Method for catalyzing Knoevenagel condensation reaction through tilapia mossambica scale collagen

Abstract

The invention provides a method for catalyzing a Knoevenagel condensation reaction by using tilapia scale collagen, which is characterized in that collagen in tilapia scale is selectively extracted and separated by a hot solvent extraction method, and is used for catalyzing the Knoevenagel condensation reaction between an aromatic aldehyde compound and an active methylene compound. According to the method, the tilapia mossambica scale collagen is used as a green catalyst for the Knoevenagel condensation reaction to prepare the high-added-value organic platform chemicals, a catalyst system for the Knoevenagel condensation reaction is enriched, the yield of a target product can reach 90% or above, and the method has good substrate universality.

Description

Technical Field

[0001] The present invention belongs to the field of resource utilization of fishery by - products, and particularly relates to a method for catalyzing the Knoevenagel condensation reaction with tilapia scale collagen. Background Technique

[0002] The tilapia industry is an important pillar of the agricultural economy in South China and has become an important supporting industry in the local area. At present, the tilapia aquaculture area reaches more than 260,000 mu, with a yield close to 8 million tons, accounting for about 50% of the national tilapia production. It is the core advantageous production area of the world tilapia industry, with an output value reaching tens of billions of yuan and hundreds of thousands of job positions. In recent years, after years of exploration and efforts, the deep - processing industry in the tilapia industry in South China has developed rapidly and occupied the high point of the national tilapia deep - processing industry. For example, in 2023, there are 15 tilapia processing and export enterprises in Maoming City, Guangdong Province, with an annual processing and export capacity of nearly 480,000 tons. They mainly export through frozen whole fish or slices to more than 20 countries and regions such as the United States, Russia, and Canada. The export volume is 99,000 tons, and the value of goods is 2.197 billion yuan, achieving a historic leap from a large aquaculture city to a large export city. However, in the process of tilapia deep - processing, tens of thousands of tons of waste such as fish scales (fish scales account for about 3% of the fresh fish quality) are still inevitably generated. If disposed of randomly, it will not only pollute the environment but also cause waste of resources and reduce the overall economic benefits of the tilapia industry. Therefore, how to efficiently utilize tilapia scales and other by - products has become an important problem faced by the green and healthy development of the tilapia industry in South China.

[0003] Fish scales are bone derivatives formed after the evolution of collagen in the dermal layer of fish. Studies have found that fish scales are rich in collagen (36 - 50%) and hydroxyapatite (HAP, 35 - 43%). Collagen is a white, opaque, unbranched fibrous protein (Formula 1), with a molecular weight of 300,000 daltons. Fish scales can be used as raw materials for collagen extraction, and the collagen in fish scales has a similar structure to that of livestock collagen and can be used to replace livestock - sourced collagen to solve potential food safety problems brought by livestock - sourced collagen such as foot - and - mouth disease and mad cow disease, meeting people's needs. At present, it has become an important substitute for livestock - sourced collagen. However, as a food - grade fish scale collagen, the quality requirements are relatively high, and the preparation process is complex, resulting in the processing capacity of fish scale by - products not meeting the actual production needs, and the processing volume is much lower than the output volume. Therefore, it is urgent to develop application technologies for low - quality fish scale collagen.

[0004]

[0005] It can be seen from the molecular structural formula of fish scale collagen (Formula 1) that it contains a weakly basic amino group (-NH 2) and imino (-NH-) functional groups can be used as effective weak base catalysts to catalyze the Knoevenagel condensation reaction between aromatic aldehydes and active methylene compounds (Equation 2), and find applications in the field of organic synthesis. Therefore, by means of a simple and effective separation method, the collagen in tilapia scales is extracted and separated, and applied to the field of catalytic organic synthesis, which will further broaden the application scope of tilapia by-products, increase the added value of the fishery in South China and even the whole country, and promote the green and healthy development of our fishery resources.

[0006] Summary of the Invention

[0007] Aiming at the above deficiencies in the prior art, the purpose of the present invention is to provide a method for catalyzing the Knoevenagel condensation reaction with tilapia scale collagen. The present invention selectively separates the collagen in tilapia scales by means of hot solvent extraction, and uses it as a catalyst to catalyze the Knoevenagel condensation reaction to prepare high-value-added organic platform chemicals, thereby improving the high-value utilization method of tilapia by-products.

[0008] To achieve the above purpose, the present invention provides the following technical solution: A method for catalyzing the Knoevenagel condensation reaction with tilapia scale collagen, selectively extracting and separating the collagen in tilapia scales by hot solvent extraction method, and using it to catalyze the Knoevenagel condensation reaction between aromatic aldehydes and active methylene compounds.

