Sheep colostrum, polypeptide, preparation method of sheep colostrum and polypeptide, and application of sheep colostrum and polypeptide

Abstract

The invention provides a polypeptide, and the sequence of the polypeptide is SVDGKENLI. The polypeptide is separated from sheep colostrum after fermentation of the lactobacillus rhamnosus D8A. The invention further provides application of the polypeptide, and the polypeptide can be applied to preparation of drugs for inhibiting neuraminidase, resisting viruses and promoting lung repair. The invention further provides sheep colostrum which comprises the polypeptide. The invention also provides a preparation method of the sheep colostrum, which comprises the following steps: filtering and pre-sterilizing the sheep colostrum, then carrying out secondary homogenization on the sheep colostrum, carrying out flash sterilization and cooling on the sheep colostrum subjected to secondary homogenization, then adding fructose, carrying out sterilization, inoculating a leavening agent comprising the rhamnosus lactobacillus D8A, and carrying out fermentation, so as to obtain the sheep colostrum. And then carrying out primary homogenization, concentration and freeze-drying to obtain the product.

Description

Technical Field

[0001] This invention relates to the field of polypeptides, specifically to a polypeptide obtained from sheep colostrum fermentation that has the function of inhibiting neuraminidase, and the application of this polypeptide. This invention also relates to sheep colostrum and its preparation method. Background Technology

[0002] Peptides in fermented sheep colostrum are a class of functional components with diverse biological activities. They are primarily released from sheep milk proteins through microbial fermentation and exhibit broad potential in promoting health. Sheep milk itself is rich in high-quality proteins such as α-, β-, and κ-casein. During fermentation by probiotics such as Kefir and Lactobacillus, these proteins are progressively hydrolyzed by proteases produced by the microorganisms, generating bioactive peptides with specific amino acid sequences. This process mimics the human digestive mechanism but releases functional short peptide fragments more efficiently. Multiple studies have shown that peptides isolated from fermented sheep milk can effectively reduce the level of tumor necrosis factor-α in mice, indicating their anti-inflammatory capabilities. In particular, peptides fermented from sheep milk with a compound strain containing Lactobacillus acidophilus and Bifidobacterium exhibit strong anti-inflammatory properties. The types of peptides isolated from sheep colostrum and fermented milk are generally more numerous than those isolated from cow milk, and the addition of probiotics further increases the variety of peptides in fermented sheep milk. This suggests that sheep milk is a more suitable base for developing functional fermented dairy products with high bioactive peptide content.

[0003] In their paper "A Novel Natural Influenza: A H1N1 Virus Neuraminidase Inhibitory Peptide Derived from Cod Skin Hydrolysates and Its Antiviral Mechanism," Jianpeng Li et al. disclosed a peptide, PGEKGPSGEAGTAGPPGTPGPQGL. This natural peptide effectively inhibits the H1N1 influenza A virus by competitively binding to neuraminidase (NA). Experiments showed that its EC50 was 471 ± 12 μg / mL, and it significantly reduced viral replication in a cell model, playing a role in the early stages of infection. However, this peptide is easily broken down by pepsin. Summary of the Invention

[0004] The first objective of this invention is to provide a polypeptide isolated from sheep colostrum fermented by Lactobacillus rhamnosus D8A with antiviral function.

[0005] A second objective of this invention is to provide an application of the aforementioned polypeptide in the field of antiviral therapy.

[0006] A third objective of this invention is to provide a sheep colostrum comprising the aforementioned polypeptide.

[0007] A fourth objective of this invention is to provide a method for preparing the aforementioned sheep colostrum.

[0008] This invention is achieved through the following technical solution: This invention provides a polypeptide with the sequence SVDGKENLI.

[0009] The polypeptide can be used to prepare drugs that inhibit neuraminidase; or to prepare antiviral drugs; or to prepare drugs that promote lung repair.

[0010] This invention provides a sheep colostrum containing the aforementioned polypeptide.

[0011] A method for preparing sheep colostrum includes the following steps: The fresh sheep colostrum was filtered, pre-sterilized, and then homogenized in two stages. After homogenization, the fresh sheep colostrum was flash-sterilized and cooled. After adding fructose to sterilized sheep colostrum and performing a first sterilization, it is inoculated with a starter culture including Lactobacillus rhamnosus D8A for fermentation. After primary homogenization, concentration, and first freeze-drying, the product is obtained.

