CN117327759A

CN117327759A - Zanthoxylum seed protein polypeptide and application thereof as feed additive

Info

Publication number: CN117327759A
Application number: CN202311305305.9A
Authority: CN
Inventors: 尚贤毅; 桑春艳; 杨军丽
Original assignee: Longnan Fumin Industrial Development Co ltd; Lanzhou Institute of Chemical Physics LICP of CAS
Current assignee: Longnan Fumin Industrial Development Co ltd; Lanzhou Institute of Chemical Physics LICP of CAS
Priority date: 2023-10-10
Filing date: 2023-10-10
Publication date: 2024-01-02

Abstract

The invention provides a pricklyash seed protein polypeptide with an antibacterial effect, which is prepared by performing enzymolysis on pricklyash seed protein by pepsin and then performing enzymolysis by papain. And the enzymolysis product of papain is separated and purified by a Sephadex G-25 gel column, deionized water is used as eluent, the elution speed is 0.5ml/min, and the components separated for 20-30min are collected. The pricklyash seed protein polypeptide provided by the invention can be used for preparing an anti-salmonella product. The pricklyash seed protein provided by the invention has an antibacterial effect, and can improve the immunity of organisms, improve the growth performance, reduce the cost and increase the economic benefit when used as a feed additive.

Description

Zanthoxylum seed protein polypeptide and application thereof as feed additive

Technical Field

The invention belongs to the technical field of feed additives, and particularly relates to a pricklyash seed protein polypeptide and application thereof as a feed additive.

Background

The pricklyash peel is pericarp of pricklyash peel of Rutaceae plant of Rutaceae, is one of main flavoring in each vegetable system of China, and can also be used as Chinese medicine. The Chinese is the first large-scale production country of the Chinese prickly ash, the current cultivation area reaches 100 ten thousand hectares, the annual output is about 50 ten thousand tons, the Chinese prickly ash seeds are main economic byproducts of the Chinese prickly ash, the output is higher than the Chinese prickly ash peel, however, the Chinese prickly ash seeds are not fully and effectively utilized for a long time, and most of the Chinese prickly ash seeds are burnt or scattered into the field to serve as crop fertilizers and are also often discarded as waste at will.

More researches have shown that the pricklyash seeds are rich in a large amount of grease, protein and amino acid, and have the potential of being used as a good animal feed raw material. The data result shows that the pepper seeds are added into the commercial laying hen diet, so that the laying rate of the laying hen can be improved; proper amount of pricklyash seeds are added into the feed, so that the growth performance of meat chickens and carps can be improved. However, in piglet cultivation, the direct addition of pepper seeds to piglet feed can cause obvious stress reaction of piglets, so that the cultivation effect is not good.

In the prior art, a pricklyash seed protein antibacterial peptide is reported, and is prepared by taking pricklyash seed protein prepared by alkali treatment as a raw material and then carrying out enzymolysis by pepsin. However, the pepper seed protein antibacterial peptide has resistance to escherichia coli only, and has poor antibacterial effect on salmonella which is another main pathogen causing diarrhea of piglets. In addition, the antibacterial peptide prepared by the method contains more antagonistic factors, so that stronger stress reaction can occur to piglets, and the growth of the piglets is influenced.

Disclosure of Invention

The invention provides a pricklyash seed protein polypeptide with an antibacterial effect and application thereof as a feed additive, and the protein peptide can be used for improving the body immunity of cultured animals.

The invention firstly provides a pricklyash seed protein polypeptide, and the preparation method thereof is as follows:

1) Dissolving semen Zanthoxyli protein in ethanol solution, treating at 30-35deg.C to obtain ethanol treatment solution,

wherein the adding concentration of the pricklyash seed protein in the ethanol solution is 50-100mg/ml;

2) Adjusting the pH value of the treatment solution to 2-3 by using hydrochloric acid solution, then adding pepsin for enzymolysis, inactivating enzyme after 2-3 hours of enzymolysis, and cooling to obtain enzymolysis solution;

3) And (3) carrying out ultrasonic treatment on the cooled enzymolysis liquid, wherein the ultrasonic power is 150W, the ultrasonic treatment time is 5min, centrifuging for 10min at 8000r/min after ultrasonic treatment, and taking supernatant, and carrying out vacuum freeze drying to obtain the pricklyash seed protein peptide.

