Method for enriching collagen from eel meat leftover material

Abstract

The invention discloses a method for enriching collagen from eel meat leftover materials, and belongs to the field of deep processing of aquatic products. According to the method, the eel meat leftover materials are used as raw materials, the eel source collagen is obtained through separation according to the solubility of different proteins, the method for enriching the collagen in the eel meat leftover materials is provided, and a new effective utilization method and a new utilization direction are provided for the eel meat leftover materials. The collagen prepared by the method disclosed by the invention is low in cost and high in purity, and waste utilization of eel meat leftover materials is realized. In addition, the eel meat obtained in the preparation process of the method can be further processed to enrich protein or prepare minced fillet products, and high-value utilization of eel resources is achieved.

Description

Technical Field

[0001] This invention relates to a method for enriching collagen from eel meat scraps, belonging to the field of deep processing of aquatic products. Background Technology

[0002] Eels, also known as eels, mainly live in tropical and temperate waters. Their flesh is tender and nutritious, rich in high-quality protein and various essential amino acids, making them popular with consumers. Currently, eel farming and eel product processing are the main focus of research in the eel industry. However, as eel products become increasingly diverse and widespread, the waste of leftover scraps after processing is becoming more and more serious.

[0003] To better develop the edible and economic value of eels and make high-value use of the scraps left over from eel production, eel scraps are now often reprocessed for use in food additives, feed, reconstituted meat products, fish oil, and collagen extraction. Among these, using eel scraps for collagen extraction can maximize the recycling and utilization of the scraps and generate significant economic benefits.

[0004] Collagen is a natural high-molecular-weight protein, mostly obtained from the skin, bones, and ribs of animals. With the increasing demand for collagen, in addition to terrestrial animals such as pigs and cattle, the skin and bones of aquatic animals such as fish have also become targets for collagen extraction. For example, the optimization of the extraction process and physicochemical property analysis of collagen from Japanese eel skin (Li Xiaomin, Yang Chenyu, Deng Yun, Tao Ningping, Li Li, & Qiu Weiqiang et al. (2023). Optimization of the extraction process and physicochemical property analysis of collagen from Japanese eel skin. Journal of Shanghai Ocean University, 32(6), 1144-1154.) mentions the use of acid-enzyme extraction method for extracting collagen from eel skin, with a maximum collagen yield of 32.78%; in patent CN112961894B, an eel bone collagen peptide is provided. The extraction process can use eel bones as raw materials to finally prepare collagen peptide powder; in the response surface methodology optimization of the extraction process of acid-soluble eel swim bladder collagen (Li Hangting, Mo Yiwen, Li Nuoying, Tang Yunping, Yang Zuisu. Journal of Guangdong Ocean University, 2022, 42(6):114-121.DOI:10.3969 / j.issn.1673-9159.2022.06.015.), eel swim bladder was used as raw material, and the extraction rate of eel swim bladder collagen was 65.32%.

[0005] However, most existing technologies only recycle and reuse single parts of eels, such as eel skin, bones, and swim bladders, which have high requirements for eel scraps. They do not extract and utilize collagen from the fish meat and internal organs, which account for a significant portion of the scraps. This results in a certain amount of waste because the eel scrap resources are not utilized and developed efficiently to the greatest extent. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention employs simple and effective steps and methods to enrich high-purity eel-derived collagen from skinned eel meat scraps. This not only facilitates the high-value utilization of eel meat scraps and solves the problem of waste, but also provides a new direction for the development and utilization of eel collagen.

[0007] To achieve the above objectives, the present invention provides a method for enriching collagen from eel meat scraps, comprising the following steps:

[0008] (1) After processing the scrap into small pieces / particles of 1cm*1cm, add eight times the volume (v / w) of phosphate buffer, stir and crush at 200rpm for 1min; after centrifuging at 4℃ and 10000rpm for 20min, a floating film appears on the upper layer of the solution. Separate and remove the floating film, soak the floating film in petroleum ether for 9h to remove the oil (the ratio of petroleum ether to floating film v / m is 10:1), dry at 45℃ for 20min, then crush it with a mixer for 2min, pass it through a 40-mesh sieve to obtain skinned eel meat scrap powder;

[0009] (2) Add 0.6M acetic acid (material-to-liquid ratio of 1:10) and 1wt% (mass percentage) pepsin to the skinned eel meat scrap powder obtained in step (1). After reacting at 37°C for 24 hours, centrifuge at 10,000 rpm for 20 minutes and collect the supernatant. Extract the remaining precipitate again with acetic acid and pepsin of the same concentration and material-to-liquid ratio, centrifuge at 10,000 rpm for 20 minutes and collect the supernatant.