[0012] The filter used for filtration has a pore size of 80-100 mesh. The pre-sterilization temperature is 70°C; The pre-sterilization time is 18 seconds; The temperature of the secondary homogenizer shall not exceed 60°C; The primary pressure of the secondary homogenizer is 15 MPa, and the secondary pressure is 8 MPa. The flash sterilization time is 12 seconds; The heat source temperature for the flash sterilization is 76°C. The cooling temperature is 4°C; The vacuum degree of the flash sterilization is 0.5 MPa; During the flash sterilization process, the temperature of the fresh sheep colostrum does not exceed 65°C.

[0013] The amount of fructose added is 7-8 wt%; The first sterilization temperature is 95-100℃; The first sterilization time is 30-60 seconds; The pressure of the primary homogenizer is 20-25 MPa; The concentration ratio is 20%-25% of the original volume; The first freeze-drying agent used in the first freeze-drying process includes 3-4 wt% sorbitol, 5-10 wt% trehalose, 0.2-0.5 wt% ascorbic acid and 0.8-1.2 wt% monosodium glutamate, with the balance being water; The temperature for the first freeze-drying process is between -80°C and -60°C.

[0014] The inoculum amount of the fermenting agent is 0.1%; The fermentation temperature was 37℃, and the time was 16-25 hours. The vacuum level of the concentration is less than 10 Torr; The concentration temperature is 40-45℃.

[0015] The preparation method of the fermentation agent includes the following steps: Lactobacillus rhamnosus D8A was streaked onto MRS solid medium and incubated at 37°C until plump single colonies were formed; single colonies were picked and inoculated into MRS liquid medium for two consecutive passages for activation. The activated and expanded Lactobacillus rhamnosus D8A was inoculated into optimized MRS medium at 2%-2.5%, and fermented for 20-28 hours at a controlled temperature of 36℃±0.5℃, pH 6.8, and dissolved oxygen of 20%-25% to obtain the fermentation broth. After fermentation, centrifuge the fermentation liquid and discard the supernatant to obtain bacterial sludge; Mix the bacterial sludge with the second freeze-drying protectant evenly, perform a second freeze-drying until dehydrated, and grind evenly to obtain the fermentation agent; The optimized MRS medium comprises 11.0 g tryptone, 12.0 g beef extract, 4.0 g yeast extract, 25.0 g glucose, 1.5 mL Tween 80, 3.0 g dipotassium hydrogen phosphate, 1.5 g magnesium sulfate, 1.75 g manganese sulfate, and 1 L water.

[0016] The second freeze-drying process uses a second freeze-drying protectant comprising 10-13 wt% skim milk powder, 5-8 wt% trehalose, 3-6 wt% maltose, 2-4 wt% mannitol, with the remainder being water. The weight ratio of the mycelium sludge to the second freeze-drying protectant is 1:2-2.5; The second freeze-drying temperature is -50°C to -55°C.

[0017] The centrifugation acceleration is 5000-6000g; The centrifugation temperature is 4-6℃.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: The polypeptide isolated from fermented sheep colostrum provided by this invention can inhibit H5N1 neuraminidase, thereby reducing the release of the virus.

[0019] The sheep colostrum containing the aforementioned polypeptide provided by this invention helps elderly people with weakened immune systems resist H5N1 virus infection.

[0020] The method for preparing sheep colostrum provided by this invention is simple and can be industrialized.

[0021] The polypeptide provided by this invention can inhibit H5N1 neuraminidase, and therefore can be used to prepare a variety of pharmaceuticals. Attached Figure Description

[0022] Figure 1 The chemical structure of the polypeptide provided by the present invention is shown; Figure 2 The HPLC chromatogram of the polypeptide provided by the present invention is shown.

[0023] Figure 3 The results of weight changes in each group of mice infected with influenza virus provided in Example 6.

[0024] Figure 4 The image shows a photograph of cells sampled from the lungs of mice in the negative control group (PBS group) that were killed after 21 days of culture.

[0025] Figure 5 The image shows a photograph of cells sampled from the lungs of mice in the experimental group (peptide group) that were killed after 7 days of culture.

[0026] Figure 6 The image shows lung cells of mice in the experimental group (peptide group) after 20 days of culture.

[0027] Figure 7 The image shows a photograph of cells sampled from the lungs of mice in the oseltamivir group that were killed after 7 days of culture.

