A fish meal substitute, its preparation method and application

Abstract

The present invention provides a fish meal substitute, a preparation method thereof and an application thereof, belonging to the technical field of aquatic product breeding feeds. The fish meal substitute described in the present invention comprises chlorella powder and compound Chinese herbal medicine. The fish meal substitute provided by the present invention can completely replace the fish meal in the traditional largemouth bass feed, improve the body color and flesh color of the largemouth bass, improve the muscle nutritional components, increase the muscle pH, increase the muscle texture parameters and muscle fiber density and down-regulate the expression of the MSTN gene, thereby improving the muscle quality of the largemouth bass.

Description

Technical Field

[0001] The present invention belongs to the technical field of aquaculture feeds, and particularly relates to a fish meal substitute, a preparation method thereof, and an application thereof. Background Art

[0002] The largemouth bass (Micropterus salmoides), belonging to Perciformes and Centrarchidae, has delicious meat and high economic value. In recent years, with the success of artificial breeding of the largemouth bass, its artificial aquaculture industry has developed rapidly. As a carnivorous fish, the largemouth bass has a high demand for dietary protein. The protein content in commercial feeds for the largemouth bass is as high as 45-50%. Fish meal, as a high-quality protein in aquaculture feeds, is an important raw material to meet the high-protein nutritional requirements of the largemouth bass. However, with the depletion of global fishery resources and the continuous growth of aquaculture scale, the supply of fish meal falls short of demand and its price climbs. Therefore, finding a new protein source to replace fish meal and reducing the use of fish meal in feeds has become an important direction to promote the sustainable development of aquaculture.

[0003] Chlorella is a spherical single-celled microalga, belonging to Trebouxiophyceae, Chlorellales, and Chlorellaceae. It is the earliest microalga to achieve commercial production. Because of its high protein content, containing various vitamins, trace elements, and polyunsaturated fatty acids such as linoleic acid and α-linolenic acid, Chlorella has been widely used in aquaculture feeds. In these applications, it has been found that partial or complete replacement of fish meal with Chlorella powder has no adverse effects on the growth and health of pearl gentian grouper, rainbow trout, zebrafish, and African catfish. However, research on the impact of Chlorella replacing fish meal on the muscle quality of fish has found that after the level of Chlorella powder replacing fish meal exceeds 60%, the total antioxidant capacity, muscle hardness, and shear force of Pacific white shrimp muscle are significantly reduced. When the level of Chlorella powder replacing fish meal exceeds 43.6%, the crude protein content of crucian carp muscle is also significantly reduced. When Chlorella completely replaces fish meal in the feed, the crude protein content of largemouth bass muscle also decreases significantly. Therefore, increasing the proportion of Chlorella powder replacing fish meal while slowing down the decline in fish muscle quality caused by Chlorella powder replacing fish meal has become an urgent problem to be solved.

[0004] Studies have shown that Chinese herbal medicines can increase the content of muscle nutrients in aquatic animals, improve the muscle antioxidant capacity, and thus enhance the muscle quality of fish. In the studies on Acipenser schrenckii, Ctenopharyngodon idella, Pelteobagrus fulvidraco, and Micropterus salmoides, it was found that the addition of Chinese herbal medicines increased the cohesiveness of fish muscle, the content of umami amino acids, the protein content of muscle, and the water-holding capacity of muscle, reduced the cooking loss rate of muscle, and increased the content of umami amino acids and the water-holding capacity of muscle. Thus, it can be seen that Chinese herbal medicines can enhance the muscle quality of aquaculture animals. However, in order to improve the muscle quality of different species of fish, the types of Chinese herbal medicines added are different, and there are no reports on the impact of adding Chinese herbal medicines under the substitution of Chlorella vulgaris for fish meal on the muscle quality of cultured fish. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a fish meal substitute, which can improve the body color and flesh color of Micropterus salmoides and enhance the muscle quality of Micropterus salmoides.

[0006] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0007] A fish meal substitute, the fish meal substitute includes Chlorella vulgaris powder and compound Chinese herbal medicine, and the compound Chinese herbal medicine includes, by weight: 3-5 parts of Atractylodes lancea, 3-5 parts of Atractylodes macrocephala, 3-5 parts of Magnolia officinalis, 3-5 parts of Citrus reticulata Blanco, 3-5 parts of Aucklandia lappa Decne., 3-5 parts of Amomum villosum Lour., 15-20 parts of stir-fried Hawthorn, Medicated Leaven and Malt, 6-10 parts of Endothelium Corneum Gigeriae Galli, 6-10 parts of Dioscorea opposita Thunb., 3-5 parts of Fructus Aurantii Immaturus, 1-3 parts of Glycyrrhiza uralensis Fisch., 5-8 parts of Poria cocos (Schw.) Wolf, 5-8 parts of Codonopsis pilosula (Franch.) Nannf., 3-5 parts of Astragalus membranaceus (Fisch.) Bunge.

