Preparation method and application of recombinant probiotics and bacterial powder thereof

Abstract

The invention discloses a preparation method and application of a recombinant probiotic and its bacterial powder, wherein the recombinant probiotic generates indigo through the expression of exogenous genes; the exogenous genes include a indigo synthase gene and a 4'-phosphopantetheinyl transferase synthesis gene. By preparing the recombinant probiotic into bacterial powder and adding it to feed, the incidence probability of diarrhea in farmed animals can be reduced, the intestinal health of the animals can be improved, the production performance can be improved, and the economic losses can be reduced.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a preparation method and application of a recombinant probiotic and bacterial powder thereof. Background Art

[0002] In recent years, the incidence of diarrheal infectious diseases has increased in large-scale farms, and has gradually become the main cause of growth retardation and high mortality in farmed animals. Taking piglets as an example, there are many pathogens that cause diarrheal infectious diseases in piglets, mainly including viral infections, bacterial infections, parasitic infections, etc. Among them, bacterial infections are the most common. About 50% of the deaths of piglets each year are related to Escherichia coli. At present, vaccines and probiotic preparations are the most eye-catching for prevention and treatment. However, due to the large number of serotypes of enterotoxigenic Escherichia coli, large variability, and complex antigens, the actual use of vaccines has different effects, and there are high costs, high requirements for transportation and storage conditions, and the use of attenuated live vaccines has the risk of in vivo reversion. Therefore, the large-scale promotion of vaccines is difficult.

[0003] As a kind of live bacterial preparation, probiotics can promote the metabolism and absorption of nutrients, stimulate the animal immune system, and enhance the animal's disease resistance. Intestinal probiotics, in particular, can promote the development of the animal's intestinal immune system and have the advantages of being green and residue-free, and have attracted widespread attention from researchers from all over the world.

[0004] Taking Bacillus subtilis as an example, the protease and amylase produced by itself can degrade the nutrients in the feed, such as starch and protein, which are difficult to absorb in the intestine, into small molecules such as glucose and amino acids, which can promote the digestion and absorption of feed by farmed animals and improve feed utilization; at the same time, substances such as lactic acid produced by Bacillus subtilis itself can have a good inhibitory effect on Escherichia coli.

[0005] At present, there are studies on using probiotics as feed additives to reduce the diarrhea rate of farmed animals. Microecological preparations formed by the addition of probiotics are also an important research direction for optimizing animal husbandry. However, it is known that probiotics are mostly used for human health care, and the development of probiotics fortified for animal needs is currently relatively vacant.

[0006] Gulan (CAS#: 2435-59-8) is a natural blue product of microorganisms. It is often used in textile dyeing processes because of its bright color and non-toxicity. However, Gulan itself also has good antioxidant activity, and has a good scavenging effect on 1,1-diphenyl-2-picrylhydrazyl free radicals (DPPH) and superoxide free radicals; Gulan can also play an anti-inflammatory effect by intervening in the expression of some inflammation-related molecules; Gulan can also significantly inhibit pathogenic bacteria such as Staphylococcus aureus, Escherichia coli, and Candida albicans by producing superoxide and hydroxyl free radicals. However, because Gulan is a non-water-soluble substance, the natural strains that can be expressed are mostly pathogenic bacteria or marine microorganisms, and cannot be directly used for animal feeding.

[0007] In view of this, the present invention is proposed. Summary of the invention

[0008] The purpose of the present invention is to provide a preparation method and application of a recombinant probiotic and its bacterial powder. By preparing the recombinant probiotic into bacterial powder and adding it to feed, the incidence of diarrhea in farmed animals can be reduced, their intestinal health can be improved, production performance can be improved, and economic losses can be reduced.

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] In a first aspect, the present invention provides a recombinant probiotic that produces indigo blue by expressing exogenous genes; the exogenous genes include a indigo blue synthase gene and a 4'-phosphopantetheinyl transferase synthesis gene.

[0011] In some embodiments, the blue pigment synthase gene includes indigoidine synthase IndC, Blue-pigment synthetase, and putative indigoidine synthase IndC.

[0012] In some embodiments, the blue pigment synthase gene is selected from at least one of indigoidine synthase IndC (NCBI.Gene.ID: 61359656), Blue-pigment synthetase (NCBI.Gene.ID: 15402259) or putative indigoidine synthase IndC (NCBI.Gene.ID: 10467378).