[0010] (3) Combine the supernatants obtained in step (2), adjust the pH of the solution to 7, add sodium chloride solid to a concentration of 2.5M, and salt out for 24 hours. Centrifuge at 10,000 rpm for 20 minutes and remove the precipitate. Redissolve the precipitate with an appropriate amount of 0.5M acetic acid, and then dialyze it with 0.1M acetic acid and deionized water as external solutions for 48 hours. Freeze-dry the dialyzed sample solution to obtain the finished collagen product.

[0011] The results showed that the prepared product contained approximately 7.78% hydroxyproline, 86.22% collagen in the freeze-dried powder, and 22.35% collagen in the skinned eel meat scraps. The collagen extraction rate was 36.85%, the collagen molecular weight was 110 kDa, and the collagen contained two bands, 95 and 110 kDa, which are consistent with the characteristics of type I collagen.

[0012] The present invention also provides a method for enriching collagen from eel meat scraps, the method comprising the following steps:

[0013] (1) Add phosphate buffer to eel meat scraps, stir, crush and centrifuge, and take the floating film on the top layer of the solution; soak the floating film in petroleum ether for 9-12 hours, dry and crush to obtain skinless eel meat scrap powder.

[0014] (2) Add acetic acid and enzyme to the peeled eel meat scrap powder obtained in step (1) for enzymatic hydrolysis, centrifuge, and collect the supernatant; the enzyme is pepsin, or a mixture of pepsin and papain;

[0015] (3) Adjust the pH of the supernatant obtained in step (2) to 2-5, add sodium chloride solid for salting out, centrifuge and take out the precipitate; redissolve the precipitate with acetic acid, and dialyze with acetic acid and deionized water as external solution for 48-72 hours in sequence; freeze-dry the dialyzed sample solution to obtain the finished collagen.

[0016] In one embodiment of the present invention, in step (1), the pH of the phosphate buffer is 7.0-7.5.

[0017] In one embodiment of the present invention, the pH of the phosphate buffer is 7.

[0018] In one embodiment of the present invention, the amount of phosphate buffer added is: added at a ratio of (1-3):(8-25) of material to liquid.

[0019] In one embodiment of the present invention, the amount of petroleum ether added is based on a ratio of (1-3):(10-20) of floating film to petroleum ether.

[0020] In one embodiment of the present invention, in step (2), the concentration of acetic acid is 0.5-0.7M; the amount of acetic acid added is: added according to a material-to-liquid ratio of (1-3):(10-20);

[0021] In one embodiment of the present invention, the amount of enzyme added is 1 to 1.5 wt%; the enzyme activity of the pepsin is 1200 U / g and the enzyme activity of the papain is 6000 U / g.

[0022] In one embodiment of the present invention, the pepsin and papain are compounded in a ratio of (1-2):(1-2);

[0023] In one embodiment of the present invention, the enzymatic hydrolysis reaction is carried out at 30-40°C for 24-36 hours.

[0024] In one embodiment of the present invention, in step (3), the concentration of sodium chloride is 2-3M;

[0025] In one embodiment of the present invention, the salting-out time is 12-36 hours, preferably 24 hours;

[0026] In one embodiment of the present invention, the dialysis is performed with a molecular weight cutoff of 7000 to 10000 Da.

[0027] In one embodiment of the present invention, the centrifugation is carried out at a rate of 8000-15000 rpm for a time of 20-50 min.

[0028] In one embodiment of the present invention, in step (1), the drying conditions are: 40-50°C;

[0029] Preferably, the pulverization is performed by using a mixer for 2 to 5 minutes at a speed of 200 to 500 rpm.

