Preparation method of fishbone collagen tripeptide

Abstract

The invention discloses a preparation method of fishbone collagen tripeptide, which comprises the following steps: soaking fishbone dust (30-60 meshes) in a choline-arginine eutectic solvent, stirring, centrifuging or filtering, and collecting supernate; adding pure water with the volume 3-6 times that of the supernatant into the supernatant, standing for more than 24 hours, centrifuging or filtering again, and collecting the supernatant; carrying out tangential filtration treatment by adopting a nanofiltration membrane with the molecular weight cutoff of 200Da, recovering the eutectic solvent in the filtrate through vacuum concentration, adding pure water into the trapped fluid, carrying out repeated nanofiltration treatment to remove solvent residues, and collecting the trapped fluid; and carrying out spray drying on the trapped fluid to obtain a powder product with the collagen tripeptide content of more than 50%. Compared with a traditional enzyme method for preparing fishbone collagen tripeptide, the method disclosed by the invention has the advantages that the extraction rate is high, a deliming step is not needed, the enzyme cost is saved, an extraction solvent is green and can be recycled, and the like.

Description

Technical Field

[0001] The invention relates to a method for preparing fishbone collagen tripeptide, belonging to the technical field of polypeptide preparation. Background Art

[0002] Collagen peptide is a bioactive peptide that protects joints, skin, bones and cardiovascular function, and is one of the most important functional food ingredients in the world. The raw materials of aquatic collagen peptide are mainly fish scales and fish skins, but with the rapid development of the aquatic collagen peptide market, the prices of fish scales and fish skins have risen sharply, bringing more and more raw material pressure to production companies.

[0003] Collagen tripeptide is a popular high-end collagen peptide product on the market. Compared with ordinary collagen peptide products, this type of product has a smaller relative molecular weight, higher biological efficacy, greater technical difficulty and higher added value. Collagen tripeptide products are currently mainly produced by complex enzymatic hydrolysis of multiple proteases. However, due to the limited hydrolysis ability of proteases on small molecular oligopeptides, the preparation of collagen tripeptide products with a purity of >50% (relative molecular weight 189~500 Da) requires a high amount of enzyme and the production cost is too high.

[0004] Fish bones are one of the main by-products of fish processing. They are rich in collagen and widely available, so they are bound to be an important source of raw materials for aquatic collagen peptides in the future. In the current aquatic processing industry, fish bones are mostly discarded or made into feed, and the added value of the products is relatively low, and there is still a lot of room for growth. Fish bones are multi-layer dense self-assembled structures composed of collagen bundles and hydroxyapatite nano-platelet crystals in an orderly manner, making it difficult to directly extract or enzymatically hydrolyze its collagen components. At present, the traditional process of preparing collagen peptides from fish bones requires de-ashing pre-treatment, which requires a large amount of acidic medium consumption, which will bring huge environmental pressure to the company.

[0005] In view of the above-mentioned industry status quo, the present invention has developed a preparation process of fishbone collagen tripeptide based on thermal extraction of a new green low eutectic solvent. The process does not require deashing pretreatment of the fishbone raw material, nor does it require the use of proteases. The collagen tripeptide content in the product can reach more than 50%. The solvent is non-toxic and can be recycled, so it has great application potential in the preparation of fishbone collagen tripeptide. Summary of the invention

[0006] The invention aims to develop a non-enzymatic method for efficiently preparing fishbone collagen tripeptide.

[0007] The technical solution adopted by the present invention is:

[0008] (1) After the fish fillets are steamed to remove the attached meat, wet fish bones are obtained, which are then dried, ball-milled and sieved (30-60 mesh) to produce fish bone powder;

[0009] (2) The fish bone powder is soaked in a choline-arginine low eutectic solvent (choline: arginine molar ratio of 1:1, water content of 20-50%), at a solid-liquid ratio of 1:10-1:80 and a temperature of 80-140°C, stirred and extracted for 2-8 hours, and then centrifuged or filtered to collect the supernatant;

[0010] (3) using a nanofiltration membrane with a molecular weight cutoff of 200 Da for tangential filtration, recovering the low eutectic solvent in the filtrate by vacuum concentration, adding pure water to the retentate for repeated nanofiltration to remove the residual solvent, and collecting the retentate;

[0011] (4) The retentate is spray-dried to obtain a powder product with a collagen tripeptide content of more than 50%.

[0012] The mesh size of the fish bone powder in step (1) of the present invention cannot be higher than 60 meshes. Fish bone powder that is too fine will easily float above the solvent and reduce the extraction rate.

[0013] Preferably, the process conditions in step (2) of the present invention are solid-liquid ratio of 1:40, temperature of 120°C, and extraction time of 3 h.

[0014] Furthermore, under the optimal process conditions, the collagen extraction rate can reach 86%, and the purity of collagen tripeptide can reach more than 50%.

