Application of sea cucumber peptide to preparation of feed for relieving high-temperature heat stress of lateolabrax japonicus

Abstract

The invention discloses application of sea cucumber peptide in preparation of feed for relieving high-temperature heat stress of lateolabrax japonicus. The sea cucumber peptide added into the feed can improve the growth performance of the lateolabrax japonicus; the antioxidant capacity of serum of lateolabrax japonicus is improved; the non-specific immunocompetence of serum of lateolabrax japonicus is improved; the intestinal digestive enzyme activity of lateolabrax japonicus is improved; energy metabolism related indexes of lateolabrax japonicus are improved; the organelle stability of the lateolabrax japonicus liver cells is improved.

Description

Technical Field

[0001] This invention relates to the field of fish feed, and more particularly to the use of sea cucumber peptides in the preparation of feed to alleviate high-temperature heat stress in sea bass. Background Technology

[0002] Spotted sea bass Lateolabrax maculatus The Chinese spotted sea bass (Perciformes), also known as the spotted perch, belongs to the order Perciformes, family Perciformes, and genus Perciformes in taxonomy. Its muscle is rich in essential high-quality amino acids, polyunsaturated fatty acids, vitamins, and trace elements beneficial to human health. Over the past two decades, the production of farmed Chinese spotted sea bass has increased year by year, reaching 246,900 tons in 2023, making it an important economic fish species in my country. Widely farmed in southern China, the optimal water temperature range for its growth as a temperate fish is 16-28℃. However, with global warming and summer water temperatures in southern China frequently exceeding 30℃, Chinese spotted sea bass are kept in high-temperature farming environments for extended periods. This leads to a decline in their antioxidant and immune functions, affecting their growth performance and even causing diseases, which seriously hinders the healthy and sustainable development of the Chinese spotted sea bass farming industry.

[0003] To address this issue, there is an urgent need for a low-cost, highly feasible, effective feed additive that can effectively alleviate heat stress in sea bass. Heat stress is a major factor leading to oxidative stress, decreased immune function, gut microbiota imbalance, mitochondrial dysfunction, and abnormal energy metabolism in sea bass, undoubtedly impacting their growth. Using feed additives is one of the more feasible methods to alleviate this stress. Current research indicates that sea cucumber byproducts account for approximately 50% of sea cucumber biomass, are abundant, and contain nutrients similar to those found in the body wall. During processing, these byproducts are often discarded, resulting in significant resource waste and environmental pollution. Sea cucumber byproducts can be processed into sea cucumber peptides with specific physiological functions through enzymatic hydrolysis and other methods. Sea cucumber peptides are widely used in the pharmaceutical, anti-aging cosmetic, and food processing industries. Furthermore, numerous studies have confirmed that sea cucumber peptides possess advantages such as antioxidant properties, improved gut microbiota, blood sugar reduction, enhanced immunity, improved blood lipids, and improved mitochondrial function. Therefore, adding sea cucumber peptides could be a potentially effective way to alleviate the negative effects of high-temperature heat stress on sea bass and improve its growth performance by enhancing immunity, reducing oxidative stress, and improving mitochondrial function. Summary of the Invention

[0004] The purpose of this invention is to provide a feed additive, sea cucumber peptide, to alleviate the high-temperature heat stress of sea bass.

[0005] To achieve the above objectives, the present invention provides the use of sea cucumber peptides in the preparation of feed to alleviate high-temperature heat stress in sea bass.

[0006] Further, the added amount of the sea cucumber peptide is 250-2000 mg sea cucumber peptide per 100 g feed.

[0007] Further, the added amount of the sea cucumber peptide is 250-1500 mg sea cucumber peptide per 100 g feed.

[0008] Further, the added amount of the sea cucumber peptide is 250-1000 mg sea cucumber peptide per 100 g feed.

[0009] Further, the added amount of the sea cucumber peptide is 500-1000 mg sea cucumber peptide per 100 g feed.

