Feed for epinephelus malabaricus and preparation method thereof

Abstract

This invention belongs to the field of feed processing technology, specifically relating to a feed for *Sinocyclocheilus spp.* and its preparation method. The *Sinocyclocheilus spp.* feed provided by this invention is made from a mixture of *Chlorella vulgaris* polysaccharide, fish meal, soybean meal, wheat flour, brewer's yeast powder, fish oil, soybean oil, soybean lecithin, compound vitamin mineral salts, choline chloride, vitamin C phosphate, and calcium dihydrogen phosphate. The *Sinocyclocheilus spp.* feed provided by this invention can improve the growth performance and feed efficiency of *Sinocyclocheilus spp.*, reduce the crude fat content in muscle, and increase the crude protein content of the whole fish and muscle, effectively improving the muscle quality of *Sinocyclocheilus spp.* Simultaneously, this *Sinocyclocheilus spp.* feed can also improve the antioxidant capacity and immunity of *Sinocyclocheilus spp.*, reduce fat deposition, and improve liver structure, thus having a liver-protective effect and solving a key technical problem urgently needing breakthroughs in the current development of aquaculture.

Description

Technical Field

[0001] This invention belongs to the field of feed processing technology, specifically relating to a feed for grouper and its preparation method. Background Technology

[0002] China is a major aquaculture country, accounting for as much as 70% of the world's aquaculture production. Grouper is an important marine aquaculture fish, and the striped grouper (Epinephelus coioides), also known as the spotted grouper or green grouper, is currently one of the most widely farmed species and an important component of my country's aquaculture industry. Currently, with the continuous expansion of aquaculture scale, in order to increase fish production and make full use of resources and space, farming density and quantity are constantly increasing, and high-density intensive aquaculture has become an important development trend.

[0003] High-density aquaculture makes the water bodies susceptible to pollution, causing oxidative stress in fish, leading to frequent fish diseases, and consequently, the overuse of antibiotics, increasing the risk of antibiotic residues and bacterial resistance in farmed aquatic products. At the same time, fish growth and immune function are also affected by factors such as environmental pollution, disease, nutrition, and growth cycles. Therefore, finding a safe and efficient feed additive to improve fish growth performance and immune function has become one of the important research directions in the fish farming industry.

[0004] Patent document CN104186960A discloses a feed additive for oblique-banded grouper and its application method. This feed additive consists of photosynthetic bacteria liquid, Bacillus pumilus powder, and Lactobacillus acidophilus liquid. 10g to 50g of the additive is added per 1kg of basal feed, mixed thoroughly, and then coated with emulsified fish oil. This feed additive for oblique-banded grouper is suitable for the healthy aquaculture of oblique-banded grouper, significantly increasing the activity of SOD, AKP, and lysozyme in the fish, thereby effectively promoting the growth of oblique-banded grouper.

[0005] Patent document CN105995183A discloses an ecological feed formula and preparation method for *Sinocyclocheilus scoparia*, which is made from fish meal, mealworm powder, sandworm powder, earthworm powder, silkworm pupa powder, shrimp head powder, blood meal, squid viscera powder, brewer's yeast, soybean meal, soybean lecithin, α-starch, wheat flour, alfalfa powder, moringa powder, mixed powder of large seaweed, fish oil, mixed powder of shells, dried ginger powder, choline chloride, compound multivitamin powder, and phytase preparation. This feed can improve the feed utilization rate of *Sinocyclocheilus scoparia* and enhance its growth performance.

[0006] Patent document CN105995183A discloses a feed for improving the muscle quality and growth performance of grouper, which is composed of allicin, betaine, and dried Eucommia ulmoides leaf powder. This feed improves the muscle quality and growth performance of grouper, significantly increasing weight gain and survival rate, resulting in a substantial improvement in grouper growth performance. Simultaneously, this feed can reduce the water content of grouper muscle and increase muscle protein and fat content, thereby enhancing the muscle quality of grouper.

[0007] High-density intensive and aquacultured fish often suffer from nutritional and metabolic diseases, such as excessive fat accumulation, fatty liver, cirrhosis, and leptomeningopathy. However, current feeds or feed additives for the grouper (Siniperca spp.) are insufficient to address these nutritional and metabolic diseases. Therefore, researching and developing feed additives with hepatoprotective effects is of significant practical importance for improving the quality, safety, and overall quality of grouper. Summary of the Invention

[0008] To address the shortcomings of existing technologies, the present invention aims to provide a feed for *Chlorella vulgaris* and its preparation method. The feed for *Chlorella vulgaris* provided by this invention uses a self-made protein-nucleated *Chlorella vulgaris* polysaccharide as a feed additive. Studies have found that this protein-nucleated *Chlorella vulgaris* polysaccharide can enhance the immunity of *Chlorella vulgaris*, reduce fat deposition, improve antioxidant function, and has liver-protective effects.

[0009] This invention provides a feed for grouper, comprising the following components and their mass percentages:

[0010] The formula contains: Chlorella polysaccharide 0.01–0.16%, fish meal 35–52%, soybean meal 1–20%, wheat flour 18–22%, brewer's yeast mixture 1–2%, fish oil 1–5%, soybean oil 1–5%, soybean lecithin 0.8–1%, complex vitamin and mineral salts 1–2%, choline chloride 0.4–0.6%, vitamin C phosphate 0.4–0.6%, and calcium dihydrogen phosphate 1–2%.