[0008] Preferably, the compound Chinese herbal medicine includes, by weight: 4 parts of Atractylodes lancea, 4 parts of Atractylodes macrocephala, 4 parts of Magnolia officinalis, 4 parts of Citrus reticulata Blanco, 4 parts of Aucklandia lappa Decne., 4 parts of Amomum villosum Lour., 18 parts of stir-fried Hawthorn, Medicated Leaven and Malt, 8 parts of Endothelium Corneum Gigeriae Galli, 8 parts of Dioscorea opposita Thunb., 4 parts of Fructus Aurantii Immaturus, 2 parts of Glycyrrhiza uralensis Fisch., 6 parts of Poria cocos (Schw.) Wolf, 6 parts of Codonopsis pilosula (Franch.) Nannf., 4 parts of Astragalus membranaceus (Fisch.) Bunge.

[0009] Preferably, the compound Chinese herbal medicine is a decoction.

[0010] Preferably, the mass ratio of the compound Chinese herbal medicine to the Chlorella vulgaris powder is 1-4:35.

[0011] Another purpose of the present invention is to provide a preparation method of the fish meal substitute, including the following steps: weighing each raw material according to the proportion, mixing the compound Chinese herbal medicine raw materials and steaming them with water, and mixing the steaming liquid with the Chlorella vulgaris powder to obtain the fish meal substitute.

[0012] Preferably, the steaming time is 20-40 min.

[0013] Another purpose of the present invention is to provide the application of the fish meal substitute and the fish meal substitute obtained by the preparation method in aquaculture.

[0014] Preferably, the feed obtained by mixing the fish meal substitute with the fish meal-free basal feed is used to feed Micropterus salmoides.

[0015] Preferably, the fish meal substitute accounts for 60-80% of the mass of the mixed feed.

[0016] Preferably, the Micropterus salmoides are fish from 3 months old to before market.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention provides a fish meal substitute, which includes chlorella powder and compound Chinese herbal medicine. The fish meal substitute provided by the present invention can completely replace the fish meal in the traditional Micropterus salmoides feed, reduce the feeding cost of Micropterus salmoides, and can improve the body color and meat color of Micropterus salmoides, improve the muscle nutritional components, increase the muscle pH, improve the muscle texture parameters and muscle fiber density, and down-regulate the MSTN gene expression, thereby improving the muscle quality of Micropterus salmoides. Description of the Drawings

[0019] Figure 1 : Sampling points for the body color and meat color of Micropterus salmoides;

[0020] Figure 2 : Effects of fish meal substitute on the muscle cross-section and muscle fiber structure of Micropterus salmoides, A: HE staining of muscle cross-section; B: Muscle fiber diameter and muscle fiber density;

[0021] Figure 3 : Effects of fish meal substitute on the expression of genes related to muscle development of Micropterus salmoides. Detailed Embodiments

[0022] The present invention provides a fish meal substitute, which includes chlorella powder and compound Chinese herbal medicine. The compound Chinese herbal medicine includes the following raw materials: Atractylodes lancea, Atractylodes macrocephala, Magnolia officinalis, Citrus reticulata Blanco, Aucklandia lappa Decne, Amomum villosum Lour., stir-fried Hawthorn, stir-fried Medicago sativa, Chinese yam, Fructus Aurantii Immaturus, Glycyrrhiza uralensis Fisch., Poria cocos, Codonopsis pilosula, and Astragalus membranaceus.

[0023] The chlorella powder in the present invention is purchased from Demeter Biotechnology (Zhuhai) Co., Ltd.

[0024] Chlorella has a high protein content and contains various vitamins, trace elements, and polyunsaturated fatty acids such as linoleic acid and α-linolenic acid. Chinese herbal medicine has the effect of increasing the content of muscle nutritional components in aquatic animals and improving the muscle antioxidant capacity, thereby enhancing the muscle quality of fish. The present invention creatively combines chlorella and Chinese herbal medicine to replace the fish meal in aquaculture feed, which can reduce the breeding cost while improving the fish meat quality.

[0025] The compound Chinese herbal medicine of the present invention comprises, by weight parts: Atractylodes lancea 3 - 5 parts, Rhizoma Atractylodis Macrocephalae 3 - 5 parts, Magnolia officinalis 3 - 5 parts, Citrus reticulata Blanco 3 - 5 parts, Aucklandia lappa Decne. 3 - 5 parts, Amomum villosum Lour. 3 - 5 parts, Stir-fried Hawthorn Fruit, Stir-fried Medicated Leaven and Stir-fried Malt 15 - 20 parts, Endothelium Corneum Gigeriae Galli 6 - 10 parts, Dioscorea opposita Thunb. 6 - 10 parts, Fructus Aurantii Immaturus 3 - 5 parts, Glycyrrhiza uralensis Fisch. 1 - 3 parts, Poria cocos 5 - 8 parts, Codonopsis pilosula (Franch.) Nannf. 5 - 8 parts, Astragalus membranaceus (Fisch.) Bunge 3 - 5 parts. Preferably, the compound Chinese herbal medicine comprises, by weight parts: Atractylodes lancea 4 parts, Rhizoma Atractylodis Macrocephalae 4 parts, Magnolia officinalis 4 parts, Citrus reticulata Blanco 4 parts, Aucklandia lappa Decne. 4 parts, Amomum villosum Lour. 4 parts, Stir-fried Hawthorn Fruit, Stir-fried Medicated Leaven and Stir-fried Malt 18 parts, Endothelium Corneum Gigeriae Galli 8 parts, Dioscorea opposita Thunb. 8 parts, Fructus Aurantii Immaturus 4 parts, Glycyrrhiza uralensis Fisch. 2 parts, Poria cocos 6 parts, Codonopsis pilosula (Franch.) Nannf. 6 parts, Astragalus membranaceus (Fisch.) Bunge 4 parts.