[0013] Specifically, the blue-pigment synthase gene indigoidine synthase IndC (NCBI.Gene.ID: 61359656) is derived from the gene of DSM 6842 of the genus Pseudoalteromonas rubra; the blue-pigment synthetase gene Blue-pigment synthetase (NCBI.Gene.ID: 15402259) is derived from the gene of DSM 40593 of the genus Streptomyces fulvissimus; the blue-pigment synthase gene putative indigoidine synthase IndC (NCBI.Gene.ID: 10467378) is derived from the gene of BSR3 of the genus Burkholderia gladioli.

[0014] In some embodiments, the 4'-phosphopantetheinyl transferase synthetic gene is putativeindigoidine synthase IndC (NCBI. Gene. ID: 56652119).

[0015] Specifically, the 4′-phosphopantetheinyl transferase synthesis gene putative indigoidine synthase IndC (NCBI.Gene.ID: 56652119) is derived from the gene of Bacillus tropicus BSR3.

[0016] In some embodiments, the host of the recombinant probiotic is selected from any one of Lactobacillus plantarum, Bacillus subtilis, Bacillus coagulans or Clostridium butyricum.

[0017] In the present invention, the Lactobacillus plantarum selected is Lactobacillus plantarum SK151; the Bacillus subtilis selected is Bacillus subtilis WB800; the Bacillus coagulans selected is Bacillus coagulans TQ33; the Clostridium butyricum selected is Clostridium butyricum CDC51208, but it is not limited to the above strains. Other strains of the same species or other probiotics with similar effects are also within the protection scope of the present invention.

[0018] In some embodiments, the recombinant probiotics are constructed by connecting the blue pigment synthase gene and the 4'-phosphopantetheinyl transferase synthesis gene to an expression vector, and then introducing the recombinant expression vector into the host probiotics to obtain the recombinant probiotics.

[0019] In some embodiments, the expression vectors used in the construction of the above-mentioned recombinant probiotics include prokaryotic plasmids such as pET28a(+), pET23a, and pGEX.

[0020] In the present invention, regarding the method of introducing a plasmid carrying the above-mentioned genes, these genes may be present on the same plasmid or on different plasmids.

[0021] In some embodiments, the cyanine synthase gene and the 4'-phosphopantetheinyl transferase synthetic gene are placed on one plasmid for expression.

[0022] In some embodiments, the above-mentioned culturing method of recombinant probiotics includes: inoculating a suspension of recombinant probiotics into a culture medium for activation, then reactivating the activated bacterial solution for a second time, and then continuing to ferment the culture solution after the second activation until the strain in the fermentation solution reaches an appropriate concentration, adding an inducer, and then continuing to culture.

[0023] Specifically, the culture conditions in the above activation, secondary activation and fermentation steps are: temperature range of 36-38°C, rotation speed of 150-250rpm; inducer is isopropyl thiogalactoside; the final concentration of isopropyl thiogalactoside solution is 180-220uM; the continuous culture time after adding the inducer is 12h.

[0024] In a second aspect, the present invention provides a method for preparing recombinant probiotic powder, which comprises fermenting and culturing the above-mentioned recombinant probiotics, then adding a protective agent to the fermented bacterial liquid to prepare a feed liquid, and then spray drying.

[0025] In some embodiments, the volume ratio of the bacterial liquid to the protective agent added in the feed liquid is 1:1-3:1.

[0026] In the present invention, the protective agent comprises skim milk, maltodextrin, gum arabic, trehalose, soy protein and whey powder.

[0027] In some embodiments, the protective agent comprises, by weight, 5-40 parts of skim milk, 10-80 parts of maltodextrin, 5-40 parts of gum arabic, 1-10 parts of trehalose, 5-30 parts of soy protein, and 1-20 parts of whey powder;

[0028] The inventors further optimized the components of the protective agent. By weight, the protective agent includes 10-30 parts of skim milk, 15-40 parts of maltodextrin, 10-30 parts of gum arabic, 1-5 parts of trehalose, 10-20 parts of soy protein, and 1-10 parts of whey powder;

[0029] More preferably, the protective agent includes 20 parts of skim milk, 30 parts of maltodextrin, 20 parts of gum arabic, 5 parts of trehalose, 15 parts of soy protein and 10 parts of whey powder in parts by weight.

[0030] In some embodiments, the concentration of the protectant is 300-500 g / L.

[0031] In some embodiments, the spray drying conditions are: an inlet air temperature of 160° C., an outlet air temperature of 80° C., and a feed rate of 1200 mL / h.

