A method for preparing high content rTG type fish oil using mackerel processing by-products

Abstract

The application discloses a method for preparing high-content rTG type fish oil by using mackerel processing by-products, relates to the technical field of high-value processing of aquatic by-products and preparation of functional fish oil, and specifically comprises the following steps: after mackerel by-products are subjected to low-temperature mincing and natural antioxidant treatment, the mackerel by-products are subjected to composite enzymolysis, pH-salt synergistic demulsification and centrifugal separation to obtain crude oil, ceramic membrane refining and dehydration, ethanolysis to obtain ethyl ester, two-stage molecular distillation enrichment, immobilized lipase catalytic re-esterification into rTG type, short-path distillation impurity removal, nitrogen filling and sealing, and finally high-purity rTG fish oil products are obtained; the method takes mackerel by-products as raw materials, adopts low-temperature pretreatment and natural antioxidant, carries out oxygen isolation by using nitrogen gas in the whole process, retains the structure of unsaturated fatty acids by composite enzymolysis, improves separation efficiency by pH-salt synergistic demulsification and ceramic membrane refining, carries out ethanolysis and two-stage molecular distillation enrichment, and then carries out immobilized lipase catalytic re-esterification into rTG type, short-path distillation impurity removal, nitrogen filling and sealing, so that the method is environmentally friendly, safe and has high bioavailability.

Description

Technical Field

[0001] This invention relates to the field of high-value processing of aquatic by-products and preparation of functional fish oil, specifically a method for preparing high-content rTG-type fish oil using mackerel processing by-products. Background Technology

[0002] The seafood processing industry occupies an important position in the food industry system. Mackerel, as an economically important marine fish with stable production and large-scale fishing, generates a large amount of by-products during processing, such as fish heads, bones, skin, viscera, and scraps of fish meat. Currently, most seafood processing enterprises sell these by-products at low prices or dispose of them directly. Only a small amount of by-products are used for simple fishmeal processing. The abundant unsaturated fatty acid resources within these by-products are not fully utilized, resulting in a serious waste of high-quality lipid resources. Fish oil is a functional oil rich in polyunsaturated fatty acids, among which EPA and D... HA has multiple physiological benefits, such as regulating blood lipids and maintaining cardiovascular health. The market demand for high-purity, high-absorption-efficiency fish oil products continues to increase. Conventional fish oil raw materials mostly use whole fish meat, resulting in high raw material procurement costs and production capacity being easily limited by the fishing cycle. Compared with fatty acid ethyl ester fish oil, structured triglyceride fish oil has significant advantages in human digestion and absorption, and is the mainstream development direction of the high-end functional fish oil market. The industry urgently needs a complete set of processing technology that can stably produce high-quality structured triglyceride fish oil using aquatic processing waste.

[0003] Existing conventional processing methods for extracting fish oil from aquatic byproducts mostly employ high-temperature steaming and pressing. The high-temperature environment directly damages the molecular structure of unsaturated fatty acids, causing oil oxidation and deterioration. The finished product often exceeds oxidation limits, requiring the addition of large amounts of synthetic antioxidants to extend shelf life, posing a potential food safety hazard. Single-protease hydrolysis has low oil release efficiency and severe emulsification after enzymatic hydrolysis. Simply relying on static separation of crude fish oil results in low oil recovery rates and high impurity content. Subsequent refining processes require large amounts of acid and alkali reagents for decolorization and degumming, producing high concentrations of... Salt-containing organic wastewater presents significant environmental challenges. Traditional fish oil purification relies solely on single distillation to enrich unsaturated fatty acids, resulting in limited enrichment and failing to meet the active substance content standards required for high-end fish oil. Most processing techniques can only produce fatty acid ethyl ester fish oil. To convert it into triglyceride products, chemical catalytic esterification is often employed, which involves harsh reaction conditions, numerous byproducts, unrecoverable catalysts, high raw material losses, and difficulty in controlling residual ethyl ester content in the finished product. Furthermore, the entire processing lacks systematic oxygen-barrier protection measures, and the step-by-step operation of each process is poorly integrated, making it unsuitable for continuous, large-scale industrial production. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for preparing high-rTG type fish oil using mackerel processing by-products. First, low-temperature pretreatment combined with a natural compound antioxidant isolates oxidation. Then, nitrogen-protected enzymatic hydrolysis fully releases the oil. Combined with pH-salt synergistic demulsification and ceramic membrane refining, the crude fish oil is purified. After ethanol hydrolysis and two-stage molecular distillation to enrich unsaturated fatty acids, immobilized lipase catalyzes vacuum re-esterification, followed by short-path distillation for impurity removal and nitrogen sealing to obtain the finished product. The entire process is carried out under controlled oxygen and low temperature conditions, reducing the loss of unsaturated fatty acids, improving oil extraction rate and product purity. The immobilized enzyme is recyclable, reducing production costs, and the resource utilization of waste alleviates the environmental pressure on aquatic product processing.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for preparing high-rTG type fish oil using mackerel processing by-products, the specific steps of which are as follows: S1, take mackerel processing by-products, which include fish head, fish bones, fish skin, fish belly meat and fish viscera, of which fish belly meat and fish viscera account for 25%-55% of the total mass of the by-products, and fish head, fish bones and fish skin account for 45%-75%; wash, drain and mince to 3-8mm at 0-8℃, add a natural antioxidant compound of mixed tocopherol and rosemary extract, and mix well; S2, add water to the pretreated raw material to a material-to-liquid mass ratio of 1:(1.0-1.4), purge with nitrogen, add a compound protease consisting of neutral protease and papain, adjust the pH to 6.8-7.2, enzymatically hydrolyze at 46-50℃ for 2-3 hours, and then heat to 78-82℃ to inactivate the enzyme for 8-12 minutes. S3, cool the enzyme hydrolysate after enzyme inactivation to 48-52℃, add food-grade sodium chloride to dissolve, adjust the pH to 5.3-5.6, let stand for 20-35 minutes, centrifuge, and collect the upper crude fish oil phase; S4. Crude fish oil is filtered through an inorganic ceramic membrane in a cross-flow manner. The clarified permeate is collected and dehydrated under low temperature and vacuum to obtain refined mackerel oil. S5, refined fish oil is mixed with anhydrous ethanol and sodium ethoxide catalyst, and carried out an alcoholysis reaction under nitrogen protection; after the reaction, water is added for washing, and the mixture is allowed to stand for separation. The upper fatty acid ethyl ester phase is collected, and residual ethanol and water are removed under reduced pressure to obtain fish oil ethyl ester; the fish oil ethyl ester is enriched by two-stage molecular distillation to obtain enriched ethyl ester. S6 involves re-esterifying the enriched ethyl ester with food-grade glycerol under vacuum catalysis by immobilized lipase. After filtering to separate the immobilized lipase, the product is purified by short-path distillation, sealed and stored under high-purity nitrogen to obtain the rTG type fish oil product.