[0026] In the present invention, Atractylodes lancea is bitter, dry, pungent and dispersing, with aromatic and warm nature, capable of drying dampness and strengthening the spleen, and is used for dampness obstructing the middle-jiao and abdominal distension. Rhizoma Atractylodis Macrocephalae is sweet, tonifying, promoting diuresis, bitter, warm and dry, good at tonifying qi and strengthening the spleen, drying dampness and promoting diuresis, and treating qi deficiency of the spleen and stomach and edema due to spleen deficiency. Magnolia officinalis is bitter, dry, purging and descending, pungent, dispersing, warm and dredging, capable of treating dampness and stasis in the gastrointestinal tract, food retention, and regulating qi stagnation in the gastrointestinal tract, and is a medicine for treating abdominal distension caused by dampness obstruction, food retention and qi stagnation. Citrus reticulata Blanco is pungent, fragrant, dispersing, bitter, dry, warm and resolving dampness, capable of regulating the ascending and descending of qi mechanism of the spleen and lung to regulate qi and harmonize the middle-jiao, and drying dampness and regulating qi to resolve phlegm turbidity. Aucklandia lappa Decne. is pungent, fragrant, warm and relieving pain, bitter, dry and descending, dredging the triple energizer, especially good at promoting qi movement in the stomach, and also strengthening the spleen and promoting digestion. Amomum villosum Lour. is pungent and capable of promoting dispersion, with aromatic and warm nature, and is used for treating various syndromes such as dampness obstruction in the middle-jiao, qi stagnation of the spleen and stomach, cold-damp diarrhea and fetal restlessness. Stir-fried Hawthorn Fruit, Stir-fried Medicated Leaven and Stir-fried Malt are compounded by Stir-fried Hawthorn Fruit, Stir-fried Medicated Leaven and Stir-fried Malt in a mass ratio of 1:1:1, and the combination of the three medicines has a good effect of promoting digestion and removing stagnation. Endothelium Corneum Gigeriae Galli is the horny inner wall of the gizzard of the Phasianidae animal. As an implementable way, in the present invention, it is Endothelium Corneum Gigeriae Galli, good at promoting the movement of the spleen and stomach, promoting digestion and removing accumulation. Dioscorea opposita Thunb. has the functions of tonifying the spleen and stomach, promoting the production of body fluid and benefiting the lung, and tonifying the kidney and arresting seminal emission. Fructus Aurantii Immaturus is good at breaking qi and removing accumulation to relieve distension, and is also good at promoting qi movement and resolving phlegm to relieve stuffiness, and is an important medicine for treating gastrointestinal accumulation and phlegm stagnation in chest impediment. Glycyrrhiza uralensis Fisch. is sweet, tonifying, moistening, emollient and cool in nature, capable of tonifying qi and replenishing the middle-jiao, relieving spasm and pain, and moderating the properties of other medicines. Poria cocos promotes diuresis by promoting the excretion of dampness, strengthens the spleen and harmonizes the stomach, and calms the mind and soothes the nerves. Codonopsis pilosula (Franch.) Nannf. is sweet, tonifying and mild, not dry or greasy, and is mostly used for mild cases of qi deficiency of the spleen and lung. Astragalus membranaceus (Fisch.) Bunge is dry, warm, tonifying and ascending, sweet, light and promoting diuresis, benefiting the stomach and strengthening the superficial resistance, and treating qi deficiency of the spleen and lung.

[0027] The compound Chinese herbal medicine of the present invention has both nutritional and medicinal dual functions, can enhance immunity, and promote the digestion and absorption of nutrients.

[0028] In the fish meal substitute of the present invention, the compound Chinese herbal medicine is added in the form of decoction.

[0029] In the fish meal substitute provided by the present invention, the mass ratio of the compound Chinese herbal medicine to the chlorella powder is 1 - 4:35. In the fish meal substitute provided by the present invention, the chlorella powder and the compound Chinese herbal medicine are formulated in a specific ratio. Substituting fish meal to feed aquatic products can improve their digestion ability of feed, enhance immunity, and improve the muscle quality of fish.

[0030] The present invention also provides a method for preparing the fish meal substitute, which includes the following steps: Weigh each raw material according to a proportion, mix the compound Chinese herbal medicine raw materials and steam them with water. As an implementable way, the water added in the present invention is preferably enough to submerge the Chinese medicine raw materials. The steaming time is 20 - 40 min, and the preferred time is 25 - 35 min. Mix the steaming liquid with the chlorella powder to obtain the fish meal substitute.