[0009] Further, it specifically includes the following steps:

[0010] (1) Mix tilapia scale powder with a mesh size of 40 - 60 with a solvent, and carry out extraction at 40 - 100 °C for 2.0 - 8.0 h;

[0011] (2) After the extraction is completed, filter while it is hot to remove the solid residue. Drop the filtrate into absolute ethanol, and the dissolved collagen in the extract rapidly precipitates in the form of white flocs. Filter and wash the flocs with absolute ethanol 3 times, dry them in a vacuum drying oven at 60 °C for 24 h, and then obtain tilapia scale collagen. Finally, use a traditional Chinese medicine grinder to crush it to 40 - 60 mesh to obtain tilapia scale collagen powder catalyst, and store it for later use;

[0012] (3) Add 0.01 - 1.0 g of tilapia scale collagen powder catalyst and aromatic aldehyde and active methylene compound with a molar ratio of 1:1.2 to an ethanol solution with a concentration of 50% - 95% for Knoevenagel condensation reaction, and the reaction temperature is 25 - 75 °C.

[0013] Further, in step (1), the solvent is water or a deep eutectic solvent containing a proton acceptor and a proton donor.

[0014] Further, in step (1), the mass-to-volume ratio of tilapia scale powder to the solvent is (5 - 30) g: 100 mL.

[0015] Further, the aromatic aldehyde compound is furfural or a benzaldehyde derivative with substituents.

[0016] Further, in step (2), the collagen in the extract is separated and purified by the anti-solvent method.

[0017] Further, the anti-solvent method refers to a method of separating and purifying the collagen in the extract by mixing an anti-solvent with the tilapia scale collagen extract and taking advantage of the difference in their solubility.

[0018] Further, in step (3), the mass-to-volume ratio of tilapia scale collagen powder catalyst to the ethanol solution is (0.1 - 20) g: 100 mL.

[0019] Further, in step (3), the active methylene compound is malononitrile containing a strong electron-withdrawing group.

[0020] Further, the temperature of the Knoevenagel condensation reaction is 25 °C.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The present invention selectively separates the collagen in tilapia scales by hot solvent extraction and uses it as a catalyst to catalyze the Knoevenagel condensation reaction, expanding the application field of tilapia by-products, enhancing the added value of the tilapia industry, and promoting its green and healthy development.

[0023] 2. The present invention uses tilapia scale collagen as a green catalyst for the Knoevenagel condensation reaction to prepare high-value-added organic platform chemicals, enriching the catalyst system for the Knoevenagel condensation reaction. The yield of the target product can reach over 90%, and it has good substrate generality. Description of the Drawings

[0024] Figure 1 is a photo of tilapia scales;

[0025] Figure 2 is the tilapia scale collagen powder extracted with water as the solvent in Example 1;

[0026] Figure 3 is the Fourier transform infrared spectrum of the scale collagen extracted in Example 1;

[0027] Figure 4 Photograph of the representative Knoevenagel condensation reaction product for Example 1

[0028] Figure 5 1H NMR spectrum of the representative Knoevenagel condensation reaction product for Example 1 Detailed implementation manners

[0029] The method of the present invention will be described in detail below in conjunction with specific embodiments. All tilapia scales used in the present invention are from a tilapia deep processing aquatic products company in South China. After separating the tilapia scales from other scraps and washing them clean, they are naturally air-dried ( Figure 1 ), and then the dried tilapia scales are pulverized to 40-60 mesh with a traditional Chinese medicine pulverizer to obtain tilapia scale powder, which is stored for later use.

[0030] I. Method for catalyzing Knoevenagel condensation reaction with tilapia scale collagen

[0031] Example 1

[0032] A method for catalyzing Knoevenagel condensation reaction with tilapia scale collagen comprises the following steps:

[0033] (1) Add 300 mL of deionized water and 30 g of tilapia scale powder to a 500 mL round-bottom flask, stir magnetically, heat in an oil bath, and extract at 100 °C for 5.0 h. After the extraction is completed, filter while it is hot, collect the filtrate, and cool it to room temperature. The cooled tilapia scale collagen solution is viscous;

[0034] (2) Drop the cooled filtrate into 500 mL of anhydrous ethanol anti-solvent. The tilapia scale collagen rapidly precipitates in the form of flocs and stands for half an hour; after the collagen is completely precipitated, filter, wash it 3 times with anhydrous ethanol, and then transfer the solid to a vacuum drying oven and dry it in a 60 °C vacuum drying oven for 24 h to obtain 9.9 g of tilapia scale collagen with an extraction rate of 33%. Use a traditional Chinese medicine pulverizer to pulverize it to 40-60 mesh to obtain tilapia scale collagen powder catalyst ( Figure 2 ), which is stored for later use;

