阅读说明：本技术 具有抑菌功能的生物活性肽、复合生物活性肽及其制备方法和用途 (Biological active peptide and composite biological active peptide with bacteriostatic function and preparation method and application thereof ) 是由 高峰 刘迎春 訾阳 杜贺阳 王珍如 格日勒图 于 2021-03-12 设计创作，主要内容包括：本发明公开了具有抑菌功能的生物活性肽、复合生物活性肽及其制备方法和用途。本发明首先公开了具有抑制细菌功能的生物活性肽,所述生物活性肽的制备方法包括：以羊血为原料利用酶水解法处理得到羊血血红蛋白,其中,所述的酶选自胃蛋白酶、中性蛋白酶、木瓜蛋白酶或胰蛋白酶中的任何一种。本发明进一步公开了具有更佳抑制细菌功能的复合生物活性肽,该复合生物活性肽由采用胃蛋白酶对羊血进行酶水解法处理得到的生物活性肽以及采用木瓜蛋白酶对羊血进行酶水解法处理得到的生物活性肽复合组成,体外和体内抑制试验结果表明,本发明所提供的生物活性肽或复合生物活性肽对于沙门氏菌和大肠杆菌具有显著的抑菌和抗炎作用。(The invention discloses a biological active peptide with a bacteriostatic function, a composite biological active peptide, a preparation method and application thereof. The invention firstly discloses a bioactive peptide with the function of inhibiting bacteria, and the preparation method of the bioactive peptide comprises the following steps: sheep blood is taken as a raw material and treated by an enzymatic hydrolysis method to obtain sheep blood hemoglobin, wherein the enzyme is selected from any one of pepsin, neutral protease, papain or trypsin. The invention further discloses a composite bioactive peptide with better bacteria inhibiting function, which is formed by compounding a bioactive peptide obtained by carrying out enzymatic hydrolysis on sheep blood by adopting pepsin and a bioactive peptide obtained by carrying out enzymatic hydrolysis on the sheep blood by adopting papain, and in-vitro and in-vivo inhibition test results show that the bioactive peptide or the composite bioactive peptide provided by the invention has obvious antibacterial and anti-inflammatory effects on salmonella and escherichia coli.)

1. A method for preparing a bioactive peptide having a function of inhibiting bacteria, the method comprising: processing sheep blood serving as a raw material by using an enzymatic hydrolysis method to obtain sheep blood hemoglobin; wherein the enzyme is selected from one of pepsin, neutral protease, papain or trypsin.

2. The bioactive peptide according to claim 1, wherein the temperature for enzymatic hydrolysis of sheep blood with pepsin is 37 ℃, the temperature for enzymatic hydrolysis of sheep blood with neutral protease is 45 ℃, the temperature for enzymatic hydrolysis of sheep blood with papain is 37 ℃ and the temperature for enzymatic hydrolysis of sheep blood with trypsin is 55 ℃.

3. The bioactive peptide according to claim 1, characterized in that in the enzymatic hydrolysis treatment of sheep blood with enzyme, the amount of enzyme added is 0.5-8 wt%, preferably 3 wt% of the mass of hemoglobin powder in sheep blood.

4. Bioactive peptide according to claim 1, characterised in that in the enzymatic hydrolysis of sheep blood with an enzyme the enzymatic hydrolysis is carried out for a period of 0.5-10h, preferably 1-6h, most preferably 3 h.

5. The bioactive peptide of claim 1 wherein the sheep blood hemoglobin obtained by enzymatic hydrolysis is purified by a process comprising: centrifuging the crude extractive solution of sheep blood hemoglobin obtained by enzyme hydrolysis, filtering the supernatant with sterilizing filter to obtain filtrate, sequentially placing the filtrate into ultrafiltration membranes of 10kDa, 5kDa and 2kDa, filtering, and collecting filtrate to obtain purified bioactive peptide.

6. The composite bioactive peptide with the function of inhibiting bacteria is characterized by being compounded by bioactive peptide obtained by carrying out enzymatic hydrolysis on sheep blood by pepsin and bioactive peptide obtained by carrying out enzymatic hydrolysis on the sheep blood by papain.

7. The complex bioactive peptide of claim 6, wherein said complex bioactive peptide is prepared from bioactive peptide obtained by enzymatic hydrolysis of sheep blood with pepsin and bioactive peptide obtained by enzymatic hydrolysis of sheep blood with papain in accordance with (1-5): (1-5), preferably, in a volume ratio of 1: 1 in volume ratio.

8. Use of a biologically active peptide according to any one of claims 1 to 5 in the manufacture of a medicament for inhibiting bacteria.

9. Use of the complex bioactive peptide of claim 6 or 7 in the manufacture of a medicament for inhibiting bacteria.

10. Use according to claim 8 or 9, wherein the bacteria are salmonella or escherichia coli.

Technical Field

The invention relates to a biological active peptide, in particular to a biological active peptide and a compound biological active peptide which are derived from sheep blood and have a bacteriostatic function and a preparation method thereof, and further relates to application of the biological active peptide and the compound biological active peptide in preparation of bacteriostatic drugs, belonging to the field of biological active peptides and preparation thereof.

Background

Bioactive Peptides are defined as specific protein fragments that have a positive effect on body function and condition and may ultimately affect health (Dziuba M, Darewicz M. Food Proteins as recursors of Bioactive Peptides-Classification activities [ J ]. Food & Technology International,2007,13(6): 393-404.). Bioactive peptides have the potential to modulate a range of physiological functions in the body. They can be encrypted in the polypeptide chain of the protein and can be released by proteolysis, where they can interact with appropriate receptors, exhibiting hormone-like activity (FitzGerald R, Murray B.A. biological peptides and fatty references [ J ]. International Journal of Dairy Technology,2006, (59): 118-. Bioactive peptides typically contain 3 to 20 amino acids, while bioactive peptides extracted from food typically contain 2 to 9 amino acids. Their activity depends on amino acid composition and sequence, and bioactive active peptides that have been reported to date include: antihypertensive peptides (antihypertensive peptides), antithrombotic peptides, opioid peptides, casein phosphopeptides (CPP), antibacterial peptides, cell regulatory peptides, and immunoregulatory peptides. According to their functional property classifications, bioactive peptides can be classified as antimicrobial peptides, immunomodulatory peptides, antioxidant peptides, antihypertensive peptides, and the like. These peptides play an important role in human health. Bioactive peptides have great potential for development, particularly in functional foods, by increasing their concentration in the food, causing them to produce measurable biological effects, or by introducing them into foods naturally free of peptides. Bioactive peptides as signaling molecules play important roles in physiological function and pathogenesis, and antimicrobial peptides and proteins are considered to have potential agricultural uses in current drug research. Initial studies have found that some naturally occurring bioactive peptides have the undesirable characteristics of non-specificity, toxicity, low stability, and low tendency to degrade, and the low effectiveness of these bioactive peptides can affect their application to agricultural crops. Advanced research techniques on extraction, separation, characterization, and molecular synthesis of molecular structures of bioactive peptides have been greatly developed and improved in the latter half of the last century, and thus, bioactive peptides have come into force. In recent years, a variety of polypeptides having excellent biological activity have been synthesized, they can be used in scientific fields such as medical treatment and immunity, and peptides and small proteins having antibacterial activity have been isolated from many organisms, their role in defense has been determined, and their use in agriculture has been excavated as soon as they are found. However, some natural peptides have undesirable properties, complicating their use, which has also led to advances in polypeptide synthesis and high throughput activity screening, providing opportunities for redesign and rational design of new peptides.

