Crucian feed with soybean meal replaced by cottonseed protein concentrate and application of crucian feed

Abstract

The invention discloses a crucian feed with soybean meal replaced by cottonseed protein concentrate and application of the crucian feed. Cottonseed protein concentrate is used as a main plant protein source and completely replaces soybean meal in a feed formula, and the mass percent of the cottonseed protein concentrate is 19.78%. The feed further comprises the following components: fish meal, casein, flour, fish oil, soybean oil, vitamins, mineral premix and the like. The feed provided by the invention can significantly improve the ingestion speed and the ingestion amount of crucian carps and effectively promote the growth of crucian carps, and the weight gain rate, the specific growth rate and the feed efficiency of the feed are significantly superior to those of a feed containing soybean meal. The feed can regulate and control lipid metabolism related gene expression of crucian muscle and liver, enhance fat synthesis and catabolism, promote muscle protein and fat deposition and increase muscle polyunsaturated fatty acid content, and has no negative influence on liver health and intestinal flora homeostasis. The invention provides an efficient solution for soybean meal decrement substitution, and has important application value.

Description

Technical Field

[0001] This invention relates to the field of aquaculture feed technology, specifically to a crucian carp feed that uses cottonseed concentrated protein as a substitute for soybean meal and its application. Background Technology

[0002] With the intensive development of aquaculture, traditional protein sources such as fishmeal and soybean meal are becoming increasingly scarce and their prices are fluctuating wildly. Developing new, efficient, and sustainable alternative protein sources has become an urgent research and development direction for the industry. As an important freshwater economic fish in my country, optimizing the feed formulation for crucian carp is crucial for reducing farming costs and ensuring industry profitability.

[0003] Soybean meal, due to its high protein content and stable yield, is currently the most important plant protein source in animal feed. However, soybean meal contains anti-nutritional factors such as antigenic proteins and trypsin inhibitors, and excessive use may affect the intestinal health and nutrient utilization rate of fish. Meanwhile, the competition for land resources in soybean cultivation and its dependence on international market prices are prompting research and industry to seek more diversified alternatives.

[0004] Cottonseed protein concentrate is a product made from cottonseed after dehulling and delinting, followed by extraction to remove oils and some harmful substances such as gossypol. It has a high crude protein content (usually ≥60%), a relatively balanced amino acid composition, and low levels of oligosaccharides that are difficult for fish to digest. Furthermore, its resource supply is relatively stable, and it has been studied for application in pig and poultry feed. However, its application in aquatic animals, especially crucian carp, remains relatively weak. Existing research mainly focuses on primary products such as cottonseed meal, whose use in aquatic feed is strictly limited due to the presence of toxic substances such as free gossypol. As a deeply processed, low-gossypol product, the feasibility, appropriate proportion, and mechanisms of influence on crucian carp growth performance, nutrient metabolism, liver health, and overall physiological state through the systematic replacement of soybean meal with cottonseed protein concentrate in crucian carp feed are still unclear, lacking systematic scientific data and theoretical support.

[0005] Therefore, it is urgent to conduct a systematic invention study to clarify the scientific solution for replacing soybean meal with cottonseed concentrate protein in crucian carp feed, so as to broaden the sources and applications of feed protein, reduce the dependence of feed production on soybean meal, improve the production performance and efficiency of farmed fish, and provide key technical support for the development of efficient and environmentally friendly compound feed for crucian carp.

[0006] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0007] The purpose of this invention is to provide a crucian carp feed that replaces soybean meal with cottonseed concentrated protein and its preparation method. In the field of crucian carp farming, it can significantly promote the growth performance of crucian carp, enhance nutrient deposition, improve fat metabolism, and not affect health.

[0008] To achieve the above objectives, the present invention provides a crucian carp feed in which cottonseed protein concentrate replaces soybean meal, wherein cottonseed protein concentrate completely replaces soybean meal, and the mass percentage of cottonseed protein concentrate in the feed is 19-20%.

