enzyme concentrations above 2% were required tolower residual protein levels to below 5%. The effects of temperature were less pronounced,but slightly less protein residue was observed at the upper-temperature limit, supportingthe findings in Figure 1.Mar. Drugs 2024, 22, 445 4 of 18Mar. Drugs 2024, 22, x FOR PEER REVIEW 4 of 18 Figure 1. SEB Pro FL100: Pareto chart of standardized effects (24 factorial design). p-value is the probability used to assess the statistical significance of the result. Figure 2 shows that the enzyme works best at the lower end of the pH range and drastically decreases as the pH increases. Enzyme concentrations above 2% were required to lower residual protein levels to below 5%. The effects of temperature were less pro-nounced, but slightly less protein residue was observed at the upper-temperature limit, supporting the findings in Figure 1. Figure 2. SEB Pro FL100: Prediction and optimization plot for mean of protein vs. pH, temperature, and enzyme concentration. The residual plot of multiple regression analysis was randomly distributed (Figure 3), indicating that the model is unbiased (R2 = 99.54). Figure 1. SEB Pro FL100: Pareto chart of standardized effects (24 factorial design). p-value is ther a ilit se t assess t e statistical si ifica ce f t e res lt.Mar. Drugs 2024, 22, x FOR PEER REVIEW 4 of 18 Figure 1. SEB Pro FL100: Pareto chart of standardized effects (24 factorial design). p-value is the probability used to assess the statistical significance of the result. Figure 2 shows that the enzyme works best at the lower end of the pH range and drastically decreases as the pH increases. Enzyme concentrations above 2% were required to lower residual protein levels to below 5%. The effects of temperature were less pro-nounced, but slightly less protein residue was observed at the upper-temperature limit, suppo ting the findings in Figure 1. Figure 2. SEB Pro FL100: Prediction and optimization plot for mean of protein vs. pH, temperature, and enzyme concentration. The residual plot of multiple regression analysis was randomly distributed (Figure 3), indicating that the model is unbiased (R2 = 99.54). Figure 2. SEB Pro FL100: Prediction and optimization plot for mean of protein vs. pH, temperature,and enzyme concentration.The residual plot of multiple regression analysis was randomly distributed (Figure 3),indicating that the model is unbiased (R2 = 99.54).Based on the data obtained, the levels for reaction time were chosen too broadly, andthey did not correctly bracket the early phase of deproteination (<2 h), during which thedynamics of deproteination would be best-observed. The pH and enzyme concentrationstrongly influenced the reaction efficiency, and the levels chosen to study their impact weretoo wide. To further narrow down the optimal pH, reaction time, and enzyme concentration,a factorial design was created using tighter levels, as described in the materials and methodssection. Ten additional experiments were selected from a full factorial design investigatingthe factors of time, pH, and enzyme concentration only, bracketing the optimum morenarrowly than the initial 24 factorial design (Table 2).Mar. Drugs 2024, 22, 445 5 of 18Mar. Drugs 2024, 22, x FOR PEER REVIEW 5 of 18 Figure 3. SEB Pro FL100: Residuals vs. fitted values. Based on the data obtained, the levels for reaction time were chosen too broadly, and they did not correctly bracket the early phase of deproteination (<2 h), during which the dynamics of deproteination would be best-observed. The pH and enzyme concentration strongly influenced the reaction efficiency, and the levels chosen to study their impact were too wide. To further narrow down the optimal pH, reaction time, and enzyme con-centration, a factorial design was created using tighter levels, as described in the materials and methods section. Ten additional experiments were selected from a full factorial design investigating the factors of time, pH, and enzyme concentration only, bracketing the op-timum more narrowly than the initial 24 factorial design (Table 2). Table 2. Additional multifactorial design experiments performed for SEB Pro FL100 with improved level settings. ID Time (h) Conc (%) pH Mean Total N (%) SD N Protein N (%) Residual Protein (%) 1 8 4 4.2 7.54 0.06 0.82 5.15 2 8 2 4.7 8.32 0.02 1.60 9.98 3 1 4 4.7 8.16 0.01 1.44 8.97 4 1 2 4.2 8.09 0.03 1.37 8.54 5 2 1 4.7 8.72 0.06 2.00 12.52 6 4 1 4.2 8.46 0.02 1.74 10.90 7 2 2 4.7 8.39 0.00 1.67 10.42 8 4 4 4.7 8.05 0.04 1.33 8.28 9 2 4 4.2 7.77 0.02 1.05 6.58 10 1 1 4.7 8.73 0.05 2.01 12.56 The model predicted that the lowest residual protein using SEB Pro FL100 could be achieved at pH 4.2 and an enzyme concentration of 4%. At these conditions, the predicted residual protein content is 6.18%. The Pareto chart of the standardized effects (Figure 4) confirms the results of the initial factorial design, where the enzyme concentration and the pH are the most important factors, as indicated by the low P-value of 0.006 and 0.003, respectively. Furthermore, the reaction time is not statistically significant, indicating that the reaction occurs rapidly within the first hour. Due to the small sample size, the validity of the p-value should be interpreted with caution. Marzieh et al. [19] used the proteases Trypsin (pH 7.7) and Ficin (pH 7.5) to deproteinate Litopennaeus vannamei shells at room temperature for 1–4 h, using varying enzyme concentrations ranging from 0.1–0.033%. Sodium metabisulfite was used as an additive because of its ability to reduce disulfide bonds, increasing protein solubility. Agreeing with our study, the reaction time was found Figure 3. SEB Pro FL100: Residuals vs. fitted values.Table 2. Additional multifactorial design experiments performed for SEB Pro FL100 with improvedlevel settings.ID Time (h) Conc (%) pH Mean Total N (%) SD N Protein N (%) Residual Protein (%)1 8 4 4.2 7.54 0.06 0.82 5.152 8 2 4.7 8.32 0.02 1.60 9.983 1 4 4.7 8.16 0.01 1.44 8.974 1 2 4.2 8.09 0.03 1.37 8.545 2 1 4.7 8.72 0.06 2.00 12.526 4 1 4.2 8.46 0.02 1.74 10.907 2 2 4.7 8.39 0.00 1.67 10.428 4 4 4.7 8.05 0.04 1.33 8.289 2 4 4.2 7.77 0.02 1.05 6.5810 1 1 4.7 8.73 0.05 2.01 12.56The model predicted that the lowest residual pr using SEB Pro FL100 could beachieved at pH 4.2 and