Application of nucleotide and fructo-oligosaccharide, low-fish-meal aquatic feed and preparation method of low-fish-meal aquatic feed

Abstract

The invention discloses application of nucleotide and fructo-oligosaccharide, low-fish-meal aquatic feed and a preparation method of the low-fish-meal aquatic feed, and relates to the technical field of feed. The content of the fish meal in the feed formula system is only 15%, and the low-fish-meal feed has the breeding effect equivalent to or even better than that of the traditional 35% fish meal feed through reasonable matching and combination of nucleotide and fructo-oligosaccharide. The nucleotide and the fructo-oligosaccharide can synergistically regulate the expression of appetite-related genes, optimize the intestinal micro-ecological environment, improve the digestive enzyme activity and activate multiple metabolic pathways, so that the food calling property and the palatability of the low-fish-meal feed are remarkably improved, the feeding rate of the eriocheir sinensis is increased, the growth is promoted, and the feed coefficient is reduced. Under the condition that a large amount of plant protein replaces fish meal, through the synergistic effect of nucleotide and fructo-oligosaccharide, the appetite of the eriocheir sinensis is not reduced, the growth is not slow, the protein deposition rate of the feed is high, the cost of each ton of the feed can be saved by about 1030 yuan, the reduction amplitude reaches 15.7%, and the economic requirements of large-scale culture of the eriocheir sinensis are met.

Description

Technical Field

[0001] The present invention belongs to the field of feeds, and particularly relates to the application of nucleotides and fructooligosaccharides, a low-fishmeal aquatic feed and a preparation method thereof. Background Art

[0002] Fish meal is the most important protein source in aquatic feeds. Due to the limited global fishery resources, the supply of fish meal is difficult to meet the rapidly developing demand of the aquaculture industry, resulting in continuous price increases. Developing and applying low-fishmeal aquatic feeds has become an important development direction in the aquatic feed industry.

[0003] The Chinese mitten crab (Eriocheir sinensis) is one of the characteristic economic aquaculture varieties in China, ranking third in the global crustacean aquaculture production. It has excellent flavor quality and rich nutritional value, and has strong market demand in East Asian regions such as China, Japan and South Korea. As the world's largest producer of Chinese mitten crabs, China's production reached 782,200 tons in 2022, showing a stable growth trend. With the improvement of the scale and intensification of Chinese mitten crab farming, the demand for feed and the dependence on fish meal have also increased. At present, the feed cost accounts for about 70% of the total farming cost. As the main cost component, the price of fish meal has risen from about 8,000 yuan / ton in 2010 to about 12,500 yuan / ton in 2022, and there is still an upward trend, seriously restricting the economic benefits of the industry. The wide application of fish meal in feeds is mainly due to its high-quality amino acid composition, high content of essential fatty acids, moderate carbohydrate content, good palatability, low content of anti-nutritional factors and high digestibility and absorption rate.

[0004] However, in the process of replacing fish meal with a large amount of plant-based protein sources, there are usually multiple systemic problems: one is the decline in palatability, which causes the Chinese mitten crab to refuse to eat, resulting in a significant reduction in the feeding rate; the second is that the anti-nutritional factors in plant proteins interfere with the digestion and absorption process; the third is that the unbalanced amino acid composition of plant proteins limits the effective utilization of nutrients; the fourth is the lack of specific functional components in fish meal, leading to metabolic regulation imbalance. The combined effect of these problems is ultimately manifested as the slow growth of Chinese mitten crabs, an increase in the feed conversion ratio and a decline in immune function, seriously affecting the breeding efficiency and the sustainable development of the industry.

[0005] As a key strategy to improve the effects of low-fishmeal diets, functional components in feeds have attracted extensive research attention. Such components can not only improve the flavor characteristics of feeds and increase the feed intake of aquatic animals, but also promote growth, enhance feed conversion efficiency, and even reduce water pollution. There is a wide variety of feed functional components, including nitrogen-containing compounds, sulfur-containing compounds, and various plant and animal extracts. Existing studies have shown that different functional components exhibit specific effects in aquatic animals. For example, squid paste has a significant feeding attraction effect in black tiger shrimp diets due to its unique fishy and fragrant aroma; the addition of glutamic acid can effectively improve the palatability of grass carp diets; dimethyl-β-propiothetin has an obvious effect on increasing the feeding rate in tilapia farming. Nucleotides, especially guanylic acid (GMP) and inosinic acid (IMP), due to their unique umami characteristics and the ability to participate in various physiological metabolic processes, show effects of promoting feeding, accelerating growth, and improving health levels in some aquatic animals.

[0006] Fructooligosaccharide (FOS), as a non-digestible functional oligosaccharide, has achieved fruitful research results in the field of aquatic feeds in recent years. This compound can selectively stimulate the growth and reproduction of beneficial intestinal flora and optimize the microbial ecological balance. Existing studies have confirmed that adding fructooligosaccharide to the diets of various aquatic animals can produce positive effects. Research has shown that adding 2-3% fructooligosaccharide to the diets of Caspian salmon can significantly enhance the immune response, improve stress resistance, and increase digestive enzyme activity. In the study of Litopenaeus vannamei diets, it was found that the addition of fructooligosaccharide had positive effects on growth performance, feed utilization efficiency, intestinal microbial composition, and immune function. Particularly important is that fructooligosaccharide can significantly improve the adaptability of Sparus latus to plant protein diets and promote the protein deposition process by regulating the structure of the microbial flora. Further research revealed that fructooligosaccharide effectively alleviates the negative effects of high-plant-protein diets on the digestive and absorption systems of Macrobrachium rosenbergii by promoting the proliferation of beneficial bacteria and strengthening the intestinal barrier function.