[0009] Preferably, the mass percentage of cottonseed protein concentrate in the crucian carp feed provided by the present invention is 19.78%.

[0010] Preferably, the crucian carp feed provided by the present invention further includes the following components by weight percentage: 8-12% fish meal, 13-17% casein, 23-27% wheat flour, 2-3% fish oil, 2-3% soybean oil, 4-6% mineral salt premix, 0.3-0.5% vitamin premix, 0.1-0.2% choline chloride, 2-4% carboxymethyl cellulose, and 15-18% cellulose.

[0011] More preferably, the mass percentage of each component in the crucian carp feed provided by the present invention is as follows: 10% Peruvian fish meal, 15% casein, 25% wheat flour, 2.5% fish oil, 2.5% soybean oil, 5% mineral salt premix, 0.39% vitamin premix, 0.11% choline chloride, 3% carboxymethyl cellulose, 19.78% cottonseed protein concentrate, and 16.72% cellulose.

[0012] The crucian carp feed provided by this invention can be applied to the field of crucian carp farming, including improving crucian carp feed intake, weight gain rate, specific growth rate and feed efficiency.

[0013] The crucian carp feed provided by this invention can also be used to promote the deposition of muscle protein and fat and the synthesis of polyunsaturated fatty acids in crucian carp.

[0014] The crucian carp feed provided by this invention can also be used to regulate the expression of genes related to lipid metabolism in crucian carp muscle and liver, and enhance fat synthesis and decomposition metabolism.

[0015] The present invention also provides a method for raising crucian carp, by feeding the above-mentioned feed twice a day for a period of not less than 56 days.

[0016] Preferably, the crucian carp farming method provided by the present invention is implemented in a recirculating aquaculture system with a photoperiod of 12 hours of light and 12 hours of darkness.

[0017] The present invention has the following advantages: This invention provides a crucian carp feed that uses cottonseed concentrated protein as a substitute for soybean meal, which can be applied to crucian carp feeding and has the following technical effects and advantages: Feeding stimulation effect: Increased food intake and eating speed; Growth-promoting effects: Significantly improves weight gain rate, specific growth rate, and feed efficiency; Quality improvement effects: Increases muscle protein and fat content, and optimizes fatty acid composition; Metabolic activation effect: Upregulates the expression of growth and lipid metabolism genes, and enhances protein turnover; Health protection effects: Maintains gut microbiota homeostasis, and ensures normal liver function and tissue structure; Industrial application value: It can solve the problem of soybean meal dependence and achieve the industrial goal of reducing and replacing soybean meal consumption. Attached Figure Description

[0018] Figure 1 The effects of cottonseed protein concentrate as a substitute for soybean meal on the growth performance of crucian carp in different feeding groups.

[0019] Figure 2 The effects of cottonseed protein concentrate as a substitute for soybean meal on the whole fish and muscle components of crucian carp in different feeding groups.

[0020] Figure 3 The effects of cottonseed protein concentrate as a substitute for soybean meal on the feeding behavior of crucian carp in different feeding groups.

[0021] Figure 4 The effects of cottonseed protein concentrate as a substitute for soybean meal on plasma biochemical indicators in crucian carp in different feeding groups.

[0022] Figure 5 The effect of cottonseed protein concentrate as a substitute for soybean meal on the liver oil of crucian carp in different feeding groups.

[0023] Figure 6 The effects of cottonseed protein concentrate as a substitute for soybean meal on HE sections of crucian carp livers in different feeding groups.

[0024] Figure 7 The effects of cottonseed protein concentrate as a substitute for soybean meal on amino acids and fatty acids in crucian carp muscle in different feeding groups.

[0025] Figure 8 The effects of cottonseed protein concentrate as a substitute for soybean meal on the gut microbiota of crucian carp in different feeding groups.

[0026] Figure 9 The effects of cottonseed protein concentrate as a substitute for soybean meal on genes related to muscle growth and lipid metabolism in crucian carp in different feeding groups.

