Bioactive peptide capable of improving holothurian summer passing ability and application thereof

Abstract

The application discloses a kind of biological active peptide capable of improving sea cucumber summer passing capacity and its preparation method and application.The biological active peptide is obtained by extracting, enzyme hydrolysis, separation and purification using tuna meat as raw material, and the amino acid sequence of the active peptide is MADFFTRL.Test shows that the biological active peptide can improve the survival rate of sea cucumber under high temperature stress, enhance the activities of SOD, CAT and other antioxidant enzymes, reduce the content of MDA, reduce the oxidative damage caused by high temperature, thereby improving the heat resistance and safe summer passing capacity of sea cucumber.The biological active peptide can be used for preparing sea cucumber feed additive or functional feed, and has good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of functional peptide preparation technology, specifically relating to a bioactive peptide that can improve the summer survival ability of sea cucumbers and its application. Background Technology

[0002] Sea cucumbers possess high nutritional value and potential for medicinal development, playing a vital role in marine ecosystems. They participate in organic matter decomposition and nutrient cycling, contributing positively to maintaining benthic ecological balance. As a key aquaculture species, sea cucumbers have become an important commodity in the marine aquaculture industry, following fish, shrimp, shellfish, and algae, occupying a crucial position in multi-trophic level integrated aquaculture and marine ranching enhancement models. In recent years, the sea cucumber aquaculture industry has developed rapidly, playing a positive role not only in promoting the adjustment of fishery structure in coastal areas, expanding income channels for fishermen, and driving local economic development, but also in promoting the transformation of marine aquaculture towards green and ecological directions.

[0003] However, with the continuous expansion of sea cucumber farming, the industry has gradually exposed problems such as germplasm degradation, frequent diseases, increased pressure on the farming environment, and rising difficulty in product quality control. These issues have become important factors restricting the sustainable and healthy development of the sea cucumber industry. Sea cucumbers are typical cold-temperate marine organisms, with an optimal growth temperature generally ranging from 3℃ to 18℃, of which 10℃ to 16℃ is the most suitable range. Temperature changes have a significant impact on sea cucumbers' feeding, growth, and physiological metabolism. Especially under high summer temperatures, the increased temperature of the farming water can easily trigger stress responses in sea cucumbers, such as vomiting and skin dissolution, which can even lead to death in severe cases.

[0004] In recent years, some regions have experienced frequent periods of sustained high temperatures and muggy weather during the summer. Environmental factors such as temperature and dissolved oxygen in the aquaculture water have fluctuated significantly, exceeding the range that sea cucumbers can withstand for normal survival and growth. This has led to phenomena such as sea cucumber detachment from reefs, evisceration, skin dissolution, and even mass mortality, causing substantial economic losses to aquaculture production. Therefore, how to effectively ensure the safe survival of sea cucumbers during the high-temperature summer and reduce aquaculture losses has become an urgent technical problem to be solved in sea cucumber farming, and a key area of ​​continuous attention and research for those skilled in the art. Summary of the Invention

[0005] This invention provides a bioactive peptide that can improve the ability of sea cucumbers to survive the summer and its application. This peptide can improve the high temperature resistance of sea cucumbers, making them more suitable for surviving the high temperatures of summer and increasing their survival rate.

[0006] This invention first provides a bioactive peptide, which is obtained by freeze-drying tuna meat, grinding it into powder, adding distilled water to dissolve and adjust the pH, transferring it to an ultrasonic cleaner and a hot reflux extraction device to extract crude protein, adding protease to the crude protein, adjusting the pH, enzymatically hydrolyzing it at a suitable temperature, inactivating the enzyme in the mixture, centrifuging the enzymatic hydrolysate, collecting the supernatant, and freeze-drying it. The pH adjustment mentioned above involves using a 0.1 mol / L NaOH solution to adjust the pH of the solution to 8.0. The protease in question is trypsin with 6000 U / mL added; The preferred enzymatic hydrolysis temperature is 35°C; Furthermore, the amino acid sequence of the bioactive peptide is MADFFTRL (SEQ ID NO:2). In another aspect, this invention provides an application of bioactive peptides in the preparation of feed additives that enhance the heat resistance of sea cucumbers. The present invention also provides a sea cucumber feed, wherein the above-mentioned bioactive peptides are added.