[0035] At the same time, Fourier transform infrared spectroscopy (FT-IR) is used to characterize its molecular structure ( Figure 3 ). Figure 3 The broad peak at a wavenumber of 3428.65 cm -1 is the characteristic peak corresponding to the stretching vibration of the N-H bond in collagen (association hydrogen bond of amino group and imino group); 1667.21 cm -1The peak at [specific position] is caused by the stretching vibration of C=O (amide I band) characteristic peak; 1555.26 cm -1 The peak at [specific position] is the characteristic peak of the planar twisting and folding of N-H (amide II band), and overlaps with the characteristic peak of C-N stretching vibration; 1408.16 cm -1 Characteristic absorption peak of collagen carboxyl residue at [specific position]. FT-IR results show that the hot solvent method can extract the corresponding collagen from tilapia scales.

[0036] (3) Add 0.05 g of catalyst (tilapia scale collagen powder), 2.5 mmol of benzaldehyde, 3.0 mmol of malononitrile, and 10 mL of 95% ethanol to a 25 mL round-bottom flask, and react at room temperature (25 °C) (magnetic stirring, 200 rpm). Use thin-layer chromatography (TLC) to monitor the reaction progress (the developing agent is a mixture of petroleum ether and ethyl acetate, and the volume ratio of the two is 5:1).

[0037] (4) After the reaction is completed, filter the catalyst and wash it 3 times with 95% ethanol (5 mL each time), and combine the filtrate;

[0038] (5) Transfer the combined filtrate (~25 mL) to a 50 mL round-bottom flask. After recovering ethanol by a rotary evaporator, the product is enriched in the residual aqueous phase of the round-bottom flask in crystalline form;

[0039] (6) After cooling, filter the product and wash it 3 times with deionized water (5 mL each time, the raw materials are all soluble in water). Transfer the product to a vacuum drying oven at 50 °C and dry it for 24 h to obtain the crystalline target product ( Figure 4 ), and its structure is identified by nuclear magnetic resonance hydrogen spectrum ( 1 HNMR) ( Figure 5 ). In Figure 5 ( 1 H NMR, ppm, CDCl 3 is the solvent), the singlet at δ = 7.81 is the hydrogen on the 1st position of the double bond (CCH=), the doublet at δ = 7.92 - 7.93 is the hydrogen on the 2nd position of the benzene ring (CCH=), the triplet at δ = 7.55 - 7.59 is the hydrogen on the 3rd position of the benzene ring (CCH=), the triplet at δ = 7.64 - 7.68 is the hydrogen on the 4th position of the benzene ring (CCH=), and the area ratio of each peak is 1:2:2:1, indicating that the prepared product is consistent with the target product.

[0040] Example 2

[0041] (1) Add 300 mL of deionized water and 90 g of tilapia scale powder to a 500 mL round-bottom flask, stir magnetically, heat in an oil bath, and extract for 8.0 h at 90 °C. After the extraction is completed, filter while it is hot, collect the filtrate, and cool it to room temperature. The cooled tilapia scale collagen solution is viscous.

[0042] (2) The regeneration and separation process of tilapia scale collagen is the same as that in Example 1, and the extraction rate of tilapia scale collagen is 18%.

[0043] (3) The raw materials and experimental procedures for the Knoevenagel condensation reaction catalyzed by the tilapia scale collagen powder extracted under this condition are the same as those in Example 1.

[0044] Example 3

[0045] (1) Add 300 mL of deionized water and 15 g of tilapia scale powder to a 500 mL round-bottom flask, stir magnetically, heat in an oil bath, and extract for 2.0 h at 80 °C. After the extraction is completed, filter while it is hot, collect the filtrate, and cool it to room temperature. The cooled tilapia scale collagen solution is viscous.

[0046] (2) The regeneration and separation process of tilapia scale collagen is the same as that in Example 1, and the extraction rate of tilapia scale collagen is 13%.

[0047] (3) The raw materials and experimental procedures for the Knoevenagel condensation reaction catalyzed by the tilapia scale collagen powder extracted under this condition are the same as those in Example 1.

[0048] Example 4

[0049] (1) Mix 0.1 mol of tetraethylammonium chloride and 0.5 mol of ethylene glycol in a 100 mL round-bottom flask, and heat at 50 °C for 24 h to prepare a deep eutectic solvent with tetraethylammonium chloride as the proton acceptor and ethylene glycol as the proton donor.