[0015] The technical advantages of the present invention are mainly reflected in:

[0016] (1) The method disclosed in the present invention does not require deashing pretreatment of fish bone raw materials, does not consume acidic medium, is environmentally friendly, and does not bring environmental pressure.

[0017] (2) The method disclosed in the present invention does not require the use of protease, thus saving the cost of enzymes.

[0018] (3) The method disclosed in the present invention can produce a collagen tripeptide product with a purity higher than 50%, which exceeds the product purity of most traditional enzymatic hydrolysis processes at an acceptable cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The specific implementation modes of the present invention will be further described in detail below in conjunction with the accompanying drawings.

[0020] Figure 1 Viscosities of various choline-organic acid and choline-amino acid deep eutectic solvents at 25°C and 80°C. (A) Deep eutectic solvents based on choline-organic acid system, CA: citric acid, LA: lactic acid, OA: oxalic acid, HAc: acetic acid, CC: choline chloride; (B) Deep eutectic solvents based on choline-amino acid system, Arg: arginine, Asp: aspartic acid, His: histidine, Lys: lysine, Glu: glutamic acid, Gly: glycine, CH: choline hydroxide.

[0021] Figure 2 Viscosity and fish bone collagen extraction rate at 80°C after adding 30% water to various choline-organic acid and choline-amino acid deep eutectic solvents. (A) Deep eutectic solvent based on choline-organic acid system, CA: citric acid, LA: lactic acid, OA: oxalic acid, HAc: acetic acid, CC: choline chloride; (B) Deep eutectic solvent based on choline-amino acid system, Arg: arginine, Asp: aspartic acid, His: histidine, Lys: lysine, Glu: glutamic acid, Gly: glycine, CH: choline hydroxide.

[0022] Figure 3 Single factor optimization of the extraction process of fish bone collagen tripeptide using choline-arginine (Arg-CH) low eutectic solvent. (A) Water content; (B) Solid-liquid ratio; (C) Extraction temperature; (D) Extraction time. The results are expressed as mean ± standard deviation (n=3). Different letters indicate significant differences between the data (p < 0.05).

[0023] Figure 4 Diagram of the precipitation state of fish bone hydroxyapatite after adding different precipitants to the supernatant of the extract.

[0024] Figure 5 Fourier transform infrared spectrum of fish bone collagen tripeptide extracted by choline-arginine (Arg-CH).

[0025] Figure 6 Size exclusion chromatogram and molecular weight distribution of fish bone collagen tripeptide extracted by choline-arginine (Arg-CH). DETAILED DESCRIPTION

[0026] Example 1

[0027] This embodiment provides a method for evaluating the extraction rate of fish bone collagen. The extraction rate is the ratio of the content of hydroxyproline specific to collagen in the extract to the total content in the fish bone raw material.

[0028] The specific technical solution steps are as follows:

[0029] The hydroxyproline content was detected by the chloramine T method hydroxyproline kit. The sample was digested to determine the total hydroxyproline content: 200 μL of fish bone extract was transferred into an ampoule or an equal mass of fish bone raw material calculated according to the solid-liquid ratio, and 2.0 mL of 6 mol / L hydrochloric acid was added to acidify the sample, and then the ampoule was sealed. Digested in an oven at 110°C for 6 h until no large lumps were visible, and after cooling, filtered through a 0.22 μm microporous filter membrane for the determination of the free hydroxyproline content in the hydrolyzate.

[0030] The pH value of the sample was adjusted to neutral with a 10 mol / L NaOH solution. The volume was made up to 4 mL with distilled water, and the supernatant was taken. The sample to be tested, chloramine T, dimethylaminobenzaldehyde and ultrapure water were added in order according to a ratio of 1:1:1:2 and mixed. The sample was placed in a water bath at 60°C for 20 min, and the absorbance of the sample at a wavelength of 562 nm was measured using an enzyme marker. The total hydroxyproline content and free hydroxyproline content of the sample were determined by this method.

[0031] Hydroxyproline standard solutions with concentrations of 7.500, 3.750, 1.875, 0.938, 0.469, 0.234, and 0.117 μg / mL were prepared, and the same method was used to experiment and establish a standard curve. With absorbance as the dependent variable and hydroxyproline concentration as the independent variable, the linear regression equation for hydroxyproline was: y = 0.0495 x + 0.0531, R 2 = 0.9997.

[0032] The collagen extraction rate E (%) is calculated by the following formula: the percentage of hydroxyproline contained in the extract to the total hydroxyproline in the fish bone raw material.

[0033] (1);

[0034] (1) Where: E represents the collagen extraction rate, %; c1 represents the hydroxyproline concentration in the extract sample, μg / mL; c2 represents the hydroxyproline concentration in the fish bone sample of the same mass as the raw material, μg / mL.

[0035] Example 2

[0036] This embodiment provides a method for extracting collagen tripeptides from tilapia bones using a choline-arginine deep eutectic solvent.

