Method for preparing oligomeric chitin by using procambarus clarkii shells as matrix

Abstract

The invention discloses a method for extracting chitin from shrimp shells of procambarus clarkia and decomposing the chitin into oligomeric chitin and taking a byproduct as an organic fertilizer. The method comprises the following steps: (1) calcium element removal: mixing procambarus clarkii shells with an acetic acid solution, fully reacting, carrying out solid-liquid separation, and retaining a solid; (2) protein removal: adding protease into the solid obtained in the step (1), carrying out solid-liquid separation after full reaction, and retaining the solid; and (3) preparing oligomeric chitin, namely reacting the shrimp shells in the steps (1) and (2) to obtain chitin, and decomposing the chitin by using chitinase. The method has the beneficial effects that acidic or alkaline waste liquid is not generated in the process of obtaining the chitin extraction byproducts by adopting the method, so that the method is more environment-friendly, and the yield of the prepared oligomeric chitin is higher.

Description

Technical Field

[0001] The invention relates to the technical field of extracting chitin and decomposing it into oligochitoxin, and in particular to a method for preparing oligochitoxin by taking Procambarus clarkii shells as a matrix. Background Art

[0002] Procambarus clarkii (commonly known as crayfish) is a widely cultivated economic aquatic product with promising aquaculture prospects due to its strong adaptability, high reproductive capacity, low disease risk, wide-ranging diet, rapid growth, and tolerance to high temperatures and low oxygen levels. In recent years, with the booming development of the Procambarus clarkii industry, its processing has also gradually gained attention. However, current processing of Procambarus clarkii is mostly primary, with a large amount of shrimp heads and shells as by-products. These by-products contain a wealth of useful components, such as chitin, calcium ions, and protein, which can be used in industrial production, medical materials, food processing, and other fields.

[0003] Chitin, a natural polymer widely found in the shells of shrimp and crabs, is chemically known as β-(1,4)-2-acetylamino-2-deoxy-D-glucose. Its excellent biocompatibility, biodegradability, and antimicrobial properties make it valuable for applications in a variety of fields, including food, medicine, agriculture, and textiles. Chitin is abundant in the shells of Procambarus clarkii, reaching levels of 20% to 30% in dried shells. However, current industrial methods for extracting chitin from Procambarus clarkii shells primarily use strong acids and alkalis, which pose significant environmental risks.

[0004] Furthermore, chitin can be converted into chitosan after deacetylation, which has higher biological activity and wider application prospects. Oligochitosan is a chitin oligomer with higher biological activity and solubility, and has higher economic value in the fields of medicine, cosmetics, and food additives.

[0005] Although Procambarus clarkii shells are rich in chitin and other usable components, these byproducts are currently largely underutilized. Direct disposal not only wastes resources but also pollutes the environment. Therefore, developing an efficient and environmentally friendly method for processing Procambarus clarkii shells that can not only extract high-value chitin and oligochitosan but also convert the byproducts into organic fertilizer, achieving comprehensive resource utilization and environmentally friendly production, is of great economic and ecological significance.

[0006] Chinese patent application publication number CN111647097A discloses a method for extracting chitosan from discarded shrimp shells. The method comprises first deproteinizing shrimp shell powder using a protease to obtain a protein hydrolyzate, then decalcifying the deproteinized shrimp shell powder using dilute hydrochloric acid to obtain crude chitosan, then adding a sodium acetate solution to the decalcified solution to obtain solid calcium acetate and a sodium chloride solution, and then concentrating the sodium chloride solution to a saturated solution and salting out the protein hydrolyzate to obtain solid protein. Finally, the crude chitosan is decolorized using ethanol and distilled to obtain chitosan of higher purity. The chitosan extraction rate of this patent is low, and oligomeric chitosan is not produced, so further improvement is needed. Summary of the Invention

[0007] The technical problem to be solved by the present invention is how to provide a green and low-cost method for preparing oligochitosan, while being able to use the extracted by-products such as calcium ions and proteins as organic fertilizers.

[0008] The present invention solves the above technical problems through the following technical means:

[0009] The present invention provides a method for preparing oligochitoxin using Procambarus clarkii shells as a matrix, comprising the following steps:

[0010] (1) Removing calcium: mixing the crayfish shells with acetic acid solution, allowing the mixture to react fully, and then separating the solid and liquid to retain the solid;

[0011] (2) Protein removal: adding protease to the solid obtained in (1), and after sufficient reaction, separating the solid and the liquid, and retaining the solid;

[0012] (3) Preparation of oligochitosan: Add [C2mim][OAc] (1-ethyl-3-methylimidazolium acetate) solution to the solid obtained in (2), heat it, cool it in an ice bath, and then add it to the chitinase solution for reaction.