[0007] Compared with existing technologies, this invention uses bioactive peptides derived from tuna as functional components, which can significantly improve the tolerance and survival rate of sea cucumbers under high-temperature stress conditions. At the same time, it enhances the activity of antioxidant enzymes SOD and CAT in sea cucumbers, reduces MDA content, and alleviates oxidative damage caused by high temperature, thereby helping sea cucumbers to safely survive the summer. These bioactive peptides can be further used to prepare feed additives or functional feeds that improve the heat resistance of sea cucumbers, and have good application and promotion value. Attached Figure Description

[0008] Figure 1 Hydrolytic capacity diagram of different proteases; Figure 2 : Effect of pH on protein hydrolysis rate; Figure 3 : Effect of enzyme addition on protein hydrolysis rate; Figure 4 : Effect of temperature on protein hydrolysis rate; Figure 5 : Effect of time on protein hydrolysis rate; Figure 6 : Gel chromatography chromatogram of tuna polypeptide G-25 dextran; Figure 7 : Liquid phase distribution diagram of component F2 obtained by reversed-phase high-performance liquid chromatography; Figure 8 : Diagram showing the scavenging effect of tuna peptides on DPPH free radicals; Figure 9 Survival rates of sea cucumbers in each group during the experiment; Figure 10 : CAT content in sea cucumbers of each group; Figure 11 : Graphs showing the SOD content in each sea cucumber; Figure 12 : MDA content in sea cucumbers of each group. Detailed Implementation

[0009] The present invention will now be described in detail with reference to specific embodiments and accompanying drawings.

[0010] Example 1: Extraction of bioactive peptides 1. Extraction of tuna protein Cleaned tuna meat was freeze-dried and ground into powder. 5g of tuna powder was placed in a 250ml Erlenmeyer flask. A suitable amount of distilled water was added at a material-to-liquid ratio of 1:20 (g:mL), and the mixture was soaked for 0.5 hours. Then, 0.1 mol / L NaOH solution was slowly added dropwise to adjust the pH to 8.0. Subsequently, the pH-adjusted solution was transferred to both an ultrasonic cleaner (set to 100W) and a hot reflux extraction apparatus, both set to 50°C for extraction. Each extraction lasted 0.5 hours, and this process was repeated three times to ensure complete protein extraction.

[0011] After extraction, the three extracts were combined, and the pH was adjusted to 5.0 using 0.5 mol / L hydrochloric acid solution. After standing for 0.5 hours, the mixture was centrifuged at 5000 r / min for 10 min, and the supernatant was collected to obtain the tuna protein extract.

[0012] 2. Enzymatic hydrolysis of tuna protein Tuna protein extract was freeze-dried under vacuum to obtain crude protein powder. This powder was dissolved in distilled water to prepare an aqueous solution with a mass concentration of 20 g / L. Subsequently, five enzymes—alkaline protease, neutral protease, papain, trypsin, and pepsin—were added to this aqueous solution at a dosage of 3000 U / mL each. Based on the optimal operating conditions for each enzyme, the pH and temperature of the solution were adjusted to initiate the enzymatic hydrolysis reaction, thereby screening for the optimal protease.

[0013] Five proteases were used to enzymatically hydrolyze tuna protein solutions under their respective optimal reaction conditions. The results are shown in the figure. Figure 1 .Depend on Figure 1 It can be seen that the five enzymes have significantly different effects on the enzymatic hydrolysis of tuna protein. For tuna protein, trypsin has the best enzymatic hydrolysis effect and the highest content of tuna polypeptide is 15.37%.

[0014] 3. Optimization of protein enzymatic hydrolysis conditions A crude protein aqueous solution with a mass concentration of 20 g / L was enzymatically hydrolyzed under the conditions of pH 7.0, temperature 50℃, enzyme dosage of 6000 U / mL, and hydrolysis time of 1 hour. Subsequently, all reaction conditions except for the factor under investigation were fixed, and the effects of different pH values ​​(6.5 to 9.0), enzyme dosages (2000 to 10000 U / mL), hydrolysis temperatures (30 to 55℃), and hydrolysis times (1 to 5 hours) on the content of tuna peptides were investigated. Before hydrolysis, protease was added to the solution and shaken thoroughly, then the solution was placed in a water bath to complete the hydrolysis. After hydrolysis, the mixture was heated in a boiling water bath for 10 minutes to inactivate the enzyme. The hydrolysate was centrifuged at 3500 r / min for 15 minutes, and the supernatant was collected. Subsequently, the supernatant was treated with trichloroacetic acid (TCA) to precipitate unhydrolyzed protein, and centrifuged again at 3500 r / min for 15 minutes. The final collected supernatant was used for further analysis.