[0032] The preparation method of the recombinant probiotic powder provided by the invention is harmless, pollution-free, easy to operate, and has mild culture conditions.

[0033] In a third aspect, the present invention provides the use of the recombinant probiotics or the recombinant probiotic powder prepared by the preparation method of the recombinant probiotic powder in the preparation of antibacterial and anti-diarrhea feed.

[0034] Through research, it was found that Gulan has a synergistic effect with the recombinant probiotics constructed by the present invention and has a more excellent antibacterial effect; it was also found that the recombinant probiotic powder, as a feed additive, has a good promoting effect on the growth of farmed animals and greatly reduces the diarrhea rate.

[0035] The present invention has the following beneficial effects:

[0036] The present invention provides a recombinant probiotic for synthesizing gulan. After being prepared into bacterial powder, it has a high number of viable bacteria, strong antibacterial activity, can produce neutral protease and amylase, and has no significant difference in performance from fermentation broth. It can be used as a microbial agent in feed for poultry and livestock. Gulan, as a heterologous expression product, also has very excellent antibacterial activity and can further inhibit the growth of harmful Escherichia coli in farmed animals. At the same time, combined with the efficacy of gulan in intervening in some inflammatory signal molecules, the bacterial powder can effectively improve the intestinal health of farmed animals as a feed additive. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0038] Example 1

[0039] This embodiment provides a construction of a recombinant probiotic and a method for culturing the same.

[0040] The exogenous genes in the recombinant probiotics are indigoidine synthase IndC (NCBI.Gene.ID: 61359656) and putative indigoidine synthase IndC (NCBI.Gene.ID: 56652119); the host is Bacillus subtilis WB800; and the vector is pET28a (+).

[0041] The construction method is:

[0042] (1) Take 100 μL of seed solution and inoculate it into 50 mL of LB3 liquid medium. Cultivate at 37°C and 250 rpm until the OD600 is about 0.4. Transfer the bacterial solution to a pre-cooled 50 mL centrifuge tube and place it on ice for 5 min.

[0043] (2) The mixture was centrifuged at 2000 × g for 5 min at 4°C, the supernatant was discarded, and the bacterial pellet was gently resuspended in 1 mL of pre-cooled 1 mol / L sorbitol buffer and centrifuged at 5000 × g for 5 min at 4°C.

[0044] (3) Wash the bacterial pellet twice (3 times in total) with 1 mL of pre-cooled 1 mol / L sorbitol buffer. Gently resuspend the bacterial pellet with 250 μL of pre-cooled 1 mol / L sorbitol buffer and dispense 50 μL into each tube. If not used immediately, store in a -80°C refrigerator.

[0045] (4) Add about 100 ng of plasmid to the competent cells, mix gently, transfer to a pre-cooled 1 mm electroporation cuvette, let stand for 3 min, and then transform at a voltage of 0.7–0.9 kV (Bio-Rad, MicroPulserTM).

[0046] (5) Immediately after electroporation, add 1 mL of pre-cooled LB3 medium and transfer the bacterial solution to a 2 mL centrifuge tube. Resuscitate the culture at 37°C, 250 rpm for 45 min or at 30°C, 250 rpm for 1.5 h.

[0047] (6) Spread the bacterial solution onto LB3 solid medium of corresponding resistance and culture at 30°C overnight.

[0048] The cultivation method is:

[0049] (1) 10% of the inoculum of the above recombinant probiotics was selected and inoculated into 5 mL of LB medium, and activated at 37°C and 200 rpm for 6 h; 10% of the inoculum of the above activated bacterial solution was inoculated into 50 mL of LB medium, and a second activation was performed under the same conditions.

[0050] (2) The recombinant probiotics after secondary activation were added to 1 L of LB medium at an inoculum rate of 10% for cultivation, and the absorbance of the fermentation broth was detected at a wavelength of 600 nm using a spectrophotometer. When the absorbance of the fermentation broth at a wavelength of 600 nm reached 0.6, IPTG with a final concentration of 200 uM was added to the fermentation broth; the fermentation broth was then placed in a shaker at 37° C. and 200 rpm for continuous cultivation for 12 h.

[0051] Example 2

[0052] This embodiment provides a construction of a recombinant probiotic and a method for culturing the same.

[0053] The exogenous genes in the recombinant probiotics are: Blue-pigment synthetase (NCBI.Gene.ID: 15402259) and putative indigoidine synthase IndC (NCBI.Gene.ID: 56652119); the host is: Lactobacillus plantarum SK151; and the vector is pET28a (+).

