Fish oil, preparation method and application thereof, and fish oil emulsion

Abstract

The invention relates to the technical field of fish oil preparation, and discloses fish oil, a preparation method and application thereof and a fish oil emulsion. The preparation method of the fish oil comprises the following steps: crushing carassius auratus gibelio, and adding water to prepare homogenate; adding one or more than two enzymes selected from flavourzyme, compound protease and alkaline protease into the homogenate for enzymolysis, and separating grease after enzymolysis to obtain the fish oil. The carassius auratus gibelio serves as a preparation raw material, protease suitable for the carassius auratus gibelio is adopted for enzymolysis to prepare the fish oil, meanwhile, the optimal enzymolysis process parameters are determined, compared with commercially available fish oil and Hunan crucian carp oil, the carassius auratus gibelio oil has the advantages that the blood fat improving effect is achieved, more unsaturated fatty acids are obtained, the pancreatic lipase activity inhibition rate and the cholesterol micelle solubility inhibition rate are remarkably increased, and the fish oil has a good application prospect. Therefore, the invention establishes a process method for preparing the hemlock crucian carp oil with a remarkable blood fat reducing effect, and the application range of hemlock crucian carp processing byproducts is further expanded.

Description

Technical Field

[0001] The present application relates to the technical field of fish oil, and in particular to fish oil prepared using Hefang crucian carp, and a preparation method and application thereof. Background Art

[0002] Freshwater fisheries, a key industrial sector in my country, generate a significant amount of by-products as the proportion of freshwater fish product processing increases. Their development and utilization are crucial for increasing industrial value-added and reducing resource depletion. Aquatic product processing by-products contain nutrients such as protein, amino acids, minerals, oils, and organic calcium. Fish fat, in particular, has attracted widespread attention due to its high concentration of unsaturated fatty acids, such as EPA and DHA, which have pharmacological activity against certain diseases. This has led to extensive research focused on the high-value utilization of lipids in fish by-products.

[0003] The most commonly used method for extracting fish oil is enzymatic hydrolysis. This method, characterized by mild reaction conditions, environmental friendliness, safety, high oil quality, and high-value byproducts, makes it an ideal method for recovering oil and protein from fish and their processing byproducts. The quality and efficacy of fish oil are closely related to its preparation process. Different processes often produce varying yields, nutritional value, appearance, amino acid composition, fatty acid profile, and hypoglycemic, lipid-lowering, and anti-hypertensive effects, as well as antioxidant activity. Summary of the Invention

[0004] In view of this, the purpose of this application is to provide a fish oil and a preparation method thereof, so that the prepared fish oil can significantly improve its blood lipid improvement effect and has higher nutritional value and quality;

[0005] Another purpose of the present application is to provide a use of the above-mentioned fish oil in the preparation of a product having the effect of improving blood lipids and to provide an emulsion product based on the above-mentioned fish oil.

[0006] In order to solve the above technical problems / achieve the above objectives or at least partially solve the above technical problems / achieve the above objectives, as a first aspect of the present application, a method for preparing fish oil is provided, comprising:

[0007] S1. Crush the crucian carp and add water to make a homogenate;

[0008] S2. Add one or more enzymes selected from flavor protease, composite protease, and alkaline protease to the homogenate for enzymatic hydrolysis, and separate the oil after enzymatic hydrolysis to obtain the fish oil.

[0009] Optionally, the enzymatic hydrolysis is performed using a combination of flavor protease and any one of a composite protease and an alkaline protease. Further optionally, the ratio of the flavor protease to the composite protease or alkaline protease to the total enzyme activity is 1:(1-3).

[0010] Optionally, the enzymatic hydrolysis temperature is 50-60° C., and the added amount is 1500±500 U / g.

[0011] Optionally, ultrasonic treatment is added in S2 to assist enzymatic hydrolysis.

[0012] As a second aspect of the present application, provided is fish oil prepared by the preparation method.

[0013] As a third aspect of the present application, provided is the use of the fish oil in preparing a product having the effect of improving blood lipids.

[0014] Optionally, the product comprises an emulsion.

[0015] As a fourth aspect of the present application, a fish oil emulsion having the effect of improving blood lipids is provided, comprising the fish oil described in the present application.

[0016] Optionally, emulsifiers and co-emulsifiers are also included.

