Method for extracting taurine from egg yolk

Abstract

The invention provides a method for extracting taurine from egg yolk, and belongs to the technical field of deep processing of agricultural products, the method comprises the following steps: unfreezing frozen egg yolk, and extracting with an extraction reagent; and centrifuging the extraction reaction liquid, taking supernate, standing, and centrifuging again to obtain a product containing taurine. The method has the advantages of simplicity and easiness in operation, mild extraction conditions, low equipment requirements, low cost, small pollution and the like, can remarkably improve the extraction rate of the taurine in the egg yolk, and is of great significance to industrial production of the taurine and high value-added development of eggs.

Description

Technical Field

[0001] The present invention relates to the technical field of deep processing of agricultural products, and in particular to a method for extracting taurine from egg yolk. Background Art

[0002] Taurine, a sulfur-containing, non-protein amino acid with the chemical name 2-aminoethanesulfonic acid, exists in a free form in the human body and is an essential nutrient with multiple physiological functions. It promotes the metabolism of proteins, lipids, amino acids, and carbohydrates, facilitates the absorption of other minerals such as Cu, Fe, Zn, and Mn, and indirectly regulates lipid metabolism. Taurine protects lymphocytes and is closely related to the total white blood cell count in the blood. Taurine deficiency can even lead to abnormalities in cell morphology and function. Studies have also shown that taurine improves the immunomodulatory and antioxidant capacity of immunosuppressed mice of different ages and has significant effects on reproductive performance, immune system development, and antioxidant capacity in certain poultry species. Taurine is a potent liver protectant, improving vision, promoting brain development, relieving fatigue, lowering cholesterol, and providing anti-inflammatory and analgesic benefits. It also possesses some anti-tumor activity. Taurine is widely added to foods for infants, young children, and the elderly. With the continuous improvement of living standards and the growing awareness of taurine's functions, the market potential for taurine production is enormous, with considerable economic benefits.

[0003] Currently, the industrial production of taurine relies primarily on chemical synthesis methods, such as the ethylene oxide method and the ethyleneimine method. However, these methods are associated with high energy consumption, environmental pollution, and the production of numerous byproducts. Natural taurine is widely found in the muscle tissue of marine animals, reaching levels of 3562 mg / kg dry weight. Taurine is found in low concentrations in everyday foods such as vegetables, milk, and eggs, with taurine primarily found in the yolk. However, the extraction of natural taurine is costly.

[0004] With the rapid growth of poultry and egg production, demand for eggs as a daily food is gradually reaching saturation. The deep development and utilization of eggs, particularly the enhancement of their added value through comprehensive processing, has become a topic of growing interest. Therefore, the use of advanced technologies to extract taurine from eggs not only increases the added value of poultry eggs but also provides a new approach to taurine production. However, due to the complex composition and solid structure of egg yolk, the extraction and separation of taurine from eggs presents significant challenges. Summary of the Invention

[0005] In view of the technical problems existing in the background technology, the present invention provides a method for extracting taurine from egg yolk, aiming to solve the technical problems of low efficiency, high cost and low product activity in extracting taurine from egg yolk.

[0006] The technical solutions of the present invention are as follows: A method for extracting taurine from egg yolk comprises the following steps: S1. Thaw the frozen egg yolk and extract it with an extraction reagent; S2. Centrifuge the extraction reaction solution, take the supernatant, let it stand, and then centrifuge it again to obtain a product containing taurine.

[0007] Compared with extracting taurine directly from fresh egg yolks, the present invention first freezes the egg yolks. After freezing, the egg yolks become gelled, and larger ice crystals are formed during the freezing process, which promotes the concentration of taurine, making the taurine in the egg yolks easier to extract under the matching extraction solvent.

[0008] Preferably, in the above method, the egg yolk is frozen at -80°C for 48-72 hours.

[0009] Preferably, in the above method, the extraction reagent is ethanol or ultrapure water.

[0010] More preferably, in the above method, the mass volume ratio of egg yolk to extraction reagent is 1g:(3-6)ml, and the extraction time is 5-8min.

[0011] Preferably, in the above method, the centrifugal conditions for extracting the reaction solution are: 8000-12000 r / min, centrifugation at 4°C for 15-20 min, repeated 2-3 times.

