Method for efficiently extracting and separating protein from cooked and degreased mussel powder

Abstract

The invention relates to the technical field of food processing, in particular to a method for efficiently extracting and separating protein from cooked and degreased mussel powder. The invention provides a method for efficiently extracting isolated protein from cooked and degreased mussel powder. The isolated protein is extracted by adopting a ball milling-ultrasonic synergistic auxiliary alkali-solution and acid-isolation method. The method has the advantages of simple process, low cost, short process time and the like, the purity of the extracted mussel protein is greater than 80%, the protein extraction rate is greater than 85%, the high-valued utilization of the cooked and degreased mussel powder is realized, the efficiency of extracting and separating the protein by alkali dissolution and acid precipitation is improved, an innovative path is provided for deep processing of the mussels, and the method is suitable for popularization and application. The method has important industrial value for efficient comprehensive utilization of byproducts of aquatic products.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, specifically to a method for efficiently extracting and separating proteins from boiled and defatted mussel powder. Background Technology

[0002] Mussels are bivalve mollusks that attach themselves to their shells via byssal threads. They are high-yielding and vigorous growers, making them one of my country's main farmed shellfish. Known for their delicious and nutritious meat, they are often called "eggs of the sea." They are high in protein, rich in eight essential amino acids including lysine, and minerals such as zinc. They also contain unsaturated fatty acids like EPA and DHA, as well as active ingredients such as taurine, offering health benefits such as boosting immunity and reducing the risk of cardiovascular disease. They are an important source of marine foods and functional ingredients. However, fresh mussels, due to their high water content and strong endogenous enzyme activity, are prone to spoilage after harvest due to microbial contamination and enzymatic hydrolysis. Therefore, processing them into steamed, boiled, and frozen semi-finished products is often used to extend their shelf life. Mussel cooking liquid, rich in protein and other nutrients, is commonly used in the preparation of seafood soy sauce. The oil extracted from the mussels during cooking can be solvent-extracted to produce high-value-added mussel oil, which is high in unsaturated fatty acids EPA and DHA and possesses anti-inflammatory and antioxidant properties.

[0003] However, mussel powder, even after steaming and defatting, still retains a high protein content (60%-70%). But due to the effects of heat treatment and extraction solvents, the protein undergoes partial denaturation, significantly reducing its solubility and other functional properties. It also contains residual polysaccharides, ash, and other impurities, limiting its direct utilization value. Common protein extraction methods, such as isoelectric point precipitation and salting out, suffer from low extraction rates, poor protein purity, and high desalting costs, making them unsuitable for the demands of high-end food or biomaterial fields.

[0004] Alkali dissolution and acid precipitation is a classic industrial method for extracting plant or animal proteins. It involves breaking down intermolecular forces under alkaline conditions to increase protein solubility, followed by acidic conditions to adjust to the isoelectric point for protein separation. However, when applied alone to cooked, defatted mussel powder, the protein extraction rate is low due to poor protein functional properties and interference from impurities, failing to meet the demands of industrial production. Therefore, process optimization or combining it with other technologies is urgently needed to improve protein extraction efficiency and achieve high-value utilization of mussel resources. As described in Chinese Patent CN109090334A, "A Method for Recovering and Separating Proteins from the Gonads of Yesso scallops," functional proteins are recovered and separated from the gonads of Yesso scallops using an alkali-dissolution and acid-precipitation method. However, this method has two shortcomings: ① It is only applicable to fresh Yesso scallop gonads, where the proteins have not been denatured by heat treatment and extraction solvents, resulting in high solubility. This method is unsuitable for the 'denatured, poorly soluble cooked and defatted mussel powder' described in this application. Direct application would lead to extremely low protein extraction rates due to incompatibility with the raw materials. ② Alkali dissolution relies solely on stirring, which is unsuitable for the low-solubility cooked and defatted mussel powder described in this application. It is difficult to fully dissolve the powder, reducing both the extraction rate and the purity of the separated proteins. Therefore, how to improve the protein extraction rate and product purity in cooked and defatted mussel powder is the main research problem of this invention. Summary of the Invention

[0005] The technical problem this invention aims to solve is that current alkaline dissolution and acid precipitation methods are mostly used for proteins in fresh materials with high solubility. However, the proteins in mussel powder after cooking and defatting undergo partial denaturation due to the effects of heat treatment and extraction solvents, resulting in a significant reduction in their solubility and other functional properties. At the same time, impurities such as polysaccharides and ash remain. Ordinary alkaline dissolution and acid precipitation methods cannot meet the requirements for protein extraction rate and product purity. There is an urgent need to improve the protein extraction effect through process optimization or by combining other technical means, so as to realize the high-value utilization of mussel resources.

