Method for extracting fish oil from squid viscera

Abstract

This invention relates to a method for extracting fish oil using squid viscera, belonging to the field of biotechnology for oil extraction, and includes the following steps: (1) Rinse fresh squid viscera with clean water and crush the viscera into a uniform slurry for later use; (2) Grind citrus peel to 40-60 mesh for later use; (3) Add 3-5g of citrus peel, 2-4g of kombucha, and 1×10⁶-1×10⁸ CFU / g of Aspergillus spore powder per kilogram of slurry, mix evenly, and heat at 28-30℃ at a speed of 80-100 r / min. n, ferment for 18-24 hours; (4) add an appropriate amount of water to the fermentation product, with a material-to-water volume ratio of 1:1-1:2, stir evenly, and preheat to the enzymatic hydrolysis temperature; (5) use neutral protease and lipase for compound enzymatic hydrolysis. After the enzymatic hydrolysis is completed, raise the temperature of the material to 90-95℃ and maintain it for 10-15 minutes to destroy the enzyme activity and terminate the reaction; (6) the enzymatic hydrolysate is separated by a three-phase centrifuge to obtain three components: the upper layer is crude squid oil, the lower layer is protein hydrolysate, and the middle layer is solid residue. This invention uses the synergistic effect of fermentation pretreatment and compound enzymatic hydrolysis to significantly improve the fish oil extraction rate and protect its quality.

Description

Technical Field

[0001] This invention relates to a method for extracting fish oil using squid viscera, belonging to the field of biotechnology for oil extraction. Background Technology

[0002] Squid, also known as cuttlefish or squid, belongs to the phylum Mollusca, class Cephalopoda, order Decapoda, and family Cuttlefishidae. It is mainly distributed in the Atlantic, Indian, and Pacific Oceans. Squid meat is delicate and nutritious, with a delicious flavor. It is rich in protein and macroelements such as calcium, iron, and phosphorus, and contains trace elements such as selenium, copper, manganese, and iodine. It is considered a high-protein, low-fat, and low-calorie high-quality seafood, highly favored by consumers. With the increasing scale of squid fishing in my country, squid processing has gradually become an important part of the domestic aquatic product processing industry. The byproducts generated during squid processing (viscera, skin, ink sac, etc.), except for some processed into fishmeal, are mostly directly buried, which not only hinders the high-value utilization of squid but also causes a certain degree of environmental pollution. Research has found that squid viscera contain abundant nutrients such as protein, carbohydrates, amino acids, taurine, trace elements, and polyunsaturated fatty acids. Therefore, how to develop and utilize squid viscera in a high-value manner has become a key research direction for improving its overall economic benefits. Currently, squid viscera are being developed and utilized. Squid viscera are rich in EPA and DHA, which provide good raw materials for fish oil. After oil extraction from squid viscera, the hydrolysate contains rich peptides and amino acids, which can be used as a seasoning. However, squid viscera have a distinct fishy and bitter taste, which leads to the fact that its extracted and separated products are generally used in the feed industry, resulting in low added value. Summary of the Invention

[0003] This invention provides a method for extracting fish oil from squid viscera, which solves the problems of severe fishy and bitter taste, low extraction rate, and low added value in existing methods for extracting fish oil from squid viscera.

[0004] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: A method for extracting fish oil from squid viscera includes the following steps: (1) Rinse the fresh squid viscera with clean water to remove any mud, sand, shell fragments and other impurities that may be mixed in. Use a tissue homogenizer to crush the viscera into a uniform paste for later use. (2) Grind the citrus peel to 40-60 mesh and set aside; (3) Add 3-5g of citrus peel, 2-4g of kombucha, and 1×10g of Aspergillus spore powder per kilogram of slurry. 6 - 1×10 8 Mix CFU / g thoroughly, ferment at 28-30℃ at 80-100 r / min for 18-24 hours; (4) Add an appropriate amount of water to the fermentation product, with a material-to-water volume ratio of 1:1 to 1:2, stir evenly, and preheat to the enzymatic hydrolysis temperature; (5) Use neutral protease and lipase for compound enzymatic hydrolysis. After the enzymatic hydrolysis is completed, heat the material to 90-95℃ and maintain it for 10-15 minutes to destroy the enzyme activity and terminate the reaction. (6) The enzymatic hydrolysate is separated by a three-phase centrifuge to obtain three components: the upper layer is crude squid oil, which is refined into clear, odorless squid oil after standard oil refining steps; the lower layer is protein hydrolysate, which is concentrated and spray-dried to obtain protein powder; the middle layer is solid residue, which is dried and pulverized to be used as a high-protein feed ingredient and can be used to formulate aquatic or livestock feed.