[0011] Furthermore, the aforementioned grouper feed comprises the following components and their mass percentages:

[0012] The formula contains: Chlorella polysaccharide 0.04-0.08%, fish meal 46%, soybean meal 19%, wheat flour 20.92-20.96%, brewer's yeast mixture 2%, fish oil 3.5%, soybean oil 3.5%, soybean lecithin 1%, complex vitamin and mineral salts 2%, choline chloride 0.5%, vitamin C phosphate 0.5%, and calcium dihydrogen phosphate 1%.

[0013] The basic feed for grouper provided by this invention has the following composition: crude protein 46.84-47.27%, crude fat 11.39-11.76%, crude ash 12.48-12.51%, and moisture 5.88-5.92%.

[0014] Furthermore, the preparation method of the protein-nucleated Chlorella polysaccharide is as follows:

[0015] Weigh the Chlorella proteinensis powder into an ethanol brown bottle, add distilled water, stir well, and place it in a constant temperature water bath shaker for 8-10 hours. Remove it, filter it with a gauze bag, rotary evaporate it, freeze it at -80℃, and vacuum dry it to obtain the final product.

[0016] Furthermore, the ratio of the protein-nucleated Chlorella to distilled water is 1g:25mL.

[0017] Furthermore, the processing conditions of the constant temperature water bath shaker are: 8-9 hours in a water bath at a temperature of 55-65℃ and a rotation speed of 100-200 r / min.

[0018] Furthermore, the brewer's yeast mixture is composed of brewer's yeast powder and brewer's yeast cell wall powder in a mass ratio of 1:(0.4-0.6).

[0019] Furthermore, the brewer's yeast powder contains ≥3% mannan oligosaccharides and ≥40% protein.

[0020] Furthermore, the content of mannan in the brewer's yeast cell wall powder is ≥20%, the content of β-glucan is ≥20%, and the crude protein content is ≤35%.

[0021] In addition, the present invention also provides a method for preparing the aforementioned grouper feed, comprising the following steps:

[0022] The raw materials are crushed and sieved. First, fish meal, soybean meal, and flour are mixed and stirred evenly. Then, brewer's yeast mixture, compound vitamin mineral salts, choline chloride, vitamin C phosphate, calcium dihydrogen phosphate, and chlorella polysaccharide are added and stirred evenly. Then, fish oil, soybean oil, and soybean lecithin are added and stirred evenly to obtain a mixture. Next, water is added and mixed thoroughly. The amount of water added is 30% of the total mass of the mixture. The mixture is then made into granules, dried, and the final product is obtained.

[0023] The *Chlorella proteoglycans* polysaccharide provided by this invention is a polysaccharide with an average molecular weight of 77.310 kDa. This polysaccharide is composed of rhamnose, arabinose, galactose, glucose, xylose, mannose, and glucuronic acid in a molar ratio of 0.342:0.270:0.242:0.026:0.046:0.041:0.025. When used as an additive in the feed of *Epinephelus coioides*, this polysaccharide has a particularly beneficial effect on improving liver immune function, enhancing antioxidant function, and protecting the liver. Specifically, this polysaccharide is added to the basal feed of *Epinephelus coioides*. Depending on growth performance, the appropriate addition amount is 0.04–0.08% of the basal feed mass. Excessive addition will affect the growth performance, liver structure, and immunity of *Epinephelus coioides*, while insufficient addition will not achieve the desired effect.

[0024] The inventors have been dedicated to the research of grouper feed or feed additives, and have achieved many research results. This invention is the first to propose the application of self-made Chlorella polysaccharide as a feed additive for grouper. The effects of this Chlorella polysaccharide on the growth performance, morphological parameters, whole fish and muscle nutrient composition, and muscle texture of grouper were studied. Simultaneously, the inventors established a fatty liver model induced by a high-fat diet at the animal level, further exploring the effects of Chlorella polysaccharide on the liver morphology and structure of grouper, the expression levels of hepatocyte immune inflammation-related genes, the expression levels of antioxidant-related genes in the liver, and the expression levels of inflammation-related genes in liver tissue. To further verify the hepatoprotective effect of Chlorella proteoglycans on the liver of grouper, the inventors established a ConA-induced spleen cell (GS cell) model of grouper at the cellular level. After intervention with Chlorella proteoglycans, the effects of Chlorella proteoglycans on GS cell survival rate, oxidative stress level, morphological protection, apoptosis rate, and expression level of immune inflammation-related genes in GS cells were investigated. This verified the protective effect and molecular mechanism of Chlorella proteoglycans on hepatocyte damage in grouper.

[0025] Furthermore, the oblique-banded grouper is a warm-water, mid-to-lower-level, narrow-temperate fish, exhibiting extremely high sensitivity to temperature stress. Its growth rate, feeding rhythm, and immune disease resistance are all closely related to temperature. The optimal growth temperature for the oblique-banded grouper is 15–32℃, with an optimum temperature of 22–28℃. Below or above this temperature range, its feeding ability weakens, and it may even die. Therefore, improving the temperature tolerance of the oblique-banded grouper is crucial for enhancing its growth adaptability and survival rate.