[0007] Although the effectiveness of single functional components has been relatively well verified, the synergistic mechanism among functional components in aquatic feeds still needs to be further explored. There may be complementary or synergistic effects among multiple components, resulting in a comprehensive effect that exceeds the addition of single components. For example, some studies have found that the combined application of seaweed extract and yeast β-glucan can synergistically regulate the expression patterns of immune-related genes in sea bream at the molecular level. Research has shown that the combined use of probiotics and oligosaccharides can significantly improve the adaptability of grass carp to plant protein diets, mainly manifested as an increase in intestinal microbial diversity and enhanced digestive and absorption functions.

[0008] However, there is currently a lack of research on the synergistic effects of two functional components with different mechanisms of action, nucleotides and fructooligosaccharide, in low-fishmeal diets for crustaceans, especially the effects and molecular mechanisms in the farming application of Eriocheir sinensis have not been systematically elucidated. Summary of the Invention

[0009] The present invention provides an application of nucleotides and fructooligosaccharides, a low-fishmeal aquatic feed and a preparation method thereof. By adding specific doses of nucleotide and fructooligosaccharide functional components to a feed with the fish meal content reduced to 15%, through reasonable collocation of plant protein sources and adjustment of the balance of key amino acids, a set of overall schemes with synergistic effects are formed, which can significantly reduce the feed cost while ensuring the breeding effect.

[0010] In order to achieve the above technical objectives, the present invention mainly adopts the following technical solutions:

[0011] The present invention discloses the application of nucleotides and fructooligosaccharides in synergistically activating the thyroid hormone synthesis and glycerophospholipid metabolism pathways of aquatic products.

[0012] The present invention discloses the application of nucleotides and fructooligosaccharides in synergistically improving the activity of digestive and absorption enzymes of aquatic products.

[0013] The present invention discloses the application of nucleotides and fructooligosaccharides in synergistically enhancing the antioxidant and immune capabilities of aquatic products.

[0014] The present invention discloses the application of nucleotides and fructooligosaccharides in synergistically enhancing the feeding rate of aquatic products, promoting growth and reducing the feed coefficient.

[0015] Preferably, the nucleotides include adenylic acid, guanylic acid, inosinic acid, uridylic acid and cytidylic acid, and the mass ratio of adenylic acid, guanylic acid, inosinic acid, uridylic acid and cytidylic acid is 1-3:1-3:1-3:1-3:1-3; the fructooligosaccharide is fructan FOS.

[0016] The present invention also discloses a low-fishmeal aquatic feed, which includes nucleotides and fructooligosaccharides. The addition amount of nucleotides in the feed is 0.6-1.2 g / kg, and the addition amount of fructooligosaccharides in the feed is 0.1-0.3%.

[0017] In a preferred embodiment of the present invention, by weight, it includes the following components: 120-180 parts of fish meal, 200-250 parts of soybean meal, 200-250 parts of cottonseed meal, 10-30 parts of fish oil, 10-30 parts of soybean oil, 5-15 parts of lecithin, 3-8 parts of cholesterol, 3-8 parts of choline chloride, 150-200 parts of pregelatinized starch, 30-50 parts of compound vitamins, 10-30 parts of compound minerals, 10-30 parts of sodium alginate, 5-15 parts of calcium dihydrogen phosphate, 6-12 parts of nucleotides, and 10-30 parts of fructooligosaccharides.

[0018] Preferably, by weight parts, it comprises the following components: 150 parts of fish meal, 240 parts of soybean meal, 240 parts of cottonseed meal, 20 parts of fish oil, 20 parts of soybean oil, 10 parts of lecithin, 5 parts of cholesterol, 5 parts of choline chloride, 180 parts of pregelatinized starch, 40 parts of compound vitamins, 20 parts of compound minerals, 20 parts of sodium alginate, 10 parts of calcium dihydrogen phosphate, 8.5 parts of nucleotides, and 20 parts of fructooligosaccharide.

[0019] The present invention also discloses a preparation method of the low-fish-meal aquatic feed as described above, comprising the following steps:

[0020] (1) Weigh the remaining feed raw materials except fish oil and soybean oil according to the formula ratio, crush them with a pulverizer and then sieve them, and mix them evenly;

[0021] (2) Add fish oil, soybean oil and an appropriate amount of water to the powder in step (1), stir into a dough, and extrude it into pellets;

[0022] (3) Air-dry the pellets under natural conditions until the moisture content is 6-15%;

[0023] (6) Seal and package, and store frozen.

[0024] Preferably, the aquatic product is a crustacean aquaculture organism.

[0025] Compared with the prior art, the present invention has at least the following beneficial effects:

[0026] 1. The nucleotides and fructooligosaccharide functional components in the present invention, as key parts of the overall formula system, comprehensively improve the feeding rate and utilization efficiency of Eriocheir sinensis to the low-fish-meal feed by synergistically regulating appetite and stimulating the senses, optimizing the intestinal microecological environment, and participating in various metabolic pathways, giving full play to the synergistic effect of each component, making up for each other's advantages and disadvantages, and achieving the best overall effect. It can not only increase the palatability of the feed, improve the feeding rate of Eriocheir sinensis, but also improve its digestion and absorption ability, promote the growth of Eriocheir sinensis, and reduce the feed coefficient.

[0027] 2. The combination of functional components in the present invention can not only improve the palatability of the low-fish-meal feed, but also improve the activity of digestive and absorption enzymes of Eriocheir sinensis through synergistic effects, improve intestinal health, enhance antioxidant and immune capabilities, and comprehensively promote the healthy growth of Eriocheir sinensis. In particular, the synergistic effect of nucleotides and fructooligosaccharide can significantly activate key metabolic pathways such as thyroid hormone synthesis and glycerophospholipid metabolism, and promote the growth, development and molting process of Eriocheir sinensis.

