CN116218705A - Lactobacillus plantarum JYLP-375 and microbial inoculum thereof and application of microbial inoculum in preparation of silage for preventing and improving calf diarrhea - Google Patents

Abstract

The invention relates to the technical field of microorganisms, in particular to lactobacillus plantarum JYLP-375, a bacterial agent thereof and application of preparing silage for preventing and improving calf diarrheaLactobacillus plantarum) JYLP-375 was deposited in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at the date of 27, 6, and the deposit address is Hospital No. 3, north Chen West Lu 1, the Korean region of Beijing, and the deposit number: CGMCC No.18039. The lactobacillus plantarum JYLP-375 provided by the invention is used for harvesting corn stalks and sweet potato seedlings after autumn harvestThe silage obtained by silage fermentation treatment of agricultural wastes such as seedlings and the like has good effects of improving the constitution of calves and preventing and improving the diarrhea of the calves.

Description

Lactobacillus plantarum JYLP-375 and microbial inoculum thereof and application of microbial inoculum in preparation of silage for preventing and improving calf diarrhea

Technical Field

The invention relates to the technical field of microorganisms, in particular to lactobacillus plantarum JYLP-375 and a microbial inoculum thereof and application of the lactobacillus plantarum JYLP-375 in preparing silage for preventing and improving calf diarrhea.

Background

Silage refers to fresh green plants fermented by microorganisms under closed anaerobic conditions. The silage has long preservation time, can preserve juice and most of original nutritional ingredients in green raw materials, the damage of the nutritional ingredients is usually not more than 15%, and the nutrient loss in the natural drying process of the silage is about 30%, and the nutrient loss after mildew is about 50%. Research shows that the silage can store about 83% of dry matters, reduce the loss of nutrients such as carotene, crude protein and the like in the silage, and has higher nutritive value. The best hay can only maintain about 70% of the dry matter of the raw material. The silage with good quality has main nutrition quality similar to that of silage raw materials, and is mainly characterized in that the silage has good palatability, the feed intake, the organic matter digestibility and the effective energy value of ruminants are similar to those of the silage raw materials, the vitamin content and the energy level of the silage are higher, and the nutrition quality is better. The silage has faint scent and succulent taste, proper taste and rich nutrition, and the silage can provide an excellent feed source for domestic animals in North China, northwest China, northeast China and other areas in winter by adopting a silage storage method.

The North China plain area at Shandong belongs to temperate zone monsoon climate, is clear in four seasons, is a great agricultural province, and is developed in animal husbandry. Autumn grains mainly comprise corn, sweet potato, sorghum, peanut and the like. At present, after autumn grains are finished, many agricultural byproducts such as corn sorghum straw, sweet potato seedling particles, peanut seedling and the like are naturally stored for preparing calf feeds in winter, pathogen microorganism pollution is easy to occur to the farm animals by the naturally stored agricultural straw and seedling particles, and intestinal diseases such as diarrhea and the like are induced in winter or early spring cold seasons, so that intestinal peristalsis is high, intestinal absorption is difficult and the like, and intestinal contents and moisture are discharged. Calves are calves six months before birth, the gastrointestinal system is weak, and once diarrhea occurs, the calves are extremely easy to die, and huge losses are caused.

Disclosure of Invention

In order to improve the quality of stored feed and reduce the diarrhea incidence of calves in cold seasons, the invention provides lactobacillus plantarum JYLP-375 and a microbial inoculum thereof and application of silage for preparing the silage for preventing and improving calf diarrhea.

In a first aspect, the invention provides a lactobacillus plantarum JYLP-375, lactobacillus plantarum @ or @ mLactobacillus plantarum) JYLP-375 was preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at the date of 27, 6.A preservation address is 1# 3 of North Chen West Lu of the Korean area of Beijing city, and a preservation number is CGMCC No.18039.

In a second aspect, the present invention provides a microbial inoculum comprising Lactobacillus plantarum JYLP-375.

Further, the microbial inoculum comprises lactobacillus plantarum JYLP-375 freeze-dried powder.

Further, the preparation method of the lactobacillus plantarum JYLP-375 freeze-dried powder comprises the following steps:

(1) Activating preserved lactobacillus plantarum JYLP-375 on an MRS flat-plate culture medium, inoculating the activated bacteria into the MRS liquid culture medium according to an inoculum size of 1%, and culturing for 24 hours at 37 ℃ to obtain a bacterial liquid;

(2) Centrifuging the bacterial liquid, collecting bacterial cells, washing the bacterial cells by using sterile physiological saline, and re-suspending the bacterial cells in 15% of recovered skim milk to obtain suspension;

(3) The concentration of the suspension is adjusted to 1.0-2.0X10 ¹⁰ cfu/mL is used for obtaining bacterial suspension, and after the bacterial suspension is frozen and dried, the lactobacillus plantarum JYLP-375 freeze-dried powder is obtained.