[0027] Figure 10 The effects of cottonseed protein concentrate as a substitute for soybean meal on genes related to liver growth and lipid metabolism in crucian carp in different feeding groups. Detailed Implementation

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Note: Unless otherwise specified, the experimental methods in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0030] Example 1: Crucian carp feed formulation using cottonseed concentrated protein as a substitute for soybean meal This embodiment provides a method for preparing six isonitrogenous and isolithic experimental feeds by replacing different proportions of soybean meal (0%, 15%, 30%, 45%, 75%, and 100%) with cottonseed concentrate, denoted as C0, C15, C30, C45, C75, and C100, respectively. The experimental feed formulations (% dry matter) are as follows:

[0031] Basic fixed components: Peruvian TASA fish meal 10%, casein 15%, flour 25%, fish oil 2.5%, soybean oil 2.5%, mineral salt premix 5%, vitamin premix 0.39%, choline chloride 0.11%, carboxymethyl cellulose 3%.

[0032] Gradient substitution components: Group C0 (0% substitution): 24% soybean meal, 0% cottonseed protein concentrate, 12.5% ​​cellulose; Group C15 (15% substitution): Soybean meal 20.4%, cottonseed protein concentrate 2.97%, cellulose 13.13%; Group C30 (30% replacement): Soybean meal 16.8%, cottonseed protein concentrate 5.93%, cellulose 13.77%; Group C45 (45% replacement): Soybean meal 13.2%, cottonseed protein concentrate 8.9%, cellulose 14.4%; Group C75 (75% replacement): 6% soybean meal, 14.84% cottonseed protein concentrate, and 15.66% cellulose; Group C100 (100% replacement): 0% soybean meal, 19.78% cottonseed protein concentrate, and 16.72% cellulose.

[0033] Different feed formulations were used to feed crucian carp, and relevant growth indicators of crucian carp in each group were measured to further optimize the optimal feed formula.

[0034] Example 2: Verification of Aquaculture Effect Three hundred and sixty healthy crucian carp of similar size were selected and randomly divided into 18 rearing tanks, with 20 fish per tank and three replicates per group. The experiment was conducted in a recirculating aquaculture system with a 12-hour light cycle followed by a 12-hour dark cycle. Fish were fed twice daily, with saturation feedings recorded, along with the number and weight of dead fish. The experiment lasted 56 days. Feces were removed and approximately 10% of the water was replaced daily to ensure the safety of the aquaculture water.

[0035] 1. Sample index collection: After the crucian carp growth experiment, the fish were fasted for 24 hours. All fish in each tank were then removed, counted, and weighed. Weight gain rate, feed efficiency, and specific growth rate were calculated. Five fish from each tank were randomly selected to measure body length and weight to calculate condition factor. The liver and visceral mass were also weighed to calculate hepatomegaly-to-body ratio (HSI) and viscera-to-body ratio (VSI). Dorsal muscle samples were collected for nutrient composition analysis. Three additional fish were anesthetized with MS-222, and blood was collected from the tail vein before collecting liver, intestinal, and muscle samples. Two fish from each tank were randomly selected for crude component analysis. Blood samples were centrifuged at 3500 rpm / min for 10 min, and the supernatant was collected. Plasma, liver, intestinal, and muscle samples were stored at -80℃. The remaining crucian carp were fed for another 3 days. Five hours after the last feeding, three fish from each tank were dissected to collect intestinal contents. The hindgut contents of each fish were collected in 1.5 mL sterile centrifuge tubes, flash-frozen in liquid nitrogen, and used for intestinal microbial analysis.

[0036] The initial and final samples of whole fish were steamed at 120℃ for 30 min in an autoclave, then mashed and mixed, and dried in a 75℃ oven. The resulting powder was then dried at 105℃ to constant weight, and the moisture content was calculated using the weight loss method. Moisture content was calculated after freeze-drying of the muscle. Crude protein content was determined using the Kjeldahl method (2300-Auto-analyzer, FOSS, Hillerød, Denmark); fat was extracted from the samples using ether based on the Soxhlet extraction method (36680-analyzer, BUCHI, Flawil, Switzerland).