Antimicrobial peptides were first found in insect larvae by the Karolinska research institute Dr. Hans Berman (Boman H G, Hultmark D. cell-free immunity in insects. Annu [ J ]. Rev Microbiol,1987,41(1): 103-. Antimicrobial peptides can be divided into two broad categories according to their molecular structure: linear peptides and disulfide-bonded rich cysteine peptides. Cysteine-rich antimicrobial peptides were first found in circulating phagocytic cells of mammals, and one function of these cells is to recognize foreign microorganisms, take them up and kill them. One mechanism by which cells kill ingested microorganisms is the intracellular release of antimicrobial peptides. In mammals, there are two main families of antimicrobial peptides: defensins and cathelicidins. After confirming the presence of defensins in phagocytes, other scientists have also found cysteine-rich peptides similar to linear peptides and found such peptides in the mucous-coated epithelium of the trachea and intestines, which can be considered equivalents on mammalian skin where bacteria are often seen and provide a good environment for their reproduction. Thus, these broad spectrum antibiotics are particularly important as a first line of defense against inhaled pathogens in air, food and water. Antimicrobial peptides have now been identified in nature in plant seeds, fish skin secretions and crab blood. Peptide-based defense mechanisms are widely available in the animal kingdom, and further studies have identified antimicrobial peptides in every species from bacteria to plants to humans. Antimicrobial peptides play an important role in the evolution of complex multicellular organisms, and have a powerful broad spectrum of antimicrobial properties for combating a wide variety of microorganisms, including bacteria, fungi, viruses, and protozoa. Its biological diversity is also determined by its widespread existence in animals and plants.

Antibacterial peptides usually consist of 30-60 amino acids, have the advantages of good thermal stability, stable protease activity and broad-spectrum bacteriostasis, and can quickly and effectively kill fungi and bacteria (Srensen OE, Borregaard N, et al. antibacterial peptides in amino acid microorganisms [ J ]. Contrib Microbiol,2008, (15): 61-77.). As a new approach to anti-infective therapy, Robert Hancock and Hans Georg Sah exist in almost all life forms and are important components of innate immune defense as short cationic amphiphilic peptides with antibacterial and immunomodulatory activity. These peptides provide templates for two different classes of antibacterial drugs, and the direct-acting antimicrobial host defense peptides have rapid, highly potent, and exceptionally broad-spectrum activity. In mammals, these peptides generally have very weak antibacterial activity under physiological conditions, but their ability to modulate the immune response through various mechanisms is particularly important in the organism, a function which is an integral part of the process of innate immunity, which itself has many of the characteristics of successful anti-infective therapy, namely rapid action and broad spectrum antibacterial activity. Therefore, antimicrobial peptides are considered as a new generation of antibiotics, and also as natural immunomodulators.

Chinese animal blood has abundant resources, wide sources, easy obtainment and the like, and the value of the animal blood is not fully utilized due to single by-product. However, with the development of modern technology, animal blood can be hydrolyzed into active peptides, and the peptides have the effects of preventing and treating diseases and regulating human physiological functions. In the existing research, the research on the bioactive peptides of pig blood and cow blood is more, but the research on the source of sheep blood is less, and the research system is incomplete. Therefore, animal blood is utilized to decompose hemoglobin in blood cells into globin and heme, and then the globin is subjected to enzymolysis to obtain peptide substances with biological activity, the peptide substances are used in the food industry and the pharmaceutical industry, the ever-increasing requirements of people on the functionality of food are greatly met, the pollution of blood to the environment when animals are slaughtered is solved, and the utilization rate of protein resources is improved. In order to enable the bioactive peptide to better exert the bacteriostatic activity per se and enable the diarrhea of livestock and poultry animals to be treated more efficiently and quickly in the practical and suitable production, the research on the bacteriostatic activity of the bioactive peptide has the practical production significance for solving the problem.

The inner Mongolia area is a province where mutton production and consumption in China are great, sheep blood is used as a mutton slaughtering byproduct and can be processed into conventional protein feed, but the sheep blood is often not approved as animal feed due to the characteristics of safety, complex treatment steps and poor palatability of animal sources, so that the phenomenon of waste is very serious, and the problem of serious environmental pollution is caused by improper discharge of blood. Livestock diarrhea is a common multiple disease in animal husbandry production, and the diarrhea caused by high morbidity and mortality becomes a big problem in animal husbandry production.

Disclosure of Invention

One of the purposes of the invention is to provide a biological active peptide or composite biological active peptide which is derived from sheep blood and has a bacteriostatic function;

the second purpose of the invention is to provide a method for preparing the biological active peptide or the compound biological active peptide with the bacteriostatic function;

the third purpose of the invention is to use the biological active peptide or the compound biological active peptide to prepare bacteriostatic drugs.

The above object of the present invention is achieved by the following technical solutions:

the invention firstly provides a bioactive peptide with the function of inhibiting bacteria, and the preparation method comprises the following steps: sheep blood hemoglobin obtained by treating sheep blood as a raw material by using an enzymatic hydrolysis method, wherein the enzyme is selected from any one of pepsin, neutral protease, papain or trypsin; preferably, the enzyme is pepsin. Wherein, when pepsin is used for carrying out the enzymolysis treatment on the sheep blood, the temperature of the enzymolysis method is preferably 37 ℃, when neutral protease is used for carrying out the enzymolysis treatment on the sheep blood, the temperature of the enzymolysis method can be 45 ℃, when papain is used for carrying out the enzymolysis treatment on the sheep blood, the temperature of the enzymolysis method is preferably 37 ℃, and when trypsin is used for carrying out the enzymolysis treatment on the sheep blood, the temperature of the enzymolysis method is preferably 55 ℃.

As a preferred embodiment, in the enzymatic hydrolysis treatment of sheep blood with enzyme, the enzyme is added in an amount of 0.5-8 wt%, most preferably 3 wt% based on the mass of hemoglobin powder.

As a preferred embodiment, when the sheep blood is treated by enzymatic hydrolysis with an enzyme, the time for the enzymatic hydrolysis may be 0.5 to 10 hours, preferably 1 to 6 hours, and most preferably 3 hours.

In order to realize better bacteriostatic effect, the sheep blood hemoglobin obtained by the enzyme hydrolysis method can be purified, which comprises the following steps: centrifuging the crude extractive solution of sheep blood hemoglobin obtained by enzyme hydrolysis, filtering the supernatant with sterilizing filter, sequentially filtering the filtrate with ultrafiltration membranes of 10kDa, 5kDa and 2kDa, and collecting the filtrate to obtain purified bioactive peptide.

The invention further provides a composite biological active peptide with better bacteria inhibiting effect, which is composed of two sheep blood hemoglobins obtained by respectively carrying out enzymatic hydrolysis treatment on sheep blood by using pepsin and papain as raw materials.

Wherein, the two sheep blood hemoglobins can be composed according to any proportion, preferably, the two sheep blood hemoglobins are (1-5): (1-5), more preferably, the two sheep hemoglobins are present in a volume ratio of 1: 1 in volume ratio. The bacterium described in the present invention is preferably salmonella or escherichia coli.

Detailed description of the specific embodiments of the invention

The method comprises the steps of firstly mixing bioactive peptides obtained by carrying out enzymolysis on sheep blood hemoglobin by pepsin, trypsin, neutral protease and papain for 1-6h with normal saline according to the volume ratio of 7:3, diluting the bioactive peptides, and carrying out bacteriostatic tests on salmonella, common escherichia coli and E.coli K88 by using the bioactive peptides diluted by the normal saline. The bacteriostatic activity of the 4 protease enzymatic biological active peptides on salmonella, common escherichia coli and E.coli K88 is preliminarily verified by a drug sensitive paper sheet method. Wherein, the bioactive peptides which are subjected to enzymolysis for 3h, 4h, 5h and 6h by pepsin have no inhibition zone on salmonella, common escherichia coli and E.coli K88, but have inhibition zones with higher sensitivity on salmonella, common escherichia coli and E.coli K88 at 1h and 2h of enzymolysis, and the diameters of the inhibition zones are respectively 12.05mm (1h, salmonella), 11.99mm (2h, salmonella), 15.89mm (1h, escherichia coli), 15.05mm (2h, escherichia coli), 14.58mm (1h, E.coli K88) and 14.83mm (2h, E.coli K88); neutral protease enzymolysis 2h, 3h, 4h, 5h and 6h of bioactive peptide has no inhibition zone on salmonella, common escherichia coli and E.coli K88, but neutral protease enzymolysis 1h of bioactive peptide has an inhibition zone highly sensitive to common escherichia coli, and the diameter of the inhibition zone is 14.81 mm; no bacteriostatic zone appears in the trypsin enzymolysis and the papain enzymolysis for 1h-6 h. Can preliminarily judge that the biological active peptide with better antibacterial performance can be prepared by the pepsin enzymolysis method.

