Compound additive for grouper feed with high vegetable protein content and application of compound additive

Abstract

The invention discloses a compound additive for grouper feed with high vegetable protein content and application of the compound additive, the compound additive is bovine cholate and taurochenodeoxycholate, and the mass ratio of the bovine cholate to the taurochenodeoxycholate is (1-3): (1-3). The composite additive can be applied to preparation of high-vegetable-protein feed for groupers, preparation of high-vegetable-protein feed for improving growth performance of the groupers and preparation of high-vegetable-protein feed for improving liver tissue structures of the groupers. The high-vegetable-protein feed for improving the liver health of the groupers is prepared or the utilization rate of the groupers to the high-vegetable-protein feed is increased.

Description

Technical Field

[0001] The present invention belongs to the technical field of feed additives, and particularly relates to a compound additive for high-plant-protein feed for groupers and its application. Background Art

[0002] The pearl gentian grouper (Epinephelus fuscoguttatus♀×E. ) is an important economic fish in the southern waters of China and has great development potential. However, the cost of commercial feed for groupers remains high, mainly due to the high proportion of fish meal in the feed (about 50%), which directly leads to the increase in production costs. With the rapid development of mariculture and the increasing depletion of wild fish resources, the supply of fish meal has become increasingly tight and the price has continued to rise, which greatly restricts the development of the grouper aquaculture industry. To reduce the feed cost, it has become an urgent task to develop low-cost plant protein sources to replace fish meal. Since plant proteins contain relatively high anti-nutritional factors, their impact on fish is not obvious when replacing fish meal at a low level, but when the replacement ratio exceeds a certain threshold, it often damages liver health, resulting in slow growth or even death of groupers. Therefore, how to improve the utilization rate of high-plant-protein feed by groupers has become an important challenge faced by the grouper aquaculture industry.

[0003] In recent years, it has been found that adding bile acids to feed can alleviate the liver and pancreas damage of aquatic animals caused by high-plant-protein. As one of the important components of bile, bile acids are a general term for a class of steroidal carboxylic acids produced by cholesterol metabolism in the liver. Free bile acids, that is, unconjugated bile acids, after being synthesized in the liver, combine with glycine or taurine to form conjugated bile acids. The conjugation process reduces the hydrophilicity and toxicity of bile acids and enhances their acidity, thereby exerting specific biological functions. At present, there are no reports and applications on using conjugated bile acids or their salts to improve the utilization rate of high-plant-protein feed by groupers. Summary of the Invention

[0004] The purpose of the present invention is to provide a compound additive for high-plant-protein feed for groupers.

[0005] The present invention also aims at the application of the above compound additive in the preparation of high-plant-protein feed for groupers, in the preparation of high-plant-protein feed for improving the growth performance of groupers, in the preparation of high-plant-protein feed for improving the liver tissue structure of groupers, in the preparation of high-plant-protein feed for improving the liver health of groupers, or in the application of improving the utilization rate of high-plant-protein feed by groupers.

[0006] The above first object of the present invention can be achieved by the following technical solution: A high-plant-protein feed composite additive for groupers, wherein the composite additive is cholate and taurochenodeoxycholate, and the mass ratio of cholate to taurochenodeoxycholate is 1-3:1-3.

[0007] Preferably, the cholate and the taurochenodeoxycholate in the present invention are sodium salts of conjugated bile acids.

[0008] Preferably, the cholate is sodium cholate, and the taurochenodeoxycholate is sodium taurochenodeoxycholate.

[0009] Preferably, the mass ratio of sodium cholate to sodium taurochenodeoxycholate is 1:1.

[0010] The above second object of the present invention can be achieved by the following technical solution: The application of the above composite additive in the preparation of a high-plant-protein feed for groupers.

[0011] The present invention also provides the application of the above composite additive in the preparation of a high-plant-protein feed for improving the growth performance of groupers.

[0012] The present invention also provides the application of the above composite additive in the preparation of a high-plant-protein feed for improving the liver tissue structure of groupers.

[0013] The present invention also provides the application of the above composite additive in the preparation of a high-plant-protein feed for improving the liver health of groupers.

[0014] Preferably, the improvement of the liver health of groupers includes improving the liver tissue structure of groupers, reducing liver damage of groupers, reducing the inflammatory response of groupers' livers, enhancing the oxidative stress response of groupers' livers, reducing endoplasmic reticulum stress in groupers' livers, and reducing apoptosis of groupers' liver cells.

[0015] The present invention also provides the application of the above composite additive in improving the utilization rate of high-plant-protein feed by groupers.

