A method for green production of high-stable pufa type fish oil from rainbow trout viscera

Abstract

The application discloses a method for preparing high-stable PUFA fish oil from rainbow trout viscera by a solubilization pretreatment assisted biological enzymatic hydrolysis technology, which comprises the following steps: placing frozen rainbow trout viscera after low-temperature thawing in 0.1% sodium bicarbonate aqueous solution for incubation, instantaneously cooling the mixture of the viscera and the solution for 5 min, and homogenizing the frozen tissue sample by using a tissue homogenizer; oscillating the homogenized viscera liquid in a warm water bath, adding protease for biological enzymatic hydrolysis, and low-temperature centrifuging the enzymatic hydrolysate; taking fish oil after centrifugation; placing the obtained fish oil in a special glass bottle, sealing the glass bottle after flushing in nitrogen, and storing the glass bottle in dark. The yield of the prepared fish oil is up to 90% or more, the color is bright yellow or light yellow, the fish oil is clear and transparent, has no obvious fishy smell and fish oil rancidity, and contains rich polyunsaturated fatty acids (>40%), which is obviously higher than that of commercially available fish oil; the acid value is low, the free fatty acid content is lower than 0.1%, the oxidation induction time is long, and the fish oil has good oxidation stability.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for green preparation of high-stable PUFA type fish oil from rainbow trout viscera, belonging to the technical field of food processing. BACKGROUND

[0002] Lipids are essential nutrients for maintaining the life activities of organisms, and play an important structural and physiological function in the process of biological survival and growth. As an important component unit of functional lipids, polyunsaturated fatty acids (PUFAs) refer to fatty acids containing two or more unsaturated double bond structures, which have unique physiological activity and health care function. Numerous studies have shown that PUFAs regulate lipid metabolism, promote the development of nervous system and visual system, and play an important role in stabilizing cell membrane function, regulating gene expression, maintaining cytokine and lipoprotein balance, immune regulation, anti-cardiovascular disease, and promoting growth and development. The unique biological activity of PUFAs makes it a research focus in the field of food nutrition, and the demand for PUFAs type lipids is increasing. Therefore, it is imperative to develop nutritional lipid products rich in PUFAs, which can help meet the urgent needs of consumers for nutritional and healthy food.

[0003] Rainbow trout is a high-yield and excellent high-grade freshwater fish promoted by the Food and Agriculture Organization of the United Nations to the world. The annual production of rainbow trout in China is about 40,000 tons, and it has become one of the main freshwater aquaculture fish in China. Only about 60-70% of the whole adult is used in the processing process of rainbow trout, and the by-products such as viscera have high fat content and are rich in PUFAs, but they are often directly discarded or exist in the form of primary processing such as feed, with low utilization rate of high-value processing, causing great waste of resources and environmental pollution. Therefore, extracting oil from rainbow trout viscera is a direct way to turn waste into treasure and increase value efficiently.

[0004] Current fish oil preparation technologies mainly include pressing, cooking, supercritical fluid extraction, chemical hydrolysis, organic solvent extraction and biological enzymatic hydrolysis, and the main principle is to destroy the organization structure of raw materials by external force or reagent to separate fish oil. The extraction rate of pressing technology is low, the cooking method involves high temperature which can easily promote the oxidation of PUFAs, the supercritical fluid extraction technology requires expensive equipment and harsh operating conditions, and the chemical hydrolysis and organic solvent extraction technology can easily damage the oil and cause environmental pollution. Biological enzymatic hydrolysis technology is favored due to its mild operating conditions, green low carbon and environmental friendliness, but the low oil extraction rate and high cost greatly limit its industrial application. In addition, rainbow trout viscera is easily damaged during transportation, frozen storage and other links, and the high temperature process involved in biological enzymatic hydrolysis process aggravates the oxidation of PUFAs, resulting in serious decline in fish oil quality, and even harm to human health in severe cases. Therefore, how to obtain oil with high PUFA content and good oxidation stability from rainbow trout viscera is a problem to be solved in the industrial application of biological enzymatic hydrolysis technology. SUMMARY

[0005] Technical problem: The technical problem to be solved by the present application, the goal to be achieved.

[0006] To solve the problems of low extraction rate, easy oxidation, high acid value and poor quality of rainbow trout viscera oil, the present application uses rainbow trout viscera as raw material to prepare fish oil through solubilization pretreatment and auxiliary biological enzymolysis technology. The fish oil prepared by the method is a high-value unsaturated glyceride product or base oil, which is rich in PUFAs, has high oil yield and low acid value, and has good oxidation stability.