[0030] In one embodiment of the present invention, the concentration of acetic acid used for dialysis is 0.1 to 0.15 M; the concentration of reconstituted acetic acid is 0.3 to 0.5 M.

[0031] The present invention also provides collagen prepared by the above method.

[0032] Beneficial effects

[0033] (1) This invention uses eel meat scraps as raw materials and separates eel-derived collagen based on the solubility of different proteins, providing a method for enriching collagen in eel meat scraps, and providing a new effective utilization method and a new utilization direction for eel meat scraps.

[0034] (2) The precipitate after enriching collagen from eel meat scraps can still be used for further processing of fish paste or extraction of eel protein. This not only facilitates the high-value utilization of eel meat scraps and solves the problem of waste, but also proves that the invention is highly practical and can realize the high-value utilization of eel scraps.

[0035] (3) The hydroxyproline content in the product obtained by the method of the present invention is about 7.78%, the collagen content in the skinless eel meat scraps is 22.35%, the collagen extraction rate is 36.85%, the collagen molecular weight is 110kDa, and the collagen contains two bands, 95 and 110kDa respectively, which are consistent with the characteristics of type I collagen. Attached Figure Description

[0036] Figure 1 This is a schematic diagram of the floating film powder of the present invention.

[0037] Figure 2 This is a schematic diagram illustrating collagen extraction after adding buffer solution according to the present invention.

[0038] Figure 3 This is a flowchart of the experimental process of the present invention.

[0039] Figure 4 The figures show the distribution of collagen content and extraction rate in the examples and comparative examples.

[0040] Figure 5 This is a schematic diagram of collagen electrophoresis according to the present invention. Detailed Implementation

[0041] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0042] The methods involved in the following embodiments are as follows:

[0043] I. Preparation method of eel meat scraps

[0044] (1) After processing the scrap into small pieces / particles of 1cm*1cm, add eight times the volume (v / w) of buffer solution, stir and crush at 200rpm for 1min; after centrifuging at 4℃ and 10000rpm for 20min, a floating film appears on the upper layer of the solution. Separate and remove the floating film, soak the floating film in petroleum ether for 9h to remove the grease (the ratio of petroleum ether to floating film v / m is 10:1), dry at 45℃ for 20min, and then crush it with a mixer for 2min to obtain skinned eel meat scrap powder;

[0045] (2) Add 0.6M acetic acid (material-to-liquid ratio of 1:10) and 1wt% (mass percentage) pepsin to the skinned eel meat scrap powder obtained in step (1). After reacting at 37°C for 24 hours, centrifuge at 10,000 rpm for 20 minutes and collect the supernatant. Extract the remaining precipitate again with acetic acid and pepsin of the same concentration and material-to-liquid ratio, centrifuge at 10,000 rpm for 20 minutes and collect the supernatant.

[0046] (3) Combine the supernatants obtained in step (2), adjust the pH of the solution to 7, add sodium chloride solid to a concentration of 2.5M, and salt out for 24 hours. Centrifuge at 10,000 rpm for 20 minutes and remove the precipitate. Redissolve the precipitate with an appropriate amount of 0.5M acetic acid, and then dialyze it with 0.1M acetic acid and deionized water as external solutions for 48 hours. Freeze-dry the dialyzed sample solution to obtain the finished collagen product.

[0047] II. Methods for Detecting Collagen Content

[0048] Collagen content was determined using hydroxyproline, a specific component of collagen, following the method described in GB / T 9696.23-2008, "Determination of Hydroxyproline Content in Meat and Meat Products," where the hydroxyproline standard curve was y = 0.0484x + 0.0223. The conversion between collagen content and hydroxyproline content in aquatic products was calculated using equation (1):

[0049] X(%)=MX1.1

[0050] In the formula:

[0051] X: Collagen content (%);

[0052] M: Hydroxyproline content (%);

[0053] 1.1: Conversion factor.

[0054] III. Calculation Method for Collagen Extraction Rate

[0055] The collagen extraction rate is calculated according to formula (2):

[0056]

[0057] In the formula:

[0058] Y: Collagen extraction rate (%);

[0059] M1: Mass (g) of collagen in freeze-dried powder obtained from eel scraps;

[0060] M2: Mass (g) of collagen in each gram of eel scraps.