[0026] The inventors have also made significant progress in their research on the temperature tolerance of grouper. They discovered that the combined use of pomelo juvenile fruit polysaccharide and theaflavins can improve the temperature tolerance of pearl grouper. However, in practical applications, it was found that theaflavins have poor stability, making it difficult to guarantee their effectiveness. Through continuous in-depth research, the inventors unexpectedly discovered that the self-made Chlorella polysaccharide and the optimized brewer's yeast mixture provided in this invention can synergistically improve the temperature tolerance of grouper. In particular, the combination of Chlorella polysaccharide and brewer's yeast mixture (composed of brewer's yeast powder and brewer's yeast cell wall powder at a mass ratio of 1:(0.4-0.6)) can significantly improve the temperature tolerance of grouper and effectively enhance its adaptability to temperature variations.

[0027] Compared with the prior art, the grouper feed provided by the present invention has the following advantages:

[0028] (1) The feed for grouper provided by the present invention can not only improve the growth performance and feed efficiency of grouper, but also reduce the crude fat content of muscle and increase the crude protein content of whole fish and muscle, which can effectively improve the muscle quality of grouper.

[0029] (2) The feed for grouper provided by the present invention can also improve the antioxidant capacity and immunity of grouper, reduce fat deposition and improve liver structure of grouper, and has no toxic side effects and is environmentally friendly, solving the key technical problems that urgently need to be solved in the development of aquaculture. Attached image description:

[0030] Figure 1 Figure showing the effect of adding different amounts of Chlorella polysaccharide to the feed on the liver of grouper.

[0031] Figure 2 The effect of adding different amounts of Chlorella polysaccharide to the feed on the enzyme activity in the liver of grouper; where: A: Aspartate aminotransferase (AST), B: Alanine aminotransferase (ALT), C: Malondialdehyde (MDA), D: Superoxide dismutase (SOD).

[0032] Figure 3 The effect of adding different amounts of Chlorella polysaccharide to the feed on the expression of immune-related genes in the liver of grouper; where: A: interleukin-1β (IL-1β), interleukin-8 (IL-8), interleukin-12 (IL-12); B: caspase-3, caspase-8, B-lymphoma-2 (Bcl-2).

[0033] Figure 4The effect of adding different amounts of Chlorella polysaccharide to the feed on inflammatory markers in the liver supernatant of grouper; where: A: interleukin-1β (IL-1β), B: interleukin-6 (IL-6), C: tumor necrosis factor-α (TNF-α).

[0034] Figure 5 The effect of adding different amounts of Chlorella polysaccharide to the feed on the expression of immune inflammation-related genes in hepatocytes of the grouper induced by ConA protein; where: A: CAT, GPX, GR; B: interleukin-1β (IL-1β); interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α); C: caspase 3, caspase 8, caspase 9.

[0035] Figure 6 The figure shows the effect of Chlorella polysaccharide on the survival rate of ConA-induced spleen cells (GS cells) of grouper.

[0036] Figure 7 Figure showing the effect of Chlorella polysaccharide on ConA-induced oxidative stress in oblique band spleen cells (GS cells).

[0037] Figure 8 The diagram shows the protective effect of Chlorella polysaccharide on the morphology of ConA-induced spleen cells (GS cells) of grouper; where: A: control group, B: model group, C: CP100 group, D: CP200 group, E: CP400 group.

[0038] Figure 9 The figure shows the effect of Chlorella polysaccharide on the apoptosis rate of spleen cells (GS cells) of grouper induced by ConA; where: A: control group, B: model group, C: CP100 group, D: CP200 group, E: CP400 group, F: cell apoptosis rate.

[0039] Figure 10 Figure 1 shows the effect of Chlorella polysaccharide on the expression of immune inflammation-related genes in ConA-induced spleen cells (GS cells) of grouper; where: A: interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α); B: caspase-3, caspase-8, caspase-9; C: glutathione peroxidase (GPx), manganese superoxide dismutase (MnSOD). Detailed Implementation

[0040] The present invention will be further described below through specific embodiments, but this is not intended to limit the invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the invention, but as long as they do not depart from the basic idea of ​​the invention, they are all within the scope of the invention. The materials involved in this invention can all be obtained through commercially available or conventional techniques in the art.

[0041] Example 1: Preparation and structural analysis of Chlorella polysaccharide with protein nucleus

[0042] I. Preparation method of Chlorella polysaccharide with protein nucleus:

[0043] Weigh 16g of Chlorella pyrenoidosa powder into an ethanol brown bottle, add 400mL of distilled water, stir well, and place in a constant temperature water bath shaker at 60℃ and 150r / min for 9h. Remove, filter with a gauze bag, rotary evaporate, freeze at -80℃, and vacuum dry to obtain the final product.

[0044] II. Structural analysis results of Chlorella polysaccharides:

[0045] 2.1 Monosaccharide composition analysis of Chlorella polysaccharide (CP)

[0046] Table 1 Monosaccharide composition of Chlorella polysaccharide (CP)

[0047]

[0048] Ion chromatography analysis of the monosaccharide composition of CP identified six monosaccharides, as shown in Table 1, including rhamnose (34.2%), arabinose (27%), galactose (24.2%), xylose (4.6%), mannose (4.1%), and glucose (2.6%). Chlorella polysaccharide (CP) also contains 2.5% glucuronic acid.

[0049] 2.2 Composition of methylated glycosidic bonds in Chlorella polysaccharide (CP)

[0050] Table 2. Glycosidic bond composition of methylated Chlorella polysaccharide (CP).