[0006] Furthermore, in S1, the mass ratio of mixed tocopherols to rosemary extract in the natural antioxidant is 1:(0.3-0.8), and the total amount added accounts for 0.04%-0.08% of the mass of the by-product.

[0007] Furthermore, in S2, the mass ratio of neutral protease to papain in the complex protease is 1:(0.6-0.9), and the total amount added accounts for 0.9%-1.4% of the mass of the byproduct; the enzymatic hydrolysis reaction time is 2-3 hours, and the enzyme inactivation incubation time is 8-12 minutes.

[0008] Furthermore, in S2, after nitrogen purging, a slight positive pressure of 0.02-0.05 MPa is maintained inside the reactor, and the headspace oxygen content inside the reactor is reduced to no more than 0.5%; the stirring rate during enzymatic hydrolysis is 50-80 r / min.

[0009] Furthermore, in step S3, the amount of food-grade sodium chloride added is 2.0%-3.0% of the total mass of the enzymatic hydrolysate; the standing demulsification time is 20-35 minutes; the centrifugation speed is 3000-4000 r / min, and the centrifugation time is 10-15 minutes.

[0010] Furthermore, in step S4, the inorganic ceramic membrane is made of alumina with a pore size of 0.3-0.6 μm; the cross-flow filtration temperature is 30-38℃, the transmembrane pressure difference is 0.05-0.15 MPa, and the filtration is repeated 2-3 times; the low-temperature vacuum dehydration temperature is 35-45℃, the vacuum degree is -0.08 MPa to -0.095 MPa, and the dehydration is carried out until the water content of the fish oil is ≤0.25%.

[0011] Furthermore, in S5, the mass ratio of refined mackerel oil to anhydrous ethanol is 100:(35-55), the amount of sodium ethoxide catalyst added is 0.3%-0.8% of the mass of refined fish oil, the alcohol hydrolysis reaction temperature is 42-52℃, and the reaction time is 1-2 hours.

[0012] Furthermore, in step S5, the ethyl ester of fish oil obtained after the ethanololysis reaction has a total EPA+DHA content of 30%-50%; the two-stage molecular distillation enrichment adopts a scraped-film device with nitrogen protection throughout the process; the first-stage distillation temperature is 95-115℃, the second-stage distillation temperature is 120-145℃, the system absolute pressure is 0.1-5Pa, and the residence time of single-stage material is ≤60 seconds; the total EPA+DHA content in the enriched ethyl ester obtained after two-stage molecular distillation enrichment is 68%-76%.

[0013] Furthermore, in S6, the mass ratio of enriched ethyl ester to food-grade glycerol is 100:(7-10); the immobilized lipase is immobilized Candida antarcticis lipase, and the amount added is 4%-8% of the mass of enriched ethyl ester; the re-esterification reaction temperature is 50-55℃, the absolute pressure of the system is 0.5-2kPa, and the reaction time is 8-12 hours.

[0014] Furthermore, in S6, the conditions for short-path distillation are: temperature 90-135℃, system absolute pressure 0.5-10Pa; the rTG type fish oil product obtained after the short-path distillation meets the following indicators: total EPA+DHA content is 65%-75%, triglyceride lipids account for ≥80% of the total oil content, and residual ethyl ester content is ≤15%.