[0028] Figure 8 The image shows lung cells of mice in the oseltamivir group after 20 days of culture. Detailed Implementation

[0029] The Lactobacillus rhamnosus D8A involved in this invention has been disclosed in Chinese invention application with publication number CN118389363A.

[0030] Example 1 This embodiment provides a method for preparing sheep colostrum. The specific preparation method is as follows: Cryopreserved Lactobacillus rhamnosus D8A was streaked onto MRS solid medium and cultured at 37°C until plump single colonies formed. Subsequently, single colonies were picked and inoculated into liquid medium for two consecutive passages for activation.

[0031] Then, the activated and expanded Lactobacillus rhamnosus D8A was inoculated into optimized MRS medium at an inoculum size of 2%. Fermentation was then carried out at 36°C, pH 6.8, stirring speed of 150 r / min, and dissolved oxygen content of 20% for 25 h to obtain the fermentation broth.

[0032] The optimized MRS liquid culture medium formulation is as follows: 11.0 g tryptone, 12.0 g beef extract, 4 g yeast extract, 25.0 g glucose, 1.5 mL Tween 80, 3.0 g dipotassium hydrogen phosphate, 1.5 g magnesium sulfate, 1.75 g manganese sulfate, and 1 L water. The pH of this optimized MRS liquid culture medium is 6.8. The optimized MRS liquid culture medium was sterilized at 0.1 MPa and 115 °C for 20 min before use.

[0033] Place the fermentation broth in a centrifuge and centrifuge at 4℃ and 5000g for 15 minutes to collect the bacterial sludge.

[0034] A freeze-drying protectant was added to the mycelium slurry. After the mycelium slurry and freeze-drying protectant were mixed evenly, the mixture was pre-frozen at -35°C for 13 hours, and then freeze-dried under vacuum at -52°C and a vacuum degree of 0.08 mBar for 40 hours until completely dry, yielding the freeze-dried starter culture (live bacteria preparation). In the above method, the weight ratio of mycelium slurry to freeze-drying protectant was 1:2. The freeze-drying protectant consisted of 10 wt% skim milk powder, 8 wt% trehalose, 4 wt% maltose, 2 wt% mannitol, and the remainder being water.

[0035] Fresh sheep colostrum was filtered using a filter with a mesh size of 80-100. The filtered colostrum was then refrigerated at 4°C. Next, it was pre-sterilized at 70°C for 18 seconds; followed by secondary homogenization at a temperature not exceeding 60°C (first stage pressure 15 MPa, second stage pressure 8 MPa); and then flash-sterilized in a flash tank for 12 seconds. The inlet heat source temperature of the flash tank was 76°C, the vacuum degree of the flash tank was 0.5 MPa, and the center temperature of the fresh sheep colostrum in the flash tank did not exceed 65°C. Finally, the sterilized milk was cooled to 4°C with ice water to obtain sterilized sheep colostrum. Flash sterilization ensures sterilization effectiveness while reducing protein denaturation during the sterilization process. The sterilization data after completion and the data after refrigeration at 4°C for five days are as follows: The specific sterilization data is shown in Table 1 below.

[0036] Table 1 Add sterilized sheep colostrum to a mixing tank, followed by 8% (by weight of the colostrum) of fructose, and mix thoroughly. Sterilize at 95-100℃ for 30 seconds. After sterilization, rapidly cool to 4℃ for storage. While thoroughly stirring, inoculate the sterilized and cooled sheep colostrum with 0.1% of freeze-dried starter culture (Lactobacillus rhamnosus D8A starter culture) and incubate at 37℃ for 25 hours. After fermentation, perform primary homogenization at 25 MPa. To avoid inactivation of bioactive substances in the fermented sheep milk, use vacuum centrifugation with a vacuum level below 10 Torr and a centrifugation temperature controlled at 45℃. The fermented milk prepared in the previous step was then concentrated to 20% (v / v) of its original volume, and a freeze-drying protectant (3wt% sorbitol, 7wt% trehalose, 0.2wt% ascorbic acid, and 1.2wt% monosodium glutamate, with the balance being water) was added. After thorough mixing and grinding, the mixture was pre-frozen at -80℃ for 2 hours, and then transferred to a freeze dryer for vacuum freeze-drying at -80℃ for 24 hours, finally obtaining a fluffy, porous, sponge-like freeze-dried cake. Under aseptic conditions, the cake was crushed and sieved to obtain fermented sheep colostrum.