[0013] Preferably, in step (1), the concentration of the acetic acid solution is 0.4 to 0.6 mol / L, more preferably 0.4 to 0.45 mol / L.

[0014] Preferably, in step (1), the ratio of Procambarus clarkii shell to acetic acid solution is 1 g: (6-10) mL, more preferably 1 g: (8-10) mL.

[0015] Preferably, in step (1), the reaction temperature is 30-60° C., and the reaction time is 7-11 h.

[0016] Preferably, in step (2), the reaction temperature is 45-50° C., the reaction time is 16-20 h, the reaction pH is 7-7.5, and the ratio of protease to substrate is 8-12 u / mg.

[0017] Preferably, in step (3), the concentration of the [C2mim][OAc] (1-ethyl-3-methylimidazolium acetate) solution is 5-20% (v / v).

[0018] Preferably, in step (3), the heating treatment temperature is 90 to 120° C. and the time is 30 to 60 minutes.

[0019] Preferably, in step (3), the reaction temperature is 40-55° C., the reaction time ranges from 12 h to 24 h, and the reaction pH value is 6.

[0020] The beneficial effects of the present invention are:

[0021] 1. The present invention uses organic weak acid and protease to extract chitin. Compared with the traditional strong acid and strong alkali process, this method is more environmentally friendly and maintains the demineralization rate and deproteinization rate at a high level.

[0022] 2. The present invention uses [C2mim][OAc] to pretreat chitin. [C2mim][OAc] can destroy the crystal structure of chitin, causing chitin to expand from a granular state to a lamellar structure, thereby allowing chitinase to contact chitin more fully and improving the enzymatic hydrolysis efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the infrared spectrum of the oligomeric chitosan obtained in Example 6 of the present invention. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Unless otherwise defined, the professional terms used below have the same meaning as those understood by professional and technical personnel in this field.

[0025] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources or prepared by known methods.

[0026] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the quantitative tests in the following examples were repeated three times and the results were averaged.

[0027] The neutral protease used in the following examples was purchased from Shanghai Yuanye Biotechnology Co., Ltd., model: Yuanye S10013.

[0028] Example 1:

[0029] The method for extracting chitin from Procambarus clarkii shells comprises the following steps:

[0030] (1) Removal of calcium: Acetic acid was prepared into a solution with a molar concentration of 0.4 mol / L, and then added to the crayfish shells at a ratio of 1 g:10 mL. The reaction temperature was set at 30° C. After reacting for 11 hours, solid-liquid separation was performed to obtain a filtrate 1 containing calcium ions and the crayfish shells from which the calcium ions were removed.

[0031] (2) Protein Removal: Using a pH 7.5 buffer solution, neutral protease (Source Leaf S10013) and the Procambarus clarkii shells obtained in step (1) were added to the buffer solution at a ratio of enzyme to substrate of 8 u / mg. The reaction temperature was set at 45°C. After 16 hours of reaction, solid-liquid separation was performed to obtain filtrate 2 containing amino acids and chitin solid.

[0032] In this example, the calcium and protein extraction rates from Procambarus clarkii shells were 83.33% for calcium and 93.58% for protein. The higher the extraction rate, the less calcium and protein impurities there are, the looser the chitin structure, and the more efficient the subsequent enzymatic hydrolysis of chitin into oligomeric chitin.

[0033] Example 2:

[0034] The difference between this embodiment and embodiment 1 is that:

[0035] Change the reaction temperature in step (1) from 30°C to 45°C.

[0036] The enzyme amount: substrate amount in step (2) was changed to "9u / mg", and the rest was the same as in Example 1.

[0037] In this embodiment, the extraction rates of calcium and protein extracted from the shell of Procambarus clarkii are: the calcium extraction rate is 85.24%, and the protein extraction rate is 95.13%.

[0038] Example 3:

[0039] The difference between this embodiment and embodiment 1 is that:

[0040] The concentration of acetic acid solution in step (1) was changed from 0.4 mol / L to 0.45 mol / L; the dosage ratio was changed from 1 g:10 mL to 1 g:8.88 mL; the reaction temperature was changed from 30 ° C to 50 ° C,

[0041] The enzyme amount in step (2): substrate amount = 8u / mg was changed to "10u / mg", and the reaction temperature was changed from 45°C to "50°C". The rest was the same as in Example 1.

[0042] In this embodiment, the extraction rates of calcium and protein extracted from the shell of Procambarus clarkii are: the calcium extraction rate is 85.67%, and the protein extraction rate is 96%.