[0015] Compared with existing technologies, this method for preparing high-rTG type fish oil using mackerel processing by-products has the following advantages: I. This invention utilizes various by-products from mackerel processing as raw materials, broadening the sources of fish oil raw materials, reducing the environmental pressure caused by the direct discharge of aquatic processing waste, and realizing the high-value recycling of aquatic processing resources. It combines low-temperature pretreatment with compound natural antioxidants and a nitrogen-based oxygen-isolation protection system throughout the entire process. From raw material processing, enzymatic hydrolysis, esterification to distillation, the entire process inhibits oil oxidation and rancidity, significantly reducing the generation of peroxides and aldehydes during fish oil processing. It eliminates the need for large amounts of artificially synthesized antioxidants, improving the safety of the finished product. The invention employs a compound protease-assisted mild enzymatic hydrolysis process, which, compared to traditional high-temperature cooking and oil extraction methods, can completely preserve the unsaturated fatty acid structure in fish oil. Combined with pH-salt synergistic demulsification and ceramic membrane cross-flow refining processes, it improves the separation efficiency and clarity of crude fish oil, simplifies subsequent refining steps, reduces the excessive use of acids, alkalis, and organic solvents, lowers production wastewater treatment costs, and balances economic efficiency and environmental protection.

[0016] II. This invention utilizes a series of processes combining ethanol hydrolysis, two-stage molecular distillation enrichment, and enzymatic vacuum re-esterification to convert ordinary fish oil into high-purity structural triglyceride-type fish oil. This effectively increases the total content of unsaturated fatty acids in the product while optimizing the oil molecular structure. Compared to ordinary ethyl ester-type fish oil, it better aligns with the human lipid absorption and metabolism pathway, improving the bioavailability of nutrients. Immobilized lipase is used to catalyze the esterification reaction, and the catalyst can be filtered, recycled, and reused, reducing the continuous investment cost of catalytic materials. Short-path distillation removes residual ethyl esters and small molecule impurities, precisely controlling the proportion of lipid components in the finished product. The entire process employs low-pressure, low-temperature distillation combined with a nitrogen-sealed storage system to avoid high-temperature, long-term processing that damages active unsaturated fatty acids, resulting in a more stable finished product with a longer storage period. The entire process is suitable for large-scale continuous production, with smooth process connections, eliminating the need for complex intermittent operations, and stably producing high-quality structural triglyceride-type fish oil products.

[0017] Other advantages, objectives and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination or study, or may be learned from the practice of the invention. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0019] Figure 1 This is a flowchart of an overall process for preparing high-rTG type fish oil using mackerel processing by-products. Figure 2 Flowchart of nitrogen-protected compound enzymatic hydrolysis process for mackerel by-products; Figure 3 Flowchart of the process for demulsifying and extracting crude fish oil by centrifugation with pH-salt synergistic effect of enzymatic hydrolysate. Detailed Implementation

[0020] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structure, features and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0021] Example 1: Weigh 20 kg of mackerel processing by-products, which include fish head, bones, skin, belly meat, and viscera. The fish head accounts for 25%, fish bones 18%, fish skin 12%, belly meat 25%, and viscera 20%. The belly meat and viscera together account for 45% of the total by-product mass, and the fish head, bones, and skin together account for 55%. Specific steps are as follows... Figure 1 As shown.

[0022] The raw materials are washed with clean water at 4℃ to remove blood and impurities from the surface. After washing, they are laid flat to drain until no more water droplets fall. Then they are fed into a crushing equipment to be crushed to a particle size of 3mm to 8mm.

[0023] Prepare a natural antioxidant mixture by mixing tocopherol and rosemary extract in a mass ratio of 1:0.5. The total amount of antioxidant added is 0.06% of the total mass of by-products. Sprinkle the antioxidant evenly into the shredded material and stir continuously until it is evenly mixed.

[0024] Add purified water to the mixture, controlling the material-to-liquid mass ratio to 1:1.2. Transfer all materials to a sealed reactor, purge the air inside the reactor with nitrogen to replace it, maintaining a slight positive pressure of 0.03 MPa until the headspace oxygen content inside the reactor drops below 0.5%. Figure 2 As shown.

[0025] A compound protease was prepared with a mass ratio of neutral protease to papain of 1:0.8. The total amount of compound protease added was 1.2% of the total mass of by-products. The protease was added to the material in the reactor, and the pH of the system was adjusted to 7.0 using food-grade dilute hydrochloric acid and sodium bicarbonate. The stirring device was turned on, the stirring speed was 60 r / min, and the temperature was controlled at 48℃ for continuous enzymatic hydrolysis for 2.5 h.

[0026] After the enzymatic hydrolysis process is completed, the temperature is raised to 80℃ and maintained for 10 minutes to complete the enzyme inactivation operation and terminate the protease activity.

[0027] Cool the enzyme-inactivated hydrolysate to 50°C, weigh out food-grade sodium chloride and add it to the system. The amount of sodium chloride added is 2.5% of the total mass of the hydrolysate. Continue stirring until the sodium chloride is completely dissolved. Adjust the pH of the system to 5.5, and let it stand for 30 minutes to complete the pH-salt synergistic demulsification. Figure 3 As shown.

