Feed additive for promoting xanthophyll deposition of micropterus salmoides and detection method

Abstract

The invention relates to the technical field of feed additives, in particular to a feed additive for promoting xanthophyll deposition of largemouth bass and a detection method. An extracting solution, a 40% KOH-methanol solution and a 10% Na2SO4 solution are prepared; the method comprises the following steps: pretreating a sample by using an extracting solution and 40% KOH-methanol; carrying out saponification treatment on the pretreated sample, and then cooling; treating the sample subjected to saponification treatment by using a Na2SO4 solution; and measuring the absorbance of the treated sample. By optimizing the formula of the extracting solution and improving the pretreatment and saponification conditions, the extraction efficiency of the xanthophyll is remarkably improved, so that the accuracy of a detection result is ensured. Tedious steps and potential error sources are reduced, so that the detection result is more reliable. Meanwhile, the adopted materials and reagents are relatively low in cost, high-end professional equipment is not needed in the operation process, and the detection cost is reduced. Therefore, the problems that an existing xanthophyll deposition detection method is high in cost and complex in operation are solved.

Description

Technical Field

[0001] The invention relates to the technical field of feed additives, in particular to a feed additive for promoting lutein deposition of largemouth bass and a detection method thereof. Background Art

[0002] Lutein is an important natural pigment that is widely present in plant and animal tissues, especially in fish. During the breeding process of largemouth bass (Micropterus salmoides), the deposition of lutein not only affects the appearance and color of the fish, but is also closely related to its antioxidant capacity, immunity and growth performance. Therefore, accurate detection of the deposition of lutein in largemouth bass is of great significance for evaluating the effect of feed additives, optimizing breeding conditions and improving breeding benefits.

[0003] Traditional lutein detection methods mostly use high performance liquid chromatography (HPLC), but these methods are complex to operate, costly, and require professional technicians and equipment support, which limits their widespread application in grassroots farms. Therefore, the development of a simple, fast, and accurate detection method is of great significance to promote the research and application of lutein deposition in largemouth bass. Summary of the invention

[0004] The purpose of the present invention is to provide a feed additive and a detection method for promoting lutein deposition in largemouth bass, aiming to solve the problems of high cost and complicated operation of the existing lutein deposition detection method.

[0005] To achieve the above object, in a first aspect, the present invention provides a method for detecting the promotion of lutein deposition in largemouth bass, comprising the following steps:

[0006] Prepare extract, 40% KOH-methanol solution and 10% Na2SO4 solution;

[0007] The samples were pretreated using extraction solution and 40% KOH-methanol;

[0008] The pretreated sample is subjected to saponification treatment and then cooled;

[0009] The saponified samples were treated with Na2SO4 solution;

[0010] The absorbance of the treated samples was measured.

[0011] Among them, in "preparing the extract, 40% KOH-methanol solution and 10% Na2SO4 solution", the extract is prepared with n-hexane, anhydrous ethanol, acetone and toluene, and the ratio is 10:7:6:6; the 40% KOH-methanol solution is prepared by placing 40g KOH in a 250ml beaker, adding 15ml pure water and stirring rapidly, adding 50ml methanol and stirring until clarified and constant volume; the 10% Na2SO4 solution is prepared with anhydrous sodium sulfate and pure water, and the ratio is 1:9.

[0012] Among them, in "pre-treating the sample using the extraction solution and 40% KOH-methanol", the following steps are included:

[0013] Weigh 0.5g of sample and put it into a 50ml constant volume flask;

[0014] After adding 25 ml of the extract, ultrasonic treatment was performed and then the extract was added to make up the volume;

[0015] Take 5 ml of the diluted solution into a 100 ml volumetric flask and add 25 ml of the extract solution, shake well, then add 2 ml of 40% KOH-methanol solution into the volumetric flask, cover it and shake gently for 1 min.

[0016] Among them, in "performing saponification treatment on the pretreated sample", the duration of the saponification treatment is 20 minutes and the cooling time is 10 minutes.

