Application of polypeptide protein complex in inhibiting Vibrio harveyi and Vibrio maxima

Abstract

The present invention relates to the use of a polypeptide protein complex for inhibiting Vibrio harveyi and Vibrio maxima, belonging to the field of aquatic animal nutrition. The polypeptide protein complex is prepared by first adding 2.4L of alkaline protease Alcalase to cod muscle at an amount of 1.8-2.0ml per 100g of fish meat, hydrolyzing for 55-60 minutes, then adding Flavourzyme 500MG at an amount of 1.2-1.5g per 100g of fish meat, and continuing hydrolysis for 90-100 minutes; during the entire enzymatic hydrolysis process, the reaction temperature is maintained at 54-56°C, and the enzymatic solution is rotary evaporation and freeze-dried to obtain a freeze-dried powder of the polypeptide protein complex. The polypeptide protein complex has an antibacterial effect on Vibrio harveyi and Vibrio maxima. The preparation method of the present invention eliminates the complex process of isolating, purifying, and identifying antimicrobial peptides, reducing costs while ensuring antimicrobial activity, thereby ensuring large-scale use as an antimicrobial agent in aquatic feed.

Description

Technical Field

[0001] The invention belongs to the field of aquatic animal nutrition, and particularly relates to the application of a polypeptide protein complex in inhibiting Vibrio harveyi and Vibrio turbot. Background Art

[0002] Antibiotics, as feed additives, were once widely used throughout the entire aquaculture industry, including aquaculture, to promote growth and reduce morbidity and mortality in farmed animals. However, excessive use of antibiotics has also led to problems such as drug residues and drug resistance in farmed animals, ultimately endangering human health. Consequently, many countries around the world have enacted bans on the use of antibiotics in feed.

[0003] The "antibiotic ban" for feed companies has led to an increase in their demand for antibiotic alternatives, among which antimicrobial peptides are an important choice. They can kill certain pathogens and prevent pathogen infections, but their separation, purification and identification processes are complicated. Therefore, selecting a polypeptide protein complex with antimicrobial effects can not only play the antibacterial role of antimicrobial peptides, but also eliminate the complicated separation, purification and identification steps, becoming a very promising method.

[0004] Vibrio spp., including Vibrio harveyi, Vibrio maxima, and Vibrio anguillarum, can infect farmed animals such as Litopenaeus vannamei, turbot, and grouper. They are a common pathogen causing vibriosis in marine aquaculture, often leading to symptoms such as exophthalmos, gastroenteritis, and muscle ulcers, ultimately impacting animal survival and economic returns. Therefore, research into the antibacterial effects of Vibrio spp. is crucial for reducing disease and improving survival rates in marine aquaculture. Summary of the Invention

[0005] The technical problem addressed by the present invention is to provide a polypeptide-protein complex for use in inhibiting Vibrio harveyi and Vibrio maxima. This invention addresses the frequent outbreaks of fish and shrimp diseases caused by Vibrio harveyi and Vibrio maxima following the ban on antibiotics. By improving existing processes for preparing polypeptide-protein complexes, the present invention has developed a polypeptide-protein complex that effectively inhibits Vibrio harveyi and Vibrio maxima.

[0006] The present invention is achieved according to the following technical solutions:

[0007] The polypeptide protein complex is used to inhibit Vibrio harveyi and Vibrio turbot. The polypeptide protein complex is prepared by enzymatically hydrolyzing cod muscle. The specific method is as follows: first, 2.4L of alkaline protease Alcalase is added to the cod muscle at an amount of 1.8-2ml per 100g of fish meat, and the hydrolysis time is 55-60min. Then, Flavourzyme 500MG is added at an amount of 1.2-1.5g per 100g of fish meat, and the hydrolysis is continued for 90-100min. During the entire enzymatic hydrolysis process, the reaction temperature is maintained at 54-56°C. 。

[0008] Furthermore, the ratio of cod muscle to water added during the enzymatic hydrolysis of the cod muscle is as follows: 1.5 ml of water is added to every 1 g of cod muscle.

[0009] Furthermore, the initial pH of the enzymatic hydrolysis was 9.0.

[0010] Furthermore, the enzymatic hydrolyzate obtained after enzymatic hydrolysis was rotary evaporated at 50° C. until the water content reached 50%, and then freeze-dried to obtain a polypeptide protein complex freeze-dried powder.