[0028] After demulsification, the mixed liquid is sent to a tubular centrifuge, set to 3500 r / min, centrifuged for 12 min, and after separation, the upper crude fish oil phase is collected, and the lower protein aqueous phase and intermediate solid residue are removed.

[0029] An inorganic ceramic membrane made of alumina with a pore size of 0.45μm was selected. The crude fish oil was sent into a cross-flow filtration device, the filtration temperature was controlled at 35℃, the transmembrane pressure difference was 0.10MPa, and the filtration was repeated twice. The clarified fish oil that permeated through the ceramic membrane was collected.

[0030] The clarified fish oil was transferred to a vacuum dehydration device, and the dehydration temperature was set to 40℃ and the vacuum degree to -0.09MPa. The dehydration was continued until the moisture content of the fish oil was ≤0.25%, resulting in low-oxidation mackerel oil. The intermediate product had a total EPA and DHA content of 28.1%, a peroxide value of 2.3 meq O2 / kg, and a moisture content of 0.22%.

[0031] Take all the low-oxidation mackerel oil and mix it with anhydrous ethanol at a mass ratio of 100:45. Add sodium ethoxide catalyst, with the amount of catalyst added being 0.5% of the total mass of refined fish oil. Nitrogen gas is introduced throughout the process to isolate oxygen, and the alcoholysis reaction is carried out at a controlled temperature of 48℃ for 1.5 hours.

[0032] After the reaction was completed, pure water was added to the system and the mixture was stirred and washed. After standing and separating into layers, the upper fatty acid ethyl ester phase was collected. The residual ethanol and free water in the system were removed by vacuum distillation to obtain fish oil ethyl ester. The total content of EPA and DHA in fish oil ethyl ester was 39.2%.

[0033] Ethyl fish oil was fed into a scraped-film two-stage molecular distillation apparatus, with nitrogen protection throughout the process. The system absolute pressure was 1 Pa, and the residence time of each stage was 45 s.

[0034] The first-stage distillation temperature is 105℃ to remove short-chain fatty acids and light component impurities. The first-stage heavy phase material is collected and enters the second-stage molecular distillation temperature of 132℃ to separate long-chain saturated fatty acids, finally obtaining enriched ethyl ester. The total content of enriched ethyl ester EPA plus DHA is 72.8%, the peroxide value is 3.0 meq O2 / kg, and the moisture content is 0.08%.

[0035] Weigh the enriched ethyl ester and food-grade glycerol in a mass ratio of 100:8. Add immobilized Candida antarcticis lipase to the mixture at a rate of 6% of the total mass of the enriched ethyl ester. Transfer the material to a vacuum reactor and control the absolute pressure of the system at 1.2 kPa and the temperature at 52°C. Stir continuously for 10 hours and continuously remove the generated ethanol under reduced pressure during the reaction.

[0036] After the reaction is complete, the immobilized lipase is separated by filtration, and the mixed oil is sent to a short-path distillation device. The distillation temperature is 115℃ and the system absolute pressure is 3Pa to remove free fatty acids and residual ethyl esters from the system.

[0037] After distillation, the mixture is filled with high-purity nitrogen, sealed, and stored in a light-proof environment to obtain the rTG type fish oil product.

[0038] Finished product testing indicators: EPA plus DHA total content 70.6%, triglyceride lipids account for 83.2% of total oil content, residual ethyl ester content 8.8%, peroxide value 3.2 meq O2 / kg, p-anisidine value 8.6, TOTOX value 15.0, acid value 2.2 mg KOH / g, moisture content 0.19%, turbidity 4.8 NTU.

[0039] Example 2: Take 20kg of mackerel processing by-products, and keep the mass ratio of each component of the by-products consistent with that in Example 1. Rinse thoroughly with clean water at 6℃, drain, and then mince to a particle size range of 3mm to 8mm.

[0040] The natural antioxidant is a mixture of tocopherols and rosemary extract in a 1:0.4 ratio. The total amount of antioxidant added is 0.05% of the total mass of by-products. The mixture is thoroughly stirred and homogenized.

[0041] Add water to adjust the feed-to-liquid mass ratio to 1:1.2, replace the air inside the reactor with nitrogen to maintain a slight positive pressure of 0.02 MPa inside the reactor, and keep the headspace oxygen content below 0.5%.

[0042] The complex protease was prepared by mixing neutral protease and papain at a mass ratio of 1:0.7, with the total addition amount being 1.0% of the total mass of the raw materials. The pH of the system was adjusted to 6.9, the stirring rate was 50 r / min, and the enzyme was enzymatically hydrolyzed at 47℃ for 3 h.

[0043] After enzymatic hydrolysis, raise the temperature to 78°C and incubate for 12 minutes to complete the enzyme inactivation process.

[0044] Cool the enzymatic hydrolysate to 48°C, add 2.2% (by weight of total enzymatic hydrolysate) of food-grade sodium chloride, and after complete dissolution, adjust the pH to 5.6. Let it stand for 20 minutes to complete demulsification.