[0017] Among them, in “treating the saponified sample by Na2SO4 solution”, the following steps are included:

[0018] Add 30 ml of n-hexane to the constant volume flask, shake gently for 1 min, then add 10% Na2SO4 solution to make up the volume;

[0019] Place the volumetric flask on a shaker and shake for 2 min, then place the volumetric flask in the dark for 1 h.

[0020] In a second aspect, a feed additive for promoting lutein deposition in largemouth seabass, used in the method for detecting a feed additive for promoting lutein deposition in largemouth seabass as described in the first aspect, comprises silkworm feces.

[0021] The detection method for promoting lutein deposition in largemouth black bass of the present invention comprises the following steps: preparing an extract, a 40% KOH-methanol solution and a 10% Na2SO4 solution; using the extract and the 40% KOH-methanol to pretreat a sample; performing saponification treatment on the pretreated sample and then cooling it; treating the saponified sample with a Na2SO4 solution; and measuring the absorbance of the treated sample. The present invention significantly improves the extraction efficiency of lutein by optimizing the extract formula, improving the pretreatment and saponification conditions, thereby ensuring the accuracy of the detection results. Compared with the traditional detection method, the scheme is simpler to operate, reduces cumbersome steps and potential sources of error, and makes the detection results more reliable. At the same time, the cost of the materials and reagents used is relatively low, and the operation process does not require high-end professional equipment, which reduces the detection cost. This makes the scheme more popular and practical in grassroots farms and scientific research institutions, and helps to promote the widespread application of largemouth black bass lutein deposition detection technology. Thereby solving the problem of high cost and complex operation of the existing lutein deposition detection method. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 The present invention provides a flow chart of a method for detecting the promotion of lutein deposition in largemouth bass.

[0024] Figure 2 is a flow chart of sample pretreatment using extraction solution and 40% KOH-methanol.

[0025] Figure 3 It is a flow chart of treating the sample after saponification by Na2SO4 solution.

[0026] Figure 4 It is the overall body color control group of largemouth bass.

[0027] Figure 5 This is the view of the largemouth bass in each treatment group after removing the scales from the body and observing under a 100x oil microscope.

[0028] Figure 6 This is a view of the largemouth bass in each treatment group observed under a 10x optical microscope after removing the scales on the body surface.

[0029] Figure 7 This is a view of the middle fin ray of the caudal fin of a largemouth bass observed under a 4x optical microscope. DETAILED DESCRIPTION

[0030] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.

[0031] See also Figures 1 to 7 In a first aspect, the present invention provides a method for detecting the promotion of lutein deposition in largemouth bass, comprising the following steps:

[0032] S1 is prepared with extracting solution, 40% KOH-methanol solution and 10% Na2SO4 solution;

[0033] The extract is prepared using n-hexane, anhydrous ethanol, acetone and toluene in a ratio of 10:7:6:6. The 40% KOH-methanol solution is prepared by placing 40g KOH in a 250ml beaker, adding 15ml pure water and stirring rapidly, adding 50ml methanol and stirring until clarified and constant volume. The 10% Na2SO4 solution is prepared using anhydrous sodium sulfate and pure water in a ratio of 1:9.

[0034] Specifically, before the experiment begins, prepare the following reagents in advance: extraction solution (n-hexane: anhydrous ethanol: acetone: toluene = 10:7:6:6); 40% KOH-methanol solution (put 40g KOH in a 250ml beaker, add 15ml pure water and stir quickly, add 50ml methanol, stir until clear, transfer to a 100ml volumetric flask, and make up to volume with methanol); 10% Na2SO4 solution (mass of anhydrous sodium sulfate: mass of pure water = 1:9).

[0035] S2 pretreated the samples using extraction solution and 40% KOH-methanol;

[0036] S21 weigh 0.5g of sample and put it into a 50ml constant volume bottle;

[0037] Specifically, when sampling, use tweezers to carefully remove the scales on the body surface of the largemouth bass, and carefully remove the back skin. After mixing the samples, weigh 0.5 g of the sample (accurate to 0.0001 g) and add it to a 50 ml volumetric flask.

