Yeast polypeptide derived from saccharomyces boulardii as well as preparation method and application thereof
阅读说明：本技术 源自布拉迪酵母的酵母多肽及其制备方法和应用 (Yeast polypeptide derived from saccharomyces boulardii as well as preparation method and application thereof ) 是由 胡汪洋 连姝凝 刘波静 于 2021-01-22 设计创作，主要内容包括：本发明公开了一种源自布拉迪酵母的酵母多肽及其制备方法和应用。一种源自布拉迪酵母的酵母多肽,名为FTP-21,其全序列为：SWLSGGFKLKKSVVKRFKLCYCRRFCVF,SEQ ID NO:1。所述的多肽的制备方法,化学人工合成或者提取自布拉迪酵母CNCM I-1079的发酵产物。多肽的用途,用于选择性的杀菌或者抑菌,包括E.coli K 88、E.coli K 12、Salmonella ATCC13076、S.aureus ATCC25923、C.perfringens ATCC13124；选择性的保留益生菌,包括Lactobacillus acidophilus ATCC4356、Bifidobacterium longum ATCC27533。所述的多肽的用途,用于调理犬猫肠道菌群以及促进营养吸收的饲料添加剂。所述的多肽的用途,用于制备改善犬猫炎症的免疫调控饲料添加剂。本发明酵母多肽分子量小,净电荷高,稳定性好,溶解性强,易消化吸收,并具有选择性的抗菌活性和安全性高等特点。(The invention discloses a yeast polypeptide derived from saccharomyces boulardii and a preparation method and application thereof. A yeast polypeptide derived from Saccharomyces boulardii, designated FTP-21, having the full sequence: SWLSGGFKLKKSVVKRFKLCYCRRFCVF, SEQ ID NO: 1. The preparation method of the polypeptide comprises the step of chemically and artificially synthesizing or extracting the polypeptide from a fermentation product of the Saccharomyces boulardii CNCM I-1079. Use of a polypeptide for selective bactericidal or bacteriostatic action, comprising E.coli K 88、 E.coli K 12 、 Salmonella ATCC13076 、 S.aureus ATCC25923、 C.perfringens ATCC13124； SelectingThe probiotics can be kept in a sexual mode, Included Lactobacillus acidophilus ATCC4356、 Bifidobacterium longum ATCC27533。 the polypeptide is used as a feed additive for conditioning canine/feline intestinal flora and promoting nutrient absorption. The application of the polypeptide in preparing an immune regulation feed additive for improving inflammation of dogs and cats. The yeast polypeptide has the characteristics of small molecular weight, high net charge, good stability, strong solubility, easy digestion and absorption, selective antibacterial activity, high safety and the like.)
1. A yeast polypeptide derived from saccharomyces boulardii, characterized in that: named FTP-21, the full sequence of which is: SWLSGGFKLKKSVVKRFKLCYCRRFCVF, SEQ ID NO: 1.
2. A method for producing a polypeptide according to claim 1, characterized in that: chemically synthesized or extracted from the fermentation product of Saccharomyces boulardii CNCM I-1079.
3. The method of claim 2, wherein: the extraction procedure of the fermentation product from Saccharomyces boulardii CNCMI-1079 was as follows: selecting Saccharomyces boulardii CNCMI-1079 as a fermenting microorganism, selecting corn and pomegranate rind extracts as fermenting substrates, and adjusting the fermentation parameters as follows: fermenting for 24-48 hours in a fermentation tank with the temperature of 25 ℃, the humidity of 75% and neutral and acidic pH value; freeze-drying in a vacuum freeze-drying drier to obtain yeast powder, crushing yeast cells by mechanical grinding, performing heat treatment and ethanol precipitation to extract yeast protein substances, and purifying by anion exchange to obtain the polypeptide FTP-21.
4. Use of a polypeptide according to claim 1, characterized in that: for selective bactericidal or bacteriostatic action, includingE. coli K 88、 E. coli K 12、Salmonella ATCC13076、S. aureus ATCC25923、C. perfringens ATCC13124；The probiotics can be selectively retained, and the probiotics can be selectively retained,including Lactobacillus acidophilus ATCC4356、Bifidobacterium longum ATCC27533。
5. Use according to claim 4, characterized in that: the feed additive is used for conditioning the intestinal flora of dogs and cats and promoting nutrient absorption.