[0051]

[0052] Twenty glycan residues were identified from the Chlorella polysaccharide (CP) fraction. As shown in Table 2, the galactose residues consisted of 10.8% Galp-(1→, 18.7% →4)-GALP-(1→, 3.3% →3)-GALP-(1→, 4.2% →6)-GALP-(1→, and 10.5% 3,6)-GALP-(1→). The arabinose residues consisted of 18.2% Araf-(1→, 2.6% Arap-(1→, 3.4% →2)-Araf-(1→, 4.2% →5)-Araf-(1→, and 1.0% →2,5)-Araf-(1→). The glucose residues consisted of 5.2% →3,4)- Glcp-(1→, 2.5% →4,6)-Glcp-(1→ and 2.3% →2,4,6)-Glcp-(1→). CP contains low proportions of rhamnose, xylose, and mannose, with sugar chain contents of 2.6% →2)-Rhap-(1→, 1.4% →3)-Rhap-(1→, 0.6% →2,4)-Rhap-(1→, 1.4% →4)-Xylp-, 2.1% Manp-(1→ and 2.6% →2)-Manp-(1). Furthermore, based on methylation analysis of CP, the molar ratio of sugar components is similar to that of monosaccharide composition analysis.

[0053] Experimental Example 1: Effects of Chlorella polysaccharide on growth performance and muscle quality of grouper.

[0054] 1. Test method:

[0055] To investigate the effects of adding Chlorella polysaccharide to the feed on the growth performance and muscle quality of grouper, grouper were fed a basal diet supplemented with Chlorella polysaccharide at five different concentrations: 0%, 0.02%, 0.04%, 0.08%, and 0.16%. The experimental culture period was 8 weeks.

[0056] 1.1 Preparation of compound feed for grouper: The specific feed formula is shown in Table 3.

[0057] Table 3. Composition and Nutritional Components of the Feed (%)

[0058]

[0059] The brewer's yeast mixture is composed of brewer's yeast powder and brewer's yeast cell wall powder in a mass ratio of 1:0.6; the Chlorella polysaccharide (CP) is the Chlorella polysaccharide obtained in Example 1.

[0060] The preparation method of the compound feed for the oblique grouper is as follows:

[0061] The raw materials are crushed and sieved. First, fish meal, soybean meal, and flour are mixed and stirred evenly. Then, brewer's yeast mixture, compound vitamin mineral salts, choline chloride, vitamin C phosphate, calcium dihydrogen phosphate, and chlorella polysaccharide are added and stirred evenly. Then, fish oil, soybean oil, and soybean lecithin are added and stirred evenly to obtain a mixture. Next, water is added and mixed thoroughly. The amount of water added is 30% of the total mass of the mixture. The mixture is then made into granules, dried, and the final product is obtained.

[0062] 1.2 Determination of growth performance and morphological parameters of *Sinocyclocheilus scoparia*:

[0063] At the beginning and end of the experiment, the grouper in each group were counted and weighed. Then, the growth indicators of the grouper in each group were calculated according to the following formula.

[0064] Weight gain rate (WGR,%) = 100 × (final average weight - initial average weight) / initial average weight;

[0065] Body fatness (CF, g·cm) -3 Body condition = 100 × body mass / body length;

[0066] Visceral mass ratio (VSI,%) = 100 × visceral mass weight / body mass;

[0067] Liver-to-body weight ratio (HSI,%) = 100 × liver weight / body weight.

[0068] 1.3 Determination of whole fish and muscle nutritional components of *Sinocyclocheilus scoparia*:

[0069] The samples were dried to constant weight in a 105℃ constant temperature drying oven, and the chemical composition of the whole fish and muscle was analyzed. After digestion with concentrated sulfuric acid, the crude protein content (N×6.25) was determined by the Kjeldahl method. The crude fat content was determined using a Soxhlet extractor. The ash was ashed in a muffle furnace at 550℃ for 24 h.

[0070] 1.4 Determination of the muscle texture characteristics of *Sinocyclocheilus scoparia*:

[0071] The non-spiky muscle was removed from the sample and cut into small cubes with sides of 2.5 cm. A 75 mm diameter disc probe with a range of 250 N was used. The sample rise height was 25 mm, and the deformation percentage was 30%. The detection speed was set to 30, and the initial applied force was 0.375 N.

[0072] 2. Test Results:

[0073] The experimental results are shown in Tables 4 to 6.

[0074] 2.1 The results of the measurement of growth performance and morphological parameters of the oblique-banded grouper are shown in Table 4:

[0075] Table 4. Effects of Chlorella polysaccharide on growth performance and morphological parameters of grouper.

[0076]

[0077]

[0078] As shown in Table 4, feeding grouper with the addition of Chlorella polysaccharide provided by the present invention for 8 weeks resulted in significant differences in the weight gain (WGR) among different treatment groups. The WGR showed a trend of first increasing and then decreasing, and all reached their maximum value when the amount of Chlorella polysaccharide added was 0.08%.

[0079] 2.2 The results of the determination of the whole fish and muscle nutritional components of the oblique grouper are shown in Table 5:

[0080] Table 5. Effects of Chlorella polysaccharide on the nutritional composition of whole grouper and muscle (%)

[0081]

[0082] As shown in Table 5, adding 0.02-0.08% Chlorella polysaccharide to the feed can significantly reduce the crude fat content of the muscle; adding 0.08% Chlorella polysaccharide can significantly increase the crude protein content of the whole fish and muscle.