[0061] IV. Methods for Determining the Molecular Weight of Collagen

[0062] The molecular weight of collagen was analyzed by SDS-PAGE. The specific steps were as follows: 5 mg of collagen was pretreated; 10 μL of the protein solution was loaded onto a pre-cast gel; and the parameters were set before electrophoresis: 80 V for the first 20 minutes, followed by 100 V until the electrophoresis was complete. Markers with molecular weights ranging from 20 to 250 kDa were used to estimate the protein molecular weight.

[0063] Raw materials involved in the examples:

[0064] The buffer solution involved in the following examples is:

[0065] (1) 0.025 mol / L sodium chloride, 0.005 mol / L tris(hydroxymethyl)aminomethane, 0.05 mol / L disodium ethylenediaminetetraacetate, pH 7.5;

[0066] (2) is a phosphate buffer with a concentration of 0.01-0.05 mol / L and a pH of 7-7.5, preferably with a concentration of 0.02 mol / L and a pH of 7.

[0067] Unless otherwise specified, all m / v in the following examples are in g / mL.

[0068] The pepsin, flavor protease, alkaline protease, neutral protease, and papain involved in the following examples were purchased from Sinopharm Chemical Reagent Co., Ltd. and Beijing Solarbio Technology Co., Ltd., with model numbers 9001-75-6, 9001-92-7, 9014-01-1, 9068-59-1, and 9001-73-4, respectively, and enzyme activities of 1200 U / g, 10 U / mg, 200 U / mg, 50 U / mg, and 6000 U / g, respectively.

[0069] Example 1: A method for enriching collagen from eel meat scraps

[0070] Collagen extraction after adding buffer solution is as follows Figure 2 As shown, the experimental procedure is as follows: Figure 3 As shown, the specific steps are as follows:

[0071] (1) After processing the scraps into small pieces / particles of 1cm*1cm, add eight times the volume (m / v = 1:8) of phosphate buffer (pH: 7), stir and crush at 200rpm for 1min; after centrifugation at 4℃ and 10000rpm for 20min, a floating film appears on the upper layer of the solution. Separate and remove the floating film, soak it in petroleum ether for 9h to remove the grease (the ratio of floating film to petroleum ether is m / v = 1:10), dry it at 45℃ for 20min, and then crush it with a mixer (speed: 200rpm) for 2min to obtain skinned eel meat scrap powder. Figure 1 );

[0072] (2) Add 0.6M acetic acid (material-liquid ratio m / v is 1:10) and 1wt% (mass percentage) pepsin (enzyme activity is 1200U / g) to the skinned eel meat scrap powder obtained in step (1). After reacting at 37℃ for 24h, centrifuge at 10000rpm for 20min and collect the supernatant.

[0073] The precipitate was extracted once again with acetic acid and pepsin of the same concentration and material-to-liquid ratio (reacted at 37°C for 24 h), centrifuged at 10,000 rpm for 20 min, and the supernatant was collected.

[0074] (3) Combine the supernatants obtained in step (2), adjust the pH of the solution to 7, add sodium chloride solid to a concentration of 2.5M, and salt out for 24 hours. Centrifuge at 10,000 rpm for 20 minutes and remove the precipitate. Redissolve the precipitate with an appropriate amount of 0.5M acetic acid, and then dialyze it sequentially with 0.1M acetic acid and deionized water as external solutions for 48 hours (the molecular weight cutoff is 7000 Da). Freeze-dry the dialyzed sample solution to obtain the finished collagen product.

[0075] The results are as follows: The hydroxyproline content in the product obtained in this embodiment is about 7.78%, the collagen content in the freeze-dried powder is 86.22%, the collagen content in the skinless eel meat scraps is 22.35%, the collagen extraction rate is 36.85%, the collagen molecular weight is 110kDa, and the collagen contains two bands, 95 and 110kDa, which are consistent with the characteristics of type I collagen.