[0007] Technical solution: The complete technical means and method of the present application.

[0008] The present application provides a method for green preparation of high-stability PUFAs type fish oil from rainbow trout viscera, which mainly comprises the following steps:

[0009] (1) Raw material preparation: during the processing of rainbow trout, collect fresh fish viscera tissue as raw material for extracting oil, or thaw frozen rainbow trout viscera;

[0010] (2) Tissue solubilization: add a tissue solubilization aid to the raw material in step (1), and the mass ratio between the two is maintained at 2:1-1:2, then mix uniformly and place in a temperature reactor for incubation;

[0011] (3) Instantaneous rapid cooling: the tissue sample obtained in step (2) is instantaneously cooled, and the viscera tissue releases oil during the short heat and cold alternation process to obtain a tissue sample;

[0012] (4) Physical homogenization: use a tissue homogenizer to homogenize the tissue sample obtained above to obtain a rainbow trout viscera slurry;

[0013] (5) Biological enzymolysis: the viscera slurry obtained in step (4) is placed in a temperature shaker and preheated to the desired temperature of the protease, and then an appropriate amount of protease is added for biological enzymolysis;

[0014] (6) Centrifugal separation: after enzymolysis, immediately perform low-temperature centrifugal separation;

[0015] (7) Storage and preservation: the PUFAs type fish oil prepared above is placed in a sealed storage device, inert gas is injected to exclude air in the bottle, and it is stored at room temperature in the dark to reduce the influence of oxygen, light and other factors on the oxidation stability of unsaturated glycerides as much as possible.

[0016] In an embodiment of the present application, the rainbow trout viscera includes all viscera tissues such as liver, spleen, digestive tract, gallbladder and swim bladder, which need to be uniformly sampled.

[0017] In one embodiment of the present application, the thawing is placing the rainbow trout viscera at ~4°C overnight.

[0018] In one embodiment of the present application, in step (2), the tissue solubilizer is one or more of salt, sodium bicarbonate, and water.

[0019] In one embodiment of the present application, in step (2), the concentration of the tissue solubilizer is 0-1.2%.

[0020] In one embodiment of the present application, in step (2), the temperature of the temperature reactor is controlled at 30-80°C, and the incubation time is 3-15 min.

[0021] In one embodiment of the present application, in step (2), the incubation is a process of placing the raw material from room temperature into the temperature reactor and slowly increasing the temperature to the set temperature of the temperature reactor, and the incubation time is the time for temperature increase and maintenance at the set temperature.

[0022] In one embodiment of the present application, in step (3), the equipment required for the instant quenching is a low-temperature refrigerator, a rapid cooling machine, a vacuum cooling machine, or a liquid nitrogen tank, and the cooling time varies depending on the equipment used.

[0023] In one embodiment of the present application, in step (3), the temperature of the instant quenching environment is -20 to -80°C, and the time is 1-5 min.

[0024] In one embodiment of the present application, in step (4), the homogenization is controlled at a rotation speed of 5000-15000 r / min for 0.5-5 min.

[0025] In one embodiment of the present application, in step (5), the protease can be one or more of neutral protease, animal protease, complex protease, alkaline protease, and trypsin, and the concentration is 1-4.0%, and the enzyme activity is 1000 u-4000 U / g.

[0026] In one embodiment of the present application, in step (5), the pH is controlled at 5-9, the temperature of the temperature shaker is controlled at 35-65°C, the enzyme digestion time is controlled at 0.5-3 h, and the shaking speed is preferably 100-250 r / min.

[0027] In one embodiment of the present application, in step (6), the low-temperature separation is performed at a temperature of 2-8°C and a rotation speed of 5000-12000 r / min for 5-15 min.

[0028] In one embodiment of the present application, in step (7), the closed storage device is a plastic bottle, a glass bottle, a coated metal can, etc., and the best is opaque packaging; the inert gas is one or a mixed form of nitrogen or carbon dioxide.

[0029] The present application uses the above method to prepare high-stability PUFA type fish oil.

[0030] The second object of the present application is to apply the above high-stability PUFA type fish oil in the fields of functional food, food material preparation and cosmetics.

[0031] Beneficial effects: the benefits brought by the present application, the indicators achieved.