[0083] 2.3 The results of the determination of the muscle texture characteristics of the oblique-banded grouper are shown in Table 6:

[0084] Table 6. Results of the determination of the textural properties of the muscle of the grouper (n=4)

[0085]

[0086]

[0087] As shown in Table 6, compared with the CP0 group, the first cycle hardness of the grouper muscle in the CP200 group was the greatest, and the difference was significant (P<0.05); the muscle elasticity was the greatest in the CP0 group and the least in the CP400 group, and the difference was significant (P<0.05).

[0088] The above results indicate that the feed sugar for grouper provided by this invention can improve the growth performance and muscle quality of grouper.

[0089] Experimental Example 2: Effects of Chlorella polysaccharide on the morphology, structure, and histochemistry of the liver of the grouper.

[0090] 1. Test method:

[0091] To investigate the effects of adding Chlorella polysaccharide to the feed on the morphology, structure, and histochemistry of the liver of *Sinocyclocheilus scoparia*, five concentration gradients of Chlorella polysaccharide were added to the basal diet at concentrations of 0%, 0.02%, 0.04%, 0.08%, and 0.16% (as shown in Table 3). After 8 weeks of culture, liver tissue was subjected to HE staining and enzyme activity assays.

[0092] 1.1. HE staining of liver tissue:

[0093] A suitable amount of 4% paraformaldehyde solution was placed in a centrifuge tube for fixation. The liver sample was then removed, the tissue was minced, and dehydrated in alcohol. The dehydrated liver tissue was embedded in paraffin and cut into thin paraffin sections of approximately 6 μm using a microtome. These sections were then stained with hematoxylin and eosin (H&E). The sections were observed under an optical microscope, photographed, and saved. Image analysis was then performed using software.

[0094] 1.2 Liver tissue enzyme activity detection:

[0095] Take a liver sample of about 1g, cut it into small pieces, and then homogenize it using a homogenizer to prepare a 10% liver homogenate. Place the prepared liver homogenate in a low-temperature, low-speed centrifuge at 3000 rpm. -1 Centrifuge at 1000 rpm for 10 min, and take the supernatant after centrifugation. Measure the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), superoxide dismutase (SOD), and malondialdehyde (MDA) according to the instructions on the kit.

[0096] 2. Test Results:

[0097] The test results are as follows Figure 1 and Figure 2 As shown.

[0098] 2.1 The effects of adding different amounts of Chlorella polysaccharide to the feed on the liver of the grouper are as follows: Figure 1 As shown, the liver changes in grouper fed with Chlorella proteoglycans (CP) were as follows: The CP200, CP400, and CP800 groups were similar to the CP0 group, with clear cell structure, very few apoptotic or necrotic cells, and oval nuclei. The CP1600 group showed vacuolation and inflammatory infiltration in liver cells, nuclear condensation, and more severe damage to cell structure. This indicates that adding 0.04–0.08% Chlorella proteoglycans to the diet can significantly improve the liver structure of grouper. In the untreated group, hepatocytes showed nuclear displacement and numerous vacuoles; with increasing Chlorella proteoglycan concentration, nuclear displacement and vacuolation were significantly improved.

[0099] 2.2 Effects of adding different amounts of Chlorella polysaccharide to the feed on enzyme activity in the liver of grouper are as follows: Figure 2 As shown, compared with the CP0 group, the contents of ALT and AST were significantly reduced in the CP200, CP400 and CP800 groups (P<0.05); the contents of MDA in the CP400 and CP800 groups were significantly lower than those in the CP0 group (P<0.05); and the contents of SOD in each CP addition group were significantly higher than those in the CP0 group (P<0.05).

[0100] The above results demonstrate that the feed for *Sinocyclocheilus scoparia* provided by this invention can significantly improve hepatocyte structure. Example 3: Effects of *Chlorella vulgaris* polysaccharide on immune inflammation-related genes in *Sinocyclocheilus scoparia* hepatocytes.

[0101] 1. Test method:

[0102] To investigate the effects of adding Chlorella proteoglycans to the feed on immune inflammation-related genes in the liver cells of *Sinocyclocheilus bream*, Chlorella proteoglycans were added to the basal diet at five different concentrations: 0%, 0.02%, 0.04%, 0.08%, and 0.16% (as shown in Table 3). After 8 weeks of culture, liver tissue was collected for quantitative fluorescence analysis.

[0103] Quantitative fluorescence analysis: Total RNA was extracted from hepatocytes using the TRIzol lysis method. QSelect RT Super Mix was used for reverse transcription. The cDNA was diluted 5-fold and then spotted into a 96-well plate. In a 20 μL reaction mixture, 0.4 μL each of the upper and lower primers, 10 μL of Mix, and 4 μL of cDNA were added to each well. The mixture was then diluted to 20 μL with water. The plate was then placed in the instrument, and the quantitative PCR reaction program was: 94℃ for 30 s, 94℃ for 5 s, 50-60℃ for 15 s, and 72℃ for 10 s, for 40 cycles. The expression levels of the relevant genes were detected using QRT-PCR. -ΔΔCT The relative expression level of genes can be calculated.

[0104] 2. Test Results:

[0105] The test results are as follows Figure 3 As shown, after an 8-week culture trial, liver tissue was collected to detect the expression levels of immune-related genes. Compared with the CP0 group, the CP400 group significantly inhibited the expression of IL-1β, caspase-3, and caspase-8 genes (P<0.05) and promoted the expression of Bcl-2 gene (P<0.05). All CP addition groups significantly downregulated the expression of IL-8 and IL-12 genes (P<0.05). These results indicate that the grouper feed provided by this invention can significantly improve the expression of immune inflammation-related genes in grouper.

