Application of hyodeoxycholic acid in improving growth performance and muscle nutrition quality of micropterus salmoides

Abstract

The invention belongs to the field of aquatic feed nutriology, and particularly relates to application of hyodeoxycholic acid to improvement of growth performance and muscle nutrition quality of largemouth bass. According to the method, 500-1500 mg / kg of hyodeoxycholic acid is added into largemouth bass feed, the optimal adding amount is 1000 mg / kg, and the hyodeoxycholic acid is added into the largemouth bass feed. The invention also provides a largemouth bass feed added with hyodeoxycholic acid and a feed additive. The hyodeoxycholic acid is added into the feed for the micropterus salmoides, so that the growth performance and the nutritional quality of the micropterus salmoides in the growing period can be improved.

Description

Technical Field

[0001] This invention belongs to the field of aquatic feed nutrition, specifically relating to the application of porcine deoxycholic acid in improving the growth performance and muscle nutritional quality of largemouth bass. Background Technology

[0002] Largemouth bass is one of the main farmed fish species in China, and it is highly favored by the market due to its rapid growth, tender meat, and high nutritional value. However, with consumers' increasing demands for the nutritional quality of aquatic products, optimizing the nutritional composition of its muscle to enhance its commercial value and market competitiveness has become an important issue for the aquaculture industry. Although the high-fat diets commonly used in aquaculture can meet the energy requirements of largemouth bass, they have problems such as uneven fat deposition, low content of functional fatty acids such as EPA and DHA, and poor protein nutritional value, which directly affect the nutritional value and consumer quality of the muscle.

[0003] Currently, largemouth bass farming faces numerous shortcomings. Traditional diets have limitations in fat digestion, absorption, and metabolism, leading to inefficient nutrient utilization. Furthermore, the muscle contains low levels of long-chain polyunsaturated fatty acids such as EPA and DHA, and there is room for improvement in the composition and content of essential amino acids, as well as enhancing the nutritional value of protein. Existing farming techniques primarily focus on improving growth performance, with relatively limited methods for improving nutritional quality. While porcine deoxycholic acid and similar substances have been proven to improve growth performance and carcass quality in livestock and poultry, their application in fish farming remains unexplored. Summary of the Invention

[0004] To address the aforementioned problems, this invention proposes adding porcine deoxycholic acid to a high-fat diet for largemouth bass. By leveraging the biological functions of cholic acid in regulating lipid metabolism and promoting nutrient absorption, multiple objectives can be achieved: improving the growth performance of largemouth bass, increasing the content of amino acids such as glutamic acid in muscle, and increasing the content of unsaturated fatty acids such as oleic acid and linoleic acid in muscle, thereby providing an innovative, effective, and economical nutritional quality improvement solution for aquaculture.

[0005] This invention is achieved through the following technical solution: The application of porcine deoxycholic acid in improving the growth performance and muscle nutritional quality of largemouth bass is described by adding 500-1500 mg / kg of porcine deoxycholic acid to the basal diet of largemouth bass.

[0006] Furthermore, the amount of porcine deoxycholic acid added is 1000 mg / kg.

[0007] Furthermore, the basic feed described herein can meet the healthy and normal growth requirements of largemouth bass or comply with the feed standards for largemouth bass.

[0008] Furthermore, the method of adding the substance involves dissolving porcine deoxycholic acid in edible oil and then spraying it evenly onto the surface of the largemouth bass feed.

[0009] Furthermore, the oil mentioned is soybean oil.

[0010] Furthermore, the amount of soybean oil used is less than 50 mL / kg of feed.

[0011] The present invention also provides an optimal technical solution for feeding largemouth bass with added porcine deoxycholic acid, wherein the amount of porcine deoxycholic acid added is 1000 mg / kg.

[0012] The present invention also provides a feed additive for largemouth bass, wherein the feed additive contains porcine deoxycholic acid.