[0032] Compared with the prior art, the fish oil with high content of polyunsaturated fatty acids provided by the present application has the following advantages:

[0033] (1) The present application relates to a method for green preparation of high-stability PUFA type fish oil from rainbow trout viscera, and the selected raw material is rainbow trout viscera, which is abundant in resources, and the waste by-products are deeply developed to greatly improve the added value of rainbow trout viscera; the preparation process of high-stability PUFA type fish oil is summarized as "tissue solubilization - transient rapid cooling - physical homogenization - biological enzymolysis - centrifugal separation", the required instruments and equipment for the preparation process are simple and feasible to operate, the preparation technology is green, safe and environmentally friendly, the required processing aids are low in cost and widely available, and the prepared fish oil product is expected to be industrialized, thereby providing scientific and technological support for high-value utilization of rainbow trout processing by-products.

[0034] (2) In the tissue solubilization and transient rapid cooling method adopted by the present application, the tissue solubilization agent used helps to accelerate the separation of oil and tissue protein, and the temperature difference between heat and cold stimulates further acceleration of the release of visceral oil, thereby improving the extraction rate of visceral oil and ensuring the integrity of fish oil nutritional components, especially polyunsaturated glycerides.

[0035] (3) The present application uses biological enzymolysis to prepare fish oil, and the enzymolysis reaction conditions are mild, the reagents are green, safe and environmentally friendly, compared with the existing enzymolysis method, the high-temperature enzyme inactivation step with high energy consumption and easy oxidation is omitted, which can effectively avoid the oxidation of polyunsaturated fatty acids in fish oil caused by high-temperature enzyme inactivation; compared with the traditional oil preparation method, the biological enzymolysis oil preparation technology not only can ensure the integrity of triglycerides, but also can effectively retain the trace nutrients in fish oil, which has high nutritional value and meets the demands of consumers for nutritional and healthy oil products.

[0036] (4) The fish oil prepared by the present application has high yield, is rich in polyunsaturated fatty acids, and is rich in trace nutrients such as fat-soluble vitamins, has high nutritional value and oxidation stability, and does not need to add artificial antioxidants and other additives, the product is natural, green and additive-free, and meets the current concept of "clean label".

[0037] (5)The fish oil product prepared by the present application presents a transparent orange yellow color, low acid value, good oxidation stability, and product quality comparable to commercially available fish oil, and can be directly consumed, processed into functional food or used as medicine, while the oil by-product can be directly dried into protein or peptide powder and applied in multiple fields such as food and feed.

[0038] (6)The acid value of the rainbow trout visceral oil prepared by the extraction method is less than 1.0 mg / g. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 The process flow chart of the high-stability PUFAs type fish oil prepared by the present application and the final fish oil product are shown in the figure.

[0040] Figure 2 The high-stability PUFAs type fish oil product prepared by Example 1 is shown in the figure.

[0041] Figure 3 The fish oil products prepared by Example 2 and Comparative Examples 1-3 are shown in the figure. DETAILED DESCRIPTION

[0042] The following examples are used to illustrate the present application, but are not used to limit the scope of the present application. Modifications or replacements of the methods, steps or conditions of the present application, without departing from the spirit and essence of the present application, all belong to the scope of the present application.

[0043] If not specifically indicated, the experimental materials, reagents and instruments used in the examples of the present application can be commercially available, and if not specifically indicated, the technical means used in the examples are conventional means well known to those skilled in the art.

[0044] 1. Measurement of oxidation induction time

[0045] The prepared fish oil was used as the experimental object, 3 g of fish oil sample was placed in a special test tube, and the oxidation induction time was measured by using a Rancimat oil oxidation stability instrument, and the parameters were set as follows: 120℃, air flow rate 20.0 L / h.

[0046] 2. Measurement of acid value

[0047] (1) Preparation of reagents: KOH solution (0.1 mol / L); ether-isopropanol mixed solution (1:1, v / v); phenolphthalein indicator (1 g dissolved in 100 mL 95% ethanol);

[0048] (2) Experimental method: take a clean 250 mL conical flask, according to the requirements of the balance 3 g or so of rainbow trout fish oil, add ether-isopropyl alcohol mixed solution 60 mL and 3 drops of phenolphthalein solution, shake well. Again with a standard titration solution (i.e. 0.1 mol / L of KOH) of the graduated buret to the sample solution manual titration, when the sample solution appears slightly red, and 15 s no obvious fading, as the end point of titration. Immediately stop titration, record the consumption of standard titration solution of milliliter number.

[0049] (3) Calculation formula:

[0050]

[0051] X = —— (1) wherein: X AV acid value, unit of milligrams per gram (mg / g);

[0052] V - the sample determination of the volume of standard titration solution, units of milliliters (mL);

[0053] V0 - the corresponding blank determination of the volume of standard titration solution, units of milliliters (mL);

[0054] c - the molar concentration of standard titration solution, units of moles per liter (mol / L);

[0055] 56.1 - the molar mass of potassium hydroxide, units of grams per mole (g / mol);

[0056] m - the sample weight of oil and fat, units of grams (g).

