Lactobacillus plantarum 6PC and application thereof in preparation of deodorization feed

Abstract

The invention belongs to the technical field of microorganisms, and provides lactobacillus plantarum 6PC and application thereof in preparation of deodorization feed, the lactobacillus plantarum 6PC is preserved in China General Microbiological Culture Collection Center (CGMCC) on September 29, 2024, the preservation number is CGMCC No.32103, and the lactobacillus plantarum 6PC has cholate resistance, can survive in simulated gastric juice, and can be used for preparing deodorization feed. The strain can inhibit escherichia coli, can also inhibit escherichia coli and can grow in a heterotrophic nitrification culture medium, the ammonia nitrogen concentration is 75 mg / l or below, and the ammonia nitrogen removal rate is kept 80% or above. After the bacterial powder is added into the pig feed, the diarrhea rate is obviously reduced; the ammonia nitrogen content in the excrement is reduced by 87.09%; the ammonia concentration in the sheep house can also be reduced by more than 80%, and the ammonia can be used as feed and a feed additive to reduce the emission of harmful gas, and can also be directly sprayed in a feeding shed to enhance the health of animals and improve the green living environment.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a strain of Lactobacillus plantarum 6PC and application thereof in preparing deodorized feed. Background Art

[0002] Deodorized feed refers to feed that can reduce the odor of animal excrement and other excreta through special raw material selection, processing technology or adding specific additives. Its purpose is to improve the air quality around the farm, reduce the impact on nearby residents, and reduce environmental disputes caused by odor problems. Reducing the production of odorous substances means that the microbial community in the intestine is in a relatively healthy state, and animals are less susceptible to intestinal diseases. At the same time, due to the improved feed utilization rate, the growth rate of animals may be accelerated, and the output and quality of livestock products such as meat, eggs, and milk may also be improved. With increasingly stringent environmental regulations, the use of deodorized feed can help farms better meet environmental standards, avoid penalties due to environmental problems, and ensure the sustainable operation of farms.

[0003] As people's requirements for environmental quality and livestock product quality continue to increase, the research and development of deodorized feed will pay more attention to the use of natural, green, and efficient raw materials and additives. The preparation of existing deodorized feed is mainly through optimizing the nutritional components in the feed, adding specific enzyme preparations, such as proteases, cellulases, etc.; or adding probiotics to change the structure of intestinal microbial communities, reducing the abnormal fermentation of harmful bacteria on nutrients such as protein, thereby reducing the production of odorous gases such as ammonia and hydrogen sulfide. For example, the invention patent CN1930282B discloses a new type of lactic acid bacteria with antifungal activity, which can also reduce ammonia concentration and can be used as a feed additive, feed, etc. The invention patent CN117229980B discloses a sinus-eating Weissella, which has a deodorizing effect on waste gas in farms and has good development and application prospects. The sinus-eating Weissella provides a new reagent or deodorizing method for reducing / removing odor in farms. Therefore, probiotics are of great significance in the preparation of deodorized feed.

[0004] The inventor screened and obtained a strain of Lactobacillus plantarum during the research process. The Lactobacillus plantarum has good bile salt tolerance, can effectively survive in simulated gastric juice, can inhibit the growth of Escherichia coli, has a significant ammonia removal effect, and the ammonia nitrogen removal rate is maintained at more than 80%. The strain can be used as feed and feed additive. Summary of the invention

[0005] The primary purpose of the present invention is to provide a Lactobacillus plantarum 6PC, wherein the Lactobacillus plantarum 6PC is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on September 29, 2024, with a deposit number of CGMCC No.32103.

[0006] The second object of the present invention is to provide a bacterial agent containing the Lactobacillus plantarum 6PC.

[0007] The third object of the present invention is to provide the use of the Lactobacillus plantarum 6PC or the bacterial agent in the preparation of a functional product, wherein the functional product has one or more of the following effects:

[0008] (1) Having bile salt tolerance;

[0009] (2) It has antibacterial effect;

[0010] (3) It has the effect of removing ammonia;

[0011] (4) Prevent or treat diarrhea.