[0006] To address the problems of existing technologies, this invention provides a method for efficiently extracting and separating proteins from cooked and defatted mussel powder. The method employs a ball milling-ultrasound synergistic alkali dissolution and acid precipitation technique for protein extraction. This method offers advantages such as simple process, low cost, and short processing time. The extracted mussel protein has a purity greater than 80% and a protein extraction rate greater than 85%, enabling high-value utilization of cooked and defatted mussel powder, improving the efficiency of alkali dissolution and acid precipitation protein extraction, and providing an innovative path for the deep processing of mussels. It also has significant industrial value for the efficient and comprehensive utilization of aquatic product by-products.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a method for efficiently extracting and separating protein from cooked defatted mussel powder, comprising the following steps:

[0008] (1) The cooked and defatted mussel powder is sieved, dissolved in water, and ball-milled to obtain a suspension of cooked and defatted mussel powder. The cooked and defatted mussel powder has poor solubility and is very easy to precipitate in water. The homogenization treatment by ball milling can make the mussel powder more dispersed in water, thereby improving the efficiency of alkali dissolution and acid precipitation.

[0009] (2) Adjust the pH of the steamed defatted mussel powder suspension to alkaline, use ultrasonic crushing and extraction, and centrifuge for the first time to obtain the crude extract of mussel protein; wherein, the ultrasonic power is 390-650 W, and the crushing and extraction time is 20-30 min; the role of ultrasonic crushing: the steamed defatted mussel protein has undergone denaturation and aggregation, and the ultrasonic cavitation effect can destroy the intermolecular forces of aggregated protein, break the protein aggregates, and improve its solubility in alkaline solution; if the ultrasonic efficiency is too low, the cavitation effect is insufficient, the protein extraction efficiency is low, and if the ultrasonic efficiency is too high, the equipment energy consumption will increase, and it may significantly affect the functional properties of the protein.

[0010] (3) Adjust the pH of the crude extract of mussel protein isolate to acidic, let it stand at 4°C, centrifuge it a second time to collect the precipitate, adjust the pH of the precipitate to neutral, freeze dry it to obtain mussel protein isolate.

[0011] Furthermore, in step (1), the cooked and defatted mussel powder is passed through an 80-mesh sieve and dissolved in water at a material-to-liquid ratio of 1:40 to 1:100 (w:v), preferably 1:100. In the alkali-dissolving and acid-precipitating process, wet nano-grinding is better than solid dry grinding because it can more precisely control particle size, effectively control temperature to prevent protein denaturation, reduce particle aggregation, and has wider applicability. The above parameters are the optimal process parameters obtained through process optimization, based on the results of mass yield and protein extraction rate in Table 1-2. If the material-to-liquid ratio is too low, the alkali penetration is weak, resulting in insufficient protein extraction efficiency; if the material-to-liquid ratio is too high, it easily leads to resource waste.

[0012] Furthermore, in step (1), the rotation speed of the ball mill rotor is 3000 rpm, the flow rate of the suspension is 110 rpm, and the ball milling time is 5-10 min, preferably 10 min. If the ball milling time is too long or too short, it will affect the particle size of the defatted mussel powder, thereby affecting the protein extraction efficiency.

[0013] Furthermore, in step (2), the pH value is adjusted to 11.5-12.5, preferably pH 12. Too low a pH will result in insufficient protein solubility, while too high a pH will easily cause changes in the protein structure.

[0014] Furthermore, in step (2), the ultrasonic power is 520 W and the ultrasonic time is 30 min. Both excessively high and excessively low ultrasonic power will affect the protein extraction efficiency.