[0028] 3. For the overall formula system of the low-fish-meal feed in the present invention, under the condition of low fish meal, the feed still has good palatability, the appetite of Eriocheir sinensis does not decrease, feed waste is reduced, and the feed coefficient is low and the protein deposition rate is high.

[0029] 4. The overall formula system of the present invention has a specific effect on Eriocheir sinensis, and can effectively replace the various functions lost when the fish meal content in the feed is reduced from 35% to 15%, meeting the requirements of large-scale cultivation of Eriocheir sinensis.

[0030] 5. The feed of the present invention has significant economic benefits. Compared with the traditional 35% fish meal formula, the low fish meal formula (15% fish meal) of the present invention can save about 1,030 yuan per ton of feed, with a reduction rate of 15.7%. Calculated based on an annual output of 780,000 tons of Eriocheir sinensis and an average feed coefficient of 1.8, the annual feed consumption is about 1.4 million tons, and it can save about 1.44 billion yuan in feed costs for the industry every year. At the same time, due to the high protein deposition rate and low feed coefficient of the feed of the present invention, compared with the traditional high fish meal feed, it can further reduce the breeding cost and improve the breeding economic benefit. Description of the Drawings

[0031] Figure 1 It is a diagram showing the effect of the economic low fish meal feed of the present invention on the growth performance of Eriocheir sinensis;

[0032] Figure 2 It is a diagram showing the effect of the economic low fish meal feed of the present invention on the feed coefficient, one-hour food intake and expression of appetite-related genes of Eriocheir sinensis;

[0033] Figure 3 It is a diagram showing the effect of the economic low fish meal feed of the present invention on the digestive enzyme activity and related gene expression of Eriocheir sinensis;

[0034] Figure 4 It is a diagram showing the effect of the economic low fish meal feed of the present invention on the expression of antioxidant-related genes of Eriocheir sinensis;

[0035] Figure 5 It is a diagram showing the effect of the economic low fish meal feed of the present invention on the significantly enriched pathways of the hepatopancreas transcriptome of Eriocheir sinensis;

[0036] Figure 6 It is a diagram showing the effect of the economic low fish meal feed of the present invention on the expression of key genes in the thyroid hormone synthesis and phospholipid metabolism pathways of Eriocheir sinensis. Detailed Embodiments

[0037] In order to make those skilled in the art more clearly understand the technical solution of the present invention, the following examples are listed for illustration. It should be noted that the following examples do not limit the scope of protection required by the present invention. The technical solution of the present invention, unless otherwise specified, is a conventional solution in the art.

[0038] In the present invention, fructooligosaccharide, as a non-digestible oligosaccharide, optimizes the intestinal microecological environment by selectively promoting the growth of beneficial intestinal flora. Previous studies have found that the addition of 0.2% fructooligosaccharide can significantly increase the abundances of Bifidobacterium and Lactobacillus in the intestine of Chinese mitten crabs and reduce the proportion of harmful bacteria such as Clostridium perfringens. This change in the microbial flora improves the digestion and absorption efficiency of nutrients in the feed on the one hand and may affect host metabolism and immune function through microbial metabolites such as short-chain fatty acids on the other hand.

[0039] There is a multi-level synergistic relationship between nucleotides and fructooligosaccharide. First, the improved intestinal environment by fructooligosaccharide may promote the absorption of nucleotides; second, nucleotides, as the basic units for nucleic acid synthesis, can provide necessary precursor substances for the proliferation of intestinal epithelial cells promoted by fructooligosaccharide; third, the two functional components may jointly affect key metabolic pathways, such as the thyroid hormone synthesis and glutathione metabolism pathways, through different routes. This multi-level synergistic effect enables the combination of nucleotides and fructooligosaccharide to produce a comprehensive effect that exceeds the effects of their individual addition, especially under the condition of low fish meal, it can more effectively make up for the nutritional and functional defects caused by the reduction of fish meal.

[0040] In the present invention, the low fish meal feed specifically refers to a formulation system with a fish meal content of only 15%, which significantly reduces the amount of fish meal used compared with the traditional 35% fish meal feed. In this formulation, the reduced fish meal protein is mainly replaced by plant protein sources (such as soybean meal and cottonseed meal). Since plant proteins lack the unique flavor substances and key functional components in fish meal, specific functional components need to be added to maintain the palatability and nutritional utilization efficiency of the feed.

[0041] The present invention selects nucleotides and fructooligosaccharide as the key functional components in the low fish meal feed formulation for Chinese mitten crabs and synergistically optimizes them with other formulation components for the following technical reasons:

[0042] 1. Structure-function correlation: Nucleotides, as the basic components of nucleic acids, participate in various physiological processes. In particular, the interaction between the phosphate groups in the structures of guanylic acid (GMP) and inosinic acid (IMP) and the umami receptors produces a significant umami taste, which is suitable for stimulating the chemoreceptors of aquatic crustaceans;

[0043] 2. Special metabolic characteristics of crustaceans: Crustaceans such as Chinese mitten crabs have a high demand for exogenous nucleotides during growth and molting processes, so they have a relatively high demand for exogenous nucleotides, especially under the condition of low fish meal feed;

[0044] 3. Intestinal regulatory effect of fructooligosaccharide: As a non-digestible oligosaccharide, fructooligosaccharide can selectively promote the growth of beneficial intestinal bacteria, improve the intestinal microecological environment, and enhance the digestion and absorption efficiency of nutrients, especially promoting the utilization of plant proteins;