[0011] Furthermore, the chemical composition of the polypeptide protein complex is as follows: crude protein content is more than 85%, crude fat content is less than 0.5%, and ash content is less than 10%; the molecular weight distribution of the polypeptide protein complex is: >10,000Da, 0.12%; 10,000-5,000Da, 0.89%; 5,000-3,000Da, 1.94%; 3,000-2,000Da, 4.13%; 2,000-1,000Da, 21.86%; 1,000-500Da, 21.59%; 500-200Da, 43.29%; <200Da, 6.23%.

[0012] The beneficial effects of the present invention compared with the prior art are as follows:

[0013] (1) Compared with the prior art, the present invention eliminates the complicated process of separation, purification and identification of antimicrobial peptides, thereby reducing costs while ensuring antimicrobial effects, thereby ensuring large-scale application as an antimicrobial agent in aquatic feed.

[0014] (2) Compared with existing antibiotics, although the antibacterial effect of the present invention is not as obvious as that of antibiotics, it will not produce drug resistance in aquatic animals, will not produce super-resistant bacteria in aquatic animals, and will not be enriched in the human body as humans eat aquatic animals, thereby ensuring the safety of aquatic products.

[0015] (3) Compared with the enzymatic fish slurry commonly used in aquatic feed, the process of preparing polypeptide protein in the present invention is more gentle, which not only retains the antimicrobial peptide function, but also does not increase the cost too much, thereby realizing its large-scale use in aquatic feed, with stronger operability and controllable cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Punch method was used to determine the inhibitory effect of 500 and 1000 mg / ml of the polypeptide protein complex solution on Vibrio harveyi;

[0017] Figure 2 Punch method was used to determine the inhibitory effect of 1000 and 1500 mg / ml of the polypeptide protein complex solution on Vibrio harveyi;

[0018] Figure 3 Punch method was used to test the inhibitory effect of 500, 1000 and 1500 mg / ml of polypeptide protein complex solution on Vibrio maxima;

[0019] Figure 4 The punch method was used to determine the inhibitory effects of 500, 1000 and 1500 mg / ml of the polypeptide protein complex solution on Vibrio anguillarum. Specific implementation plan

[0020] The technical solution of the present invention is further illustrated below by specific examples, but the protection scope of the present invention is not limited in any form by the examples:

[0021] Example 1 Preparation process of polypeptide protein complex

[0022] Thaw the frozen Pacific cod in a 50°C oven, then remove the fish head, viscera, skin, bones, etc., and clean the fish muscle with water. Crush the fish muscle in a grinder, then add 150ml of purified water to every 100g of muscle (wet weight), and heat it to 90°C in a fermenter for 15 minutes to inactivate the endogenous enzymes in the muscle. Cool the muscle and water mixture in the fermenter to room temperature, adjust the pH so that the pH of the mixture reaches 9.0, then add 2ml of alkaline protease Alcalase 2.4L to every 100g of muscle, mix thoroughly, and enzymatically hydrolyze at 55°C for 60 minutes; open the fermenter, add Flavourzyme 500MG, and add 1.5g of flavor protease to every 100g of fish meat, and continue enzymatic hydrolysis for 90 minutes. The fermenter was rapidly heated to 90°C and maintained for 10 minutes to inactivate 2.4L of Alcalase and 500mg of Flavorzyme. The inactivated enzymatic hydrolysate was distilled at 50°C using rotary evaporation. When the water content of the hydrolysate was reduced to 50%, it was freeze-dried for 10 hours.

[0023] The chemical composition of the polypeptide protein complex is as follows: crude protein content is more than 85%, crude fat content is less than 0.5%, and ash content is less than 10%.

[0024] Through this preparation process, more than 99% of the molecular weight of the polypeptide protein complex is below 10,000 Da, and 50% is below 500 Da. The specific molecular weight distribution is: >10,000 Da, 0.12%; 10,000-5,000 Da, 0.89%; 5,000-3,000 Da, 1.94%; 3,000-2,000 Da, 4.13%; 2,000-1,000 Da, 21.86%; 1,000-500 Da, 21.59%; 500-200 Da, 43.29%; <200 Da, 6.23%.

[0025] Example 2 Observation of the antibacterial effect of the polypeptide protein complex on Vibrio harveyi, Vibrio maxima and Vibrio anguillarum using the punch method

[0026] (1) Preparation of bacterial cultures of Vibrio harveyi, Vibrio turbot, and Vibrio anguillarum

[0027] First, take out the stored Vibrio harveyi, Vibrio maxima, and Vibrio anguillarum from the -80°C refrigerator, activate the corresponding Vibrio, and then inoculate them into 5 ml of TSB culture medium, culture at 37°C and 120 rpm with shaking for 5-6 hours until the OD600 value of the bacterial solution reaches about 0.3, and store at 4°C.