[0057] 3, fatty acid composition determination

[0058] Take 20 mg or so of milk fat in a glass stoppered test tube, add 2 mL of 0.5 mol / L KOH-CH3OH solution, placed in a 65℃ constant temperature water bath for 30 min for saponification. Then add 2 mL of BF3-CH3OH (1:3, v / v) solution, and then placed in a 70℃ constant temperature water bath, heated for 10 min and cooled to room temperature. Add 2 mL of chromatographic grade n-hexane and shake vigorously for 3-4 min, after the layering, take the supernatant, add anhydrous Na2SO4 to absorb a small amount of water, after membrane filtration, gas chromatography analysis.

[0059] Gas chromatography conditions: Thermo Fisher Trace TR-FAME column, 60 m x 0.25 mm x 0.25 pm, temperature program: 0 min, 130 °C, hold for 3 min; 5 °C / min to 200 °C, hold for 10 min; 2 °C / min to 220 °C, hold for 5 min; injection volume: 1 pL; split ratio: 20; column flow: 1.8 mL / min. A 52-fatty acid methyl ester mix was used for fatty acid qualitative and relative quantitative analysis.

[0060] The reagents and raw materials used in the following examples can be purchased on the market, and the alkaline protease has an enzyme activity of 1000 u-4000 U / g.

[0061] Example 1:

[0062] This example mainly includes the following steps:

[0063] (1) Raw material preparation: 500 g of frozen fish internal organs were weighed as raw materials for extracting oil and grease, and were placed in a 1.5 L reaction container for overnight thawing at low temperature;

[0064] (2) Tissue solubilization: an equal amount of 0.1% concentration sodium bicarbonate aqueous solution was added to the raw materials, and after being mixed uniformly, the mixture was placed in a 60 °C temperature reactor for incubation for 12 min;

[0065] (3) Instantaneous rapid cooling: the tissue sample obtained in step (2) was placed in a -40 °C environment for instantaneous cooling for 5 min, so as to reduce the destruction of nutrients and release the oil and grease from the internal organs as much as possible;

[0066] (4) Physical homogenization: the tissue sample obtained above was homogenized by using a tissue homogenizer, the homogenization speed was controlled at 6000 r / min, and the time was controlled at 2 min, to obtain rainbow trout internal organ slurry;

[0067] (5) Biological enzymolysis: the internal organ slurry obtained in step (4) was placed in a temperature shaker and preheated to 50 °C, the slurry pH was adjusted to 8, and then 2% alkaline protease was added for biological enzymolysis, the enzymolysis time was 2 h, and the shaking speed was 200 r / min;

[0068] (6) Centrifugal separation: after the enzymolysis was completed, low-temperature centrifugal separation was immediately performed, the temperature was 4 °C, the speed was 8000 r / min, and the centrifugal time was 10 min;

[0069] (7) Storage and preservation: the PUFA type fish oil prepared above was placed in a special glass bottle, nitrogen was flushed in, and then the bottle was sealed and stored in the dark.

[0070] The fish oil product prepared above, for example, Figure 2As shown, the color presents light yellow, clear and transparent, slightly fish oil specific smell, high content of polyunsaturated fatty acids, low acid value, no rancid taste, good oxidation stability.

[0071] Example 2:

[0072] This example mainly includes the following steps:

[0073] (1) Raw material preparation: 500g of fresh fish viscera tissue as the raw material for extracting oil, placed in a 1.5L reaction container;

[0074] (2) Tissue solubilization: add an equal amount of distilled water solution to the raw material, mix well and incubate in a 60℃ temperature reactor for 12min;

[0075] (3) Instantaneous cooling: place the tissue sample obtained in step (2) in a -80℃ environment for instantaneous cooling for 2min, while reducing the damage to nutrients, and release the oil from the viscera tissue as much as possible;

[0076] (4) Physical homogenization: use a tissue homogenizer to homogenize the above obtained tissue sample, the homogenization speed is controlled at 6000r / min, and the time is controlled at 2min, to obtain rainbow trout viscera slurry;

[0077] (5) Biological enzymolysis: place the viscera slurry obtained in step (4) in a temperature shaker and preheat to 55℃, then add 2.5% neutral protease for biological enzymolysis, the enzymolysis time is 1.5h, and the shaking speed is 150r / min;

[0078] (6) Centrifugal separation: after enzymolysis, immediately perform low-temperature centrifugal separation, temperature 4℃, speed 8000r / min, centrifugal time 10min;

[0079] (7) Storage: place the above prepared PUFA type fish oil in a special glass bottle, flush with nitrogen and seal to avoid light.