[0045] 4. Synergistic mechanism: Fructooligosaccharide can enhance the absorption and utilization of nucleotides by regulating the intestinal microbial composition. At the same time, nucleotides can provide necessary nucleic acid precursors for the growth of beneficial bacteria promoted by fructooligosaccharide, forming a mutually promoting synergistic relationship between the two;

[0046] 5. Synergistic regulation of metabolic pathways: Through systematic research, it is found in the present invention that the co-addition of nucleotides and fructooligosaccharide can synergistically activate key pathways such as thyroid hormone synthesis and glycerophospholipid metabolism, enhance the body's ability to digest and absorb plant proteins, and promote growth and development;

[0047] 6. Formulation synergistic effect: In the present invention, through the optimized combination of plant protein sources (soybean meal, cottonseed meal) with functional components of nucleotides and fructooligosaccharide, and by supplementing appropriate amounts of coated lysine and methionine, a set of formulation system with synergistic effect is formed, making up for the various nutritional limitations brought about by simply reducing fish meal;

[0048] 7. Technical feasibility: The industrial production of high-purity nucleotides and fructooligosaccharide has been mature, and the cost-effectiveness is suitable for large-scale application in aquatic feeds.

[0049] In addition to the above technical reasons, the low-fish meal formulation system of the present invention can exert its effects through multiple action mechanisms through the synergistic effect of functional components of nucleotides and fructooligosaccharide, far exceeding the effects of adding any one functional component alone. The formulation of the present invention comprehensively improves the digestion and absorption ability of Eriocheir sinensis to plant proteins by regulating multiple metabolic pathways, including thyroid hormone synthesis and phosphate and phosphoester metabolism, etc., makes up for the nutritional limitations brought about by low-fish meal, and enables the low-fish meal feed plan to achieve a cultivation effect equivalent to or even better than that of high-fish meal feeds.

[0050] The following is illustrated by specific examples.

[0051] Example 1 Preparation of experimental feeds

[0052] In the present invention, the protein and fat contents of the overall formulation of the low-fish meal feed are equivalent to those of the feed containing 35% fish meal, and nutritional balance is achieved through precise proportioning.

[0053] In this experiment, a normal fish meal control diet containing 35% fish meal was set up, and based on a low fish meal diet containing 15% fish meal, six diets supplemented with different doses of nucleotide functional components and fructooligosaccharide combinations were designed, resulting in a total of seven diet formulations. The specific design is as follows: control group (35% fish meal), experimental group 1 (15% fish meal), experimental group 2 (15% fish meal + 0.3 g / kg nucleotide), experimental group 3 (15% fish meal + 0.6 g / kg nucleotide), experimental group 4 (15% fish meal + 0.9 g / kg nucleotide), experimental group 5 (15% fish meal + 1.2 g / kg nucleotide), and experimental group 6 (15% fish meal + 0.9 g / kg nucleotide + 0.2% fructooligosaccharide). The experimental diet formulations and their approximate chemical compositions are shown in Table 1, where the compound vitamins and compound minerals are both prior arts, and their raw material ratio compositions only need to meet the growth requirements of Chinese mitten crabs.

[0054] The nucleotides used in this experiment were a commercially available nucleotide mixture, composed of purified adenosine monophosphate (AMP), disodium guanylate (GMP), disodium inosinate (IMP), disodium uridylate (UMP), and cytidine monophosphate (CMP) in a ratio of 1:1:1:1:1, with a purity of over 98%. The fructooligosaccharide (FOS) used was purchased from Shanghai Yuanye Bio-Technology Co., Ltd., with a purity of 95%. Based on the pre-test results, 0.2% was determined to be the optimal addition level for fructooligosaccharide.

[0055] Table 1 Composition formulations and chemical compositions of the experimental diets (g / kg, dry matter basis)

[0056]

[0057]

[0058] 1Vitamin premix: Retinol acetate 0.043 g, Thiamin hydrochloride 0.15 g, Riboflavin 0.0625 g, Ca pantothenate 0.3 g, Niacin 0.3 g, Pyridoxine hydrochloride 0.225 g, Para-aminobenzoic acid 0.1 g, Ascorbic acid 0.5 g, Biotin 0.005 g, Folic acid 0.025 g, Cholecalciferol 0.0075 g, α-tocopherol acetate 0.5 g, Menadione 0.05 g, Inositol 1 g, filled up to 100 g with cellulose (All ingredients are filled with α-cellulose to 100 g);

[0059] 2 Mineral premix: KH2PO4 21.5 g, NaH2PO4 10.0 g, Ca(H2PO4)2 26.5 g, CaCO3 10.5 g, KCl 2.8 g, MgSO4·7H2O 10.0 g, AlCl3·6H2O 0.024 g, ZnSO4·7H2O 0.476 g, MnSO4·6H2O 0.143 g, KI 0.023 g, CuCl2·2H2O 0.015 g, CoCl2·6H2O 0.14 g, Calcium lactate 16.50 g, Fe-citrate 1 g, filled up to 100 g with cellulose (All ingredients are diluted with α-cellulose to 100 g).

[0060] After the feed was prepared, the contents of five nucleotides in the feed were measured by high performance liquid chromatography with reference to the standard (GB 5413.40-2016), as shown in Table 2. The analysis showed that compared with the normal fish meal feed, the contents of the five nucleotides in the low fish meal feed without added nucleotides all decreased, and the contents of IMP and UMP had a relatively large difference.