[0017] This application uses Hefang crucian carp as the preparation raw material, adopts protease suitable for Hefang crucian carp for enzymatic hydrolysis to prepare fish oil, and at the same time establishes the optimal enzymatic hydrolysis process parameters. Compared with commercially available fish oil and Hunan crucian carp oil, the fish oil has more unsaturated fatty acids in improving blood lipids, and the inhibition rate of pancreatic lipase activity and the inhibition rate of cholesterol micelle solubility are significantly improved. Therefore, this application establishes a process method for preparing Hefang crucian carp oil with significant blood lipid-lowering effect, further expanding the application range of Hefang crucian carp processing by-products. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The drawings constituting a part of the present application are used to provide a further understanding of the present application. The exemplary embodiments and descriptions of the present application are used to explain the present application and do not constitute an improper limitation on the present application.

[0019] Figure 1 Shown are the results of fish oil extraction rates from crucian carp surimi by different single enzyme hydrolysis combinations;

[0020] Figure 2 The figure shows the fish oil extraction rate of crucian carp paste enzymatically hydrolyzed with different proportions of flavor protease and compound protease in the total enzyme activity;

[0021] Figure 3 The figure shows the fish oil extraction rate of crucian carp paste by single enzyme hydrolysis of flavor protease and compound protease and multi-enzyme combined hydrolysis;

[0022] Figure 4 The figures show the fish oil extraction rates of crucian carp raw materials using different methods; "dual enzyme hydrolysis to extract fish oil" (Group 1), "dual enzyme hydrolysis and simultaneous ultrasound extraction of fish oil" (Group 2), "ultrasound first and then dual enzyme hydrolysis to extract fish oil" (Group 3);

[0023] Figure 5 Shown are the health index results of different fish oils; XJ represents Xiang crucian carp oil, HFJ represents Hefang crucian carp oil;

[0024] Figure 6 Shown is a comparison of the lipid-lowering effects of different fish oils. DETAILED DESCRIPTION

[0025] The present application discloses a fish oil, a preparation method and application thereof, and a fish oil emulsion. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the desired results. It should be noted in particular that all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in this application. The products, processes and applications described in this application have been described through preferred embodiments. It is obvious that relevant personnel can modify or appropriately change and combine the methods described herein without departing from the content, spirit and scope of this application to implement and apply the technology of this application. Obviously, the described embodiments are part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0026] It should be noted that, in this document, if relational terms such as "first" and "second", "step 1" and "step 2", and "(1)" and "(2)" appear, they are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply that there is any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "including a..." do not exclude the presence of other identical elements in the process, method, article or device comprising the elements. At the same time, the embodiments in this application and the features in the embodiments can be combined with each other in the absence of conflict.

[0027] Hefang Crucian Carp is a new high-quality crucian carp variety bred by Hunan Normal University through distant hybridization. It is bred using Japanese white crucian carp (♀) as the female parent and red crucian carp (♂) as the male parent. It has the characteristics of fast growth, strong stress resistance, and excellent meat quality. It is a new high-quality freshwater fish variety in the field of aquaculture. Through research, this application found that using Hefang Crucian Carp as a preparation raw material can produce fish oil with higher lipid-lowering activity:

[0028] Therefore, in a first aspect of the present application, a method for preparing fish oil is provided, comprising:

[0029] S1. Crush the crucian carp and add water to make a homogenate;

[0030] S2. Add one or more enzymes selected from flavor protease, composite protease, and alkaline protease to the homogenate for enzymatic hydrolysis, and separate the oil after enzymatic hydrolysis to obtain the fish oil.

[0031] In certain embodiments of the present application, the crucian carp of the combined recipe is pre-processed, including descaling, de-eviscerating, cleaning, chopping into small pieces, and crushing into a minced state. In other embodiments of the present application, the material-liquid ratio of the crucian carp minced recipe to water is 1:(1-5), for example, 1:1, 1:2, 1:3, 1:4, or 1:5.

[0032] In certain embodiments of the present application, compound protease, flavor protease, neutral protease, papain, and alkaline protease were used to enzymatically hydrolyze the crucian carp. The results showed that flavor protease, compound protease, and alkaline protease had higher fish oil extraction rates, while neutral protease and papain had lower fish oil extraction rates; among them, flavor protease had the best fish oil extraction rate of nearly 50%.