[0015] The results of single-factor experiments on the enzymatic hydrolysis of tuna protein are presented in Figures 2 to 5 middle. Figure 2 The results showed that pH affects the enzymatic hydrolysis of tuna proteins, with the best hydrolysis effect achieved at pH 8.0, resulting in a peptide content as high as 15.89%. Subsequently, increasing pH led to a decrease in peptide yield, attributed to the inactivation of the enzyme by extreme pH, which in turn affected the dissociation of active sites and substrates. Figure 3 This revealed the relationship between enzyme dosage and peptide content: as the enzyme dosage increased, the peptide content increased, reaching a peak of 15.68% at 6000 U / mL, and then tended to plateau. To optimize cost, 6000 U / mL was determined to be the optimal enzyme dosage.

[0016] The peptide content is closely related to the temperature of enzymatic hydrolysis (Figure 4). The peptide content is highest (15.88%) when the enzymatic hydrolysis temperature is 35℃. As the temperature continues to rise, the peptide content shows a downward trend. This may be because the enzyme is a protein, and when the temperature rises to a certain level, the enzyme activity will decrease or even become inactive.

[0017] In the initial stage of enzymatic hydrolysis, the peptide content increased with the extension of hydrolysis time (Figure 5). When the hydrolysis time reached 3 h, the peptide content reached the highest value (15.69%). However, as the time continued to extend, the peptide content showed a downward trend, which may be related to the complete hydrolysis of the substrate.

[0018] In summary, the optimal conditions for enzymatic hydrolysis of tuna protein are: pH 8.0, enzyme dosage of 6000 U / mL, temperature of 35℃, and time of 3 hours.

[0019] 4. Isolation, purification, and bioactivity determination of tuna peptides 1) Isolation of polypeptides The tuna protein hydrolysate was sequentially separated using ultrafiltration membranes (10, 5, and 1 kDa). The permeate fraction of the filtrate (relative molecular mass ≤ 1 kDa) was collected, freeze-dried, pulverized, and passed through a 40-mesh sieve to obtain tuna peptide powder. The initially screened small molecule peptide mixture (molecular weight less than 1 kDa) was further separated and purified using Sephadex G-25 column chromatography. This process successfully obtained small molecule peptide complexes derived from tuna. The chromatographic results are shown below. Figure 6 As shown, the low molecular weight peptide F2 was selected as the subject of subsequent experiments because it was eluted later.

[0020] 2) Purification of peptides 10 mg of tuna small molecule peptide complex was dissolved in sterile water to prepare a solution with a concentration of 10 mg / mL. The solution was then filtered through a 0.45 μm microporous membrane and purified by reversed-phase high-performance liquid chromatography (RP-HPLC). A single characteristic chromatographic peak was collected to obtain high-purity tuna small molecule peptides. Figure 7 As shown, peaks 2, 3, and 6 are particularly large, indicating that these components are present in high amounts and have strong responsiveness in the complex, and are likely the main active substances. Therefore, these fractions rich in components 2, 3, and 6 were selected for further in-depth analysis.

[0021] 3) Determination of peptide bioactivity 100 μg of purified tuna small molecule polypeptide powder was placed in a 10 mL test tube, followed by the addition of 4 mL of DPPH methanol solution containing 0.004% (v / v), and the mixture was vigorously shaken to ensure thorough mixing. The mixture was allowed to stand in the dark for 30 min, followed by centrifugation at 5000 r / min for 10 min. The supernatant was collected, and its absorbance was measured at 517 nm using a UV-Vis spectrophotometer. The scavenging efficiency of this solution for DPPH free radicals was calculated according to formula (1).

[0022] (1) In the formula: R1 is the DPPH radical scavenging rate, %; A1 is the absorbance of the sample; A2 is the absorbance without DPPH radicals; and A0 is the absorbance of the blank control group.

[0023] As shown in Figure 8, within the mass concentration range of 0–0.8 g / L, the scavenging effect of tuna small molecule peptides on DPPH free radicals showed an increasing trend with the increase of sample mass concentration. Subsequently, with the increase of mass concentration, the antioxidant growth trend slowed down until it stabilized. Tuna small molecule peptides have a significant scavenging effect on DPPH free radicals.

[0024] 148 small peptides were identified from components 2, 3, and 6 using liquid chromatography-tandem mass spectrometry (LC-MS / MS), with molecular weights ranging from 0.4 to 1.1 kDa. The molecular activity of each peptide was predicted using PeptideRanker software, and two peptides with high activity were identified, with amino acid sequences MFLFRCAD (SEQ ID NO:1) and MADFFTRL (SEQ ID NO:2). Example 2: Effects of bioactive peptides on the high-temperature resistance of sea cucumbers Healthy sea cucumbers were purchased from a sea cucumber farm in Liaoning Province and raised in a laboratory. The water quality needed to be kept clean and stable, with a salinity of 15‰, a pH of 8, a dissolved oxygen of 5mL / L, a light intensity of 2000lux, 14 hours of light per day, and a water temperature of 15℃. During this period, an appropriate amount of seaweed was fed.