[0076] Example 2: The Influence of Different Experimental Conditions

[0077] 1. The effect of floating film and petroleum ether

[0078] Compared with Example 1, the ratio (m / v) of the floating film to petroleum ether in step (1) was adjusted to 1:5, while the other conditions remained the same as in Example 1. The collagen product was prepared and tested according to the method of Example 1.

[0079] The results showed that the hydroxyproline content in the obtained product was 7.68%, the collagen content in the freeze-dried powder was 85.24%, the collagen content in the skinless eel meat scraps was 22.35%, and the collagen extraction rate was 34.60%.

[0080] 2. The effects of different feed-to-liquid ratios and acidification times

[0081] Compared with Example 1, the material-to-liquid ratio m / v in step (2) was adjusted to 1:5 and the acidification time was adjusted to 18 hours (reaction at 37°C for 18 hours). That is, step (2) is:

[0082] Add 0.6M acetic acid (material-liquid ratio m / v of 1:5) and 1wt% (mass percentage) pepsin (total enzyme activity of 1200 U / g) to the skinned eel meat scrap powder obtained in step (1). After reacting at 37℃ for 18h, centrifuge at 10000rpm for 20min and collect the supernatant.

[0083] The remaining precipitate was extracted once again with acetic acid and pepsin of the same concentration and material-to-liquid ratio (reacted at 37°C for 18 h), centrifuged at 10,000 rpm for 20 min, and the supernatant was collected.

[0084] The remaining conditions were kept the same as in Example 1, and the collagen products were prepared and tested according to the method of Example 1.

[0085] The results showed that the hydroxyproline content in the obtained product was 7.60%, the collagen content in the freeze-dried powder was 84.38%, the collagen content in the skinless eel meat scraps was 22.35%, and the collagen extraction rate was 30.92%.

[0086] 3. Effects of different sodium chloride concentrations and salting-out times

[0087] Compared with Example 1, the salt concentration in step (3) was adjusted to 2M and the salting-out time was adjusted to 20 hours. That is, step (3) is:

[0088] (3) Combine the supernatant obtained in step (2), adjust the pH of the solution to 7, add sodium chloride solid to a concentration of 2.0M, salt out for 20 hours, centrifuge at 10,000 rpm for 20 minutes, and take out the precipitate.

[0089] The remaining conditions were kept the same as in Example 1, and the collagen products were prepared and tested according to the method of Example 1.

[0090] The results showed that the hydroxyproline content in the obtained product was 7.73%, the collagen content in the freeze-dried powder was 85.83%, the collagen content in the skinless eel meat scraps was 22.35%, and the collagen extraction rate was 31.17%.

[0091] Example 3: Effects of different enzymatic hydrolysis conditions

[0092] 1. Single-enzyme hydrolysis effect

[0093] The specific implementation method is the same as in Example 1, except that the pepsin in step (2) is adjusted to be: flavor protease, alkaline protease, neutral protease, and papain; the added enzyme activity is the same as in Example 1.

[0094] All other conditions remained the same as in Example 1. The collagen products were prepared and tested according to the method in Example 1, and the results are shown in Table 1 below:

[0095] Table 1: Effects of different types of enzymes

[0096] Types of enzymes Hydroxyproline content in the product Collagen content in freeze-dried powder Collagen extraction rate Flavor proteases 5.98 66.378 30.69 alkaline protease 4.89 54.279 22.03 neutral protease 5.32 59.052 21.64 Papain 6.42 71.262 28.44

[0097] 2. The effect of dual-enzyme combination enzymatic hydrolysis

[0098] The specific implementation method is the same as in Example 1, except that the pepsin in step (2) is adjusted as follows:

[0099] Pepsin: Flavor protease (added at an enzyme activity ratio of 1:1);

[0100] Pepsin: Alkaline protease (added at an enzyme activity ratio of 1:1);

[0101] Pepsin: Neutral protease (added at an enzyme activity ratio of 1:1);

[0102] Pepsin: Papain (added at an enzyme activity ratio of 1:1);

[0103] The total amount of added enzyme activity remained the same as in Example 1;

[0104] All other conditions remained the same as in Example 1. The collagen products were prepared and tested according to the method in Example 1, and the results are shown in Table 2 below:

[0105] Table 2: Effects of different types of enzymes

[0106]

[0107]

[0108] 3. The effect of three-enzyme combination enzymatic hydrolysis

[0109] The specific implementation method is the same as in Example 1, except that the pepsin in step (2) is adjusted to:

[0110] Pepsin: Flavor protease: Papain (added at an enzyme activity ratio of 1:1:1); The total amount of added enzyme activity (total added enzyme activity of 1200 U / g) is consistent with that in Example 1;

[0111] The remaining conditions were kept the same as in Example 1. The collagen products were prepared and tested according to the method of Example 1. The results showed that the hydroxyproline content in the product was 7.67%, the collagen content in the freeze-dried powder was 85.14%, the collagen content in the skinless eel meat scraps was 22.35%, and the collagen extraction rate was 29.56%.

[0112] Comparative Example 1:

[0113] The specific implementation method is the same as in Example 1, except that the petroleum ether used to remove the oil from the eel scraps in step (1) is changed to isopropanol. All other conditions are the same as in Example 1. The collagen product was prepared and tested according to the method of Example 1.

[0114] The results showed that the hydroxyproline content in the product obtained in this comparative example was 6.37%, the collagen content in the freeze-dried powder was 70.76%, the collagen content in the skinless eel meat scraps was 22.35%, and the collagen extraction rate was 22.22%.

[0115] Comparative Example 2:

[0116] The specific implementation method is the same as in Example 1, except that step (1) is adjusted so that the eel meat scraps powder is not crushed, that is, step (1) is:

[0117] After processing the scraps into small pieces / particles of 1cm*1cm, add eight times the volume (m / v = 1:8) of phosphate buffer (pH: 7), stir and crush at 200rpm for 1min; after centrifugation at 10000rpm for 20min at 4℃, a floating film appears on the upper layer of the solution. Separate and remove the floating film, soak it in petroleum ether for 9h to remove grease (the ratio of floating film to petroleum ether is m / v = 1:10), and dry at 45℃ for 20min to obtain skinned eel meat scrap powder;

[0118] The remaining conditions were kept the same as in Example 1, and the collagen products were prepared and tested according to the method of Example 1.

[0119] The results showed that the hydroxyproline content in the obtained product was 6.29%, the collagen content in the freeze-dried powder was 69.79%, the collagen content in the skinless eel meat scraps was 22.35%, and the collagen extraction rate was 22.31%.

[0120] Comparative Example 3:

[0121] The specific implementation method is the same as in Example 1, except that the acid in step (2) is changed to citric acid, that is, step (2) is:

[0122] Add 0.6M citric acid (material-liquid ratio m / v is 1:10) and 1wt% (mass percentage) pepsin (enzyme activity is 1200U / g) to the skinned eel meat scrap powder obtained in step (1). After reacting at 37℃ for 24h, centrifuge at 10000rpm for 20min and collect the supernatant.

[0123] The remaining precipitate was extracted once again with citric acid and pepsin of the same concentration and material-to-liquid ratio (reacted at 37°C for 24 h), centrifuged at 10,000 rpm for 20 min, and the supernatant was collected.

[0124] All other conditions remain the same as in Example 1.

[0125] The results showed that the hydroxyproline content in the obtained product was 5.74%, the collagen content in the freeze-dried powder was 63.67%, the collagen content in the skinless eel meat scraps was 22.35%, and the collagen extraction rate was 19.95%.

[0126] Comparative Example 4:

[0127] The specific implementation method is the same as in Example 1, except that the enzyme in step (2) is changed to trypsin, that is, step (2) is:

[0128] Add 0.6M acetic acid (material-to-liquid ratio m / v of 1:10) and 1wt% (mass percentage) trypsin (enzyme activity of 1200U / g) to the skinned eel meat scrap powder obtained in step (1). After reacting at 37℃ for 24h, centrifuge at 10000rpm for 20min and collect the supernatant.

[0129] The remaining precipitate was extracted once again with acetic acid and trypsin of the same concentration and material-to-liquid ratio (reacted at 37°C for 24 h), centrifuged at 10,000 rpm for 20 min, and the supernatant was collected.