[0012] Preferably, the functional product is one or more of feed, feed additives, food, food additives, dietary supplements, health products or medicines.

[0013] The fourth object of the present invention is to provide the use of the Lactobacillus plantarum 6PC or the bacterial agent in the preparation of a breeding shed cleaner.

[0014] The fifth object of the present invention is to provide the use of the Lactobacillus plantarum 6PC or the bacterial agent in the preparation of a water cleaner.

[0015] The sixth object of the present invention is to provide an antibacterial agent, comprising the Lactobacillus plantarum 6PC or the bacterial agent.

[0016] The seventh object of the present invention is to provide a probiotic agent for preventing and treating diarrhea, comprising the Lactobacillus plantarum 6PC or the bacterial agent.

[0017] The invention has the following beneficial effects: the invention provides a plant lactobacillus 6PC, the plant lactobacillus 6PC has bile salt tolerance, can survive in simulated gastric juice, can inhibit Escherichia coli, can grow in heterotrophic nitrification culture medium, has an ammonia nitrogen concentration of less than 75 mg / l, and maintains an ammonia nitrogen removal rate of more than 80%; animal experiment results show that after adding bacterial powder to pig feed, the diarrhea rate is significantly reduced; the ammonia nitrogen content in feces is also significantly reduced, and can be reduced by up to 87.09%; the ammonia concentration in the sheep house can also be reduced by more than 80%, and the plant lactobacillus 6PC can be used as feed and feed additives to reduce the emission of harmful gases, and can also be directly sprayed in feeding sheds to enhance animal health and contribute to a green living environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Phylogenetic tree of strains

[0019] Figure 2Survival rate of Lactobacillus plantarum 6PC in simulated gastric fluid

[0020] Figure 3 Inhibition zone of Lactobacillus plantarum 6PC DETAILED DESCRIPTION

[0021] The following embodiments are for better understanding of the present invention, but are not limited to the present invention. The experimental methods in the following examples, unless otherwise specified, are all routine laboratory methods, and the experimental materials used in the following examples, unless otherwise specified, are all conventional biochemical reagents. The quantitative tests in the following examples are all repeated three times, and the results are averaged.

[0022] In the following examples, the culture medium formula used is as follows:

[0023] MRS solid culture medium: peptone 10g, beef extract 10g, yeast powder 5g, K2HPO22g, diammonium citrate 2g, sodium acetate 2g, glucose 20g, Tween 80 1ml, MgSO4·7H2O 0.58g, MgSO4·4H2O 0.25g, H2O 1000ml.

[0024] Heterotrophic nitrification medium (g / L): sodium acetate 2, MgSO4·7H2O 0.05, K2HPO40.2, NaCl 0.12, MnSO4·4H2O0.01, FeSO40.01, pH 7.0–7.2, sterilization at 121℃ for 20min.

[0025] Example 1. Screening and identification of strains

[0026] 1. Strain Isolation and Purification

[0027] Under sterile conditions, appropriate amount of kimchi (from a market in Lanzhou) was taken into MRS broth and cultured in a 37°C constant temperature incubator for 12 hours. Then, a standard 10-fold concentration series gradient dilution was performed. The dilutions were 10 -3 , 10 -4 , 10 -5 , 10 -6 100 μL of the bacterial solution was applied to MRS solid medium, and 3 parallel tests were performed for each dilution. The medium was inverted and placed in an anaerobic incubator at 37°C for 48 hours. A single colony was picked and purified using the three-step streak method on MRS solid medium. A single colony was picked and purified and inoculated into MRS broth for proliferation and culture until the logarithmic growth phase for later use, and it was named 6PC.

[0028] 2. Strain Identification

[0029] 1 mL of spare bacterial culture solution was extracted and centrifuged at 10000 r / min for 1 min. The culture solution was discarded, the precipitate was collected, and the genomic DNA of the isolated strain was extracted using a bacterial genome extraction kit, which was used as a template for PCR amplification of the strain 16SrRNA gene. The sequence was sent for sequencing and the obtained sequence was subjected to BLAST comparison analysis in NCBI to construct a phylogenetic tree.