The minimum inhibitory concentration of the goat blood-derived bioactive peptide on salmonella, common escherichia coli and E.coli K88 is determined, wherein the minimum inhibitory concentration of the pepsin bioactive peptide on the salmonella after 3 hours of enzymolysis is 18.27 mu g/mL; the minimum inhibitory concentration of the pepsin bioactive peptide on salmonella after 3 hours of enzymolysis is 18.27 mug/mL; the minimum inhibitory concentration of the pepsin bioactive peptide on E.coli K88 after 3 hours of enzymolysis is 36.54 mu g/mL. The trypsin bioactive peptide has no inhibiting effect on salmonella, common escherichia coli and E.coli K88 after being subjected to enzymolysis for 1-6h, namely has no MIC value; the neutral protease bioactive peptide has no inhibition effect on salmonella, common escherichia coli and E.coli K88 after being subjected to enzymolysis for 1-6h, namely has no MIC value; the papain bioactive peptide has no inhibitory effect on salmonella, common escherichia coli and E.coli K88 after being subjected to enzymolysis for 1-6h, namely has no MIC value. In conclusion, the bioactive peptide prepared by the 4 proteases has bacteriostatic activity on salmonella, common escherichia coli and E.coli K88 only after being subjected to enzymolysis for 1-6h and 3h, and has no bacteriostatic effect on the salmonella, the common escherichia coli and E.coli K88 at other times. Can preliminarily judge that the biological active peptide with better antibacterial performance can be prepared by the enzymolysis of pepsin for 3 hours.

The invention further investigates the in vitro bacteriostatic effect of 3 groups of sheep blood-derived composite bioactive peptides, wherein the 3 groups of composite bioactive peptides are respectively as follows: (1) the pepsin enzymatic hydrolysis bioactive peptide and the neutral protease enzymatic hydrolysis bioactive peptide are prepared according to the following steps of 1: 1 in volume ratio; (2) the pepsin enzymatic hydrolysis bioactive peptide and the papain enzymatic hydrolysis bioactive peptide are prepared according to the following steps of 1: 1 in volume ratio; (3) the papain enzymolysis bioactive peptide and the neutral protease enzymolysis bioactive peptide are prepared according to the following steps of 1: 1 in volume ratio; the bacteriostatic activity of the composite protease enzymatic biological active peptide on salmonella and common escherichia coli is preliminarily verified by a drug sensitive paper sheet method.

According to the test results, the following results can be seen: the composite bioactive peptide prepared by enzymolysis of pepsin and neutral protease for 3h, 4h, 5h and 6h has no inhibition zone on salmonella and common escherichia coli, but has inhibition zones with higher sensitivity on salmonella and common escherichia coli in enzymolysis for 1h and 2h, and the diameters of the inhibition zones are respectively 11.68mm (1h, salmonella), 13.71mm (2h, salmonella), 10.48mm (1h, escherichia coli) and 12.32mm (2h, escherichia coli); the composite bioactive peptide obtained by enzymolysis of pepsin and papain for 3 hours has no inhibition zone on salmonella and common escherichia coli, but the composite bioactive peptide obtained by enzymolysis of pepsin and neutral protease for 1 hour, 2 hours, 4 hours, 5 hours and 6 hours has an inhibition zone highly sensitive to salmonella and common escherichia coli, and the diameter of the inhibition zone is 9.36mm (1 hour, salmonella), 12.85mm (2 hour, salmonella), 9.21mm (4 hour, salmonella), 11.86mm 5(5 hour, salmonella), 15.06mm (6 hour, salmonella), 10.86mm (1 hour, escherichia coli), 12.38mm (2 hour, escherichia coli), 11.84mm (4 hour, escherichia coli), 10.01mm (5 hour, escherichia coli), 9.89mm (6 hour, escherichia coli); the papain and the neutral protease are subjected to enzymolysis for 1h-6h, and the bioactive peptide compounded by the pepsin and the papain enzymolysis method is preliminarily judged to be capable of preparing the bioactive peptide with better antibacterial performance.

The invention further inspects the in vivo bacteriostasis effect of the sheep blood source biological active peptide, and the test result proves that the sheep blood source biological active peptide has the in vivo bacteriostasis and anti-inflammation functions.

The invention takes sheep blood as a raw material and utilizes an enzymatic hydrolysis method to process sheep blood hemoglobin to obtain the bioactive peptide with antibacterial property, researches the antibacterial property of the peptide, aims to provide certain basic data for the anti-diarrhea pathogenic bacteria of the bioactive peptide from the sheep blood in animal husbandry, provides certain theoretical basis for the development and utilization of the bioactive peptide from the sheep blood, and has certain economic value and social benefit for solving the pollution of blood to the environment when animals are slaughtered and improving the application of animal blood feed resources in the animal husbandry production.

Drawings

FIG. 1 results for inhibition of Salmonella by pepsin, neutral protease, papain and trypsin drug sensitive paper sheets.

FIG. 2 results of inhibition of common Escherichia coli by pepsin, neutral protease, papain and trypsin drug sensitive paper

Fig. 3 results for inhibition of e.coli K88 by pepsin, neutral protease, papain and trypsin drug sensitive paper sheets.

FIG. 4 shows the result of inhibiting Salmonella by pepsin and neutral protease complex enzyme drug sensitive paper.

FIG. 5 shows the result of inhibiting Salmonella by pepsin and papain complex enzyme drug sensitive paper.

FIG. 6 shows the results of inhibiting Escherichia coli by pepsin and neutral protease complex enzyme drug sensitive paper.

FIG. 7 shows the results of inhibiting Escherichia coli by pepsin and papain complex enzyme drug sensitive paper.

FIG. 8 duodenum sections of blank control group, negative control group, positive control group, test 1 group and test 2 group.

FIG. 9 jejunal sections of blank control group, negative control group, positive control group, test 1 group and test 2 group.

Figure 10 ileal sections of blank control, negative control, positive control, test 1 and test 2 groups.

FIG. 11 is a graph showing the effect of bioactive peptides on the expression level of TNF-A gene in rat small intestine.

FIG. 12 is a graph showing the effect of bioactive peptides on the expression level of IL-6 gene in rat small intestine.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. It is to be understood that the described embodiments are exemplary only and are not limiting upon the scope of the invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.

Test example 1 preparation of crude extract of biologically active peptide derived from sheep blood and in vitro antibacterial test

1. Sample source

Fresh sheep blood was collected from a certain slaughterhouse of the four kingdoms.

2. Test method

2.1 separation of hemoglobin from sheep blood

Adding anticoagulant prepared by mixing 24.5g of glucose, 8g of citric acid and 22g of sodium citrate with 1L of distilled water into fresh sheep blood, centrifuging at 4 ℃ for 10min at 10000r/min, collecting hemoglobin precipitate, adding deionized water with the same volume, mixing uniformly, performing ultrasonic crushing for 10min, centrifuging at 4 ℃ for 10min at 4000r/min, collecting clean hemoglobin, freeze-drying in a freeze dryer for 24h, and grinding into powder at 4 ℃ for storage.

2.2 preparation of crude extract of sheep blood-derived bioactive peptide

Calculating the volume of the bioactive peptide according to the required amount, dissolving the hemoglobin powder with water (the mass of the hemoglobin powder is 5 percent of the added water amount), adding the enzyme in an amount which is 3 percent of the mass of the hemoglobin powder, and respectively adding pepsin, neutral protease, papain and trypsin for hydrolysis under the optimal action conditions of the enzymes. The 4 sets of experiments were performed simultaneously, and were dosed as required.

Hydrolyzing the mixture for 6h (shown in Table 1) at 100r/min in a gas bath shaker according to the optimal temperature and pH value of pepsin activity, trypsin activity, neutral protease activity and papain activity, measuring the pH value of the reaction system every 1h during the test, maintaining the pH value of the reaction system within a specified range, dropwise adding NaOH (1moL/L) when the reaction system is too acid, and dropwise adding HCl (1moL/L) when the reaction system is too alkaline.

Sampling at 1h, 2h, 3h, 4h, 5h and 6h in the reaction process, heating in boiling water bath for 10min after reaction, centrifuging at 4 deg.C 5000r/min for 10min to obtain crude bioactive peptide extractive solution, and refrigerating at 4 deg.C for use.

