Clostridium butyricum and application of clostridium butyricum in pig mother-child integrated feeding

Abstract

The invention discloses clostridium butyricum with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No. 29591 and discloses application of the clostridium butyricum CGMCC No. 29591 in integrated feeding of mother and child pigs, and experiments find that the clostridium butyricum CGMCC No. 29591 has obvious effects in pig breeding, such as improvement of breeding of sows, improvement of growth of piglets and the like, and the PSY (Particle Swarm Yarn) index is obviously improved.

Description

Technical Field

[0001] The invention relates to the field of breeding technology, and in particular to Clostridium butyricum and application thereof in integrated pig mother-and-kidney feeding. Background Art

[0002] The survey shows that the PSY index in European and American countries is 25 heads, and in some Nordic countries the PSY even reaches 30 heads, while the PSY index of large-scale pig farms in my country is around 18 heads. Only a few breeding pig companies have a PSY of around 20 heads, but their improved technology is kept confidential.

[0003] In order to improve the PSY index, domestic pig breeding companies directly introduced sow breeds from abroad, but the farrowing rate and piglet survival rate of localized pigs have never met expectations, indicating that there are shortcomings in the technology of sow breeding. In recent years, with the introduction of the concept of "mother-child integration", there has been an increasing number of studies on targeted regulation of the nutritional and metabolic needs of mothers and children to improve the reproductive performance of sows.

[0004] As some studies have pointed out, sows will undergo dramatic physical changes during pregnancy and lactation to adapt to the requirements of pregnancy and lactation. These two stages will directly affect the sow's piglet size, lactation performance, and the piglet's survival rate, birth weight, and weaning weight.

[0005] As some studies have pointed out, there is a significant correlation between the intestinal flora of piglets and sows. Maternal microorganisms determine the early intestinal flora colonization of suckling piglets. The impact of maternal microorganisms on piglets is mainly reflected in the intestines of piglets, which is crucial to the growth and development of the offspring's immune system.

[0006] According to the above research theory, some companies add specific preparations, such as Chinese medicine compositions, Western medicine preparations, and bacterial agents, to the feed of sows or piglets during specific time periods such as pregnancy and lactation, in order to improve the PSY index by improving the physique of sows and the survival rate of piglets. Relatively speaking, the effect is not ideal and needs to be improved. Summary of the invention

[0007] To solve at least one of the above technical defects, the present invention provides the following technical solutions:

[0008] The first aspect of the present application document discloses a Clostridium butyricum, with the deposit number being CGMCCNo29591.

[0009] The second aspect of this application document discloses the application of Clostridium butyricum CGMCCNo29591 in the integrated feeding of pig mother and piglet.

[0010] By chance, the inventors discovered a strain in the soil of a farm, and obtained the strain after separation and purification, and then obtained the preserved strain through measures such as mutagenesis. After biological property detection, 16sDNA sequencing analysis and other means, it was finally identified as Clostridium butyricum with a preservation number of CGMCC No 29591. In subsequent experiments, it was found that it had significant effects in pig breeding, such as improving the breeding of sows and the growth of piglets, and the PSY index was significantly improved.

[0011] As for the use of Clostridium butyricum, it can be used in direct feed, microecological preparations, and powdered bacteria.

[0012] In addition, the butyric Clostridium is effective in inhibiting the growth of harmful bacteria, such as Escherichia coli, Staphylococcus aureus, Salmonella, and Clostridium perfringens.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] 1. The Clostridium butyricum in the present invention is suitable for use in pig breeding, and has the following advantages: the litter weight and average litter weight of weaned piglets are increased, the back fat loss of sows during the weaning period is reduced, the constipation rate of sows and the diarrhea rate of piglets are reduced, the piglet survival rate is high, the sow's delivery process is shortened, the estrus interval is shortened, the PSY index is significantly improved, and the economic benefits are significantly improved.

[0015] Preservation Information

[0016] A butyric Clostridium butyricum of the present invention is deposited in the China Center for Type Culture Collection (CGMCC for short), the address of which is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit number is CGMCCNo29591, and the deposit date is January 15, 2024. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0018] Figure 1 It is the inhibition zone diagram;

[0019] Figure 2 It is a liquid phase detection diagram;

[0020] Figure 3 This is a picture of sow feces. DETAILED DESCRIPTION

[0021] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0022] First, isolation, culture and detection of Clostridium butyricum

[0023] 1) Separation step

[0024] The soil from the pig farm was counted using the dilution plate method, and single colonies were isolated by streaking on a culture dish. The culture medium composition (g / L): 20g cornmeal, 10g soybean meal, 5g glucose, 5g yeast extract, 5g sodium chloride, 3g K2HPO4, 3g KH2PO4, 0.3g MnSO4, and 17 agarose with a pH of 7.0. Sterilize at 121°C, and after cooling to 55°C, pour the plate, ferment at 36°C, put it in an anaerobic tank, add an oxygen repellent, and culture it statically. Subculture single colonies for later use.