[0030] Phylogenetic tree of strains Figure 1 As shown, the comparison results show that the similarity between strain 6PC and Lactobacillus plantarum strain ABRIIFBI-36 is as high as 98%. Therefore, the strain 6PC is identified as Lactobacillus plantarum, with the Latin name Lactobacillus plantarum, and is named Lactobacillus plantarum 6PC. The Lactobacillus plantarum 6PC was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on September 29, 2024, with a deposit number of CGMCC No.32103, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Graduate School of Microbiology, Chinese Academy of Sciences, Tel: 010-64807355, Fax: 010-64807288, Postal Code: 100101.

[0031] In the following examples, Lactobacillus plantarum 6PC is abbreviated as Lactobacillus plantarum 6PC. Example 2: Bile salt tolerance experiment of Lactobacillus plantarum 6PC

[0032] The purified Lactobacillus plantarum 6PC single colony was inoculated into MRS broth for proliferation and culture, and then inoculated into MRS medium containing 0.1%, 0.2%, 0.4%, and 0.6% bile salts at a ratio of 1%, respectively, and cultured in an anaerobic incubator at 37°C for 48 h. The OD 600 .

[0033] The experimental results are shown in Table 1. At a bile salt concentration of 0.1%, the OD 600 With the increase of bile salt concentration, 6PC still maintained a fast growth rate. When the bile salt concentration reached 0.6%, the OD 600 It reached 2.154, proving that Lactobacillus plantarum 6PC has good bile salt tolerance.

[0034] Table 1 Bile salt tolerance experiment of Lactobacillus plantarum 6PC

[0035]

[0036] Example 3: Survival experiment of Lactobacillus plantarum 6PC in simulated gastric fluid

[0037] Preparation of simulated gastric fluid: Pepsin (1:10000) was prepared into a pepsin solution with a concentration of 1 mg / ml using sterilized PBS, and the pH was adjusted to 3.0 using 1 mol / ml HCL solution, and then sterilized by filtration through a 0.22 μM microporous filter membrane.

[0038] 1 ml of activated Lactobacillus plantarum 6PC bacterial solution was added to 9 ml of simulated gastric fluid and cultured at 37°C. Samples were taken after 0, 0.5, 1, 1.5, 2, 2.5, and 3 hours, and the number of effective viable bacteria was detected. The survival rate of Lactobacillus plantarum 6PC in simulated gastric fluid was calculated using the following formula:

[0039] Survival rate (%) = (number of viable bacteria after reaction / number of viable bacteria before reaction) × 100

[0040] The experimental results are as follows Figure 2 As shown, the survival rate of Lactobacillus plantarum 6PC can reach 90.7±0.3% after 3 hours in simulated gastric fluid, indicating that Lactobacillus plantarum 6PC has a good survival rate in gastric fluid.

[0041] Example 4: Antibacterial Experiment of Lactobacillus plantarum 6PC

[0042] 100 μL of Escherichia coli was evenly spread on MRS solid culture medium, and the Lactobacillus plantarum 6PC liquid was adsorbed on a sterilized filter paper with a diameter of 5 mm. The plate was placed on a plate coated with Escherichia coli (from the Gansu Branch of the Industrial Microbiological Culture Collection Center), and cultured at 37°C for 24 hours to observe the inhibition zone.

[0043] The experimental results are as follows Figure 3 As shown, it can be seen that Lactobacillus plantarum 6PC can inhibit Escherichia coli.