[0013] The beneficial effects of this invention compared to existing technologies are as follows: Adding porcine deoxycholic acid to the feed of largemouth bass improves the growth performance and nutritional quality of the growing largemouth bass, with the optimal addition rate of 1000 mg / kg. This achieves a synergistic effect of promoting growth and maintaining feed utilization. While maintaining stable levels of core conventional nutrients such as crude protein and crude fat in muscle, the total amount of 17 amino acids, the total amount of essential amino acids, and the total amount of flavor amino acids are significantly higher than those in the control group and the 500 mg / kg and 1500 mg / kg groups. Detailed Implementation

[0014] The technical solution of the present invention will be further explained below, but the scope of protection of the present invention is not limited in any way by the embodiments.

[0015] (I) Basic Principles Porcine deoxycholic acid, as a secondary bile acid, has multiple biological functions. It can not only emulsify with fat to form micelle structures, promoting the digestion, absorption and metabolism of fat, but also regulate the expression of lipid metabolism-related genes by activating nuclear receptors such as TGR5 and FXR to optimize fatty acid composition. At the same time, it can improve overall digestion and absorption efficiency, increase the utilization efficiency of nutrients such as protein and fat, and improve digestive function to enhance feed intake, thereby promoting growth performance.

[0016] (II) Requirements for seedlings, feed, and deoxycholic acid supplementation in pigs The method described in this patent is applicable to earthen pond aquaculture or facility-based aquaculture using formulated feed. Seedlings should be products from reputable seedling manufacturers that have passed quarantine inspection; stocking density and aquaculture cycle should be flexibly adjusted according to local conditions and aquaculture methods; the formulated feed used at each aquaculture stage must meet the requirements of GB / T 22919.9-2024 Aquatic Compound Feed Part 9: Largemouth Bass Compound Feed.

[0017] The recommended addition amount of porcine deoxycholic acid is 1000 mg / kg (based on feed weight). Two methods of addition are possible: one is to add it together with the concentrated premix during feed formulation; the other is to fully dissolve it in vegetable oils such as soybean oil and then spray it evenly onto the surface of the finished pelleted feed. The amount of soybean oil used to dissolve the porcine deoxycholic acid must be controlled below 50 mL / kg (based on feed weight) to prevent the crude fat content in the feed from exceeding the aforementioned feed standard requirements.

[0018] (III) Feeding and Management During the aquaculture process, management should be tailored to local conditions and standardized according to local standards. Specific management includes water quality management, feeding management, and input management. Water quality management directly affects growth rate, disease control, and product quality and safety. This is especially crucial in high-density aquaculture. In pond aquaculture during the high-temperature season, water should be changed every 10-15 days, with the water volume controlled at 10%-20% of the pond's depth. Modern ecological aquaculture models such as pond recirculation systems and land-based container aquaculture should rely on internal purification systems to reduce the frequency of water changes. Regarding dissolved oxygen, pond aquaculture should be equipped with paddlewheel or impeller aerators. Facility-based aquaculture requires 24-hour continuous water aeration supplemented with micro-pore bottom aeration to ensure dissolved oxygen levels remain above 5 mg / L. During hot, humid weather or nighttime periods of low dissolved oxygen, liquid oxygen equipment should be used to supplement pure oxygen and prevent fish from surfacing due to oxygen deficiency. Key water quality indicators need regular monitoring and control; the pH value should be maintained between 7.0 and 8.5. When the levels are too low, alkaline water conditioners should be used to adjust them. Ammonia nitrogen, nitrite and other indicators need to be adjusted by regularly applying microecological preparations, or by raising filter-feeding fish such as silver carp and bighead carp, as well as shellfish such as snails and clams in the purification area for biological purification.

[0019] The feed should be a complete artificial compound feed with high protein (≥40%) and low starch (≤150 g / kg). The daily feeding amount and frequency need to be dynamically adjusted according to the size of the fish, water temperature and weather conditions: the daily feeding amount should be controlled at 4%~6% of the fish body weight during the juvenile stage (less than 100 grams), and fed 3~5 times a day; the feeding rate should be reduced to 1%~2% during the adult stage (more than 100 grams), and fed 2~3 times a day.