[0033] In other embodiments of the present application, the enzymatic hydrolysis is performed using flavor protease and a combination of enzymes selected from a composite protease and an alkaline protease. In other embodiments of the present application, the ratio of the enzyme activity of the flavor protease to the composite protease or alkaline protease is 1:(1-3), for example, 1:1, 1:2, or 1:3. Although flavor protease exhibits significantly higher fish oil extraction efficiency than other proteases, when used in combination with the composite protease and alkaline protease, its usage should not exceed 50% of the total enzyme activity, with a 1:1 ratio being optimal. If the ratio of flavor protease exceeds 50%, for example, at a ratio of 2:1 or 3:1, the fish oil extraction efficiency will be significantly reduced, which reflects a certain synergistic effect between flavor protease, the composite protease, and the alkaline protease.

[0034] In certain embodiments of the present application, the enzymatic hydrolysis temperature is 50-60°C, for example, 50°C, 55°C, or 60°C, and the addition amount is 1500±500 U / g. In other embodiments of the present application, the enzymatic hydrolysis pH is 7.0-8.5, for example, 7.0, 7.5, 8.0, or 8.5; and the enzymatic hydrolysis time is 1-5 hours, for example, 1 hour, 3 hours, or 5 hours.

[0035] In certain embodiments of the present application, in order to further improve the fish oil extraction rate, an ultrasonic treatment is further added in S2 to assist enzymolysis, wherein the power of the ultrasonic treatment is 200-300W and the ultrasonic treatment time is 0.5-5h, for example, 30min, 50min, 1h, 2h, 3h, 4h or 5h. In other embodiments of the present application, the homogenate is subjected to ultrasonic treatment and enzymolysis simultaneously. In other embodiments of the present application, the homogenate is first subjected to ultrasonic treatment and then subjected to enzymolysis. Experimental verification shows that in terms of fish oil extraction rate, the process of simultaneously subjecting the homogenate to ultrasonic treatment and enzymolysis is significantly higher than the process of first subjecting the homogenate to ultrasonic treatment and then subjecting the homogenate to enzymolysis, while the process of only subjecting the homogenate to enzymolysis has the lowest fish oil extraction rate.

[0036] In certain embodiments of the present application, the oil is separated by centrifugation or centrifugation + organic solvent extraction, and the organic solvent includes but is not limited to n-hexane. In other embodiments of the present application, the step of separating the oil includes:

[0037] The enzymatically hydrolyzed solution is centrifuged to separate the upper clear oil and the middle emulsion layer; the middle emulsion layer is extracted with n-hexane and allowed to stand for stratification; the organic phase is evaporated and then centrifuged to separate the upper clear oil; the two separated clear oils are combined to obtain the fish oil.

[0038] In a second aspect of the present application, fish oil prepared by the preparation method described herein is provided. In certain embodiments of the present application, the prepared fish oil is dark yellow, has a higher iodine value, a lower peroxide value and acid value, and has higher EPA and DHA content, with a better health index. In experiments on pancreatic lipase inhibition rate and cholesterol micellar solubility inhibition rate, two indicators of lipid-lowering activity, the fish oil prepared by the present application method showed significant improvement compared to commercially available fish oil and fish oil prepared from Hunan crucian carp.

[0039] Based on the efficacy and activity of the fish oil described above, a third aspect of this application provides the use of the fish oil in preparing a product with the effect of improving blood lipids. In certain embodiments of this application, the product is a fish oil emulsion; however, the product may also be prepared into any other suitable dosage form, such as capsules, granules, and the like.

[0040] In a fourth aspect of the present application, a fish oil emulsion having the effect of improving blood lipids is provided, comprising fish oil prepared by the preparation method described herein. In certain embodiments of the present application, the fish oil emulsion further comprises an emulsifier and an emulsifier aid, and the fish oil emulsion can be prepared by ultrasonic emulsification.

[0041] In some other embodiments of the present application, the emulsifier is soy lecithin, and the co-emulsifier is sucrose ester and vitamin E.

[0042] In some other embodiments of the present application, the fish oil emulsion comprises 10% fish oil, 1.2% soy lecithin, 0.1% sucrose ester and 0.02% vitamin E, with the balance being water.