[0050] (2) Add 2.0 g of tilapia scale powder and 20 mL of the "tetraethylammonium chloride - ethylene glycol" deep eutectic solvent to a 50 mL round-bottom flask, and react in an oil bath at 40 °C for 8.0 h. After the reaction is completed, filter while it is hot. The tilapia scale collagen dissolved in the filtrate is regenerated, separated, and purified by the ethanol antisolvent method. The subsequent treatment method is the same as that in Example 1. The extraction rate of tilapia scale collagen is 12%.

[0051] (3) The raw materials and experimental procedures for the Knoevenagel condensation reaction catalyzed by the tilapia scale collagen powder extracted under this condition are the same as those in Example 1.

[0052] Example 5

[0053] (1) Mix 0.1 mol of 1-butyl-3-methylimidazolium chloride with 0.5 mol of ethylene glycol in a 100 mL round-bottom flask, and heat at 50 °C for 24 h to prepare a deep eutectic solvent with 1-butyl-3-methylimidazolium chloride as the proton acceptor and ethylene glycol as the proton donor.

[0054] (2) Add 2.0 g of tilapia scale powder and 20 mL of the "1-butyl-3-methylimidazolium chloride - ethylene glycol" deep eutectic solvent to a 50 mL round-bottom flask, and react in an oil bath at 40 °C for 8.0 h. After the reaction, filter while it is hot. The tilapia scale collagen dissolved in the filtrate is regenerated, separated, and purified by the ethanol anti-solvent method. The subsequent treatment method is the same as that in Example 1. The extraction rate of tilapia scale collagen is 15%.

[0055] (3) The raw materials and experimental procedures for the Knoevenagel condensation reaction catalyzed by the tilapia scale collagen powder extracted under this condition are the same as those in Example 1.

[0056] Example 6

[0057] (1) Mix 0.1 mol of choline bromide with 0.5 mol of ethylene glycol in a 100 mL round-bottom flask, and heat at 50 °C for 24 h to prepare a deep eutectic solvent with choline bromide as the proton acceptor and ethylene glycol as the proton donor.

[0058] (2) Add 2.0 g of tilapia scale powder and 20 mL of the "choline bromide - ethylene glycol" deep eutectic solvent to a 50 mL round-bottom flask, and react in an oil bath at 40 °C for 8.0 h. After the reaction, filter while it is hot. The tilapia scale collagen dissolved in the filtrate is regenerated, separated, and purified by the ethanol anti-solvent method. The subsequent treatment method is the same as that in Example 1. The extraction rate of tilapia scale collagen is 18%.

[0059] (3) The raw materials and experimental procedures for the Knoevenagel condensation reaction catalyzed by the tilapia scale collagen powder extracted under this condition are the same as those in Example 1.

[0060] Example 7

[0061] (1) Mix 0.1 mol of choline chloride with 0.5 mol of ethylene glycol in a 100 mL round-bottom flask, and heat at 50 °C for 24 h to prepare a deep eutectic solvent with choline chloride as the proton acceptor and ethylene glycol as the proton donor.

[0062] (2) Add 2.0 g of tilapia scale powder and 20 mL of the "choline chloride - ethylene glycol" eutectic solvent into a 50 mL round - bottom flask, and react in an oil bath at 40 °C for 8.0 h. After the reaction, perform hot filtration while it is hot. The tilapia scale collagen dissolved in the filtrate is regenerated, separated, and purified by the ethanol antisolvent method. The subsequent treatment method is the same as that in Example 1. The extraction rate of tilapia scale collagen is 20%.

[0063] (3) The raw materials and experimental procedures for the Knoevenagel condensation reaction catalyzed by the tilapia scale collagen powder extracted under this condition are the same as those in Example 1.

[0064] Example 8

[0065] (1) Mix 0.1 mol of betaine and 0.5 mol of ethylene glycol in a 100 mL round - bottom flask, and heat at 50 °C for 24 h to prepare a eutectic solvent with betaine as the proton acceptor and ethylene glycol as the proton donor.

[0066] (2) Add 2.0 g of tilapia scale powder and 20 mL of the "betaine - ethylene glycol" eutectic solvent into a 50 mL round - bottom flask, and react in an oil bath at 40 °C for 8.0 h. After the reaction, perform hot filtration while it is hot. The tilapia scale collagen dissolved in the filtrate is regenerated, separated, and purified by the ethanol antisolvent method. The subsequent treatment method is the same as that in Example 1. The extraction rate of tilapia scale collagen is 15%.

[0067] (3) The raw materials and experimental procedures for the Knoevenagel condensation reaction catalyzed by the tilapia scale collagen powder extracted under this condition are the same as those in Example 1.

[0068] Example 9

[0069] (1) Mix 0.1 mol of trimethylamine hydrochloride and 0.5 mol of 1,2 - propanediol in a 100 mL round - bottom flask, and heat at 50 °C for 24 h to prepare a eutectic solvent with trimethylamine hydrochloride as the proton acceptor and 1,2 - propanediol as the proton donor.