TABLE 14 optimal reaction conditions for proteases

2.3 separation and purification of sheep blood-derived bioactive peptides

After obtaining the crude extract of the bioactive peptide, centrifuging the crude liquid at 3500r/min for 5min at room temperature, firstly passing through a 0.45 mu m sterilizing filter and then passing through a 0.22 mu m sterilizing filter, sequentially putting the obtained filtrate into ultrafiltration membranes with the specifications of 10kDa, 5kDa and 2kDa for filtering, and then filtering, wherein the obtained liquid is the final bioactive peptide.

2.4 in vitro antibacterial test of sheep blood-derived bioactive peptides

2.4.1 culture and isolation of test strains

Collecting test strains: salmonella separated and identified in a chick diarrhea excrement sample, escherichia coli separated and identified in a sheep excrement sample and a standard escherichia coli strain (serotype O149: K88)

② weighing 1g of the collected fresh excrement sample, placing the weighed fresh excrement sample into an EP tube, adding 10ml of normal saline, and uniformly mixing the weighed fresh excrement sample with the normal saline by oscillation. And sucking 200 mul of the mixed solution, and placing the mixed solution in a sorbitol MacConKai agar culture medium to be cultured for 18h at the constant temperature of 37 ℃. Selecting a single bacterial colony from a culture dish, selecting the single bacterial colony into physiological saline to prepare bacterial suspension, sucking 200 mul of the bacterial suspension, placing the bacterial suspension in a sorbitol MacconKate agar culture medium for constant-temperature culture at 37 ℃ for 18h, and preliminarily determining the bacterial colony to be escherichia coli and salmonella according to the color development indication of the eosin methylene blue culture medium. Repeating the above steps for 3 times to obtain separated and purified salmonella, escherichia coli and a standard escherichia coli strain (serotype O149: K88).

2.4.2 determination of zone of inhibition diameter (drug sensitive paper method)

Mixing bioactive peptides obtained by carrying out enzymolysis on sheep blood hemoglobin by 4 proteases including pepsin, trypsin, neutral protease and papain for 1-6h with normal saline according to the volume ratio of 7:3, diluting the bioactive peptides, and carrying out bacteriostatic tests on salmonella, common escherichia coli and E.coli K88 by using the bioactive peptides diluted by the normal saline. The test method is as follows:

preparation of drug sensitive paper sheet

Using a puncher to punch qualitative filter paper into circular paper sheets with the diameter of 6mm, putting the filter paper into sterilized wide-necked bottles, filling 20 sheets of the filter paper into each bottle, pouring 70% of liquid medicine into the corresponding bottles respectively, and putting the wide-necked bottles into an autoclave for autoclaving (121 ℃, 27 minutes). Soaking in shaking table at 37 deg.C for 18 hr at 100r/min, pouring out the excessive medicinal liquid, oven drying at 37 deg.C in oven, and storing in refrigerator at 4 deg.C for use.

② drug sensitive test procedure

Cultured salmonella, common escherichia coli and e.coli K88 were picked into a sterile saline tube with a sterile pipette tip, 200 μ l of bacterial suspension (0.5 mm turbidimetric tube concentration) was aspirated into a petri dish and poured over a sorbitol mcanckei agar plate, and the bacterial suspension was thoroughly mixed with the liquid medium while the medium was not yet solidified. After the water on the plate is completely absorbed by the agar, the drug sensitive paper is stuck on the culture medium by using sterile tweezers. Each plate is pasted with 6 drug sensitive paper sheets, the paper sheets are fully contacted with the surface of the agar and cannot be taken up after being pasted, the distance between the paper sheets is not less than 24mm, and the distance between the centers of the paper sheets and the edge of the plate is not less than 15 mm. The position of various drug sensitive paper sheets was recorded. And (3) placing the flat plate at a constant temperature of 37 ℃ for culturing for 18-24h, taking out, photographing, measuring the diameter of the inhibition zone by using ImageJ1.8.0 for five times, and calculating the average value to obtain the experimental result.

2.4.3 determination of minimum inhibitory concentration (MIC method)

Picking single bacterial colony of cultured salmonella, common escherichia coli and E.coli K88 by using a pipette tip, and diluting the bacterial colony in a test tube by using normal saline until the turbidity degree of the diluted normal saline is the same as that of a 0.5 McLeod turbiditube.

The minimum inhibitory concentration of the biological active peptide obtained by 6h enzymolysis of sheep blood hemoglobin by 4 enzymes to three bacteria is determined by adopting a trace broth two-fold dilution method. Adding 100 μ l of EC broth into 1-11 th well of 96-well plate, adding 100 μ l of liquid bioactive peptide stock solution into 1 st well, blowing, mixing, sucking 100 μ l to 2 nd well, sucking 100 μ l to 3 rd well, diluting at multiple times to 11 th well, sucking 100 μ l from 11 th well, discarding, adding 100 μ l into each well, and concentrating to 1 × 10⁸CFU/ml bacterial suspension, 100. mu.l EC broth alone and 100. mu.l concentration 1X 10 were added to well 12⁸CFU/ml bacterial suspension served as blank control. In this case, the concentrations of the drugs in each well are 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625, 0.0078125, 0.00390625, 0.001953125 and 0.0009765 times of the concentration of the bioactive peptide protein in sequence. Placing the 96-well plate in a constant temperature incubator at 37 deg.C for incubation for 18h, observing colony growth, and optionally adding5 mul of TTC color developing agent is added, and the color developing agent is added to be cultured for 3 hours at the constant temperature of 37 ℃ and then the color change condition is observed.

2.4.4 Effect of sheep blood-derived bioactive peptides on the permeability of cell membranes of 3 bacteria

Selecting single bacterial colony of cultured salmonella, common escherichia coli and E.coli K88 with pipette tip, diluting with normal saline, and adjusting concentration of E.coli K88 bacterial liquid to 1 × 10⁶CFU/mL. 200. mu.L of the bacterial suspension was added to a blank control well of a 96-well plate, followed by 10. mu.L of 0.035mol/LONPG, respectively, and then 10. mu.L of pepsin bioactive peptide (MIC concentration of E.coli K88) was added.

Incubation at 37 ℃ and OD determination of each well solution at 10, 30, 60, 90 and 120min₄₁₅3 parallel experiments were performed at each time point, and the experimental protocol is shown in Table 2.

TABLE 2 test scheme for influence of sheep blood-derived bioactive peptide on cell membrane permeability of 3 bacteria

In addition, when the culture was carried out for 60min, the resultant ONPG content per minute in the supernatant was measured by centrifugation at 4000r/min at room temperature for 20 min. The formula is (A)₄₁₅X 1000/sample volume (. mu.L)/reaction time (min). times.4.86, A₄₁₅Represents the OD value of the supernatant, and 4.86 is the ONPG extinction coefficient (cm/mM)).

2.4.5 Effect of sheep blood-derived bioactive peptides on surface hydrophobicity of 3 bacterial cells

The experimental scheme for the influence of the sheep blood-derived bioactive peptide on the surface hydrophobicity of 3 bacterial cells is shown in Table 3. Weighing 37.5g EC broth, dissolving in 1L distilled water, mixing thoroughly to dissolve, autoclaving (121 deg.C, 15 min.) to culture Salmonella, Escherichia coli and E.coli K88 in nutrient broth to logarithmic phase, centrifuging at 5000r for 10min, discarding supernatant and leaving thalli precipitate, and washing with 0.1mol/L sterile PBS 3 times. Then the pellet was resuspended in 0.1mol/L KNO₃In (b), the adjusted concentration (AO) is 1X 10⁶CFU/mL is ready for use. (as per Table 3 addAdding into each solution) at room temperature for 10min, vortex oscillating for 2min to mix each solution thoroughly, standing for 15min, collecting upper layer liquid, placing in 96-well plate, repeating each group for 3 times, and determining OD₄₀₀(A1) The value is obtained. The hydrophobicity (adhesiveness) of 3 kinds of bacteria to each solvent was calculated.

Calculating the formula: coli hydrophobicity (adhesiveness) ═ 1-a1/a0 × 100%.