[0043] In the comparative experiments provided in this application, unless otherwise specified, other experimental conditions and materials were kept consistent, except for the differences noted in each group, to facilitate comparability. In addition, all materials used in this application were commercially available, for example, each protease was purchased from Nanning Pangbo Bioengineering Co., Ltd., and Hefang crucian carp and Xiang crucian carp were purchased from a local aquatic market.

[0044] The following further describes the fish oil provided in this application, its preparation method and application, and fish oil emulsion.

[0045] Example 1:

[0046] (1) Single enzyme enzymatic extraction of fish oil

[0047] Take fresh Hefang crucian carp or Xiang crucian carp, remove the scales and internal organs, clean it, cut it into small pieces, and crush it until it becomes a paste.

[0048] Take 20g of surimi and add distilled water at a 1:1 material-liquid ratio to form a homogenate. Pour the mixture into a 50ml conical flask and heat it in a water bath to the specified reaction temperature. Add dilute hydrochloric acid or sodium hydroxide to adjust the pH (Table 1). Add 1500U / g of enzyme based on the weight of the surimi, and allow enzymatic hydrolysis to proceed at the reaction temperature. After a 3-hour reaction, heat in a 95°C water bath for 5 minutes to inactivate the enzyme. After cooling, centrifuge at 8000 rpm for 20 minutes to separate the supernatant oil and the middle emulsion. Add n-hexane to the emulsion at a 1:2 ratio and allow the emulsion to stand at 35°C to separate. Rotary evaporate the separated organic phase and centrifuge to obtain the fish oil. Combine the two oil fractions.

[0049] Table 1

[0050]

[0051] (2) Double enzyme hydrolysis to extract fish oil

[0052] Take fresh Hefang crucian carp or Xiang crucian carp, remove the scales and internal organs, clean it, cut it into small pieces, and crush it until it becomes a paste.

[0053] Take 20g of surimi, add distilled water at a 1:1 material-liquid ratio to make a homogenate, pour into a 50ml conical flask, heat it in a water bath to 55°C, add dilute hydrochloric acid or dilute sodium hydroxide to adjust the pH to 7.5, add 1500U / g of surimi with different enzyme activity ratios (flavor protease: composite protease = 1:2, 1:3, 1:1, 2:1 or 3:1), and perform enzymatic hydrolysis at 55°C. After 3h of enzymatic hydrolysis, heat in a 95°C water bath for 5min to inactivate the enzymes. After cooling, centrifuge at 8000r / min for 20min to separate the upper clear oil layer and the middle emulsion layer. Add n-hexane to the emulsion layer at a ratio of 1:2 and let it stand at 35°C to separate the layers. The separated organic phase is rotary evaporated and centrifuged a second time. The two oil fractions are combined to obtain the fish oil.

[0054] (3) Dual enzyme hydrolysis and simultaneous ultrasonic extraction of fish oil

[0055] Take fresh Hefang crucian carp or Xiang crucian carp, remove the scales and internal organs, clean it, cut it into small pieces, and crush it until it becomes a paste.

[0056] Take 20g of surimi, add distilled water at a 1:1 material-liquid ratio to form a homogenate, pour into a 50ml conical flask, heat it in a water bath to 55°C, add dilute hydrochloric acid or dilute sodium hydroxide to adjust the pH to 7.5, add 1500U / g of surimi at the optimal enzyme activity ratio of dual enzyme (flavor protease: compound protease = 1:1), and place it in a CNC ultrasonic cleaner at 55°C and 300W for 3 hours. After the specified time, heat it in a 95°C water bath for 5 minutes to inactivate the enzymes. After cooling, centrifuge at 8000 rpm for 20 minutes to separate the upper clear oil layer and the middle emulsion layer. Add n-hexane at a 1:2 ratio to the emulsion layer and let it stand at 35°C to separate the layers. The separated organic phase is rotary evaporated and centrifuged a second time. The two oil fractions are combined to obtain the fish oil.

[0057] (4) Ultrasound followed by double enzyme hydrolysis to extract fish oil

[0058] Take fresh Hefang crucian carp or Xiang crucian carp, remove the scales and internal organs, clean it, cut it into small pieces, and crush it until it becomes a paste.