[0043] Example 4:

[0044] The difference between this embodiment and embodiment 3 is that:

[0045] The concentration of acetic acid solution in step (1) was changed from 0.4 mol / L to 0.45 mol / L; the dosage ratio was changed from 1 g:10 mL to 1 g:8.88 mL; the reaction temperature was changed from 50 ° C to 60 ° C,

[0046] The enzyme amount: substrate amount in step (2) was changed to "11u / mg", and the rest was the same as in Example 1.

[0047] In this embodiment, the extraction rates of calcium and protein extracted from the shell of Procambarus clarkii are: the calcium extraction rate is 86.43%, and the protein extraction rate is 98.43%.

[0048] Example 5:

[0049] The difference between this embodiment and embodiment 4 is that:

[0050] The enzyme amount: substrate amount in step (2) was changed to "12u / mg", and the rest was the same as in Example 4.

[0051] In this embodiment, the extraction rates of calcium and protein extracted from the shell of Procambarus clarkii are: the calcium extraction rate is 86.67%, and the protein extraction rate is 99.56%.

[0052] Comparative Example 1:

[0053] The difference between this comparative example and Example 1 is:

[0054] Change the reaction temperature of 30°C in step (1) to "70°C",

[0055] The pH value in step (2) was changed from 7.5 to 6.5, and the reaction temperature from 45°C to 55°C. The rest was the same as in Example 1.

[0056] In this comparative example, the extraction rates of calcium and protein extracted from the shell of Procambarus clarkii were: the calcium extraction rate was 78.1%, and the protein extraction rate was 66%.

[0057] Table 1 Comparison of calcium extraction rate and protein extraction rate under different reaction conditions

[0058]

[0059] As can be seen from Table 1, Example 5 has the highest calcium extraction rate and protein extraction rate.

[0060] Example 6:

[0061] The preparation method of oligomeric chitosan comprises the following steps:

[0062] The chitin obtained in step (2) of Example 5 was used as a raw material and pretreated in a 5% (v / v) [C2mim][OAc] solution at 110°C for 40 minutes. The treated chitin was then cooled in an ice bath. The pretreated chitin was added to a chitinase solution and reacted at 40°C and pH 6 for 24 hours.

[0063] The obtained product was characterized by Figure 1 Infrared spectrum visible: 1615.18 and 1582.66cm -1 The corresponding band is the amide I band of N-acetylglucosamine; 1537.2 cm -1 Amide II bands; 3280.06 and 3093.07 cm -1 The characteristic absorption peaks of OH and NH are shown in Figure 2. The yield of N-acetylglucosamine and N,N'-diacetylchitobiose were 82.26 mg / g and 558.4 mg / g, respectively, determined by HPLC.

[0064] Example 7:

[0065] The difference between this embodiment and embodiment 6 is that:

[0066] The concentration of [C2mim][OAc] solution was changed from 5% (v / v) to "10% (v / v)", and the rest was the same as in Example 6.

[0067] In this example, the yield of N-acetylglucosamine was determined to be 73.38 mg / g, and the yield of N,N'-diacetylchitobiose was determined to be 570.16 mg / g by HPLC.

[0068] Example 8:

[0069] The difference between this embodiment and embodiment 6 is that:

[0070] The concentration of [C2mim][OAc] solution was changed from 5% (v / v) to "15% (v / v)", and the rest was the same as in Example 6.

[0071] In this example, the yield of N-acetylglucosamine was measured by HPLC to be 59.69 mg / g, and the yield of N,N'-diacetylchitobiose was 516.33 mg / g.

[0072] Example 9:

[0073] The difference between this embodiment and embodiment 6 is that:

[0074] The concentration of [C2mim][OAc] solution was changed from 5% (v / v) to "20% (v / v)", and the rest was the same as in Example 6.

[0075] In this example, the yield of N-acetylglucosamine was measured by HPLC to be 42.84 mg / g, and the yield of N,N'-diacetylchitobiose was 450.7 mg / g.

[0076] Comparative Example 2:

[0077] The difference between this comparative example and Example 6 is:

[0078] The concentration of [C2mim][OAc] solution was changed from 5% (v / v) to "30% (v / v)", and the rest was the same as in Example 6.

[0079] In this comparative example, the yield of N-acetylglucosamine was determined to be 9.97 mg / g, and the yield of N,N'-diacetylchitobiose was determined to be 86.72 mg / g by HPLC.

[0080] Table 2 Comparison of oligochitosan yields under different reaction conditions

[0081]

[0082] As shown in Table 2, the total amount of oligomeric chitosan obtained in Example 7 is the highest.