[0005] Furthermore, preferably: the amount of neutral protease added is 400-600 U / g of raw material and the amount of lipase added is 800-1000 U / g of raw material.

[0006] Furthermore, preferably: the enzymatic hydrolysis temperature is 45-50℃, the time is 7-10 hours, and the rotation speed is 80-100 r / min.

[0007] Furthermore, preferably: in step (5), the compound enzymatic hydrolysis is first performed by adding 100-200 U / g of lipase and 400-600 U / g of protease, hydrolyzing at 45-50℃ and 80-100 r / min for 6-8 hours; then adding 600-900 U / g of lipase, hydrolyzing at 45-50℃ and 80-100 r / min for 1-2 hours.

[0008] Furthermore, preferably: in step (6), the three-phase centrifugal separation temperature is 80-85℃, the rotation speed is 4000-5000r / min, and the separation time is 5-8 min.

[0009] Furthermore, preferably, the enzymatic hydrolysate is mixed with a separation stabilizer at 0.1-0.3% of the raw material weight before the three-phase separation.

[0010] Furthermore, preferably, the separation stabilizer comprises, by weight, 10 parts soybean lecithin, 7 parts konjac gum, and 33 parts calcium chloride.

[0011] The beneficial effects of this invention are: This invention constructs a complete resource recycling chain, fully utilizing squid viscera. The main products are: the upper layer is crude squid oil, which is clear, has no obvious fishy or bitter taste, and has a high crude fat recovery rate. After standard oil refining steps, it can be used as a raw material for health products, pharmaceuticals, or high-end feed, possessing high economic value; the lower layer is protein hydrolysate, which is clear and has no obvious fishy or bitter taste. After concentration, the protein hydrolysate is spray-dried to obtain protein powder; the middle layer is solid residue, which, after drying and pulverizing, can be used as a high-protein feed ingredient for formulating aquatic or livestock feed. This invention converts all intermediate products into valuable products, and the entire process does not generate waste residue or wastewater requiring additional treatment, thus eliminating environmental pollution at the source. Detailed Implementation

[0012] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative effort are also described.

[0013] The basic components of the squid viscera described in this invention are as follows: moisture 56.68%, crude protein 19.36%, crude fat 21.03%, and ash 2.93%.

[0014] The citrus peel is made from fresh orange peel, dried and crushed. The kombucha is purchased from the official store of Shandong Zhishi Kombucha Weibao Vinegar Moth, using a 1:1 weight ratio of old kombucha to kombucha liquid, which is then thoroughly mixed before use.

[0015] Aspergillus was produced using Rhizopus koji from Angel Yeast, activated to a concentration of 1×10⁻⁶. 6 - 1×10 8 CFU / ml, for later use; The yeast used is Angel Yeast's low-sugar yeast, activated to a concentration of 1×10⁻⁶. 6 - 1×10 8 CFU / ml, for later use; The lactobacillus used is Angel Yeast's 2-strain (classic) yogurt starter, activated to a concentration of 1×10⁻⁶. 6 -1×10 8 CFU / ml, for later use; Acetic acid bacteria were obtained from Shanghai Preservation Microbiology Co., Ltd., and activated to a concentration of 1×10⁻⁶. 6 -1×10 8 CFU / ml, for later use; Other raw materials were purchased from the market and there were no special requirements.

[0016] Examples 1-5 investigated the effects of different fermentation bacteria on the extraction of fish oil from squid viscera.