[0031] As an implementable way, the present application uses a high-temperature and high-pressure cooking pot to steam the Chinese medicine raw materials, collect the steaming liquid, dilute or concentrate it to make 5 mL of the decoction equivalent to 1 g of the compound Chinese herbal medicine in mass, and then mix it with the chlorella powder.

[0032] The present invention also provides the application of the fish meal substitute in aquatic product breeding. After the fish meal substitute provided by the present invention is mixed with the fish meal-free basic feed, it can be used to feed Micropterus salmoides. Preferably, the fish meal substitute accounts for 60 - 80% of the mass of the mixed feed, and more preferably 65 - 75%. As an implementable way, the present invention weighs the fish meal substitute and the basic feed according to a proportion, mixes them evenly, air-dries them, makes them into expanded pellet feeds with a diameter of 4 mm, and stores them at -20 °C for standby.

[0033] The fish meal-free feed containing the fish meal substitute of the present invention can meet the protein nutrition requirements of Micropterus salmoides and improve the muscle quality of Micropterus salmoides.

[0034] The Micropterus salmoides described in the present invention is the fish from 3 months old to before listing. When the Micropterus salmoides is 3 months old and weighs 100.20 ± 0.29 g per tail, the fish meal substitute of the present invention can be used to replace the fish meal, greatly reducing the cost in the artificial feeding process of Micropterus salmoides.

[0035] In the specific embodiment of the present invention, the original compound Chinese herbal medicines are all purchased from Yuanshengtang Pharmacy in Yangling District (Yangling District, Xianyang City, Shaanxi Province).

[0036] The following combines the embodiments to elaborate on the technical solutions provided by the present invention in detail, but they cannot be understood as limiting the protection scope of the present invention.

[0037] Example 1

[0038] A fish meal substitute

[0039] The mass ratio of chlorella powder to compound Chinese herbal medicine is 35:1.

[0040] The compound Chinese herbal medicine includes, by weight: 4 parts of Atractylodes lancea, 4 parts of Atractylodes macrocephala, 4 parts of Magnolia officinalis, 4 parts of Citrus reticulata Blanco, 4 parts of Aucklandia lappa Decne., 4 parts of Amomum villosum Lour., 18 parts of stir-fried Hawthorn, Medicago sativa and Crataegus pinnatifida, 8 parts of Endothelium corneum gigeriae galli, 8 parts of Dioscorea opposita Thunb., 4 parts of Fructus aurantii Immaturus, 2 parts of Glycyrrhiza uralensis Fisch., 6 parts of Poria cocos Wolf., 6 parts of Codonopsis pilosula (Franch.) Nannf., and 4 parts of Astragalus membranaceus (Fisch.) Bunge.

[0041] The preparation method is as follows:

[0042] Weigh the Chlorella powder and each raw material of the compound Chinese herbal medicine proportionally. After mixing the Chinese medicine raw materials, use a high-temperature and high-pressure cooking pot to cook the Chinese medicine raw materials for 30 minutes. Collect the cooking liquid, dilute it to 5 mL of decoction equivalent to 1 g of the mass of the compound Chinese herbal medicine, and then mix it with the Chlorella powder to obtain the fish meal substitute.

[0043] Example 2

[0044] A fish meal substitute

[0045] The difference between this example and Example 1 is that the mass ratio of Chlorella powder to the compound Chinese herbal medicine is 35:2, and the rest are the same as in Example 1.

[0046] Example 3

[0047] A fish meal substitute

[0048] The difference between this example and Example 1 is that the mass ratio of Chlorella powder to the compound Chinese herbal medicine is 35:3, and the rest are the same as in Example 1.

[0049] Example 4

[0050] A fish meal substitute

[0051] The mass ratio of Chlorella powder to the compound Chinese herbal medicine is 35:3.

[0052] The compound Chinese herbal medicine includes, by weight: Atractylodes lancea 5 parts, Atractylodes macrocephala 5 parts, Magnolia officinalis 5 parts, Citrus reticulata Blanco 5 parts, Aucklandia lappa Decne. 5 parts, Amomum villosum Lour. 5 parts, stir-fried Hawthorn, stir-fried Medicago sativa and stir-fried Hovenia dulcis Thunb. 15 parts, Endothelium corneum gigeriae galli 10 parts, Dioscorea opposita Thunb. 6 parts, Fructus aurantii Immaturus 3 parts, Glycyrrhiza uralensis Fisch. 2 parts, Poria cocos Wolf 5 parts, Codonopsis pilosula (Franch.) Nannf. 5 parts, Astragalus membranaceus (Fisch.) Bunge 3 parts.

[0053] The preparation method is the same as that in Example 1.

[0054] Example 5

[0055] This example compared the effects of different fish meal substitutes

[0056] This study has been reviewed by the Experimental Animal Ethics Committee of Northwest A&F University, conforms to the principles of animal protection, animal welfare and ethics, and complies with the relevant regulations of national experimental animal welfare ethics (license number: NWAFU-314021274). All experimental procedures involving animal care supplies were strictly carried out in accordance with the standard operating procedures (SOP) of the "Guide for the Care and Use of Laboratory Animals" of Northwest A&F University.