[0037] Indicator Testing The sheep colostrum was reconstituted to a concentration of 1 g / mL, centrifuged at 4℃ and 6000g for 15 min, and the supernatant was collected for microbial safety testing. Specific data are shown in Table 2.

[0038] Table 2

[0039] Example 2 This embodiment provides a method for extracting polypeptides.

[0040] Specifically, fermented sheep colostrum samples were extracted using 40% ethanol solution under acidic conditions (pH 6). After extraction, the samples were centrifuged at 10,000 rpm for 10 min at 4°C to remove large molecular proteins, bacterial cells, and insoluble impurities, obtaining the supernatant. The supernatant was then concentrated 5-fold and extracted twice with 4 times its volume of n-butanol. The organic phase was then concentrated under vacuum to obtain a crude extract. The crude extract was further concentrated 2.5-fold and then purified by chromatography. Chromatographic purification included multiple stages. In the first stage of purification, a Sephadex LH-20 column was used with an injection volume of 1.5 mL. After injection, the constant flow pump was turned on, the flow rate was adjusted to 3 mL / 8 min, and elution was performed using 80% (v / v) chromatographic methanol as the mobile phase. After elution, fractions were collected at different time points. These fractions were then subjected to the next stage of chromatographic purification. In the multi-stage chromatographic purification, the Sephadex LH-25 column was used for secondary purification. The sample used for secondary purification was the fraction obtained from primary purification, concentrated 15-fold. The mobile phase used for secondary purification was distilled water, and the flow rate was controlled at approximately 3 ml / 10 min. After elution, the fraction was collected using an automatic collector. The fraction was then concentrated 15-fold and further purified using an RP-C18 column for tertiary purification. The tertiary purification conditions were as follows: the mobile phase was water and acetonitrile solution containing 0.1% trifluoroacetic acid; the elution parameters were as follows: 10 min 95% A, 20 min 70% A, 25 min 65% A, 30 min 40% A, 40 min 15% A, 50 min 5% A; the flow rate was 0.3 ml / min; and the detection wavelength was 259 nm. After elution, the fraction was collected, and each fraction corresponded to only one chromatographic peak. A high-purity eluent was obtained, and the powdered product was obtained by vacuum freezing for subsequent identification and functional verification.

[0041] Example 3 Polypeptide Function Identification Experimental instruments and materials Multifunctional fluorescent microplate reader, SP-Max 3500FL; pipettes; 96-well microplate; H5N1 neuraminidase; fluorescent substrate 2'-(4-methylumbelliferone)-α-D-acetylneuraminidide sodium hydrate (4-MUNANA), purchased from Sigma-Aldrich; 2-(N-morpholino)ethanesulfonic acid (MES), calcium chloride, sodium hydroxide, anhydrous ethanol; positive control was oseltamivir carboxlate (OSC).

[0042] Experimental methods The positive control and the frozen powder products corresponding to the fractions separated in Example 2 were dissolved in DMSO to prepare a solution with a concentration of 1000 μmol / L. This solution was then serially diluted to six concentration gradients: 500 μmol / L, 250 μmol / L, 125 μmol / L, 62.5 μmol / L, 31.25 μmol / L, and 15.625 μmol / L, with three sets prepared for each gradient. 60 μL of buffer solution was added to each well of a 96-well microplate. The buffer solution contained 30 mM 2-(N-morpholino)ethanesulfonic acid and 5 mM CaCl2. Then, 12 μL of neuraminidase and 12 μL of neuraminidase substrate were added to each well.

[0043] The wells were divided into three groups: a blank group, in which no test sample was added, but only 10 μL of DMSO solution was added; a control group, in which 10 μL of neuraminidase inhibitor control standard was added; and a test group, in which 10 μL of the test sample corresponding to each distillate obtained in Example 2 was added.