[0038] S22 was added with 25 ml of extract solution, subjected to ultrasonic treatment, and then the extract solution was added to make up the volume;

[0039] Specifically, add about 25 ml of the extract, and ultrasonicate for 2 minutes until the color of the liquid no longer changes; then add the extract to make up to volume and shake well.

[0040] S23 Take 5 ml of the diluted solution in a 100 ml volumetric flask and add 25 ml of the extract solution, shake well, then add 2 ml of 40% KOH-methanol solution to the volumetric flask, cover it and shake gently for 1 min.

[0041] Specifically, take 5 ml of the diluted solution into a 100 ml volumetric flask, add 25 ml of the extract, and shake gently to mix; add 2 ml of 40% KOH-methanol solution into the volumetric flask, cover it and shake gently for 1 min.

[0042] S3 performs saponification treatment on the pretreated sample and then cools it;

[0043] The duration of the saponification treatment is 20 minutes, and the cooling time is 10 minutes.

[0044] Specifically, saponify in a water bath at 56°C for 20 minutes. During the saponification process, a condensing device should be added to condense the bottleneck of the volumetric flask to prevent the solution in the volumetric flask from evaporating. After the saponification is completed, the volumetric flask is immediately placed in cold water to cool for 10 minutes until it reaches room temperature.

[0045] S4 treated the saponified sample with Na2SO4 solution;

[0046] S41 Add 30 ml of n-hexane to the constant volume flask, shake gently for 1 min, and then add 10% Na2SO4 solution to make up the volume;

[0047] Specifically, add 30 ml of n-hexane to the volumetric flask, shake gently for 1 min, and then add 10% Na2SO4 solution to make up to volume.

[0048] S42 Place the volumetric flask on a shaker and shake for 2 minutes, then place the volumetric flask in the dark for 1 hour.

[0049] Specifically, the volumetric flask was placed on a shaker and shaken vigorously at 200 times / min for 2 min, and then the volumetric flask was treated in the dark for 1 h;

[0050] S5 measures the absorbance of the treated sample.

[0051] Specifically, 2 ml of the supernatant was transferred to a new 50 ml volumetric flask and the volume was fixed with n-hexane; the absorbance A was measured at a wavelength of (473 ± 1) nm using an ultra-micro spectrophotometer, with n-hexane used as a blank control; the calculation and results were performed according to the following formula:

[0052] X=2.11864×A 473 ×K / M

[0053] Where: X——mass fraction of lutein (C40H56O2) in the sample (g / kg);

[0054] K——dilution multiple of sample supernatant;

[0055] M——sample mass (g);

[0056] A473——Absorbance.

[0057] The measurement results are shown in the following table:

[0058] Table 1 Comparison of different silkworm excrement additions on lutein content in the skin of largemouth bass

[0059]

[0060] Note: Data are expressed as mean ± standard error (n=3). Different letters in the same row indicate statistically significant differences (P<0.05).

[0061] After testing, it was found that when silkworm feces were added to the feed of largemouth bass, the lutein content in the skin of largemouth bass in the MF1.5 group did not increase significantly compared with the group fed with ordinary commercial feed (P>0.05), but there was an upward trend compared with the group fed with ordinary commercial feed; and the lutein content in the skin of largemouth bass in the MF3.0 and MF4.5 groups increased significantly compared with the group fed with ordinary commercial feed (P<0.05). As the amount of silkworm feces added to the feed increased, the lutein content in the skin of largemouth bass also showed an increasing trend. This also shows that when silkworm feces are added to the feed of largemouth bass, the skin color can be significantly yellowed, and the degree of yellowing can increase with the increase of the amount of silkworm feces added to the feed.