[0106] Experiment Example 4: Effects of Chlorella polysaccharide on antioxidant capacity and immunity of grouper.

[0107] 1. Test method:

[0108] To investigate the effects of adding Chlorella polysaccharide to the feed on the antioxidant capacity and immunity of grouper. Chlorella polysaccharide was added to the basal diet at five different concentrations: 0%, 0.02%, 0.04%, 0.08%, and 0.16% (as shown in Table 3). After 8 weeks of culture, a stress test was conducted using concanavalin A (ConA). Liver tissue was collected 24 hours later to detect the activity of antioxidant-related indicators and the mRNA expression of immune-related genes.

[0109] 1.1 Determination of antioxidant activity in liver tissue: Take liver samples from -80℃, weigh 0.1g of tissue, add 1mL of extraction solution and 2 grinding beads, set the grinder temperature to 4℃ and 60HZ, grind the sample in the grinder for 2min, take it out and observe if there is no obvious lumpy precipitate, put it in a centrifuge, set the temperature to 4℃ and 4000rpm, centrifuge for 15min, then aspirate the supernatant into a new EP tube, and then perform the detection according to the instructions of the selected Nanjing Jiancheng enzyme activity kit.

[0110] 1.2. The mRNA expression of immune-related genes in liver tissue was determined using quantitative real-time analysis: Total RNA was extracted from liver tissue using the TRIzol lysis method. Q Select RT Super Mix was used for reverse transcription. The cDNA was diluted 5-fold and then spotted into a 96-well plate. In a 20 μL reaction mixture, 0.4 μL each of the upper and lower primers, 10 μL of Mix, and 4 μL of cDNA were added to each well. The volume was then adjusted to 20 μL with water. The plate was then placed in the instrument, and the quantitative PCR reaction program was: 94℃ for 30 s, 94℃ for 5 s, 50-60℃ for 15 s, and 72℃ for 10 s, for 40 cycles. The expression levels of related genes were detected using QRT-PCR. -ΔΔCT The relative expression level of genes can be calculated.

[0111] 2. Test Results:

[0112] The test results are as follows Figure 4 and Figure 5 As shown.

[0113] 2.1 The effect of adding different amounts of Chlorella polysaccharide to the feed on inflammatory markers in the liver supernatant of grouper is as follows: Figure 4As shown, ConA induces inflammation in hepatocytes. Compared with the control group, the levels of inflammatory factors such as IL-1β, IL-6 and TNF-α were significantly increased in the model group (P<0.05). After treatment with different concentrations of CP, the levels in the CP400 and CP800 groups were significantly lower than those in the model group (P<0.05).

[0114] 2.2 The effects of adding different amounts of Chlorella polysaccharide to the feed on the expression of immune inflammation-related genes in hepatocytes of the oblique-banded grouper induced by concanavalin A are as follows: Figure 5 As shown:

[0115] (1) As Figure 5 As shown in Figure A, compared with the control group, the expression levels of CAT, GPx, and GR mRNA in the model group were significantly decreased (P<0.05). Compared with the model group, the pre-feeding CP diet group significantly increased the expression levels of CAT and GPx mRNA in a dose-dependent manner (P<0.05).

[0116] (2) mRNA expression of inflammation-related genes in liver tissue, such as Figure 5 As shown in Figure B, compared with the control group, the levels of inflammation-related genes IL-1β, IL-6, and TNF-α in the model group were upregulated by 27.39-fold, 4.62-fold, and 3.24-fold, respectively. Compared with the model group, feeding 200, 400, and 800 mg / kg CP groups significantly inhibited the increase in the mRNA expression levels of IL-1β, IL-6, and TNF-α. However, the expression level of TNF-α in the CP1600 group was significantly higher than that in the model group (P<0.05), while there was no significant difference in IL-6 (P>0.05).

[0117] (3) mRNA expression of apoptosis-related genes in liver tissue, such as Figure 5 As shown in Figure C, compared with the control group, the expression levels of caspase-3, caspase-8 and caspase-9 mRNA in the model group increased by 2.93-fold, 4.16-fold and 6.71-fold, respectively, and the CP group significantly inhibited the increase in these expression levels (P<0.05).

[0118] The above results indicate that the grouper feed provided by the present invention can significantly enhance the antioxidant capacity of grouper, reduce inflammatory response, improve hepatocyte apoptosis response, and reduce liver damage induced by chemical substances in grouper.

[0119] Experimental Example 5: Effects of Chlorella polysaccharide on ConA-induced spleen cells of grouper. 1. Experimental Methods:

[0120] To investigate the effects of Chlorella polysaccharide on ConA-induced spleen cells (GS cells) of the grouper, cells were seeded into 6-well or 96-well plates and cultured at 25°C with 5% (v / v) CO2. The cells were changed every 2–3 days in L-15 medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibiotics. When the cells reached 80–90% confluence, 5 μg / mL ConA or CP (100, 200, and 400 μg / mL) was added and incubated for 24 h. The experiment consisted of five groups: a control group (GS cells cultured in medium without CP or ConA), a model group (GS cells cultured in medium with only ConA and incubated for 24 h), and a CP pretreatment group (cell cultured in medium with 100, 200, and 400 μg / mL CP and incubated for 24 h, followed by ConA treatment for 24 h). After drug treatment, cell pellets and supernatants were collected to determine the survival rate, oxidation level, morphology, apoptosis rate, and expression levels of immune inflammation-related genes in GS cells induced by ConA. Among these:

[0121] 1.1. GS cell viability determination:

[0122] Cell viability was determined using the CCK-8 assay. Cells were resuspended and seeded into 96-well plates with 100 μL of culture medium added to each well, resulting in a cell count of 5 × 10⁶ cells / well. 4 Cells were incubated for 48 hours in a 5% CO2 incubator at 25°C. Subsequently, the medium was replaced with serum-free and antibiotic-free culture medium (90 μL per well), and 10 μL of CCK-8 reagent was added to each well for incubation for 2–4 hours. The absorbance was measured at 450 nm using a microplate reader. The cell proliferation rate was calculated using the formula: Cell proliferation rate (%) = (Experimental group A450 / Control group A450) × 100%.