[0045] Centrifuge at 3000 r / min for 15 min and collect the upper crude fish oil phase.

[0046] A 0.3μm alumina ceramic membrane was selected, the cross-flow filtration temperature was 30℃, the transmembrane pressure difference was 0.08MPa, and the filtration was repeated 3 times to collect the clarified fish oil.

[0047] Vacuum dehydration was carried out at 35℃ and a vacuum degree of -0.08MPa until the moisture content was ≤0.25%, resulting in low-oxidation mackerel oil.

[0048] The mass ratio of refined fish oil to anhydrous ethanol was 100:40, the amount of sodium ethoxide catalyst added was 0.4%, and the ethanololysis reaction was carried out at 42℃ for 2 hours.

[0049] Ethanol was removed by water washing and layering to obtain ethyl ester of fish oil. The ethyl ester was then sent to a two-stage molecular distillation system with a pressure of 1 Pa, a material residence time of 50 s, a first-stage distillation temperature of 95 ℃, and a second-stage distillation temperature of 120 ℃ to obtain enriched ethyl ester. The enriched ethyl ester had a total EPA and DHA content of 70.9%, a peroxide value of 2.9 meq O2 / kg, and a moisture content of 0.09%.

[0050] The ethyl ester to glycerol mass ratio was 100:7, the immobilized lipase was added at 5%, the reaction was carried out at 50℃ and 0.5 kPa for 12 h, and the ethanol was removed under reduced pressure during the reaction.

[0051] The enzyme preparation was separated by filtration, and impurities were removed by short-path distillation at 90℃ and a system pressure of 5Pa. The finished product, rTG fish oil, was obtained by sealing with high-purity nitrogen.

[0052] Finished product testing indicators: Total EPA and DHA content 68.9%, triglyceride content 81.7%, residual ethyl ester 10.6%, peroxide value 3.5 meq O2 / kg, p-anisidine value 9.4, TOTOX 16.4, acid value 2.4 mg KOH / g, moisture 0.22%, turbidity 5.4 NTU.

[0053] Example 3: Weigh 20kg of mackerel processing by-products, with the same component ratio as in Example 1, wash and drain at 5℃, and mince into 3mm to 8mm particles.

[0054] A mixture of tocopherols and rosemary extracts in a mass ratio of 1:0.7 was formulated as an antioxidant, with a total addition amount of 0.07% of the raw material mass. The mixture was stirred and mixed thoroughly.

[0055] The water-to-liquid mass ratio is controlled at 1:1.4. Nitrogen is used to replace the air in the reactor, and the reactor is kept under a slight positive pressure of 0.05 MPa, with the headspace oxygen content below 0.5%.

[0056] The compound protease, neutral protease, and papain were mixed in a 1:0.9 ratio, with a total addition amount of 1.4% of the raw material mass. The pH was adjusted to 7.1, the stirring rate was 80 r / min, and the enzyme was enzymatically hydrolyzed at 50℃ for 2 h.

[0057] Heat to 82℃ and hold for 8 minutes to complete enzyme inactivation.

[0058] Cool the enzymatic hydrolysate to 52°C, add 2.8% (by weight of food-grade sodium chloride) of the total enzymatic hydrolysate, dissolve, adjust the pH to 5.4, and let stand for 35 minutes to break the emulsion.

[0059] Centrifuge at 4000 r / min for 10 min and collect the upper crude fish oil phase.

[0060] A 0.6μm alumina ceramic membrane was selected, the filtration temperature was 38℃, the transmembrane pressure difference was 0.12MPa, and the filtration was repeated twice.

[0061] Vacuum dehydration was performed at 45℃ and a vacuum degree of -0.095MPa until the moisture content was ≤0.25%, yielding low-oxidation mackerel oil.

[0062] The refined fish oil and anhydrous ethanol were mixed at a mass ratio of 100:50, with sodium ethoxide added at 0.6%, and the mixture was ethanolically hydrolyzed at 52°C for 1 hour.

[0063] Fish oil ethyl ester was obtained by washing and de-alcoholizing with water. The two-stage molecular distillation system was operated at a pressure of 1 Pa, with a material residence time of 40 s. The first-stage distillation temperature was 115 °C, and the second-stage distillation temperature was 145 °C. The total content of EPA and DHA in the enriched ethyl ester was 73.6%, with a peroxide value of 3.2 meq O2 / kg and a moisture content of 0.07%.

[0064] The ethyl ester to glycerol mass ratio was 100:10, the immobilized lipase was added at 7%, the reaction was carried out at 55℃ and 2 kPa for 8 h, and ethanol was continuously removed during the reaction.

[0065] Lipase was removed by filtration, and residual ethyl esters and free fatty acids were removed by short-path distillation at 135°C and a system pressure of 10 Pa. The product was then sealed with nitrogen to obtain the final product.

[0066] Finished product testing indicators: EPA plus DHA total content 71.4%, triglyceride content 84.0%, residual ethyl ester 7.9%, peroxide value 3.6 meq O2 / kg, p-anisidine value 9.8, TOTOX 17.0, acid value 2.5 mg KOH / g, moisture 0.24%, turbidity 5.9 NTU.