[0062] From a macroscopic perspective, after the breeding experiment, the largemouth bass in each treatment group were anesthetized and placed on a white board to observe the changes in body color with the naked eye. The body color of the largemouth bass in the control group was overall bluish-black (Figure A); after 1.5% silkworm feces were added to the feed, the overall color of the fish was yellowish, including the scales, skin and fins, which were slightly yellow (Figure B); after 3.0% and 4.5% silkworm feces were added to the feed, the largemouth bass, including the overall color, scales, skin and fins, became more and more yellow (Figures C and D). As the amount of silkworm feces added to the feed increased, the degree of yellowing of its body color also showed an increasing trend.

[0063] The scales at the base of the dorsal fin of largemouth black bass in each treatment group were placed on a slide, 0.65% Ringer's solution was added, and a coverslip was placed on it. The scales were then observed under a 100x oil microscope. The results showed that the pigment in the scales of the control group was mainly melanin, and the melanin was star-shaped, had a strong color development ability, occupied a large volume, and the fish body was mainly black (Figure E); after adding 1.5% silkworm feces to the feed, the melanin in the scales changed from star-shaped to dot-shaped, and the color development ability weakened. At the same time, the number and volume of xanthophyll cells continued to increase (as shown by the arrows), and they were distributed around the melanocytes, which enhanced the color development ability of the xanthophyll cells, resulting in a slightly yellowish color of the fish body (Figure F); after adding 3.0% and 4.5% silkworm feces to the feed, the morphology of melanin in the scales of largemouth black bass also changed, and the number and volume of xanthophyll cells further increased, and the color development ability of xanthophyll cells was further enhanced, making the fish body color yellow (Figures G and H).

[0064] After carefully removing the scales on the body surface of the largemouth seabass in each treatment group with tweezers, the skin of the middle part of the back was taken on a glass slide, 0.65% Ringer's solution was added, and then the skin was observed under a 10x optical microscope. The results showed that the skin of the largemouth seabass in the control group was mainly black, and the skin was mainly composed of melanocytes, accompanied by a small number of xanthophyll cells (Figure I); after adding silkworm feces to the feed, a large number of xanthophyll cells were filled around the melanocytes on the surface of the fish skin (indicated by the arrows), and the color of the fish skin turned yellow. As the silkworm feces content in the feed increased, the yellowing degree of the fish skin gradually increased (Figures J, K and L).

[0065] The fin rays in the middle of the caudal fin of largemouth black bass in each treatment group were cut with surgical scissors and placed on a glass slide. After adding 0.65% Ring's solution, the rays were observed under a 4x optical microscope. The results showed that the pigment of the caudal fin rays of the control group was mainly melanin, and the overall color of the fin rays was black (Figure M). After adding silkworm feces to the feed, the number and volume of xanthophyll cells in the caudal fin rays increased greatly, and with the increase in the amount of silkworm feces added, the proportion of xanthophyll cells in the fin rays increased step by step, and the yellowing degree of the fin rays gradually increased (Figures N, O and P).

[0066] During the experiment, under the action of an optical microscope, the scales, skin, fins and other tissues of the fish can be magnified, so that the changes in the amount and distribution of pigments in each tissue can be intuitively observed. According to relevant literature, the main component of yellow pigment cells is lutein, so the content of yellow pigment cells in various tissues of the fish can be quantified by measuring the content of lutein. Lutein is easily soluble in n-hexane and insoluble in water, so lutein can be dissolved in the extract through this property, and then the absorbance of lutein is measured using an ultraviolet spectrophotometer based on the characteristic that lutein has a maximum absorption peak at a wavelength of 473±1nm, and the absorbance is converted into a numerical value representing the content using a formula, thereby achieving quantitative determination of yellow pigment cells.

[0067] In a second aspect, a feed additive for promoting lutein deposition in largemouth seabass, used in the method for detecting a feed additive for promoting lutein deposition in largemouth seabass as described in the first aspect, comprises silkworm feces.