[0037] Formulas for calculating some growth performance and morphology indicators: Weight gain rate (WGR; %) = 100 × [final weight (g) - initial weight (g)] / initial weight (g); Feed efficiency (FE; %) = 100 × [final body weight (g) - initial body weight (g)] / feed intake; Specific growth rate (SGR; % / d) = 100 × (ln final body weight - ln initial body weight) / number of days.

[0038] 2. The results of data collection, processing, and analysis are as follows: After statistical analysis and processing, the effects of cottonseed concentrate protein replacing soybean meal on the growth performance of crucian carp were obtained in the following figures. Figure 1 As shown, where, Figure 1 In the table, A, B, C, and D represent final body weight, feed efficiency, weight gain rate, and specific growth rate, respectively. It can be concluded that the final body weight, weight gain rate, feed efficiency, and specific growth rate of crucian carp show an increasing trend with increasing substitution level, with group C100 exhibiting the highest final body weight, weight gain rate, feed efficiency, and specific growth rate. P <0.05).

[0039] Further analysis was conducted on the effects of cottonseed protein concentrate replacing soybean meal on the whole fish and muscle components of crucian carp. The results are shown in [the table below]. Figure 2 As shown, where, Figure 2 In the table, A, B, C, and D represent crude fat in muscle, crude protein in muscle, crude fat in whole fish, and crude protein in whole fish, respectively. It can be seen that the crude protein and crude fat content of the crucian carp muscle in the replacement group were higher than those in the control group, and the crude protein and fat content of the whole fish showed a similar trend.

[0040] Further statistical analysis was conducted on the effects of different substitution levels on the feeding amount and feeding rate of crucian carp. The results are shown in [the table below]. Figure 3 As shown, where, Figure 3 In the table, A, B, C, D, and E represent feeding rate, feed intake, survival rate, first feeding time (morning feeding), and second feeding time (afternoon feeding), respectively. The results showed that as the substitution level increased, the feeding rate of crucian carp tended to increase, the average feed intake increased significantly, and the survival rate also increased in group C100. Feeding time showed a significant decreasing trend, indicating that cottonseed protein concentrate exhibited a feeding-inducing effect.

[0041] Based on the trend of gradually increasing growth and feed efficiency in crucian carp, four representative gradient groups (C0, C15, C45, and C100) from the six groups were selected for subsequent experiments to conduct more in-depth research. The effects of cottonseed concentrate protein replacing soybean meal on the plasma biochemistry of crucian carp were further analyzed. The results are shown below. Figure 4 As shown, where, Figure 4 In the table, A, B, C, D, and E represent alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein, albumin, and free amino acids, respectively. It can be seen that AST shows an increasing trend in groups C0 to C100. P <0.05), ALT showed no significant difference ( P>0.05). There were no significant differences in TP and ALB levels among the groups. P> 0.05). The FAA content in the plasma of the C100 group showed a decreasing trend compared with the control group. These results may indicate that free amino acids in the blood of the treated fish were rapidly absorbed and used for tissue protein synthesis, leading to a decrease in the level of free amino acids in the blood, and an intensified turnover metabolism of proteins and amino acids in the body, ultimately promoting rapid growth of the fish.

[0042] To further verify the effect of cottonseed protein concentrate replacing soybean meal on the liver health of crucian carp, liver samples from each group were subjected to Oil Red and HE staining analysis. The Oil Red results for each group are shown below. Figure 5 As shown, from Figure 5 As can be seen, lipid droplets in liver tissues from different treatment groups (C0, C15, C45, C100) showed a significant increasing trend. The number of lipid droplets remained relatively stable when the replacement level exceeded 45%. HE staining results for each group are shown below. Figure 6 As shown, from Figure 6 As can be seen, the overall structure of liver tissue in groups C0, C15, C45 and C100 was intact, the arrangement of hepatocytes and the morphology of hepatic sinusoids were basically the same, and no obvious histological damage or differences between groups were observed.