[0025] After 7 days of acclimatization, 40 uniformly shaped sea cucumbers were randomly divided into 4 groups (10 per group). One group remained unchanged as a blank control. The temperature of the other three groups was gradually increased to 30°C at a rate of 1°C per hour. Two of these groups were fed small tuna peptides with sequences SEQ ID NO:1 and SEQ ID NO:2, respectively, and were labeled as peptide group 1 and peptide group 2. The group without peptides served as the control group. Each group had 3 replicates. The experiment lasted for 7 days, during which the mortality rate of sea cucumbers in each group was observed and recorded. After the experiment, the surviving sea cucumbers were euthanized, and muscle tissue was collected. The contents of catalase (CAT) and malondialdehyde (MDA) in the sea cucumbers were determined using a micro-method; the contents of superoxide dismutase (SOD) in the sea cucumbers were determined using an enzyme-linked immunosorbent assay (ELISA).

[0026] The survival rates of sea cucumbers in each group are shown below. Figure 9 The survival rate of sea cucumbers in the blank group was over 95%, while the survival rate of sea cucumbers in the experimental group under high temperature was only 15%. The survival rate of sea cucumbers fed with tuna small molecule peptides in peptide groups 1 and 2 was significantly improved compared with the experimental group (P < 0.05), and the survival rate of peptide group 2 could reach 90%. This confirms that the tuna small molecule peptide with sequence SEQ ID NO:2 obtained in this invention can improve the high temperature resistance of sea cucumbers.

[0027] like Figure 10 and Figure 11 As shown, the SOD and CAT enzyme activities in the control group were significantly lower than those in the blank group (p<0.05). After peptide intervention, compared with the control group, the antioxidant enzyme activities in the sea cucumbers of peptide 1 and peptide 2 groups were significantly increased compared with the model group (p<0.05); the SOD and CAT enzyme activities in the sea cucumbers of peptide 2 group were significantly increased compared with the control group. This confirms that the tuna small molecule peptide with the sequence SEQ ID NO:2 can improve the antioxidant capacity and enzyme activity of sea cucumbers under high temperature.

[0028] like Figure 12 As shown, the content of MDA peroxidation product in the control group was significantly higher than that in the normal group at high temperature (p<0.05), while the MDA content in peptide 1 and peptide 2 groups was significantly lower than that in the control group, and peptide 2 had a slightly better effect than peptide 1.

[0029] In summary, the tuna small molecule polypeptide provided by this invention has antioxidant capabilities, which can reduce the formation of free radicals and alleviate oxidative damage to the body, thereby improving the high-temperature resistance of sea cucumbers. It can be applied and promoted as a functional feed additive.

Claims

1. A bioactive peptide, characterized in that, The bioactive peptides are obtained by freeze-drying tuna meat, grinding it into powder, dissolving it in distilled water to adjust the pH, transferring it to an ultrasonic cleaner and a hot reflux extraction device to extract crude protein, adding protease to the crude protein, adjusting the pH and performing enzymatic hydrolysis, inactivating the enzyme in the mixture, centrifuging the hydrolysate, collecting the supernatant and freeze-drying it.

2. The bioactive peptide according to claim 1, characterized in that, The pH adjustment mentioned above involves using a 0.1 mol / L NaOH solution to adjust the pH of the solution to 8.

0.

3. The bioactive peptide according to claim 1, characterized in that, The protease mentioned is obtained by enzymatic hydrolysis using trypsin.

4. The bioactive peptide according to claim 1, characterized in that, The enzymatic hydrolysis described herein is carried out at 35°C.

5. The bioactive peptide according to claim 1, characterized in that, The bioactive peptide described herein has the amino acid sequence SEQ ID NO:

2.

6. The use of the bioactive peptide of claim 1 in the preparation of a feed additive for improving the heat resistance of sea cucumbers.

7. A sea cucumber feed, characterized in that, The sea cucumber feed contains the bioactive peptide described in claim 1.

8. A method for improving the summer survival ability of sea cucumbers, characterized in that, The method described herein involves feeding the sea cucumbers with the sea cucumber feed described in claim 7 during the sea cucumber farming process.

Images