[0067] Comparative Example 1: Take 20 kg of mackerel processing by-products that are exactly the same as those in Example 1, wash them with room temperature water and drain them, then put them directly into the cooking equipment and cook them at a constant temperature of 95°C for 20 minutes. After cooking, use a screw press to press and separate the oil. After pressing, let the crude oil stand to remove solid residue.

[0068] The crude oil is pressed without the addition of antioxidants or nitrogen protection. All subsequent operating parameters, including pH salt demulsification, ceramic membrane filtration, vacuum dehydration, ethanol hydrolysis, two-stage molecular distillation, lipase re-esterification, and short-path distillation and sealing, are replicated from Example 1.

[0069] Finished product testing indicators: fish oil recovery rate 78.1%, total EPA and DHA content 69.4%, triglyceride content 82.5%, residual ethyl ester 9.5%, peroxide value 5.8 meq O2 / kg, p-anisidine value 13.2, TOTOX 24.8, acid value 2.8 mg KOH / g, moisture 0.27%, turbidity 7.6 NTU.

[0070] Comparative Example 2: Example 1 replicates all operating parameters for raw material low-temperature pretreatment, nitrogen-enzymatic hydrolysis, and enzyme inactivation.

[0071] After enzyme inactivation, the addition of sodium chloride and pH adjustment are omitted. After the enzyme hydrolysate is cooled to 50°C, it is allowed to stand for 30 minutes. Then, it is centrifuged at 3500 r / min for 12 minutes to collect the crude fish oil phase.

[0072] The ceramic membrane refining, ethanol hydrolysis, molecular distillation, enzymatic re-esterification, and short-path distillation sealing were all performed according to the parameters in Example 1.

[0073] Finished product testing indicators: fish oil recovery rate 75.8%, total EPA and DHA content 68.7%, triglyceride content 81.1%, residual ethyl ester 11.8%, peroxide value 4.2 meq O2 / kg, p-anisidine value 10.7, TOTOX 19.1, acid value 2.6 mg KOH / g, moisture 0.31%, turbidity 9.2 NTU.

[0074] Comparative Example 3: The steps of low-temperature pretreatment, nitrogen-enzymatic hydrolysis, enzyme inactivation, pH-salt synergistic demulsification, and centrifugation to separate crude fish oil are completely consistent with those in Example 1.

[0075] The crude fish oil obtained by centrifugation is directly fed into a vacuum dehydration device, omitting the alumina ceramic membrane cross-flow filtration process. The vacuum dehydration parameters are consistent with those in Example 1.

[0076] Example 1 replicates the process of ethanol hydrolysis, two-stage molecular distillation, enzymatic re-esterification, and short-path distillation.

[0077] Finished product testing indicators: fish oil recovery rate 82.4%, total EPA and DHA content 69.8%, triglyceride content 80.6%, residual ethyl ester 12.7%, peroxide value 4.6 meq O2 / kg, p-anisidine value 11.9, TOTOX 21.1, acid value 2.8 mg KOH / g, moisture 0.33%, turbidity 14.8 NTU.

[0078] Comparative Example 4: Raw material pretreatment, nitrogen enzymatic hydrolysis, pH salt demulsification, ceramic membrane filtration, vacuum dehydration, ethanol hydrolysis, and two-stage molecular distillation were all performed according to the process in Example 1 to obtain enriched ethyl ester.

[0079] Ethyl esters are enriched and no longer mixed with glycerol and immobilized lipase for re-esterification. Instead, light component impurities are removed by direct short-path distillation, and the product is stored under nitrogen-sealed conditions. The final product is ethyl ester-type fish oil.

[0080] Finished product testing indicators: fish oil recovery rate 84.1%, total EPA and DHA content 72.8%, triglyceride lipid content 9.4%, residual ethyl ester content 86.5%, peroxide value 3.4 meq O2 / kg, p-anisidine value 9.1, TOTOX 15.9, acid value 2.6 mg KOH / g, moisture 0.21%, turbidity 5.1 NTU.

[0081] Comparative Example 5: The low-temperature pretreatment, nitrogen-enzymatic hydrolysis, and enzyme inactivation process are exactly the same as in Example 1.

[0082] After the enzyme-inactivating solution is cooled to 50°C, without adding food-grade sodium chloride, the pH of the system is adjusted to 5.5 using only dilute hydrochloric acid. The mixture is allowed to stand for 30 minutes to break the emulsion, and then centrifuged at 3500 r / min for 12 minutes to collect the crude fish oil.

[0083] The ceramic membrane refining, ethanol hydrolysis, molecular distillation, re-esterification, and short-path distillation all followed the parameters of Example 1.

[0084] The crude fish oil phase moisture content was 0.63%. The finished product test indicators were: fish oil recovery rate 79.4%, finished product moisture content 0.28%, turbidity 8.7 NTU, and peroxide value 3.7 meq O2 / kg.

[0085] Comparative Example 6: The raw material pretreatment, nitrogen-protected compound enzymatic hydrolysis, and enzyme inactivation steps are consistent with those in Example 1.