6. Use according to claim 4, characterized in that: is used for preparing the immune regulation feed additive for improving the inflammation of dogs and cats.
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a yeast polypeptide derived from saccharomyces boulardii, and a preparation method and application thereof.
Background
The Saccharomyces boulardii is a tropical yeast, also called Saccharomyces boulardii, which was first isolated from litchi and mangosteen in 1923 by France scientist Henli, and belongs to Saccharomyces cerevisiae subspecies. Compared with saccharomyces cerevisiae, the survival rate of the saccharomyces boulardii is higher at 37 ℃, 39 ℃, 45 ℃ and 1h in a gastric acid environment, the tolerance of the saccharomyces boulardii in a bile salt environment is obviously higher than that of the saccharomyces cerevisiae, and in addition, the antioxidant function of the saccharomyces boulardii is also stronger. The saccharomyces boulardii is rich in metabolites, and polyamines secreted by the saccharomyces boulardii are beneficial to improving the activity of digestive enzymes, so that the absorption of nutrients by intestinal tracts is promoted. The saccharomyces boulardii can secrete at least 1500 substances in the process of passing through the intestinal tract, and has the functions of promoting the proliferation of beneficial bacteria in the intestinal tract, inhibiting the growth of harmful bacteria, regulating the balance of intestinal flora, enhancing the immunity of the organism and the like. Therefore, the saccharomyces boulardii is widely applied to medicine, food, health care products and feed processing.
The polypeptide is a small molecular protein substance widely existing in organisms in the nature, the source of the polypeptide is very wide, and at present, 3250 various peptides are found in organisms such as animals, plants, microorganisms and the like. Among them, 21 fungal species have been found to be capable of producing polypeptides by fermentation. The partial immune polypeptide has a broad-spectrum antibacterial effect, for example, the partial immune polypeptide has a strong bactericidal effect on common pathogenic bacteria such as escherichia coli, staphylococcus aureus and the like on cats and dogs, the unique membrane-breaking bactericidal mechanism enables the pathogenic bacteria not to easily generate drug resistance, and the partial immune polypeptide is one of potential antibiotic substitutes, for example, the partial immune polypeptide can be used for substituting antibiotics to a great extent in the infection caused by pet diarrhea, postoperative wounds and bacterial diseases, and is beneficial to the long-term health of the cats and dogs. At present, most of the polypeptides reported in the research are secreted by bacteria (gram-positive bacteria and gram-negative bacteria), and although polypeptide products secreted by part of probiotics have a good effect of treating diarrhea of animals such as cats and dogs, long-term feeding of the polypeptide products can easily cause the colonization of exogenous probiotics in intestinal tracts, so that the balance of inherent flora in the intestinal tracts is damaged, and the risk of generating certain dependence on the animals exists when the polypeptide products are fed with the bacterial probiotics for a long time. Compared with bacterial probiotics, the saccharomyces boulardii belongs to 'over-gastric bacteria' as a few yeast probiotics, cannot be planted in intestinal tracts, is metabolized within 48 hours generally, and has the inherent advantage of small side effect. However, there is currently little research on polypeptides of Saccharomyces boulardii.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a yeast polypeptide derived from saccharomyces boulardii, and a preparation method and application thereof.
A yeast polypeptide derived from Saccharomyces boulardii, designated FTP-21, having the full sequence: SWLSGGFKLKKSVVKRFKLCYCRRFCVF, SEQ ID NO: 1.
The preparation method of the polypeptide comprises the step of chemically and artificially synthesizing or extracting the polypeptide from a fermentation product of the Saccharomyces boulardii CNCM I-1079.
The preparation method comprises the following steps of extracting a fermentation product of the Saccharomyces boulardii CNCMI-1079: selecting Saccharomyces boulardii CNCMI-1079 as a fermenting microorganism, selecting corn and pomegranate rind extracts as fermenting substrates, and adjusting the fermentation parameters as follows: fermenting for 24-48 hours in a fermentation tank with the temperature of 25 ℃, the humidity of 75% and neutral and acidic pH value; freeze-drying in a vacuum freeze-drying drier to obtain yeast powder, crushing yeast cells by mechanical grinding, performing heat treatment and ethanol precipitation to extract yeast protein substances, and purifying by anion exchange to obtain the polypeptide FTP-21.