[0020] The use and management of inputs should be standardized throughout the entire process. Feed, fish medicines, etc. should be purchased from compliant suppliers. It is recommended that dedicated personnel and warehouses be used for storage and use, and complete inbound and outbound and usage ledgers should be established to ensure that input information is traceable throughout the entire process.

[0021] (iv) Testing on improvement in growth performance and nutritional quality After the breeding cycle ended, in order to clarify the effect of porcine deoxycholic acid supplementation on the growth performance and nutritional quality of largemouth bass, the relevant detection and analysis were divided into four modules: growth performance index determination and calculation, routine nutrient composition analysis, amino acid composition analysis, and fatty acid composition analysis. The specific contents are as follows: 1. Measurement and calculation of growth performance indicators Weight gain rate, specific growth rate, feed conversion ratio, condition factor, liver body index, and visceral body index need to be measured and calculated. The calculation methods for each indicator are as follows: Weight gain rate (WGR, %) = (Final weight - Initial weight) / Initial weight × 100; Specific growth rate (SGR, %) = (ln final body weight - ln initial body weight) / t × 100; Feed conversion rate (FCR) = Feeding amount / (Final gross weight - Initial gross weight + Weight of dead fish); Condition factor (CF, g / cm³) = Final body weight (g) / Final body length 3 (cm) 3 ) × 100; Hepatosomatic index (HSI, %) = Liver weight (g) / Body weight (g) × 100; Viserosomatic index (VSI, %) = Visceral weight (g) / Body weight (g) × 100; 2. Routine nutritional composition analysis The moisture, crude protein, crude fat, ash, and crude fiber content of the muscle samples were tested according to national standard methods. The corresponding testing standards for each indicator are as follows: Moisture content: GB 5009.3-2016 National Food Safety Standard - Determination of Moisture in Food; Ash content: GB 5009.4-2016 National Food Safety Standard - Determination of Ash Content in Food; Crude protein: GB 5009.5-2025 National Food Safety Standard - Determination of Protein in Food; Crude fat: GB 5009.6-2025 National Food Safety Standard - Determination of fat in food.

[0022] 3. Amino acid composition analysis Following the "GB 5009.124-2016 National Food Safety Standard - Determination of Amino Acids in Food", the amino acid composition of muscle was analyzed, and the following parameters were calculated: the absolute content of various essential and non-essential amino acids, the total amount of essential amino acids, and the total amount of non-essential amino acids.

[0023] 4. Fatty acid composition analysis Following the "GB 5009.168-2016 National Food Safety Standard - Determination of Fatty Acids in Food", the relative content of various major fatty acids in muscle was calculated, and the following indicators were statistically analyzed: total saturated fatty acids, total monounsaturated fatty acids, total polyunsaturated fatty acids, the sum of DHA and EPA, as well as the ratio of unsaturated fatty acids to saturated fatty acids and the ratio of polyunsaturated fatty acids to saturated fatty acids.

[0024] To verify the effectiveness of the aforementioned technology, a 60-day controlled feeding experiment was conducted on largemouth bass with a body weight of approximately 200g during their rearing period. Combined with growth performance measurements, the dose-effect of porcine deoxycholic acid was systematically evaluated.

[0025] Experimental group design The experiment consisted of one control group and four treatment groups supplemented with porphyrin (deoxycholic acid). The control group was fed a basal high-fat diet (without porphyrin), while the treatment groups received porphyrin supplemented with 500 mg / kg, 1000 mg / kg, and 1500 mg / kg of porphyrin, respectively, on their basal high-fat diets. The specific method of supplementation was as follows: the corresponding dose of porphyrin was fully dissolved in soybean oil and then evenly sprayed onto the surface of the finished pelleted feed. The amount of soybean oil used for dissolving the porphyrin was controlled at 50 mL / kg (by feed weight) to ensure that the crude fat content of the feed did not exceed the aforementioned feed standard requirements. Each group had three biological replicates, with 20 uniformly sized and healthy experimental fish in each replicate.