[0061] Content of Five Nucleotides in Test Feed in Table 2

[0062]

[0063] Cultivation of Chinese Mitten Crab in Example 2

[0064] The cultivation experiment was carried out at the comprehensive experimental base of Zhejiang Institute of Freshwater Fisheries. The juvenile crabs used were purchased from Chongming Farm (Shanghai, China). Before the start of the experiment, 35 independent 300L aerated water tanks (100cm×50cm×60cm) were divided into 7 groups, randomly numbered, and 1 six-layer crab nest (30cm×30cm) and 2 groups of plastic pipes (each group had 5 plastic pipes, tied with cable ties) were placed as crab shelters. 1400 healthy Chinese mitten crabs with sound appendages and similar weights (0.58±0.01g) were randomly assigned to the water tanks, with 40 crabs in each tank. The cultivation experiment lasted for 8 weeks, and the crabs were fed twice a day until full (8:30, 16:30), and the number of molted crabs was recorded for subsequent statistical analysis. During the whole cultivation period, one-third of the water in each water tank was changed every morning, and specific parameters were maintained, including water temperature of 25±1°C, dissolved oxygen concentration >7.0mg / L, pH value in the range of 7.4 - 7.8, and ammonia nitrogen concentration <0.05mg / L. The uneaten feed in the water tanks after 1h of feeding was collected at the 8th week, dried in an oven and weighed, and the feed intake of juvenile crabs in each water tank within 7 days was recorded for subsequent statistical analysis.

[0065] Among them, the relevant parameters of growth performance and feed utilization were calculated as follows:

[0066] Weight gain rate (%) = 100×(final weight - initial weight) / initial weight;

[0067] Survival rate (%) = 100×number of final crabs / number of initial crabs;

[0068] Feed coefficient = feed intake / (final weight - initial total weight + weight of dead individuals);

[0069] Protein efficiency = (final weight - initial weight) / protein content of the ingested feed;

[0070] Protein deposition rate (%) = 100×(protein content of final crabs - protein content of initial crabs) / protein content of the ingested feed;

[0071] 1-hour food intake (feed weight / crab weight) = [fed feed weight - unfed feed weight × (1 + feed leaching rate)] / final weight. Determination of feed leaching rate: Weigh a portion of the feed and soak it in a water tank (with the same amount of water as the feeding test tank and no crabs). After 1 hour, collect and weigh it again after drying. Feed leaching rate (%) = 100 × (feed weight before soaking - dry weight of feed after soaking) / feed weight before soaking.

[0072] The primer sequences of key genes related to appetite, digestion, antioxidant, and thyroid hormone synthesis and phospholipid metabolism pathways are shown in Table 3.

[0073] Table 3 Gene primer sequences used for real-time fluorescence quantitative PCR

[0074]

[0075]

[0076] All data are expressed as mean ± standard error of the mean (SEM), and statistical analysis of the data was performed using SPSS 20.0 software. One-way ANOVA was used to determine the significant differences between the low-fishmeal groups. If the one-way ANOVA indicated significant differences, the Duncan multiple comparison method was used to evaluate the significance between groups. In addition, a t-test was used to compare the differences between the control group and experimental group 1. When p < 0.05, the differences were considered statistically significant. Values with different superscripts on the same row indicate significant differences, and asterisks indicate the significance level (* indicates p < 0.05; ** indicates 0.001 < p < 0.01; *** indicates p < 0.001).

[0077] Test Example 1 examined the effect of the economical low-fishmeal feed of the present invention on the growth performance of Chinese mitten crabs

[0078] The results are as Figure 1 shown. It can be Figure 1 seen that the economical low-fishmeal feed formula of the present invention is suitable for the cultivation of Chinese mitten crabs. After adding nucleotide functional components to the 15% fishmeal feed, the weight gain rate and survival rate of Chinese mitten crabs were significantly increased, and the protein efficiency and protein deposition rate were significantly increased. In particular, when the addition amount of the nucleotide functional component reached 0.9 g / kg, the weight gain rate of Chinese mitten crabs was equivalent to that of the traditional 35% fishmeal feed. After adding the combination of nucleotides and fructooligosaccharides to experimental group 6, the weight gain rate was significantly higher than that of other groups, indicating that the synergistic effect of the two functional components could further promote growth. In terms of survival rate, protein efficiency, and protein deposition rate, experimental group 6 also showed the best or equivalent level to the control group. This shows that the low-fishmeal formula system of the present invention has successfully achieved cost reduction while maintaining or even exceeding the cultivation effect of traditional high-fishmeal feeds, with obvious economic advantages.

[0079] Test Example 2: Investigate the effects of the economical low-fishmeal feed of the present invention on the feed coefficient, one-hour food intake, and the expression of appetite-related genes of Chinese mitten crabs

[0080] The results are as Figure 2 shown. It can be seen from Figure 2 that the overall formula system of the low-fishmeal feed of the present invention can significantly reduce the feed coefficient and increase the food intake. After adding the nucleotide functional component, the feed coefficient is significantly lower than that of the non-added group, and the one-hour food intake is significantly increased. Especially when the addition amount is 0.9 g / kg, the one-hour food intake of Chinese mitten crabs reaches a relatively high level, comparable to that of traditional high-fishmeal feeds. And the feed coefficient of Experimental Group 6 (the combination of nucleotide and fructooligosaccharide) is the lowest, and the one-hour food intake is the highest, significantly superior to the groups with only nucleotide added.

[0081] In addition, the low-fishmeal feed formula of the present invention plays a role in promoting feeding by regulating the expression of appetite-related genes of Chinese mitten crabs. After adding the nucleotide functional component to the low-fishmeal feed, the expression levels of appetite-promoting genes such as NPYR, GABA2, and Ghrelin are significantly up-regulated, while the expression levels of appetite-inhibiting genes such as LEPR and CCKR are significantly down-regulated. Especially in Experimental Group 6, the expression levels of NPYR and GABA2 genes are significantly increased, while the expression level of CCKR gene is significantly decreased, which is consistent with the result of the increased food intake of Chinese mitten crabs. This indicates that the overall formula system of the low-fishmeal feed of the present invention can effectively solve the problem of poor palatability of low-fishmeal feeds, improve the feeding desire of Chinese mitten crabs for feeds, and thus achieve efficient farming.