[0059] Take 20g of surimi, add distilled water at a 1:1 material-liquid ratio to form a homogenate, pour into a 50ml conical flask, and place it in a digitally controlled ultrasonic cleaner at 55°C and 300W for 3 hours. After the end, add dilute hydrochloric acid or dilute sodium hydroxide to adjust the pH to 7.5. Add 1500U / g of surimi with a dual enzyme (flavor protease: compound protease = 1:1) at the optimal enzyme activity ratio, and perform enzymatic hydrolysis at the reaction temperature for 3 hours. After the specified time, heat in a 95°C water bath for 5 minutes to inactivate the enzymes. After cooling, centrifuge at 8000 rpm for 20 minutes to separate the upper clear oil layer and the middle emulsion layer. Add n-hexane at a 1:2 ratio to the emulsion layer and let it stand at 35°C to separate the layers. The separated organic phase is rotary evaporated and centrifuged a second time. The two oil fractions are combined to obtain the fish oil.

[0060] (5) Preparation of fish oil emulsion

[0061] 10% fish oil, 1.2% soy lecithin, 0.1% sucrose ester, and 0.02% vitamin E, with the balance being water, were vortexed and then placed in an ultrasonic cleaner at 55°C, 500W, and 20 minutes to obtain a fish oil emulsion.

[0062] Example 2:

[0063] The fish oil extraction rate is calculated according to formula (1):

[0064]

[0065] In formula I: R is the fish oil extraction rate (%); m1 is the mass of the obtained fish oil (g); m2 is the mass of the crude fat in the raw material (g).

[0066] (1) Extraction rate of fish oil by single enzyme hydrolysis

[0067] Table 2

[0068]

[0069] Note: Different letters indicate significant differences (p < 0.05), the same below;

[0070] Combined with Table 2 and Figure 1The results show that the performance of various single enzymes on the fish oil extraction rate is as follows: flavor protease > compound protease > alkaline protease > neutral protease > papain. The optimal enzyme is flavor protease. The difference between alkaline protease and compound protease is very small, and there is no difference in significance analysis. However, considering that compound protease has higher enzyme activity and less dosage, it is more economical, so the optimal enzyme flavor protease and compound protease are selected for subsequent dual enzyme compounding; and the pH value of the fish paste homogenate itself is around 6.90 (verified), close to 7.0, among which the optimal pH value of compound protease is 7.0. Compared with alkaline protease without adding sodium hydroxide to adjust the pH, the fish oil produced is greener. Therefore, considering the comprehensive consideration, it is more appropriate to use flavor protease and compound protease for the subsequent compound dual enzyme enzymatic hydrolysis experiment.

[0071] (2) Extraction rate of fish oil by double enzyme hydrolysis

[0072] Table 3

[0073]

[0074] Combined with Table 3 and Figure 2 The results show intuitively that when flavor protease and compound protease are compounded according to five enzyme activity ratios, the fish oil extraction rate is the highest when the flavor protease and compound protease are 1:1, and the extraction rates are similar under the compound ratios of 1:2 and 1:3. However, when the ratio of flavor protease exceeds 1:1 and its dosage is gradually increased, the fish oil extraction rate decreases significantly. This indicates that flavor protease and compound protease have a synergistic effect in improving the fish oil extraction rate within a certain ratio range.

[0075] Table 4

[0076]

[0077] Combined with Table 4 and Figure 3 The results clearly show that a 1:1 ratio of flavor protease to compound protease activity yielded superior fish oil extraction results compared to other conditions. Therefore, the optimal subsequent enzymatic hydrolysis process was determined to be a temperature of 55°C, a pH of 7.5, a 1:1 solid-liquid ratio, a hydrolysis time of 3 hours, an enzyme dosage of 1500 U / g, and a 1:1 ratio of the two enzyme activities. The optimal process achieved a fish oil extraction yield of 56.92%.