[0086] Cool the enzyme inactivation solution to 50°C, add 2.5% sodium chloride (by total mass of the enzyme hydrolysate) and dissolve completely. Do not adjust the pH of the system. Let it stand for 30 minutes and then centrifuge to collect the crude fish oil phase.

[0087] The subsequent ceramic membrane filtration, ethanol hydrolysis, molecular distillation, enzymatic re-esterification, and distillation sealing operations were completely replicated in Example 1.

[0088] The crude fish oil phase moisture content was 0.69%. The finished product test indicators were: fish oil recovery rate 78.7%, finished product moisture content 0.30%, turbidity 10.1 NTU, and peroxide value 3.9 meq O2 / kg.

[0089] Performance testing: Fish oil recovery rate was calculated as the ratio of the actual mass of low-oxidized mackerel oil obtained to the total extractable oil mass of the same batch of raw materials; peroxide value was determined by iodometric titration; acid value was determined by acid-base titration; EPA and DHA content were determined by gas chromatography; the proportion of triglycerides and residual ethyl esters were determined by high performance liquid chromatography; moisture content was determined by moisture determination method; turbidity was determined by turbidimeter; p-anisidine value was determined according to the oil oxidation evaluation method; TOTOX value was calculated as 2 × peroxide value + p-anisidine value.

[0090] Table 1 shows the main indicators of intermediate products during the process.

[0091]

[0092] Table 2 shows the performance measurement comparison data of the examples and comparative examples 1 to 4.

[0093]

[0094] Table 3 shows the comparative test data of different demulsification methods.

[0095]

[0096] Experimental conclusion: The complete process in Examples 1 to 3 includes low-temperature nitrogen-protected enzymatic hydrolysis, pH-salt synergistic demulsification, ceramic membrane coalescence clarification, ethanol hydrolysis conversion, two-stage molecular distillation enrichment, and immobilized lipase re-esterification. The fish oil recovery rate ranges from 82.7% to 84.6%, the total EPA plus DHA content of the finished product ranges from 68.9% to 71.4%, the proportion of triglyceride lipids ranges from 81.7% to 84.0%, the residual ethyl ester content ranges from 7.9% to 10.6%, the peroxide value ranges from 3.2 to 3.6 meq O2 / kg, the p-anisidine value ranges from 8.6 to 9.8, the TOTOX value ranges from 15.0 to 17.0, the acid value ranges from 2.2 to 2.5 mg KOH / g, and the moisture content ranges from 0.19% to 0.24%.

[0097] Compared with the cooking and pressing oil extraction process in Comparative Example 1, the low-temperature nitrogen complex enzymatic hydrolysis of this invention can significantly reduce the peroxide value, p-anisidine value and TOTOX value of oil while keeping the subsequent enrichment and re-esterification parameters unchanged, and effectively inhibit the oxidation of polyunsaturated fatty acids.

[0098] Compared with Comparative Example 2, which omitted the pH-salt synergistic demulsification process, the synergistic demulsification method of simultaneously adjusting pH and adding sodium chloride can improve oil recovery efficiency, reduce the moisture content of the finished product, and improve oil separation effect.

[0099] Compared with Comparative Example 3, which omits the ceramic membrane coalescence clarification process, the alumina ceramic membrane cross-flow filtration can remove raw material protein colloids and fine suspended matter, reduce the turbidity and moisture residue of the finished product, and reduce the interference of impurities on subsequent distillation and re-esterification reactions.

[0100] Compared with Comparative Example 4, which omitted the immobilized lipase re-esterification process, the high-content ethyl ester raw material can be converted into rTG-type fish oil with triglycerides as the main component after the enzyme-catalyzed re-esterification reaction, which meets the product index requirements for the absorption advantage of edible fish oil. The products that have not undergone re-esterification treatment are mostly ethyl ester structures and do not meet the high rTG fish oil standard.

[0101] Comparing Comparative Example 5 and Comparative Example 6 with single demulsification methods, neither adjusting pH alone nor adding sodium chloride alone could achieve efficient demulsification. The fish oil recovery rate decreased, the crude fish oil contained a large amount of water, and the final product showed significant deterioration in turbidity, moisture content, and peroxide value. This proves that the synergistic effect of pH adjustment and sodium chloride combination is a key condition for ensuring efficient oil-water separation.

[0102] All experimental data prove that the complete and defined continuous process combination of this invention can stably prepare high-content rTG-type fish oil with excellent indicators using mackerel processing by-products as raw materials.