[0082] Test Example 3: Investigate the effects of the economical low-fishmeal feed of the present invention on the digestive enzyme activities and related gene expressions of Chinese mitten crabs

[0083] Pre-treat the hepatopancreas samples, that is, accurately weigh the hepatopancreas, then put the hepatopancreas into pre-cooled 0.85% physiological saline for homogenization at a ratio of 1:10, and then centrifuge the extract at 4°C at 1500 g for 15 minutes using a centrifuge (5415R, Eppendorf, Germany). Collect the supernatant and store it at -80°C for subsequent determination of the activities of related digestive and absorption enzymes. Use a commercial kit from Nanjing Jiancheng Bioengineering Institute (China) to test the activities of related digestive and absorption enzymes. According to the manufacturer's protocol, use an enzyme-labeling instrument (BioTek Instruments, Inc., USA) to measure the activities of glutathione peroxidase (A005-1), α-amylase (C016-1), trypsin (A080-2), lipase (A054-2), and γ-glutamyltransferase (C017-2) at 412 nm, 660 nm, 253 nm, 580 nm, and 415 nm respectively. The results are as Figure 3 shown.

[0084] It can be seen from Figure 3 that the overall formula system of the low-fishmeal feed of the present invention can significantly improve the activities of digestive enzymes in the hepatopancreas of Chinese mitten crabs. The low-fishmeal feed can cause a decrease in the activities of amylase, trypsin, lipase and γ-glutamyltransferase in Chinese mitten crabs, while the activities of these enzymes are significantly increased after adding the nucleotide functional component. With the increase of the addition amount of the nucleotide functional component, the amylase activity gradually increases and reaches a relatively high level when the addition amount is 0.9 g / kg. The amylase activity of experimental group 6 (the combination of nucleotide and fructooligosaccharide) is the highest, significantly higher than that of other groups, and even exceeds that of the control group. The trypsin activity reaches a relatively high level in experimental group 4 and shows the best performance in experimental group 6. The lipase activity and γ-glutamyltransferase activity also show a similar trend and reach the highest values in experimental group 6.

[0085] In addition, the overall formula system of the low-fishmeal feed of the present invention can significantly improve the expression levels of genes related to digestion in the hepatopancreas of Chinese mitten crabs. After adding the nucleotide functional component to the low-fishmeal feed, compared with the non-added group, the overall expression levels of genes related to digestion in the hepatopancreas of Chinese mitten crabs increase significantly. The carbonic anhydrase genes (CarB1 and CarB2) have relatively high expression levels in experimental group 4 and the strongest expression in experimental group 6. The expression level of cathepsin L gene (CatL) in experimental group 6 is significantly higher than that of other groups. These genes play important roles in the digestion process of Chinese mitten crabs, and the increase in their expression levels provides a molecular basis for the enhancement of digestive enzyme activity, thereby promoting the digestion and absorption efficiency of nutrients in the feed.

[0086] Test example 4 examines the effect of the economical low-fishmeal feed of the present invention on the expression of antioxidant-related genes in Chinese mitten crabs

[0087] Total RNA was extracted from the preserved thoracic ganglion tissue samples using TRIzol reagent. The RNA was reverse transcribed into cDNA using a reverse transcription kit. The reaction volume was 20 μL, including 10 μL of 2×SYBR Green PCR Master Mix, 0.8 μL of upstream and downstream primers (10 μM), 1 μL of cDNA template and 8.2 μL of nuclease-free water. qRT-PCR detection was performed using a real-time fluorescence quantitative PCR system, and S27 was used as the internal reference gene to compare the relative mRNA expression levels between groups.

[0088] It can be seen from Figure 4It can be seen that the overall formula system of the low-fishmeal feed of the present invention can effectively improve the antioxidant capacity of Chinese mitten crabs. After adding nucleotide functional components to the low-fishmeal feed, the expression level of the antioxidant-related gene Nrf2 was significantly up-regulated. The expression level in experimental group 4 was relatively high, while that in experimental group 6 was the highest, significantly higher than that of other groups. The expression level of the gene Keap1, which inhibits antioxidant function, was significantly down-regulated with the increase of nucleotide addition amount, especially in experimental group 6, where the decrease was the most significant, lower than the control group level. The activity of glutathione peroxidase was also significantly increased after adding nucleotides. The activity levels in experimental group 4 and experimental group 6 were relatively high, comparable to that of the control group. These results indicate that the overall formula system of the low-fishmeal feed of the present invention effectively enhances the antioxidant capacity of Chinese mitten crabs, improves the health status, and thus promotes growth and increases survival rate by regulating the expression of key antioxidant genes and increasing the activity of antioxidant enzymes.

[0089] Test example 5 investigated the effect of the economical low-fishmeal feed of the present invention on the significantly enriched pathways of the hepatopancreas transcriptome of Chinese mitten crabs

[0090] The results are as Figure 5 shown. It can be Figure 5 seen that the overall formula system of the low-fishmeal feed of the present invention can regulate multiple important metabolic pathways in Chinese mitten crabs. Through KEGG pathway enrichment analysis, among the 6 significantly enriched pathways, the glutathione metabolism, thyroid hormone synthesis, phosphoric acid and phosphate metabolism, and glycerophospholipid metabolism pathways were all significantly up-regulated, while the tyrosine metabolism and galactose metabolism pathways were significantly down-regulated. These results are from the differential gene analysis between experimental group 6 and experimental group 1, reflecting the regulatory effect of the synergistic action of nucleotides and fructooligosaccharides on the metabolic pathways of Chinese mitten crabs.