[0054] The construction method and culture method are the same as in Example 1.

[0055] Example 3

[0056] This embodiment provides a construction of a recombinant probiotic and a method for culturing the same.

[0057] The exogenous genes in the recombinant probiotics are: putative indigoidine synthase IndC (NCBI.Gene.ID: 10467378) and putative indigoidine synthase IndC (NCBI.Gene.ID: 56652119); the host is: Bacillus coagulans TQ33; and the vector is pET28a (+).

[0058] The construction method and culture method are the same as in Example 1.

[0059] Example 4

[0060] This embodiment provides a construction of a recombinant probiotic and a method for culturing the same.

[0061] The exogenous genes in the recombinant probiotics are indigoidine synthase IndC (NCBI.Gene.ID: 61359656) and putative indigoidine synthase IndC (NCBI.Gene.ID: 56652119); the host is Clostridium butyricum CDC51208; and the vector is pET28a (+).

[0062] The construction method and culture method are the same as in Example 1.

[0063] Example 5

[0064] This embodiment provides a method for preparing recombinant probiotic powder:

[0065] A spray drying protective agent with a total concentration of 300 g / L was prepared with different weight portions of ingredients: 20 weight portions of skim milk, 15 weight portions of soy protein, 5 weight portions of trehalose, 30 weight portions of maltodextrin, 20 weight portions of gum arabic, and 10 weight portions of whey powder.

[0066] The fermentation liquid of the recombinant probiotics obtained in Example 1 and the protective agent were mixed in a volume ratio of 1:1, and spray-dried under the conditions of an inlet temperature of 160° C., an outlet temperature of 80° C., and a feed rate of 1200 mL / h to obtain bacterial powder.

[0067] Example 6

[0068] This embodiment provides a method for preparing recombinant probiotic powder:

[0069] A spray drying protective agent with a total concentration of 300 g / L was prepared with different weight portions of ingredients: 5 weight portions of skim milk, 30 weight portions of soy protein, 10 weight portions of trehalose, 49 weight portions of maltodextrin, 5 weight portions of gum arabic, and 1 weight portion of whey powder.

[0070] The fermentation liquid of the recombinant probiotics obtained in Example 2 and the protective agent were mixed in a volume ratio of 1:1, and spray-dried under the conditions of an inlet temperature of 160° C., an outlet temperature of 80° C., and a feed rate of 1200 mL / h to obtain bacterial powder.

[0071] Example 7

[0072] This embodiment provides a method for preparing recombinant probiotic powder:

[0073] A spray drying protective agent with a total concentration of 300 g / L was prepared with different weight portions of ingredients: 40 weight portions of skim milk, 5 weight portions of soy protein, 1 weight portion of trehalose, 14 weight portions of maltodextrin, 20 weight portions of gum arabic, and 20 weight portions of whey powder.

[0074] The fermentation liquid of the recombinant probiotics obtained in Example 3 and the protective agent were mixed in a volume ratio of 1:1, and spray-dried under the conditions of an inlet temperature of 160° C., an outlet temperature of 80° C., and a feed rate of 1200 mL / h to obtain bacterial powder.

[0075] Example 8

[0076] This embodiment provides a method for preparing recombinant probiotic powder:

[0077] A spray drying protective agent with a total concentration of 300 g / L was prepared with different weight portions of ingredients: 20 weight portions of skim milk, 15 weight portions of soy protein, 5 weight portions of trehalose, 30 weight portions of maltodextrin, 20 weight portions of gum arabic, and 10 weight portions of whey powder.

[0078] The fermentation liquid of the recombinant probiotics obtained in Example 4 and the protective agent were mixed in a volume ratio of 1:1, and spray-dried under the conditions of an inlet temperature of 160° C., an outlet temperature of 80° C., and a feed rate of 1200 mL / h to obtain bacterial powder.

[0079] Comparative Example 1

[0080] The difference between this comparative example and Example 1 is that the host of the recombinant probiotics is Saccharomyces cerevisiae.

[0081] Comparative Example 2

[0082] The difference between this comparative example and Example 2 is that the host of the recombinant probiotics is Saccharomyces cerevisiae.

[0083] Comparative Example 3

[0084] The difference between this comparative example and Example 3 is that the host of the recombinant probiotics is Escherichia coli.

[0085] Comparative Example 4

[0086] The difference between this comparative example and Example 5 is that the recombinant probiotics are the recombinant probiotics constructed in Comparative Example 1.