[0078] (3) Fish oil extraction rate under different ultrasonic treatment methods

[0079] Using Hefang crucian carp and Xiang crucian carp as raw materials, fish oil was extracted with the optimal ratio of double enzymes according to the methods of "double enzyme hydrolysis and extraction of fish oil" (Group 1), "double enzyme hydrolysis and simultaneous ultrasonic extraction of fish oil" (Group 2), and "ultrasound followed by double enzyme hydrolysis and extraction of fish oil" (Group 3) in Example 1. The results are shown in Tables 5 and 6, as well as the summary. Figure 4 ;

[0080] Table 5

[0081]

[0082]

[0083] Table 6

[0084]

[0085] Combining Tables 5 and 6, and Figure 4 The results showed that, no matter whether Xiang crucian carp or Hefang crucian carp was used, the extraction rate of fish oil by dual enzyme hydrolysis and simultaneous ultrasonic extraction was the highest, and was significantly higher than the fish oil extraction rates of the other two groups.

[0086] Example 3:

[0087] Using Hefang crucian carp and Xiang crucian carp as raw materials, the fish oil was extracted at the optimal ratio of the two enzymes according to the method of "dual-enzyme hydrolysis and simultaneous ultrasonic extraction of fish oil" in Example 1, and the fish oil prepared from the two raw materials was tested for physical and chemical indicators and analyzed for fish oil health index;

[0088] (1) Fish oil physical and chemical index detection:

[0089] Iodine value: refer to GB / T 5532-1995 Determination of iodine value of animal and vegetable fats and oils.

[0090] Peroxide value: refer to GB / T 5538-2005 Determination of peroxide value of animal and vegetable oils and fats.

[0091] Saponification value: refer to GB / T 5534-2008 Determination of saponification value of animal and vegetable fats and oils.

[0092] Acid value: refer to GB / T 5530-2005 Determination of acid value and acidity of animal and vegetable oils and fats.

[0093] Fatty acid composition: refer to GB / T 5009.168 gas chromatography method.

[0094] The results are shown in Tables 7-11 below.

[0095] Table 7

[0096]

[0097] Table 8

[0098]

[0099]

[0100] Table 9

[0101]

[0102] Table 10

[0103]

[0104] Table 11

[0105]

[0106]

[0107] In terms of physical and chemical properties, the iodine value of fish oil reflects the content of unsaturated fatty acids in the oil. The iodine value of Hefang crucian carp oil is significantly higher than that of Hunan crucian carp oil, indicating that the unsaturated fatty acid content may be higher and more beneficial to human health. Combined with the fatty acid composition results, this conclusion is confirmed. The unsaturated fatty acid content of Hefang crucian carp oil is higher, especially in terms of EPA and DHA.

[0108] The higher the acid value, the higher the content of free fatty acids in the oil, which usually means that the degree of hydrolysis of the oil is higher.

[0109] The peroxide value mainly reflects the degree of oxidation of oils and fats, especially the content of primary oxidation products (such as peroxides). The higher the peroxide value, the higher the degree of oxidation of the oils and fats, which usually means that the unsaturated fatty acids in the oils and fats have been oxidized to form peroxides. The peroxide value of Hunan crucian carp oil is significantly higher than that of Hefang crucian carp oil, while its acid value is lower. This is because in the early stages of oxidation, peroxides are mainly generated. At this time, the peroxide value will increase significantly, while the acid value may not change much. This is because the peroxides have not yet been further decomposed into secondary oxidation products such as aldehydes, ketones, and acids;

[0110] A low saponification value means that the fatty acid carbon chain of triglycerides in fish oil is longer. Long-chain fatty acids usually include linoleic acid and other essential fatty acids for the human body and have high nutritional value. There is no significant difference between the two in this regard.

[0111] (2) Fish Oil Health Index

[0112] Table 12

[0113]

[0114] According to Table 12 and Figure 5The PUFA / SFA ratio is an index commonly used to assess the impact of a diet on cardiovascular health. This index assumes that PUFAs can suppress LDL cholesterol and cholesterol levels, while SFAs help raise serum cholesterol levels. The PUFA / SFA ratio for fish generally ranges from 0.50 to 1.62, with a higher ratio indicating better nutritional value. Hefang crucian carp oil has a higher ratio of 1.77 than Xiang crucian carp oil, confirming its superior ability to suppress LDL cholesterol and cholesterol levels.

[0115] The atherogenicity index (IA) is used to characterize the atherogenic potential of fatty acids (FAs). The IA formula represents the relationship between the sum of SFAs and the sum of unsaturated fatty acids. SFAs are considered pro-atherogenic because they facilitate the adhesion of lipids to cells in the circulation and immune system. Unsaturated fatty acids, on the other hand, inhibit plaque accumulation and reduce levels of phospholipids, cholesterol, and esterified fatty acids. The IA of fish oils ranges from 0.21 to 1.41, with both fish oils near the lowest IA values. The Hefang crucian carp oil has a lower IA than the Xiang crucian carp oil, indicating that it is less likely to cause vascular atherosclerosis and is healthier.