[0028] (2) Antibacterial test

[0029] Inject 150 μL of the corresponding Vibrio solution into the solid culture medium, spread evenly, and let it dry for 3-5 minutes. Then, using the plate punch method, evenly punch several 6 mm holes in the culture medium and mark them. Slightly aim the bottom of the culture dish at the flame of an alcohol burner and heat the bottom. Prepare peptide protein complex solutions at concentrations of 500, 1000, and 1500 mg / ml using sterile water. Inject different concentrations of peptide protein complex liquid into different wells. Use antibiotics as positive controls and 1.5% saline as negative controls. Incubate at 37°C upright for 24 hours, observe the results, and record them with photos.

[0030] (3) Experimental results

[0031] Depend on Figure 1 It can be seen that the inhibitory effect of 500 and 1000 mg / ml polypeptide protein complex solutions on Vibrio harveyi is not as strong as that of antibiotics, but compared with the normal saline group, there is a significant inhibitory effect. Figure 2 It can be seen that when the concentration of the polypeptide protein complex solution is further increased to 1500 mg / ml, the inhibition zone does not increase further compared with 1000 mg / ml, indicating that excessively high concentration does not increase the antibacterial effect of the polypeptide protein complex.

[0032] Depend on Figure 3 As can be seen, unlike the results of the Vibrio harveyi inhibition experiment, the polypeptide protein complex solution had no inhibitory effect on Vibrio maxima at concentrations of 500 and 1000 mg / ml. However, when the solubility of the polypeptide protein complex was increased to 1500 mg / ml, it had a certain inhibitory effect on Vibrio maxima. Although the inhibitory effect was still not as strong as that of the antibiotic, it was significantly more inhibitory than the saline group. This indicates that the polypeptide protein complex provided by the present invention has species-specific inhibitory effects on Vibrio, with a strong inhibitory effect on Vibrio harveyi but a weaker inhibitory effect on Vibrio maxima.

[0033] Depend on Figure 4 As can be seen, unlike the results of the Vibrio harveyi inhibition experiment in Example 2 and the Vibrio maxima turbot inhibition experiment in Example 3, the polypeptide protein complex solution at three concentrations of 500, 1000, and 1500 mg / ml showed no significant inhibitory effect on Vibrio anguillarum, consistent with the saline control group. This result further demonstrates that the polypeptide protein complex provided by the present invention has species-specific inhibitory effects on pathogens, and even for Vibrio at the same genus level, there are differences. In this experiment, the inhibitory effect on Vibrio harveyi was the strongest, followed by Vibrio maxima turbot, and there was no inhibitory effect on Vibrio anguillarum.

[0034] Example 3 Verification of the Antibacterial Effect of the Polypeptide Protein Complex on Vibrio harveyi, Vibrio maxima and Vibrio anguillarum Using the Plate Count Method

[0035] (1) Preparation of different gradient peptide protein complex culture medium

[0036] Based on TSB medium, the nitrogen source (tryptone, soy peptone) in TSB liquid medium was replaced with 0%, 25%, 50%, 75%, and 100% of polypeptide protein complex nitrogen, respectively, to prepare 5 culture media with equal protein content. The specific preparation method is shown in Table 1.

[0037] Table 1 Peptide protein complex culture medium formula (taking 1L as an example)

[0038] 0％ 25％ 50％ 75％ 100％ Tryptone (g) 15.00 11.25 7.50 3.75 0.00 Soy peptone (g) 5.00 3.75 2.50 1.25 0.00 Peptide-protein complex (g) 0.00 5.80 11.60 17.40 23.20 Protein content (%) 17.81 17.81 17.81 17.81 17.81

[0039] (2) Plate count

[0040] Three types of Vibrio harveyi, Vibrio turbot and Vibrio anguillarum were inoculated into TSA medium and cultured at 37°C for 24 hours for recovery. Single colonies were picked and dissolved in 1.5% saline. 3 mL of the above experimental culture medium containing different gradients of peptide protein complexes was added to each centrifuge tube, and 200 μl of bacterial solution was inoculated respectively. The culture was shaken at 37°C until the OD600nm value of the fastest growing group reached 1.0, and then gradually diluted to 10 with sterile water. -5 , 10 -6 , 10 -7 (approximately 1000, 100, and 10 CFU, respectively).

[0041] Take 200 μL of each of the three dilutions and spread them on a plate counting medium. Incubate the plate upside down at 37° C. for 24 h, and count the number of colonies on the medium (select the results with CFU between 30 and 300 for counting).