TABLE 3 influence test scheme of sheep blood-derived bioactive peptide on surface hydrophobicity of 3 bacteria cells

2.4.6 Effect of sheep blood-derived bioactive peptides on protein concentration of 3 bacterial culture solutions

Selecting single bacterial colony of cultured salmonella, common escherichia coli and E.coli K88 with pipette tip in test tube, diluting with normal saline, and adjusting bacterial suspension concentration to 1 × 10⁶CFU/mL. Is prepared to have a concentration of 1 × 10⁶CFU/mL bacterial suspension, adjusting the concentration of the bacterial suspension and the bioactive peptide to be 1MIC in an EP tube, continuously culturing for 6h in a gas bath shaking table with the temperature of 37 ℃ and the rmp/min, timing after putting the bacterial suspension into the shaking table, sampling once every 1h, determining the protein concentration of the sample by using a BCA protein concentration determination kit, and repeating three times in each group.

3. Test results

3.1 influence of sheep blood-derived bioactive peptide on diameter of inhibition zone of 3 bacteria

The bacteriostatic activity of the 4 protease enzymatic biological active peptides on salmonella, common escherichia coli and E.coli K88 is preliminarily verified by a drug sensitive paper sheet method.

TABLE 44 inhibition zone diameters of 3 bacteria by protease enzymatic hydrolysis of bioactive peptides: (_mm)

Note: the diameter R of the bacteriostatic circle is more than or equal to 15_mmIs extremely sensitive; r is more than or equal to 10 and less than 15_mmHigh sensitivity; r is more than or equal to 8 and less than 10_mmLow sensitivity; r < 8_mm(ii) a Is 6 in the insensitive table_mmIs the diameter of the drug sensitive paper sheet

The bacteriostatic results according to table 4 and figures 1-3 show that: the bioactive peptides which are subjected to enzymolysis for 3h, 4h, 5h and 6h by pepsin have no inhibition zone on salmonella, common escherichia coli and E.coli K88, but have inhibition zones with higher sensitivity on salmonella, common escherichia coli and E.coli K88 in enzymolysis for 1h and 2h, and the diameters of the inhibition zones are respectively 12.05mm (1h, salmonella), 11.99mm (2h, salmonella), 15.89mm (1h, escherichia coli), 15.05mm (2h, escherichia coli), 14.58mm (1h, E.coli K88) and 14.83mm (2h, E.coli K88); neutral protease enzymolysis 2h, 3h, 4h, 5h and 6h of bioactive peptide has no inhibition zone on salmonella, common escherichia coli and E.coli K88, but neutral protease enzymolysis 1h of bioactive peptide has an inhibition zone highly sensitive to common escherichia coli, and the diameter of the inhibition zone is 14.81 mm; no bacteriostatic zone appears in the trypsin enzymolysis and the papain enzymolysis for 1h-6 h. Can preliminarily judge that the biological active peptide with better antibacterial performance can be prepared by the pepsin enzymolysis method.

3.2 minimum inhibitory concentration of sheep blood-derived bioactive peptide on 3 bacteria

The minimum inhibitory concentration (namely MIC) test results of the bioactive peptide prepared by 4 proteases on salmonella, common escherichia coli and E.coli K88 are shown in Table 5, and the minimum inhibitory concentration of the pepsin bioactive peptide on the salmonella after 3 hours of enzymolysis is 18.27 mu g/mL; the minimum inhibitory concentration of the pepsin bioactive peptide on salmonella after 3 hours of enzymolysis is 18.27 mug/mL; the minimum inhibitory concentration of the pepsin bioactive peptide on E.coli K88 after 3 hours of enzymolysis is 36.54 mu g/mL. The trypsin bioactive peptide has no inhibiting effect on salmonella, common escherichia coli and E.coli K88 after being subjected to enzymolysis for 1-6h, namely has no MIC value; the neutral protease bioactive peptide has no inhibition effect on salmonella, common escherichia coli and E.coli K88 after being subjected to enzymolysis for 1-6h, namely has no MIC value; the papain bioactive peptide has no inhibitory effect on salmonella, common escherichia coli and E.coli K88 after being subjected to enzymolysis for 1-6h, namely has no MIC value. In conclusion, the bioactive peptide prepared by the 4 proteases has bacteriostatic activity on salmonella, common escherichia coli and E.coli K88 only after being subjected to enzymolysis for 1-6h and 3h, and has no bacteriostatic effect on the salmonella, the common escherichia coli and E.coli K88 at other times. Can preliminarily judge that the biological active peptide with better antibacterial performance can be prepared by the enzymolysis of pepsin for 3 hours.

TABLE 5 minimum inhibitory concentration of sheep blood-derived bioactive peptide on 3 bacteria

3.3 Effect of sheep blood-derived bioactive peptides on the permeability of cell membranes of 3 bacteria

The results of the effect of pepsin enzymolysis for 3h on the permeability of the cell membranes of 3 bacteria are shown in Table 6. As can be seen from table 6, since the e.coli K88 negative control group only added the bacterial suspension and the physiological saline without adding the substance for destroying the cell membrane permeability, it was shown that the addition of the physiological saline to the bacterial suspension did not destroy the cell membrane of serotype e.coli K88, resulting in the lowest β -galactosidase activity content in the culture solution (P < 0.01). The positive control group added 2% T-ritonX, which is extremely destructive to cell membrane, resulted in the rupture of E.coli K88 cell membrane and the release of beta-galactosidase into the culture medium, resulting in the highest beta-galactosidase activity content (P < 0.01) in the culture medium. The added pepsin bioactive peptide group has a very significant difference (P is less than 0.01) from a negative control and a positive control due to the damage effect on the cell membrane of E.coli K88. The more beta-galactosidase is released into the culture over time, and therefore the more active pepset of pepsin is due to the destructive effect on the cell membrane of e.coli K88. The release amount of beta-galactosidase is shown in the sequence of positive control group > pepsin bioactive peptide test group > negative control group. As can be seen from tables 7 and 8, the effect of pepsin enzymolysis for 3h on the cell membrane permeability of Salmonella and Escherichia coli was consistent with the above results.

TABLE 6 influence of sheep blood-derived bioactive peptides on Salmonella cell membrane permeability

Note: data are shown as (Mean ± Sd) where upper case letters indicate temporal differences and lower case letters indicate differences between groups. The same row of numbers with different shoulder marks represent significant difference (P <0.05), and the same shoulder marks represent insignificant difference (P > 0.05).

TABLE 7 influence of sheep blood-derived bioactive peptides on the permeability of common Escherichia coli cell membranes

Note: data are shown in the table as (Mean ± Sd), where upper case letters indicate time differences and lower case letters indicate differences between groups. The same row of numbers with different shoulder marks represent significant difference (P <0.05), and the same shoulder marks represent insignificant difference (P > 0.05).

Table 8 effect of sheep blood-derived bioactive peptides on e.coli K88 cell membrane permeability

Note: data are shown as (Mean ± Sd) where upper case letters indicate temporal differences and lower case letters indicate differences between groups. The same row of numbers with different shoulder marks represent significant difference (P <0.05), and the same shoulder marks represent insignificant difference (P > 0.05).

3.4 Effect of sheep blood-derived bioactive peptides on the hydrophobicity of E.coli K88 cell surface

The acid-base property of the pepsin bioactive peptide is determined according to the principle of similar compatibility. As can be seen from table 9, the hydrophobicity of the pepsin experimental group to e.coli K88 is 99.18%, the hydrophobicity to e.coli K88 is all greater than that of the blank control group (25.15%), the acidic control group (75.85%), and the alkaline control group (32.24%), the difference between groups is very significant (P is less than 0.01), which indicates that the adhesiveness of the pepsin bioactive peptide to e.coli K88 is higher than that of acidic chloroform to e.coli K88, and the hydrophobicity of the salmonella and the common escherichia coli experimental group in table 9 is greater than that of the blank control group, and the alkaline control group and the acidic control group are not different from that of the acidic control group, so it is determined that the pepsin bioactive peptide has strong acidity.