[0103] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for preparing high-rTG-type fish oil using mackerel processing by-products, characterized in that, The specific steps of this method are as follows: S1, take mackerel processing by-products, which include fish head, fish bones, fish skin, fish belly meat and fish viscera, of which fish belly meat and fish viscera account for 25%-55% of the total mass of the by-products, and fish head, fish bones and fish skin account for 45%-75%; wash, drain and mince to 3-8mm at 0-8℃, add a natural antioxidant compound of mixed tocopherol and rosemary extract, and mix well; S2, add water to the pretreated raw material to a material-to-liquid mass ratio of 1:(1.0-1.4), purge with nitrogen, add a compound protease consisting of neutral protease and papain, adjust the pH to 6.8-7.2, enzymatically hydrolyze at 46-50℃ for 2-3 hours, and then heat to 78-82℃ to inactivate the enzyme for 8-12 minutes. S3, cool the enzyme hydrolysate after enzyme inactivation to 48-52℃, add food-grade sodium chloride to dissolve, adjust the pH to 5.3-5.6, let stand for 20-35 minutes, centrifuge, and collect the upper crude fish oil phase; S4. Crude fish oil is filtered through an inorganic ceramic membrane in a cross-flow manner. The clarified permeate is collected and dehydrated under low temperature and vacuum to obtain refined mackerel oil. S5, refined fish oil is mixed with anhydrous ethanol and sodium ethoxide catalyst, and carried out an alcoholysis reaction under nitrogen protection; after the reaction, water is added for washing, and the mixture is allowed to stand for separation. The upper fatty acid ethyl ester phase is collected, and residual ethanol and water are removed under reduced pressure to obtain fish oil ethyl ester; the fish oil ethyl ester is enriched by two-stage molecular distillation to obtain enriched ethyl ester. S6 involves re-esterifying the enriched ethyl ester with food-grade glycerol under vacuum catalysis by immobilized lipase. After filtering to separate the immobilized lipase, the product is purified by short-path distillation, sealed and stored under high-purity nitrogen to obtain the rTG type fish oil product.

2. The method for preparing high-rTG type fish oil using mackerel processing by-products according to claim 1, characterized in that, In step S1, the mass ratio of mixed tocopherols to rosemary extract in the natural antioxidant is 1:(0.3-0.8), and the total amount added accounts for 0.04%-0.08% of the mass of the by-product.

3. The method for preparing high-rTG type fish oil using mackerel processing by-products according to claim 1, characterized in that, In S2, the mass ratio of neutral protease to papain in the complex protease is 1:(0.6-0.9), and the total amount added accounts for 0.9%-1.4% of the mass of the byproduct; the enzymatic hydrolysis reaction time is 2-3 hours, and the enzyme inactivation incubation time is 8-12 minutes.

4. The method for preparing high-rTG type fish oil using mackerel processing by-products according to claim 1, characterized in that, In step S2, after nitrogen purging, a slight positive pressure of 0.02-0.05 MPa is maintained inside the reactor, and the headspace oxygen content inside the reactor is reduced to no more than 0.5%; the stirring rate during enzymatic hydrolysis is 50-80 r / min.

5. The method for preparing high-rTG type fish oil using mackerel processing by-products according to claim 1, characterized in that, In step S3, the amount of food-grade sodium chloride added is 2.0%-3.0% of the total mass of the enzymatic hydrolysate; the standing demulsification time is 20-35 minutes; the centrifugation speed is 3000-4000 r / min, and the centrifugation time is 10-15 minutes.

6. The method for preparing high-rTG type fish oil using mackerel processing by-products according to claim 1, characterized in that, In step S4, the inorganic ceramic membrane is made of alumina with a pore size of 0.3-0.6 μm; the cross-flow filtration temperature is 30-38℃, the transmembrane pressure difference is 0.05-0.15 MPa, and the filtration is repeated 2-3 times; the low-temperature vacuum dehydration temperature is 35-45℃, the vacuum degree is -0.08 MPa to -0.095 MPa, and the dehydration is carried out until the water content of the fish oil is ≤0.25%.

7. The method for preparing high-rTG type fish oil using mackerel processing by-products according to claim 1, characterized in that, In step S5, the mass ratio of refined mackerel oil to anhydrous ethanol is 100:(35-55), the amount of sodium ethoxide catalyst added is 0.3%-0.8% of the mass of refined fish oil, the alcohol hydrolysis reaction temperature is 42-52℃, and the reaction time is 1-2 hours.

8. The method for preparing high-rTG type fish oil using mackerel processing by-products according to claim 1, characterized in that, In step S5, the fish oil ethyl ester obtained after the ethanololysis reaction has a total EPA+DHA content of 30%-50%; the two-stage molecular distillation enrichment adopts a scraped film device with nitrogen protection throughout the process; the first-stage distillation temperature is 95-115℃, the second-stage distillation temperature is 120-145℃, the system absolute pressure is 0.1-5Pa, and the single-stage material residence time is ≤60 seconds; the enriched ethyl ester obtained after two-stage molecular distillation enrichment has a total EPA+DHA content of 68%-76%.

9. A method for preparing high-rTG type fish oil using mackerel processing by-products according to claim 1, characterized in that, In S6, the mass ratio of enriched ethyl ester to food-grade glycerol is 100:(7-10); the immobilized lipase is immobilized Candida antarcticis lipase, and the amount added is 4%-8% of the mass of enriched ethyl ester; the re-esterification reaction temperature is 50-55℃, the absolute pressure of the system is 0.5-2kPa, and the reaction time is 8-12 hours.

10. A method for preparing high-rTG type fish oil using mackerel processing by-products according to claim 1, characterized in that, In S6, the conditions for short-path distillation are: temperature 90-135℃, system absolute pressure 0.5-10Pa.

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