A probiotic postbiotic of Clostridium butyricum with high selenium-loading activity and its application

Abstract

The present invention provides a postbiotic of a Clostridium butyricum bacterium preparation with high selenium-loading activity and its application, which is prepared by inoculating Clostridium butyricum with the preservation number of CGMCC No. 30181 into a culture medium for fermentation culture. The present invention prepares postbiotics from the fermentation broth of Clostridium butyricum with a high selenium-loading effect. The prepared postbiotics have polypeptides with antibacterial effects, and can promote the growth and development of meat ducks, improve feed conversion rate, increase antioxidant and immune capabilities, and improve meat quality. The compound biological preparation containing postbiotics prepared by the present invention can effectively solve the problem of excessive addition of antibiotic products in current feeds, and will also play an important role in reducing drug residues in poultry products and reducing bacterial drug resistance; in addition, the compound biological preparation contains selenium ions in synergy with prebiotics, which will have a good biological intervention effect on poultry.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological products, and particularly relates to a postbiotic of a Clostridium butyricum bacterium preparation with high selenium-loading activity and its application. Background Art

[0002] With the rapid development of today's social economy, in order to obtain more benefits, more and more farms use the method of adding hormones or drugs to the feed to make animals grow rapidly. However, the improper use of hormones and drugs will damage the stability of the immune system of livestock and poultry, reduce their immunity, and produce drug resistance and drug residues. Selenium not only supports the growth and development of animals, but also prevents myocardial degeneration and necrosis, participates in protein synthesis, maintains normal visual function, improves the body's immunity, enhances its resistance to the invasion of foreign pathogens, and reduces the occurrence of diseases. It can also be used to prevent cardiovascular diseases and diabetes, and scavenge free radicals in the body to achieve the effect of anti-aging.

[0003] Research shows that Clostridium butyricum, used alone or in combination with other probiotics and prebiotics, can effectively treat diarrhea, enteritis, and constipation caused by dysbacteriosis, promote the absorption of vitamin E, decompose bile acids, and improve antioxidant capacity. As a feed additive, Clostridium butyricum can produce antibacterial peptides and small molecule organic acids, fight pathogenic bacteria in the animal digestive tract, maintain and regulate the microbial balance, improve the immune function of animals, and produce digestive enzymes and vitamins to promote animal digestion and food intake and nutritional effects. Therefore, it can be widely used to replace antibiotics in animals such as chickens, pigs, and cows, promote animal growth, prevent and treat dysentery and diarrhea, prevent and treat liver diseases, and improve the survival rate of newborn animals. Summary of the Invention

[0004] The purpose of the present invention is to provide a postbiotic of a Clostridium butyricum bacterium preparation with high selenium-loading activity and its application, and to be used in the preparation of a feed additive for poultry.

[0005] The present invention first provides a postbiotic of a Clostridium butyricum bacterium preparation, which is prepared by inoculating Clostridium butyricum with the preservation number of CGMCC No. 30181 into a culture medium for fermentation culture;

[0006] As a specific record of the embodiment, the culture medium has a composition ratio as follows: peptone 5 - 10 g / L, beef extract powder 5 - 10 g / L, sodium acetate 3 g / L, Se 4+ concentration 40 - 400.0 mg / L, pH value 6.8 - 7.0 ± 0.2.

[0007] Preferably, the Se 4+ concentration is 360.0 mg / L.

[0008] For the fermentation culture described above, the culture temperature is 37 - 38 °C and the time is 24 h.

[0009] Furthermore, the postbiotics of the bacterial preparation contain selenium ions, polypeptides, α - amylase, lipase, lactic acid, butyric acid, acetic acid, and propionic acid;

[0010] As a specific record of the example, the amino acid sequences of the polypeptides are LEEIEEE (SEQ ID NO:1, molecular weight: 889.39), LTEEE (SEQ ID NO:2, molecular weight: 619.27), TIVDDK (SEQ ID NO:3, molecular weight: 689.36), SELEEE (SEQ ID NO:4, molecular weight: 734.30), GVQGAAKMKEA (SEQ ID NO:5, molecular weight: 1088.56), IIKELPTV (SEQ ID NO:6, molecular weight: 911.60), ENFTPLEK (SEQ ID NO:7, molecular weight: 976.49);

[0011] Furthermore, the polypeptides IIKELPTV (SEQ ID NO:6) and ENFTPLEK (SEQ ID NO:7) provided by the present invention are used for preparing antibacterial products.