[0057] 1. Rearing of Micropterus salmoides

[0058] Purchased experimental largemouth bass from Heyang Fish Farm in Weinan City, Shaanxi Province. First, they were temporarily raised in an indoor recirculating aquaculture tank. After 2 weeks, 225 healthy largemouth bass with uniform specifications (100.20 ± 0.29 g / fish) were selected and randomly divided into 5 groups, with 3 replicates in each group and 15 fish in each replicate. They were respectively raised in 15 recirculating aquaculture tanks (600 L / tank).

[0059] The 5 groups of largemouth bass were fed the following feeds respectively: basal feed + fish meal, basal feed + chlorella powder, basal feed + fish meal substitute of Example 1, basal feed + fish meal substitute of Example 2, basal feed + fish meal substitute of Example 3, which were respectively denoted as the FM group, CM group, CMH2 group, CMH4 group, and CMH6 group.

[0060] The 5 groups of feeds fed in this invention were five groups of experimental feeds with equal nitrogen (CP: 52%) and equal lipid (CL: 10%). After the raw materials of each group of experimental feeds were crushed and mixed evenly in proportion, they were air-dried and transported to the Feed Research Institute of the Chinese Academy of Agricultural Sciences (Beijing, China) to make expanded pellet feeds with a diameter of 4 mm, and stored for standby at -20°C. The formula and nutritional components of each group of feeds are shown in Table 1, and the fatty acid composition of the feeds is shown in Table 2. The 5 groups of largemouth bass were fed twice a day (9:00, 18:00), and the feeding rate was 2% - 3%. During the test period, the water temperature was 24 ± 1°C, pH was 7.2 - 7.4, dissolved oxygen > 5 mg / L, and ammonia nitrogen concentration < 0.05 mg / L. The culture period was 80 days.

[0061] Table 1 Formulas and nutritional levels of experimental feeds (g / kg; air-dried matter basis)

[0062]

[0063]

[0064] Note: *: Purchased from Beijing Envigo Biotech Co., Ltd. The content per kilogram of the mixed feed is as follows: VA 810000 IU, VD3 198000 IU, VE 4500 mg, VK3 1440 mg, VB1 900 mg, VB2 1350 mg, VB6 830 mg, VC 13000 mg, VB3 110 mg, inositol 12000 mg, iron 12000 mg, copper 400 mg, zinc 3000 mg, manganese 1500 mg, magnesium 60000 mg, cobalt 40 mg, iodine 100 mg, selenium 30 mg.

[0065] Table 2 Fatty acid composition of experimental feeds (percentage of total fatty acids, %)

[0066]

[0067]

[0068] Note: 1 SFA: Saturated fatty acid; 2 MUFA: Monounsaturated fatty acid; 3 PUFA: Polyunsaturated fatty acid, the same as the following table.

[0069] 2. Sample collection

[0070] After the aquaculture experiment ended, the fish were fasted for 24 h, and then anesthetized with 200 mg / L MS-222 (3-Aminobenzoic acid ethyl ester methanesulfonate) for sampling. First, three fish were randomly selected from each aquaculture tank, and the body colors of their dorsal, lateral line, and abdominal parts were measured with a colorimeter. Then, the dorsal muscles on both sides of the fish body were dissected with a scalpel. One side of the dorsal muscle was stored in a -20 °C refrigerator for the determination of conventional muscle nutrient components, fatty acid, and amino acid compositions, and the other side of the dorsal muscle was detected for its muscle color, texture, and physicochemical properties within 24 h. Another three fish were randomly selected from each aquaculture tank, anesthetized according to the above method, and then the dorsal muscle on one side was dissected and stored in 4% formaldehyde solution for standby. The dorsal muscle on the other side was first quickly frozen in liquid nitrogen and then stored in a -80 °C ultra-low temperature refrigerator for the detection of relative gene expression levels.

[0071] Data were expressed as mean ± standard deviation (mean ± SD), and variance test and one-way analysis of variance (One-Way ANOVA) were performed using SPSS 22.0 software (SPSS, IL, USA). The Tukey method was used for multiple comparison tests, with P < 0.05 considered significantly different. Pearson's correlation test was used for correlation analysis, and GraphPad Prism 9 (Graphpad Software, San Diego, CA) software was used for data plotting.

[0072] 3. Effects on the body color and muscle color of fish

[0073] The colorimetric values of the dorsal skin, lateral line skin, abdominal skin, and dorsal muscle of Micropterus salmoides were detected using a spectrophotometric colorimeter D65 / 10° (CS-820, Hangzhou Color Spectrum Technology Co., Ltd., Hangzhou, China), and the colorimetric values were brightness (L*), redness (a*), and yellowness (b*). The detection sites of the body color and muscle color of the fish are shown in Figure 1 . The measurement results are shown in Table 3.

[0074] Table 3 Effects of fish meal substitutes on the body color and muscle color of Micropterus salmoides

[0075]

[0076]

[0077] Note: For data in the same row, the same superscript letter or no superscript letter indicates no significant difference (P>0.05), and different superscript letters indicate significant difference (P<0.05). The same applies hereinafter.