[0010] Further, the added amount of the sea cucumber peptide is 500 mg sea cucumber peptide per 100 g feed.

[0011] Further, the high-temperature heat stress refers to the heat stress of Epinephelus akaara caused by breeding at a temperature higher than the suitable breeding temperature. Generally, the suitable breeding temperature of Epinephelus akaara is 16-28℃, and Epinephelus akaara bred at a temperature higher than this temperature (such as, but not limited to, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, and the like) will cause high-temperature heat stress. The high-temperature heat stress in the embodiment of the present application is the heat stress of Epinephelus akaara caused by breeding at a high temperature of 33℃ for 56 days.

[0012] Further, the alleviation of the high-temperature heat stress of Epinephelus akaara is evaluated by at least one of the following indexes: improving the growth performance of Epinephelus akaara; improving the antioxidant capacity of the serum of Epinephelus akaara; improving the non-specific immune capacity of the serum of Epinephelus akaara; improving the digestive enzyme activity in the intestinal tract of Epinephelus akaara; improving the energy metabolism related indexes of Epinephelus akaara; improving the stability of organelles of hepatocytes of Epinephelus akaara.

[0013] Further, the improvement of the growth performance of Epinephelus akaara refers to improving the average weight of Epinephelus akaara, and / or improving the weight gain rate, and / or improving the feed efficiency; the improvement of the antioxidant capacity of the serum of Epinephelus akaara refers to reducing the content of malondialdehyde in the serum, and / or increasing the content of superoxide dismutase in the serum, and / or increasing the content of catalase in the serum, and / or increasing the total antioxidant capacity, and / or increasing the content of glutathione peroxidase in the serum; the improvement of the non-specific immune capacity of the serum of Epinephelus akaara refers to increasing the content of alkaline phosphatase in the serum of Epinephelus akaara, and / or increasing the content of acid phosphatase, and / or increasing the content of fish complement protein 3, and / or increasing the content of fish complement protein 4, and / or increasing the total protein content; the improvement of the digestive enzyme activity in the intestinal tract of Epinephelus akaara refers to increasing the activity of trypsin and amylase in the intestinal tract of Epinephelus akaara; The improved energy metabolism related indexes of the Lateolabrax japonicus refer to reduced insulin content, and / or reduced triglyceride content, and / or reduced lactic acid, and / or reduced total cholesterol content, and / or increased high-density lipoprotein. The improved organelle stability of the hepatocytes of the Lateolabrax japonicus refers to relatively reduced number of lipid droplets observed in transmission electron microscopy in the hepatocytes of the Lateolabrax japonicus, and relieved endoplasmic reticulum swelling.

[0014] On one hand, the sea cucumber peptide added in the feed can alleviate the negative effects of high temperature stress on the Lateolabrax japonicus, so as to regulate the feed efficiency of the Lateolabrax japonicus, and improve the growth performance of the Lateolabrax japonicus, and on the other hand, the sea cucumber peptide added in the feed can improve the organelle stability of the hepatocytes of the Lateolabrax japonicus.

[0015] The embodiment of the present application shows that the sea cucumber peptide added in the feed can not only improve the feed utilization rate, antioxidant capacity and non-specific immune capacity of the Lateolabrax japonicus, but also has the effects of improving the activity of some digestive enzymes in the intestine of the Lateolabrax japonicus and maintaining the stability of organelles. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is a diagram of the effects of different levels of sea cucumber peptides added in the feed on the growth performance of the Lateolabrax japonicus.

[0017] Figure 2 is a diagram of the effects of different levels of sea cucumber peptides added in the feed on the antioxidant indexes in the serum of the Lateolabrax japonicus.

[0018] Figure 3 is a diagram of the effects of different levels of sea cucumber peptides added in the feed on the non-specific immune indexes in the serum of the Lateolabrax japonicus.

[0019] Figure 4 is a diagram of the effects of different levels of sea cucumber peptides added in the feed on the intestinal health related indexes of the Lateolabrax japonicus.

