Method for improving phosphorus utilization of lateolabrax japonicus and application of stevioside of lateolabrax japonicus

Abstract

The invention discloses a method for improving phosphorus utilization of lateolabrax japonicus and application of stevioside of the lateolabrax japonicus. The phosphorus utilization of lateolabrax japonicus is improved by adjusting a PI3K / AKT pathway. The method is simple to operate, and raw materials are easy to obtain, safe and harmless. Meanwhile, a PI3K / AKT pathway can be effectively activated, insulin resistance and lipid metabolism of lateolabrax japonicus are improved, so that the phosphorus utilization rate of lateolabrax japonicus is increased, and the growth performance of lateolabrax japonicus is improved.

Description

Technical Field

[0001] The invention relates to the field of feed, and in particular to a method for improving phosphorus utilization of Lateolabrax japonicus and application of stevioside thereof. Background Art

[0002] Lateolabrax maculatus, also known as Chinese seabass, belongs to the order Perciformes, family Euperciidae, and genus Lateolabrax in taxonomy. Lateolabrax muscle is rich in essential amino acids, polyunsaturated fatty acids, vitamins, and trace elements that are beneficial to human health. In the past two decades, the production of Lateolabrax aquaculture has increased year by year, reaching 246,900 tons in 2023, making it an important economic fish in my country.

[0003] Phosphorus is considered to be a very important nutrient and an important nutrient in aquatic feed. It directly promotes the formation of bones and is also an important component of the nucleus and cell membrane of animal cells. In freshwater fish mainly ingest phosphorus through feed, but its utilization rate is only 50%-70%. In recent years, with the increase in breeding density and the expansion of breeding scale, as well as the excessive feeding of feed by farmers in pursuit of efficiency, the problem of phosphorus loss in the breeding process of sea bass has become increasingly serious. A large amount of phosphorus has entered the water body, and the phosphorus element in the tail water is precisely one of the most difficult elements to deal with, resulting in frequent eutrophication of breeding waters in various places, which seriously hinders the healthy and sustainable development of sea bass breeding. In order to change this situation, low-phosphorus feed came into being. However, under low-phosphorus conditions, sea bass generally show decreased growth performance and lipid metabolism disorders.

[0004] Therefore, there is an urgent need to provide a method for improving phosphorus utilization of Lateolabrax japonicus with low cost, high feasibility and good effect. Summary of the invention

[0005] The object of the present invention is to provide a method for improving phosphorus utilization of Lateolabrax japonicus and application of stevioside thereof.

[0006] To achieve the above object, the present invention provides a method for improving phosphorus utilization of Lateolabrax japonicus by regulating the PI3K / AKT pathway.

[0007] Furthermore, the method described is to add steviol glycosides.

[0008] Furthermore, the added amount of steviol glycoside is 150 mg / kg to 450 mg / kg.

[0009] Furthermore, the added amount of steviol glycoside is 450 mg / kg.

[0010] Furthermore, the regulation of the PI3K / AKT pathway refers to regulating the expression of the PI3K / AKT pathway genes insr, and / or irs1, and / or irs2, and / or pi3k.

[0011] Furthermore, the improving phosphorus utilization of striped seabass means that the growth performance of striped seabass under low phosphorus levels reaches the growth performance of striped seabass under appropriate phosphorus levels; wherein the low phosphorus level means that the phosphorus content does not exceed 0.38%, and the appropriate phosphorus level means that the phosphorus content is 0.76±0.1%.

[0012] The present invention also protects the use of stevioside in preparing low-phosphorus feed for feeding sea bass; wherein the low-phosphorus feed refers to a feed with a phosphorus content not exceeding 0.38%.

[0013] The inventors found through transcriptomics that the PI3K / AKT pathway of Lateolabrax fed with low-phosphorus diets was significantly inhibited compared with Lateolabrax fed with normal-phosphorus diets. As an insulin-mediated pathway, the PI3K / AKT pathway is crucial for regulating glucose homeostasis in the insulin signaling system and is closely related to glucose lipid metabolism and insulin resistance. In previous work, metabolomics showed that the content of glycosides in the serum of Lateolabrax fed with low-phosphorus diets was significantly reduced compared with Lateolabrax fed with normal-phosphorus diets.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] 1) The present invention provides a method for improving phosphorus utilization of Lateolabrax japonicus. The method is simple to operate, and the raw materials are readily available and safe and harmless.