[0078] As can be seen from Table 4, the skin brightness values (L*) of the lateral line part of Micropterus salmoides in the CMH2, CMH4, and CMH6 groups were significantly higher than those in the FM group and the CM group (P<0.05). However, there was no significant difference in the skin redness values (a*) of Micropterus salmoides among all groups (P>0.05). The skin yellowness value (b*) of the abdominal skin of Micropterus salmoides in the CM group was significantly higher than that in the FM group (P<0.05). The yellowness value (b*) of the dorsal muscle of Micropterus salmoides in the CM group was significantly higher than that in the FM group, while the yellowness values (b*) of the dorsal muscles of Micropterus salmoides in the CMH2, CMH4, and CMH6 groups were significantly higher than those in the CM group (P<0.05).

[0079] It can be seen that the fish meal substitute provided by the present invention can better improve the body color and meat color of Micropterus salmoides.

[0080] 4. Effects on the crude components of Micropterus salmoides

[0081] The determination of the crude components of the feed and fish muscle referred to the AOAC (1995) method. Among them, the determination of crude protein used the Kjeldahl method, the determination of crude fat used the Soxhlet extraction method, the determination of moisture used the direct drying method, and the determination of ash content used the dry ashing method at 550°C. The results are shown in Table 4.

[0082] Table 4 Effects on the crude components of Micropterus salmoides muscle (%, wet weight)

[0083]

[0084] As can be seen from Table 4, there was no significant difference in the crude protein, moisture, and crude ash of Micropterus salmoides muscle among all groups (P>0.05), while the crude fat of Micropterus salmoides muscle in the CMH2, CMH4, and CMH6 groups was significantly lower than that in the FM group (P<0.05).

[0085] 5. Effects on the amino acid composition of fish muscle

[0086] The amino acid composition of fish muscle was determined by an amino acid analyzer (Hitachi, L-8800, Hitachi, Tokyo) with reference to the method of GB / T 5009.124-2016. The determination process was completed by Sichuan Weil Testing Technology Co., Ltd. (Chengdu, China). The results are shown in Table 5.

[0087] Table 5 Effects on the amino acid composition of Micropterus salmoides muscle (mg / g, wet weight)

[0088]

[0089] Note: 1 Flavor amino acids (FAA): Include aspartic acid (Asp), glutamic acid (Glu), glycine (Gly), and alanine (Ala).

[0090] As can be seen from Table 5, the threonine content in the muscle of largemouth bass in the CM group was significantly higher than that in the FM group, while the leucine content was significantly lower than that in the FM group (P < 0.05). The glutamic acid, methionine, leucine, and arginine in the muscle of largemouth bass in the CMH6 group were all significantly higher than those in the CM group, and the contents of flavor amino acids, essential amino acids, and total amino acids in the muscle of this group of fish were all significantly higher than those in the FM group and the CM group (P < 0.05).

[0091] 6. Effects on the fatty acid composition of fish muscle

[0092] The fatty acid compositions of the feed and fish muscle were determined by gas chromatography. During the determination, first weigh 0.3 - 0.5 g of muscle sample and place it in a 10 mL centrifuge tube. Add 5 mL of methanol:chloroform (1:2; v / v) to the tube, and perform tissue homogenization for 15 s under a high-speed disperser (XHF-D, Ningbo), then let it stand for 1 - 2 h. Filter the upper clear liquid with quantitative filter paper. Add 4 mL of distilled water to the supernatant, centrifuge at 3000 rpm for 5 min, discard the supernatant, dry the lower layer solution under negative pressure at 40 °C to obtain muscle lipids. Then add 1 mL of chromatographic pure n-hexane to dissolve the oil, add 1 mL of 0.4 M KOH-methanol solution, mix well and let it stand for 30 min for methylation. Then add 2 mL of distilled water and mix well. After standing and separating layers, extract 0.5 mL of the upper layer solution. Use a microsyringe to aspirate 1 μL of the upper clear liquid and inject it into the injection port of a gas chromatograph (Agilent 7820a, Agilent Technologies, USA) for fatty acid determination. Compare and identify the measured results with fatty acid standards (47015-U, Sigma-Aldrich, Inc., St. Louis, USA), calculate the content of each fatty acid by the area normalization method, and express the content of each fatty acid in the form of a percentage of the total fatty acids. The results are shown in Table 6.

[0093] Table 6 Effects on the fatty acid composition of largemouth bass muscle (percentage of total fatty acids, %)

[0094]

[0095] As shown in Table 6, the levels of 14:0, 20:4n-6 and 18:3n-3 in the muscle of largemouth bass in the CM group were significantly lower than those in the FM group, and the levels of 16:1n-7, 18:2n-6, 20:5n-3, 22:5n-3 and 22:6n-3 were significantly higher than those in the FM group (P<0.05). The level of n-6PUFA in the muscle of largemouth bass in the CM group was significantly higher than that in the FM group, while the level of n-3PUFA was significantly lower than that in the FM group (P<0.05). The level of 16:1n-7 in the muscle of largemouth bass in the CMH4 group was significantly higher than that in the CM group (P<0.05).