[0017] A method for extracting fish oil from squid viscera includes the following steps: (1) Rinse the fresh squid viscera with clean water to remove any mud, sand, shell fragments and other impurities that may be mixed in. Use a tissue homogenizer to crush the viscera into a uniform paste for later use. (2) Grind the citrus peel to 40-60 mesh and set aside; (3) Add 4g of citrus peel, 3g of kombucha and 1ml of Aspergillus per kilogram of slurry, mix well, ferment at 28-30℃ and 100r / min for 24h; (4) Add an appropriate amount of water to the fermentation product, with a material-to-water ratio of 1:1, stir evenly, and preheat to 45-50°C. (5) Enzymatic hydrolysis using a combination of protease and lipase is performed. The specific steps are as follows: First, add 500 U / g of protease, at a temperature of 45-50℃ and a rotation speed of 100r / min, and hydrolyze for 6 hours; then add 1000 U / g of lipase, at a temperature of 45-50℃ and a rotation speed of 100r / min, and hydrolyze for 2 hours. After the hydrolysis is completed, raise the temperature of the material to 90-95℃ and maintain it for 15 minutes to destroy the enzyme activity and terminate the reaction. (6) The enzymatic hydrolysate is separated by a three-phase centrifuge. The three-phase centrifugation temperature in step (6) is 80-85℃, the centrifugation speed is 4000r / min, and the separation time is 8 min. Three components are obtained: the upper layer is crude squid oil, the lower layer is protein hydrolysate, and the middle layer is solid residue.

[0018] The microbial strains used in different embodiments are shown in Table 1.

[0019] Table 1. Strain combinations of different embodiments

[0020] At the end of the experiment, the indicators in the crude squid oil and protein hydrolysate were measured. The specific results are shown in Table 3, and the specific measurement methods are as follows: (1) Determination of moisture content: Refer to GB / T 5009.3—2003; (2) Determination of crude protein: Refer to GB / T 5009.5—2003; (3) Determination of crude fat: Refer to GB / T 5009.6—2003; (4) Determination of ash content: GB / T 5009.4—2003; (5) Sensory evaluation: The crude squid oil and protein hydrolysate were evaluated according to the standards in Table 2.

[0021] Table 2 Sensory Evaluation Criteria

[0022] Overall score calculation formula: Overall score = Σ(Score of each indicator × Weight of that indicator) Fat recovery rate (%) = Weight of crude fat in crude squid oil / Weight of crude fat in squid viscera × 100 Protein recovery rate (%) = weight of crude protein in protein hydrolysate / weight of crude protein in squid viscera × 100.

[0023] Table 3. Results of the determination of the effects of different fermentation bacteria on fish oil extraction from squid viscera.

[0024] As shown in Table 3, the method of this invention achieves a fat recovery rate of 76.32% and a protein recovery rate of 70.32% in the crude squid oil, indicating a high overall recovery rate. Furthermore, the sensory evaluation scores of both the crude squid oil and the protein hydrolysate are above 82 points, significantly higher than other experimental methods.

[0025] This invention utilizes the principles of citrus peel, kombucha, and Aspergillus as follows: Before enzymatic hydrolysis, a micro-fermentation step involving citrus peel, kombucha, and Aspergillus is performed. During its growth, Aspergillus secretes various enzymes that can break down the tough cell wall structure of squid viscera cells. Simultaneously, the addition of citrus peel and kombucha may regulate the pH of the fermentation system and introduce more active substances, creating more favorable conditions for the subsequent enzymatic hydrolysis reaction, allowing for a more complete release of the encapsulated oil. This biological pretreatment method is gentler and more thorough than simple physical crushing. Enzymatic hydrolysis employs a combination of protease and lipase. Protease efficiently degrades the protein network surrounding the oil droplets, while lipase directly hydrolyzes some fats. Their synergistic effect maximizes the release of oil. Furthermore, the mild hydrolysis conditions (temperature 45-50℃, pH adjusted according to enzyme characteristics) effectively avoid the damage of unsaturated fatty acids (such as DHA and EPA) caused by high temperatures, ensuring the freshness and nutritional value of the fish oil.