[0091] Among them, the up-regulation of the glutathione metabolism pathway further confirms that the low-fishmeal feed formula of the present invention can enhance the antioxidant capacity of Chinese mitten crabs. The up-regulation of the thyroid hormone synthesis and glycerophospholipid metabolism pathways is related to promoting growth and energy metabolism. This indicates that the overall formula system of the low-fishmeal feed of the present invention can not only improve the feeding rate and digestion and absorption ability of Chinese mitten crabs to the low-fishmeal feed, but also comprehensively regulate the body functions from the metabolic level, promoting growth and development and health status.

[0092] Test example 6 investigated the effect of the economical low-fishmeal feed of the present invention on the expression of key genes in the thyroid hormone synthesis and phospholipid metabolism pathways of Chinese mitten crabs

[0093] The results are as Figure 6 shown. It can be Figure 6It can be seen that the overall formula system of the low-fishmeal feed of the present invention can significantly regulate the expression levels of genes related to thyroid hormone synthesis and phospholipid metabolism in Chinese mitten crabs. In the thyroid hormone synthesis pathway, the TSH receptor (TSHR) and thyroid peroxidase (TPO), as key regulatory factors, showed obvious differences in their expression patterns among the experimental groups. The results showed that in each group with nucleotide added alone (experimental groups 2-5), as the added concentration increased, the expression levels of TSHR and TPO showed an increasing trend, but the ideal effect was not achieved. However, in experimental group 6 (the group with nucleotide and fructooligosaccharide added synergistically), the expression levels of TSHR and TPO were significantly higher than those in other experimental groups (P<0.05), and even exceeded those in the traditional high-fishmeal control group, indicating that nucleotide and fructooligosaccharide have a significant synergistic activation effect on the thyroid hormone synthesis pathway.

[0094] At the same time, the key enzymes cytidine phosphotransferase 1A (PCYT1A) and cytidine phosphotransferase 2 (PCYT2) in the phospholipid metabolism pathway also showed similar expression characteristics. In experimental groups 1-5, the expression levels of these two enzymes gradually increased with the increase in the nucleotide addition amount, but the increase amplitude was limited. The expression levels of the two enzymes in experimental group 6 were significantly higher than those in all other low-fishmeal experimental groups, and were significantly higher than the control group level (P<0.01), further confirming that the synergistic effect of nucleotide and fructooligosaccharide can significantly activate the phospholipid metabolism pathway.

[0095] These gene expression analysis results provide direct evidence for the molecular mechanism of action of the formula system of the present invention. As an important endocrine signal regulating basal metabolism and growth and development, the activation of the thyroid hormone synthesis pathway can directly promote the growth and development and molting process of Chinese mitten crabs. The up-regulation of the expression levels of PCYT1A and PCYT2 significantly enhanced the phospholipid synthesis ability, promoted the renewal and functional optimization of the digestive tract epithelial cell membrane, and thus improved the digestion and absorption efficiency of nutrients such as plant proteins in the feed. The synergistic activation of these two key metabolic pathways provides a molecular-level mechanistic explanation for the optimal growth performance and feed utilization efficiency observed in experimental group 6, and confirms the scientificity and innovation of the formula system of the present invention.

[0096] As can be seen from the above analysis, the low-fishmeal high-efficiency feed formula system of the present invention realizes the synergistic effect of multiple action mechanisms by precisely adding functional components of nucleotides and fructooligosaccharides: First, by regulating appetite-related genes (upregulating NPYR, GABA2, Ghrelin, and downregulating LEPR, CCKR), it directly promotes feeding behavior; second, by optimizing the intestinal microecology with fructooligosaccharides and synergistically enhancing the activity of digestive enzymes and the expression of related genes with nucleotides, it improves the digestion and absorption efficiency of plant protein sources in the feed; third, by jointly activating the glutathione metabolism and thyroid hormone synthesis pathways, it promotes growth and development and improves stress resistance; fourth, by optimizing the metabolism of phosphoric acid and phosphates and glycerophospholipid metabolism, it improves energy utilization efficiency. The combined action of these series of action mechanisms enables the low-fishmeal feed formula system of the present invention to maintain or even exceed the breeding effect of traditional high-fishmeal feeds while significantly reducing the fishmeal usage, achieving a win-win situation in economic benefits and breeding effects.

[0097] Example 2: Economic Benefit Analysis

[0098] To evaluate the economic benefits of the present invention, according to the market raw material prices in 2022, a detailed comparative analysis of the costs of traditional 35% fishmeal feed and the 15% fishmeal feed of the present invention was carried out, and the results are shown in Table 4.

[0099] Table 4 Comparative Analysis of the Costs of Economical Low-Fishmeal Feed and Traditional High-Fishmeal Feed (Calculated According to the Market Prices in 2022)

[0100]

[0101]

[0102] As can be seen from Table 4, by adopting the low-fishmeal feed formula of the present invention, the cost per ton of feed can be saved by 1028.35 yuan, and the cost reduction rate reaches 15.7%. The main savings come from the reduced fishmeal usage. Although the usage of plant protein sources (soybean meal, cottonseed meal) increases and functional components of nucleotides and fructooligosaccharides are added, the overall cost is still significantly reduced. Considering that the annual output of Chinese mitten crabs is about 780,000 tons and the annual feed consumption of the industry is about 1.4 million tons, the full adoption of the present invention's formula can save about 1.44 billion yuan in feed costs for the industry. More importantly, due to the synergistic effect formula of nucleotides and fructooligosaccharides of the present invention reducing the feed conversion ratio (judging from the experimental data, the feed conversion ratio of experimental group 6 is 8.6% lower than that of the control group), the improvement of the actual breeding economic benefits will be more significant.