[0015] Furthermore, in step (2), the first centrifugation speed is 10,000 rpm, and the time is 20-25 min. Too low a speed will affect the acquisition of the clarified supernatant, while too high a speed will increase the energy consumption of the equipment.

[0016] Furthermore, in step (3), the pH value is adjusted to 4.0-5.0, preferably pH 4.5, and the mixture is allowed to stand at 4°C for 1 hour. Too low a pH will cause protein denaturation, while too high a pH will reduce the protein extraction efficiency.

[0017] Furthermore, in step (3), the second centrifugation speed is 10,000 rpm, and the time is 15-20 min. Too low a speed will affect the acquisition of the clarified supernatant, while too high a speed will increase the energy consumption of the equipment.

[0018] A mussel protein isolate prepared by the above method, with a purity ≥80%.

[0019] The beneficial effects of this invention are as follows:

[0020] (1) This invention uses high-protein cooked and defatted mussel powder as raw material and innovatively adopts a ball milling-ultrasound synergistic alkali dissolution and acid precipitation method to prepare mussel protein isolate. The coupling effect of the three methods significantly improves the purity of the isolated protein from 68.11% of the raw material to more than 80% (up to 90.48%), and the protein extraction rate reaches more than 85% (up to 95.96%). The efficiency is more than 200% higher than the single alkali dissolution and acid precipitation process, and more than 150% higher than the ball milling-assisted alkali dissolution and acid precipitation process. It also effectively shortens the alkali dissolution time and realizes the efficient recovery and high-value utilization of protein in mussel powder.

[0021] (2) The mussel protein isolate obtained by the present invention has high protein purity and excellent quality. Its preparation process is simple, low cost, easy to achieve large-scale production and industrial application, and has significant economic and social benefits. It has broad prospects and market potential in many related fields such as health food and sports nutrition supplements. Attached Figure Description

[0022] Figure 1 pH optimization of acid precipitation process in alkali dissolution and acid precipitation.

[0023] Figure 2 Quality yield of ball mill-assisted alkali dissolution and acid precipitation process optimization.

[0024] Figure 3 Protein purity optimized by ball milling-assisted alkali dissolution and acid precipitation process.

[0025] Figure 4 Protein extraction rate optimized by ball milling-assisted alkali dissolution and acid precipitation process.

[0026] Figure 5Quality yield of ball milling-ultrasound synergistic assisted alkali dissolution and acid precipitation process optimization.

[0027] Figure 6 Protein purity improved by ball milling-ultrasound synergistic alkali dissolution and acid precipitation process optimization.

[0028] Figure 7 Protein extraction rate optimized by ball milling-ultrasound synergistic alkali dissolution and acid precipitation process. Detailed Implementation

[0029] The following description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0030] The following embodiments can be understood as illustrating only a part of the structure or method of the present invention, or as a combination of embodiments explaining the broader structure or method of the present invention. Unless otherwise specified, all raw materials of the present invention are commercially available.

[0031] Unless otherwise specified, all raw materials used in the following embodiments were purchased from the market.

[0032] Example 1: Determination of protein content in cooked defatted mussel powder

[0033] Steamed mussels (provided by Qingdao Marine Food Nutrition and Health Innovation Research Institute; preparation process as follows: purple mussels were placed in 90℃ hot water, boiled for 6 minutes after the water boiled again, shelled, cooled, and frozen) were freeze-dried (steamed mussels were pre-frozen at -40℃ for 12 hours, then freeze-dried at -40℃ under vacuum of 1-10 Pa for 72 hours), ground into powder, passed through a 40-mesh sieve, and defatted for 2 hours with 95% ethanol at a material-to-liquid ratio of 1:15 (w / v). The powder was then centrifuged at 4000 rpm for 20 minutes, and the precipitate was collected. The defatting process was repeated twice more. The ethanol in the defatted precipitate was evaporated to obtain defatted mussel powder. The protein content of the defatted mussel powder was determined using the Kjeldahl method according to the National Food Safety Standard GB 5009.5-2016. The results showed that the protein content of the defatted mussel powder was 68.11%, making it a good high-protein byproduct suitable for the preparation of mussel protein isolate.