[0026] Another advantage of this invention is the proactive elimination or transformation of fishy and bitter odors during the production process. During fermentation, microorganisms such as Aspergillus and kombucha metabolize and transform the fishy precursors in squid viscera as food, thereby reducing the production of fishy substances at the source. Natural essential oils (such as limonene) contained in citrus peels have a refreshing aroma and can effectively neutralize and mask unpleasant odors. During the enzymatic hydrolysis stage, a combination of neutral protease and lipase is used to specifically hydrolyze short peptides at the protein ends that produce bitterness, thus significantly reducing the fishy and bitter odor of the final product.

[0027] Example 6 investigated the effect of enzymatic hydrolysis process on the extraction of fish oil from squid viscera.

[0028] The process is basically the same as in Example 1, except that the enzymatic hydrolysis steps are as follows: (5) Enzymatic hydrolysis is performed using a combination of protease and lipase. The specific steps are as follows: First, add 200 U / g of lipase and 500 U / g of protease, at a temperature of 45-50℃ and a rotation speed of 100r / min, and hydrolyze for 6 hours; then add 800 U / g of lipase, at a temperature of 45-50℃ and a rotation speed of 100r / min, and hydrolyze for 2 hours. After the hydrolysis is completed, raise the temperature of the material to 90-95℃ and maintain it for 15 minutes to destroy the enzyme activity and terminate the reaction.

[0029] After completion, the fat recovery rate of crude squid oil was measured to be 83.14%, which was 8.94% higher than that of Example 1, with a sensory evaluation score of 83.06. The protein recovery rate was 76.28%, which was 8.53% higher than that of Example 1, showing a significant improvement, with a sensory evaluation score of 84.26.

[0030] In squid visceral cells, lipids are often tightly encapsulated by protein networks and cell membranes. In the first enzymatic hydrolysis step, lipase and protease coexist, enabling simultaneous hydrolysis and breakdown: protease degrades the protein matrix, releasing the encapsulated lipids; simultaneously, the initial lipase (200 U / g) immediately acts on these newly exposed lipid interfaces, initiating hydrolysis and preventing oil droplets from re-aggregating or being encapsulated by incompletely hydrolyzed protein fragments, effectively improving protein recovery. In the second step, supplementing with a high dose of lipase (800 U / g), the enzyme acts more efficiently on these refined substrates, thereby increasing the degree of hydrolysis and recovery of total fat.

[0031] Example 7 A method for extracting fish oil from squid viscera includes the following steps: (1) Rinse the fresh squid viscera with clean water to remove any mud, sand, shell fragments and other impurities that may be mixed in. Use a tissue homogenizer to crush the viscera into a uniform paste for later use. (2) Grind the citrus peel to 40-60 mesh and set aside; (3) Add 3g of citrus peel, 4g of kombucha and 1ml of Aspergillus per kilogram of slurry, mix well, ferment at 28-30℃ and 80r / min for 18h; (4) Add an appropriate amount of water to the fermentation product, with a material-to-water ratio of 1:2, stir evenly, and preheat to 45-50°C. (5) Enzymatic hydrolysis using a combination of protease and lipase is performed. The specific steps are as follows: First, add 100 U / g of lipase and 400 U / g of neutral protease to the raw material, at a temperature of 45-50℃ and a rotation speed of 80 r / min, and hydrolyze for 6 hours; then add 700 U / g of lipase, at a temperature of 45-50℃ and a rotation speed of 80 r / min, and hydrolyze for 2 hours. After the hydrolysis is completed, raise the temperature of the material to 90-95℃ and maintain it for 15 minutes to destroy the enzyme activity and terminate the reaction. (6) The enzymatic hydrolysate is separated by a three-phase centrifuge. The three-phase centrifugation temperature in step (6) is 80-85℃, the centrifugation speed is 5000r / min, and the separation time is 5min. Three components are obtained: the upper layer is crude squid oil, the lower layer is protein hydrolysate, and the middle layer is solid residue.