In a third aspect, the invention provides an application of lactobacillus plantarum JYLP-375 in preparing silage for preventing and improving calf diarrhea, in particular to silage corn straw using a microbial inoculum containing lactobacillus plantarum JYLP-375.

Further, the method for ensiling corn stalks by using the microbial inoculum comprises the following steps:

(1) Preparing Cheng Yi raw bacteria aqueous solution by thawing the Lactobacillus plantarum JYLP-375 freeze-dried powder with well water, and standing for 30min after the preparation;

(2) Fully crushing corn stalks by using a crusher, uniformly spreading the crushed corn stalks in a fermentation tank, layering and spraying a probiotic aqueous solution into the corn stalks, and rapidly covering and compacting each layer of probiotic aqueous solution; after the probiotics water solution is sprayed, the fermentation tank is sealed immediately, and the fermentation time is 100 days.

Further, 100 g of lactobacillus plantarum JYLP-375 freeze-dried powder silage corn straw 20 tons.

The invention has the beneficial effects that:

the lactobacillus plantarum JYLP-375 provided by the invention has very high survival rate in an in-vitro simulated gastrointestinal environment and has stronger cell colonization capability. The lactobacillus plantarum JYLP-375 is used for silage fermentation treatment of agricultural wastes such as corn stalks, sweet potato seedlings and the like after autumn harvest, and the obtained silage has good effects of improving the constitution of calves and preventing and improving the diarrhea of the calves.

Detailed Description

In order to better understand the technical solutions of the present invention, the following description will clearly and completely describe the technical solutions of the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.

EXAMPLE 1 screening, purification and characterization of Lactobacillus plantarum JYLP-375

1. Fungus source

And collecting self-made pickle liquid from farmers in Tuo town in the Hechuan district of Chongqing in 2018, 10 months and 16 days.

2. Strain screening

(1) Randomly sampling pickle liquid, diluting with sterilized normal saline with dilution gradient of 10 ^-1 、10 ^-2 、10 ^-3 、10 ^-4 、10 ^-5 、10 ^-6 、10 ^-7 The reference numerals are 1#, 2#, 3#, 4#, 5#, 6#, 7#, respectively, for standby;

(2) Preparation of MRS plate medium: taking 10g of peptone, 5g of beef powder, 5g of sodium acetate trihydrate, 2g of dipotassium phosphate heptahydrate, 1mL of Tween 80, 0.05g of manganese sulfate tetrahydrate, 2g of triammonium citrate, 20g of glucose, 0.2g of magnesium sulfate heptahydrate, 15g of agar and 1000mL of distilled water; mixing the above raw materials, naturally adjusting pH, stirring the bacterial liquid, sterilizing at 121deg.C under 0.1MPa for 20min, pouring sterilized culture medium into a plate, and cooling;

(3) Culturing: coating the solution 1#, 2#, 3#, 4#, 5#, 6#, 7# of the step (2) in the MRS plate culture medium by using a coater, and culturing for 48 hours at 37 ℃ under anaerobic conditions;

(4) Colonies were selected according to the following colony characteristics: the diameter is 1-2mm, the colony is round, the edge is neat, and the middle of the micro white is provided with a bulge;

(5) Separating and purifying, selecting 5 single colonies according to the colony characteristics of the step (4), inoculating to the culture medium of the step (3) by a streaking method, culturing for 48 hours at 37 ℃ under anaerobic conditions, selecting single colonies, and placing in a glycerol tube for preservation at-70 ℃.

2. Authentication

Single colony after separation and purification is sent to identification unit: bioengineering (Shanghai) Co., ltd.