[0043] Further analysis of the amino acid and fatty acid composition of the muscle revealed the effect of cottonseed protein concentrate replacing soybean meal on the amino acid and fatty acid composition of crucian carp muscle. Figure 7 As shown, where, Figure 7 In the analysis, A and B represent the principal components of muscle fatty acids and muscle amino acids, respectively. It can be seen that the fatty acid content in muscle varies significantly. P< 0.05), while the amino acid composition in the muscles of the treated group was not significantly different from that of the control group ( P> 0.05). Further analysis of the fatty acid and amino acid content in crucian carp muscle was conducted, and the results are shown in Tables 1 and 2. As can be seen from Tables 1 and 2, when the substitution level exceeded 45%, the content of monounsaturated and polyunsaturated fatty acids in the muscle increased significantly, while the content of single amino acids in the muscle was similar among the groups.

[0044] Table 1. Fatty acid content of crucian carp muscle (g / 100g, wet weight) Table 2. Amino acid content of crucian carp muscle (g / 100g, wet weight) Further analysis of the gut microbiota under each treatment group revealed the effect of cottonseed protein concentrate replacing soybean meal on the gut microbiota of crucian carp. Figure 8 As shown, where, Figure 8In the table, A, B, and C represent alpha diversity analysis, phylum-level species composition, and genus-level species composition, respectively. It can be seen that with increasing substitution levels, gut microbial alpha diversity and species / genus-level microbial community composition were not significantly affected. However, the beneficial gut microbiota Firmicutes phylum (mainly...) ZOR006 The abundance of the genus Aeromonas showed an increasing trend, while the abundance of the potential pathogenic microorganism Aeromonas showed a decreasing trend, indicating that cottonseed protein concentrate replaced soybean meal to maintain the intestinal homeostasis of crucian carp while promoting the colonization of beneficial microorganisms.

[0045] Further analysis of the effects of cottonseed concentrate protein replacing soybean meal on genes related to muscle growth and lipid metabolism in crucian carp in different treatment groups is shown in the following results. Figure 9 As shown, where, Figure 9 In the diagram, A, B, and C represent muscle growth genes, muscle energy metabolism genes, and muscle lipid synthesis and breakdown genes, respectively. The results showed significant differences in gene expression among C0, C15, C45, and C100. Groups C45 and C100 exhibited higher expression levels in multiple genes, particularly growth-related genes (such as...). tor , 4ebp1 Significantly upregulated expression in () P< 0.05), tor Genes regulate protein synthesis and cell growth. 4ebp1 Inhibition of translation initiation factors, and their increased expression, may reflect the activation of growth regulation mechanisms. In lipid metabolism-related genes (such as...) acc , scd1 The expression levels of C45 and C100 groups also showed an increasing trend. P< 0.05). acc and scd1 These genes respectively promote lipid synthesis and the synthesis of unsaturated fatty acids. Among the genes related to lipid breakdown, fat Mediating fatty acid transport, hsl Promotes lipolysis. lpl It promotes lipoprotein energy supply, and its relative expression level increased in the C45 and C100 groups. P< The value of 0.05 indicates that the liver's lipid synthesis and catabolism capabilities are simultaneously enhanced.