[0032] Example 8 A method for extracting fish oil from squid viscera includes the following steps: (1) Rinse the fresh squid viscera with clean water to remove any mud, sand, shell fragments and other impurities that may be mixed in. Use a tissue homogenizer to crush the viscera into a uniform paste for later use. (2) Grind the citrus peel to 40-60 mesh and set aside; (3) Add 4g of citrus peel, 3g of kombucha and 1ml of Aspergillus per kilogram of slurry, mix well, ferment at 28-30℃ and 100r / min for 24h; (4) Add an appropriate amount of water to the fermentation product, with a material-to-water ratio of 1:1, stir evenly, and preheat to 45-50°C. (5) Enzymatic hydrolysis using a combination of protease and lipase is performed. The specific steps are as follows: First, add 200 U / g of lipase and 500 U / g of neutral protease. The temperature is 45-50℃ and the rotation speed is 100 r / min. The enzymatic hydrolysis is performed for 6 hours. Then, add 700 U / g of lipase. The temperature is 45-50℃ and the rotation speed is 100 r / min. The enzymatic hydrolysis is performed for 2 hours. After the enzymatic hydrolysis is completed, the material is heated to 90-95℃ and maintained for 15 minutes to destroy the enzyme activity and terminate the reaction. (6) The enzymatic hydrolysate is separated by a three-phase centrifuge. The three-phase centrifugation temperature in step (6) is 80-85℃, the centrifugation speed is 5000r / min, and the separation time is 5 min. Three components are obtained: the upper layer is crude squid oil, the lower layer is protein hydrolysate, and the middle layer is solid residue.

[0033] Example 9 A method for extracting fish oil from squid viscera includes the following steps: (1) Rinse the fresh squid viscera with clean water to remove any mud, sand, shell fragments and other impurities that may be mixed in. Use a tissue homogenizer to crush the viscera into a uniform paste for later use. (2) Grind the citrus peel to 40-60 mesh and set aside; (3) Add 4g of citrus peel, 3g of kombucha and 1ml of Aspergillus per kilogram of slurry, mix well, ferment at 28-30℃ and 100r / min for 24h; (4) Add an appropriate amount of water to the fermentation product, with a material-to-water ratio of 1:2, stir evenly, and preheat to 45-50°C. (5) Enzymatic hydrolysis using a combination of protease and lipase is performed. The specific steps are as follows: First, add 200 U / g of lipase and 500 U / g of neutral protease to the raw material at a temperature of 45-50℃ and a rotation speed of 100 r / min for 6 hours; then add 600 U / g of lipase at a temperature of 45-50℃ and a rotation speed of 100 r / min for 2 hours. After the enzymatic hydrolysis is completed, raise the temperature of the material to 90-95℃ and maintain it for 15 minutes to destroy the enzyme activity and terminate the reaction. (6) The enzymatic hydrolysate is separated by a three-phase centrifuge. The three-phase centrifugation temperature in step (6) is 80-85℃, the centrifugation speed is 5000r / min, and the separation time is 5min. Three components are obtained: the upper layer is crude squid oil, the lower layer is protein hydrolysate, and the middle layer is solid residue.

[0034] The fat recovery rate, protein recovery rate, sensory scores of crude squid oil, and sensory scores of protein hydrolysate in Examples 7-9 were determined, as detailed in Table 4.

[0035] Table 4 Measurement results of different embodiments

[0036] Example 10: Investigating the effect of separation stabilizers on the extraction of fish oil from squid viscera. The process is essentially the same as in Example 6, except that 0.2% of a separation stabilizer is added to the enzymatic hydrolysate before the three-phase separation. The separation stabilizer, by weight, comprises 10 parts soybean lecithin, 7 parts konjac gum, and 23 parts calcium chloride.

[0037] After completion, the fat recovery rate of the crude squid oil was measured to be 82.37%, the protein recovery rate was 78.34%, the sensory evaluation score of the crude squid oil was 95.26, and the sensory evaluation score of the protein hydrolysate was above 97.36, which is a significant improvement compared to Example 6.