[0020] Figure 5 is a diagram of the effects of different levels of sea cucumber peptides added in the feed on the energy metabolism related indexes in the serum of the Lateolabrax japonicus.

[0021] Figure 6 is an electron microscope ultrastructure diagram of the hepatocytes of the Lateolabrax japonicus in the S500 group and the S0 group.

[0022] The abbreviations S0, S250, S500, S1000, S2000 in the drawings refer to 0, 250 mg / 100 g, 500 mg / 100 g, 1000 mg / 100 g, and 2000 mg / 100 g of sea cucumber peptide added to the basic formula feed of the control group, respectively. DETAILED DESCRIPTION

[0023] Embodiments of the present application are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout the drawings denote the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to explain the present application, and cannot be understood as limiting the present application. If a specific technique or condition is not specified in the embodiments, the technique or condition described in the literature in the art or according to the product manual is used. If the manufacturer of the reagent or instrument is not specified, it is a conventional product that can be commercially available.

[0024] The sea cucumber peptide used in the following examples can be commercially available, such as that purchased from Lain (Xiamen) Molecular Biotechnology Co., Ltd.

[0025] Example 1 1. Feed preparation Test feed preparation: According to the nutritional requirements of Epinephelus akaara, fish meal, chicken meal, soybean meal, and casein were used as the main protein source, and fish oil and soybean oil were used as the main fat source to prepare a basic feed with a crude protein content of 43.40% and a crude fat content of 12.0%. Five sea cucumber peptide addition groups (S0, S250, S500, S1000, S2000) were designed. Five formula feeds with different levels of sea cucumber peptide were prepared in the basic feed. Each group correspondingly added 0 mg / 100 g (i.e. S0), 250 mg / 100 g (i.e. S250), 500 mg / 100 g (i.e. S500), 1000 mg / 100 g (i.e. S1000), and 2000 mg / 100 g (i.e. S2000) of sea cucumber peptide.

[0026] 2. Epinephelus akaara breeding test The trial was conducted at Zhongshan Tongwei Special Aquaculture Research Base, and the fish were purchased from the same place. The fish were acclimated to the environment by placing them in a 1000 L tank and feeding them commercial feed for 2 weeks. Then, 225 fish with a healthy body and uniform size (140 ± 0.03 g) were selected and randomly divided into 15 500 L freshwater tanks. Each tank was fed with one of the five experimental feeds, with three replicates for each feed. During the 8-week trial, the fish were fed twice a day (6:00 and 18:00) until they were full, and the tank was siphoned to remove waste. During the trial, a recirculating water system was used to control the water temperature, which was maintained at 33 ± 1°C, with dissolved oxygen ≥ 6.9 mg / L, ammonia nitrogen < 0.1 mg / L, and pH 7.0-7.5.

[0027] Sample collection: After the trial, the fish were starved for 24 hours to ensure that the digestive tract was empty, and then weighed to calculate growth parameters. Before sampling, 12 fish were randomly selected from each tank and immersed in MS-222 (Sigma, Ronkonkoma, NY, USA) for anesthesia. The remaining 3 fish were placed in a self-sealing bag and stored at -20°C for whole body composition analysis. For the 12 live fish, blood samples were taken from the tail vein using a 2-mL sterile disposable syringe, and allowed to clot overnight at 4°C. After centrifugation (3500 rpm, 10 min, 4°C), the serum was collected and stored at -80°C until use. After collecting the blood, the internal organs were weighed, and the liver and abdominal fat tissues were separated and weighed separately for subsequent calculation of liver-somatic index, visceral-somatic index, and abdominal fat rate. After weighing, the liver and intestinal tissues were placed in 2.0 mL cryotubes and immediately frozen in liquid nitrogen, then stored in a -80°C freezer for subsequent analysis.

[0028] Determination of whole body proximate composition: The moisture, crude protein, crude lipid, and ash content of the experimental diets and whole body samples were analyzed by standard procedures (AOAC, 2002).