(1) In the identification process, the primers used were as follows:

27F：5'-AGAGTTTGATCCTGGCTCAG-3'；

1492R：5'- GGTTACCTTGTTACGACTT-3'。

(2) The gene sequences identified were as follows:

TCTAAAAGGTTACCCCACCGACTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAATGGCTTTAAGAGATTAGCTTACTCTCGCGAGTTCGCAACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCACCAGAGTGCCCAACTTAATGCTGGCAACTGATAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTATCCATGTCCCCGAAGGGAACGTCTAATCTCTTAGATTTGCATAGTATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCCAGGCGGAATGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCATTCATCGTTTACGGTATGGACTACCAGGGTATCTAATCCTGTTTGCTACCCATACTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCGATGCACTTCTTCGGTTGAGCCGAAGGCTTTCACATCAGACTTAAAAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAAATACCGTCAATACCTGAACAGTTACTCTCAGATATGTTCTTCTTTAACAACAGAGTTTTACGAGCCGAAACCCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGTCGGCTACGTATCATTGCCATGGTGAGCCGTTACCCCACCATCTAGCTAATACGCCGCGGGACCATCCAAAAGTGATAGCCGAAGCCATCTTTCAAGCTCGGACCATGCGGTCCAAGTTGTTATGCGGTATTAGCATCTGTTTCCAGGTGTTATCCCCCGCTTCTGGGCAGGTTTCCCACGTGTTACTCACCAGTTCGCCACTCACTCAAATGTAAATCATGATGCAAGCACCAATCAATACCAGAGTTCGTTCGACT。

(3) Through identification, the strain is Lactobacillus plantarumLactobacillus plantarum) The strain is named as Lactobacillus plantarum JYLP-375 and is sent to China general microbiological culture Collection center for preservation of the microorganism and the preservation address in 2019, 6 and 27 days: beijing city, chaoyang district, north Chenxi lu 1, 3, accession number: CGMCC No.18039.

EXAMPLE 2 preparation of Lactobacillus plantarum JYLP-375 lyophilized powder

(1) Preparation of MRS liquid medium: 10g of peptone, 5g of beef powder, 5g of sodium acetate trihydrate, 2g of dipotassium phosphate heptahydrate, 1mL of Tween 80, 0.05g of manganese sulfate tetrahydrate, 2g of triammonium citrate, 20g of glucose, 0.2g of magnesium sulfate heptahydrate and 1000mL of distilled water are mixed, the pH is adjusted to 6.8, and after the bacterial liquid is stirred, the bacterial liquid is sterilized for 20min at 121 ℃ and 0.1 MPa.

(2) Activating preserved lactobacillus plantarum JYLP-375 on the MRS flat-plate culture medium prepared in the example 1, inoculating the activated lactobacillus plantarum JYLP-375 into the MRS liquid culture medium according to an inoculum size of 1%, and then culturing for 24 hours at 37 ℃ to obtain bacterial liquid.

(3) Centrifuging a bacterial liquid, collecting bacterial cells, washing the bacterial cells with sterile physiological saline, and re-suspending the bacterial cells in 15% by mass of reconstituted skim milk to obtain a suspension; the concentration of the suspension is adjusted to be 1.0-2.0X10 ¹⁰ cfu/mL to obtain bacterial suspension, and freeze-drying the bacterial suspension to obtain the lactobacillus plantarum JYLP-375 freeze-dried powder.

In this example, the prepared Lactobacillus plantarum JYLP-375 lyophilized powder had a cell number of 1×10 ¹⁰ cfu/g，2×10 ¹⁰ cfu/g。

EXAMPLE 3 Lactobacillus plantarum JYLP-375 pass simulation of gastrointestinal digestive juice test

1. Preparation of simulated gastric fluid and test treatments

(1) The simulated gastric fluid is prepared according to the following method: diluting with 9.5% hydrochloric acid and distilled water to pH 1.5, adding 1.0g pepsin per 100mL, mixing, and filtering with 0.22 μm sterile filter membrane.

(2) Test treatment: weighing several parts of the Lactobacillus plantarum JYLP-375 obtained in example 2 to obtain a viable count of 2×10 ¹⁰ cfu/g of product was used as samples, each weighing 1g, transferred to pre-heated tubes containing 9mL of simulated gastric fluid, and treated at 37℃for 1h, 2h, 3h, 4h, respectively, 3 replicates each.

2. Preparation of simulated intestinal juice and test treatment

(1) The simulated intestinal juice is prepared according to the following method: 6.8g KH ₂ PO ₄ Dissolving in 500mL distilled water, adding 3g bile salt and 10g trypsin, and concentratingThe pH value of the solution is regulated to 6.8 by 4g/L NaOH solution, distilled water is used for constant volume to 1L, and the solution is filtered by a sterile filter membrane with the thickness of 0.22 mu m after being uniformly mixed, so that the solution is prepared for use.