[0044] The 96-well microplate was then placed in a multi-functional fluorescent microplate reader and vortexed for 1 minute. After incubation at 37°C for 5 minutes, the plate was removed, and 10 μL of neuraminidase fluorescent substrate was added to each well. The plate was then placed back in the multi-functional fluorescent microplate reader and vortexed for 1 minute. After incubation at 37°C for 40 minutes, the plate was removed, and 100 μL of stop solution (15 mM NaOH in 80% ethanol aqueous solution) was added to each well. The plate was then placed back in the multi-functional fluorescent microplate reader and vortexed for 1.5 minutes. The excitation wavelength was set to 355 nm, and the emission wavelength to 460 nm. After incubation, fluorescence intensity (RFU) was measured. Three parallel experiments were performed, and the average inhibition rate of the sample at each concentration gradient was calculated for each parallel experiment. The corresponding IC50 value was then fitted. The fitted IC50 value for the control group was 0.33 μM. One fraction in the test group had an IC50 value of 0.47 μM. This indicates that the peptide in this fraction has an inhibitory effect on neuraminidase.

[0045] Example 4 Polypeptide molecular identification The fraction corresponding to the test sample with an IC50 value of 0.47 μM was vacuum-frozen to obtain a powder product, which was then reconstituted with 0.1% formic acid aqueous solution. Mass spectrometry was performed using a C18 capillary column, and fragment ion data were obtained by dissociating the parent ion. The raw data was imported into the UniProt database, and the category was selected as sheep. A search was performed in the sheep protein reference database, and finally, a hydrolyzed fragment (UniProt number A0A836CWA4) that highly overlapped with the peptide was found in amino acids 278-286 of sheep lactoferrin.

[0046] The specific tests are as follows: Mass spectrometer: QTRAP 5500; Chromatograph; LC-30AD; Chromatographic column: Agilent 653750-902 column( AdvanceBio Peptide Map 150mm×2.1mm, 2.7μm) Washing conditions: The flow rate was 0.3 mL / min; the column temperature was set to 27 °C. Eluent A is an aqueous solution of 0.1% formic acid, and eluent B is an acetonitrile solution of 0.1% formic acid. The elution methods are as follows: 0-2 min, 5% B and 95% A; 2-5 min, 5-40% B and 95-60% A; 5-8 min, 40-70% B and 60-30% A; 8-10 min, 75-90% B and 25-10% A; 10-12 min, 95% B and 5% A; 12-14 min, 95-5% B and 5-95% A; 14-15 min, 5% B and 95% A. The spray voltage is 5500V; The air pressure of the curtain air is 0.25 MPa; The ion source temperature is 575℃.

[0047] The measured spectrum is as follows Figure 2 As shown.

[0048] Then, the amino acid sequence of the polypeptide was obtained by using the Edman degradation method, and the final amino acid sequence was SVDGKENLI.

[0049] The structural formula of the polypeptide SVDGKENLI is C41H71N11O16, and its molecular weight is 974.08.

[0050] m / z: 973.5080 (100.0%), 974.5114 (44.4%), 975.5147 (9.5%), 974.5051(4.0%), 975.5123 (3.3%), 975.5084 (1.8%), 976.5156 (1.4%), 976.5181 (1.4%).

[0051] Example 5 Cell experiments After resuscitation, A549 cells were cultured in MEM medium containing 10% fetal bovine serum and 1% dual antibiotics (penicillin 100 U / mL and streptomycin 100 μg / mL) and then placed in an incubator at 37°C and 5% CO2 until the cells reached the logarithmic growth phase.

[0052] Virus strain: Influenza A virus (H1N1) strain was amplified in SPF-grade chicken embryos: The allantoic cavity of 10-11 day old chicken embryos was inoculated with the virus and incubated at 35°C for 48 hours. The allantoic fluid was then collected. The viral titer in each allantoic fluid sample was then adjusted to 1×10⁻⁶. 6 TCID 50 / mL to ensure consistent viral concentration in experiments. The method for determining viral titer is TCID. 50 Law.

[0053] Cell seeding: A549 cells in logarithmic growth phase were seeded into 96-well plates (100 μL per well) for CCK-8 assay. The concentration of A549 cells in each well was adjusted to 5 × 10⁻⁶. 4 The cells / mL were then incubated at 37°C with 5% CO2 for 24 hours.

[0054] Viral infection and administration: After aspirating the culture medium from the wells, wash twice with PBS to remove residual serum.

[0055] Experimental Groups: Blank control group: containing only A549 cells and normal culture medium, without viral infection or peptide treatment.

[0056] Virus control group: Inoculated with influenza A virus (H1N1) strain (MOI=1), without any added drugs.