[0026] Feed source: Tongwei largemouth bass feed. Ingredients: wheat flour, soybean, fish meal, spirulina powder, rapeseed meal, yeast powder, sodium chloride, vitamins, and minerals. Protein ≥35-38%, crude fat ≥7%, crude fiber ≤10%, crude ash ≤11%, moisture ≤10%, total phosphorus ≥1.5%, lysine ≥1.95%, calcium 0.8-2.2%, sodium chloride 0.3-1.2%. Feeding and Management All experimental fish underwent a 7-day acclimatization period in the experimental culture system, fed a basal diet during this time, successfully eliminating the stress effects of environmental changes. In the formal experimental phase, all experimental groups adopted a uniform feeding strategy: feeding twice daily at 8:00 AM and 4:00 PM, using the apparent satiety method and recording feed intake. Simultaneously, water temperature and quality were monitored daily, ensuring all environmental and nutrient factors remained consistent, effectively eliminating interference from irrelevant variables.

[0027] Sample collection and analysis After the experiment, the growth indicators of fish in each replicate group were measured according to the aforementioned method. Subsequently, six fish were randomly selected from each replicate, and muscle samples were collected. Nutritional components were analyzed according to the described detection method. Finally, through data integration, the dose-response relationship between the amount of porcine deoxycholic acid added and the growth performance and nutritional quality of largemouth bass was clarified.

[0028] Experimental results Table 1 shows that the average final weight (288.81 g) and weight gain rate (52.63%) of the group supplemented with 1000 mg / kg porcine deoxycholic acid were significantly higher than those of the control group, while the feed conversion ratio (FCR) was not significantly different from that of the control group. However, the growth indicators of the 500 mg / kg group showed no statistically significant difference from the control group, and the FCR of the 1500 mg / kg group was higher than that of the control group and other supplemented dose groups. Body composition and visceral development indicators showed that the condition factor (2.65–2.72 g / cm³), liver-to-body ratio (2.68–2.86%), and visceral-to-body ratio (10.88–11.92%) of all experimental groups were significantly higher than those of the control group, with no significant differences among the experimental groups. This indicates that porcine deoxycholic acid can promote fat deposition and liver and visceral organ development in largemouth bass. In conclusion, 1000 mg / kg is the appropriate supplementation dose of porcine deoxycholic acid in largemouth bass formulated feed, which can significantly improve growth performance without reducing feed utilization.

[0029] Table 2 shows that, in terms of conventional nutritional indicators, the addition of porcine deoxycholic acid at various doses did not significantly change the core nutritional components (crude protein, crude fat, and ash) of largemouth bass muscle. Only the moisture content of the 500 mg / kg group was slightly affected, and the moisture and nutritional components of the muscle remained stable at the appropriate growth dose (1000 mg / kg). This indicates that the additive promotes growth without damaging the basic nutritional value of the fish meat and has good application safety.

[0030] Table 3 shows that adding different doses of porcine deoxycholic acid to the feed had a significant effect on the amino acid content of largemouth bass muscle, with the 1000 mg / kg dose showing the best improvement. The total amount of 17 amino acids, the total amount of essential amino acids, the total amount of non-essential amino acids, and the total amount of flavor amino acids were all highest in the 1000 mg / kg group, and were significantly higher than the control group and the 500 mg / kg and 1500 mg / kg groups. There were no significant differences in the above indicators between the 500 mg / kg and 1500 mg / kg groups, but both were significantly higher than the control group. Among individual amino acids, only the flavor amino acid glutamic acid content was significantly higher in the 1000 mg / kg group (3.04) than the control group (2.72) and other experimental groups. Although the values ​​of other individual essential amino acids (threonine, valine, etc.), flavor amino acids (aspartic acid, glycine, etc.), and non-essential amino acids (serine, proline, etc.) fluctuated between groups, there were no significant differences, and the content of most individual amino acids in the 1000 mg / kg group was higher than that in other groups.