an enzyme concentration of 4%. At these conditions, the predictedresidual protein content is 6.18%. The Pareto chart of the standardized effects (Figure 4)confirms the results of the initial factorial design, where the enzyme concentration andthe pH are the most important factors, as indicated by the low P-value of 0.006 and 0.003,respectively. Furthermore, the reaction time is not statistically significant, indicating thatthe reaction occurs rapidly within the first hour. Due to the small sample size, the validityof the p-value should be interpreted with caution. Marzieh et al. [19] used the proteasesTrypsin (pH 7.7) and Ficin (pH 7.5) to deproteinate Litopennaeus vannamei shells at roomtemperature for 1–4 h, using varying enzyme concentrations ranging from 0.1–0.033%.Sodium metabisulfite was used as an additive because of its ability to reduce disulfidebonds, increasing protein solubility. Agreeing with our study, the reaction time was foundto be relevant only in the first 2 h. The lowest residual protein content was 5.31% for Trypsinand 6.1% for Ficin.The main effects plot (Figure 5) suggests that even higher enzyme concentrations inthe range of 4 vol% may be needed to achieve optimal protein removal, confirming earlierresults that a lower pH improved protein removal.Mar. Drugs 2024, 22, 445 6 of 18Mar. Drugs 2024, 22, x FOR PEER REVIEW 6 of 18 to be relevant only in the first 2 h. The lowest residual protein content was 5.31% for Tryp-sin and 6.1% for Ficin. Figure 4. SEB Pro FL100: Additional experiments, Pareto chart of the standardized effects. p-value is the probability used to assess the statistical significance of the result. The main effects plot (Figure 5) suggests that even higher enzyme concentrations in the range of 4 vol% may be needed to achieve optimal protein removal, confirming earlier results that a lower pH improved protein removal. Figure 5. SEB Pro FL100: Additional experiments, main effects plot for mean of protein vs. enzyme concentration (EC) and pH. The residual vs. fitted plot (Figure 6) showed randomly distributed values, indicating that our model was non-biased. However, one outlier was detected for experiment ID 1 at 5.15% residual protein, where the result for residual protein was significantly lower than the predicted value. Moreover, Table 3 lists the five best alternative solutions for SEB Pro FL100, producing theoretical residual protein contents between 8–13%. Figure 4. SEB Pro FL10 : Ad itional experiments, Pareto chart of the standardized eff cts. p-value isthe probability used to asse the statistical sign fican e of the result.Mar. Drugs 2024, 22, x FOR PEER REVIEW 6 of 18 to be relevant only in the first 2 h. The lowest residual protein content was 5.31% for Tryp-sin and 6.1% for Ficin. Figure 4. SEB Pro FL100: Additional experiments, Pareto chart of the standardized effects. p-value is the probability used to assess the statistical significance of the result. The main effects plot (Figure 5) suggests that even higher enzyme concentrations in the range of 4 vol% may be need d to achieve optimal pro ein removal, con irming earlier r sults th a lower pH improved protein removal. Figure 5. SEB Pro FL100: Additional experiments, main effects plot for mean of protein vs. enzyme concentration (EC) and pH. The residual vs. fitted plot (Figure 6) showed randomly distributed values, indicating that our model was non-biased. However, one outlier was detected for experiment ID 1 at 5.15% residual protein, where the result for residual protein was significantly lower than the predicted value. Moreover, Table 3 lists the five best alternative solutions for SEB Pro FL100, producing heoretical residual prot in contents between 8–13%. Figure 5. SEB Pro FL100: Additional experiments, main effects plot for mean of protein vs. enzymeconcentration (EC) and pH.The residual vs. fitted plot (Figure 6) showed randomly distributed values, indicatingthat our model was non-biased. However, one outlier was detected for experiment ID 1 at5.15% residual protein, where the result for residual protein was significantly lower thanthe predicted value. Moreover, Table 3 lists the five best alternative solutions for SEB ProFL100, producing theoretical residual protein contents between 8–13%.Mar. Drugs 2024, 22, x FOR PEER REVIEW 7 of 18 Figure 6. SEB Pro FL100: Multifactorial design, residuals vs. fitted. Table 3. Five best alternative solutions for SEB Pro FL100 deproteination. Enzyme Concentration pH Residual Protein Content 2 4.2 8.23 4 4.7 8.31 2 4.7 10.36 1 4.2 10.58 1 4.7 12.70 2.3. Sea-B-Zyme L200 Table 4 shows the residual protein content for the 24 factorial design. Based on the results, the theoretical residual protein content after treatment with the papain-based commercial protease is 2.3% at the following optimized conditions: pH 7; temperature, 70 °C; enzyme concentration, 1.5%; and reaction time, 2 h. Table 5 shows the top five alter-native solutions. Table 4. Kjeldahl analytical results of 24 factorial design for Sea-B-Zyme L200. ID pH Temperature Conc Time Mean Total N (%) SD Total N Protein N Residual Protein (%) 1 7.00 70 0.50 2 7.31 0.03 0.59 3.68 2 7.00 60 1.50 8 7.19 0.02 0.47 2.95 3 7.00 60 1.50 2 7.32 0.04 0.60 3.75 4 7.00 60 0.50 8 7.37 0.06 0.65 4.04 5 5.50 60 0.50 2 8.09 0.01 1.37 8.59 6 6.25 65 1.00 5 7.44 0.00 0.72 4.51 7 5.50 70 1.50 2 7.53 0.04 0.81 5.07 8 5.50 60 0.50 8 7.87 0.06 1.15 7.19 9 5.50 70 0.50 8 7.53 0.05 0.81 5.03 10 5.50 60 1.50 2 7.75 0.01 1.03 6.47 11 7.00 60 0.50 2 7.60 0.01 0.88 5.47 12 7.00 70 1.50 8 7.22 0.04 0.50 3.10 13 5.50 70 0.50 2 7.85 0.10 1.13 7.09 14 6.25 65 1.00 5 7.38 0.04 0.66 4.13 15 5.50 70 1.50 8 7.73 0.02 1.01 6.33 16 7.00 70 1.50 2 7.05 0.57 0.33 2.06 17 6.25 65 1.00 5 7.48 0.03 0.76 4.72 Figure 6. SEB Pro FL100: ultifactorial design, residuals vs. fitted.Mar. Drugs 2024, 22, 445 7 of 18Table 3. Five best alternative solutions for SEB Pro FL100 deproteination.Enzyme Concentration pH Residual Protein Content2 4.2 8.234 4.7 8.312 4.7 10.361 4.2 10.581 4.7 12.702.3. Sea-B-Zyme L200Table 4 shows the residual protein content for the 24 factorial design. Based on theresults, the theoretical residual protein content after treatment with the papain-basedcommercial protease is 2.3% at the following optimized conditions: pH 7; temperature,70 ◦C; enzyme concentration, 1.5%; and reaction time, 2 h. Table 5 shows the top fivealternative solutions.Table 4. Kjeldahl analytical results of 24 factorial design for Sea-B-Zyme L200.ID pH Temperature Conc Time MeanTotal N (%)SDTotal N Protein NResidualProtein (%)1 7.00 70 0.50 2 7.31 0.03 0.59 3.682 7.00 60 1.50 8 7.19 0.02 0.47 2.953 7.00 60 1.50 2 7.32 0.04 0.60 3.754 7.00 60 0.50 8 7.37 0.06 0.65 4.045 5.50 60 0.50 2 8.09 0.01 1.37 8.596 6.25 65 1.00 5 7.44 0.00 0.72 4.517 5.50 70 1.50 2 7.53 0.04 0.81 5.078 5.50 60 0.50 8 7.87 0.06 1.15 7.199 5.50 70 0.50 8 7.53 0.05 0.81 5.0310 5.50 60 1.50 2 7.75 0.01 1.03 6.4711 7.00 60 0.50 2 7.60 0.01 0.88 5.4712 7.00 70 1.50 8 7.22 0.04 0.50 3.1013 5.50 70 0.50 2 7.85 0.10 1.13 7.0914 6.25 65 1.00 5 7.38 0.04 0.66 4.1315 5.50 70 1.50 8 7.73 0.02 1.01 6.3316 7.00 70 1.50 2 7.05 0.57 0.33 2.0617 6.25 65 1.00 5 7.48 0.03 0.76 4.7218 5.50 60 1.50 8 7.58 0.07 0.86 5.3619 7.00 70 0.50 8 7.20 0.05 0.48 3.03Table 5. Sea-B-Zyme L200: Top five alternative solutions.pH Temp (◦C) EnzymeConcentration (%)ReactionTime (h)Predicted ResidualProtein Content (%)7 70 0.5 8 2.777 60 1.5 8 2.897 70 1.5 8 2.947 60 1.5 2 3.347 70 0.5 2 3.61As shown in Figure 7, the parameter with the most significant effect was the pH,followed by the enzyme concentration and reaction temperature. The reaction time hadan insignificant influence on the response factor within the tested limits (Figure 7). Theonly significant two-way interaction was the enzyme concentration × reaction time, with ap-value of 0.044.Mar. Drugs 2024, 22, 445 8 of 18Mar. Drugs 2024, 22, x FOR PEER REVIEW 8 of 18 18 5.50 60 1.50 8 7.58 0.07 0.86 5.36 19 7.00 70 0.50 8 7.20 0.05 0.48 3.03 Table 5. Sea-B-Zyme L200: Top five alternative solutions. pH Temp (°C) Enzyme Concentration (%) Reaction Time (h) Predicted Residual Protein Content (%) 7 70 0.5 8 2.77 7 60 1.5 8 2.89 7 70 1.5 8 2.94 7 60 1.5 2 3.34 7 70 0.5 2 3.61 As shown in Figure 7, the parameter with the most significant effect was the pH, followed by the enzyme concentration and reaction temperature. The reaction time had an insignificant influence on the response factor within the tested limits (Figure 7). The only significant two-way interaction was the enzyme concentration × reaction time, with a p-value of 0.044. Figure 7. Sea-B-Zyme L200: Pareto chart of the standardized effects. p-value is the probability used to assess the statistical significance of the result. The main effects plot (Figure 8) results indicate that increases in the pH result in a sharp drop in the residual protein content. Temperature, concentration, and time also saw sharp decreases between low and centre points, but only minimal further reduction be-tween centre points and upper levels. Figure 7. Sea-B-Zyme L200: Pareto chart of the standardized effects. p-value is the probability usedto assess the statistical significance of the result.The main effects plot (Figure 8) results indicate that increases in the pH result in asharp drop in the residual protein content. Temperature, concentration, and time alsosaw sharp decreases between low and centre points, but only minimal further reductionbetween centre points and upper levels.Mar. Drugs 2024, 22, x FOR PEER REVIEW 8 of 18 18 5.50 60 1. 8 7.58 0.07 0.86 5.36 19 7.00 70 0.50 8 7.20 0.05 0.48 3.03 Table 5. Sea-B-Zyme L200: Top five alternative solutions. pH Temp (°C) Enzyme Concentration (%) Reaction Time (h) Predicted Residual Protein Content (%) 7 70 0.5 8 2.77 7 60 1.5 8 2.89 7 70 1.5 8 2.94 7 60 1.5 2 3.34 7 70 0.5 2 3.61 s s o n in Figure 7, the parameter with the most significant effect was the pH, f l y the enzyme concentration and reaction temperature. Th reaction time had a ificant influence on the response factor within the tested limits (Figure 7). The o l si ificant two-way interaction was the enzyme concentration × reaction time, with a p-value of 0.044. Figure 7. Sea-B-Zyme L200: Pareto chart of the standardized effects. p-value is the probability used to assess the statistical significance of the result. The main effects plot (Figure 8) results indicate that increases in the pH result in a sharp drop in the residual protein content. Temperature, concentration, and time also saw sharp decreases between low and centre points, but only minimal further reduction be-twee centre points and upper levels. Figure 8. Enzymatic deproteination with Sea B Zyme L200: Main effects plot for protein (data means).The residuals plot shows some clustering and is not randomly scattered around zero,indicating that our model is not completely unbiased (Figure 9). At various places alongthe predicted vs. actual plot, the model over- or under-predicts the response, despite thehigh R2 value of 99.65. Values have a normal distribution, and no unusual data pointswere detected.Mar. Drugs 2024, 22, 445 9 of 18Mar. Drugs 2024, 22, x FOR PEER REVIEW 9 of 18 Figure 8. Enzymatic deproteination with Sea B Zyme L200: Main effects plot for protein (data means). The residuals plot shows some clustering and is not randomly scattered around zero, indicating that our model is not completely unbiased (Figure 9). At various places along the predicted vs. actual plot, the model over- or under-predicts the response, despite the high R2 value of 99.65. Values have a normal distribution, and no unusual data points were detected. Figure 9. Sea-B-Zyme L200: Residuals vs. fitted values. 