[0070] (2) Add 2.0 g of tilapia scale powder and 20 mL of the "trimethylamine hydrochloride - 1,2 - propanediol" eutectic solvent into a 50 mL round - bottom flask, and react in an oil bath at 40 °C for 8.0 h. After the reaction, perform hot filtration while it is hot. The tilapia scale collagen dissolved in the filtrate is regenerated, separated, and purified by the ethanol antisolvent method. The subsequent treatment method is the same as that in Example 1. The extraction rate of tilapia scale collagen is 18%.

[0071] (3) The raw materials and experimental procedures for the Knoevenagel condensation reaction catalyzed by the tilapia scale collagen powder extracted under this condition are the same as those in Example 1.

[0072] Example 10

[0073] (1) Mix 0.1 mol of betaine with 0.5 mol of 1,3 - propanediol in a 100 mL round - bottom flask and heat at 50 °C for 24 h to prepare a deep - eutectic solvent with betaine as the proton acceptor and 1,3 - propanediol as the proton donor.

[0074] (2) Add 2.0 g of tilapia scale powder and 20 mL of the "betaine - 1,3 - propanediol = 1:5" deep - eutectic solvent to a 50 mL round - bottom flask and react in an oil bath at 40 °C for 8.0 h. After the reaction, filter while it is hot. The tilapia scale collagen dissolved in the filtrate is regenerated, separated, and purified by the ethanol antisolvent method. The subsequent treatment method is the same as that in Example 1. The extraction rate of tilapia scale collagen is 22%.

[0075] (3) The raw materials and experimental procedures for the Knoevenagel condensation reaction catalyzed by the tilapia scale collagen powder extracted under this condition are the same as those in Example 1.

[0076] Example 11

[0077] (1) Mix 0.1 mol of choline chloride with 0.5 mol of 1,2 - propanediol in a 100 mL round - bottom flask and heat at 50 °C for 24 h to prepare a deep - eutectic solvent with choline chloride as the proton acceptor and 1,2 - propanediol as the proton donor.

[0078] (2) Add 2.0 g of tilapia scale powder and 20 mL of the "choline chloride - 1,2 - propanediol" deep - eutectic solvent to a 50 mL round - bottom flask and react in an oil bath at 40 °C for 8.0 h. After the reaction, filter while it is hot. The tilapia scale collagen dissolved in the filtrate is regenerated, separated, and purified by the ethanol antisolvent method. The subsequent treatment method is the same as that in Example 1. The extraction rate of tilapia scale collagen is 21%.

[0079] (3) The raw materials and experimental procedures for the Knoevenagel condensation reaction catalyzed by the tilapia scale collagen powder extracted under this condition are the same as those in Example 1.

[0080] Example 12

[0081] (1) Mix 0.1 mol of betaine with 0.5 mol of urea in a 100 mL round - bottom flask and heat at 50 °C for 24 h to prepare a deep - eutectic solvent with betaine as the proton acceptor and urea as the proton donor.

[0082] (2) Add 2.0 g of tilapia scale powder and 20 mL of the "betaine-urea" eutectic solvent to a 50 mL round-bottom flask, and react in an oil bath at 40 °C for 8.0 h. After the reaction, perform suction filtration while it is hot. The tilapia scale collagen dissolved in the filtrate is regenerated, separated, and purified by the ethanol anti-solvent method. The subsequent treatment method is the same as that in Example 1. The extraction rate of tilapia scale collagen is 19%.

[0083] (3) The raw materials and experimental procedures for the Knoevenagel condensation reaction catalyzed by the tilapia scale collagen powder extracted under this condition are the same as those in Example 1.

[0084] Example 13

[0085] (1) Mix 0.1 mol of choline chloride and 0.5 mol of 1,3-propanediol in a 100 mL round-bottom flask, and heat at 50 °C for 24 h to prepare a eutectic solvent with choline chloride as the proton acceptor and 1,3-propanediol as the proton donor.

[0086] (2) Add 2.0 g of tilapia scale powder and 20 mL of the "choline chloride-1,3-propanediol" eutectic solvent to a 50 mL round-bottom flask, and react in an oil bath at 40 °C for 8.0 h. After the reaction, perform suction filtration while it is hot. The tilapia scale collagen dissolved in the filtrate is regenerated, separated, and purified by the ethanol anti-solvent method. The subsequent treatment method is the same as that in Example 1. The extraction rate of tilapia scale collagen is 24%.

[0087] (3) The raw materials and experimental procedures for the Knoevenagel condensation reaction catalyzed by the tilapia scale collagen powder extracted under this condition are the same as those in Example 1.