[0025] 2) Cultivation and purification

[0026] The selected Clostridium butyricum was inoculated into the culture medium, the culture medium components: 40g glucose, 30g yeast extract powder, 0.15g calcium chloride, 3g potassium dihydrogen phosphate, 3g dipotassium hydrogen phosphate, 5g sodium chloride, 5g calcium carbonate, 1L water, sodium hydroxide was used to adjust the pH value to 7.0, and then 3g sodium pyrosulfite was added to form the culture medium, and the culture was cultured in an anaerobic tank at a temperature of 33°C for 24h to obtain the relevant strains. Liquid fermentation broth The selected Clostridium butyricum was inoculated into the liquid culture medium, the culture medium components: culture medium (g / L): 20g cornmeal, 10g soybean meal, 5g glucose, 5g yeast extract, 5g sodium chloride, 2g sodium pyrosulfite, 3g KH2PO4, 5g taurine, pH was adjusted to 8.5. Sterilized at 121°C. The fermentation temperature was 36°C, and the culture was statically cultured to obtain the fermentation broth of different strains.

[0027] 3) Identify the isolated strains

[0028] 3.1. Physiological and biochemical identification: Use the purchased Clostridium butyricum biochemical identification strips and follow the prescribed steps to conduct identification, as shown in the following table

[0029] Table 1. Physiological and biochemical reaction results

[0030] Anaerobic growth lactose maltose sucrose Glucose (anaerobic) fructose + + + + + + starch Litmus Melezitose Xylose Gas production Butyrate production + + - + + +

[0031] The morphological characteristics of the Clostridium brevebutyricum are as follows: the bacteria are in the shape of thin rods, with both ends being spindle-shaped, generally 0.8-1.2 μm×1.5-2.0 μm in size, and are Gram-positive.

[0032] 3.2. DNA extraction of Clostridium butyricum and sequencing of 16sDNA fragments amplified by PCR

[0033] 3.21. Purpose of the experiment: Through PCR, the isolated strains were amplified in vitro using 16sDNA specific tag sequences, and the PCR products were sent to a biological company for sequencing. The sequencing results were compared by bl ast to identify the species of the strains.

[0034] 3.22 DNA extraction

[0035] Add 400uL of sterile pure water to the sterilized 1.5mL centrifuge tube, and at the same time, pick up a full loop of the strain to be tested from the culture dish in the clean bench and add it to the centrifuge tube. Place the centrifuge tube on an oscillator for oscillation and mixing, insert it into the float, and place it in an ultrasonic instrument. Sonicate for 5 minutes at 40KHZ and 600w power. Take it out and shake it again, and sonicate it for 5 minutes again. Sonicate it 3 times in total, each time for 5 minutes. Then put the bacterial solution after ultrasonic disruption into the refrigerator as a DNA template for standby use.

[0036] 3.23 DNA extraction from cell-disrupted strains

[0037] During the experiment, it was found that for a small number of strains, it was difficult to effectively release the DNA in the cells, whether it was ultrasonic disruption or high-temperature heating to denature the cells. Therefore, for such strains, a DNA extraction kit was used to extract the bacterial DNA, which was used as a template for PCR amplification. The kit used in this process is Shanghai Shenggong, Bacterial Genomic DNA Rapid Extraction Kit (Product No.: B518225) The standard operating steps are:

[0038] 3.231. Take 1 ml of overnight cultured bacterial solution and add it to a 1.5 ml centrifuge tube. Centrifuge at 8,000 rpm for 1 min at room temperature. Discard the supernatant and collect the bacteria. Add 500 μl of Buffer Digest ion and shake to mix. Incubate in a 65°C water bath for 2 h until the cells are completely lysed.

[0039] 3.232. During the water bath, invert and mix once every 10 minutes to promote sample lysis. When the mixed solution becomes clear and transparent, the lysis is complete. If the solution does not become clear, it means that the sample is not completely lysed and the water bath time should be appropriately extended.