2.4. Confirmation Runs, Sea B Zyme L200 Confirmation runs were performed for Sea B Zyme only, due to its superior results over SEB Pro FL100. Twenty confirmation runs were performed at the optimal predicted conditions of 1.5% enzyme concentration, 70 °C reaction temperature, and 2 h reaction time. All confirmation experiments were performed using the same shell material that was used for optimization experiments. The residual protein content was determined us-ing both the Kjeldahl nitrogen estimation and the amino acid analysis using GC-MS. All tests were performed in duplicate, and the results are shown in Table 6. Table 6. Residual protein content in confirmation runs performed at optimal conditions as deter-mined by the factorial design experiment: pH 7, 70 °C, and 1.5% Sea B Zyme for 2 h. Residual protein (%) Kjeldahl Approach Amino Acid Analysis Sample ID Mean ±SD (%) Mean ± SD (%) Before deproteination 17.28 ± 0.04 17.38 ± 0.18 1 2.25 ± 0.08 2.24 ± 0.14 2 0.00 ± 0.00* 2.50 ± 0.10 3 1.40 ± 0.13 4.65 ± 0.25* 4 0.93 ± 0.70 2.73 ± 0.58 5 5.43 ± 1.14* 2.77 ± 1.14 6 1.56 ± 0.26 2.39 ± 0.11 7 0.75 ± 0.61 1.79 ± 0.12 Figure 9. Sea-B-Zyme L200: Residuals vs. fitted values.2.4. Confirmation Runs, Sea B Zyme L200Confirmation runs were performed for Sea B Zyme only, due to its superior resultsover SEB Pro FL100. Twenty confirmation runs were performed at the optimal predictedconditions of 1.5% enzyme concentration, 70 ◦C reaction temperature, and 2 h reactiontime. ll fi i ex eri ents were performed using the same shell material that wasused for optimization experiments. The r sidual protein content was det rmined usingboth the Kjeldahl nitrogen estimation and the amino acid analysis using GC-MS. All testswere performed in uplicate, and the results are shown in Table 6.The average residual protein content was 1.65 and 2.01%, as determined by the Kjeldahlmethod an GC-MS analysis, respectively. This was slightly lower than the value of 2.3%residual protein that was predicted by the 24 factorial design. The Kjeldahl approach hada slightly higher standard deviation than amino analysis, indicating that the latter mayhave higher precision. Overall, the results of the two methods are in agreement. Thequantification of residual protein in chitin is not yet standardized, and research groups useseveral different methods for evaluating deproteination success. One of the most commonapproaches described in the literature is the estimation via the Kjeldahl method, whichwe also used in this study. This approach relies on the assumption that the chemicallydeproteinated chitin reference is perfectly pure, yet fully acetylated. This is an inherentsource of error, as intensive deproteination conditions also cause partial deacetylation, andsmall amounts of residual protein may still be present. Amino acid analysis via GC-MS isconsidered the most precise method for the determination of protein content in chitin, sinceit directly measures amino acid concentration, and is especially useful when determiningvery small amounts of residual protein residue in chitin, where precision is relevant [20].We, therefore, double-tested the samples of the confirmation run with both methods andcompared the results. There was a very good alignment between values obtained from theGC-MS amino analysis and the Kjeldahl estimation. Our results show that the Kjeldahlmethod, as described here, is a reliable method to quickly estimate the residual proteincontent and identify optimal deproteination conditions.Mar. Drugs 2024, 22, 445 10 of 18Table 6. Residual protein content in confirmation runs performed at optimal conditions as determinedby the factorial design experiment: pH 7, 70 ◦C, and 1.5% Sea B Zyme for 2 h.Residual protein (%)Kjeldahl Approach Amino Acid AnalysisSample ID Mean ±SD (%) Mean ± SD (%)Before deproteination 17.28 ± 0.04 17.38 ± 0.181 2.25 ± 0.08 2.24 ± 0.142 0.00 ± 0.00 * 2.50 ± 0.103 1.40 ± 0.13 4.65 ± 0.25 *4 0.93 ± 0.70 2.73 ± 0.585 5.43 ± 1.14 * 2.77 ± 1.146 1.56 ± 0.26 2.39 ± 0.117 0.75 ± 0.61 1.79 ± 0.128 0.84 ± 0.13 1.47 ± 0.019 2.87 ± 0.08 1.67 ± 0.1710 2.50 ± 2.20 1.49 ± 0.2211 1.09 ± 0.57 1.69 ± 0.0312 1.59 ± 0.13 1.85 ± 0.0813 1.18 ± 0.35 1.76 ± 0.2014 2.37 ± 1.32 2.86 ± 0.7615 1.87 ± 0.70 2.05 ± 1.0616 2.68 ± 0.70 1.55 ± 0.5017 1.09 ± 0.22 1.61 ± 0.1118 1.56 ± 0.97 2.46 ± 0.2619 1.78 ± 0.39 1.53 ± 0.1520 1.31 ± 0.08 1.83 ± 0.37Grand average 1.65 ± 0.53 2.01 ± 0.34* Excluded from average and SD calculation.In terms of enzymes, papain was able to produce higher-purity chitin at a lowerenzyme concentration and, therefore, provided the most efficient and economical depro-teination solution. Both papain and fungal proteases have previously been reported for usein chitin deproteination. Earlier studies described the deproteination of chitin at pH 5.6–6.0and 37.5 ◦C for 60 h [21]. Boonkam et al. [22] reported optimal conditions for papain-catalyzed deproteination at pH 8 and 40 ◦C for 1 h, using 1 unit of papain/ g of shrimp.Rohyami et al. [23] had a different research focus and investigated papain’s ability to causepartial deacetylation of chitin. In their study, shrimp shells were deproteinated with papainat concentrations ranging from 0–25%. The best results were achieved at 25% papain, whichcould result in a DDA of 41%. Valdez-Pena et al. [24] screened several industrial proteasesfor deproteination efficiency in shrimp heads (Litopennaeus vannamei) before demineral-ization, and achieved the lowest levels of residual protein, at 2.75%, using Alcalase, andbetween 3–4% for papain and a fungal protease from Aspergillus oryzae. Moreover, Denget al. [25] used a two-step enzymatic treatment to deproteinate Litopenaeus vannamei shells.They tested aspartic protease P6281, saccharopepsin, Alcalase, and papain, and foundthat P6281 and saccharopepsin produced the best deproteination results. The degree ofdeproteination for papain was only 42.3% compared to 71.2% for protease P6281. Residualprotein content was determined by incubating the shrimp shell sample in 1 M NaOH at40 ◦C for 3 h, and then quantifying the protein content of the supernatant using the Kjeldahlapproach. The degree of deproteination was calculated from the Kjeldahl result, and theobserved weight reduction was calculated during deproteination.On the other hand, Gagne and Simpson [26] used chymotrypsin and papain to depro-teinate shrimp waste obtained from Eastern Quebec, Canada. They achieved the lowestresidual protein contents of 1.8% using chymotrypsin and 2.8% using papain. The pa-pain reaction was conducted at 38 ◦C and pH 8.7, with an enzyme/waste ratio of 10:1000.While the reaction was run at a much lower temperature, the final protein residue wascomparable to our study. Vazquez et al. [27] deproteinated squid pens (Illex argentines)Mar. Drugs 2024, 22, 445 11 of 18using a NaOH-based process, as well as an enzymatic approach using the three industrialproteases, namely Alcalase, Esperase, and Neutrase. After deproteination, both solid andliquid fractions were analyzed. Protein in the chitin sample was calculated as the sum ofamino acids analyzed by the ninhydrin method using an amino acid