[0012] Another aspect of the present invention also provides a use of the postbiotics of the bacterial preparation, which is an application as a feed additive.

[0013] The present invention also provides a poultry feed, which is added with the postbiotics of the bacterial preparation.

[0014] The present invention prepares postbiotics from the fermentation broth of Clostridium butyricum with a high selenium - loading effect. The prepared postbiotics have polypeptides with antibacterial effects and can promote the growth and development of meat ducks, improve feed conversion rate, and improve meat quality, such as improving the fatty acid composition of meat ducks, the antioxidant performance of meat ducks, and the immune ability of meat ducks. The postbiotics prepared by the present invention effectively solve the problem of excessive addition of antibiotic products in current feeds and also make significant contributions to reducing drug residues and reducing bacterial drug resistance. In addition, if selenium and Clostridium butyricum act synergistically, it can not only supplement the selenium requirement of livestock and poultry bodies but also balance the intestinal microecology. Description of the Drawings

[0015] Figure 1 : Diagram of the cumulative selenium content enriched by Clostridium butyricum in liquid media with different selenium contents. The control group was fermented in the above - mentioned liquid medium without adding selenium, and the experimental groups (addition amount 40 - 400 mg / L) were fermented by Clostridium butyricum continuously in liquid media containing different selenium.

[0016] Figure 2 : Inhibitory effect diagrams of postbiotics of Clostridium butyricum on Escherichia coli before and after domestication.

[0017] Figure 3 : Inhibitory effect diagrams of postbiotics of Clostridium butyricum on Riemerella anatipestifer before and after domestication.

[0018] Figure 4 : Inhibitory effect diagrams of postbiotics of Clostridium butyricum on Salmonella before and after domestication.

[0019] Figure 5 , 6 , 7, 8: Flow cytometry diagrams of Escherichia coli, Riemerella anatipestifer, and Salmonella when the addition amount of postbiotics of Clostridium butyricum with high selenium load is 1:1.

[0020] Figure 9 : Electron microscopy diagrams of the morphological effect of postbiotics of Clostridium butyricum with high selenium load on Escherichia coli.

[0021] Figure 10 : Electron microscopy diagrams of the morphological effect of postbiotics of Clostridium butyricum with high selenium load on Riemerella anatipestifer.

[0022] Figure 11 : Secondary mass spectrometry and planar structure diagrams of the IIKELPTV sequence.

[0023] Figure 12 : Secondary mass spectrometry and planar structure diagrams of the ENFTPLEK sequence.

[0024] Figure 13 : 3D structure diagrams of the IIKELPTV sequence.

[0025] Figure 14 : 3D structure diagrams of the ENFTPLEK sequence.

[0026] Figure 15 : Histogram of the effect of Clostridium butyricum with high selenium ions on the fatty acids in the breast muscle of meat ducks. Detailed implementation manners

[0027] Based on obtaining Clostridium butyricum with high selenium load (preservation number: CGMCC No. 30181), the present invention prepares a fermentation broth with bactericidal and growth-promoting effects through the optimization of fermentation conditions, and analyzes the components in the broth.

[0028] The starting strain Clostridium butyricum used in the present invention is cultured in a medium containing high-concentration Se 4+ ions, and a Clostridium butyricum strain capable of being rich in high-concentration Se 4+ is obtained through domestication and screening. The screened strain is in high Se 4+It can grow stably under certain concentration conditions, improving the ability of Clostridium butyricum to carry a high load of selenium. When the addition amount of selenium ions in the culture medium is 360 mg / L, the selenium ion content in the bacterial cells can reach 21.30 μg / L. This Clostridium butyricum was deposited on March 28, 2024 at the General Microbiology Center of the China Microbial Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 30181.

[0029] The culture medium used in the embodiments of the present invention has a specific composition as follows: peptone 5 - 10 g / L, beef extract powder 5 - 10 g / L, sodium acetate 3 g / L, Se 4+ concentration 40 - 400.0 mg / L, pH value 6.8 - 7.0 ± 0.2. The culture temperature is 37 - 38 °C and the time is 24 h.