[0040] 3.233. Add 200 μl Buffer PB, mix thoroughly by inversion, and place on ice for 5 min. Centrifuge at 10,000 rpm for 5 min at room temperature, and transfer the supernatant (500-550 μl) to a new 1.5 ml centrifuge tube.

[0041] 3.234. Add an equal volume of isopropanol, invert 5-8 times to mix thoroughly, and place at room temperature for 2-3 minutes. Centrifuge at 10,000 rpm for 5 minutes at room temperature and discard the supernatant. Add 1 ml of 75% ethanol, invert and rinse for 1-3 minutes, centrifuge at 10,000 rpm for 2 minutes, and discard the supernatant (twice).

[0042] 3.235. Open the lid and invert at room temperature for 5-10 minutes until the residual ethanol evaporates completely. Dissolve the obtained DNA with 50-100 μl TEBuffer. The extracted DNA can be used for the next experiment immediately or stored at -20℃.

[0043] 3.24. Establishment of PCR system and control of related parameters

[0044] The PCR system is shown in Table 2, where the PCR process parameters are set as follows:

[0045] 1. 94°C for 10 minutes (to increase the amount of DNA released).

[0046] 2. {94°C for 30 s, 55°C for 30 s, 72°C for 90 s} × 30 cycles.

[0047] 3. Extend at 72°C for 10 minutes.

[0048] 4. Store at 10℃.

[0049] Table 2. PCR system

[0050]

[0051] The PCR product was subjected to gel electrophoresis using 1% agarose. During the gel process, the color developer was added at 1uL / 100mL (Shanghai Biotechnology, 4s green color developer). 2uL of the PCR product was mixed with 1uL of loading buffer and spotted into the agarose gel electrophoresis tank. The voltage was set to 170V and the time was 20 minutes.

[0052] 3.25. Alignment of Sequencing Data

[0053] Copy the 16s fragment sequence and the base sequence in the nucleic acid sequence txt file sent back by Bioengineering after sequencing. At the same time, log in to the NCBI gene database, enter the Blast comparison mode, select the nucleic acid comparison database, enter the sequenced seq file sequence into the comparison window, adjust the comparison parameters, and select the strain with a sequence similarity of more than 90% as the reference strain for identification.

[0054] 3.26 Results

[0055] The value of DNA concentration depends on the nucleic acid analyzer for detection, so the gradient dilution method is adopted to explore through experiments, and the dilution is 10 times, 100 times, 150 times and 200 times respectively. Finally, it is detected that the DNA template concentration diluted 10 times is suitable for the current PCR system. The bl ast comparison results show that the 16s sequence of this strain has a similarity of 98% with a variety of Clostridium butyricum strains, so it is determined that this strain belongs to Clostridium butyricum.

[0056] Second, antibacterial test

[0057] 2.1. Strain culture

[0058] Cultivate the fermentation broth of Clostridium butyricum using the RCM medium and verify the antibacterial zone. (RCM ingredient list: 10g peptone, 10g beef powder, 3g yeast extract powder, 5g glucose, 1g soluble starch, 3g sodium chloride, 3g sodium acetate, 0.5g L-cysteamine hydrochloride, 1L water, adjust the pH value to 7.0 with sodium hydroxide, and then add 3g sodium bicarbonate). According to the RCM medium, in the clean bench, inoculate two full rings of Clostridium butyricum into the culture medium, tie the 4 layers of sealing film tightly, seal it tightly, put it in the incubator, 37℃, and culture it for 24h.

[0059] 2.2. Use inhibition zone to verify strains

[0060] The inhibition zone experiment uses Escherichia coli, Staphylococcus aureus, Salmonella, Clostridium perfringens, etc. from the bacterial culture collection center as indicator bacteria. First, prepare a 1.5% agar medium, sterilize it at 121°C, and pour it into a culture dish as the lower plate. After solidification, place Oxford cups on the surface, with each Oxford cup about 5 cm apart, for standby use. Then sterilize the nutrient agar medium and place it in a 52°C water bath for standby use. Then add the bacterial solutions of ten pathogenic bacteria, including Staphylococcus aureus, Escherichia coli, Salmonella, Clostridium perfringens, Listeria suis, Streptococcus, Pseudomonas aeruginosa, Corynebacterium pyogenes, Klebsiella, and Haemophilus parasuis, to the 52°C nutrient agar medium to dilute the concentration of the pathogens to 10 4 -10 5 CFU / mL level. Then pour about 20mL on top of the lower plate, wait for 3-4 minutes for the upper plate to solidify, use tweezers to pull out the Oxford cup for standby use. Weigh 2.5g of the prepared experimental sample, pour it into a 10mL centrifuge tube, then add 5mL of normal saline (0.85%), dilute it 2 times, shake it evenly, then draw 150-200uL of the sample dilution, add it to the agar hole of the Oxford cup, first put it in a 4℃ refrigerator, diffuse for 4 hours, then put it in an incubator at 37℃ and culture it for 24h. Anaerobic bacteria need to be cultured in anaerobic tanks, and then check the inhibition zone and detect the diameter of the inhibition zone.