[0016] 2) This method can effectively activate the PI3K / AKT pathway, improve the insulin resistance and lipid metabolism of the sea bass, thereby increasing the phosphorus utilization rate and improving the growth performance of the sea bass. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a graph showing the effect of adding different levels of stevioside on the growth performance of sea bass.

[0018] Figure 2 This is a graph showing the effects of adding different levels of stevioside on the body shape indicators of sea bass.

[0019] Figure 3 This is a graph showing the effect of adding different levels of stevioside on serum total triglycerides in seabass.

[0020] Figure 4 This is a graph showing the effect of adding different levels of stevioside on the expression of genes related to the PI3K / AKT pathway in Lateolabrax.

[0021] Figure 5 This is a graph showing the effect of adding different levels of stevioside on the expression of genes related to lipid metabolism in sea bass. DETAILED DESCRIPTION

[0022] Embodiments of the present invention are described in detail below, and the examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals represent the same or similar elements or elements with the same or similar functions from beginning to end. The embodiments described below by reference to the accompanying drawings are exemplary, are intended to be used to explain the present invention, and are not to be construed as limitations of the present invention. Those who do not indicate specific techniques or conditions in the embodiments are carried out according to the techniques or conditions described in the documents in this area or according to the product specification. Those who do not indicate the manufacturer of reagents or instruments used are all conventional products that can be obtained commercially.

[0023] 1. Feed preparation

[0024] According to the nutritional needs of striped seabass, five kinds of feeds with crude protein of about 46% and crude fat of 12.44% were prepared: normal phosphorus positive control group NP (normal phosphorus 0.76%), low phosphorus negative control group LP (low phosphorus is 0.38%) and three experimental group feeds 150STV, 300STV and 450STV consisting of three different levels (150mg / kg, 300mg / kg and 450mg / kg) of stevioside added to the LP basic feed.

[0025] 2. Experiment on breeding of Lateolabrax japonicus

[0026] The culture experiment of L. japonicus was conducted in the seawater test field of Longzhou Pond in Jimei, Xiamen. The experimental fry were purchased from a farm in Zhangzhou. The fry were placed in a 1000L culture tank and fed with commercial feed for 2 weeks to adapt to the environment. Then 450 L. japonicus with strong physique and uniform size (1.28±0.02g) were selected and randomly distributed to 15 500L freshwater culture barrels. The fry were fed with 6 kinds of experimental feeds, with 3 replicates for each feed. During the 8-week culture period, the fry were fed twice a day (8:00 and 17:00) and the dirt in the tank was siphoned out after feeding. During the culture period, the water temperature was 27±2℃, the dissolved oxygen was ≥6.0mg / L, the ammonia nitrogen was <0.1mg / L, and the pH was 7.0-7.5.

[0027] 3. Sample collection:

[0028] After the experiment, the fish were deprived of feed for 24 hours to ensure that the digestive tract was empty, and they were weighed to calculate growth parameters. Before sampling, 15 fish were randomly selected from each tank and injected with 60 mg / L of MS-222 (Sigma, Ronkonkoma, NY, USA) to anesthetize the selected fish. Three randomly selected fish were taken from each tank and placed in a ziplock bag and stored at -20°C for whole body composition analysis; for the remaining 12 fish, blood samples were taken from the tail vein of the fish using a 27-gauge needle and a 1 ml syringe and coagulated overnight at 4°C. Serum was collected after centrifugation (3500 rpm, 10 minutes, 4°C) and stored at -80°C until use. After collecting blood, the visceral mass of the fish was weighed, and then the liver tissue and abdominal fat tissue were separated and weighed separately for the subsequent calculation of liver-to-body ratio, viscera-to-body ratio, and abdominal fat rate. After weighing, the liver tissue and abdominal fat tissue were placed in 2.0 mL cryovials and immediately frozen in liquid nitrogen, and then stored in a -80°C refrigerator for subsequent analysis.

[0029] 4. Index determination

[0030] 1) Determination of the overall approximate composition:

[0031] The experimental diets and whole samples were analyzed for moisture, crude protein, crude lipids, and ash content using standard procedures (AOAC, 2002).