[0030] The present invention will be described in detail below in conjunction with specific embodiments and drawings.

[0031] Example 1: Cultivation and domestication of Clostridium butyricum with high selenium ion load

[0032] The Clostridium butyricum was domesticated by gradually increasing the Se 4+ concentration in the culture medium. After the domesticated strain was continuously cultured for several generations, the cell morphology, growth activity, and selenium content of the strain were compared with those of the Clostridium butyricum before domestication to further determine the characteristics of the domesticated Clostridium butyricum and its ability to be stably passaged.

[0033] A domesticated strain of Clostridium butyricum (CGMCC No. 30181) that can tolerate high concentrations of selenium ions (Se 4+ )(360 mg / L) was deposited on March 28, 2024 at the General Microbiology Center of the China Microbial Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 30181. The domesticated selenium-rich Clostridium butyricum has the ability to carry selenium ions. When the addition amount of selenium ions is 360.0 mg / L, the organic selenium content in the bacterial cells is 52.41 μg / L (see Figure 1 ), which is greater than the selenium content of ordinary Lactobacillus plantarum from ducks.

[0034] Example 2: Preparation of postbiotics from Clostridium butyricum before and after domestication and analysis of active ingredients

[0035] 1) Preparation of postbiotics rich in small peptides: Absorb 200 μl of the strain and inoculate it into 100 ml of RCM medium. Under anaerobic conditions, culture at 37 °C for 24 h, then centrifuge at 4 °C and 6000 r / min for 10 min. Take the supernatant and filter it through a biological organic filter membrane to obtain postbiotics, which are rich in small peptides.

[0036] The specific composition of the medium used is as follows: peptone 5 - 10 g / L, beef extract powder 5 - 10 g / L, sodium acetate 3 g / L, Se 4+ concentration 40 - 400.0 mg / L, pH value 6.8 - 7.0 ± 0.2.

[0037] 2) Component analysis of postbiotics: The amylase content in the postbiotics of Clostridium butyricum after domestication is 0.22 U / mL, which is 4.76% higher than that of the postbiotics prepared from Lactobacillus plantarum before domestication; the lipase content is 35.66 U / L, which is 60.12% higher than before domestication; the small peptide content is 20.98 mg / mL, which is 12.25% higher than before domestication; the acetic acid content is 0.107 mg / mL, which is 16.30% higher than before domestication; the butyric acid content is 0.762 mg / mL, which is 14.93% higher than before domestication; the isovaleric acid content is 0.119 mg / mL, which is increased by 10.92%; the amino acid content is 563.98, which is 2.35% higher than before domestication.

[0038] Example 3: Comparison of the antibacterial effects of postbiotics of Clostridium butyricum before and after domestication

[0039] 1) Determination of the antibacterial curve at different concentrations of postbiotics rich in small peptides

[0040] After culturing the high-load selenium Clostridium butyricum (CGMCC No. 30181) in RCM medium for 24 h, extract the postbiotics and mix them with the medium inoculated with harmful bacteria at a ratio of 1:3 and 1:1, and then put them into a microbial growth analyzer to measure the growth curve respectively.

[0041] Figure 2 、 Figure 3 、 Figure 4 The results show that before and after domestication, when the addition amount of Clostridium butyricum to harmful bacteria is 1:3, there is no inhibitory effect on harmful bacteria. Before and after domestication, when the addition amount of Clostridium butyricum to harmful bacteria is 1:1, there is an obvious inhibitory effect on the growth and reproduction of Escherichia coli, Riemerella anatipestifer, and Salmonella; Figure 2 It shows that Escherichia coli added with postbiotics of Clostridium butyricum did not show growth phenomenon within 36 h continuously, Figure 3 It shows that Riemerella anatipestifer added with postbiotics of Clostridium butyricum did not show growth phenomenon within 36 h continuously, Figure 4It was shown that Salmonella with the addition of postbiotics of Clostridium butyricum did not grow within 24 consecutive hours. The above measurement results indicated that when the addition ratio of Clostridium butyricum postbiotics was 1:1, it had a good inhibitory effect on harmful bacteria, and the antibacterial effect of the domesticated Clostridium butyricum (CGMCC No. 30181) was enhanced.