[0087] Comparative Example 5

[0088] The difference between this comparative example and Example 6 is that the recombinant probiotics are the recombinant probiotics constructed in Comparative Example 2.

[0089] Comparative Example 6

[0090] The difference between this comparative example and Example 7 is that the recombinant probiotics are the recombinant probiotics constructed in Comparative Example 3.

[0091] Experimental Example 1

[0092] In vitro antibacterial effect assay

[0093] The bacterial powders, antibiotics, Bacillus subtilis fermentation broth, indigo plant, and distilled water prepared in Examples 5-8 and Comparative Examples 4-6 were used as the experimental group and the control group to perform an in vitro Escherichia coli inhibition experiment and compare the antibacterial abilities.

[0094] Take the intestinal contents of the piglet and streak it on a MacConkey plate with an inoculation loop, incubate at 37℃ for 24h, pick the brick-red smooth colonies on the MacConkey plate, gram stain, and examine under a microscope. If it is a G-bacterium, pick the colonies and incubate them in LB liquid medium at 37℃ and 200rpm for 24h, and store at 4℃ for later use.

[0095] The bacterial powder prepared in Examples 5-8 and Comparative Examples 4-6, the indigo plant standard, the fermentation broth of the strain in Example 1 (based on the indigo plant content in the fermentation broth), and penicillin were prepared into solutions with a content of 500 mg / L, and antibacterial experiments were carried out with distilled water as the control group.

[0096] The above products were tested for their in vitro antibacterial effects according to the coating plate method mentioned in the Chinese literature "Comparative Study of Inhibition Zone Method in Antibacterial Test [J]. Food Industry, 2016, 37(11): 122-125." The results are as follows:

[0097] Table 1 In vitro antibacterial effect

[0098] Detection object Inhibition zone size (cm) Example 5 23 Example 6 19 Example 7 21 Example 8 16 Comparative Example 4 10 Comparative Example 5 9 Comparative Example 6 7 Valley Blue 14 Fermentation Broth 20 penicillin 11 Distilled water -

[0099] As can be seen from Table 1, the size of the inhibition zone of the recombinant probiotic powder prepared in Examples 5-8 is significantly larger than that of the standard product of Gulan and penicillin, proving that Gulan and the recombinant probiotics have a synergistic effect, and the powder prepared by the recombinant probiotics constructed by the present invention has a better antibacterial effect; and compared with the recombinant probiotic powder prepared in Comparative Examples 4-6, the recombinant probiotic powder prepared by the present invention has a better antibacterial effect. The in vitro antibacterial effect of the fermented liquid and the recombinant probiotic powder prepared in Example 5 is compared. Although the solution concentrations of the antibacterial experiments of both are 500 mg / L, the fermented liquid is calculated as the solution concentration based on the Gulan content, so the Gulan concentration in the solution prepared by the fermented liquid is only 500 mg / L; while the solution prepared by the powder of Example 5 is 500 mg / L prepared by the powder, the Gulan content in the bacteria is higher, and there is a protective agent, the loss is smaller, and the relative concentration is higher than the fermented liquid, so the in vitro antibacterial effect of the recombinant probiotic powder prepared in Example 5 is better than the in vitro antibacterial effect of the fermented liquid.

[0100] Experimental Example 2

[0101] Diarrhea inhibition effect test

[0102] Take dry senna leaves, grind them into fine powder (discard the veins), grind them with boiling distilled water, and then add cold distilled water to make an 8% suspension for use. When modeling, take purebred healthy mice weighing (20±2)g, generally give them the test drug first, and then gavage them with 8% senna leaf powder suspension at 2g / L. Put the mice in an observation box padded with white paper, change the paper every 1h, and record the number of abnormal bowel movements of the mice within 1-6h after gavage of senna leaves.

[0103] Mouse feces can be divided into four types: normal feces, soft feces with normal appearance and more content, soft feces with abnormal appearance, watery feces and mucus feces. The first two are considered normal feces, and the latter two are considered abnormal feces.