analyzer. Proteinin the liquid fraction was determined using the Lowry assay. The results showed thatEsperase was the best enzyme for the deproteination of squid pens, followed by Alcalaseand Neutrase. Dhanabalan et al. [28] investigated a combination of enzymatic and chemicaldeproteination of Acetes shell, a tiny, soft, shrimp-like Arctic krill. Controlled enzymaticdeproteination with Alcalase at an enzyme/substrate ratio of 1.53 at 53.5 ◦C for variouslengths of time was performed until the specified degree of hydrolysis was reached (5, 15,and 30%). This was followed by a mild deproteination in NaOH to lower the residual pro-tein to approximately 1%. These samples were compared to chitin which was deproteinatedin 3% NaOH. The deproteination success was assessed using the Kjeldahl approach, likethe current study. The lowest residual protein content achieved enzymatically was 3.33%.The authors note that, unlike the enzymatic process, the secondary NaOH treatment couldremove a large portion of lipophilic pigments. Enzymatically deproteinated chitin has ahigher degree of acetylation than chemically extracted chitin.In addition, Younes et al. [29] investigated five different crude protease extracts for thedeproteination of shrimp shells (Metapenaeus monoceros). The best results were achievedwith protease from Bacillus mojavensis (A21), with a residual protein content that was5.8% higher than the chemically deproteinated control sample, as determined by Kjeldahlanalysis. The performance of the crude enzyme preparation was also found to be superiorto the control enzyme bromelain, and Alcalase. In addition, six microbial and nine fishalkaline crude proteases were used in deproteinating shrimp shells (Metapenaeus monoceros).The best results were achieved for Bacillus mojavensis (A21) and Balistes capriscus (greytriggerfish) proteases with 77 ± 3 and 78 ± 2%, respectively. The degree of deproteinationwas calculated from the Kjeldahl crude protein content of the solid sample and the observedweight reduction during deproteination [30,31].The studies above demonstrate that fast protein removal from chitin within a fewhours is possible with several proteases. No protease clearly stands out, but Alcalase hasbeen used in many studies and has become a common enzyme used as a performancereference. The studies have in common that there is always low residual protein contentafter treatment, ranging from 1.8–6.1% in most studies. The results of papain deproteinationpresented in this study align well with the most successful deproteination studies. Theprotein residue found in enzymatically deproteinated chitin has been attributed to covalentbonds between protein and chitin, hydrogen bonds, and hydrophilic interactions. Lastly,spatial restrictions limiting access to the substrate inside the chitin network are thought tocontribute to incomplete deproteination [4,6,15,32,33].2.5. Calculation of Total Deproteination during Chitin ExtractionThroughout the chitin extraction process, protein is removed step by step. While thedeproteination step focuses on removing protein bound to shells, some protein is removedprior to the deproteination step by simply grinding and washing the shells. This proteinincludes meat protein particles, contents of the cephalothorax, and protein loosely attachedto the outside of the shell. Figure 10 displays the proximate composition of the shrimpshells throughout the chitin extraction process.The chart compares the main shell constituents, protein, minerals, lipids, and chitinon a dry basis. In earlier studies on processing shrimp shells using the same pre-treatmentand demineralization process (unpublished), we found lipid content of 1.99% in untreatedshells, and once pre-treated, the lipid content was undetectable by the Soxhlet extraction.This finding confirmed published values for lipids in shrimp by-products, ranging from2–3.15% in untreated Penaeus kerathurus and Metapenaeus monoceros shells [34] to 0.3–0.5%in Pandalus borealis [35]. Lipid quantification of the deproteinated sample was thereforenot performed, and we assumed a value of 2% for raw shrimp shells and zero for allMar. Drugs 2024, 22, 445 12 of 18subsequent process samples to visualize the general composition. The residual proteincontent determined via GC-MS, which was slightly higher than the Kjeldahl value, is usedfor these calculations to present the worst-case scenario. Pre-treatment removes the looselyattached proteins. Demineralization removes minerals, leaving a very small amount ofresidual ash. Finally, deproteination reduces the residual protein content to 2.01% (theaverage residual protein content from the 20 confirmation runs). A final chitin purity of97.12% is achieved.Mar. Drugs 2024, 22, x FOR PEER REVIEW 12 of 18 attached to the outside of the shell. Figure 10 displays the proximate composition of the shrimp shells throughout the chitin extraction process. Figure 10. Compositional changes throughout chitin extraction (assuming lipid concentration is 0% and * indicates unpublished data). The chart compares the main shell constituents, protein, minerals, lipids, and chitin on a dry basis. In earlier studies on processing shrimp shells using the same pre-treatment and demineralization process (unpublished), we found lipid content of 1.99% in untreated shells, and once pre-treated, the lipid content was undetectable by the Soxhlet extraction. This finding confirmed published values for lipids in shrimp by-products, ranging from 2–3.15% in untreated Penaeus kerathurus and Metapenaeus monoceros shells [34] to 0.3–0.5% in Pandalus borealis [35]. Lipid quantification of the deproteinated sample was therefore not performed, and we assumed a value of 2% for raw shrimp shells and zero for all sub-sequent process samples to visualize the general composition. The residual protein con-tent determined via GC-MS, which was slightly higher than the Kjeldahl value, is used for these calculations to present the worst-case scenario. Pre-treatment removes the loosely attached proteins. Demineralization removes minerals, leaving a very small amount of residual ash. Finally, deproteination reduces the residual protein content to 2.01% (the average residual protein content from the 20 confirmation runs). A final chitin purity of 97.12% is