[0032] 2) Determination of biochemical indicators:

[0033] Total triglycerides (TG) in serum were determined using a commercial kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

[0034] 3) Real-time fluorescence quantitative PCR (qPCR) determination of PI3K / AKT pathway-related genes and lipid metabolism-related genes in the liver:

[0035] Total RNA was extracted using a commercial kit (RC101–01, Vazyme Biotech Co., Ltd., Nanjing, China) according to the instructions and dissolved in RNase-free water. RNA purity and integrity were then tested. cDNA was synthesized using a commercial kit (R211–01, Vazyme Biotech Co., Ltd., Nanjing, China) according to the instructions. Afterwards, cDNA was subjected to real-time quantitative PCR to detect specific mRNA levels as described previously. The relative expression of genes was determined by 2 -ΔΔCt method.

[0036] 5. Data statistics and analysis:

[0037] The experimental data were analyzed by one-way analysis of variance using SPSS26.0 statistical software, and Tukey's test was used for multiple comparisons of the experimental groups. The significant difference level was P<0.05. All experimental data were expressed as mean±standard error (Mean±SEM).

[0038] 6. Results

[0039] 1) Effects of adding different levels of stevioside on the growth performance of Lateolabrax japonicus

[0040] like Figure 1 As shown in the data, with the increase of steviol glycoside levels in the feed, the final body weight (FBW), weight gain rate (WGR) and specific growth rate (SGR) of the seabass in the 300STV group and 450STV group were significantly increased compared with the LP group (P<0.05), and there was no significant difference with the NP group (P>0.05).

[0041] Weight gain rate (%) = (final body weight (g) - initial body weight (g)) / initial body weight (g) * 100%.

[0042] Specific growth rate (%)==(ln(final body weight (g))-ln(initial body weight (g))) / number of culture days*100%.

[0043] 2) Effects of adding different levels of stevioside on the body shape of Lateolabrax japonicus

[0044] like Figure 2 As shown in the data, compared with the LP group, the 300STV group significantly reduced the liver-to-body ratio (HSI), visceral-to-body ratio (VSI), and abdominal fat percentage (AFP) (P<0.05), and there was no significant difference compared with the NP group (P>0.05); in terms of fatness (CF), no significant difference was found among the groups (P>0.05).

[0045] Liver to body ratio (%) = (liver weight (g) / fish body weight (g)) * 100%

[0046] Viscera to body ratio (%) = (viscera mass weight (g) / fish body weight (g)) * 100%

[0047] Abdominal fat rate (%) = (abdominal fat weight (g) / fish body weight (g)) * 100%.

[0048] 3) Effects of adding different levels of stevioside on the whole body and muscle composition of Lateolabrax japonicus, as well as serum total triglycerides

[0049] The results are shown in Table 1 and Figure 3 .

[0050] Table 1 Effects of adding different phosphorus sources to low-phosphorus feed on the overall and muscle composition of Lateolabrax japonicus (dry matter, %)

[0051] All NP LP 150STV 300STV 450STV Crude Protein <![CDATA[48.87±0.35 a ]]> <![CDATA[46.07±0.58 c ]]> <![CDATA[46.90±0.44 bc ]]> <![CDATA[48.30±0.15 ab ]]> <![CDATA[49.43±0.24 a ]]> Crude fat <![CDATA[31.90±1.00 b ]]> <![CDATA[40.22±1.71 a ]]> <![CDATA[35.46±0.76 ab ]]> <![CDATA[34.94±0.46 b ]]> <![CDATA[33.16±0.98 b <!-- 3 -->]]> Coarse ash <![CDATA[12.31±0.08 a ]]> <![CDATA[9.24±0.20 b ]]> <![CDATA[9.37±0.06 b ]]> <![CDATA[8.99±0.07 b ]]> <![CDATA[8.96±0.10 b ]]> muscle Crude Protein <![CDATA[89.47±0.03 ab ]]> <![CDATA[88.43±0.29 c ]]> <![CDATA[88.87±0.15 bc ]]> <![CDATA[89.17±0.24 abc ]]> <![CDATA[89.90±0.55 a ]]> Crude fat 5.12±0.21 5.89±0.19 5.05±0.18 5.06±0.11 5.08±0.23 Coarse ash 5.33±0.14 5.48±0.18 4.98±0.43 5.46±0.12 5.32±0.06