Further, the pricklyash seed protein peptide prepared in the step 3) is subjected to enzymolysis by papain to obtain an enzymolysis product;

wherein papain is subjected to enzymolysis at pH of 5.5-6.8 and enzymolysis temperature of 35 ℃ for 5h.

Furthermore, the pricklyash seed protein polypeptide is prepared by separating and purifying an enzymolysis product obtained by enzymolysis of papain by using a Sephadex G-25 gel column, selecting deionized water as eluent with the elution speed of 0.5ml/min, and collecting components separated for 20-30 min.

The pricklyash seed protein polypeptide provided by the invention can be used for preparing an anti-salmonella product.

The invention also provides another application of the pricklyash seed protein polypeptide, which is an application as a feed additive.

The invention also provides a piglet feed, wherein the pricklyash seed protein polypeptide is added in the feed.

The pricklyash seed protein provided by the invention has an antibacterial effect, and can improve the immunity of organisms, improve the growth performance, reduce the cost and increase the economic benefit when used as a feed additive.

Drawings

Fig. 1: a graph of the concentration and hemolytic activity of the pepper seed protein antibacterial peptide p-C1;

fig. 2: a graph of concentration of enzymatic hydrolysate of papain versus haemolytic activity;

fig. 3: sephadex G-25 gel column separation and purification diagram of enzymolysis product of papain;

fig. 4: the result diagram of the salmonella resistance of the polypeptide, wherein A is the pepper seed protein antibacterial peptide p-C1, B is the enzymolysis product of papain, C is the antibacterial circle of the salmonella resistance of the F5 component of the enzymolysis product of papain,

fig. 5: a graph of the detection result of alanine Aminotransferase (ALT) content in piglet serum;

fig. 6: a Total Protein (TP) content detection result diagram in the serum of each group of piglets;

fig. 7: a graph of the detection result of the content of Globulin (GLO) in the serum of each group of piglets;

fig. 8: a graph of the results of the detection of Glucose (GLU) content in the serum of each group of piglets;

fig. 9: a graph of the detection result of Triglyceride (TG) content in the serum of each group of piglets;

fig. 10: statistics of incidence of diarrhea in piglets in the test group and the control group.

Detailed Description

The present invention will be described in detail with reference to the following examples and the accompanying drawings.

Example 1: preparation of Zanthoxylum seed protein antibacterial peptide p-C1

The preparation method comprises the steps of preparing pepper seed protein by alkali treatment, dissolving the pepper seed protein in a sufficient amount of ethanol solution, preheating in a water bath, adjusting the temperature to 32 ℃, adjusting the pH value of the solution to 2.0 at the same time, adding pepsin for enzymolysis, maintaining the pH value of the solution in the enzymolysis process to 2.0, after enzymolysis for 3 hours, putting into a water bath at 90 ℃ for enzyme deactivation, cooling, pouring the cooled mixture into a beaker, putting into an ultrasonic cleaner with cooling water circulation, ultrasonically extracting the antibacterial peptide with ultrasonic power of 150W for 5 minutes, centrifuging for 10 minutes at 8000r/min after ultrasonic treatment, taking supernatant, and vacuum freeze-drying to obtain the pepper seed protein antibacterial peptide, which is named as p-C1.

Verifying the Minimum Inhibitory Concentration (MIC) of the prepared pricklyash seed protein antibacterial peptide p-C1 on common pathogenic bacteria in piglets, namely the concentration of the minimum pricklyash seed protein antibacterial peptide p-C1 capable of inhibiting bacterial growth and reproduction; the method comprises the following specific steps:

respectively streaking and inoculating strains of pathogenic bacteria to an LB plate culture medium, and culturing at 37 ℃ for 24 hours; inoculating the single colony into 100ml LB liquid medium, shake culturing at 37deg.C and 150r/min overnight to obtain bacterial liquid, and regulating bacterial concentration in bacterial liquid to 5×10 ⁶ CFU/mL。