[0068] In this embodiment, silkworm excrement does not need to go through commercial processing, purification and other steps. It only needs to be collected, dried and crushed before it can be added to aquatic feed as an additive, thereby reducing the cost of breeding, which is in line with the concept of building a resource-saving and environmentally friendly society; adding silkworm excrement to aquatic feed is conducive to promoting the comprehensive development and utilization of high-quality silkworm excrement resources, and can promote the development of sericulture to a certain extent. The coloring effect of silkworm excrement is significant. As shown in the figure below, after adding 3% silkworm excrement to the feed, only through an 8-week feeding experiment, the number and volume of xanthophyll cells in the scales of largemouth bass increased significantly, and the melanocytes shrank from radioactive stars to dots, and the volume was significantly reduced, causing the largemouth bass to be yellow in color, making it more satisfactory to consumers in terms of sensory perception (yellow represents fish health), and can increase its sales value. Compared with commercial color enhancers such as aurein Y, astaxanthin, etc., the price of silkworm excrement is much lower, so using silkworm excrement as a color enhancer for aquatic animals can greatly reduce breeding costs, increase commercial value, and have the important significance of "turning waste into treasure" in green ecological environmental protection. In addition, silkworm feces contain a large amount of nutrients and bioactive substances, which may promote the growth, antioxidant properties, immune and disease resistance of aquatic animals, playing an important role of "killing two birds with one stone".

[0069] The above disclosure is only a preferred embodiment of the feed additive and detection method for promoting lutein deposition in largemouth bass of the present invention. Of course, this cannot be used to limit the scope of rights of the present invention. Ordinary technicians in this field can understand that all or part of the processes of the above embodiments and equivalent changes made according to the claims of the present invention still fall within the scope of the invention.

Claims

1. A method for detecting the deposition of lutein in largemouth bass, characterized in that: The following steps are involved: Prepare extract, 40% KOH-methanol solution and 10% Na2SO4 solution; The samples were pretreated using extraction solution and 40% KOH-methanol; The pretreated sample is subjected to saponification treatment and then cooled; The saponified samples were treated with Na2SO4 solution; The absorbance of the treated samples was measured.

2. The method for detecting an agent for promoting lutein deposition in largemouth bass according to claim 1, characterized in that: In "preparing the extracting solution, 40% KOH-methanol solution and 10% Na2SO4 solution", the extracting solution is prepared with n-hexane, anhydrous ethanol, acetone and toluene in a ratio of 10:7:6:6; the 40% KOH-methanol solution is prepared by placing 40g KOH in a 250ml beaker, adding 15ml pure water and stirring rapidly, adding 50ml methanol and stirring until clarified and constant volume; the 10% Na2SO4 solution is prepared with anhydrous sodium sulfate and pure water in a ratio of 1:

9.

3. The method for detecting an agent for promoting lutein deposition in largemouth bass according to claim 2, characterized in that: In "Pretreatment of samples using extraction solution and 40% KOH-methanol", the following steps are included: Weigh 0.5g of sample and put it into a 50ml constant volume flask; After adding 25 ml of the extract, ultrasonic treatment was performed and then the extract was added to make up the volume; Take 5 ml of the diluted solution into a 100 ml volumetric flask and add 25 ml of the extract solution, shake well, then add 2 ml of 40% KOH-methanol solution into the volumetric flask, cover it and shake gently for 1 min.

4. The method for detecting an agent for promoting lutein deposition in largemouth bass according to claim 3, characterized in that: In "performing saponification treatment on the pretreated sample", the saponification treatment lasts for 20 minutes and the cooling time is 10 minutes.

5. The method for detecting an agent for promoting lutein deposition in largemouth bass according to claim 4, characterized in that: In "treating the sample after saponification by Na2SO4 solution", the following steps are included: Add 30 ml of n-hexane to the constant volume flask, shake gently for 1 min, then add 10% Na2SO4 solution to make up the volume; Place the volumetric flask on a shaker and shake for 2 min, then place the volumetric flask in the dark for 1 h.

6. A feed additive for promoting lutein deposition in largemouth bass, used in the method for detecting a feed additive for promoting lutein deposition in largemouth bass according to any one of claims 1 to 6, characterized in that: Including silkworm feces.

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