[0044] Example 5: Ammonia removal experiment

[0045] 1. Methods

[0046] 1 mL of seed solution was inoculated into a conical flask containing 100 mL of heterotrophic nitrification medium. 4+ The concentration gradient of -N was 10 mg / l, 25 mg / l, 50 mg / l, 75 mg / l, and 100 mg / l. After culturing in a shaker at 30°C and 180 r / min for 48 h, samples were taken and the bacterial growth was detected at a wavelength of 600 nm. Then, the samples were centrifuged at 10,000 r / min for 2 min and the NH in the supernatant was determined by Nessler's colorimetric method. 4+ -N concentration, set 3 parallels. The ammonia nitrogen removal rate calculation formula is as follows:

[0047] μ=(w1-w0) / w1×100%

[0048] Note: μ: ammonia nitrogen removal rate; w1: initial ammonia nitrogen concentration of culture medium; w0: final ammonia nitrogen concentration of culture medium.

[0049] The NH4+-N concentration gradient was determined by adding volumes of 0.22 μm filtered NH4Cl solution.

[0050] 2. Results

[0051] The experimental results are shown in Table 2 and Table 3. Lactobacillus plantarum 6PC can grow in heterotrophic nitrification medium. The ammonia nitrogen concentration is below 75 mg / l, and the ammonia nitrogen removal rate is maintained above 80%.

[0052] Table 2 Growth of Lactobacillus plantarum in ammonium sulfate medium

[0053]

[0054] Table 3 Ammonia removal effect of Lactobacillus plantarum in ammonium sulfate medium

[0055]

[0056] Example 6: Pig feed application experiment

[0057] 1. Methods

[0058] 40 healthy Duroc-Changda hybrid pigs of similar weight at 60 days of age were selected and randomly divided into a control group and three embodiment groups, each with 10 pigs, half male and half female, raised in the same environment, but in four pig pens far apart. The control group was fed according to the pig farm; on this basis, the experimental group added plant lactobacillus 6PC powder, and the effective viable count of plant lactobacillus 6PC powder was greater than 20 billion / g, among which, the powder of experimental group 1 was added with 0.01%, the powder of experimental group 2 was added with 0.05%, and the powder of experimental group 3 was added with 0.1%. Feeding and management were carried out according to the routine of the pig farm, and the test period was 30 days. The first 2 days were the observation period, and the last 28 days were the formal experiment. The mixed feces were collected on the 0th day, the 14th day, and the 28th day to determine the content of ammonia nitrogen, and the growth performance of meat pigs was tested every day.

[0059] 2. Results

[0060] The experimental results are shown in Table 4. In the experimental groups with 0.05% and 0.1% bacterial powder added, the daily feed intake of pigs increased and the daily weight gain also increased significantly. Compared with the control group, the diarrhea rate was significantly reduced after adding bacterial powder. The diarrhea rate of the control group was 17.14%, and the diarrhea rate of the experimental group 3 was 2.50%.

[0061] The results of the ammonia nitrogen content test in the feces on the 14th day showed that the ammonia nitrogen content in the feces of experimental groups 1 and 2 with the addition of Lactobacillus plantarum 6PC powder was reduced by 49.24% and 53.15% compared with that in the control group, and the ammonia nitrogen content in the feces of experimental group 3 was reduced by 59.75% compared with that in the feces of the control group.

[0062] The test results of ammonia nitrogen content in feces on the 28th day showed that the ammonia nitrogen content in feces of the experimental group supplemented with Lactobacillus plantarum 6PC powder was reduced by more than 80% compared with that of the control group, which was reduced by 80.65%, 85.26% and 87.09% respectively.

[0063] Table 4 Growth index of pigs using Lactobacillus plantarum as feed additive

[0064]

[0065] Table 5 Ammonia nitrogen content in pig manure

[0066]

[0067] Example 7: Application of licking bricks for cattle and sheep

[0068] 1. Methods

[0069] Various plant extracts, molasses, salt powder, minerals, and Lactobacillus plantarum 6PC powder (the effective live bacteria count of Lactobacillus plantarum powder is greater than 20 billion / g) are added, mixed evenly to obtain blanks, which are pressed into cattle and sheep probiotic lick bricks, placed in sheep sheds, and licked by sheep.