[0042] 2) Determination of the apoptosis rate of harmful bacteria when the addition ratio of postbiotics rich in small peptides was 1:1

[0043] The results in Table 2 showed that Clostridium butyricum with high selenium load (CGMCC No. 30181) could increase the apoptosis rates of Escherichia coli, Salmonella, Staphylococcus aureus, and Riemerella anatipestifer, indicating that the antibacterial effect was enhanced after domestication. Similarly, Figures 5 - 8 It was shown that after adding the postbiotics of Clostridium butyricum with high selenium load, the number of dead bacteria increased, the apoptosis rate of cells increased, and the antibacterial effect was obvious.

[0044] Table 1: Influence of postbiotics of Clostridium butyricum with high selenium load on the apoptosis rate of harmful bacteria

[0045]

[0046] 3) Morphological changes of harmful bacteria when the addition ratio of postbiotics rich in small peptides was 1:1

[0047] From Figure 9 It could be seen by scanning electron microscopy that the postbiotics of Clostridium butyricum with high selenium load would damage the structure of Escherichia coli, and obvious rupture occurred to the bacteria after adding the postbiotics. From Figure 10 It was known that obvious shrinkage and rupture occurred to the cells of Riemerella anatipestifer after adding the postbiotics of Clostridium butyricum with high selenium load. It indicated that the postbiotics of Clostridium butyricum with high selenium load could effectively inhibit the growth of Escherichia coli and Riemerella anatipestifer.

[0048] Example 4: Analysis of small peptide sequences and effect identification of postbiotics of Clostridium butyricum before and after domestication

[0049] 1) Analysis of small peptide sequences in postbiotics

[0050] Commission a commercial company to conduct protein Shotgun identification and analysis on Clostridium butyricum and Clostridium butyricum with high selenium load. The experimental method is to add an appropriate amount of 0.1% TFA to the sample, mix well, centrifuge at 20000g for 5 minutes, take the supernatant, transfer it to a 10KD ultrafiltration centrifugal tube, and centrifuge at 12000g for 15 minutes. Add 200 μL of 0.1% TFA and centrifuge at 12000g for 15 minutes, repeat 2 times, collect the filtrate, desalt it using C18 StageTip, and vacuum dry. After drying, the peptide segments are redissolved with 0.1% FA, and the peptide segment concentration is measured for LC-MS analysis. Take an appropriate amount of peptide segments from each sample and perform chromatographic separation using a nano-flow rate Easy nL C1200 chromatographic system (Thermo Scientific). After the sample is injected into the Trap Column, it undergoes gradient separation through the chromatographic analysis column at a flow rate of 300 nl / min. After peptide segment separation, DDA (data-dependent acquisition) mass spectrometry analysis is performed using a Q-Exactive HF-X mass spectrometer (Thermo Scientific). Retrieve through the mass spectrometry database retrieval software MaxQuant 2.4.14.0. After centrifuging the fermentation broth of the strain, filter it through an organic filter membrane to make postbiotics. Add 0.1% TFA to the postbiotics, mix well, centrifuge at 20000×g for 5 minutes, take the supernatant, transfer it to a 10KD ultrafiltration centrifugal tube, and centrifuge at 12000×g for 15 minutes. Add 200 μL of 0.1% TFA and centrifuge at 12000×g for 15 minutes, repeat 2 times, collect the filtrate, desalt it using C18 StageTip, and vacuum dry. The dried polypeptide is redissolved with 0.1% FA, and the peptide segment concentration is measured. Take an appropriate amount of peptide segments and perform chromatographic separation using a nano-flow rate Easy nLC 1200 chromatographic system (Thermo Scientific).

[0051] The main amino acid sequences measured are LEEIEEE, LTEEE, TIVDDK, SELEEE, GVQGAAKMKEA, IIKELPTV, ENFTPLEK; among them, the unique amino acid sequences of Clostridium butyricum with high selenium load are LEEIEEE, LTEEE, SELEEE, IIKELPTV, ENFTPLEK.