[0012] Preferably, in the above method, the standing is carried out at low temperature, specifically: standing in a -20°C refrigerator for 20-30 minutes or in a 4°C refrigerator for 8-12 hours. Standing at low temperature can promote protein and lipid aggregation and precipitation, further reducing impurities in the product.

[0013] Preferably, in the above method, the conditions for re-centrifugation are: 8000-12000 r / min, centrifugation at 4°C for 3-5 min.

[0014] Based on the above method for extracting taurine from eggs, the present invention also provides a method for detecting the taurine content in egg yolk, which specifically comprises the following steps: S1. Thaw the frozen egg yolk and extract it with an extraction reagent; S2. Centrifuging the extraction reaction solution, taking the supernatant, letting it stand, and centrifuging it again to obtain a supernatant containing taurine; S3. Detecting the taurine content in the supernatant obtained in step S2 by enzyme-linked immunosorbent assay.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The extraction method provided by the present invention is simple and easy to operate. Taurine can be efficiently obtained through freezing, vortex oscillation extraction, centrifugation, standing at low temperature, and re-centrifugation. In this process, the extractant is mild and there is no need to introduce additives such as protein precipitants, which not only reduces costs but also avoids the introduction of impurities. The activity of the obtained product is better, which is of great significance to the industrial production of taurine and the development of high-value-added eggs. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] To more clearly illustrate the technical solution of the present invention, the following briefly introduces the drawings used in the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be derived from these drawings without inventive effort.

[0017] Figure 1 This is a flow chart of a method for extracting taurine from egg yolk according to an embodiment of the present invention; Figure 2 This is a comparison chart of taurine extraction effects from egg yolk under different extraction reagent conditions in the embodiments of the present invention; Figure 3 This is a comparison chart of taurine extraction effects from egg white under different extraction reagent conditions in the embodiments of the present invention; Figure 4 This is a comparison chart of the extraction effects of taurine from unfrozen egg yolks in the comparative example of the present invention using different extraction reagents; Figure 5 This is a comparison chart of the extraction effects of taurine from unfrozen egg white in the comparative example of the present invention using different extraction reagents; Figure 6 This is a comparison chart of taurine extraction effects in egg yolk or egg white at different extraction times in the comparative examples of the present invention; Explanation of the accompanying symbols: the vertical axis Taurine content represents the taurine content, GY represents the yolk of green-shell eggs, PY represents the yolk of pink-shell eggs, GW represents the egg white of green-shell eggs, PW represents the egg white of pink-shell eggs, express p <0.05, express p <0.01, express p <0.001. DETAILED DESCRIPTION

[0018] The following embodiments of the technical solution of the present invention will be described in detail with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention and are therefore only examples and are not intended to limit the scope of protection of the present invention.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains; the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the present invention; the term "include" and any variations thereof herein are intended to cover non-exclusive inclusions.

[0020] Traditional extraction methods for natural taurine (such as acid-base hydrolysis and organic solvent introduction) are inefficient and require strong acid and alkali treatment or high-temperature evaporation of organic solvents. Extreme pH and high temperatures can lead to taurine degradation, oxidation, isomerization, and the production of byproducts, which can easily result in the loss of active substances. Furthermore, the conventional method for detecting taurine in egg yolk or egg white is as follows: weigh the egg yolk or egg white into a conical flask, add warm water at approximately 40°C, mix thoroughly, place on an ultrasonic oscillator for ultrasonic-assisted extraction, and cool to room temperature (25°C). Precipitant I (potassium ferrocyanide solution) and Precipitant II (zinc acetate solution) are then added sequentially, mixed thoroughly, and the supernatant is centrifuged, filtered through a 0.22μm microporous membrane, and analyzed by chromatography. This method is complex and requires the introduction of multiple precipitants.

[0021] In order to solve the technical problem of difficult extraction and detection of taurine in egg yolk, the present invention provides a method for extracting taurine from egg yolk, which can achieve the technical effect of efficient taurine extraction by sequentially performing freezing, vortex oscillation extraction, centrifugation, standing at low temperature and re-centrifugation.

[0022] Please refer to Figure 1 The present invention provides a method for extracting taurine from egg yolk, comprising the following steps: (1) Thaw the frozen egg yolk and extract it with an extraction reagent; (2) The extraction reaction solution is centrifuged, the supernatant is taken, allowed to stand, and then centrifuged again to obtain a product containing taurine.