[0057] Positive drug control group: After inoculation with H1N1 virus, oseltamivir at a concentration of 1 μM was added.

[0058] Experimental group: After inoculation with H1N1 virus, the polypeptide SVDGKENLI was added at a concentration of 1 μg / mL.

[0059] After grouping, 100 μL of serum-free MEM medium containing virus (MOI=1) was added to the wells of the virus control group and experimental group. The blank control group was added to serum-free MEM medium and incubated at 37°C with 5% CO2 for 2 hours.

[0060] After incubation, the virus solution was aspirated. The experimental group was added to MEM medium (100 μL / well) containing 1 μg of peptide SVDGKENLI, while the blank control group and the virus control group were added to normal MEM medium. The culture was continued for 24 hours.

[0061] After culture, 10 μL of CCK-8 reagent (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt) was added to each well of a 96-well plate, and the plate was incubated at 37°C for 2 hours. After incubation, the OD value at 450 nm was measured using a microplate reader to calculate cell viability.

[0062] The calculation formula is as follows: Cell viability (%) = (OD value of experimental group / OD value of blank control group) × 100%.

[0063] The results are shown in Table 3.

[0064] Table 3 Results of efficacy verification against influenza A virus strain (H1N1)

[0065] Example 6 animal experiments (1) Test materials Test animals: SPF grade 6-8 week old BALB / c female mice were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd.

[0066] (2) Preparation of the test solution Taking a 2 mg / mL test solution as an example: Dissolve 2 mg of oseltamivir in 20 μL of DMSO solution (final DMSO concentration 1.5%), then add 300 μL of PEG300 (final PEG300 concentration 30%) and mix well. Then add 50 μL of Tween80 (final concentration 5%) and mix well. Finally, add water to make up to 1 mL to obtain a test solution containing 2 mg / mL oseltamivir.

[0067] (3) Testing process Six- to eight-week-old female BALB / c mice were anesthetized with isoflurane and then injected intranasally with 50 μL of a solution containing 150 TCID. 50 H1N1 influenza virus was randomly divided into an experimental group and an oseltamivir group. Additionally, normal 6-8 week old BALB / c female mice were inoculated with PBS as a negative control group (PBS group).

[0068] Each group contains 10 mice.

[0069] Oseltamivir group administration: On day 3 post-infection, mice were administered a test solution containing 2 mg / mL oseltamivir at a dose of 5 mg / kg / d by gavage, twice daily for 5 consecutive days.

[0070] Administration of the negative control group (PBS group): PBS solution with pH=7.0 was administered in the same manner, cycle and volume as the drug group.

[0071] Experimental group (peptide group): 2 mg / mL of peptide SVDGKENLI solution administered in the same manner, cycle and volume as the drug group.

[0072] Weigh each group of mice daily, observe their clinical and pathological conditions, record their scores, and observe their survival status.

[0073] After administration, the mice's weight was observed and recorded daily, the bedding was changed regularly, and any dead mice were removed promptly until the experiment ended. Based on the statistical results, a curve showing the change in mouse weight was plotted.

[0074] (4) Test Results like Figure 3 As shown, on day 9 after viral infection, the average weight of mice in the polypeptide group began to gradually recover, while the average weight of mice in the oseltamivir group did not begin to recover until day 11.

[0075] Figure 4 The image shows a photograph of cells sampled from the lungs of mice in the negative control group (PBS group) that were killed after 21 days of culture.

[0076] Figure 5 The image shows a photograph of cells sampled from the lungs of mice in the experimental group (peptide group) that were killed after 7 days of culture.

[0077] Figure 6 The image shows lung cells of mice in the experimental group (peptide group) after 20 days of culture.

[0078] Figure 7 The image shows a photograph of cells sampled from the lungs of mice in the oseltamivir group that were killed after 7 days of culture.

[0079] Figure 8 The image shows lung cells of mice in the oseltamivir group after 20 days of culture.

[0080] After HE staining of the lungs in each group, observation revealed that terminal bronchioles were clearly visible in cell images from day 7 of culture in both the oseltamivir group and the experimental group, with partial thickening of the lung wall, possibly due to inflammation. Meanwhile, in cell images from day 21 of culture in both groups, alveolar ducts at all levels appeared normal, and there was no thickening of the alveolar septa. This indicates that the polypeptide disclosed in this invention has a lung-repairing effect.