[0088] Example 14

[0089] (1) The tilapia scale collagen catalyst used is from Example 1.

[0090] (2) Add 0.01 g of the catalyst (tilapia scale collagen powder), 2.5 mmol of benzaldehyde, 3.0 mmol of malononitrile, and 10 mL of 95% ethanol to a 25 mL round-bottom flask, and react at 75 °C (magnetic stirring, 200 rpm). Use thin-layer chromatography (TLC) to track the reaction progress (the developing agent is a mixture of petroleum ether and ethyl acetate, and the volume ratio of the two is 5:1).

[0091] (3) The subsequent experimental procedures are the same as those in Example 1.

[0092] Example 15

[0093] (1) The tilapia scale collagen catalyst used is from Example 1.

[0094] (2) Add 1.0 g of catalyst (tilapia scale collagen powder), 2.5 mmol of benzaldehyde, 3.0 mmol of malononitrile, and 10 mL of 95% ethanol to a 25 mL round-bottom flask, and carry out the reaction at 25 °C (magnetic stirring, 200 rpm). Use thin-layer chromatography (TLC) to monitor the reaction progress (the developing agent is a mixture of petroleum ether and ethyl acetate with a volume ratio of 5:1).

[0095] (3) The subsequent experimental procedures are the same as those in Example 1.

[0096] Example 16

[0097] (1) The tilapia scale collagen catalyst used is sourced from Example 1.

[0098] (2) Add 1.0 g of catalyst (tilapia scale collagen powder), 2.5 mmol of benzaldehyde, 3.0 mmol of malononitrile, and 5 mL of 95% ethanol to a 25 mL round-bottom flask, and carry out the reaction at 25 °C (magnetic stirring, 200 rpm). Use thin-layer chromatography (TLC) to monitor the reaction progress (the developing agent is a mixture of petroleum ether and ethyl acetate with a volume ratio of 5:1).

[0099] (3) The subsequent experimental procedures are the same as those in Example 1.

[0100] Example 17

[0101] (1) The tilapia scale collagen catalyst used is sourced from Example 1.

[0102] (2) Add 0.05 g of catalyst (tilapia scale collagen powder), 2.5 mmol of benzaldehyde, 3.0 mmol of malononitrile, and 10 mL of 60% ethanol to a 25 mL round-bottom flask, and carry out the reaction at 25 °C (magnetic stirring, 200 rpm). Use thin-layer chromatography (TLC) to monitor the reaction progress (the developing agent is a mixture of petroleum ether and ethyl acetate with a volume ratio of 5:1).

[0103] (3) The subsequent experimental procedures are the same as those in Example 1.

[0104] Example 18

[0105] (1) The tilapia scale collagen catalyst used is sourced from Example 1.

[0106] (2) Add 0.05 g of the catalyst (tilapia scale collagen powder), 2.5 mmol of benzaldehyde, 3.0 mmol of malononitrile, and 10 mL of 50% ethanol to a 25 mL round-bottom flask, and carry out the reaction at 25 °C (magnetic stirring, 200 rpm). Use thin-layer chromatography (TLC) to monitor the reaction progress (the developing agent is a mixture of petroleum ether and ethyl acetate with a volume ratio of 5:1).

[0107] (3) The subsequent experimental procedures are the same as those in Example 1.

[0108] Example 19

[0109] (1) The tilapia scale collagen catalyst used is sourced from Example 1.

[0110] (2) Add 0.05 g of the catalyst (tilapia scale collagen powder), 2.5 mmol of furfural, 3.0 mmol of malononitrile, and 10 mL of 60% ethanol to a 25 mL round-bottom flask, and carry out the reaction at 25 °C (magnetic stirring, 200 rpm). Use thin-layer chromatography (TLC) to monitor the reaction progress (the developing agent is a mixture of petroleum ether and ethyl acetate with a volume ratio of 5:1).

[0111] (3) The subsequent experimental procedures are the same as those in Example 1.

[0112] Example 20

[0113] (1) The tilapia scale collagen catalyst used is sourced from Example 1.

[0114] (2) Add 0.05 g of the catalyst (tilapia scale collagen powder), 2.5 mmol of p-methoxybenzaldehyde, 3.0 mmol of malononitrile, and 10 mL of 60% ethanol to a 25 mL round-bottom flask, and carry out the reaction at 25 °C (magnetic stirring, 200 rpm). Use thin-layer chromatography (TLC) to monitor the reaction progress (the developing agent is a mixture of petroleum ether and ethyl acetate with a volume ratio of 5:1).

[0115] (3) The subsequent experimental procedures are the same as those in Example 1.