[0023] Furthermore, in some embodiments, the egg yolk is frozen at -80°C for 48-72 hours, the extraction reagent is selected from ethanol and ultrapure water, the extraction is carried out in a vortex shaker, and the extraction time is not less than 5 minutes.

[0024] Furthermore, in some embodiments, the extraction reaction solution is centrifuged at 8000-12000 r / min for 15-20 min at 4°C, repeated 2-3 times; the extraction reaction solution is centrifuged at 8000-12000 r / min for 3-5 min at 4°C. Furthermore, in some embodiments, the standing is performed at low temperature, specifically: standing in a -20°C refrigerator for 20-30 minutes or in a 4°C refrigerator for 8-12 hours.

[0025] Some specific examples are listed below. It should be noted that the examples described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this field or the product specifications were used. Where the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be purchased commercially.

[0026] In the following examples, taurine in the extracted products obtained in each example and comparative example was detected by enzyme-linked immunosorbent assay (ELISA). The detection was performed using a YZ-20012 (Shanghai LC / MS) kit, and the following procedures were performed according to the instructions: ① Reagent preparation: dilute 20× washing buffer solution with distilled water at a ratio of 1:20 and use immediately.

[0027] ② Sample addition: The experiment sets up blank wells, standard wells and sample wells respectively. No sample is added to the blank wells. 50μl of standard of different concentrations is added to each standard well. 10μl of the sample to be tested is first added to the sample wells, and then 40μl of sample diluent is added. Except for the blank wells, 100μl of horseradish peroxidase (HRP)-labeled detection antibody is added to each standard well and sample well. After gently tapping the ELISA plate, mix evenly, seal the reaction wells with a sealing film, and place it in a 37℃ incubator for 1h.

[0028] ③ Washing: After incubation, shake off all the liquid in each well, wash each well with 350 μl of washing solution and soak for 1 minute, place the ELISA plate on absorbent paper, tap the plate to remove the remaining liquid, and repeat the washing process 5 times.

[0029] ④Add liquid: Add 50 μl of substrate A (hydrogen peroxide solution) and B (TMB solution) to each well, tap to mix, and then place in a 37°C incubator in the dark for 15 minutes.

[0030] ⑤Terminate the reaction: Maintaining the order of adding substrate solutions, add 50 μl of stop solution to each well (the solution will immediately turn from blue to yellow).

[0031] ⑥OD value measurement: After ensuring that the bottom of the ELISA plate is clean and there are no bubbles in each well, measure the OD value of each well at a wavelength of 450nm within 15 minutes using a preheated ELISA reader.

[0032] Under the above conditions, taurine Tau was measured with good accuracy in the detection range of 5.0-800 ng / mL (the correlation coefficient R value between the linear regression of the standard and the expected concentration was greater than or equal to 0.9900), and the lowest detection concentration was less than 5.0 ng / ml.

[0033] The eggs used in the following examples were purchased from the market, including green-shell eggs and pink-shell eggs; the frozen egg whites and egg yolks used in the following examples were taken from the same sample, and the preparation method was as follows: fresh eggs were taken, samples were taken out using an egg yolk and egg white separator, the ligaments were removed and the samples were mixed separately, part of the sample was placed in a -80°C refrigerator, and the remaining part was used for standby; when used, the frozen egg whites and egg yolks were taken out and placed in an ice box to thaw.

[0034] Example 1 This example provides a method for extracting taurine from egg yolk, comprising the following steps: Take 0.1 g of thawed egg yolk and place it in a 1.5 ml centrifuge tube. Add 600 μl of ultrapure water, vortex for 5 minutes, and centrifuge at 12000 r / min for 10 minutes at 4°C. After centrifugation, transfer 400 μl of the supernatant to another centrifuge tube and let it stand in a -20°C refrigerator for 30 minutes. Then, centrifuge again at 12000 r / min for 3 minutes at 4°C, and transfer the supernatant to another centrifuge tube to obtain a clear liquid containing taurine.

[0035] Example 2 This example provides a method for extracting taurine from egg yolk, comprising the following steps: Take 0.1 g of thawed egg yolk and place it in a 1.5 ml centrifuge tube. Add 600 μl of ethanol, vortex for 5 minutes, and centrifuge at 12000 r / min for 10 minutes at 4°C. After centrifugation, transfer 400 μl of the supernatant to another centrifuge tube and let it stand in a -20°C refrigerator for 30 minutes. Then, centrifuge again at 12000 r / min for 3 minutes at 4°C, and transfer the supernatant to another centrifuge tube to obtain a clear liquid containing taurine.