[0034] Example 2: Optimization of the isoelectric point of mussel protein isolate

[0035] Defatted mussel powder was sieved through an 80-mesh sieve and prepared into a 1 mg / mL aqueous solution (absorbance between 0.2 and 0.8, with higher accuracy for UV readings). The pH was adjusted to 12, and the solution was magnetically stirred at room temperature for 2 h. After centrifugation at 10,000 rpm for 20 min, the supernatant was collected, and its pH was adjusted to 4.0, 4.5, 5.0, and 5.5, respectively. The solutions were then allowed to stand at 4℃ for 1 h, and the turbidity of the supernatant was measured at a wavelength of 600 nm. The results are as follows: Figure 1 As shown, the supernatant had the lowest turbidity when the pH of the acid precipitation was 4.5, indicating that the protein precipitation efficiency was optimal under this condition.

[0036] Example 3: Optimization of the alkali dissolution and acid precipitation process for mussel protein isolate

[0037] 0.4 g of cooked, defatted mussel powder was passed through an 80-mesh sieve and accurately weighed. The sample was dispersed in water at material-to-liquid ratios of 1:10, 1:20, 1:40, and 1:100 (w / v), and the pH was adjusted to 10 or 12 respectively. After magnetic stirring at room temperature for 2 h, 3 h, and 4 h, the supernatant was collected by centrifugation at 10,000 rpm for 20 min. The pH of the supernatant was adjusted to 4.5, and the mixture was acid-precipitated at 4℃ for 1 h. The precipitate was then collected by centrifugation at 10,000 rpm for 15 min, the pH was adjusted to neutral, and the mixture was freeze-dried to obtain alkali-soluble and acid-precipitated mussel protein isolate. The yield (%) was calculated using the following formula:

[0038] Quality yield (%) = (Weight of freeze-dried isolated protein / Accurately weighed mussel powder) × 100%

[0039] Table 1. Optimization of alkali dissolution time, pH, and feed-to-liquid ratio in the alkali dissolution and acid precipitation process.

[0040] The optimization results of the alkali dissolution and acid precipitation process are shown in Table 1. Under the same material-to-liquid ratio, when the magnetic stirring time during the alkali dissolution stage is 2 h, the mass yield is higher, and more isolated protein is obtained. Furthermore, a longer solubilization stirring time does not increase the mass yield of isolated protein and cannot significantly improve the efficiency of alkali dissolution and acid precipitation. In addition, when the pH is 12 and the solubilization time is 2 h, the mass yield of isolated mussel protein is higher than 27% when the material-to-liquid ratio of cooked defatted mussel powder is 1:40-1:100 (w / v), indicating that a high material-to-liquid ratio can significantly increase the extraction efficiency of isolated protein.

[0041] Example 4: Verification of the protein purity and protein extraction rate of the optimized process in the alkaline dissolution and acid precipitation process for mussel protein separation.

[0042] The experimental samples with a yield higher than 15% in Example 3 were selected, and their protein purity was determined using the Kjeldahl method according to the National Food Safety Standard GB5009.5-2016. The formula for calculating the protein extraction rate (%) is as follows:

[0043] Protein extraction rate (%) = (Weight of freeze-dried isolated protein) × (Purity of freeze-dried isolated protein) × (Mass of precisely weighed mussel powder) × Protein purity of mussel powder × 100%

[0044] Table 2. Validation of protein purity and protein extraction rate of the optimized alkaline dissolution and acid precipitation process.

[0045]

[0046] Table 2 shows the protein purity and extraction rate results of the optimized alkaline dissolution and acid precipitation process. The optimal conditions for the alkaline dissolution and acid precipitation process are a material-to-liquid ratio of 1:100 (w / v), pH 12, and an alkaline dissolution time of 2 h. Under these conditions, the protein purity is 90.62%, and the protein extraction rate reaches the highest value of 36.37%. This indicates that the high material-to-liquid ratio process in alkaline dissolution and acid precipitation can effectively extract and separate protein from cooked and defatted mussel powder while ensuring a certain level of protein purity.