[0116] Example 21

[0117] (1) The tilapia scale collagen catalyst used is sourced from Example 1.

[0118] (2) Add 0.05 g of the catalyst (tilapia scale collagen powder), 2.5 mmol of p-nitrobenzaldehyde, 3.0 mmol of malononitrile, and 10 mL of 60% ethanol to a 25 mL round-bottom flask, and carry out the reaction at 25 °C (magnetic stirring, 200 rpm). Use thin-layer chromatography (TLC) to monitor the reaction progress (the developing agent is a mixture of petroleum ether and ethyl acetate with a volume ratio of 5:1).

[0119] (3) The subsequent experimental procedures are the same as those in Example 1.

[0120] Example 22

[0121] (1) The tilapia scale collagen catalyst used is sourced from Example 1.

[0122] (2) Add 0.05 g of the catalyst (tilapia scale collagen powder), 2.5 mmol of 3,5-dimethoxybenzaldehyde, 3.0 mmol of malononitrile, and 10 mL of 60% ethanol to a 25 mL round-bottom flask, and carry out the reaction at 45 °C (magnetic stirring, 200 rpm). Use thin-layer chromatography (TLC) to monitor the reaction progress (the developing agent is a mixture of petroleum ether and ethyl acetate with a volume ratio of 5:1).

[0123] (3) The subsequent experimental procedures are the same as those in Example 1.

[0124] Example 23

[0125] (1) The tilapia scale collagen catalyst used is sourced from Example 1.

[0126] (2) Add 0.05 g of the catalyst (tilapia scale collagen powder), 2.5 mmol of 1-naphthaldehyde, 3.0 mmol of malononitrile, and 10 mL of 60% ethanol to a 25 mL round-bottom flask, and carry out the reaction at 25 °C (magnetic stirring, 200 rpm). Use thin-layer chromatography (TLC) to monitor the reaction progress (the developing agent is a mixture of petroleum ether and ethyl acetate with a volume ratio of 5:1).

[0127] (3) The subsequent experimental procedures are the same as those in Example 1.

[0128] II. Sample Analysis

[0129] (1) The extraction rate of tilapia scale collagen is obtained by calculating according to Formula 1.

[0130]

[0131] The molecular structure of the extracted tilapia scale collagen was characterized by Fourier transform infrared spectroscopy (FT-IR, Nicolet 6700, Thermo Fisher Scientific, USA), and potassium bromide tablets were pressed.

[0132] (2) The yield of the target product of the Knoevenagel condensation reaction was calculated according to formula 2.

[0133]

[0134] The molecular structure of the current product was then identified by a nuclear magnetic resonance spectrometer ( 1 1H-NMR, Bruker Avance III HD 400 MHz, Bruker, Germany), and deuterated chloroform (CDCl3) was used as the NMR solvent.

[0135] Table 1 Knoevenagel condensation reaction catalyzed by tilapia scale collagen in Examples 1-13

[0136]

[0137]

[0138] As can be seen from Table 1, both water and deep eutectic solvents can extract collagen from tilapia scales. Both the solvent composition and the extraction temperature have a great influence on the extraction efficiency of tilapia scale collagen. Among them, when hot water is used as the solvent, the extraction rate of tilapia scale collagen is the highest at 100 °C (Example 1). In addition, the extracted tilapia scale collagen all has good catalytic activity for the Knoevenagel condensation reaction, which is related to the intrinsic properties of amine and imine active groups contained in tilapia scale collagen. The efficiency of the Knoevenagel condensation reaction between benzaldehyde and malononitrile catalyzed by tilapia scale collagen extracted under different conditions varies little, and the yield of the target product is about 90% (Examples 1-13), indicating that the extracted tilapia scale collagen can be used as a catalyst in the field of Knoevenagel condensation reaction.

[0139] Table 2 shows the influence of reaction conditions on the Knoevenagel condensation reaction catalyzed by tilapia scale collagen in Examples 1 and 14-18

[0140]

[0141]

[0142] As can be seen from Table 2, when tilapia scale collagen is used as the catalyst, the catalyst dosage, solvent dosage, solvent composition, etc. all have a great influence on its catalysis of the Knoevenagel condensation reaction. The less the catalyst dosage, the longer the complete reaction time of the Knoevenagel condensation reaction and the higher the required reaction temperature (Examples 1 and Examples 14-15). In addition, the less the reaction solvent dosage, the more likely side reactions will occur during the reaction, reducing the yield of the target product (Examples 15-16). When the water content in the reaction system is increased and the ethanol content is decreased, it is found that the increase in water content is beneficial to the reaction and can effectively shorten the complete reaction time (Examples 1 and Examples 17-18). However, due to the slightly poor water solubility of the product, when the water content is too high, the corresponding product will precipitate during the reaction, which is not conducive to the subsequent product separation and purification process.