achieved. Chitin purity depends not only on the deproteination process itself, but also on sev-eral other factors. In our study, we wanted to create ideal conditions for deproteination, not only by optimizing the deproteination step, but also by utilizing pre-treated, suitable raw materials. For example, the shell material used in our study was of exceptional fresh-ness and was processed immediately after collection from the processing plant without a drying step or interim storage. The small particle size provided a high surface area to allow the enzyme to access the proteins. Moreover, demineralization was performed prior to deproteination, as recommended by Younes et al. [6], to increase the accessibility of the protein embedded in the chitin network. Finally, Pandalus borealis is a thin-shelled crusta-cean species, and among the smaller species harvested of food. Thin shells are generally easier to process for chitin extraction due to smaller mineral deposits and better accessi-bility of the protein to chemicals and enzymes [6,36]. To determine the economic viability of enzymatic chitin purification processes, a biorefinery approach is necessary, which as-sesses not only the cost of the enzymatic deproteination step, but the entire chitin extrac-tion process, including the potential additional revenue from a protein hydrolysate prod-uct and the cost savings for energy and chemical handling safety measures. Testing of processes on pilot-scale processing is essential to obtain such feasibility assessments, but available data are minimal [37]. Most of the literature on chitin extraction cites insufficient chitin purity, high enzyme cost, and long reaction times as the main reasons why Figure 10. o positional changes throughout chitin extraction (assu ing lipid concentration is 0and * indicates unpublished data).C itin purity depends not only on the deprot ination process itself, but also o severalther factors. In ou study, w wanted to create ideal conditio s for deproteination, noo ly by optimizing the deprotei ation tep, but also by ut lizing pre-treated, suitabl rawmaterials. For exampl , the shell material used in our study was of exceptional freshnessand was processed i mediately after collection from the p ocessing plant without a dryingstep or interim storage. The small particle size rovided a high urface area to allowthe e zyme to ccess the roteins. More ver, demine alization was p rformed priorto dep oteination, as recom nded by Younes et l. [6], to incr a e the accessibi ity ofthe pro ein embedded in the chitin network. Finally, Pandalus borealis is a thin-shelledcrustacean sp cies, and among t e smaller species harvested of food. Thin hells aregenerally easier to process for chitin extraction due t smaller mineral deposits and betterccessibility of the protein to chemicals and enzymes [6,36]. To determine the ec nomicviability of enzymatic chitin purification processes, a biorefinery approach is necessary,which assesses not only the cost of the enzymatic deproteination step, but the entire chitinextraction process, including the potential additional revenue from a protein hydrolysateproduct and the cost savings for energy and chemical handling safety measures. Testing ofprocesses on pilot-scale processing is essential to obtain such feasibility assessments, butavailable data are minimal [37]. Most of the literature on chitin extraction cites insufficientchitin purity, high enzyme cost, and long reaction times as the main reasons why enzymaticdeproteination processes have not been widely implemented on an industrial scale [5].Some researchers have addressed purity issues by applying a short, gentle caustictreatment following enzymatic deproteination. For many applications, a residual proteincontent of <5% is acceptable (industrial and some food applications). The low residualprotein content may not be critical if used as raw material for chitosan production, as itis removed during caustic deacetylation. The development of internationally acceptedchitin purity standards would help in determining what level of purity is required. Thehigh cost of enzymes has been addressed in different ways. Some authors use industrialenzymes, while others use crude protease, immobilized protease, or ferment chitin withprotease-producing microorganisms [5,38]. Even still, an enzymatic approach is almostalways more expensive or time-consuming compared to the chemical process using NaOHand HCl. Some of this higher cost can be offset by creating secondary products fromMar. Drugs 2024, 22, 445 13 of 18the protein hydrolysate [25], especially in the age of society recognizing the need forhigh-quality protein sources, and the technical capability of turning a hydrolysate intouseful and appetizing forms (e.g., peptides). Shrimp protein is particularly attractivebecause it contains not only protein but also pigment astaxanthin, one of the strongestnatural antioxidants, which is responsible for the colour of egg yolks and salmon fillets.This potential is lost in the chemical process, where protein is destroyed. Enzymaticdeproteination of chitin with commercial proteases takes as long as the average causticdeproteination reaction, roughly 2 h [5]. Significantly longer reaction times of up to severaldays are only experienced when chitin is fermented with lactic acid and protease-producingbacterial strains. Lastly, the local production setting must be considered when evaluatingthe cost-effectiveness of a chitin extraction process. Strict regulations can quickly make aprocess economically unfeasible, although reagents may be cheap. For example, in somecountries with strict environmental laws and remote shrimp processing locations, the cost ofestablishing the necessary infrastructure to permit a chemical process, such as wastewatertreatment, freshwater usage, worker health and safety regulations, and emergency firstresponse capabilities, will quickly outweigh the economic benefit. Enzymatic processesare significantly more attractive for implementation in remote areas of industrializedcountries. While materials are more expensive, enzymatic processes are performed inmild and non-hazardous conditions. Waste management is significantly easier, and bulkchemical transportation and storage are eliminated. However, to