[0052] Among them, the secondary mass spectrometry diagrams and structures of the unique amino acid sequences IIKELPTV and ENFTPLEK of the postbiotics of Clostridium butyricum with high selenium load are as Figures 11 - 14 shown. Literature reports that antibacterial peptides generally have specific amino acids such as lysine, arginine, tryptophan, and glycine. The peptide segments obtained from the postbiotics of Clostridium butyricum with high selenium load contain lysine. Based on the amino acid sequence speculation, the postbiotics of Clostridium butyricum with high selenium load have higher antibacterial ability.

[0053] 2) Identification of the antibacterial effect of the mixed peptides of Clostridium butyricum with high selenium load

[0054] The small peptides with amino acid sequences of IIKELPTV and ENFTPLEK were commissioned to a company for chemical synthesis, and the micropore dilution method was used to verify the antibacterial effect of the synthetic peptides on Escherichia coli, Salmonella and Riemerella anatipestifer. The culture medium without added bacteria was set as the negative control, the culture medium with added bacteria but no antimicrobial peptides was set as the positive control, and the culture medium with added antimicrobial peptides was set as the experimental group. The culture was cultured at 37℃ for 24 hours, the absorbance was measured at a wavelength of 600nm, and the inhibition rate was calculated.

[0055] Table 2: Inhibition rate of harmful bacteria by high-load selenobutyric acid Clostridium synthetic peptides

[0056]

[0057] From the results in Table 2, it can be seen that the screened small peptides have better antibacterial effects on Escherichia coli, Salmonella and Riemerella anatipestifer.

[0058] Example 5: Effect of high-load selenobutyric acid Clostridium postbiotics on the growth and development of broiler ducks

[0059] 180 healthy one-day-old Beijing ducks with similar body weight were selected and randomly divided into 5 groups, with 6 replicates in each group and 6 ducks in each replicate. The control group was fed with a basic diet, group I was fed with a basic diet + Clostridium butyricum fermentation broth, group II was fed with a basic diet + a high-load Clostridium selenobutyricum body group, group III was fed with a basic diet + a high-load Clostridium selenobutyricum postbiotics, and group IV was fed with a basic diet + a high-load Clostridium selenobutyricum fermentation broth group. The amount of probiotic preparation added to the experimental diet was 0.3%. The experimental design is shown in Table 3, and the basic diet composition is shown in Table 4. The feeding period was 42 days.

[0060] Table 3: Design table of meat duck breeding experiment

[0061]

[0062] Table 4: Basic feed composition for broiler ducks

[0063]

[0064] 1 The vitamin premix provides the following per kilogram of feed: VA1 2000 IU; VD3 2500 IU; VE 20 mg; VK3 3 mg; VB1 3 mg; VB2 8 mg; VB6 7 mg; D-pantothenic acid 20 mg; niacin 50 mg; folic acid 1.5 mg; biotin 0.1 mg. 2) The trace element premix provides the following per kilogram of feed: copper 9 mg; zinc 110 mg; iron 100 mg; manganese 100 mg; iodine 0.6 mg. 3) Metabolizable energy is a calculated value, and the others are measured values.

[0065] Table 5 results showed that, compared with the control group, the daily feed intake and feed-to-weight ratio of the 42-day-old ducks in Group II, Group III, and Group IV were significantly reduced (P < 0.05). Among them, the feed-to-weight ratio in Group I decreased by 3.11%, in Group II by 10.12%, in Group III by 10.89%, and in Group IV by 12.45%. This indicates that high-loaded selenium butyric acid clostridium postbiotics also have biological activity. Adding high-loaded selenium butyric acid clostridium (CGMCC No. 30181) to the diet can reduce the feed-to-weight ratio, improve feed utilization rate, enhance the digestion and absorption ability of meat ducks, and thus improve the growth performance of meat ducks.

[0066] Table 5: Growth performance of 42-day-old meat ducks

[0067]

[0068]

[0069] Example 6: Effect of high-loaded selenium butyric acid clostridium postbiotics on the slaughter performance of meat ducks

[0070] Table 6 results showed that, compared with the control group, the dressing percentage and eviscerated percentage of Group II, Group III, and Group IV were significantly increased (P < 0.05). The breast muscle percentage of Group I, Group II, Group III, and Group IV was significantly increased (P < 0.05). Among them, Group III had the best slaughter performance, with the dressing percentage increased by 4.20%, the eviscerated percentage increased by 5.62%, and the breast muscle percentage increased by 39.46%. This indicates that high-loaded selenium butyric acid clostridium postbiotics can effectively improve the slaughter performance of meat ducks.