[0104] The mice with abnormal stool characteristics were randomly divided into 6 groups, with 5 mice in each group:

[0105] (1) Control group: fed with basal diet;

[0106] (2) Experimental group 1: the bacterial powder of Example 5 was added to the basic diet;

[0107] (3) Experimental group 2: the bacterial powder of Example 6 was added to the basal diet;

[0108] (4) Experimental group 3: the bacterial powder of Example 7 was added to the basal diet;

[0109] (5) Experimental group 4: the bacterial powder of Example 8 was added to the basal diet;

[0110] (6) Experimental group 5: the bacterial powder of comparative example 1 was added to the basic diet;

[0111] (7) Experimental group 6: adding the bacterial powder of comparative example 2 to the basic diet;

[0112] (8) Experimental group: 7: Adding the bacterial powder of comparative example 3 to the basic diet;

[0113] (9) Experimental group 8: Adding rice blue to the basal diet;

[0114] The mouse diarrhea inhibition effect test was conducted with reference to the relevant indicators and methods in the Chinese literature "An Experiment on the Effect of Healthy Animal Intestinal Bacteria on Mouse Weight Gain and Fattening [J]. Sichuan Animals, 1998(04): 45-47." The measurement indicators were set as the average daily feed intake, daily weight gain, and diarrhea rate of mice. The experimental time was set to 14 days. The experimental results are as follows:

[0115]

[0116]

[0117] As can be seen from Table 2, the test groups added with bacterial powder of Examples 5-8 were superior to the control group and the test groups added with bacterial powder / grain blue of Comparative Examples 4-6 in terms of average daily feed intake, daily weight gain and diarrhea inhibition.

[0118] In summary, from the results of in vitro antibacterial effect determination and diarrhea inhibition effect test, it can be seen that the bacterial powder prepared using probiotics containing indigofera / self-produced indigofera and its fermentation broth has good antibacterial effect and diarrhea inhibition effect, while the single probiotics themselves and indigofera itself have certain antibacterial effect and diarrhea inhibition effect, but they are not excellent; at the same time, when indigofera is combined with conventional strains, the effect becomes worse.

[0119] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A recombinant probiotic that heterologously expresses beneficial molecules, It is characterized in that The recombinant probiotic produces indigo blue by expressing exogenous genes; the exogenous genes include indigo blue synthase gene and 4'-phosphopantetheinyl transferase synthesis gene; The exogenous genes of the recombinant probiotics are indigoidine synthase IndC (NCBI.Gene.ID: 61359656) and putative indigoidine synthase IndC (NCBI.Gene.ID: 56652119), and the host is Bacillus subtilis WB800; or The exogenous genes of the recombinant probiotics are putative indigoidine synthase IndC (NCBI.Gene.ID: 10467378) and putative indigoidine synthase IndC (NCBI.Gene.ID: 56652119), and the host is Bacillus coagulans TQ33; or The exogenous genes of the recombinant probiotics are Blue-pigment synthetase (NCBI.Gene.ID: 15402259) and putative indigoidine synthase IndC (NCBI.Gene.ID: 56652119), and the host is Clostridium butyricum CDC51208.

2. A method for preparing recombinant probiotic powder, It is characterized in that The method comprises fermenting and culturing the recombinant probiotics according to claim 1, then adding a protective agent to the fermented bacterial liquid to prepare a feed liquid, and then spray drying.

3. The preparation method according to claim 2, It is characterized in that The volume ratio of the bacterial liquid to the protective agent added in the feed liquid is 1:1-3:

1.

4. The preparation method according to claim 3, It is characterized in that In terms of weight, the protective agent comprises 5-40 parts of skim milk, 10-80 parts of maltodextrin, 5-40 parts of gum arabic, 1-10 parts of trehalose, 5-30 parts of soy protein and 1-20 parts of whey powder.

5. The preparation method according to claim 4, It is characterized in that In terms of weight, the protective agent includes 10-30 parts of skim milk, 15-40 parts of maltodextrin, 10-30 parts of gum arabic, 1-5 parts of trehalose, 10-20 parts of soy protein and 1-10 parts of whey powder.

6. The preparation method according to claim 5, It is characterized in that In terms of weight, the protective agent includes 20 parts of skim milk, 30 parts of maltodextrin, 20 parts of gum arabic, 5 parts of trehalose, 15 parts of soy protein and 10 parts of whey powder.

7. The preparation method according to claim 6, It is characterized in that The concentration of the protective agent is 300-500 g / L.

8. The preparation method according to claim 2, It is characterized in that The spray drying conditions are as follows: an inlet air temperature of 160-180°C, an outlet air temperature of 80-100°C, and a feed rate of 1000-1200 mL / h.

9. Use of the recombinant probiotic powder prepared by the recombinant probiotic according to claim 1 or the method for preparing the recombinant probiotic powder according to any one of claims 2 to 8 in preparing feed for inhibiting Escherichia coli.

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