TABLE 9 influence of sheep blood-derived bioactive peptides on surface hydrophobicity of 3 bacterial cells

Note: data in the table are expressed by (Mean ± Sd), the difference of the same column number with shoulder mark letter is significant (P <0.05), and the difference is not significant with the same shoulder mark letter (P > 0.05). 3.5 Effect of sheep blood-derived bioactive peptides on E.coli K88 culture fluid protein concentration

The influence of the goat blood-derived bioactive peptides on the protein content in the culture solution of serotype E.coli K88, common escherichia coli and salmonella is shown in Table 10, and as can be seen from Table 10, the concentration of the bioactive peptides protein is 146.15 +/-2.56 mu g/mL after pepsin enzymolysis for 3h, the bioactive peptides can damage the integrity of cells, so that the protein in E.coli K88, common escherichia coli and salmonella thalli is leaked into the culture solution, and the protein concentration in the culture solution is higher than the bioactive peptide concentration. And the amount of the damaged E.coli K88, common escherichia coli and salmonella cell membranes is increased along with the increase of time, and the protein content in the culture solution reaches 175.77 +/-10.95 mu g/mL, 169.30 +/-0.029 mu g/mL and 173.90 +/-0.057 mu g/mL respectively when the time reaches 6 hours.

TABLE 10 Effect of sheep blood-derived bioactive peptides on protein concentration in culture of 3 bacteria

Note: data in the table are expressed by (Mean ± Sd), the difference of the same row number and shoulder mark letter indicates significant difference (P <0.05), and the difference of the same shoulder mark letter indicates insignificant difference (P > 0.05). Test example 2 in vitro inhibitory test of composite biologically active peptide derived from sheep blood

1. Test materials

Bioactive peptide, qualitative filter paper, salmonella, common Escherichia coli, sterilized normal saline, eosin methylene blue culture medium, pepsin, neutral protease, papain, trypsin, wide-mouth bottle, sterilized forceps, and sterilized gun head

2. Test method

2.1. Preparation of drug sensitive paper sheet

2.1.1 preparation of Complex enzyme liquid medicine

The pepsin enzymatic hydrolysis bioactive peptide and the neutral protease enzymatic hydrolysis bioactive peptide are prepared according to the following steps of 1: 1, volume ratio of the mixed solution: normal saline 7: 3;

the pepsin enzymatic hydrolysis bioactive peptide and the papain enzymatic hydrolysis bioactive peptide are prepared according to the following steps of 1: 1, volume ratio of the mixed solution: normal saline 7: 3;

the papain enzymolysis bioactive peptide and the neutral protease enzymolysis bioactive peptide are prepared according to the following steps of 1: 1, volume ratio of the mixed solution: normal saline 7: 3;

2.1.2 preparation of drug sensitive paper sheet

Beating qualitative filter paper into circular paper sheets with diameter of 6mm with a puncher, placing the filter paper into sterilized wide-mouth bottles, filling 20 sheets into each bottle, respectively pouring the liquid medicine of the complex enzyme into the corresponding bottles, and placing the wide-mouth bottles into an autoclave for autoclaving (121 ℃, 27 minutes). Soaking in shaking table at 37 deg.C for 18 hr at 100r/min, pouring out the excessive medicinal liquid, oven drying at 37 deg.C in oven, and storing in refrigerator at 4 deg.C for use.

2.2 drug susceptibility testing procedure

Picking a single bacterial colony of cultured salmonella and common escherichia coli by using a sterilized pipette tip in a sterilized normal saline test tube, sucking 200 mul of bacterial suspension (the concentration of a 0.5 wheat type turbidimetric tube) and placing the bacterial suspension in a culture dish, pouring a sorbitol wheat conkay agar culture medium plate, and fully mixing the bacterial suspension with a liquid culture medium when the culture medium is not solidified. After the water on the plate is completely absorbed by the agar, the drug sensitive paper is stuck on the culture medium by using sterile tweezers. Each plate is pasted with 7 drug sensitive paper sheets, the paper sheets are fully contacted with the surface of the agar and cannot be taken up after being pasted, the distance between the paper sheets is not less than 24mm, and the distance between the centers of the paper sheets and the edge of the plate is not less than 15 mm. The position of various drug sensitive paper sheets was recorded. And (3) placing the flat plate at a constant temperature of 37 ℃ for culturing for 18-24h, taking out, taking a picture, measuring the diameter of the bacteriostatic zone by using ImageJ1.8.0 for three times, and calculating the average value to obtain the experimental result.

3. Test results

The bacteriostatic activity of the composite protease enzymatic biological active peptide on salmonella and common escherichia coli is preliminarily verified by a drug sensitive paper sheet method.

TABLE 11 sheep blood-derived bioactive peptide (Complex enzyme) in vitro anti-Salmonella and Escherichia coli inhibition zone diameter_mm)

Note: wherein the value of 6 is no inhibition zone, and the value of 6 is inhibition zone

From table 11 and the test results of fig. 4-7, it can be seen that: the composite bioactive peptide prepared by enzymolysis of pepsin and neutral protease for 3h, 4h, 5h and 6h has no inhibition zone on salmonella and common escherichia coli, but has inhibition zones with higher sensitivity on salmonella and common escherichia coli in enzymolysis for 1h and 2h, and the diameters of the inhibition zones are respectively 11.68mm (1h, salmonella), 13.71mm (2h, salmonella), 10.48mm (1h, escherichia coli) and 12.32mm (2h, escherichia coli); the composite bioactive peptide obtained by enzymolysis of pepsin and papain for 3 hours has no inhibition zone on salmonella and common escherichia coli, but the composite bioactive peptide obtained by enzymolysis of pepsin and neutral protease for 1 hour, 2 hours, 4 hours, 5 hours and 6 hours has an inhibition zone highly sensitive to salmonella and common escherichia coli, and the diameter of the inhibition zone is 9.36mm (1 hour, salmonella), 12.85mm (2 hour, salmonella), 9.21mm (4 hour, salmonella), 11.86mm (5 hour, salmonella), 15.06mm (6 hour, salmonella), 10.86mm (1 hour, escherichia coli), 12.38mm (2 hour, escherichia coli), 11.84mm (4 hour, escherichia coli), 10.01mm (5 hour, escherichia coli), 9.89mm (6 hour, escherichia coli); the papain and the neutral protease are subjected to enzymolysis for 1h-6h, and no antibacterial zone appears, so that the bioactive peptide compounded by the pepsin and the papain enzymolysis method can be preliminarily judged to be capable of preparing the bioactive peptide with better antibacterial performance.

Test example 3 in vivo bacteriostatic test of bioactive peptide

1. Test animal

Kunming rats were purchased from the university of inner Mongolia laboratory animal center, and selected for animal experiments with body conditions between 180 and 210 g. 4.1.2 test reagents and consumables

No. 16 gastric lavage needle, normal saline, paraffin, formaldehyde, absolute ethyl alcohol, xylene, RNA Mini-Preps Kit, reverse transcription Kit, Bouins stationary liquid, hematoxylin, differentiation liquid, eosin, alcohol, sealing tablet, pathological grade adhesive glass slide and cover glass.

2. Test method

Preparation of a suspension of E.coli K88 bacteria

Pouring the preserved E.coli K88 into a plate, culturing at 37 deg.C for 18h, picking single colony with pipette tip, and diluting with normal saline to obtain 1 × 10⁸CFU/mL、2×10⁸CFU/mL、3×10⁸CFU/mL、4×10⁸CFU/mL、5×10⁸CFU/mL suspension of e.coli K88, labeled and stored at 4 ℃ until use.

2.2 determination of the number of colonies in the feces of diarrheal rats

Collecting about 0.1g rat feces in sterile EP tube, adding 1ml normal saline, mixing, standing for 30min, sucking clarified supernatant 100 μ L, adding 900 μ L normal saline for dilution, and making into oral liquidDiluting for 3-7 times, and sucking 10 times³-10⁶100 mu L of the feces mixed solution is poured into a plate and cultured at 37 ℃ for 18h, and the colony number in the feces of the diarrhea rats is determined by a plate counting method.