(2) Several samples of the product of the Lactobacillus plantarum JYLP-375 obtained in example 2 with a viable count of 100 hundred million/g were weighed, each sample weighing 1g, transferred to a test tube filled with 9mL of simulated intestinal fluid, and treated at 37℃for 1h, 2h, 3h, and 4h, respectively, with 3 replicates of each treatment.

3. Statistics and analysis

Performing gradient dilution on the treated sample solution and the control strain solution, counting viable bacteria by using a pouring method, and calculating the survival rate: survival rate= (number of treated bacteria/number of original bacteria) ×100%.

TABLE 1 in vitro survival rate of JYLP-375 in simulated gastrointestinal environment

The survival rate data of the lactobacillus plantarum JYLP-375 in the in-vitro simulated gastrointestinal environment is shown in the table 1, and can be seen that the survival rate of the lactobacillus plantarum JYLP-375 in the in-vitro simulated gastrointestinal environment is very high, the survival rate is still more than 90% in gastric juice and intestinal juice for 4 hours, and a foundation is laid for the subsequent strain colonization of intestinal tracts and functions.

EXAMPLE 4 adhesion control test of Lactobacillus plantarum JYLP-375 to Caco-2

(1) Test reagent:

DMEM broth, purchased from Gibco company;

MRS plate medium was prepared in the same manner as in example 1;

MRS liquid culture medium was prepared in the same manner as in example 2;

weighing KH ₂ PO ₄ 0.27g、Na ₂ HPO ₄ 1.42g, 8g NaCl and 0.2g KCl are added with about 800mL of deionized water, fully stirred and dissolved, added with concentrated hydrochloric acid to adjust the pH to 7.4, added with deionized water to fix the volume to 1L, sterilized at 121 ℃ for 20min, and the sterile PBS buffer with the pH of 7.4 is prepared.

(2) Test preparation:

caco-2 cells were placed in DMEM medium containing 10% heat-inactivated neonatal calf serum and double antibody (penicillin concentration 100U/mL, streptomycin 1.0. Mu.g/mL) at 37℃with 5% CO ₂ Incubating in a carbon dioxide incubator with a relative humidity of 95%, changing culture solution 1 time a day, passaging 1 time for 3 days, inoculating cells into 24-well culture plate after 18 days, and inoculating at a density of about 5×10 ⁵ cfu/mL, and performing adhesion test after the cells grow to a monolayer.

Inoculating activated lactobacillus plantarum JYLP-375 into MRS liquid culture medium according to the inoculum size of 1 percent of volume fraction, and culturing for 12 hours at 37 ℃ to obtain bacterial liquid.

Centrifuging the bacterial solution to collect bacterial cells (4000 r/min, 4 ℃ C., 10 min), washing with sterile PBS buffer solution for 3 times, and suspending the bacterial cells in the sterile PBS buffer solution to obtain a concentration of 2×10 ⁸ cfu/mL of the Lactobacillus plantarum JYLP-375 suspension.

(3) Test procedure:

the Caco-2 cells which had grown into monolayers were rinsed 2 times with sterile PBS buffer, 0.5mL of JYLP-375 strain suspension and 0.5mL of fresh DMEM culture solution were added to each well, and the mixture was incubated at 37℃with 5% CO ₂ Incubate in a carbon dioxide incubator at 95% relative humidity for 1h, and then rinse the cells 5 times with sterile PBS buffer to remove non-adherent bacteria.

Performing gradient dilution on the rinsing liquid, counting viable bacteria by using a pouring method, and calculating the adhesion rate: adhesion = (initial number of cells-number of cells in rinse)/initial number of cells×100%.

After rinsing, the cells were fixed with formaldehyde, stained with gram, microscopic examined, and the number of bacteria adhering to 100 cells in 20 random fields was counted under an inverted microscope. The average number of bacteria adhered to each cell was calculated using cell culture without addition of bacterial liquid as a control. Each treatment was done in 3 replicates.

TABLE 2 adhesion Rate and adhesion count of JYLP-375 on Caco-2 cells

The adhesion rate and the cell adhesion number of the strain JYLP-375 to Caco-2 cells are shown in Table 2, and it can be seen that the Lactobacillus plantarum JYLP-375 has a strong capability in cell colonisation.