Diluting the pepper seed protein antibacterial peptide, sequentially adding 10 μl of each pepper seed protein antibacterial peptide into each well of a 96-well plate, and adding 90 μl of fermentation culture solution of each strain with adjusted concentration to a total volume of 100 μl of each well; the final concentration of Zanthoxylum seed protein antibacterial peptide p-C1 in each well was made 200. Mu.g/mL, 180. Mu.g/mL, 150. Mu.g/mL, 130. Mu.g/mL, 100. Mu.g/mL, 80. Mu.g/mL, 60. Mu.g/mL, 50. Mu.g/mL, 25. Mu.g/mL, 12.5. Mu.g/mL, 6.25. Mu.g/mL, 3.12. Mu.g/mL, 1.56. Mu.g/mL, 0.78. Mu.g/mL, 0.39. Mu.g/mL and 0.195. Mu.g/mL, respectively, and after shaking culture at 37℃for 24 hours, the experimental results were measured and recorded with an enzyme-labeled instrument at 600 nm. Simultaneously using coliform bacteria liquid (5 x 10) ⁶ CFU/mL) and blank LB liquid medium served as negative and positive controls, respectively, representing bacteriostasis rates of 0 and 100%, respectively (table 1).

Table 1: minimum inhibitory concentration table of pepper seed protein antibacterial peptide p-C1 on various bacteria

As can be seen from the results in Table 1, the Zanthoxylum seed protein antibacterial peptide p-C1 has better antibacterial effect only on escherichia coli and bacillus, but has poor antibacterial effect on salmonella, streptococcus suis and haemophilus parasuis which are common in piglets.

Considering that the haemolytic activity on red blood cells is a common index for measuring the toxicity of red blood cells on eukaryotic cells, the cytotoxicity of the prepared pricklyash seed protein antibacterial peptide p-C1 is verified by the following specific method:

50. Mu.l of porcine erythrocytes dissolved in PBS solution at a concentration of 10% are added to a 96-well plate, and 50. Mu.l of Zanthoxylum seed protein antibacterial peptide p-C1 serial diluted with PBS solution are added to make the concentration of the antibacterial peptide in each well 100. Mu.g/mL, 80. Mu.g/mL, 60. Mu.g/mL, 50. Mu.g/mL, 25. Mu.g/mL, 12.5. Mu.g/mL, 6.25. Mu.g/mL, 3.12. Mu.g/mL, 1.56. Mu.g/mL, 0.78. Mu.g/mL, 0.39. Mu.g/mL and 0.195. Mu.g/m, respectively. While 100. Mu.l of 0.2% Triton X-100 was used as positive control (100% hemolysis), negative control wells were added with 100. Mu.l of PBS (0% hemolysis).

After incubation at 37℃for 1 hour, after centrifugation of the cultured 96-well plate at 3000rpm for 5 minutes, 50. Mu.l of the supernatant was aspirated from each experimental well, OD values were measured at 550nm, and the percent hemolysis was calculated from the OD values, which was calculated as follows

Percent% hemolysis = [ (antimicrobial peptide solution experimental well OD value-negative well OD value)/(positive well OD value-negative well OD value) ]x100.

Table 2: percent hemolysis table of different concentrations of pepper seed protein antibacterial peptide p-C1 on pig red blood cells

The results in Table 2 show that p-C1 antimicrobial peptides showed hemolysis to porcine erythrocytes at a concentration of 6. Mu.g/mL and 100% hemolysis at 80. Mu.g/mL (FIG. 1), which is probably the reason why the process for preparing pricklyash seed protein antimicrobial peptides had a high stress response.

Example 2: preparation of Zanthoxylum seed protein peptide with better antibacterial effect on salmonella

Based on the preparation of the pepper seed protein antibacterial peptide p-C1, protease is selected again for enzymolysis of the pepper seed protein antibacterial peptide p-C1, and the Minimum Inhibitory Concentration (MIC) of salmonella is used as a screening standard to screen polypeptides with resistance to escherichia coli and salmonella at the same time.