[0096] 7. Effects on the physicochemical properties and texture of muscle

[0097] Take 10g of back muscle (W1) and hang it in a 4℃ refrigerator so that the muscle fibers are perpendicular to the horizontal plane. After leaving it for 24 hours, aspirate the liquid on the surface of the muscle and weigh it (W2). Calculate the value of 100×(W1-W2) / W1, which is the drip loss rate.

[0098] Weigh 0.5 g of muscle and place it in a 10 mL centrifuge tube. Add 5 mL of distilled water and use a high-speed disperser (XHF-D, After sufficient homogenization, the mixture was mixed upside down for 30 min, and the pH of the homogenate was measured with a pH meter.

[0099] Cut the muscle tissue into 1 cm 3 The muscle texture of small pieces was tested by using the TPA (Texture Profile Analysis) mode in the texture analyzer (TMS-pllot, FTC, USA) with a flat-bottomed cylindrical probe p / 5 (5 mm in diameter). The test parameters were 3 mm / s before the test, 1 mm / s during the test, and 1 mm / s after the test; compression degree 50%, dwell interval 5s, load probe type Auto-5g, data collection rate 200, and ambient temperature 18-20°C. Each sample was measured twice in parallel. The measured indicators include hardness, elasticity, adhesion, cohesion, chewiness, stickiness and shear force. The results are shown in Table 7.

[0100] Table 7 Effects on muscle texture and physicochemical properties of largemouth bass

[0101]

[0102] As shown in Table 7, there were no significant differences in the muscle texture parameters of Micropterus salmoides between the FM and CM groups (P>0.05). After adding the additive provided by the present invention to the feed, the muscle hardness of Micropterus salmoides showed an upward trend, and was significantly higher in the CMH6 group than in the CM group (P<0.05); the muscle adhesiveness in the CMH2, CMH4, and CMH6 groups of Micropterus salmoides was significantly higher than that in the CM group (P<0.05); the muscle gumminess of Micropterus salmoides showed an upward trend with the increase in the addition amount of the compound Chinese herbal medicine, and was significantly higher in the CMH6 group than in the CM group (P<0.05); the muscle chewiness in the CMH4 and CMH6 groups of Micropterus salmoides was significantly higher than that in the CM group (P<0.05). After adding the compound Chinese herbal medicine, the muscle shear force of Micropterus salmoides increased significantly, and reached the highest value in the CMH4 group (P<0.05).

[0103] There was no significant difference in the muscle pH of Micropterus salmoides between the FM and CM groups (P>0.05), and the muscle pH of Micropterus salmoides in the CMH4 group was significantly higher than that in the CM group (P<0.05). There was no significant difference in the drip loss rate of the muscle of Micropterus salmoides among all groups (P>0.05).

[0104] 8. Preparation and observation of HE-stained samples of muscle tissue

[0105] Take out the muscle tissue fixed in formaldehyde solution, after rinsing, dehydration, clearing, and embedding in paraffin wax, perform transverse cutting of muscle fibers with a section thickness of 3μm. After spreading the sections on glass slides, stain them with hematoxylin and eosin (HE staining), and finally seal the prepared slides with neutral gum. The paraffin sectioning and staining of muscle tissue samples were completed by Yangling Demonstration Area Hospital (Yangling, Shaanxi). Place the stained sections under an optical microscope (OPTEC, China) for observation and photography, and analyze and measure the muscle fiber diameter and muscle fiber density of the taken photos with image view image analysis software. The muscle fiber diameter was calculated and statistically analyzed based on the number of more than 300 muscle fibers per section, and the muscle fiber density was converted by counting the number of muscle fibers in a single field of view. The results are shown in Figure 2 Table 9.

[0106] As Figure 2 shown in Figure A, the muscle fiber structure of Micropterus salmoides was intact, with normal morphology and dense arrangement among all groups, and no muscle fiber tissue lesions were observed. The muscle fiber diameter of Micropterus salmoides in the CMH4 and CMH6 groups was significantly lower than that in the CM group (P<0.05), and the muscle fiber density in the CMH2, CMH4, and CMH6 groups of Micropterus salmoides was significantly higher than that in the CM group (P<0.05).

[0107] Table 9 Correlation between muscle fibers and texture of Micropterus salmoides

[0108]

[0109] As shown in Table 9, the myofiber diameter of Micropterus salmoides was significantly negatively correlated with muscle hardness, adhesiveness, cohesiveness, gumminess, and chewiness (P<0.05), while its myofiber density was significantly positively correlated with muscle hardness, adhesiveness, gumminess, and chewiness (P<0.05), indicating that the myofiber diameter significantly affects the texture properties of fish muscle.

[0110] 9. RT-PCR Detection of Muscle Development-Related Genes

[0111] The myogenic differentiation factor (Myogenic determining factor, MyoD) and myogenin (MyoG) detected by RT-PCR belong to the myogenic regulatory factor family (Myogenic Regulatory Facotors, MRFs), which can promote the proliferation and differentiation of muscle cells. Myostatin (MSTN) is a negative regulator that inhibits muscle growth and can suppress the proliferation and differentiation of muscle cells. β-actin is used as an internal reference gene.