[0061] Depend on Figure 1 It can be seen that the Clostridium butyricum can inhibit 10 strains of pathogenic bacteria, with the diameter of the inhibition zone between 10-25mm, and has the function of inhibiting a variety of harmful bacteria.

[0062] 2.3. Extraction and detection of antibacterial substances

[0063] 2.31. First, heat inactivate the culture broth of Clostridium butyricum of different models, heat at 121℃ for 20min, inactivate protease, precipitate impurities and completely release antimicrobial peptides from the bacteria into the fermentation broth. Then centrifuge at 4000rpm, discard the precipitate, filter the supernatant with a 0.45 filter membrane, and the filtrate obtained is used to purify the antimicrobial peptides.

[0064] 2.32. Add the filtrate extracted in the previous step to a rotary evaporator and perform rotary evaporation at 70-80°C and 40 rpm. Add 200 mL of supernatant and concentrate to about 20 mL (10 times). Add an equal volume (20 mL) of acetone to perform organic solvent extraction 3 times. During the extraction, shake the organic solvent and antimicrobial peptide liquid on a shaker at 200 rpm for 3 minutes to facilitate thorough mixing. Then let it stand for 5 minutes to allow the organic phase and the aqueous phase to be completely separated. Absorb the organic phase and the mixed organic solvent extracts three times, send them to a fume hood, and volatilize them in a water bath at 70 degrees. After concentrating the antimicrobial peptide precipitate at the bottom, add 3 mL of saline to dissolve it, and perform inhibition zone experiments and high-performance liquid chromatography detection on the dissolved solution.

[0065] 2.33. Detection of Clostridium butyrate concentration by HPLC

[0066] 0.22um filter membrane was used for filtration, and methanol was used as the mobile phase for liquid phase detection. In the process of extracting antimicrobial peptides, the Clostridium butyricum with the largest inhibition zone at 48h in 3.1 above was cultured in medium 64 for the same time, and the antimicrobial peptides were purified using the same process, and then the purified solution was used as a standard for comparison.

[0067] Because this HPLC is not only a qualitative analysis, but also involves the comparison of peak cross-sectional areas, the detailed operation methods, node control, and parameter settings of HPLC are listed in detail as follows:

[0068] First, turn on the machine and select the method (BOSHIDE-LC). When setting this method, select standard injection, injection volume of 5.0μL; control flow rate of 0.400mL / min, stop time of 30min; column temperature of 70℃, stop time consistent with the pump, 30min; MWD signal A, sample 220, bandwidth 60, reference 360, bandwidth 100, stop time consistent with the pump, 30min, select UV lamp for required lamp, peak width (response time) > 0.1min (2s); select pre-run for automatic balance. Adjust balance (open the valve first, flow until there are no bubbles in the tube; then tighten the valve, and let the mobile phase flow for 30min); mobile phase A is water: formic acid = 100:0.1; mobile phase B is isopropanol: acetonitrile: formic acid = 50:50:0.8, and the reagents used are chromatographic grade and need to be filtered through a 0.45μm aqueous membrane or organic membrane. Set the pump (flow rate 0.4mL / min, column temperature 70℃). After setting, place the samples to be tested in the leftmost column, numbered from front to back as 0, 1, 2, 3, 4, 5, 6. Turn on the pump and start injecting samples (each sample takes about 30 minutes). After all samples are tested, perform data analysis. Figure 2 It can be seen that clostridium butyrate is a type of small molecule protein polypeptide substance with antibacterial function.

[0069] 3. Animal Testing

[0070] 3.1. Preparation of bacterial powder

[0071] Fermentation tank culture medium (g / L): 20g bran, 10g soybean meal, 5g glucose, 5g yeast extract, 5g sodium chloride, 3g K2HPO4, 3g KH2PO4, 0.3g MnSO4, adjust pH to 7.0.