[0047] Example 5: Optimization of the ball milling-assisted alkali dissolution and acid precipitation process for mussel protein isolate

[0048] Defatted mussel powder was passed through an 80-mesh sieve and dispersed in water at a material-to-liquid ratio of 1:100 (w / v). The suspension was homogenized using a ball mill at a rotor speed of 3000 rpm and a flow rate of 110 rpm for milling times of 5, 10, 15, 20, and 30 min. 40 mL of the milled suspension was taken, and the pH was adjusted to 12. After magnetic stirring at room temperature for 2 h, the supernatant was collected by centrifugation at 10000 rpm for 20 min. The pH of the supernatant was adjusted to 4.5, and the mixture was acid-precipitated at 4℃ for 1 h. The precipitate was then collected by centrifugation at 10000 rpm for 15 min, the pH was adjusted to neutral, and the mixture was freeze-dried to obtain mussel protein isolate obtained by ball milling-assisted alkali dissolution and acid precipitation. The yield was calculated. The protein purity was determined using the Kjeldahl method according to the National Food Safety Standard GB 5009.5-2016, and the protein extraction rate was calculated.

[0049] The optimization results of the ball milling-assisted alkali dissolution and acid precipitation process are as follows: Figure 2 , Figure 3 , Figure 4As shown, a ball milling time of 10 minutes is the optimal condition for ball milling-assisted alkali dissolution and acid precipitation process. The isolated protein purity is 87.25%, and both the mass yield and protein yield reach their highest values ​​of 38.75% and 49.63%, respectively. This is significantly better than the protein extraction efficiency of the single alkali dissolution and acid precipitation process. This may be because the cooked and defatted mussel powder has poor solubility and easily precipitates in water. The homogenization treatment of ball milling can improve the dispersibility of the mussel powder in water, thereby increasing the efficiency of alkali dissolution and acid precipitation.

[0050] Example 6: Optimization of the ball milling-ultrasound synergistic alkali dissolution and acid precipitation process for mussel protein isolate.

[0051] The cooked and defatted mussel powder was passed through an 80-mesh sieve and dispersed in water at a material-to-liquid ratio of 1:100 (w / v). The suspension was homogenized using a ball mill with a rotor speed of 3000 rpm, a suspension flow rate of 110 rpm, and a milling time of 10 min. 30 mL of the milled suspension was taken, and its pH was adjusted to 12. Ultrasonic extraction was performed at power levels of 130 W, 260 W, 390 W, 520 W, and 650 W for 10 min, 20 min, and 30 min, respectively, followed by centrifugation at 10000 rpm for 20 min to collect the supernatant. The pH of the supernatant was adjusted to 4.5, and the mixture was acid-precipitated at 4℃ for 1 h. The precipitate was then collected by centrifugation at 10000 rpm for 15 min, the pH was adjusted to neutral, and the mixture was freeze-dried to obtain mussel protein isolate obtained through a ball-milling-ultrasonic synergistic alkali dissolution and acid precipitation process. The yield was calculated.

[0052] The optimization results of the ball milling-ultrasound synergistic alkali dissolution and acid precipitation process are as follows: Figure 5 As shown, the optimal process for alkali dissolution and acid precipitation is achieved under the conditions of 520 W ultrasonic power and 30 min ultrasonic time, with the highest yield of 78.87%, significantly superior to both single alkali dissolution and acid precipitation processes and ball milling-assisted alkali dissolution and acid precipitation processes. This is because the homogeneous cooked and defatted mussel powder solution obtained after ball milling can further utilize the cavitation effect generated by ultrasonic disruption to break down the intermolecular forces of denatured aggregated proteins and dissolve protein aggregates. The synergistic effect of ball milling and ultrasound promotes more complete dissolution of proteins in the alkali solution, thereby improving the protein extraction efficiency of alkali dissolution and acid precipitation.

[0053] Example 7: Verification of protein purity and extraction rate in the optimized process of ball milling-ultrasound synergistic alkali dissolution and acid precipitation for mussel protein separation.