[0016] Preferably, the high-plant-protein feed in the present invention includes using concentrated cottonseed protein to replace at least 50% of the animal protein in the traditional grouper feed, and the animal protein is fish meal.

[0017] More preferably, the high-plant-protein feed in the present invention includes using concentrated cottonseed protein to replace 50% of the animal protein in the traditional grouper feed, and the animal protein is fish meal.

[0018] As a preferred embodiment of the present invention, the high-plant-protein feed of the present invention is made from the following raw materials in parts by mass:

[0019] Fish meal 24-26

[0020] Gluten: 9 - 11

[0021] Flour: 14 - 16

[0022] Concentrated cottonseed protein: 24.5 - 25.5

[0023] Corn protein powder: 5 - 6

[0024] Casein: 4 - 6

[0025] Gelatin: 0.8 - 1.2

[0026] Fish oil: 3.4 - 3.5

[0027] Soybean oil: 1.4 - 1.6

[0028] Soybean lecithin: 1.9 - 2.1

[0029] Calcium dihydrogen phosphate: 0.9 - 1.1

[0030] Vitamin C: 0.02 - 0.04

[0031] Choline chloride: 0.4 - 0.6

[0032] Vitamin premix: 0.4 - 0.6

[0033] Mineral premix: 0.4 - 0.6

[0034] Antioxidant: 0.04 - 0.06

[0035] Feeding attractant: 0.08 - 0.12

[0036] Microcrystalline cellulose: 2.6 - 2.8

[0037] Methionine: 0.30 - 0.32

[0038] Lysine: 0.5 - 0.6

[0039] The above composite additive: 0.08 - 0.1

[0040] More preferably, the high-plant-protein feed of the present invention is made from the following raw materials in parts by mass: Fish meal 25

[0041] Gluten 10

[0042] Flour 15

[0043] Concentrated cottonseed protein 25.20

[0044] Corn protein powder 5.5

[0045] Casein 5

[0046] Gelatin 1

[0047] Fish oil 3.45

[0048] Soybean oil 1.50

[0049] Soybean lecithin 2

[0050] Calcium dihydrogen phosphate 1

[0051] Vitamin C 0.03

[0052] Choline chloride 0.5

[0053] Vitamin premix 0.5

[0054] Mineral premix 0.5

[0055] Antioxidant 0.05

[0056] Feeding attractant 0.10

[0057] Microcrystalline cellulose 2.72

[0058] Methionine 0.31

[0059] Lysine 0.55

[0060] The above-mentioned compound additive 0.09.

[0061] The present invention has the following advantages:

[0062] (1) The compound additive provided by the present invention can effectively improve the problems of the decline in the growth performance of fish caused by high-plant-protein feed and liver damage, and significantly improve the tolerance of grouper to high-plant-protein feed;

[0063] (2) After adding the compound additive in the present invention, grouper can better adapt to high-plant-protein feed, and improve the utilization rate of high-plant-protein feed by grouper;

[0064] (3) The compound additive in the present invention can also improve the liver health of grouper by reducing liver inflammatory response, improving antioxidant performance, reducing endoplasmic reticulum stress and apoptosis. Brief description of the drawings

[0065] Figure 1 HE section of the liver of grouper in Example 1;

[0066] Figure 2 Content of liver injury markers of grouper in Example 1;

[0067] Figure 3 Expression levels of pro-inflammatory factors and anti-inflammatory factors in the liver of grouper in Example 1;

[0068] Figure 4Expression levels of grouper liver oxidative stress-related molecules in Example 1;

[0069] Figure 5 Expression levels of grouper liver endoplasmic reticulum stress-related molecules in Example 1;

[0070] Figure 6 Expression levels of grouper liver apoptosis-related molecules in Example 1. Detailed implementation manners

[0071] The technical solutions in the present invention will be clearly and completely described below in conjunction with the embodiments and drawings in the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0072] In the following embodiments, unless otherwise specified, all are conventional methods. Unless otherwise specified, the raw materials used in the following embodiments are all commercially available products.

[0073] Example 1

[0074] 1. Test method

[0075] 1.1 Feed formula

[0076] According to the feed formula in Table 1, a control diet (CD) with a crude protein level of 48.05% and a crude fat level of 9.89% was prepared.

[0077] 50% of the fish meal in CD was replaced with concentrated cottonseed protein, and the contents of lipids, methionine and lysine were balanced accordingly to prepare a high-plant-protein diet (PD) with a crude protein level of 48.09% and a crude fat level of 9.50%.