[0052] As shown in Table 1, compared with the LP group, the 300STV group and the 450STV group significantly increased the crude protein content of the whole body (P<0.05), and significantly reduced the crude fat content of the whole body (P<0.05), and there was no significant difference with normal phosphorus (P>0.05); compared with the NP group, the crude ash content of the whole body in the LP group was significantly reduced (P<0.05), while no significant changes were found in the 150STV group, the 300STV group, and the 450STV group compared with the LP group. (P>0.05); in addition, compared with the LP group, the 450STV group significantly increased the crude protein content in the muscle (P<0.05), and there was no significant difference with normal phosphorus (P>0.05); in terms of muscle crude fat, compared with the LP group, although the 150STV group, 300STV group and 450STV group did not show significant changes (P>0.05), there was a downward trend; there was no significant difference in the crude ash content in the muscle among the groups (P>0.05).

[0053] like Figure 3 As shown, compared with the LP group, the serum triglyceride (TG) in the 300STV group and the 450STV group was significantly reduced (P<0.05), and there was no significant difference with the NP group (P>0.05).

[0054] 4) Effects of adding different levels of stevioside on the expression of genes related to the PI3K / AKT pathway in Lateolabrax japonicus

[0055] like Figure 4 As shown, compared with the LP group, the expression of irs1 gene and pi3k gene in the 450STV group was significantly upregulated (P<0.05), and there was no significant difference between it and the NP group (P>0.05); compared with the LP group, the expression of insr gene in the 300STV group was significantly upregulated (P<0.05), and there was no significant difference between it and the NP group (P>0.05); there was no significant difference in the expression of irs2 gene among the groups (P>0.05).

[0056] 5) Effects of adding different levels of phospholipids on the expression of genes related to lipid metabolism in Lateolabrax japonicus

[0057] like Figure 5As shown, compared with the LP group, the expressions of lipolysis-related genes atgl, hsl and cpt1 in the 300STV group and the 450STV group were significantly upregulated (P<0.05), and had no significant difference with the NP group (P>0.05); compared with the LP group, the expressions of transcription factor srebp1 and lipid synthesis-related genes acc1 and acc2 in the three stevioside-added groups were significantly downregulated (P<0.05), and had no significant difference with the NP group (P>0.05); the expression of lipid synthesis-related gene fas in the 450STV group was significantly downregulated compared with the LP group (P<0.05), and had no significant difference with the NP group (P>0.05); the expression of transcription factor pparγ in the 300STV group and the 450STV group was significantly downregulated compared with the LP group (P<0.05), and had no significant difference with the NP group (P>0.05).

[0058] It can be seen that the method provided by the present invention of adding steviol glycosides to low-phosphorus feed activates the PI3K / AKT pathway of the japonica seabass on the one hand, thereby regulating the lipid metabolism of the japonica seabass and improving the growth performance of the japonica seabass, and on the other hand, reduces the total phosphorus content in the feed and improves the phosphorus utilization rate of the japonica seabass. The method is easy to operate, has high feasibility, can reduce the cost of japonica seabass farming, and reduces the environmental pollution caused by the loss of phosphorus in high-phosphorus feed, and is suitable for practical use.

[0059] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and intent of the present invention.

Claims

1. A method for improving phosphorus utilization in Lateolabrax japonicus by regulating the PI3K / AKT pathway.

2. The method according to claim 1, characterized in that The method described is to add steviol glycosides.

3. The method according to claim 2, characterized in that The added amount of stevioside is 150mg / kg to 450mg / kg.

4. The method according to claim 3, characterized in that The added amount of stevioside was 450 mg / kg.

5. The method according to claim 1, characterized in that The regulating PI3K / AKT pathway refers to regulating the expression of PI3K / AKT pathway genes insr, and / or irs1, and / or irs2, and / or pi3k.

6. The method according to claim 1, characterized in that Improving the phosphorus utilization of the striped seabass means that the growth performance of the striped seabass under low phosphorus levels reaches the growth performance of the striped seabass under appropriate phosphorus levels; wherein the low phosphorus level means that the phosphorus content does not exceed 0.38%, and the appropriate phosphorus level means that the phosphorus content is 0.76±0.1%.

7. Use of stevioside in preparing low-phosphorus feed for feeding Lateolabrax japonicus; wherein the low-phosphorus feed refers to a feed with a phosphorus content not exceeding 0.38%.

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