[0029] Determination of biochemical indicators: SOD (superoxide dismutase), T-AOC (total antioxidant capacity), CAT (catalase), GSH-Px (glutathione peroxidase), MDA (malondialdehyde), ACP (acid phosphatase), AKP (alkaline phosphatase), LD (lactic acid), TP (total protein), TG (triglyceride), TC (total cholesterol), LDLC (low-density lipoprotein), HDLC (high-density lipoprotein) in serum were determined using commercial kits (Jiancheng Bioengineering Institute, Nanjing, China). INS (insulin), IgM (fish immunoglobulin M) were determined using commercial kits (Jiangsu Zimei Industry Co., Ltd.). DAO (diamine oxidase), C3 (fish complement protein 3), C4 (fish complement protein 4), LZM (lysozyme) were determined using commercial kits (Yancheng Junxing Biological Technology Co., Ltd.).

[0030] Determination of intestinal enzyme activity indicators: AMS (amylase), TPS (trypsin), and LPS (lipase) in the intestine were determined using commercial kits (Jiancheng Bioengineering Institute, Nanjing, China).

[0031] Observation of hepatocyte ultrastructure: Transmission electron microscope samples were prepared and photographed by Biorun Biological Technology Co., Ltd. (Wuhan, China), with the following brief steps: Fresh liver tissue was cut into small pieces about 1 mm 3 in size and immediately immersed in 2.5% glutaraldehyde for fixation. After 48 hours of fixation, the samples were rinsed three times with 0.1 mol·L -1 phosphate buffer solution (PBS, pH 7.4) for 15 minutes each time. After rinsing, the samples were fixed with 1% osmium tetroxide at room temperature in the dark for 2 hours, and then rinsed again with 0.1 mol·L -1 phosphate buffer solution (PBS, pH 7.4) three times. After rinsing, dehydration was performed in gradient alcohol (50%, 70%, 80%, 90%, 95%, 100%, 100%) and 100% acetone. After dehydration, the samples were infiltrated and embedded with embedding agent. The embedding plate was removed from the resin block after being treated in a 60°C oven for 48 hours. Then, ultrathin sections were cut using a Leica UC7 type sectioning machine, and stained with 2% uranyl acetate and 2.6% lead citrate. After the sample preparation was completed, the samples were observed and photographed using a Hitachi HT7700 electron microscope.

[0032] Statistical analysis: The experimental data were analyzed using SPSS 26.0 statistical software. Tukey's test was used for multiple comparisons between experimental groups, with a significant difference level of P <0.05. All experimental data were expressed as mean ± standard error (Mean ± S.E.M).

[0033] 3. Results 1) Effects of adding different levels of sea cucumber peptides to feed on the growth performance of spotted sea bass Figure 1 This graph shows the effect of adding different levels of sea cucumber peptides to the feed on the growth performance of spotted sea bass. Figure 1 As shown, when 0.5% sea cucumber peptide was added to the feed (i.e., group S500), the growth performance of spotted sea bass was significantly increased compared to group S0. P <0.05). Growth performance refers to final average weight, weight gain rate, and feed efficiency. The S500 group showed significantly higher values ​​for all three growth performance indicators compared to the S0 group. P <0.05).

[0034] Growth performance indicators include: final average weight (FBW), weight gain rate (WGR), feed efficiency (FE), average feed intake (AFI), and total feed intake (TAI). The calculation formulas are as follows: Weight gain rate (WGR, %) = (FBW-IBW) / IBW×100; Feed efficiency (FE, %) = (FBW - IBW) / AFI × 100; Average food intake (AFI, g) = TAI / d In the formula: IBW and FBW are the average initial weight (g) and average final weight (g) of the experimental fish, respectively; d is the number of experimental days; AFI is the average daily feed intake (g); and TAI is the total feed intake (g) over 56 days of culture.

[0035] A higher weight gain rate indicates a faster growth rate for the spotted sea bass, and higher feed efficiency indicates a stronger ability to utilize nutrients. It can be seen that adding sea cucumber peptides to the feed improves the growth performance of the spotted sea bass.