[0080] The fish oil product prepared above is shown in Table 1 (labeled as Example 2), the color presents light yellow, clear and transparent, basically no fish oil smell, rich in polyunsaturated glycerides, low acid value, no fish oil rancid taste, good oxidation stability. Figure 3

[0081] Comparative Example 1:

[0082] Prepare the raw material according to the method of Example 2, omit the solubilization, directly perform physical homogenization and biological enzymolysis, and the rest of the conditions remain unchanged, to prepare fish oil, the specific indicators are shown in Table 1.

[0083] Comparative Example 2:

[0084] ​The raw material was prepared according to the method of Example 2, the bioenzymatic hydrolysis process was omitted, and the rest of the conditions were unchanged, and fish oil was directly prepared to obtain the fish oil, and the specific indexes are shown in Table 1.

[0085] Comparative Example 3:

[0086] The raw material was prepared according to the method of Example 2, the bioenzymatic hydrolysis process was omitted, and the rest of the conditions were unchanged, and fish oil was directly prepared to obtain the fish oil, and the specific indexes are shown in Table 1.

[0087] Comparative Example 4:

[0088] The raw material was prepared according to the method of Example 2, the bioenzymatic hydrolysis process was omitted, and the rest of the conditions were unchanged, and fish oil was directly prepared to obtain the fish oil, and the specific indexes are shown in Table 1.

[0089] Table 1 Quality comparison of fish oil prepared by different methods

[0090]

[0091] It is found through comparative analysis in Table 1 that the yield of fish oil prepared by the present application is as high as 90% or more, the color is a pleasing bright yellow or light yellow, clear and transparent, without obvious fishy smell and fish oil rancidity, and contains rich polyunsaturated fatty acids (>40%), which is significantly higher than commercially available fish oil, and the acid value is lower, the free fatty acid content is lower than 0.1%, the oxidation induction time is long, and the oxidation stability is good.

Claims

1. A method for preparing high-stable PUFAs type fish oil from rainbow trout viscera, comprising the following steps: (1) raw material preparation: collecting fresh fish viscera tissue as raw material for extracting oil during the process of rainbow trout processing, or taking frozen rainbow trout viscera and thawing; (2) tissue solubilization: adding a tissue solubilizer to the raw material in step (1), and maintaining the mass ratio between the two at 2:1-1:2, and then mixing uniformly and placing in a temperature reactor for incubation; (3) instant quick cooling: instant cooling the tissue sample obtained in step (2), and releasing the oil from the viscera tissue in a short-term heat and cold alternation process to obtain a tissue sample; the temperature of the instant quick cooling environment is -20 to -80℃, and the time is 1-5 min; (4) physical homogenization: homogenizing the tissue sample obtained by instant quick cooling using a tissue homogenizer to obtain a rainbow trout viscera slurry; (5) biological enzymolysis: placing the viscera slurry obtained in step (4) in a temperature shaker and preheating to the desired temperature of the protease, and then adding an appropriate amount of protease for biological enzymolysis; the protease is neutral protease or alkaline protease, and the concentration is 1-4.0%, and the protease enzyme activity is 1000-4000 U / g; (6) centrifugal separation: immediately performing low-temperature centrifugal separation after the enzymolysis is completed; the low-temperature separation temperature is 2-8℃, the rotation speed is 5000-12000 r / min, and the centrifugal time is 5-15 min; (7) storage and preservation: placing the PUFAs type fish oil prepared above in a sealed storage device and flushing with inert gas. In step (2), the tissue solubilizer is a 0.1% concentration sodium bicarbonate aqueous solution or water. In step (2), the temperature of the temperature reactor is controlled at 30-80℃, and the incubation time is 3-15 min. In step (4), the homogenization rotation speed is controlled at 5000-15000 r / min, and the time is controlled at 0.5-5 min. In step (5), the temperature of the temperature shaker is controlled in the range of 35-65℃, the enzymolysis time is controlled at 0.5-3 h, and the oscillation speed is 100-250 r / min.

6. The high-stable PUFAs type fish oil prepared by the method of any one of claims 1-5.

7. The high-stable PUFAs type fish oil of claim 6 for use in the fields of functional food, food material preparation, and cosmetics. ​ 2. The method of claim 1, wherein, ​ 3. The method of claim 1, wherein, ​ 4. The method of claim 1, wherein ​ 5. The method of claim 1, wherein ​ ​ ​

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