2.3 rat in vivo bacteriostasis test

2.3.1 test animal groups

30 Kunming rats with good body condition and equivalent weight at 4 weeks are randomly divided into 5 test groups (blank control group, negative control group, positive control group, test 1 group and test 2 group), and each group has 6 rats, and the specific administration scheme is shown in Table 12. Wherein the blank control group had free ingestion without any treatment; negative control group was drenched with 3X 10⁸Coli K88 bacterial suspension at CFU/mL concentration; the positive control group is drenched with pepsin bioactive peptide; the test 1 group is a bioactive peptide treatment group, and is drenched to establish a diarrhea model with an optimal concentration of escherichia coli bacterial suspension, and the bioactive peptide is used for treating the diarrhea of rats; experiment 2 group is bioactive peptide health group and bioactive peptide is drenched from the beginning of experiment period. And after the feeding period is 35d, collecting samples of feces, intestinal tracts and other tissues to detect various indexes.

TABLE 12 dosing regimen

2.3.2 method for determining and testing food intake of rat

Rats were 9 a day early: and (3) weighing the residual material amount and the feeding amount, and calculating the feed intake.

2.3.3 rat body weight determination test method

Rats were weighed every 5 days.

2.3.4 determination of the number of colonies in rat faeces

Collecting about 0.1g rat feces, adding 1ml normal saline, mixing, standing for 30min, sucking clarified supernatant 100 μ L, adding 900 μ L normal saline, diluting 3-7 times according to the above method, sucking 10 times³-10⁶100 mu L of the feces mixed solution is poured into a plate and cultured at 37 ℃ for 18h, and the colony number in the feces of the diarrhea rats is determined by a plate counting method.

2.3.5 routine determination of rat blood

The rat is broken tail to collect blood, and the blood routine is measured.

2.3.6 sampling and sample handling

Collecting feces of a rat before slaughtering, dislocating the rat to death, splitting from the center under the jaw, picking and cutting off a carotid artery to collect whole body blood, putting the whole body blood into a blood collection tube for measuring the conventional indexes of the blood, splitting open an abdominal cavity, taking out intestinal tissues, and fixing half of the intestinal tissues in 10% formalin for paraffin section; the other half is put in a freezing storage tube at the temperature of minus 80 ℃ for standby, and is subsequently used for measuring the expression quantity of TNF-alpha and IL-6 genes. The thoracic cavity was dissected open, and spleen and thymus were weighed to calculate the organ index.

2.3.7 measurement of spleen and thymus index

Weighing spleen, thymus and pre-slaughter live weight of a rat to calculate an organ index, wherein the calculation formula is as follows:

spleen index ═ spleen weight (mg)/body weight (g),

thymus weight (mg)/body weight (g).

2.3.8 sliced intestinal tract tissue

Firstly, the duodenum, jejunum and ileum of the rat are sampled and fixed in 10% formalin solution, and are embedded after being washed, and the change of the tissue form of the intestinal tract is observed under a microscope.

② soaking the collected rat intestinal tract sample in Bouins stationary liquid for 38-48 h.

Cutting the intestinal tissue into tissue blocks with the length of 1.5cm, and performing gradient dehydration, wherein the ethanol concentration and the ethanol time in the 6-hour gradient dehydration are respectively as follows: 0.5h of 75% ethanol, 1.5h of 75% ethanol, 1h of 80% ethanol, 1h of 85% ethanol, 1h of 95% ethanol and 1h of absolute ethanol.

And thirdly, the dehydrated intestinal tract tissue blocks are subjected to paraffin dipping and are placed into liquid paraffin melted in advance for tissue embedding. After paraffin is solidified and formed, the paraffin block is trimmed, the embedded tissue paraffin block is sliced (the thickness is 7 mu m) by using a slicer, the sliced paraffin section is unfolded in warm water at 40 ℃, a slide glass is used for sticking, and the paraffin block is dried at room temperature for standby.

And fourthly, HE dyeing: the adhered slices are transparent twice in dimethylbenzene, and are dewaxed for 5min each time; sequentially dewaxing the adhered slices for 2min respectively according to the sequence of 5min of absolute ethyl alcohol, 95% ethyl alcohol, 85% ethyl alcohol, 75% ethyl alcohol and distilled water; washing off xylene completely, incubating hematoxylin staining solution for 6min, washing with tap water for 10min, soaking in differentiation solution for 1min, soaking in warm water at 50 deg.C for 5min, and incubating eosin staining solution for 3-5 min. Soaking in tap water for 5min, soaking in 95% ethanol twice (each for 1 min), soaking in absolute ethanol twice (each for 1 min), soaking in xylene twice (each for 1 min), observing with a microscope using neutral gum sealing sheet, collecting images, and measuring small intestine villus length and crypt depth using ImageJ 1.8.0.

2.3.9 Effect of pepsin bioactive peptide on TNF-alpha and IL-6 gene expression level of small intestine of rat

According to the study of the effect of Y-R (Y-R, Jiang, J-Y. miRNA-130a improves cardiac function by down-regulating TNF-alpha Expression in a Rat model of heart failure [ J ]. Europe Review for Medical & Pharmacological Sciences,2018(22):8454-8461.) on the Expression of tumor necrosis factor-alpha (TNF-alpha) by Rat cardiac function in MicroRNA (miRNA) -130a and the Expression of Gwenarelle (Gwenarelle guide, enzymic Douillard, Oliver galves, et al, early Activation of Rat Skell-6/1/STAT 56 dependency Gene Expression in early Skeletal Muscle Expression of Rat (STAT-Dependent genes) (see SEQ ID NO: 11/11) and early Expression of Rat Skeletal Muscle genes expressed in Rat (STAT-13) by Rat actin genes expressed in Biostat genes, see SEQ ID NO: 11, Biostat, SEQ ID NO: 11. Biol-11. Biol 2. express genes in early Rat-2. STAT-11. and STAT Expression of Rat-11. Staphylogenetic Expression of early genes in Biogene Expression of SEQ ID No. 11.

Primer sequences for genes of interest of Table 13

2.3.9.1 extraction and identification of rat small intestine total RNA

Grinding a proper amount of small intestine tissues of rats in liquid nitrogen; using RNA Mini-Preps Kit to crack small intestine tissues and extracting total RNA; the concentration and OD value of the RNA directly detected by the Nanodrop are between 1.8 and 2.0, the integrity of the RNA is good, and the method is suitable for subsequent tests.

2.3.9.2Q-PCR determination of TNF-alpha and IL-6 gene expression quantity of rat small intestine

Reverse transcribing the extracted RNA to cDNA; fully and evenly mixed, and the amplification reaction is carried out in a real-time quantitative PCR instrument, and the reaction system is shown in Table 16. The reaction conditions are as follows: pre-denaturation at 95 ℃ for 30sec, denaturation at 95 ℃ for 5sec, 600C extension for 30sec, 40 cycles; and (3) performing extension annealing at 60 ℃. The relative quantification (2-delta. DELTA. Ct method) was used to calculate the expression level for both gene quantifications.

TABLE 14R_ealti_meReaction of

3 data analysis

Data sorting is carried out by Excel 2007 software, data are analyzed and integrated by SAS 9.2, and RT-PCR results are analyzed by Prism 6.

4 results of the test

4.1 rat feed intake determination test results

The feed intake of rats in the test period can be obtained from table 15, and the average feed intake of 5 test groups has no significant difference (P is more than 0.05) at 0-5 d; the average feed intake of 5 test groups alone has no significant difference at 6-10d (P is more than 0.05); the average feed intake of 5 test groups alone has no significant difference at 11-15d (P > 0.05); the average feed intake of 5 test groups alone has no significant difference (P >0.05) at 16-20 d; the feed intake of the 2 groups alone was significantly higher than that of the 4 other groups at 21-25 d.

TABLE 15 feed intake during the test period

Note: data in the table are expressed by (Mean + -Sd), the difference of the same column numbers and shoulder marks is obvious (P <0.05), the difference of the same shoulder marks is not obvious (P >0.05)

4.2 measurement of rat body weight

The body weights of the rats during the test period are shown in Table 16, and the body weight changes of the rats during the 35d test period. The body weight of the rats in the 5 test groups is increased along with the time, the body weight of the rats in the 5 test groups is consistent in the early stage of the test, the body weight reaches the highest at 35d, and no significant difference exists among the test groups (P > 0.05).