[0123] 1.2 Measurement of GS cell oxidation level:

[0124] The level of oxidative stress in cells was reflected by assessing ROS levels in cell lysates. Spleen cells were incubated with 5 μg / mL ConA or CP (at an appropriate concentration) for an appropriate time. The cell pellet was collected, subjected to low-temperature sonication, and then centrifuged at 12,000 rpm for 5 min at 4°C. The supernatant was used to determine the intracellular ROS content according to the kit instructions.

[0125] 1.3 Determination of the protective effect of GS on cell morphology:

[0126] HE staining was performed on GS cells. The cell slides were immersed in 75% ethanol for 10 minutes, then gently lifted with forceps and dried over an alcohol lamp (the temperature should not be too high). The dried slides were placed into the 6-well plates, and after cooling, 500 μL of cell suspension (approximately 2 × 10⁻⁶ cells) was added. 4 (Number of cells) was added to the climbing slide. After the cells adhered, 1.5 mL of culture medium was gently added, and the slide was incubated overnight at 25°C with 5% CO2 to allow the cells to adhere and grow well. The cells were fixed with 5 μg / mL ConA or CP (appropriate concentration), stained with hematoxylin and eosin (HE), mounted with neutral resin, and photographed under a microscope (MshOtMS60). Each group was repeated in triplicate.

[0127] 1.4. Apoptosis rate determination of GS cells:

[0128] Apoptosis rate was detected by flow cytometry. Spleen cells were seeded in 6-well plates and incubated for 24 h with 5 μg / mL ConA or CP (appropriate concentration). Apoptosis rate was then detected using the V-APC / 7AAD apoptosis assay kit. After drug treatment, 1 × 10⁶ cells were collected. 6 Cells were washed three times by centrifugation with pre-cooled PBS, and the supernatant was discarded. Cells were co-stained at 28°C for 15 min and then incubated on ice for 15 min. The apoptosis rate was detected by flow cytometry (BD, FACSCalibur, NY, USA), and the data were analyzed using CELLQUESTPRO software (BD).

[0129] 1.5. GS cell immune inflammation-related gene expression assay:

[0130] The expression levels of immune-inflammatory related genes in GS cells were analyzed using real-time quantitative PCR. Total RNA was extracted from hepatocytes using the TRIzol lysis method. RNA integrity was detected by 1.5% denaturing agarose gel electrophoresis, and RNA concentration and purity were determined by measuring the OD260 / 280 ratio using a nucleic acid analyzer. Total RNA was then extracted and reverse transcribed into cDNA. The cDNA was diluted 5-fold and plated into 96-well plates. In a 20 μL reaction mixture, 0.4 μL each of the upper and lower primers, 10 μL of Mix, and 4 μL of cDNA were added to each well. The mixture was then diluted to 20 μL with water. The plate was then placed in the instrument, and the real-time quantitative PCR reaction program was: 94℃ for 30 s, 94℃ for 5 s, 50-60℃ for 15 s, and 72℃ for 10 s, for 40 cycles. The expression levels of related genes were detected by QRT-PCR, and the results were analyzed using 2... -ΔΔCT The relative expression level of genes can be calculated.

[0131] 2. Test Results:

[0132] The test results are as follows Figures 6-10As shown.

[0133] 2.1 Results of GS cell viability assay: Figure 6 As shown, after ConA-induced injury, the survival rate of GS cells in the model group was significantly decreased compared with the control group (P<0.05), at 80.49±2.84%; while the survival rate of hepatocytes in the CP intervention group was significantly increased. The survival rates of GS cells in the 100, 200 and 400 μg / mL CP pretreatment groups were 92.09±1.58%, 98.33±0.32% and 94.09±2.52%, respectively, which were significantly improved compared with the model group (P<0.05).

[0134] 2.2 Results of GS cell oxidation degree measurement are as follows Figure 7 As shown, compared with the control group, the ROS index in the cell supernatant of the model group was significantly increased (P<0.05), and the increase in ROS index induced by ConA was significantly reduced in each group pretreated with CP (P<0.05).

[0135] 2.3. Results of HE staining of GS cells are as follows: Figure 8 As shown: Control group cells showed vigorous growth, clear cell structure, and normal nucleus morphology. Figure 8 A); The model group cells showed obvious swelling, disordered growth, and damaged cell structure. Figure 8 B); Pretreatment with 100 μg / mL and 200 μg / mL CP significantly slowed hepatocyte damage and gradually restored their morphology to normal. Figure 8 CD); however, in the group pretreated with a high concentration of 400 μg / mL CP, hepatocytes showed obvious damage, with cell swelling and the cells gradually changing from spindle-shaped to round (CD); Figure 8 D).