[0143] As can be seen from Table 2, when tilapia scale collagen is used as the catalyst, the catalyst dosage, solvent dosage, solvent composition, etc. all have a great influence on its catalysis of the Knoevenagel condensation reaction. The less the catalyst dosage, the longer the complete reaction time of the Knoevenagel condensation reaction and the higher the required reaction temperature (Examples 1 and Examples 14-15). In addition, the less the reaction solvent dosage, the more likely side reactions will occur during the reaction, reducing the yield of the target product (Examples 15-16). When the water content in the reaction system is increased and the ethanol content is decreased, it is found that the increase in water content is beneficial to the reaction and can effectively shorten the complete reaction time (Examples 1 and Examples 17-18). However, due to the slightly poor water solubility of the product, when the water content is too high, the corresponding product will precipitate during the reaction, which is not conducive to the subsequent product separation and purification process.

[0144] Table 3 Substrate Expansion of Aromatic Aldehydes in Examples 1 and Examples 19-23

[0145]

[0146]

[0147] As can be seen from Table 3, tilapia scale collagen has good generality for aromatic aldehydes. When furfural and aromatic aldehydes containing electron-donating groups and electron-withdrawing groups are used as raw materials (Examples 19-22), good yields of the target product (>90%) can also be obtained. In addition, when 1-naphthaldehyde is used as the raw material, the yield of the target product also reaches more than 90% (Example 23).

[0148] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the purpose and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention.

Claims

1. A method for catalyzing Knoevenagel condensation reaction of tilapia scale collagen, characterized in that: Collagen from tilapia scales was selectively extracted and separated by hot solvent extraction, and was used to catalyze the Knoevenagel condensation reaction between aromatic aldehydes and active methylene compounds.

2. The method for tilapia scale collagen catalyzed Knoevenagel condensation reaction according to claim 1, characterized in that, The specific steps include: (1) mixing 40-60 mesh tilapia scale powder with a solvent, and extracting at 40-100° C. for 2.0-8.0 h; (2) After the extraction is completed, the solid residue is removed by suction filtration while hot, and the filtrate is added dropwise to anhydrous ethanol, and the collagen dissolved in the extract is quickly precipitated in the form of white flocs; the flocs are filtered and washed with anhydrous ethanol for 3 times, and dried in a vacuum drying oven at 60° C. for 24 hours to obtain tilapia scale collagen, and finally crushed to 40-60 meshes using a traditional Chinese medicine grinder to obtain tilapia scale collagen powder catalyst, which is stored for later use; (3) 0.01-1.0 g of tilapia scale collagen powder catalyst and an aromatic aldehyde and an active methylene compound in a molar ratio of 1:1.2 are added to a 50%-95% ethanol solution to carry out a Knoevenagel condensation reaction at a reaction temperature of 25-75° C.

3. The method for tilapia scale collagen catalysis Knoevenagel condensation reaction according to claim 2, characterized in that, In step (1), the solvent is water or a low eutectic solvent containing a proton acceptor and a proton donor.

4. The method for tilapia scale collagen catalyzed Knoevenagel condensation reaction according to claim 2, characterized in that, In step (1), the mass ratio of tilapia scale powder to solvent volume is (5-30) g:100 mL.

5. The method for tilapia scale collagen catalysis Knoevenagel condensation reaction according to claim 2, characterized in that, The aromatic aldehyde is furfural or a benzaldehyde derivative containing a substituent.

6. The method for tilapia scale collagen catalyzed Knoevenagel condensation reaction according to claim 2, characterized in that, In step (2), the collagen in the extract is separated and purified by an antisolvent method.

7. The method for tilapia scale collagen catalyzed Knoevenagel condensation reaction according to claim 6, characterized in that, The antisolvent method refers to a method of using an antisolvent to mix with a tilapia phosphocollagen extract, and separating and purifying the collagen in the extract by using the difference in solubility between the two.

8. The method for tilapia scale collagen catalyzed Knoevenagel condensation reaction according to claim 2, characterized in that, In step (3), the volume ratio of the tilapia scale collagen powder catalyst mass to the ethanol solution is (0.1-20) g:100 mL.

9. The method for tilapia scale collagen catalyzed Knoevenagel condensation reaction according to claim 2, characterized in that, In step (3), the active methylene compound is malononitrile containing a strong electron-withdrawing group.

10. The method for tilapia scale collagen catalyzed Knoevenagel condensation reaction according to claim 2, characterized in that: The Knoevenagel condensation reaction temperature is 25°C.

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