The use of said polypeptide for selective bactericidal or bacteriostatic comprisingE. coli K 88、E. coli K 12、Salmonella ATCC13076、S. aureus ATCC25923、C. perfringens ATCC13124；The probiotics can be selectively retained, and the probiotics can be selectively retained,comprises Lactobacillus acidophilus ATCC4356, Bifidobacterium longum ATCC27533。
The polypeptide is used as a feed additive for conditioning canine/feline intestinal flora and promoting nutrient absorption.
The application of the polypeptide in preparing an immune regulation feed additive for improving inflammation of dogs and cats.
The invention has the beneficial effects that:
the yeast polypeptide FTP-21 is alpha helical cationic polypeptide, is extracted from a fermentation product of Saccharomyces boulardii, and has the characteristics of small molecular weight, high net charge, good stability, strong solubility, easy digestion and absorption, selective antibacterial activity, high safety and the like. The yeast polypeptide FTP-21 has wide antibacterial spectrum and is suitable forE. coli K 88、 E. coli K 12、 Salmonella ATCC13076、 S. aureus ATCC25923、C. perfringens ATCC13124The pathogenic bacteria have antibacterial and bacteriostatic effects, and can be used for treating diseasesLactobacillus acidophilus ATCC4356、Bifidobacterium longum ATCC27533And the like, the probiotics have no sterilization effect. The yeast polypeptide FTP-21 also has the advantages of low cytotoxicity, capability of playing a role in immune regulation and control, improving tear stains of cats, reducing inflammation phenomena such as eye secretion and the like, and can be used for improving soft stool and diarrhea of dogs and cats and relieving stool odor.
Drawings
FIG. 1 is an SDS-PAGE electrophoretogram and its target yeast polypeptide FTP-21;
lane 1 is Marker; lane 2 is yeast fermentation product sample 1 after anion exchange chromatography purification; lane 3 is yeast fermentation product sample 2 after anion exchange chromatography purification; lane 4 is sample 3 of the yeast fermentation product after anion exchange chromatography purification.
FIG. 2 is a diagram of the structure of the yeast polypeptide FTP-21.
FIG. 3 shows the cell morphology of E.coli K88 after yeast polypeptide FTP-21 treatment by Transmission Electron Microscopy (TEM).
FIG. 4 shows the cytotoxicity (LDH release rate) of FTP-21, a yeast polypeptide of different solubility, which is the average of 6 independent replicates.
Detailed Description
The invention is further illustrated with reference to the figures and examples.
Example 1 preparation of Yeast fermentation product and extraction and characterization of polypeptide FTP-21
Selecting Saccharomyces boulardii CNCM I-1079 as fermenting microorganism, selecting corn and pomegranate rind extract as fermenting substrate, adjusting fermenting parameters as follows: fermenting in a fermenter with temperature of 25 deg.C, humidity of 75% and neutral pH and acidity for 24-48 hr. The results of the fermentation product measurements are shown in table 1 below:
and freeze-drying by a vacuum freeze-drying dryer to obtain yeast powder. Mechanically grinding and breaking yeast cells, and then carrying out heat treatment and ethanol precipitation to extract yeast protein substances. After collecting a yeast sample, purifying polypeptide fermented by yeast powder by anion exchange, and specifically comprising the following steps:
loading the column, adding 10 volumes of low-salt buffer solution until the ultraviolet absorption value is stable, injecting the yeast sample into the column, adjusting the flow rate to 1ml/min, continuously adding 10 volumes of low-salt buffer solution until the ultraviolet absorption value is stable, performing gradient elution by using 0.5mol/L NaCl, and collecting the sample. Yeast polypeptide (protein) concentration was determined using the bradford kit and adjusted to 1mg/ml with low salt buffer, 5. mu.g samples were taken for SDS-PAGE analysis, and the remaining samples were cryogenically stored at-80 ℃.
And (4) performing small peptide protein sequencing by using an Edman method. The purified polypeptide sample FTP-21 is fixed on a PVDF membrane printed with protein spots and placed in a reactor, after editing the sample name, the circulation method, the circulation number, the standard amino acid and the sample amount, the polypeptide is degraded in the reactor by an Edman method, an HPLC system is adjusted to an optimal state, and the released amino acid derivative is identified by HPLC. The sequencing process is repeated for the entire FTP-21 until the entire measurable sequence or a predetermined number of cycles is established and the data is finally analyzed. The complete sequence of the determined FTP-21 is as follows: SWLSGGFKLKKSVVKRFKLCYCRRFCVF, SEQ ID NO: 1. Subsequently, the molecular structure of FTP-21 was modeled using the I-TASSER protein structure analysis tool.