[0042] (3) Experimental results

[0043] As shown in Table 2, the plate count results of Vibrio harveyi showed that when the polypeptide protein complex replaced 25% of the nitrogen source in TSB medium, the number of Vibrio harveyi reached a maximum of 6.2×10 8 CFU / mL, which was 3×10 higher than that of the TSB medium control group. 8 CFU / mL, which may be due to the addition of the new nitrogen source of the polypeptide protein complex, which, to a certain extent, promoted the growth of Vibrio harveyi more than the inhibitory effect of the antibacterial peptide contained in the polypeptide protein complex on its growth. However, when the polypeptide protein complex replaced more than 50% of the nitrogen source in the TSB medium, the concentration of Vibrio harveyi bacteria decreased significantly, with a decrease of 3 orders of magnitude, indicating that the antibacterial peptide in the polypeptide protein complex began to inhibit the growth of Vibrio harveyi, so the concentration was significantly reduced to 1×10 5This result once again verified the results obtained from the punching experiment, indicating that the polypeptide protein complex of the present invention has a significant antibacterial effect on Vibrio harveyi.

[0044] Table 2 Plate counts of Vibrio harveyi on five polypeptide protein complex media

[0045]

[0046] As shown in Table 3, the plate count results for Vibrio maxima show that as the proportion of nitrogen source in TSB culture medium replaced by the polypeptide protein complex increases, the number of Vibrio maxima gradually decreases. When the nitrogen source in TSB culture medium is replaced at 50%, 75%, and 100%, the number of Vibrio maxima increases by 4.9-7.0 times compared to the unsubstituted group, although not by an order of magnitude. This indicates that the antibacterial effect of the polypeptide protein complex of the present invention on Vibrio maxima, while not as pronounced as that on Vibrio harveyi, is still quite good. This result further validates the results obtained by the punch method.

[0047] Table 3 Plate counts of Vibrio turbot on five polypeptide protein complex media

[0048]

[0049] As shown in Table 4, the plate count results for Vibrio anguillarum indicate that when the peptide protein complex replaced the nitrogen source in TSB medium at 75% and 100%, the concentration of Vibrio anguillarum decreased by 1.6 and 6.2 times, respectively, compared to the control group. This suggests that the peptide protein complex inhibits the growth of Vibrio anguillarum to a certain extent. However, first, a 50% replacement did not reduce the concentration of Vibrio anguillarum, while the same replacement reduced the concentrations of Vibrio harveyi and Vibrio maximus. Second, 100% replacement of the nitrogen source in TSA medium resulted in a monolayer of peptone, which also affected the growth of Vibrio. Third, according to the results of the punch method, the peptide protein complex showed no inhibition zone against Vibrio anguillarum. Therefore, considering all factors, the peptide protein complex in this experiment has very limited, or even no, inhibitory effect on Vibrio anguillarum.

[0050] Table 4 Plate counts of Vibrio anguillarum on five polypeptide protein complex media

[0051]

[0052] In conclusion, the polypeptide protein complex provided by the present invention contains certain antimicrobial peptides, and has species-specific inhibitory effects on Vibrio, a common pathogen in marine aquaculture. It can effectively inhibit Vibrio harveyi and Vibrio maxima, but has almost no inhibitory effect on Vibrio anguillarum.

Claims

1. Application of a polypeptide protein complex in the preparation of Vibrio harveyi and Vibrio turbot inhibitors, characterized in that: The preparation method of the polypeptide protein complex comprises enzymatically hydrolyzing codfish muscle, first adding 2.4L of alkaline protease Alcalase to the codfish muscle in an amount of 1.8-2.0ml per 100g of codfish muscle, with an initial pH of 9.0 and a hydrolysis time of 55-60min, then adding 1.2-1.5g of flavor protease Flavourzyme 500MG per 100g of codfish muscle, and continuing hydrolysis for 90-100min; during the entire enzymatic hydrolysis process, the reaction temperature is maintained at 54-56°C, and the enzymatic hydrolysate obtained after enzymatic hydrolysis is rotary evaporated at 50°C to a moisture content of 50%, and then low-temperature freeze-dried to obtain a polypeptide protein complex freeze-dried powder; the application concentration of the polypeptide protein complex to Vibrio harveyi is 500-1500mg / ml, and the application concentration of the polypeptide protein complex to Vibrio maxima is 1500mg / ml.

2. Use of the polypeptide protein complex according to claim 1 in the preparation of an antibacterial agent for Vibrio harveyi and Vibrio turbot, characterized in that: The ratio of cod muscle and water added during enzymatic hydrolysis was: 1.5 ml of water was added to every 1 g of cod muscle.

Images