[0031] Table 4 shows that the effects of different doses of porcine deoxycholic acid added to the feed on the fatty acid composition of largemouth bass muscle were mainly reflected in a significant increase in the proportion of unsaturated fatty acids: there were no significant differences in the absolute contents of total fatty acids (1.51~1.73), saturated fatty acids (0.27~0.34), monounsaturated fatty acids (0.54~0.69), polyunsaturated fatty acids (0.69~0.76), and EPA+DHA (0.13~0.15) among the groups, and the total fatty acid content in the experimental groups fluctuated slightly compared with the control group but did not reach a statistically significant level; the ratio of unsaturated fatty acids to saturated fatty acids and the ratio of polyunsaturated fatty acids to saturated fatty acids both showed a significant dose-dependent increase, with the highest in the 1500 mg / kg group, followed by the 1000 mg / kg group, and the lowest in the control group. At the same time, the experimental groups showed a significant increase in monounsaturated fatty acids (such as oleic acid C18:1n9c) and polyunsaturated fatty acids (such as DHA). The absolute content of C22:6n3 was higher than that of the control group, indicating that porcine deoxycholic acid can improve the health and nutritional value of fish by optimizing the fatty acid composition ratio, and the effect is more significant at higher doses.

[0032] In summary, 1000 mg / kg of porcine deoxycholic acid showed the best effect on improving the growth performance and nutritional quality of largemouth bass. At this dosage, the final average weight and weight gain rate were significantly higher than those of the control group and other supplemented dosage groups, while the feed conversion ratio was not significantly different from that of the control group, achieving a synergistic effect of promoting growth and maintaining feed utilization. At the same time, it could maintain the stability of core conventional nutrients such as crude protein and crude fat in muscle. The total amount of 17 amino acids, the total amount of essential amino acids, and the total amount of flavor amino acids were significantly higher than those of the control group and the 500 mg / kg and 1500 mg / kg groups. Although the ratio of unsaturated fatty acids to saturated fatty acids and the ratio of polyunsaturated fatty acids to saturated fatty acids in muscle fatty acid composition were lower than those of the 1500 mg / kg group, they were significantly higher than those of the control group. The overall effect of improving growth performance and nutritional quality was the best.

[0033] Table 1. Effects of different doses of porcine deoxycholic acid on the growth performance of largemouth bass. ; Note: Different letters indicate significant differences between groups (p < 0.05). Table 2. Effects of different doses of porcine deoxycholic acid added to feed on conventional nutrient components in the muscle of largemouth bass. ; Note: Different letters indicate significant differences between groups (p < 0.05). Table 3. Effects of different doses of porcine deoxycholic acid added to feed on amino acid content in the muscle of largemouth bass. ; Note: Different letters indicate significant differences between groups (p < 0.05). * indicates essential amino acids. # indicates flavor amino acids.

[0034] Table 4. Effects of different doses of porcine deoxycholic acid added to feed on fatty acid content in the muscle of largemouth bass. ; ; Note: Different letters indicate significant differences between groups (p < 0.05). ^ represents saturated fatty acids, * represents monounsaturated fatty acids, and # represents polyunsaturated fatty acids.

Claims

1. The application of porcine deoxycholic acid in improving the growth performance and muscle nutritional quality of largemouth bass, characterized in that, The method described is to add 500-1500 mg / kg of porcine deoxycholic acid to the basic feed of largemouth bass.

2. The application according to claim 1, characterized in that, The amount of porcine deoxycholic acid added is 1000 mg / kg of basal feed.

3. The application according to claim 1, characterized in that, The basic feed described herein can meet the healthy and normal growth requirements of largemouth bass or comply with the feed standards for largemouth bass.

4. The application according to claim 1, characterized in that, The method of adding the substance involves dissolving porcine deoxycholic acid in edible oil and then spraying it evenly onto the surface of the largemouth bass feed.

5. The application according to claim 4, characterized in that, The oil mentioned is soybean oil.

6. The application according to claim 5, characterized in that, The amount of soybean oil used should be less than 50 mL / kg of feed.

7. A feed for largemouth bass containing porcine deoxycholic acid.

8. The feed according to claim 7, characterized in that, The amount of porcine deoxycholic acid added is 1000 mg / kg of feed.

9. A feed additive for largemouth bass, characterized in that, The feed additive contains porcine deoxycholic acid.