[0116] The Thrombogenicity Index (IT) characterizes the thrombogenic potential of fatty acids and their tendency to form thrombi in blood vessels. It demonstrates the relationship between pro-thrombotic FAs (C12:0, C14:0, and C16:0) and anti-thrombotic FAs (MUFAs and n-3 and n-6 fatty acids). The IT values of fish generally range from 0.14 to 0.87. The IT values of the two fish oils fall within the middle and reasonable range. The IT value of the combined crucian carp oil is lower than that of the Hunan crucian carp oil, making it less likely to cause thrombosis in blood vessels.

[0117] The hypocholesterolemia / hypercholesterolemia ratio (HH) characterizes the relationship between hypocholesterolemic fatty acids and hypercholesterolemic FAs. Compared to the PUFA / SFA ratio, the HH may more accurately reflect the impact of FA composition on cardiovascular disease. For fish, HH values range from 1.54 to 4.83. Both fish oils had HH values that were consistent with expectations, with mean values ranging from 4.44 to 4.67.

[0118] EPA and DHA are n-3 long-chain polyunsaturated fatty acids that play a role in reducing the risk of cardiovascular disease, hypertension, and inflammation in human biological processes. The sum of eicosapentaenoic acid and docosahexaenoic acid (EPA+DHA) is a globally recognized index. Due to various factors, including environmental factors, the content of EPA in freshwater fish oil is lower than that in deep-sea fish oil. However, in a comparison of the two homemade fish oils, the combined crucian carp oil showed a higher value, indicating a richer nutritional profile.

[0119] The Fish Fat Quality / Flesh Fat Quality (FLQ) index functions similarly to the EPA+DHA index, but FLQ calculates the proportion of EPA and DHA in the total fatty acids, making it more suitable for assessing aquatic product quality. The FLQ values and EPA+DHA content of the two fish oils were identical, with the combined crucian carp oil's value exceeding that of the Hunan crucian carp oil.

[0120] The degree of unsaturation (UI) is often used as a criterion for determining the content of high-quality polyunsaturated fatty acids. The value for Hefang crucian carp oil is still higher than that for Xiang crucian carp oil, providing evidence for its greater lipid-lowering effect.

[0121] Based on the above-mentioned physical and chemical properties and the fish oil health index results, the combined crucian carp oil prepared by the method of the present application is superior to the Hunan crucian carp oil in nutritional value.

[0122] Example 4:

[0123] Using Hefang crucian carp and Xiang crucian carp as raw materials, fish oil was extracted using the method of "dual-enzyme hydrolysis and simultaneous ultrasonic extraction of fish oil" in Example 1 at the optimal dual-enzyme ratio, and fish oil prepared from the two raw materials was prepared into fish oil emulsions. The differences in lipid-lowering activity between the two fish oils and commercially available fish oil (purchased from Harbin Pharmaceutical Group Co., Ltd.) were analyzed and compared;

[0124] (1) Pancreatic lipase activity inhibition rate

[0125] Reagent preparation:

[0126] 25mM pH=7.4 PBS: Weigh 4.477g disodium hydrogen phosphate dodecahydrate and 1.95g sodium dihydrogen phosphate dihydrate and dissolve them in 500ml distilled water. Mix well and adjust the pH to 7.4 with sodium hydroxide solution.

[0127] 5mg / ml pancreatic lipase: Weigh 250mg of pancreatic lipase and dissolve it in 50ml of sterile water. Centrifuge at 8000r / min for 15min, discard the precipitate and take the supernatant. Keep the enzyme in an ice box until use.

[0128] Preparation of 11.2 mol / L p-NPB solution: Use a pipette to draw 0.11715 g of p-NPB solution into 50 ml of PBS and place it in an ice box until ready to use.

[0129] Experimental process:

[0130] Mix 500 μl of sample, 500 μl of pancreatic lipase, and 500 μl of PBS, and incubate in a 37°C water bath for 10 min. Then add 500 μl of p-NPB solution, gently shake to mix, and incubate in a 37°C water bath for 20 min. Measure the absorbance at a wavelength of 405 nm and record the value.