[0037] The specific technical solution steps are as follows:

[0038] Fresh tilapia bones were boiled in boiling water for 10 min, then the minced meat was removed, washed repeatedly with pure water and air-dried at 40°C. The clean tilapia bones were coarsely crushed with a grinder, and then crushed with a ball mill at 300 r / min for 1 h, and the fish bone powder with a particle size of 30-60 mesh was collected.

[0039] The relationship between the viscosity of several choline-organic acid and choline-amino acid eutectic solvents and the collagen extraction rate is shown in Figure 2 As shown in Figure 2, the choline-arginine deep eutectic solvent showed excellent performance in the extraction of fish bone collagen tripeptide. The relationship between process parameter optimization and extraction rate is shown in Figure 2. Figure 3 As shown, the choline-arginine low eutectic solvent with the following process conditions was finally selected for extracting fish bone collagen tripeptide.

[0040] A low eutectic solvent with a water content of 30% and a molar ratio of arginine to choline of 1:1 was prepared. 2.5 g of fish bone powder was added to 100 mL of the low eutectic solvent and the extraction was carried out under continuous stirring at 120°C for 3 h. After adding 4 times the volume of pure water to the extract and standing for 24 h, hydroxyapatite ( Figure 4 ), and centrifuged at 3000 × g for 20 min to separate and collect. The supernatant enters a tangential filtration device equipped with a nanofiltration membrane with a molecular weight cutoff of 200 Da for nanofiltration treatment. The nanofiltration equipment used is numbered RNM-25G-2, and the nanofiltration membrane used is numbered RNM-301. The filtered low eutectic solvent is collected and can be reused after concentration. The retentate is then added with 100 mL of pure water for nanofiltration once, and the filtrate is discarded. Repeat 3 to 5 times to remove the residual solvent. The retentate is then spray-dried to prepare a powder to obtain a collagen tripeptide product. The infrared spectrum of the obtained collagen tripeptide product is shown in Figure 5 As shown, it has characteristic infrared absorption peaks of collagen (amide I band and amide II band).

[0041] Example 3

[0042] This embodiment provides a method for determining fish bone collagen tripeptide.

[0043] Size exclusion high performance liquid chromatography (SEC-HPLC) was used to determine the content of collagen tripeptide in collagen hydrolysate under different conditions. The chromatographic conditions were set as follows: the chromatographic column used in the experiment was TOSOH TSKgel G2000 SWXL (300 mm × 7.8 mm); the column temperature was set to 30 °C; the isocratic elution mobile phase was a mixed solution of ultrapure water: acetonitrile: trifluoroacetic acid = 40: 60: 0.05, with a flow rate of 0.5 mL / min; the injection volume was 10.00 μL; a UV detector was used, and the detection wavelength was set to 220 nm.

[0044] Cytochrome C (15 kDa), insulin (5733 Da), bacitracin (1421 Da), and reduced glutathione (307 Da) were used as molecular weight determination reference standards. Based on the molecular weight of the standard and the retention time, a standard curve of the relationship between the logarithm of the molecular weight and the retention time was drawn to estimate the molecular weight of each sample component. With the retention time as the dependent variable and the logarithm of the molecular weight as the independent variable, the standard linear regression equation was y = -0.1993 x + 6.3504, R 2 = 0.9815. Based on this, the corresponding retention time range of the collagen tripeptide with a molecular weight of 189–500 Da was inferred, and the percentage of the peak area of ​​the collagen tripeptide product of each sample to the peak area of ​​the total product was calculated, which was defined as the collagen tripeptide content. The molecular weight distribution of the product obtained in Example 2 is as follows Figure 6As shown, it was calculated that the collagen tripeptides with molecular weights between 189–500 Da accounted for 53.26%.

[0045] The above embodiments show and describe the basic principles and main features of the present invention and the advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments, and the above embodiments and descriptions are only for illustrating the principles of the present invention, and are not intended to limit the scope of the present invention in any way. Without departing from the scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the protection claimed.

Claims

1. A method for preparing a high-purity collagen tripeptide product from fish bones, characterized in that: The following steps are involved: (1) After the fish fillets are steamed to remove the attached meat, wet fish bones are obtained, which are then dried, ball-milled and sieved to produce fish bone powder; (2) The fish bone powder is soaked in a choline-arginine low eutectic solvent at a solid-liquid ratio of 1:10-1:80 and a temperature of 80-140°C. After stirring and leaching for 2-8 hours, the mixture is centrifuged or filtered to collect the supernatant. (3) using a nanofiltration membrane with a molecular weight cutoff of 200 Da for tangential filtration, recovering the low eutectic solvent in the filtrate by vacuum concentration, adding pure water to the retentate for repeated nanofiltration to remove the residual solvent, and collecting the retentate; (4) The retentate is spray-dried to obtain a powder product with a collagen tripeptide content of more than 50%.

2. The method for preparing a high-purity collagen tripeptide product from fish bones according to claim 1, characterized in that: In step (2), the molar ratio of choline to arginine is 1:1, and the water content is 20-50%.

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