[0071] Table 6: Slaughter performance of 42-day-old meat ducks

[0072]

[0073] Example 7: Effect of high-loaded selenium butyric acid clostridium postbiotics on the meat quality of meat ducks

[0074] As shown in Table 7, compared with the control group, the lightness (L*) of Group I, Group II, Group III, and Group IV was significantly reduced (P < 0.05); the redness (a*) of Group II, Group III, and Group IV was significantly increased (P < 0.05); the cooking loss of Group III was significantly reduced (P < 0.05). The cooking loss of Group III decreased by 28.30%. The lower the cooking loss, the better the water-holding capacity of the muscle. This indicates that high-loaded selenium butyric acid clostridium postbiotics can improve the meat color of meat ducks and effectively enhance the water-holding capacity of meat ducks.

[0075] Table 7: Meat quality of 42-day-old meat ducks

[0076]

[0077]

[0078] Example 8: Effect of high-loaded selenium butyric acid clostridium postbiotics on the fatty acid composition of meat ducks

[0079] As can be seen from Table 8 and Figure 15 it can be known that, compared with the control group, the contents of oleic acid (C18:1n9c) and monounsaturated fatty acids in Group I, Group II, Group III and Group IV increased, and the contents in Group II and Group IV increased significantly (P < 0.05); the content of saturated fatty acids decreased, but not significantly. Unsaturated fatty acids are involved in generating the rich aroma and flavor of duck meat and are important indicators for evaluating meat quality and nutritional value. The experiment shows that adding high-load selenium probiotics of Clostridium butyricum to the diet can improve the fatty acid structure of meat ducks.

[0080] Table 8: Fatty acid composition table of 42-day-old meat ducks

[0081]

[0082]

[0083] Example 9: Effect of high-load selenium probiotics of Clostridium butyricum on the antioxidant function of meat ducks

[0084] As can be seen from Table 9, compared with the control group, MDA in each experimental group decreased significantly (P < 0.05), GSH-PX in Group II and Group III increased significantly (P < 0.05), and T-SOD in each experimental group increased significantly (P < 0.05). Among them, the antioxidant capacity of Group IV was the highest, indicating that the antioxidant capacity of high-load selenium Clostridium butyricum is stronger than that of Clostridium butyricum.

[0085] Table 9: Antioxidant capacity table of 42-day-old meat ducks

[0086]

[0087] Example 10: Effect of high-load selenium Clostridium butyricum on the immune performance of meat ducks

[0088] As can be seen from Table 10, compared with the control group, the number of immunoglobulins in the experimental groups increased, but not significantly (P > 0.05).

[0089] Table 10: Immune performance table of 42-day-old meat ducks

[0090]

[0091] In summary, the step-by-step increase of Se in the culture medium adopted in the present invention 4+The high selenium-loaded Clostridium butyricum (CGMCC No. 30181) obtained by concentration domestication screening not only has the probiotic advantages of Clostridium butyricum, but also has the ability to highly load selenium ions, which can improve the selenium absorption rate, reduce the environmental pollution and biological toxicity of selenium ions, improve the ability of inorganic selenium to be converted into organic selenium in the cells, and greatly improve the utilization rate of selenium ions by poultry. At the same time, the postbiotics produced by the domesticated and selected high selenium-loaded Clostridium butyricum are abundant, containing small peptides (antibacterial peptides), small molecule organic acids, digestive enzymes, etc. Because of the rich targeted antibacterial peptides, it has obvious inhibitory effects on harmful bacteria such as Escherichia coli, Salmonella, and Riemerella anatipestifer. Other bioactive substances such as digestive enzymes contained can promote the growth and development of poultry, enhance the meat quality of poultry products, and improve the antioxidant and immune abilities of poultry.

Claims

1. A polypeptide, characterized in that The amino acid sequence of the polypeptide is SEQ ID NO:6 or SEQ ID NO:

7.

2. Use of the polypeptide according to claim 1 in the preparation of products against Escherichia coli, Salmonella or Riemerella anatipestifer.

Images