[0038] The results of Example 10 show that the sensory evaluation score of the product is significantly improved after adding the separation stabilizer. The specific principle is as follows: The separation stabilizer of the present invention effectively disrupts the stable oil-in-water or water-in-oil emulsion system in the enzymatic hydrolysate and promotes the flocculation and aggregation of fine solid particles, thereby allowing the oil, water, and residue phases to be separated more quickly and thoroughly under the action of centrifugal force. The amphiphilic structure of soybean lecithin can strongly insert into and replace the protein / colloidal membrane wrapped on the surface of oil droplets, disrupting the stability of the emulsion membrane, causing the tiny oil droplets to aggregate and grow larger, accelerating their floating; the long-chain polymer of konjac gum can adsorb multiple fine solid particles and colloids, forming larger flocs through bridging, while increasing the viscosity of the aqueous phase, helping the solids to settle quickly; calcium chloride can compress the double layer on the particle surface, reduce the electrostatic repulsion between particles, and promote the aggregation of fine oil droplets and solid particles. After adding the above-mentioned separation stabilizers, the moisture content and impurity content of the crude squid oil are significantly reduced, and the color is clearer, which reduces the burden on subsequent refining; the transmittance of the protein hydrolysate is increased and the solid content is reduced, which is conducive to subsequent concentration or spray drying, resulting in higher purity protein powder; the moisture content of the solid residue is significantly reduced, making it easier to dry and reducing energy consumption.

[0039] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for extracting fish oil from squid viscera, characterized in that, Includes the following steps: (1) Rinse the fresh squid viscera with clean water to remove any mud, sand, shell fragments and other impurities that may be mixed in. Use a tissue homogenizer to crush the viscera into a uniform paste for later use. (2) Grind the citrus peel to 40-60 mesh and set aside; (3) Citrus peel 3-5 g, red tea fungus 2-4 g, and Aspergillus spore powder 1 x 10 6 CFU / g were added per kg of pulp, mixed well, and fermented at 28-30 °C at a rotation speed of 80-100 r / min for 18-24 h. 8 CFU / g were added per kg of pulp, mixed well, and fermented at 28-30 °C at a rotation speed of 80-100 r / min for 18-24 h. (4) Add an appropriate amount of water to the fermentation product, with a material-to-water volume ratio of 1:1 to 1:2, stir evenly, and preheat to the enzymatic hydrolysis temperature; (5) Use neutral protease and lipase for compound enzymatic hydrolysis. After the enzymatic hydrolysis is completed, heat the material to 90-95℃ and maintain it for 10-15 minutes to destroy the enzyme activity and terminate the reaction. (6) The enzymatic hydrolysate was separated by a three-phase centrifuge to obtain three components: the upper layer was crude squid oil, which was then refined into clear, odorless squid oil after standard oil refining steps. The lower layer is protein hydrolysate. After concentration, the protein hydrolysate is spray-dried to obtain protein powder. The middle layer is solid residue. After drying and pulverizing, it can be used as a high-protein feed ingredient to formulate aquatic or livestock feed.

2. The method for extracting fish oil from squid viscera according to claim 1, characterized in that: The amount of neutral protease added is 400-600 U / g of raw material, and the amount of lipase added is 800-1000 U / g of raw material.

3. The method for extracting fish oil from squid viscera according to claim 2, characterized in that: The enzymatic hydrolysis temperature is 45-50℃, the time is 7-10 hours, and the rotation speed is 80-100 r / min.

4. The method for extracting fish oil from squid viscera according to claim 2, characterized in that: In step (5), the compound enzymatic hydrolysis involves first adding 100-200 U / g of lipase and 400-600 U / g of protease to the raw material, hydrolyzing at 45-50℃ and 80-100 r / min for 6-8 hours; then adding 600-9000 U / g of lipase, hydrolyzing at 45-50℃ and 80-100 r / min for 1-2 hours.

5. A method for extracting fish oil from squid viscera according to claim 1, characterized in that: Step (6) The three-phase centrifugal separation temperature is 80-85℃, the rotation speed is 4000-5000r / min, and the separation time is 5-8 min.

6. The method for extracting fish oil from squid viscera according to claim 1, characterized in that: The enzymatic hydrolysate is treated with 0.1-0.3% of a separation stabilizer before the three-phase separation.

7. A method for extracting fish oil from squid viscera according to claim 6, characterized in that: The separation stabilizer, by weight ratio, comprises 10 parts soybean lecithin, 7 parts konjac gum, and 33 parts calcium chloride.

Images