[0083] Example 10:

[0084] A method for preparing oligochitoxin using Procambarus clarkii shells as a matrix comprises the following steps:

[0085] (1) Removing calcium: Acetic acid was prepared into a solution with a molar concentration of 0.6 mol / L, and then added to the crayfish shells at a ratio of 1 g:6 mL. The reaction temperature was set at 60° C., and after reacting for 7 hours, solid-liquid separation was performed to obtain a filtrate 1 containing calcium ions and the crayfish shells from which the calcium ions were removed;

[0086] (2) Protein removal: Using a buffer solution with a pH of 7, neutral protease and the Procambarus clarkii shell obtained in step (1) were added to the buffer solution at a ratio of enzyme amount to substrate amount = 10 u / mg, and the reaction temperature was set to 48°C. After reacting for 20 hours, solid-liquid separation was performed to obtain filtrate 2 containing amino acids and chitin solid;

[0087] (3) Preparation of oligomeric chitosan: The chitosan solid obtained in (2) was added to an 8% (v / v) [C2mim][OAc] solution and pretreated at 90°C for 60 minutes. The treated chitosan was then cooled in an ice bath. The pretreated chitosan was added to a chitinase solution and reacted at 55°C and pH 6 for 12 hours to obtain oligomeric chitosan.

[0088] Example 11:

[0089] A method for preparing oligochitoxin using Procambarus clarkii shells as a matrix comprises the following steps:

[0090] (1) Removing calcium: Acetic acid was prepared into a solution with a molar concentration of 0.5 mol / L, and then added to the crayfish shells at a ratio of 1 g:8 mL. The reaction temperature was set at 45° C., and after reacting for 8.5 hours, solid-liquid separation was performed to obtain a filtrate 1 containing calcium ions and the crayfish shells from which the calcium ions had been removed;

[0091] (2) Protein removal: Using a buffer solution with a pH of 7.2, neutral protease and the Procambarus clarkii shell obtained in step (1) were added to the buffer solution at a ratio of enzyme amount to substrate amount = 10 u / mg, and the reaction temperature was set to 49°C. After reacting for 18 hours, solid-liquid separation was performed to obtain filtrate 2 containing amino acids and chitin solid;

[0092] (3) Preparation of oligomeric chitosan: The chitosan solid obtained in (2) was added to an 8% (v / v) [C2mim][OAc] solution and pretreated at 120°C for 30 minutes. The treated chitosan was then cooled in an ice bath. The pretreated chitosan was added to a chitinase solution and reacted at 45°C and pH 6 for 16 hours to obtain oligomeric chitosan.

[0093] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for preparing oligochitoxin using Procambarus clarkii shells as a matrix, characterized in that: The following steps are involved: (1) Removing calcium: mixing the crayfish shells with acetic acid solution, allowing the mixture to react fully, and then separating the solid and liquid to retain the solid; (2) Protein removal: adding protease to the solid obtained in (1), and after sufficient reaction, separating the solid and the liquid, and retaining the solid; (3) Preparation of oligochitosan: Add 1-ethyl-3-methylimidazole acetate solution to the solid obtained in (2), heat it, cool it in an ice bath, and then add it to the chitinase solution for reaction to obtain oligochitosan.

2. The method for preparing oligochitoxin using Procambarus clarkii shells as a matrix according to claim 1, characterized in that: In step (1), the concentration of the acetic acid solution is 0.4-0.6 mol / L.

3. The method for preparing oligochitoxin using Procambarus clarkii shells as a matrix according to claim 1, characterized in that: In step (1), the ratio of the Procambarus clarkii shell to the acetic acid solution is 1 g: (6-10) mL.

4. The method for preparing oligochitoxin using Procambarus clarkii shells as a matrix according to claim 1, characterized in that: In step (1), the reaction temperature is 30-60° C., and the reaction time is 7-11 h.

5. The method for preparing oligochitoxin using Procambarus clarkii shells as a matrix according to claim 1, characterized in that: In step (2), the reaction temperature is 45-50° C., and the reaction time is 16-20 h.

6. The method for preparing oligochitoxin using Procambarus clarkii shells as a matrix according to claim 1, characterized in that: In step (2), the pH value of the reaction is 7 to 7.

5.

7. The method for preparing oligochitoxin using Procambarus clarkii shells as a matrix according to claim 1, characterized in that: In step (2), the ratio of protease to substrate is 8 to 12 u / mg.

8. The method for preparing oligochitoxin using Procambarus clarkii shells as a matrix according to claim 1, characterized in that: In step (3), the concentration of the 1-ethyl-3-methylimidazolium acetate solution is 5 to 20% (v / v).

9. The method for preparing oligochitoxin using Procambarus clarkii shells as a matrix according to claim 1, characterized in that: In step (3), the heating treatment temperature is 90 to 120° C. and the time is 30 to 60 minutes.

10. The method for preparing oligochitoxin using Procambarus clarkii shell as a matrix according to claim 1, characterized in that: In step (3), the reaction temperature is 40-55° C., the reaction time range is 12 h to 24 h, and the reaction pH value is 6.

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