[0081] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements can be made without departing from the principle of the present invention, and these improvements should also be considered within the scope of protection of the present invention.

Claims

1. A polypeptide, characterized in that: The polypeptide sequence is SVDGKENLI.

2. The application of the polypeptide as described in claim 1, characterized in that: Used in the preparation of drugs that inhibit neuraminidase; or Used in the preparation of antiviral drugs; Drugs used to promote lung repair.

3. A type of sheep colostrum, characterized in that: Includes the polypeptide as described in claim 1.

4. The method for preparing sheep colostrum as described in claim 3, characterized in that: Includes the following steps: Fresh sheep colostrum is filtered, pre-sterilized, and then homogenized in two stages. After homogenization, the fresh sheep colostrum is flash-sterilized, cooled, and then sterilized in the first stage after adding fructose. After inoculation with a starter culture including Lactobacillus rhamnosus D8A, it is fermented, homogenized in one stage, concentrated, and then freeze-dried to obtain the final product.

5. The method for preparing sheep colostrum as described in claim 4, characterized in that: The filter used for filtration has a pore size of 80-100 mesh. The pre-sterilization temperature is 70°C; The pre-sterilization time is 18 seconds; The temperature of the secondary homogenizer shall not exceed 60°C; The primary pressure of the secondary homogenizer is 15 MPa, and the secondary pressure is 8 MPa. The flash sterilization time is 12 seconds; The heat source temperature for the flash sterilization is 76°C. The cooling temperature is 4°C; The vacuum degree of the flash sterilization is 0.5 MPa; During the flash sterilization process, the temperature of the fresh sheep colostrum does not exceed 65°C.

6. The method for preparing sheep colostrum as described in claim 4, characterized in that: The amount of fructose added is 7-8 wt%; The first sterilization temperature is 95-100℃; The first sterilization time is 30-60 seconds; The pressure of the primary homogenizer is 20-25 MPa; The concentration ratio is 20-25% of the original volume; The first freeze-drying agent used in the first freeze-drying process includes 3-4 wt% sorbitol, 5-10 wt% trehalose, 0.2-0.5 wt% ascorbic acid and 0.8-1.2 wt% monosodium glutamate, with the balance being water; The temperature for the first freeze-drying process is between -80°C and -60°C.

7. The method for preparing sheep colostrum as described in claim 4, characterized in that: The inoculum amount of the fermenting agent is 0.1%; The fermentation temperature was 37℃, and the time was 16-25 hours. The vacuum level of the concentration is less than 10 Torr; The concentration temperature is 40-45℃.

8. The method for preparing sheep colostrum as described in claim 4, characterized in that: The preparation method of the fermentation agent includes the following steps: Lactobacillus rhamnosus D8A was streaked onto MRS solid medium and incubated at 37°C until plump single colonies were formed; single colonies were picked and inoculated into MRS liquid medium for two consecutive passages for activation. The activated and expanded Lactobacillus rhamnosus D8A was inoculated into optimized MRS medium at 2-2.5% and fermented for 20-28 hours at a controlled temperature of 36℃±0.5℃, pH 6.8, and dissolved oxygen of 20-25% to obtain the fermentation broth. After fermentation, centrifuge the fermentation liquid and discard the supernatant to obtain bacterial sludge; Mix the bacterial sludge with the second freeze-drying protectant evenly, perform a second freeze-drying until dehydrated, and grind evenly to obtain the fermentation agent; The optimized MRS medium comprises 11.0 g tryptone, 12.0 g beef extract, 4.0 g yeast extract, 25.0 g glucose, 1.5 mL Tween 80, 3.0 g dipotassium hydrogen phosphate, 1.5 g magnesium sulfate, 1.75 g manganese sulfate, and 1 L water.

9. The method for preparing sheep colostrum as described in claim 8, characterized in that: The second freeze-drying process uses a second freeze-drying protectant comprising 10-13 wt% skim milk powder, 5-8 wt% trehalose, 3-6 wt% maltose, 2-4 wt% mannitol, with the remainder being water. The weight ratio of the bacterial sludge to the second freeze-drying protectant is 1:2-2.5; The second freeze-drying temperature is -50°C to -55°C.

10. The method for preparing sheep colostrum as described in claim 8, characterized in that: The centrifugation acceleration is 5000-6000g; The centrifugation temperature is 4-6℃.

Images