[0078] Meanwhile, 0.9% of unconjugated bile acid sodium (prepared by mixing sodium cholate and chenodeoxycholic acid sodium in a ratio of 1:1) was added to PD to prepare an unconjugated bile acid sodium diet (NBD) with a crude protein level of 48.55% and a crude fat level of 9.73%.

[0079] In addition, 0.9% of conjugated bile acid sodium (prepared by mixing sodium taurocholate and sodium tauroursodeoxycholate in a ratio of 1:1) was added to PD to prepare a conjugated bile acid sodium diet (CBD) with a crude protein level of 47.95% and a crude fat level of 9.75%.

[0080] The preparation process of the feed is as follows:

[0081] First, crush the raw materials used and sieve them through a 60-mesh sieve; then take fish meal, concentrated cottonseed protein (added in PD, NBD, and CBD), wheat gluten, flour, corn protein powder, casein, and gelatin, and mix them evenly to obtain mixture a;

[0082] Secondly, take calcium dihydrogen phosphate, vitamin C, choline chloride, antioxidant, attractant, microcrystalline cellulose, vitamin premix, and mineral premix. After fully mixing them evenly, add them to mixture a and mix evenly to obtain mixture b;

[0083] Next, add fish oil, soybean oil, soy lecithin, unconjugated sodium cholate (added in NBD), and conjugated sodium cholate (added in CBD) to mixture b, and stir and mix evenly to obtain mixture c;

[0084] Finally, add 30% moisture to mixture c, and then process it into pellet feed with a particle size of 2.5 mm using a twin-screw extruder (South China University of Technology). Air-dry it at room temperature until the moisture content is about 10%, and then put it in the refrigerator (-20 °C) for standby. Among them, the nutritional components in the feed are detected for crude protein by the Kjeldahl method, for crude fat by a Soxhlet extractor, for moisture by drying in an oven at 105 °C to constant weight, and for ash by drying in a muffle furnace at 550 °C.

[0085] Table 1 Feed formula

[0086]

[0087]

[0088] It should be noted here that the vitamin premix, mineral premix, antioxidant, attractant, microcrystalline cellulose, etc. used in the embodiments of the present invention are all commonly used commercially available raw materials in the art, which can be directly purchased and added according to the usage instructions.

[0089] 1.2 Aquaculture management

[0090] The present invention takes juvenile pearl gentian groupers as the research object and feeds the groupers with the four prepared feeds. Specifically, pearl gentian grouper fry with an average weight of 15.21 grams are randomly divided into 12 replicate groups, with 25 tails in each group. The experimental period is 56 days, during which feeding is carried out twice a day, at 8:00 am and 17:00 pm respectively. Throughout the experimental process, two-thirds of the water volume in the aquaculture system is changed daily to ensure stable water quality. During the aquaculture process, the water temperature is maintained at 28 ± 2 °C, the dissolved oxygen content is maintained above 7 mg / L, and at the same time, the ammonia nitrogen content is strictly controlled to be less than 0.03 mg / L, and the nitrate content is also controlled below 0.03 mg / L.

[0091] 2. Test results

[0092] 2.1 Effects of Different Diets on the Growth Performance of Groupers

[0093] The calculation formulas for growth performance are as follows:

[0094] Weight gain rate (WGR, %) = (final weight - initial weight) × 100 / initial weight;

[0095] Specific growth rate (SGR, %) = 100 × (natural logarithm of final weight - natural logarithm of initial weight) / number of days of culture;

[0096] Feed intake (FI, %) = 100 × feed intake / (final weight + initial weight / (2 × number of days of culture));

[0097] Hepatosomatic index (HSI, %) = 100 × wet weight of liver / wet weight of fish body;

[0098] The results of growth performance are shown in Table 2. Compared with the CD group, the HSI of groupers in the PD group was significantly increased, but the FBW, WGR, and SGR were significantly decreased. Compared with the PD group, the FBW, WGR, and SGR of the CBD group were significantly increased. No significant differences were observed in FBW, WGR, SGR, and HSI between the PD group and the NBD group.

[0099] These results indicate that the treatment with conjugated bile acids (a mixture of sodium cholate and sodium taurochenodeoxycholate) significantly alleviated the inhibitory effect of high-plant-protein diets on the growth performance of groupers.

[0100] Table 2 Growth Performance of Groupers Fed Different Diets

[0101]

[0102] 2.2 Effects of Different Diets on the Liver Pathology of Groupers

[0103] Figure 1 The histological characteristics of the livers of groupers under different diet treatments are shown.