[0036] Comparative Example 1 Different from Example 1, methanol was used as the extraction reagent in this example.

[0037] Comparative Example 2 The difference from Example 1 is that this example uses thawed egg white for extraction.

[0038] Comparative Example 3 The difference from Example 2 is that this example uses thawed egg white for extraction.

[0039] Comparative Example 4 The difference from Example 1 is that in this example, fresh egg yolk or egg white that has not been frozen at -80°C and is kept aside for use is used for extraction.

[0040] Comparative Example 5 The difference from Example 2 is that in this example, fresh egg yolk or egg white that has not been frozen at -80°C and is reserved for use is used for extraction.

[0041] Comparative Example 6 Different from Comparative Example 1, this example uses fresh egg yolk or egg white that has not been frozen at -80°C and is reserved for use for extraction.

[0042] Comparative Example 7 The difference from Example 2 is that the vortex time in this example was changed from 5 min to 1 min.

[0043] The taurine in the products obtained in each embodiment and comparative example was quantitatively detected by ELISA. The test results are as follows: Figure 2-6 As shown, it can be seen from these results that: after the egg yolk is frozen pretreated, the extraction rate of taurine in the egg yolk can be significantly improved under the condition of suitable extraction reagents, but the freezing pretreatment has no obvious positive effect on the extraction of taurine in the egg white; when the egg yolk is frozen pretreated, the extraction effect of taurine in the egg yolk is best when ethanol is used as the extraction reagent; the extraction time has a great influence on the extraction effect, so it is recommended to keep the vortex for 5 minutes or more under the conditions of the present invention.

[0044] In addition, the above results show that the applicable extraction methods and extraction reagents for egg white and egg yolk are different; for example, freezing treatment can significantly improve the extraction effect of taurine in egg yolk, but has no positive effect on egg white. For example, under the same treatment conditions, taurine in egg white is best extracted with ultrapure water, while taurine in egg yolk is best extracted with ethanol.

[0045] The upper and lower limits, and interval values of the raw materials, as well as the upper and lower limits, and interval values of the process parameters listed in the present invention can all implement the present invention, and the embodiments are not listed one by one here.

[0046] In summary, the extraction method provided by the present invention has the advantages of being simple and easy to operate, having mild extraction conditions, having low equipment requirements, low cost, and having little pollution, and is particularly suitable for the extraction of taurine from egg yolk.

[0047] It should be noted that the present invention is not limited to the above-mentioned embodiments. The above-mentioned embodiments are merely examples, and any embodiments having substantially the same structure and effect as the technical concept within the scope of the technical solution of the present invention are all included in the technical scope of the present invention. In addition, without departing from the scope of the present invention, other embodiments that can be conceived by those skilled in the art and that combine some of the constituent elements in the embodiments are also included in the scope of the present invention.

Claims

1. A method for extracting taurine from egg yolk, characterized in that: The following steps are involved: S1. Thaw the frozen egg yolk and extract it with an extraction reagent; S2. Centrifuge the extraction reaction solution, take the supernatant, let it stand, and then centrifuge it again to obtain a product containing taurine.

2. The method according to claim 1, characterized in that In step S1, the egg yolk is frozen at -80°C for 48-72 hours.

3. The method according to claim 1, characterized in that The extraction reagent is ethanol or ultrapure water.

4. The method according to claim 1, wherein The mass volume ratio of the egg yolk to the extraction reagent is 1 g: (3-6) ml, and the extraction time is 5-8 minutes.

5. The method according to claim 1, characterized in that The extraction reaction solution was centrifuged at 8000-12000 r / min and 4° C. for 15-20 min, which was repeated 2-3 times.

6. The method according to claim 1, characterized in that The standing is performed at low temperature.

7. The method according to claim 6, characterized in that The specific standing is: standing in a -20°C refrigerator for 20-30 minutes or standing in a 4°C refrigerator for 8-12 hours.

8. The method according to claim 6, characterized in that The conditions for the re-centrifugation are: 8000-12000 r / min, 3-5 min at 4°C.

9. Application of the method as claimed in claims 1 to 8 in detecting the taurine content in egg yolk.

10. The use according to claim 9, characterized in that The product was taken for enzyme-linked immunosorbent assay.

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