The experimental results are as follows: as shown in FIG. 1, the yeast polypeptide of interest FTP-21 is found at the marker band 3.7kDa, in triplicate for sample 1, sample 2 and sample 3. The molecule of FTP-21 is shown in FIG. 2 as a typical alpha helix structure.
Example 2 antibacterial assay (MIC) of the Yeast polypeptide FTP-21
Bacteria (Escherichia coli)E. coliK12, Escherichia coliE. coliK88, salmonellaSalmonellaStaphylococcus aureusS. aureusClostridium perfringensC. perfringensBifidobacterium, bifidobacteriumBifidobacterium longumAnd Lactobacillus acidophilusLactobacillus acidophilus) Inoculating on MH broth solid culture medium, culturing until single colony grows out, selecting single colony and inoculating in 3mL MH broth culture medium, placing into shaking table to culture until bacterial liquid is turbid, taking 30 μ L bacterial suspension and transferring to 3mL fresh MH broth culture medium, and shake culturing at constant temperature until OD600About = 0.5; transfer 10. mu.L of bacterial suspension to 10mL of fresh MH broth medium, mix by vortexing, and control the concentration at 1X 105—5×105CFU/mL, used for determination of Minimum Inhibitory Concentration (MIC) and TEM; carrying out gradient dilution on the antibacterial immune peptide until the final concentration is 1280, 640, 320, 160, 80, 40, 20, 10, 5, 2.5, 1.25, 0.625 and 0.3125 (mu g/mL), adding 90 mu L of prepared bacterial suspension into a sterile 96-hole round bottom plate, then adding 10 mu L of peptide diluent or antibiotic diluent with corresponding concentration one by one, carrying out three repetitions on each antibacterial substance, and additionally setting a positive control hole as a medium without the antibacterial substance, only adding 100 mu L of bacterial suspension and adding 100 mu L of LB culture medium into the negative control hole; and (3) placing the 96-well plate in a biochemical incubator at 37 ℃ for moisturizing and standing culture for 18-24 hours, observing whether bacterial precipitates are generated at the bottom of each well after the culture is finished, and judging the minimum concentration of the bacterial precipitates which are not visible to naked eyes as the MIC of the antibacterial substance.
Meanwhile, a part of yeast polypeptide FTP-21 samples are taken to carry out a Transmission Electron Microscope (TEM) test, escherichia coli is taken as a test object, a control group (blank group) and a yeast polypeptide FTP-21 group are taken, and the treated different groups of samples are transferred to a transmission electron microscope (model: Hitachi H-7650) for observation.
The sequences involved in the polypeptides are specifically as follows:
1) FTP-21 has the full sequence: SWLSGGFKLKKSVVKRFKLCYCRRFCVF, SEQ ID NO: 1;
2) pig-derived immune peptide PMAP-23: RIIDLLWRVRRPQKPKFVTVWVR, respectively;
3) bovine-derived immunopeptide IN: ILPWKWPWWPWRR-NH 2;
4) insect-derived immune peptide CA: KWKLFKKIEKVGQNIRDGIIKAGPAVAVVGQATQIAK, respectively;
5) human-derived immunopeptide LL-37: LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES are provided.
Note: MIC is an abbreviation of minimum inhibition concentration, which means minimum inhibitory concentration, which is an index for measuring the antibacterial activity of an antibacterial drug, and means minimum drug concentration capable of inhibiting growth of pathogenic bacteria in a culture medium after culturing bacteria in vitro for 18 to 24 hours. The smaller the value, the better the bacteriostasis effect.
The detection results are as follows: as shown in Table 2, the yeast polypeptide FTP-21 has a certain antibacterial effect, which is equivalent to LL-37, especially on salmonella and Escherichia coli, and FTP-21 has no obvious inhibitory effect on probiotics, other polypeptides such as LL-37 also have a certain antibacterial effect, but also have a certain inhibitory effect on probiotics such as bifidobacteria, and PMAP-23 has no obvious antibacterial effect, probably because the yeast polypeptide mainly plays an immune regulation function in vivo. In the aspect of antibiotics, the aureomycin has better antibacterial effect but has higher killing effect on probiotics. As shown in FIG. 3, after FTP-21 acts on Escherichia coli K88, cell membrane is ruptured, and the content leaks out, which indicates that FTP-21 kills bacteria on Escherichia coli mainly through membrane rupture.