The specific preparation and screening methods are as follows:

1) Dissolving semen Zanthoxyli protein in ethanol solution, and liquefying at 30-35deg.C to obtain ethanol treatment solution, wherein the concentration of semen Zanthoxyli protein in ethanol solution is 50-100mg/ml;

3) And (3) carrying out ultrasonic treatment on the cooled enzymolysis liquid, wherein the ultrasonic power is 150W, the ultrasonic treatment time is 5min, centrifuging for 10min at 8000r/min after ultrasonic treatment, taking supernatant, and carrying out vacuum freeze drying to obtain the pricklyash seed protein antibacterial peptide p-C1.

Adding water solution into the prepared pricklyash seed protein antibacterial peptide p-C1 to prepare 50mg/ml solution, and performing enzymolysis with trypsin, papain, subtilisin, neutral protease, bromelain and ficin at 35deg.C for 5 hr. And (3) after enzyme deactivation of the enzymolysis liquid, centrifuging at 3500rpm for 15 minutes, taking supernatant, and performing vacuum freeze drying to obtain enzymolysis products of different enzymes.

Wherein trypsin is subjected to enzymolysis at pH range of 7.9-8.5, and papain is subjected to enzymolysis at pH range of 5.5-6.8.

The enzymatic products of the different enzymes were dissolved in water to prepare mother solutions, and the mother solutions were diluted to 200. Mu.g/mL, 100. Mu.g/mL, 80. Mu.g/mL, 60. Mu.g/mL, 50. Mu.g/mL, 25. Mu.g/mL, 12.5. Mu.g/mL, and 6.25. Mu.g/mL, respectively, for Minimum Inhibitory Concentration (MIC) detection (Table 3).

Table 3: minimum Inhibitory Concentration (MIC) of different enzymatic products against Salmonella (μg/mL)

From the results in Table 3, it can be seen that the enzymatic hydrolysis product of papain has the best bacteriostasis to Salmonella.

The enzymatic hydrolysis product of papain was tested for hemolysis of swine erythrocytes by the method described in example 1. The experimental results are shown in table 4.

Table 4: table of percent hemolysis of enzymatic products of papain at different concentrations on porcine erythrocytes

The results in Table 4 show that the enzymatic product of papain exhibits hemolysis on porcine erythrocytes at a concentration of 12.5. Mu.g/mL, whereas the hemolysis at 100. Mu.g/mL is 68% (FIG. 2). Although the haemolysis is obviously reduced compared with the pepper seed protein antibacterial peptide p-C1, certain haemolysis exists.

Example 3: further purification of enzymatic products of papain

On the basis of retaining the anti-salmonella activity, the enzymolysis product of the papain is further purified, so that hemolytic substances are reduced, and components which cause stress reaction of piglets are reduced.

Preparing enzymolysis product of papain into 50mg/ml solution, separating and purifying with Sephadex G-25 gel column, eluting with deionized water at an eluting speed of 0.5ml/min, and collecting eluting peak at 220 nm. A total of five separation peaks F1-F5 were separated (FIG. 3).

The results of the anti-salmonella activity and hemolysis experiments performed on the products of the five separation peaks according to the detection method of example 2 show that the F5 component separated at 20-30min has not yet been hemolyzed at a concentration of 200 μg/mL on the basis of retaining the anti-salmonella activity. Thus, the F5 component was selected for further investigation.

1. Antibacterial property

Streaking salmonella to be inoculated to a flat-plate culture medium, and culturing at 37 ℃ for 24 hours; the single colonies were picked and inoculated into 100mL of liquid medium, respectively, and shake-cultured overnight at 37℃and 200r/min to prepare a bacterial solution.

Then inoculating 0.1mL of bacterial liquid into different plate culture mediums respectively, and uniformly coating. Round filter paper sheets soaked in 50 mug/mL of pepper seed protein antibacterial peptide p-C1 (figure 4A), enzymolysis product of papain (figure 4B), F5 component (figure 4C) solution of the enzymolysis product of papain are taken out, dried and placed in various plate culture mediums, and the plate culture mediums are placed in a 37 ℃ incubator for 12 hours.