[0112] The total RNA of the samples was extracted using Trizol reagent (Tiangen, Beijing). Subsequently, according to the method of Prime Script RT reagent kit (Vazyme, Nanjing), the total RNA was reverse-transcribed to obtain its cDNA, and then quantitative PCR was performed using a CFX 96 real-time fluorescence quantitative PCR detection system (Bio-Rad, Hercules, CA, USA). The volume of the quantitative PCR reaction was 20 μL, containing 0.6 μL of the upstream primer and 0.6 μL of the downstream primer (10 μM), 1 μL of 1:5 diluted cDNA, 10 μL of 2×SYBR Premix Ex TaqTM II (TaKaRa, Dalian, China), and 7.8 μL of sterilized double-distilled water. Real-time quantitative PCR included an initial activation step at 95°C for 30 s, followed by 40 cycles of alternating 95°C for 30 s and 60°C for 30 s to end the PCR reaction and obtain the Ct value of the sample. After the reaction, the singleness of the reaction product was confirmed by melting curve analysis. The relative gene expression was calculated according to formula (2 -ΔΔCt ). The primers used and their sequences are shown in Table 10, and the results are as Figure 3 shown.

[0113] Table 10 Primer Sequences for Real-Time Quantitative PCR

[0114]

[0115] As Figure 3As shown, the relative mRNA expression level of the MSTN gene in the muscle of Micropterus salmoides in the CM group was significantly higher than that in the FM group (P<0.05), and the relative mRNA expression level of the MSTN gene in the muscle of Micropterus salmoides in the CMH6 group was significantly lower than that in the FM group and the CM group (P<0.05). There was no significant difference in the relative mRNA expression levels of the MyoD and MyoG genes in the muscle of Micropterus salmoides among the groups (P>0.05). The fish meal substitute of the present invention can significantly reduce the relative mRNA expression level of the MSTN gene in the muscle of Micropterus salmoides, thereby promoting the growth and development of muscle fibers of Micropterus salmoides, further increasing the muscle fiber density of the fish body, and thus improving the muscle texture and quality of the fish body.

[0116] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A fish meal substitute for the feeding of Micropterus salmoides, characterized in that, The fish meal substitute is composed of chlorella powder and compound Chinese herbal medicine. The compound Chinese herbal medicine is composed of the following raw materials by weight: 3 - 5 parts of atractylodes lancea, 3 - 5 parts of atractylodes macrocephala, 3 - 5 parts of magnolia officinalis, 3 - 5 parts of citrus reticulata peel, 3 - 5 parts of Aucklandia lappa, 3 - 5 parts of amomum villosum, 15 - 20 parts of stir - fried hawthorn, medicated leaven and malt, 6 - 10 parts of endothelium corneum gigeriae galli, 6 - 10 parts of Dioscorea opposita, 3 - 5 parts of aurantii fructus immaturus, 1 - 3 parts of licorice root, 5 - 8 parts of poria cocos, 5 - 8 parts of codonopsis pilosula, 3 - 5 parts of astragalus membranaceus; the mass ratio of the compound Chinese herbal medicine to chlorella powder is 1 - 4:

35.

2. The fish meal substitute according to claim 1, wherein The compound Chinese herbal medicine is composed of the following raw materials by weight: 4 parts of atractylodes lancea, 4 parts of atractylodes macrocephala, 4 parts of magnolia officinalis, 4 parts of citrus reticulata peel, 4 parts of Aucklandia lappa, 4 parts of amomum villosum, 18 parts of stir - fried hawthorn, medicated leaven and malt, 8 parts of endothelium corneum gigeriae galli, 8 parts of Dioscorea opposita, 4 parts of aurantii fructus immaturus, 2 parts of licorice root, 6 parts of poria cocos, 6 parts of codonopsis pilosula, 4 parts of astragalus membranaceus.

3. The fish meal substitute according to claim 1 or 2, characterized in that, The compound Chinese herbal medicine is in the form of decoction.

4. The preparation method of the fish meal substitute according to any one of claims 1-3, characterized in that, It includes the following steps: Weigh each raw material according to the proportion, mix the raw materials of the compound Chinese herbal medicine and steam them with water, and then mix the steaming liquid with chlorella powder to obtain the fish meal substitute.

5. The preparation method according to claim 4, wherein, The steaming time is 20 - 40 min.

6. Application of the fish meal substitute according to any one of claims 1 - 3 and the fish meal substitute obtained by the preparation method according to claim 4 or 5 in feeding Micropterus salmoides.

7. The application according to claim 6, wherein The feed obtained by mixing the fish meal substitute with the fish - meal - free basal diet is used to feed Micropterus salmoides.

8. The application according to claim 6, wherein The fish meal substitute accounts for 60 - 80% of the mass of the mixed feed.

9. The application according to claim 6, wherein The Micropterus salmoides are fish from 3 - month - old to pre - market.

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