TABLE 16 body weight at test period

Note: data are expressed as (Mean ± Sd), with the same column numbers and shoulder letters indicating significant differences (P <0.05) and the same shoulder letters indicating insignificant differences (P > 0.05). 4.3 determination of the number of colonies in rat faeces

The colony number in the feces of the rats in the test period can be obtained from the table 17, the difference of 5 treatment groups of the colony number of the feces at the 10 th day in the test period is very obvious (P is less than 0.01), the highest difference of 32.50 +/-1.94 and the highest difference of 29.00 +/-3.27 are respectively between the negative control group and the test 1 group, and the difference is not obvious (P is no more than 1.94 and no more than 29.00 +/-3.27)>0.05), the positive control group and the test 2 group are respectively 4.61 +/-1.53 and 12.00 +/-3.33, and have no significant difference (P)>0.05), the blank control group has extremely obvious difference (P is less than 0.01) compared with the negative control group and the test 1 group, and the positive control group and the test 2 group; the differences between the 15 th and 10 th days of the test were the same. The negative control group differed significantly from the other 4 test groups at 25d and 35d of the test (P < 0.01). This is because the concentration of the first 15 days of the test group 1 and the negative control group was 3X 10⁸CFU/mL E.coli K88 bacterial suspension causes more colony number in feces, has obvious difference (P is less than 0.01) with other 3 test groups, has no obvious difference with sheep blood source bioactive peptide irrigated in the positive control group and the experiment 2 group, and has no obvious difference with the blank control group to freely feed intestinal bacteriaPopulation perfection results in the lowest number of colonies in the feces. After 15 days, the drenching concentration of only the negative control group is 3 multiplied by 10⁸CFU/mL of e.coli K88 suspension, the poor colony count difference was very significant (P < 0.01) compared to the other test groups without the suspension.

TABLE 17 determination of the number of colonies in rat faeces

Note: data are expressed as (Mean ± Sd), with the same column numbers and shoulder letters indicating significant differences (P <0.05) and the same shoulder letters indicating insignificant differences (P > 0.05).

4.4 blood routine test results

Collecting rat blood in 15d for routine blood detection, wherein the results are shown in table 18, and at 0d, the routine values of the blood have no difference at the starting point of the test; as the test proceeded, there was a trend of increasing the blood routine index values at 15d, but there was no significant difference in the blood routine values between the test groups (P >0.05), indicating that the perfusion of e.coli K88 bacterial suspension had no significant effect on the blood routine index of rats.

TABLE 18 blood routine test results

Note: data in the table are expressed by (Mean ± Sd), the difference of the same row number with shoulder letters indicates significant difference (P <0.05), and the difference of the same non-shoulder letters indicates insignificant difference (P > 0.05). 4.5 measurement of spleen and thymus indices

As can be seen from Table 19, the weights and indices of spleen and thymus of the blank control group, the negative control group, the positive control group, the test 1 group and the test 2 group were not significantly different (P >0.05), indicating that the administration of bioactive peptide to rats had no significant effect on the organ index of rats.

TABLE 19 weight and index measurement of spleen and thymus

Note: data are expressed as (Mean ± Sd), with the same column numbers and shoulder letters indicating significant differences (P <0.05) and the same shoulder letters indicating insignificant differences (P > 0.05).

4.6 intestinal tissue section observations

The observation results of the intestinal tissue section are shown in fig. 8, 9 and 10, and the jejunum villi of the blank control group is normal in length and complete in morphological structure under the low power microscope (10 × 40); the negative control group has shorter intestinal villi than other treatment groups due to the filling of the E.coli K88 bacterial suspension, and has the conditions of thin villi and obvious deletion, deformation and fracture; the positive control group has complete villus structure of duodenum, jejunum and ileum, and has no deletion and obvious fracture of villus due to the administration of the bioactive peptide; however, the jejunal villi in the test 1 group became short and the intestinal wall became thin, and the length of the jejunal villi in the test 2 group was consistent with that of the blank control group, and the morphological structure was not damaged. These slicing results indicate that the bioactive peptides have therapeutic effects on the whole small intestine, can improve the length and structural integrity of intestinal villi, and have therapeutic effects on intestinal villi structural rupture caused by e.coli K88.

The influence of the sheep blood-derived bioactive peptide on the length of the small intestinal villus of the rat is shown in table 20, as the negative control group is irrigated with the bacterial suspension to destroy the intestinal villus to a certain extent, the length of the duodenal villus is obviously lower than that of the other 4 treatment groups (P <0.05), the blank control group is not treated at all, the positive control group and the first 15 days of the test 2 group are irrigated with the sheep blood-derived bioactive peptide, and the bioactive peptide has a certain protection effect on the intestinal villus, so that no significant difference (P >0.05) exists between the 3 groups; because the negative control group is irrigated with the bacterial suspension to destroy intestinal villi to a certain extent, the lengths of the villi of the jejunum and the ileum are obviously lower than those of other 4 treatment groups (P <0.05), the blank control group is not treated at all, the lengths of the villi of the jejunum and the ileum are obviously higher than those of other 4 treatment groups (P <0.05), the goat blood source bioactive peptide is irrigated in the positive control group and the first 15 days of the test group 2, and the lengths of the villi of the jejunum and the ileum are not obviously different (P > 0.05).

TABLE 20 influence of sheep blood-derived bioactive peptides on the villus length of the small intestine of rats

Note: data are expressed as (Mean ± Sd), with the same column numbers and shoulder letters indicating significant differences (P <0.05) and the same shoulder letters indicating insignificant differences (P > 0.05).

The effect of sheep blood-derived bioactive peptides on the crypt depth of the small intestine of rats is shown in table 21, where the crypt depth of duodenum in the negative control group is significantly higher than that in the other 4 treatment groups (P <0.05), and the positive control group is numerically lowest but has no significant difference from the test 1 group and the test 2 group (P > 0.05); the crypt depth negative control group of jejunum and ileum is obviously higher than other 4 treatment groups (P <0.05), and the positive control group is obviously lower than other 4 treatment groups (P >0.05), which shows that the secretion function and cell maturity of cells can be improved by drenching the rats with the bioactive peptide, and vice versa by drenching the rats with the bacterial suspension.

TABLE 21 Effect of sheep blood-derived bioactive peptides on depth of crypts in rat small intestine

Note: data are expressed as (Mean ± Sd), with the same column numbers and shoulder letters indicating significant differences (P <0.05) and the same shoulder letters indicating insignificant differences (P > 0.05). 4.7 Effect of bioactive peptides on the expression levels of TNF-alpha and IL-6 genes in the Small intestine of rats

As can be seen from FIG. 11, the expression level of TNF- α in the negative control group was significantly higher than that in the blank control group, the positive control group, the test 1 group and the test 2 group (P <0.05), while the difference between the blank control group, the positive control group, the test 1 group and the test 2 group was not significant (P >0.05), which indicates that the administration of bioactive peptide to rats reduced the inflammation in the small intestine of rats, thereby reducing the expression level of TNF- α in the small intestine. IL-6 is the detection index of inflammatory diseases and infection degree, and as can be seen from figure 12, the expression level of TNF-alpha in the negative control group is significantly higher than that in the blank control group, the positive control group, the test 1 group and the test 2 group (P is less than 0.05), and the difference among the blank control group, the positive control group, the test 1 group and the test 2 group is not significant (P is more than 0.05), which indicates that the biological active peptide can reduce the inflammation in the small intestine of the rat by drenching the rat, thereby reducing the expression level of IL-6 in the small intestine.

SEQUENCE LISTING

<110> university of inner Mongolia agriculture

<120> biological active peptide and composite biological active peptide with bacteriostatic function, and preparation method and application thereof

<130> NM-1001-200907A

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 23

<212> DNA

<213> Artifical sequence

<400> 1

cagaccctca cactcagatc atc 23

<210> 2

<211> 22

<212> DNA

<213> Artifical sequence

<400> 2

agccttgtcc cttgaagaga ac 22

<210> 3

<211> 19

<212> DNA

<213> Artifical sequence

<400> 3

tgtatgaaca gcgatgatg 19

<210> 4

<211> 18

<212> DNA

<213> Artifical sequence

<400> 4

agaagaccag agcagatt 18

<210> 5

<211> 24

<212> DNA

<213> Artifical sequence

<400> 5

cggagtcaac ggatttggtc gtat 24

<210> 6

<211> 24

<212> DNA

<213> Artifical sequence

<400> 6

agccttctcc atggtggtga agac 24