[0036] 2) Effects of different levels of sea cucumber peptides added to feed on serum antioxidant indices of spotted sea bass Antioxidant indicators refer to T-AOC, SOD, MDA, GSH-Px, and CAT. Figure 2 This graph shows the effect of adding different levels of sea cucumber peptides to the feed on antioxidant indicators in the serum of spotted sea bass. Figure 2 As shown, with the addition of sea cucumber peptides, the MDA content in each added group was significantly lower than that in the S0 group. P <0.05). Furthermore, the SOD activity in each added group was significantly increased compared to the S0 group ( P <0.05), the GSH-Px activity in groups S500, S1000 and S2000 was significantly increased compared with group S0 ( P <0.05). It can be seen that the addition of sea cucumber peptides to the feed improved the antioxidant capacity of sea bass serum.

[0037] 3) Effects of adding different levels of sea cucumber peptides to feed on non-specific immune indicators in sea bass serum. Non-specific immune markers refer to ACP, AKP, IgM, C3, C4, LZM, and TP. Figure 3 This is a graph showing the effect of adding different levels of sea cucumber peptides to the feed on non-specific immune indicators in the serum of spotted sea bass. Figure 3 As shown, the ACP activity of S250, S500, S1000, and S2000 was significantly increased compared to the S0 group (S250, S500, S1000, and S2000). P <0.05). The AKP activity of the S500 and S1000 groups was significantly increased compared to the S0 group ( P <0.05). Compared with the S0 group, the IgM content of spotted bass in groups S1000 and S2000 was significantly increased ( P <0.05). The LZM activity and C3 content in each added group were significantly increased compared to the S0 group ( P <0.05). The C4 content in the S1000 group was significantly increased compared to the S0 group ( P <0.05), the TP content in groups S250, S500, and S1000 was significantly increased compared to group S0. This indicates that adding sea cucumber peptides to the feed enhances the non-specific immunity of sea bass serum.

[0038] 4) Effects of adding different levels of sea cucumber peptides to feed on gut health-related indicators of spotted sea bass Intestinal health-related indicators refer to LPS, TPS, AMS in the intestines of spotted bass and DAO in the serum of spotted bass. Figure 4 This is a graph showing the effect of adding different levels of sea cucumber peptides to feed on gut health-related indicators of spotted sea bass. (Example:) Figure 4 As shown, the activities of TPS and AMS in the intestine of spotted bass in group S500 were significantly increased compared with group S0. P <0.05), LPS activity showed a trend of first increasing and then decreasing, with the highest activity in the S500 group, but there was no significant difference. P >0.05), the DAO content in the serum of spotted bass in groups S250 and S500 was significantly lower than that in group S0 ( P <0.05). It can be seen that adding sea cucumber peptides to the feed improves the activity of intestinal digestive enzymes and intestinal health-related indicators in sea bass.

[0039] 5) Effects of adding different levels of sea cucumber peptides on energy metabolism-related indicators and hepatocyte organelle morphology in sea bass serum. Energy metabolism-related indicators refer to INS, TG, TC, LD, HDLC, and LDLC. Figure 5 This is a graph showing the effect of adding different levels of sea cucumber peptides to the feed on energy metabolism-related indicators in the serum of spotted sea bass. (Example:) Figure 5As shown, the TG content of S250, S500 and S1000 groups was significantly reduced compared with that of S0 group P <0.05). The HDLC content of S500 group was significantly increased compared with that of S0 group P <0.05). There was no significant difference in the TC, LDLC and INS content of each addition group compared with S0 P It can be seen that the energy metabolism related indexes of Lateolabrax japonicus are improved by adding suitable level of sea cucumber peptide in feed.