[0072] Culture conditions: 120 L of the 200 L fermentation tank liquid was inoculated with the above-isolated and purified Clostridium butyricum, with an inoculation amount of about 4%, the fermentation temperature was 36°C, the rotation speed was 100 rpm, and the fermentation was terminated until the vegetative bacteria turned into spores.

[0073] Spore induction method: After 48 hours of fermentation, the fermentation liquid was examined under a microscope. If no spores were produced, 0.3 g / L MnSO4 solution was added for induction.

[0074] Powder making by drying method: centrifuge the bacterial liquid to obtain bacterial mud, mix the bacterial mud with corn cob powder evenly, the ratio of bacterial mud to corn cob powder is 3:2 (by mass), the humidity of the wet bacterial powder obtained after mixing is about 45%, and air-dry it with hot air at 38°C until the humidity reaches about 10%. After crushing, take samples for live bacteria counting and water content determination, as shown in the following table.

[0075] Table 3

[0076]

[0077] The Clostridium butyricum powder produced in the pilot production was stored for future use in preparation for the next animal experiment verification.

[0078] 3.2 Feeding Experiment

[0079] Here are the steps:

[0080] Phase 1: Sow breeding phase

[0081] 200 pregnant binary (Landrace × Large White) hybrid sows with similar body condition and gestational age and the same specifications as the mating boars were evenly divided into two groups, with 100 in each group, the experimental group and the control group. They were placed in the same environment and raised in the same way. They were fed with sow pregnancy feed during pregnancy, and fed with sow lactation feed during the lactation period after the sows gave birth. The experimental group was fed with conventional feed for 10 days before fertilization, and the additive dosage of Clostridium butyricum powder in pregnancy feed and sow lactation feed (common models) was 500g / ton, while the control group did not add it.

[0082] The second stage: piglet rearing stage

[0083] From the 7th day after birth to the 35th day after birth, the piglets were fed with the early compound feed. The added dosage of Clostridium butyricum powder in the experimental group was 500g / ton, while no addition was made in the control group.

[0084] Piglets were fed with concentrated feed for small nursery piglets from the 36th day after birth to the 55th day after birth. The added dosage of Clostridium butyricum powder in the experimental group was 400g / ton, while no addition was made in the control group.

[0085] Piglets were fed with large nursery piglet concentrated feed from the 56th day after birth to the 70th day after birth. The added dosage of Clostridium butyricum powder in the experimental group was 300g / ton, while no addition was made in the control group.

[0086] The corresponding data of the breeding process of sows and piglets from 32 days to 75 days of age are statistically analyzed, as shown in the following table:

[0087] Table 4. Statistics of sow reproductive performance indicators

[0088]

[0089]

[0090] Table 5. Statistics of piglet growth performance results

[0091]

[0092] Table 6. Statistical table of piglet blood biochemical index results

[0093]

[0094] The PSY index is calculated and statistically analyzed as shown in the following table.

[0095] Table 7

[0096] PSY Index Experimental Group 19.7 Control group 18.4

[0097] In summary, adding Clostridium butyricum to sow pregnancy and piglet feeding can effectively increase the litter weight and average litter weight of weaned piglets, reduce the back fat loss of sows during the lactation period, and reduce the constipation rate of sows and diarrhea rate of piglets. Figure 3 , especially in the two weeks after weaning, when the zinc oxide dosage was reduced, the diarrhea rate was significantly reduced. The reason for the significant effect of Clostridium butyricum is likely to be the production of Clostridium butyricum with comprehensive antibacterial ability.

[0098] At the same time, the strain significantly improves the immunity of piglets, especially the blood IgG and IgM levels, as well as the SOD enzyme that resists oxidative stress, which has an effect on improving the disease resistance and stress resistance of farmed animals.

[0099] The most important thing is that Clostridium butyricum can effectively improve the PSY index. According to the above animal experiments, it can be inferred that the first reason is: promoting absorption capacity, effectively improving the physical fitness of sows, etc., increasing ovulation, increasing litter size, shortening the sow weaning-estrus interval, and increasing the average annual litter size; the second is: the time required for the sow's delivery process is reduced, shortening the farrowing interval, reducing damage to piglets, and improving the survival rate; the third is: the immunity of piglets is improved, thereby improving the survival rate of piglets.

[0100] The above are only preferred embodiments of the present invention. The protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A Clostridium butyricum, with a preservation number of CGMCC No. 29591.

2. Application of Clostridium butyricum CGMCCNo29591 in integrated mother-and-child feeding of pigs.

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