[0070] The experiment was conducted at the breeding base of Zhangye Huarui Agriculture Co., Ltd. in Gansu Province. Two sheep houses with 6-12 month old lambs were used as experimental sites. The sheep house was 10 meters wide and 50 meters long, divided into 10 small compartments, with 15 sheep in each compartment. A licking brick was placed in each compartment. The control group used commercially available ordinary licking bricks, and the experimental group used licking bricks with added bacterial powder and plant extracts. During the experiment, the ammonia concentration in the sheep house was monitored on the 0th day, the 14th day, and the 28th day. The monitoring locations were 15 meters, 25 meters, and 25 meters away from the door of the sheep house, 1.5 meters above the ground, and the average of the three monitoring results was used as the final result. The monitoring instrument is a handheld pump suction ammonia detector (Shenzhen Wuyanjie Technology Co., Ltd.).

[0071] The cattle and sheep licking brick includes the following components by weight: 10-20 parts of Lactobacillus plantarum 6PC powder, 23-30 parts of plant extract (yucca, licorice, hawthorn and angelica), 2-5 parts of mineral powder, 5-10 parts of salt powder, 20-30 parts of molasses and 5-10 parts of bentonite.

[0072] 2. Results

[0073] The experimental results are shown in Table 6. The test results of ammonia concentration in the sheep house on the 14th day showed that the ammonia concentration in the sheep house of the experimental group was 7.85, which was 82.36% lower than that of 44.52 in the control group. The workers clearly felt that the air in the sheep house had improved and there was no odor.

[0074] The ammonia concentration test results of the sheep house on the 28th day were similar to those on the 14th day, indicating that the lick bricks with added bacterial powder and plant extracts effectively improved the air quality of the sheep house on the 14th day, and the ammonia concentration in the sheep house dropped by more than 80%.

[0075] Table 6 Ammonia content in sheep houses

[0076]

[0077] Note: Different letters indicate significant differences

[0078] In summary, the present invention provides a plant lactobacillus 6PC. The plant lactobacillus 6PC has bile salt tolerance, can survive in simulated gastric juice, can inhibit Escherichia coli, can grow in a heterotrophic nitrification medium, has an ammonia nitrogen concentration of less than 75 mg / l, and maintains an ammonia nitrogen removal rate of more than 80%. Animal experiment results show that after adding bacterial powder to pig feed, the diarrhea rate is significantly reduced; the ammonia nitrogen content in feces is also significantly reduced, and can be reduced by up to 87.09%; the ammonia concentration in a sheep house can also be reduced by more than 80%. The plant lactobacillus 6PC can be used as feed and feed additives to reduce the emission of harmful gases, and can also be directly sprayed in a feeding shed to enhance animal health and contribute to a green living environment.

[0079] The applicant declares that the present invention illustrates the technical solution of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0080] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0081] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A Lactobacillus plantarum 6PC, characterized in that The Lactobacillus plantarum 6PC was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on September 29, 2024, with a deposit number of CGMCC No.32103.

2. A bacterial agent, characterized in that Contains the Lactobacillus plantarum 6PC according to claim 1.

3. The use of Lactobacillus plantarum 6PC as claimed in claim 1 or the bacterial agent as claimed in claim 2 in preparing functional products, characterized in that: The functional product has one or more of the following functions: (1) Having bile salt tolerance; (2) It has antibacterial effect; (3) It has the effect of removing ammonia; (4) Prevent or treat diarrhea.

4. The use according to claim 3, characterized in that The functional product is one or more of feed, feed additives, food, food additives, dietary supplements, health products or medicines.

5. Use of Lactobacillus plantarum 6PC as claimed in claim 1 or the bacterial agent as claimed in claim 2 in preparing a feeding shed cleaner.

6. Use of the Lactobacillus plantarum 6PC as claimed in claim 1 or the bacterial agent as claimed in claim 2 in the preparation of a water detergent.

7. An antibacterial agent, characterized in that It includes the Lactobacillus plantarum 6PC described in claim 1 or the bacterial agent described in claim 2.

8. A probiotic for preventing and treating diarrhea, characterized in that: It includes the Lactobacillus plantarum 6PC described in claim 1 or the bacterial agent described in claim 2.

Images