The results show that the pepper seed protein antibacterial peptide p-C1 does not have obvious antibacterial ring, but under the same concentration condition, the antibacterial ring of the F5 component of the enzymolysis product of the papain is obviously larger than that of the enzymolysis product of the papain (figure 4), which shows that the content of the salmonella-resistant component in the F5 component of the enzymolysis product of the papain is higher.

2. Influence on piglet serum index

The antibacterial peptide p-C1 (p-C1) of the pricklyash seed protein, the enzymolysis product (pa-p) of the papain and the F5 component (pa-p-F5) of the enzymolysis product of the papain are prepared into oral liquid with the concentration of 100 mug/mL respectively. Randomly dividing 40 piglets of 14 days old before weaning into 4 groups, namely a Control Group (CG) which is only an aqueous solution, a pepper seed protein antibacterial peptide p-C1 group (p-C1), an enzymolysis product group (pa-p) of papain and an F5 group (pa-p-F5) of an enzymolysis product of papain; each group had 10 piglets. Experiments are respectively drenched with homemade oral liquid/water solution, 2 mL/head/time and 1 time a day except normal feeding. The medicine is continuously infused for 5 days.

After the experiment is finished, five indexes of alanine Aminotransferase (ALT), total Protein (TP), globulin (GLO), glucose (GLU) and Triglyceride (TG) in the collected serum of the experimental piglet are detected by using a kit.

As can be seen from fig. 5, the alanine Aminotransferase (ALT) content in the serum of piglets with different interventions was not significantly different from that of the normal control group (P > 0.05). Alanine Aminotransferase (ALT) is one of the important indexes reflecting whether liver metabolism is normal or whether liver is damaged, and from the analysis result of the index, the intervention of F5 group (pa-p-F5) of enzymatic hydrolysis product of melon protease does not cause liver damage.

As can be seen from FIG. 6, compared with the normal Control Group (CG), the Total Protein (TP) content in the piglet serum of the pa-P-F5 intervention group is obviously increased (P < 0.05) after intervention, the pa-P group also has obvious effect, and the P-C1 group has no obvious effect. The Total Protein (TP) is mainly synthesized by the liver, and the Total Protein (TP) content in the piglet serum is obviously improved by the intervention of pa-p-F5, so that the pa-p-F5 is likely to improve the feed intake and the digestion and absorption capacity of the piglet, so that the liver synthesized protein is increased, and the immunity of the piglet is also improved to a certain extent.

As can be seen from fig. 7, compared with the normal control group, the serum Globulin (GLO) content of the piglets in the intervention group is significantly increased (P < 0.05) after the intervention is performed in a different manner, and the pa-P-F5 group has the best effect, the pa-P group has the inferior effect, and the P-C1 group has the worst effect, which is only higher than the CG group. And the GLO content reflects the organism immunity condition of the piglets, and the GLO is reduced, which means that the organism disease resistance is reduced. From the detection results, the interference of the pa-p-F5 and the pa-p groups leads the GLO content in the piglet serum to be obviously increased, which proves that the pa-p-F5 and the pa-p truly improve the disease resistance of the piglet before weaning.

As can be seen from fig. 8, the Glucose (GLU) content in the piglet sera of the pa-P-F5 and pa-P intervention groups was significantly increased (P < 0.05) after the different manner of dry prognosis compared to the normal control group, and the Glucose (GLU) content in the piglet sera of the P-C1 and CG groups was not significantly changed (P > 0.05) with the highest pa-P-F5 content. The blood glucose concentration in serum is relatively constant and the blood glucose is directly oxidized to supply the energy required for metabolic activity in the animal body. The blood glucose level reflects the amount of glucose absorbed into the blood by the piglet's gut and is directly related to the digestibility of the piglet diet carbohydrates. The test results show that the high-dose intervention group and the high-dose group of the creep feed can increase the glucose content in the blood of the piglets compared with the normal control group. According to analysis, the increase of the blood sugar concentration means that the digestion and absorption effects of the intestinal tracts of the piglets on carbohydrates are obviously enhanced, and the fact that pa-p-F5 and pa-p are used as feed additives can improve the digestion rate of the piglets on nutrients including starch and non-starch polysaccharide.