EXAMPLE 3 cytotoxicity of the Yeast polypeptide FTP-21
Diluting the separated and purified Peripheral Blood Mononuclear Cells (PBMCs) to 1 × 10 with culture medium RMPI16406Per mL, 96-well plates were inoculated at 90uL per well, with background control (100 uL RM)PI1640 medium), low level control wells (90 μ L cell suspension +10 μ L PBS) and high level control wells (90 μ L cell suspension +10 μ L20% triton x-100). 96-well plates were incubated at 37 ℃ with 5% CO2Standing and culturing for 2h in an incubator; 10uL of the corresponding concentrations of FTP-21 and LL-37 were added to the final concentrations of 512. mu.g/mL, 256. mu.g/mL, 128. mu.g/mL, 64. mu.g/mL, 32. mu.g/mL, 16. mu.g/mL, 8. mu.g/mL, respectively, with 6 replicates of each treatment. After culturing in an incubator for 24h, adding 10 mu L of 20% TritonX-100 into the positive control hole, blowing and uniformly mixing by using a pipette, and continuously culturing for 15 min; after the culture is finished, centrifuging at 1500rpm for 10 min; after centrifugation, 60uL of cell supernatant is carefully sucked from each well and transferred to the corresponding well of a transparent flat-bottom 96-well culture plate; and adding 30uL of diluted Lactate Dehydrogenase (LDH) detection reagent into each well, carrying out shake culture at room temperature in dark for 30min, measuring OD492nm and OD900nm by using an enzyme labeling instrument, and calculating a delta OD value.
The LDH release rate was calculated according to the following formula: LDH release rate (%) =100% (. DELTA.OD test-. DELTA.OD negative control)/(. DELTA.OD negative control-. DELTA.OD positive control).
The results of the LDH release rate of FTP-21 peripheral monocytes are shown in FIG. 4, and the LDH release rates of the yeast polypeptide FTP-21 to peripheral monocytes are less than 20% at the concentration of less than 256. mu.g/mL, which indicates that the cytotoxicity of the yeast polypeptide FTP-21 is low.
Example 4 modulation of the digestion of the gut by Yeast fermentation products and the polypeptide FTP-21
Adding 0.5% of yeast fermentation product (containing polypeptide FTP-21) into cat food, selecting 12 domestic cats to feed, and continuously feeding for 14 days. In addition, the results of example 2 show that FTP-21 at low concentrations is able to inhibit intestinal pathogens such as E.coliE. coli K 88While having no significant inhibitory effect on intestinal probiotics such as bifidobacteria.
Example 5 immunomodulation of Yeast fermentation products and the polypeptide FTP-21
The cat food is added with 0.5 percent of yeast fermentation product (containing polypeptide FTP-21), 6 domestic cats with tear stains and serious eye secretion are selected for feeding, and the feeding is continued for 14 days, compared with the prior feeding, the tear stains of 5 cats are relieved to different degrees, specifically, the frequency of the tear stains is obviously reduced, meanwhile, the quantity of the eye secretion is also obviously reduced, and the viscosity is also reduced.
The above list is only a few specific embodiments of the present invention, and it should be noted that the present invention is not limited to the above embodiments, and many other variations are possible, including other similar structures of the synthetic yeast polypeptide FTP-21, changing the number and kinds of amino acids, but not affecting the functional properties of the core sequence, and including other extraction methods of the yeast polypeptide. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Sequence listing
<110> Hangzhou Wu Tai science and technology Co Ltd
<120> Saccharomyces boulardii-derived yeast polypeptide, and preparation method and application thereof
<141> 2021-01-22
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 28
<212> PRT
<213> Saccharomyces cerevisiae boulardii
<400> 1
Ser Trp Leu Ser Gly Gly Phe Lys Leu Lys Lys Ser Val Val Lys Arg
1 5 10 15
Phe Lys Leu Cys Tyr Cys Arg Arg Phe Cys Val Phe
20 25