compete with highlypure, chemically extracted chitin, the processes must be highly optimized and find a nicheconsumer market.Overall, the optimization of reaction parameters resulted in chitin with theoreticalresidual protein contents of 6.18 and 2.3% for fungal protease and papain, respectively.Papain performance was verified in twenty confirmation runs and produced an averageresidual protein content of 2.01%, outperforming the theoretical results of the factorialdesign. The two enzymes were selected based on different characteristics. Papain wasselected for its advertised performance of producing non-bitter hydrolysates from seafood.The enzyme has a declared activity of 5500 PU/mg, and has activity over a wide pH range,from 4–10, with the optimum somewhere between 5–8 [39,40]. A drawback of papain wasthe high-temperature optimum of 65 ◦C and the heat resistance up to 80 ◦C, meaning thatmore energy-intensive heating is required [41,42]. The optimization experiments in thisstudy determined a pH and temperature optima of 7.0 and 70 ◦C, respectively, which wasslightly above the declared values. SEB Pro FL100 was sourced because of its declared max-imum activity of 100,000 HUT/g at acidic pH of 3–5, and a comparatively low-temperatureoptimum of 40–55 ◦C (manufacturer’s specifications). These conditions would be attractivefor an industrial-scale chitin extraction process because deproteination could be performedimmediately following acid demineralization, with minimal pH adjustment. A lower pro-cess temperature is advantageous, as it results in energy savings. In this study, optimizationexperiments resulted in a theoretical residual protein content of 6.18%. The performancewas best at lower pH, but activity was highest near the upper-temperature limit.3. Materials and Methods3.1. ReagentsTwo different proteases, SEB Pro FL100 and Sea-B Zyme L200, were purchased fromEnzyme Innovation (Chino, CA, USA). Analytical-grade sodium hydroxide and hydrochlo-ric acid were purchased from Fisher Scientific (Ottawa, ON, Canada) and used in 10 wt%solutions for pH adjustment. Amino acid standards were purchased from Sigma-Aldrich(St. Louis, MO, USA).3.2. Preparation of Shell and DemineralizationFresh shrimp shells (Pandalus Borealis) were collected from a shrimp processing plantin Newfoundland, Canada. The shells were chilled on ice, transported to the laboratory,and further processed on the same day. Shells were subjected to a pre-treatment andMar. Drugs 2024, 22, 445 14 of 18demineralization process performed in a pilot-scale system. During pre-treatment, 319 kgof shrimp shell was ground, washed, and pressed, resulting in 123.6 kg of pressed wetshell with a homogeneous particle size of 1.4 mm. The weight reduction observed duringpre-treatment (wet basis) was 61.27%. Demineralization was achieved by mixing 57 kgof the pre-treated shell with water to a reaction volume of 400 L. Demineralization wasachieved within 3 h at 20 ◦C under continuous mixing by the stepwise addition of 16.39 Lof 20◦ Bé (32%) hydrochloric acid, corresponding to a concentration of 4.09%. Weightreduction during demineralization was 36.67%. The resulting 36.1 kg of the pressed shellwas frozen in portions for use in laboratory-scale deproteination experiments. The moisture,ash, and residual protein contents were determined as described below.3.3. Analytical MethodsAsh and moisture contents were determined according to standard dry ashing andmoisture procedures (AOAC 938.08 and 930.15). The residual protein content was esti-mated from the nitrogen content determined using the standard Kjeldahl method AOAC954.01/988.05 (AOAC, 2012). Because both the chitin and proteins contain nitrogen, acommon approach reported in the literature is to determine the total nitrogen content ina sample, as well as the nitrogen stemming from chitin, to calculate the residual proteincontent [36,40,41]. Varying data for chitin and chitosan nitrogen concentrations can befound in the literature. Theoretically, fully acetylated and deacetylated chitins contain 6.896and 5.1% of nitrogen, respectively [27,42]. The natural nitrogen content of crustaceans isspecies-dependent, and varies between 6–7% [18].3.4. Caustic DeproteinationA “pure” chitin standard was produced by mixing 100 g of demineralized shell (78.48%moisture) in 7% NaOH solution to a total reaction volume of 1 L (1:10 shell-to-liquid ratio)and incubating the mixture under constant stirring at 70 ◦C for 2 h. For the calculation, itwas assumed that all protein was completely removed from the shell, remaining 100% chitin(acetylated). As a result, it was assumed that all nitrogen in this sample stemmed fromchitin (NChitin). Samples produced by enzymatic deproteination (described below) werecompared to this “pure” standard. The total nitrogen content was determined by Kjeldahlanalysis, and the residual protein content was calculated using the following formula:Res.ProtEnz.DP = (NEnz.DP − NChem.DP) × 6.25 (1)Where Res.Prot, residual protein; Enz.DP, enzymatic deproteination; Chem.DP, chemi-cal deproteination; and 6.25, average nitrogen content in protein.3.5. Gas Chromatography–Mass Spectrometer (GC-MS) AnalysisIn addition to the Kjeldahl protein determination, the residual protein content ofsamples from the selected samples was also determined via gas chromatography (GC)–massspectrometer (MS) (Thermo Fisher Trace 1300 GC/ISQ-LT MS) amino acid analysis, whichis recognized as the method of highest precision for protein determination [20]. All aminoacids were weighed separately and prepared in 0.1 N HCl to prepare a stock solution at aconcentration of 0.5 mg/mL. Five different stock solution concentrations were used to builda calibration curve. The DL-Norleucine was used as an internal standard, at a 0.5 mg/mLconcentration. The internal standard was added to both standards and samples. Bothstandards and samples were subjected to derivatization using N-tertbutyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA). During the derivatization process, 50 µL aliquots ofstandards/samples were taken in a 10 mL test tube, and then dried at 70 ◦C under nitrogenfor 5 min. After drying, 100 µL of neat MTBSTFA was added, followed by adding 100 µLof acetonitrile. The test tubes were tightly capped and were heated