[0104] Figure 1 The results in

[0105] The liver tissue structure of fish fed CD was normal, with small and regularly arranged hepatocytes, and round and clearly visible cell nuclei.

[0106] In contrast, the fish in the PD treatment group showed obvious characteristics of hepatocyte damage: most hepatocytes were significantly enlarged, with multiple small vacuoles or a single large vacuole visible in the cytoplasm, and at the same time, signs of displacement of some cell nuclei to the periphery or nuclear rupture were observed.

[0107] Compared with the PD group, the histological features of the liver in the CBD group were similar to those in the normal group (CD), with only mild enlargement observed in a few hepatocytes, and the overall liver structure remained in good condition.

[0108] The liver in the NBD treatment group showed pathological features similar to those in the PD group, but the degree of lesions was more significant: most hepatocytes were significantly swollen, with severe lipid droplet deposition, and some nuclei showed abnormal phenomena such as displacement or lysis.

[0109] These results indicate that the treatment with conjugated bile acids (a mixture of sodium cholate and sodium taurochenodeoxycholate) significantly improved the histological structure of the liver in groupers.

[0110] 2.3 Effects of different diets on the liver health of groupers

[0111] Figure 2 Shows the effects of different diets on the liver injury markers in groupers.

[0112] Figure 2 The results in

[0113] Compared with the CD group, in the group fed with the PD diet, the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum were significantly increased.

[0114] Further analysis found that compared with the PD group, the treatment in the CBD group significantly reduced the activities of these two enzymes, while the NBD group did not show a significant effect.

[0115] These results indicate that the treatment with conjugated bile acids (a mixture of sodium cholate and sodium taurochenodeoxycholate) significantly improved the damage to the liver in groupers caused by the high plant protein diet.

[0116] 2.4 Effects of different diets on the liver inflammatory response in groupers

[0117] Figure 3 Shows the effects of different diets on the liver inflammatory response in groupers.

[0118] Figure 3 The results in

[0119] Compared with the CD group, feeding with the PD diet significantly upregulated the mRNA levels of il1β and tnfα, and at the same time significantly downregulated the mRNA level of il10.

[0120] Compared with the PD group, the CBD treatment significantly upregulated the expression of il10, and at the same time significantly downregulated the expressions of il1β and tnfα.

[0121] It is worth noting that compared with the PD group, the NBD group not only significantly upregulated the expression of tnfα, but also significantly downregulated the expression of il10.

[0122] These results indicate that the treatment with conjugated sodium bile acids (a mixture of sodium taurocholate and sodium tauroursodeoxycholate) significantly improved the inflammatory response in the liver of groupers induced by high-plant-protein diets, as manifested by a significant downregulation of pro-inflammatory factors and a significant upregulation of anti-inflammatory factors.

[0123] 2.5 Effects of different diets on oxidative stress in the liver of groupers

[0124] Figure 4 The effects of different diets on oxidative stress in the liver of groupers are shown.

[0125] Figure 4 The results in

[0126] showed that compared with the CD group, the PD group significantly increased the expression level of keap1, accompanied by a significant decrease in the activities of superoxide dismutase (SOD) and catalase (CAT), and a significant decrease in the level of the oxidative stress-related protein ho1.

[0127] On the other hand, compared with the PD group, the CBD treatment group significantly enhanced the activities of SOD and CAT, and the expression level of ho1 increased significantly. Meanwhile, the levels of MDA and keap1 decreased significantly in the CBD treatment group.

[0128] In addition, the expression levels of nuclear factor E2-related factor 2 (nrf2) and keap1 in the NBD treatment group were significantly higher than those in the PD group.

[0129] These results indicate that the treatment with conjugated sodium bile acids (a mixture of sodium taurocholate and sodium tauroursodeoxycholate) significantly improved the oxidative stress response in the liver of groupers induced by high-plant-protein diets.

[0130] 2.6 Effects of different diets on endoplasmic reticulum stress in the liver of groupers

[0131] Figure 5 The effects of different diets on endoplasmic reticulum stress in the liver of groupers are shown.

[0132] Figure 5 The results of

[0133] showed that compared with the CD group, when PD treatment was given, the gene expression levels of chop, perk, and xbp1 increased significantly, and the level of GRP78 protein also increased significantly.

[0134] In addition, compared with the PD group, the gene expressions of chop and perk decreased significantly in the CBD treatment group, and the level of GRP78 protein also decreased significantly.