[0103] It is particularly worth noting that by adding nucleotide and fructooligosaccharide functional components, the present invention not only solves the problems of palatability and nutritional deficiencies of low-fishmeal feeds, but also improves the utilization efficiency of plant proteins in feeds through their synergistic effects. The reduction of feed coefficient and the increase of protein deposition rate are directly translated into the shortening of the culture cycle and the improvement of cost-effectiveness, which have great economic value and environmental benefits in large-scale aquaculture models.

[0104] Without departing from the spirit and scope of the present invention, those skilled in the art can make various changes and modifications to the present invention. For example, the purification method of nucleotides can adopt chromatography, crystallization method or biotransformation method, etc.; nucleotides can exist in the form of free acid, monosodium salt or other edible salts; nucleotides and fructooligosaccharides can be premixed with carriers (such as maltodextrin, corn starch, etc.) to improve their processing and storage stability; the source of fructooligosaccharides can be plant extracts such as Jerusalem artichoke, onion, garlic, etc. or enzymatically synthesized products.

[0105] Although the present invention takes Eriocheir sinensis as the main research object, the overall formula system of this low-fishmeal feed is also expected to be applied to low-fishmeal feeds for other crustacean aquaculture species, such as Litopenaeus vannamei, Procambarus clarkii, etc., except that the addition amount of nucleotide functional components may need to be adjusted according to the specific species characteristics. In addition, the low-fishmeal formula system and the method for optimizing the addition amount described in the present invention can also provide technical references for the development of low-fishmeal feeds for other aquaculture species.

[0106] In summary, the present invention constructs an overall formula system with multiple action mechanisms by synergistically adding nucleotide and fructooligosaccharide functional components to a feed formula with a fishmeal content reduced to 15%, while significantly reducing the feed cost and ensuring or improving the culture effect. This formula system acts through multiple synergistic mechanisms to improve the feeding rate of Eriocheir sinensis to low-fishmeal feeds, promote growth and development, reduce the feed coefficient, improve the protein deposition efficiency, enhance antioxidant and immune capabilities, provide an economical, efficient and technologically advanced feed solution for the Eriocheir sinensis aquaculture industry, and promote the sustainable development of the industry.

[0107] In this specification, the specific features, structures, materials or characteristics described can be combined in a suitable manner in any one or more embodiments. Those skilled in the art can combine and combine the different embodiments or examples described in the specification and the features of different embodiments. These changes and modifications should all be regarded as falling within the protection scope of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Application of nucleotides and fructooligosaccharides in synergistically activating thyroid hormone synthesis and glycerophospholipid metabolism pathways in aquatic products.

2. Application of nucleotides and fructooligosaccharides in synergistically enhancing the activities of digestive and absorption enzymes in aquatic products.

3. Application of nucleotides and fructooligosaccharides in synergistically enhancing the antioxidant and immune capabilities of aquatic products.

4. Application of nucleotides and fructooligosaccharides in synergistically enhancing the feeding rate of aquatic products, promoting growth, and reducing the feed coefficient.

5. The application according to any one of claims 1-4, characterized in that, The nucleotides include adenylic acid, guanylic acid, inosinic acid, uridylic acid, and cytidylic acid, and the mass ratio of adenylic acid, guanylic acid, inosinic acid, uridylic acid, and cytidylic acid is 1 - 3:1 - 3:1 - 3:1 - 3:1 - 3; the fructooligosaccharide is fructan FOS.

6. A low-fishmeal aquatic feed, characterized in that, It includes nucleotides and fructooligosaccharides, the addition amount of nucleotides in the feed is 0.6 - 1.2 g / kg, and the addition amount of fructooligosaccharides in the feed is 0.1 - 0.3%.

7. The low-fishmeal aquatic feed according to claim 6, wherein By weight, it includes the following components: 120 - 180 parts of fish meal, 200 - 250 parts of soybean meal, 200 - 250 parts of cottonseed meal, 10 - 30 parts of fish oil, 10 - 30 parts of soybean oil, 5 - 15 parts of lecithin, 3 - 8 parts of cholesterol, 3 - 8 parts of choline chloride, 150 - 200 parts of pregelatinized starch, 30 - 50 parts of compound vitamins, 10 - 30 parts of compound minerals, 10 - 30 parts of sodium alginate, 5 - 15 parts of calcium dihydrogen phosphate, 6 - 12 parts of nucleotides, and 10 - 30 parts of fructooligosaccharides.

8. The low-fishmeal aquatic feed according to claim 7, wherein By weight, it includes the following components: 150 parts of fish meal, 240 parts of soybean meal, 240 parts of cottonseed meal, 20 parts of fish oil, 20 parts of soybean oil, 10 parts of lecithin, 5 parts of cholesterol, 5 parts of choline chloride, 180 parts of pregelatinized starch, 40 parts of compound vitamins, 20 parts of compound minerals, 20 parts of sodium alginate, 10 parts of calcium dihydrogen phosphate, 8.5 parts of nucleotides, and 20 parts of fructooligosaccharides.

9. The preparation method of the low-fishmeal aquatic feed according to claim 7 or 8, characterized in that It includes the following steps: (1) Weigh the remaining feed raw materials except fish oil and soybean oil according to the formula ratio, crush them with a pulverizer, sieve them, and mix them evenly. (2) Add fish oil, soybean oil, and an appropriate amount of water to the powder in step (1), stir into a dough, and extrude it into pellets. (3) Air-dry the pellets under natural conditions until the moisture content is 6 - 15%. (6) Seal and package, and store frozen.

10. The application according to any one of claims 1-4, characterized in that, The aquatic product is a crustacean aquaculture organism.

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