[0054] The experimental samples with a yield higher than 67% in Example 6 were selected, and their protein purity was determined by the Kjeldahl method in the national food safety standard GB5009.5-2016, and their protein extraction rate was calculated.

[0055] The protein purity and protein extraction rate of the optimized process in the ball milling-ultrasound synergistic alkali dissolution and acid precipitation process optimization are as follows: Figure 6 , Figure 7 As shown, the optimal conditions for the ball milling-ultrasound synergistic alkali dissolution and acid precipitation process are 520 W ultrasonic power and 30 min ultrasonic time. Under these conditions, the protein purity is 82.88%, and the protein extraction rate reaches a maximum of 95.96%, which is more than 200% more efficient than the single alkali dissolution and acid precipitation process and more than 150% more efficient than the ball milling-assisted alkali dissolution and acid precipitation process. It also effectively shortens the alkali dissolution time from 2 h to 30 min. In this invention, ball milling first mechanically breaks down the raw material, destroying the cell structure and homogenizing the solution, increasing the specific surface area to facilitate alkali penetration. Ultrasound, through cavitation effect, breaks the intermolecular forces of denatured aggregated proteins, dissolving the protein aggregates. The synergistic effect of these two processes makes the protein more soluble in the alkali solution, followed by acid precipitation for separation. The coupling of these three processes significantly improves the protein extraction efficiency and purity.

[0056] All aspects, embodiments, and features of this invention should be considered illustrative in all respects and not limiting of the invention; the scope of the invention is defined only by the claims. Other embodiments, modifications, and uses will become apparent to those skilled in the art without departing from the spirit and scope of the invention as claimed.

[0057] In the preparation method of this invention, the order of the steps is not limited to the listed order. For those skilled in the art, variations in the order of the steps without creative effort are also within the scope of protection of this invention. Furthermore, two or more steps or actions can be performed simultaneously.

[0058] Finally, it should be noted that the specific embodiments described herein are merely illustrative examples of the invention and are not intended to limit the implementation of the invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them; it is neither necessary nor possible to exemplify all embodiments here. However, these obvious variations or modifications derived from the essential spirit of the invention still fall within the scope of protection of the invention, and interpreting them as any additional limitation would contradict the spirit of the invention.

Claims

1. A method for efficiently extracting and separating protein from boiled defatted mussel powder, characterized in that... Includes the following steps: (1) The cooked and defatted mussel powder was sieved, dissolved in water, and ball-milled to obtain a suspension of cooked and defatted mussel powder. (2) Adjust the pH of the steamed defatted mussel powder suspension to alkaline, use ultrasonic crushing and extraction, and centrifuge for the first time to obtain the supernatant to obtain the crude extract of mussel protein; wherein, the ultrasonic power is 390-650 W, and the crushing and extraction time is 20-30 min. (3) Adjust the pH of the crude extract of mussel protein isolate to acidic, let it stand at 4°C, centrifuge it a second time to collect the precipitate, adjust the pH of the precipitate to neutral, freeze dry it to obtain mussel protein isolate.

2. The method as described in claim 1, characterized in that: In step (1), the steamed and defatted mussel powder is passed through an 80-mesh sieve and dissolved in water at a ratio of 1:40 to 1:100 (w:v).

3. The method as described in claim 1, characterized in that: In step (1), the rotation speed of the ball mill rotor is 3000 rpm, the flow rate of the suspension is 110 rpm, and the ball milling time is 5-10 min.

4. The method as described in claim 1, characterized in that: In step (2), the pH value is adjusted to 11.5-12.

5.

5. The method as described in claim 1, characterized in that: In step (2), the ultrasonic power is 520 W and the ultrasonic time is 30 min.

6. The method as described in claim 1, characterized in that: In step (2), the first centrifugation speed is 10,000 rpm and the time is 20-25 min.

7. The method as described in claim 1, characterized in that: In step (3), the pH value is adjusted to 4.0~5.0 and left to stand at 4℃ for 1 hour.

8. The method as described in claim 1, characterized in that: Step (3) The second centrifugation speed is 10000 rpm and the time is 15-20 min.

9. The mussel protein isolate prepared by any one of claims 1-8, characterized in that: Purity ≥ 80%.

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