[0135] However, the expression levels of grp78, atf6, and xbp1 in the NBD treatment group were significantly higher than those in the PD group.

[0136] These results indicate that the treatment with conjugated bile acids (a mixture of sodium taurocholate and sodium tauroursodeoxycholate) significantly alleviated endoplasmic reticulum stress in the liver of groupers induced by a high-plant-protein diet.

[0137] 2.7 Effects of different diets on apoptosis of liver cells in groupers

[0138] Figure 6 The effects of different diets on apoptosis of liver cells in groupers are shown.

[0139] Figure 6 The results in

[0140] showed that compared with the CD group, the PD group significantly up-regulated the gene expression levels of bax, cas3, and apaf1, and at the same time, the protein level of BCL2 also increased significantly.

[0141] On the other hand, compared with the PD group, the CBD treatment significantly down-regulated the gene expression levels of bax, cas3, and apaf1, and in addition, the protein level of BCL2 also decreased significantly.

[0142] It is worth noting that in the NBD group, the gene expression levels of cas3, cas9, apaf1, and bcl2 were significantly higher than those in the PD group, while the protein level of BCL2 was significantly lower than that in the PD group.

[0143] These results indicate that the treatment with conjugated bile acids (a mixture of sodium taurocholate and sodium tauroursodeoxycholate) has a significant inhibitory effect on apoptosis of liver cells in groupers induced by a high-plant-protein diet.

[0144] Generally speaking, adding conjugated bile acids to a high-plant-protein diet improves the liver health of groupers by reducing liver inflammatory responses, enhancing antioxidant performance, decreasing endoplasmic reticulum stress and apoptosis. The addition of conjugated bile acids can improve the utilization rate and tolerance of groupers to a high-plant-protein diet, and ultimately improve the growth performance of groupers.

[0145] It should be noted that the above embodiments are only further illustrations of the present invention, rather than limitations. Any adjustment or change by those skilled in the art within the equivalent meaning and scope of the technical solution of the present invention should be considered to be included within the protection scope of the present invention.

Claims

1. A high-plant-protein feed composite additive for groupers, characterized in that, The composite additive is cholate and taurochenodeoxycholate, and the mass ratio of cholate to taurochenodeoxycholate is 1-3:1-3.

2. The high-plant-protein feed composite additive for groupers according to claim 1, characterized in that, The cholate is sodium cholate, and the taurochenodeoxycholate is sodium taurochenodeoxycholate.

3. The high-plant-protein feed composite additive for groupers according to claim 2, characterized in that, The mass ratio of sodium cholate to sodium taurochenodeoxycholate is 1:

1.

4. Use of the composite additive according to any one of claims 1-3 in preparing a high plant protein feed for groupers.

5. Use of the composite additive according to any one of claims 1-3 in preparing a high plant protein feed for improving the growth performance of groupers.

6. Use of the composite additive according to any one of claims 1-3 in preparing a high plant protein feed for improving the liver health of groupers.

7. The application according to claim 6, wherein The improvement of the liver health of groupers includes improving the liver tissue structure of groupers, reducing liver damage of groupers, reducing the inflammatory response of groupers' livers, enhancing the oxidative stress response of groupers' livers, reducing endoplasmic reticulum stress in groupers' livers, and reducing apoptosis of groupers' liver cells.

8. Use of the composite additive according to any one of claims 1-3 in improving the utilization rate of high plant protein feed by groupers.

9. The application according to any one of claims 4 - 8, characterized in that, The high plant protein feed includes replacing at least 50% of the animal protein in the traditional grouper feed with concentrated cottonseed protein, and the animal protein is fish meal.

10. The application according to claim 9, wherein The high plant protein feed is made from the following raw materials in parts by mass: Fish meal 24-26 Gluten meal 9-11 Flour 14-16 Concentrated cottonseed protein 24.5-25.5 Corn gluten meal 5-6 Casein 4-6 Gelatin 0.8-1.2 Fish oil 3.4-3.5 Soybean oil 1.4-1.6 Soybean lecithin 1.9-2.1 Calcium dihydrogen phosphate 0.9-1.1 Vitamin C 0.02-0.04 Choline chloride 0.4-0.6 Vitamin premix 0.4-0.6 Mineral premix 0.4-0.6 Antioxidant 0.04-0.06 Feeding attractant 0.08-0.12 Microcrystalline cellulose 2.6-2.8 Methionine 0.30-0.32 Lysine 0.5-0.6 The composite additive according to any one of claims 1-3 0.08-0.1.

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