[0131] Inhibition rate (%) = [1-(A4-A3) / (A2-A1)] × 100%

[0132] Control background group A1: substrate

[0133] Control group A2: enzyme and substrate

[0134] Experimental background group A3: sample + substrate

[0135] Experimental group A4: Experimental group

[0136] The total volume is 2 mL, and PBS is added to balance the insufficient volume.

[0137] (2) Cholesterol micelle solubility inhibition rate

[0138] Reagent preparation:

[0139] A 1 mL micellar solution containing 10 mmol / L sodium taurocholate, 2 mmol / L cholesterol, 5 mmol / L oleic acid, 132 mmol / L NaCl, and 15 mmol / L phosphate buffer (pH 7.4) was prepared by ultrasonic homogenization. The solution was incubated at 37°C overnight. A blank control and a specific amount of functional factors were added to the micellar solution. The mixture was then shaken at 37°C for 2 hours and centrifuged at 16,000 rpm for 20 minutes. The supernatant was collected and the cholesterol content in the micelles was determined using a kit.

[0140] Experimental process:

[0141] Table 13

[0142]

[0143] Inhibition rate = (cholesterol solubility of blank solution - cholesterol solubility of sample solution) / cholesterol solubility of blank solution

[0144] (3) Experimental results

[0145] The results are shown in Tables 14, 15 and Figure 6 ,

[0146] Table 14 Comparison of the efficacy of fish oil in lowering blood lipids (pancreatic lipase)

[0147]

[0148] Table 15 Comparison of the efficacy of fish oil in lowering blood lipids (cholesterol micelles)

[0149]

[0150] On the basis of the determination of pancreatic lipase inhibition rate, the present application adds another evaluation index for the lipid-lowering effect, the measurement of the cholesterol micelle system. According to the fact that cholesterol can be dissolved in the micelle solution, the cholesterol solubility is measured by the kit to detect the inhibition rate of the fish oil emulsion to show its lipid-lowering effect. The results show that the lipid-lowering effect results determined by the two methods echo each other. The inhibition rate of the combined crucian carp oil on pancreatic lipase and its inhibition rate in cholesterol micelles are 73.44% ± 9.21% and 69.38% ± 1.64%, respectively. The results are very close, and the other two fish oils are more than 15% lower than the effect of the combined crucian carp oil. Therefore, it can be seen that the fish oil prepared by the method of the present application using the combined crucian carp as raw material has a good lipid-lowering effect, which is inseparable from the composition and content of fatty acids in the fish oil.

[0151] The foregoing is merely a list of specific embodiments of the present application, intended to enable those skilled in the art to understand or implement the present application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application is not limited to the embodiments shown herein, but is intended to conform to the broadest scope consistent with the principles and novel features of the present application.

Claims

1. A method for preparing fish oil, characterized in that: include: S1. Crush the crucian carp and add water to make a homogenate; S2. Add one or more enzymes selected from flavor protease, composite protease, and alkaline protease to the homogenate for enzymatic hydrolysis, and separate the oil after enzymatic hydrolysis to obtain the fish oil.

2. The preparation method according to claim 1, characterized in that The enzymatic hydrolysis is performed by using flavor protease and a combination of enzymes selected from any one of composite protease and alkaline protease.

3. The preparation method according to claim 2, characterized in that The enzyme activity ratio of the flavor protease to the composite protease or alkaline protease in the total enzyme activity is 1:(1-3).

4. The preparation method according to claim 1, characterized in that The enzymatic hydrolysis temperature is 50-60° C., and the added amount is 1500±500 U / g.

5. The preparation method according to any one of claims 1 to 4, characterized in that It also includes adding ultrasonic treatment to S2 to assist enzymatic hydrolysis.

6. Fish oil prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the fish oil according to claim 6 in preparing a product having the effect of improving blood lipids.

8. The use according to claim 7, characterized in that The product comprises an emulsion.

9. A fish oil emulsion having the effect of improving blood lipids, characterized in that: Comprising the fish oil according to claim 6.

10. The fish oil emulsion according to claim 9, characterized in that Also includes emulsifiers and co-emulsifiers.

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