[0130] All other conditions remain the same as in Example 1.

[0131] The results showed that the hydroxyproline content in the obtained product was 6.94%, the collagen content in the freeze-dried powder was 77.01%, the collagen content in the skinless eel meat scraps was 22.35%, and the collagen extraction rate was 25.30%.

[0132] Comparative Example 5:

[0133] The specific implementation method is the same as in Example 1, except that the salting-out method in step (3) is changed to ultrafiltration, that is, step (3) is:

[0134] (3) Combine the supernatants obtained in step (2), adjust the pH of the solution to 7, add sodium chloride solid to a concentration of 2.5M, ultrafilter for 24 hours, centrifuge at 10,000 rpm for 20 minutes, and remove the precipitate. Redissolve the precipitate with an appropriate amount of 0.5M acetic acid, and then dialyze it sequentially with 0.1M acetic acid and deionized water as external solutions for 48 hours (the molecular weight cutoff is 7000 Da). Freeze-dry the dialyzed sample solution to obtain the finished collagen product.

[0135] All other conditions remain the same as in Example 1.

[0136] The results showed that the hydroxyproline content in the obtained product was 6.98%, the collagen content in the freeze-dried powder was 77.52%, the collagen content in the skinless eel meat scraps was 22.35%, and the collagen extraction rate was 25.58%.

[0137] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for enriching collagen from eel meat scraps, characterized in that, The method includes the following steps: (1) Add phosphate buffer to eel meat scraps, stir, crush and centrifuge, and take the floating film on the top layer of the solution; soak the floating film in petroleum ether for 9-12 hours, dry and crush to obtain skinless eel meat scrap powder. (2) Add acetic acid and enzyme to the peeled eel meat scrap powder obtained in step (1) for enzymatic hydrolysis, centrifuge, and collect the supernatant; the enzyme is pepsin, or a mixture of pepsin and papain; (3) Adjust the pH of the supernatant obtained in step (2) to 7-8, add sodium chloride solid for salting out, centrifuge and take out the precipitate; redissolve the precipitate with acetic acid, and dialyze with acetic acid and deionized water as external solution for 48-72 hours in sequence; freeze-dry the dialyzed sample solution to obtain the finished collagen.

2. The method according to claim 1, characterized in that, In step (1), the pH of the phosphate buffer is 7.0-7.5, preferably 7; the amount of phosphate buffer added is: added according to a material-to-liquid ratio of (1-3):(8-25).

3. The method according to claim 1 or 2, wherein the amount of petroleum ether added is based on a ratio of (1-3):(10-20) of floating film to petroleum ether.

4. The method according to any one of claims 1 to 3, characterized in that, In step (2), the concentration of acetic acid is 0.5-0.7M; the amount of acetic acid added is based on a material-to-liquid ratio of (1-3):(10-20). Preferably, the amount of enzyme added is 1 to 1.5 wt%.

5. The method according to any one of claims 1 to 4, characterized in that, The pepsin and papain are compounded in a ratio of (1-2):(1-2) of enzyme activity; Preferably, the enzymatic hydrolysis reaction is carried out at 30–40°C for 24–36 hours.

6. The method according to any one of claims 1 to 5, characterized in that, In step (3), the concentration of sodium chloride is 2-3 M; Preferably, the salting-out time is 12-36 hours, and more preferably 24 hours. Preferably, the molecular weight cutoff for the dialysis is 7000-10000 Da.

7. The method according to any one of claims 1 to 6, characterized in that, The centrifugation is performed at a speed of 8000-15000 rpm for a time of 20-50 min.

8. The method according to any one of claims 1 to 7, characterized in that, In step (1), the drying conditions are: 40-50℃; Preferably, the pulverization is performed by using a mixer for 2 to 5 minutes at a speed of 200 to 500 rpm.

9. The method according to any one of claims 1 to 8, characterized in that, The concentration of acetic acid used for dialysis is 0.1–0.15 M; the concentration of reconstituted acetic acid is 0.3–0.5 M.

10. Collagen prepared by the method according to any one of claims 1 to 9.

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