[0136] 2.4. Results of GS cell apoptosis rate assay are as follows: Figure 9 As shown, compared with the control group, the apoptosis rate in the model group was significantly increased (P<0.05); after the addition of CP (100, 200 and 400 μg / mL) for protection, the apoptosis rate was significantly decreased compared with the model group (P<0.05), and the CP200 group showed the greatest decrease.

[0137] 2.5. Results of expression level measurement of immune inflammation-related genes in GS cells are as follows: Figure 10As shown, compared with the control group, the expression levels of IL-1β, IL-6, and TNF-α genes in the model group were significantly upregulated (P<0.05). Pretreatment of cells with 100, 200, and 400 μg / mL CP for 24 h significantly reduced the expression of inflammation-related genes induced by ConA (P<0.05). Similarly, the expression levels of caspase-3, caspase-8, and caspase-9 genes in the model group were significantly upregulated compared with the control group (P<0.05). Pretreatment of cells with 100, 200, and 400 μg / mL CP for 24 h significantly reduced the expression of apoptosis-related genes induced by ConA, and the degree of reduction in apoptosis was related to the concentration of CP. There was no significant difference between the CP 200 and CP 400 groups (P>0.05). Compared with the control group, the expression level of MnSOD gene in the model group was significantly decreased. Compared with the model group, the CP pretreatment group significantly increased the relative expression level of MnSOD mRNA, and the expression level in the CP200 group was the highest (P<0.05).

[0138] Experimental Example 6: The Influence of Temperature Tolerance on the Grouper

[0139] 1. Test method:

[0140] The formulated feeds of Comparative Example 1 and Example 4 in Table 1 were used to feed the grouper. The high-temperature and low-temperature half-lethal temperatures of the grouper were determined according to the method published by Shao Yanxiang in "Study on the Tolerance of Grouper to Temperature Stress". The pomelo juvenile fruit polysaccharide (YZW-A800) prepared by patent document CN 112998160A was used to replace the pyrenoid polysaccharide of Chlorella vulgaris. The remaining components were the same as those in the grouper feed prepared in Example 6, which served as Comparative Example 3.

[0141] 2. Test Results

[0142] The experimental results are shown in Table 7.

[0143] Table 7 Temperature tolerance test of grouper with oblique shape

[0144] The semi-lethal temperature of high temperature (°C) Low-temperature semi-lethal temperature (°C) Comparative Example 1 34℃ 11℃ Example 4 group 40℃ 7℃ Comparative Example 3 Groups 36℃ 10℃

[0145] As shown in Table 7, feeding the grouper with the feed provided by the present invention can improve the grouper's tolerance to temperature stress and is more conducive to the growth and reproduction of the grouper.

[0146] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A feed for Epinephelus coioides, characterized by, Includes the following ingredients and their mass percentages: The formula contains: Chlorella polysaccharide 0.01-0.16%, fish meal 35-52%, soybean meal 1-20%, wheat flour 18-22%, brewer's yeast mixture 1-2%, fish oil 1-5%, soybean oil 1-5%, soybean lecithin 0.8-1%, complex vitamin and mineral salts 1-2%, choline chloride 0.4-0.6%, vitamin C phosphate 0.4-0.6%, and calcium dihydrogen phosphate 1-2%. The preparation method of the protein-nucleated Chlorella polysaccharide is as follows: Weigh the Chlorella proteinensis powder into an ethanol brown bottle, add distilled water, stir well, and place it in a constant temperature water bath shaker for 8-10 hours. Remove it, filter it with a gauze bag, evaporate it by rotary evaporation, freeze it at -80℃, and vacuum dry it to obtain the product. The brewer's yeast mixture is composed of brewer's yeast powder and brewer's yeast cell wall powder in a mass ratio of 1:(0.4~0.6); The brewer's yeast powder contains ≥3% mannan oligosaccharides and ≥40% protein. The brewer's yeast cell wall powder contains ≥20% mannan, ≥20% β-glucan, and ≤35% crude protein.

2. The Epinephelus coioides feed of claim 1, wherein, Includes the following ingredients and their mass percentages: The formula contains 0.04-0.08% Chlorella polysaccharide, 46% fish meal, 19% soybean meal, 20.92-20.96% wheat flour, 2% brewer's yeast mixture, 3.5% fish oil, 3.5% soybean oil, 1% soybean lecithin, 2% compound vitamin and mineral salts, 0.5% choline chloride, 0.5% vitamin C phosphate, and 1% calcium dihydrogen phosphate.

3. The Epinephelus coioides feed of claim 1, wherein, The ratio of the protein-nucleated Chlorella to distilled water is 1g:25mL.

4. The Epinephelus coioides feed of claim 1, wherein, The processing conditions for the constant temperature water bath shaker are: 8-9 hours in a water bath at a temperature of 55-65℃ and a rotation speed of 100-200 r / min.

5. The method for preparing the grouper feed according to any one of claims 1 to 4, characterized in that, Includes the following steps: Crush and sieve all raw materials. First, mix fish meal, soybean meal, and flour. After stirring evenly, add brewer's yeast mixture, compound vitamin mineral salts, choline chloride, vitamin C phosphate, calcium dihydrogen phosphate, and Chlorella polysaccharide. Stir evenly and then add fish oil, soybean oil, and soybean lecithin. Stir evenly to obtain a mixture. Next, add water and mix thoroughly. The amount of water added is 30% of the total mass of the mixture. Form into granules, dry, and obtain the final product.

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