[0040] Figure 6 The electron microscope ultrastructure diagram of hepatocyte of Lateolabrax japonicus in suitable level addition group S500 and control group S0 is shown in Figure 6. Figure 6 As shown in the electron microscope section of liver, the endoplasmic reticulum of S0 group is swollen and displaced, and the phenomenon of increased lipid droplets appears. The S500 group can be seen without obvious endoplasmic reticulum swelling phenomenon and endoplasmic reticulum close to the nucleus, glycogen is sufficient, and the number of mitochondria is increased. It can be seen that the addition of sea cucumber peptide in feed improves the morphological structure of hepatocyte organelles.

[0041] It can be seen that the method provided by the present application, on the one hand, relieves the negative effects of high temperature stress of Lateolabrax japonicus, thereby regulating the feed efficiency of Lateolabrax japonicus and improving the growth performance of Lateolabrax japonicus, and on the other hand, shows that the stability of the organelle of Lateolabrax japonicus is improved and the most suitable addition amount is shown. The method is easy to operate, has high feasibility, can improve the growth performance of Lateolabrax japonicus under high temperature conditions, reduces the production loss caused by high temperature breeding conditions, and is suitable for practical use.

[0042] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above embodiments without departing from the principles and purposes of the present application within the scope of the present application.

Claims

1. Use of sea cucumber peptide for preparing feed for relieving high temperature heat stress of Epinephelus akaara.

2. Use according to claim 1, characterized in that, The sea cucumber peptide is added in an amount of 250-2000 mg per 100 g of feed.

3. Use according to claim 2, characterized in that, The sea cucumber peptide is added in an amount of 250-1500 mg per 100 g of feed.

4. Use according to claim 3, characterized in that, The sea cucumber peptide is added in an amount of 250-1000 mg per 100 g of feed.

5. Use according to claim 4, characterized in that, The sea cucumber peptide is added in an amount of 500-1000 mg per 100 g of feed.

6. Use according to claim 5, characterized in that, The sea cucumber peptide is added in an amount of 500 mg per 100 g of feed.

7. The use according to claim 1, characterized in that, The high temperature heat stress refers to heat stress caused by feeding Epinephelus akaara at a temperature higher than the suitable temperature.

8. The use according to claim 1, characterized in that, The relieving of high temperature heat stress of Epinephelus akaara is evaluated by at least one of the following indexes: improving the growth performance of Epinephelus akaara; improving the antioxidant capacity of the serum of Epinephelus akaara; improving the non-specific immune capacity of the serum of Epinephelus akaara; improving the digestive enzyme activity in the intestinal tract of Epinephelus akaara; improving the energy metabolism related indexes of Epinephelus akaara; improving the organelle stability of the hepatocytes of Epinephelus akaara.

9. Use according to claim 8, characterized in that, The improvement of the growth performance of Epinephelus akaara refers to the improvement of the final average weight, and / or the improvement of the weight gain rate, and / or the improvement of the feed efficiency; The improvement of the antioxidant capacity of the serum of Epinephelus akaara refers to the decrease of malondialdehyde in the serum, and / or the increase of superoxide dismutase in the serum, and / or the increase of catalase, and / or the increase of total antioxidant capacity, and / or the increase of glutathione peroxidase content in the serum; The improvement of the non-specific immune capacity of the serum of Epinephelus akaara refers to the increase of alkaline phosphatase content, and / or the increase of acid phosphatase content, and / or the increase of fish complement protein 3 content, and / or the increase of fish complement protein 4 content, and / or the increase of total protein content in the serum of Epinephelus akaara; The improvement of the digestive enzyme activity in the intestinal tract of Epinephelus akaara refers to the increase of trypsin and amylase activity in the intestinal tract of Epinephelus akaara The improvement of the energy metabolism related indexes of Epinephelus akaara refers to the decrease of insulin content, and / or the decrease of triglyceride content, and / or the decrease of lactic acid, and / or the decrease of total cholesterol content, and / or the increase of high-density lipoprotein; The improvement of the organelle stability of the hepatocytes of Epinephelus akaara refers to the relatively reduced number of lipid droplets and the alleviated endoplasmic reticulum swelling observed in the hepatocytes of Epinephelus akaara under transmission electron microscope.

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