As can be seen from fig. 9, the Triglyceride (TG) content in the serum of the piglets in the pa-P-F5 and pa-P intervention groups was significantly reduced (P < 0.05) after the dry prognosis in a different manner compared with the normal control group, and the pa-P-F5 intervention group was most reduced and the P-C1 group was not significantly reduced. Triglycerides (TG) are derived from the breakdown of fat, low levels of TG in serum are beneficial to piglet health, and elevated Triglycerides (TG) can induce morbidity in the body.

From the aspect of blood biochemical indexes, the oral liquid of pa-p-F5 and pa-p is safe and effective, the immunity of the piglets before weaning is improved, the purpose of preventing and treating diarrhea is achieved, and the action effect of pa-p-F5 is obviously higher than that of pa-p, which accords with the test result that the piglets in the pa-p-F5 and pa-p intervention group do not have diarrhea. The biochemical indexes of pa-p-F5 and pa-p provided by the invention meet the requirements of declaration of functional feeds and feed additives.

Example 4: effect of pa-p-F on diarrhea in piglets

Preparing F5 component of enzymolysis product of papain into creep feed, randomly dividing 40 piglets into 2 groups, normal Control Group (CG) and F5 component (pa-p-F5) of enzymolysis product of papain, each group comprising 20 piglets, wherein the following steps are:

(1) Normal control group: for 14-day-old piglets before 20 weaning, a pig farm health care program is applied.

(2) Creep feed (pa-p-F5) group: for 14-day-old piglets before 20 weaning, pa-p-F5 is added into feed according to the proportion of 1% and 0.5% for feeding respectively, and the feeding is carried out 1 time a day. Feeding continuously for 5 days.

The piglets were observed daily for diarrhea and the incidence was counted. After the end of the trial (about 6 hours after the last administration), it was observed that the creep feed (pa-p-F5) intervention group did not show diarrhea phenomena in piglets. The proportion of diarrhea of the normal control group occurring in piglets is 30% (figure 10), and according to the statistical result, the oral liquid and the creep feed have better intervention effect on preventing and treating the diarrhea of the piglets before weaning. The polypeptide prepared by the invention has good effect in preventing and treating piglet diarrhea.

Claims

1. The pepper seed protein polypeptide is characterized in that the preparation method of the pepper seed protein polypeptide comprises the following steps:

1) Dissolving pricklyash seed protein in ethanol solution to obtain ethanol treatment solution,

2) Adjusting the pH value of the treatment solution to 2-3 by using hydrochloric acid solution, then adding pepsin for enzymolysis, inactivating enzyme after enzymolysis, and cooling to obtain enzymolysis solution;

3) And carrying out ultrasonic treatment on the cooled enzymolysis liquid, centrifuging after ultrasonic treatment, taking supernatant, and carrying out vacuum freeze drying to obtain the protein polypeptide.

2. The pepper seed protein polypeptide as claimed in claim 1, characterized in that the pepper seed protein is added in ethanol solution at a concentration of 50-100mg/ml.

3. The pepper seed protein polypeptide as claimed in claim 1, wherein the ultrasonic power in 3) is 150W for 5min.

4. The pepper seed protein polypeptide as claimed in claim 1, wherein said centrifugation in 3) is a centrifugation at 8000r/min for 10min.

5. The pepper seed protein polypeptide as claimed in claim 1, wherein said pepper seed protein polypeptide is prepared by subjecting the protein polypeptide obtained in 3) to enzymolysis with papain.

6. The pepper seed protein polypeptide of claim 5, characterized in that the enzymolysis with papain is performed at 5.5-6.8 at 35 ℃ for 5h.

7. The pepper seed protein polypeptide of claim 5, wherein the enzymatic hydrolysate is separated and purified by using a Sephadex G-25 gel column, the eluent is deionized water at an elution rate of 0.5ml/min, and the separated components are collected for 20-30 min.

8. Use of a pricklyash seed protein polypeptide according to any one of claims 1-7 for the preparation of an anti-salmonella preparation.

9. Use of the pricklyash seed protein polypeptide according to any one of claims 1-7 as a feed additive.

10. A piglet feed, characterized in that the piglet feed is added with the pricklyash seed protein polypeptide according to any one of claims 1-7.