[0046] The effects of cottonseed protein concentrate replacing soybean meal on genes related to liver growth and lipid metabolism in crucian carp in different treatment groups are as follows: Figure 10 As shown, where, Figure 10 In the diagram, A, B, C, and D represent liver growth genes, liver energy metabolism genes, liver lipid breakdown and transport genes, and liver lipid synthesis genes, respectively. In crucian carp liver tissue, the core genes of the mTOR signaling pathway, which are closely related to growth (…), are… tor , s6k1 , 4ebp1The expression of ) showed an upward trend in the C100 group. to r, as a key positive regulator of protein synthesis, indicates that the C100 group of crucian carp has stronger growth potential due to its high expression. Downstream genes... s6k1 and 4ebp1 The synchronous changes further confirm that the translation initiation mechanism of the C100 group is effectively activated, providing a molecular basis for rapid growth. At the energy metabolism level, key genes in the insulin signaling pathway... pi3k and akt The highest expression was observed in the C45 and C100 groups, suggesting that these two groups may have higher efficiency in the uptake and utilization of energy substances and a more active energy metabolism state. (The energy sensor...) ampk Expression remained relatively stable across groups. The liver lipid synthesis map showed a clear upward trend in genes related to lipid synthesis from group C0 to group C100. Group C100 exhibited the highest expression across all synthetic genes, especially... scd1 High expression of this enzyme indicates that its liver not only has a strong capacity for fatty acid synthesis but also tends to produce unsaturated fatty acids, which may help maintain cell membrane fluidity and ensure overall health. Finally, it is the rate-limiting enzyme for fatty acid β-oxidation in the lipid breakdown and transport pathway. cpt1 Fatty acid transport proteins ( fat , lpl ) and rate-limiting enzymes for fat breakdown hsl The gene expression level also showed an increasing trend in both the C45 and C100 groups, with the highest level in the C100 group. In summary, this indicates that the C100 crucian carp liver not only efficiently synthesizes lipids but also possesses strong lipid decomposition and transport capabilities, achieving efficient lipid metabolism and promoting fish growth.

[0047] In summary, the feed formulation of this invention achieves optimal growth performance in crucian carp under the condition of 100% soybean meal replacement, significantly improves feeding enthusiasm, enhances fat synthesis and decomposition metabolism simultaneously, improves muscle nutritional quality, and maintains good liver and intestinal health. It realizes the efficient utilization of non-grain protein sources in aquatic feed, achieves the technical goal of reducing and replacing soybean meal, and strongly supports the national action plan for reducing and replacing soybean meal.

[0048] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A crucian carp feed using cottonseed concentrated protein as a substitute for soybean meal, characterized in that, Soybean meal is completely replaced with cottonseed protein concentrate, wherein the cottonseed protein concentrate accounts for 19-20% of the feed by mass.

2. The crucian carp feed according to claim 1, characterized in that, The cottonseed protein concentrate has a mass percentage of 19.78%.

3. The crucian carp feed according to claim 1 or 2, characterized in that, The crucian carp feed also includes the following components by weight percentage: fish meal 8-12%, casein 13-17%, wheat flour 23-27%, fish oil 2-3%, soybean oil 2-3%, mineral salt premix 4-6%, vitamin premix 0.3-0.5%, choline chloride 0.1-0.2%, carboxymethyl cellulose 2-4%, and cellulose 15-18%.

4. The crucian carp feed according to claim 3, characterized in that, The specific mass percentages of each component in the crucian carp feed are as follows: Peruvian fish meal 10%, casein 15%, wheat flour 25%, fish oil 2.5%, soybean oil 2.5%, mineral salt premix 5%, vitamin premix 0.39%, choline chloride 0.11%, carboxymethyl cellulose 3%, cottonseed protein concentrate 19.78%, and cellulose 16.72%.

5. The application of the crucian carp feed as described in any one of claims 1-4 in the field of crucian carp farming.

6. The application according to claim 5, characterized in that, The application includes improving crucian carp's feed intake, weight gain rate, specific growth rate, and feed efficiency.

7. The application according to claim 5, characterized in that, The application includes promoting the deposition of muscle protein, fat, and the synthesis of polyunsaturated fatty acids in crucian carp.

8. The application according to claim 5, characterized in that, The application involves regulating the expression of genes related to lipid metabolism in crucian carp muscle and liver, and enhancing fat synthesis and catabolism.

9. A method for farming crucian carp, characterized in that, Feed the animal with the feed described in any one of claims 1-4 twice a day for a breeding cycle of not less than 56 days.

10. The method as described in claim 9, characterized in that, Implemented in a recirculating aquaculture system, the photocycle is 12 hours of light: 12 hours of darkness.

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