at 100 ◦C for 2 hours.The sample was then allowed to cool at room temperature, and 200 µL of acetonitrile wasadded again to the tubes. The samples were then transferred into GC vials for analysis. TheGC analysis was carried out using an SLB-5ms, 20 m × 0.18 mm column with an internalMar. Drugs 2024, 22, 445 15 of 18diameter of 0.18 µm. The inlet temperature was 280 ◦C and was operated at the splitlessmode. The split flow was maintained at 100 mL/min, and the splitless time was 0.3 min.The column flow was 0.5 mL/min. The GC oven was maintained at 60 ◦C for 0 min andramped up to 100 ◦C at 20 ◦C/min; held for 1 min and ramped up to 290 ◦C at 10 ◦C/min;and held for 3 min and ramped up to 340 ◦C, then held for 2 min. The MS transfer linetemperature was 320 ◦C, and the ion source temperature was 280 ◦C. The standards andsamples were scanned using MS in the range of 40–639 (m/z).3.6. Enzymatic DeproteinationDeproteination experiments were carried out in 2500 mL beakers placed in an OLS200water bath (Grant Instruments, Cambridge, UK). Agitation was provided by a EUROSTAR60 overhead stirrer (IKA Works Inc., Wilmington, NC, USA) with an AN R1382 propellerstirrer, which was 3-bladed (IKA Works Inc., NC, USA). The pH was monitored usingan HQ40D portable multi-meter (HACH, London, ON, Canada). In each experiment,200 g of pre-treated and demineralized shell (described above) was mixed with pre-heateddistilled water to a total reaction volume of 2000 mL before the enzyme addition. ThepH was adjusted to the specified level, with 10% HCl and NaOH solutions. The solutionwas stirred for at least 30 min, or until the reaction temperature was reached and the pHremained steady. Then, the enzyme was added (Table 7) and the solution was stirred forthe pre-determined reaction time. Upon completion, the shells were strained and washedfor 5 min under running water before being hand-pressed and dried at 105 ◦C for 24 h.Table 7. High and low levels for Sea-B-Zyme L200 and SEB Pro Fl100.FactorSea-B-Zyme L200 SEB Pro FL100Low High Low HighpH 5.5 7 3 6Temperature (◦C) 60 70 40 55Enzyme concentration (% ofreaction volume) 0.5 1.5 0.1 4Reaction time (h) 2 8 2 123.7. Experimental Design and Statistical AnalysisA 24 factorial design was created to investigate the factors of enzyme concentration,temperature, reaction time, and pH. A total of 16 different combinations were tested(1 replicate) in addition to 3 centre point runs for a total of 19 runs. Statistical evaluationwas performed using Minitab software. High and low levels were set based on the manufac-turer’s recommendations for the working range of the enzyme, which helps us to determinethe optimal levels for desired results, compare enzyme efficiency, and understand howdifferent enzyme concentrations impact the overall effectiveness. The high and low levelsset for the two enzymes are summarized in Table 7.An additional multifactorial design for SEB Pro FL100 was created to improve theprediction, with more narrow-level settings. A selection of 10 experiments were performedfor the design, as shown in Tables 8 and 9. Based on the initial 24 factorial design results, alladditional experiments were performed at a temperature of 55 ◦C. The reason for runningadditional experiments for SEB Pro FL100 is explained in the results section.Table 8. SEB Pro FL100: multilevel factorial design, factor information.Factor Levels ValuesReaction time (h) 4 1, 2, 4, and 8Enzyme concentration (% of reaction volume) 3 1, 2, and 4pH 2 4.2 and 4.7Mar. Drugs 2024, 22, 445 16 of 18Table 9. Additional experiments. Multilevel factorial design for SEB Pro FL100.ID Time Enzyme Concentration pH1 8 4 4.22 8 2 4.73 1 4 4.74 1 2 4.25 2 1 4.76 4 1 4.27 2 2 4.78 4 4 4.79 2 4 4.210 1 1 4.74. ConclusionsProtein removal from pre-treated and demineralized shrimp shells (Pandalus borealis)was optimized for two commercial proteases. The performance of papain (Sea B Zyme L200)was superior, leaving residual protein content of 2.01% (dry basis). The theoretic residualprotein content after fungal protease treatment (SEB Pro FL100) was 6.18%, requiring ahigh enzyme concentration (>3%). In future studies, factors such as seasonal variabilityof the shell, crustacean species, and batch-to-batch differences should be examined, alongwith the physicochemical properties of chitin. To evaluate economic viability, a reductionof the enzyme concentration from 1.5–0.5% (Table 5, second best solution), as well asthe reusability of the enzyme liquor for subsequent batches, should be assessed. Proteinrecovery from the liquid fraction should be further evaluated as an additional sourceof revenue.Author Contributions: Conceptualization, J.P. and D.D.; methodology, J.P., V.V.R. and S.T.; val-idation, J.P., V.V.R. and A.H.; formal analysis, J.P., V.V.R. and S.T.; investigation, J.P., V.V.R. andS.T.; resources, D.D.; data curation, J.P., V.V.R. and S.T.; writing—original draft preparation, J.P.and A.H.; writing—review and editing, J.P., A.H. and D.D.; visualization, J.P. and A.H.; supervi-sion, D.D.; funding acquisition, D.D. All authors have read and agreed to the published version ofthe manuscript.Funding: This research was funded by the Loblaws Weston Seeding Innovation Fund (SFI18-0217,Memorial University) and the NL Department of Industry, Energy and Technology (5404-2382-101).Institutional Review Board Statement: Not applicable.Data Availability Statement: Available upon request.Conflicts of Interest: The authors declare no conflicts of interest.References1. 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A Seasonal Study of the Chemical Composition and Chitin Quality of Shrimp ShellsObtained from Northern Shrimp (Pandalus borealis). Carbohydr. Polym. 2008, 71, 388–393. [CrossRef]Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individualauthor(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury topeople or property resulting from any ideas, methods, instructions or products referred to in the content.
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high-quality chitin
commercial proteases
Atlantic shrimp
optimization
waste utilization
Optimization of Enzymatic Deproteination of Northern Shrimp (Pandalus borealis) Shell Chitin Using Commercial Proteases
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