Example 5 Lactobacillus plantarum JYLP-375 silage corn straw method

100 g of Lactobacillus plantarum JYLP-375 freeze-dried powder (viable count 2X 10) ¹⁰ cfu/g) was melted with 50 kg of well water, and Cheng Yi aqueous bacteria-producing solution was prepared, and left to stand for half an hour after the preparation was completed. Fully crushing corn stalks to be ensiled by a crusher, uniformly spreading a layer of crushed corn stalks in a fermentation tank, spraying a probiotic water solution on the corn stalks, rapidly covering the layer of corn stalks, and compacting. Each layer of probiotic water solution is sprayed, corn stalks are immediately covered and immediately compacted. The air in the straw is exhausted as much as possible, and special attention is paid to compaction at the edges and corners. The 100 g lactobacillus plantarum JYLP-375 freeze-dried powder can silage 20 tons of corn stalks. After the probiotics water solution is sprayed, the fermentation tank is sealed immediately, and fermentation is carried out for 100 days.

Example 6 Lactobacillus plantarum JYLP-375 silage corn straw vs. calf diarrhea prevention test

1. Test time: 12 months in 2020 to 3 months in 2021.

2. Test site: the east of mountain.

3. The test contents are as follows: and randomly distributing 120 calves which are born for 0-6 months into 2 groups of 60 calves each. The silage of example 5 and the traditional feeding method are adopted for feeding respectively. The calf diarrhea status is recorded during feeding, and is judged by the livestock enterprises owners.

4. Test results: diarrhea status of 120 calves is shown in the following tables 3 and 4.

TABLE 3 calf diarrhea status

During the whole winter and early spring time, the incidence of calf diarrhea was 28.3% in the course of feeding calves with the traditional feed, while the incidence of calf diarrhea was only 8.3% in the silage prepared in example 5. Therefore, the probability of calf diarrhea can be greatly reduced by using the Lactobacillus plantarum JYLP-375 silage corn straw as the calf feed in winter and early spring. Meanwhile, for calves with diarrhea, the onset of silage groups is lighter, and the calves can be cured only by taking radix isatidis orally for 5 days, but the traditional feeding method still needs 8 days to cure according to years of experience of livestock owners by adopting a traditional treatment mode of infusion and radix isatidis administration, and still 1 calf dies due to severe diarrhea. It can thus be seen that: the corn straw with the JYLP-375 silage of the lactobacillus plantarum is used for feeding calves, so that the diarrhea probability of the calves in winter and early spring can be reduced, the constitution of the calves can be enhanced, and the quick recovery after diarrhea is facilitated.

In conclusion, the lactobacillus plantarum JYLP-375 can be used for silage, and the silaged feed has good effects of improving the constitution of calves and preventing and improving the diarrhea of the calves.

Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Various equivalent modifications and substitutions may be made in the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and it is intended that all such modifications and substitutions be within the scope of the present invention/be within the scope of the present invention as defined by the appended claims.

Claims (8)

1. A lactobacillus plantarum JYLP-375 is characterized in thatLactobacillus plantarum) JYLP-375 was preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at the date of 27, 6.A preservation address is 1# 3 of North Chen West Lu of the Korean area of Beijing city, and a preservation number is CGMCC No.18039.

2. A microbial agent comprising the lactobacillus plantarum JYLP-375 according to claim 1.

3. The microbial inoculant of claim 2, wherein the microbial inoculant comprises lactobacillus plantarum JYLP-375 lyophilized powder.

4. The microbial inoculum of claim 3, wherein the preparation method of the lactobacillus plantarum JYLP-375 freeze-dried powder comprises the following steps:

(1) Activating preserved lactobacillus plantarum JYLP-375 on an MRS flat-plate culture medium, inoculating the activated bacteria into the MRS liquid culture medium according to an inoculum size of 1%, and culturing for 24 hours at 37 ℃ to obtain a bacterial liquid;

(2) Centrifuging the bacterial liquid, collecting bacterial cells, washing the bacterial cells by using sterile physiological saline, and re-suspending the bacterial cells in 15% of recovered skim milk to obtain suspension;

(3) The concentration of the suspension is adjusted to 1.0-2.0X10 ¹⁰ cfu/mL is used for obtaining bacterial suspension, and after the bacterial suspension is frozen and dried, the lactobacillus plantarum JYLP-375 freeze-dried powder is obtained.

5. Use of lactobacillus plantarum JYLP-375 according to claim 1 for preparing silage for preventing and improving calf diarrhea.

6. The use according to claim 5, wherein corn stover is ensiled with a microbial inoculum comprising lactobacillus plantarum JYLP-375.

7. The use of claim 6, wherein the method of ensiling corn stover with a microbial inoculant comprises the steps of:

(1) Preparing Cheng Yi raw bacteria aqueous solution by thawing the Lactobacillus plantarum JYLP-375 freeze-dried powder with well water, and standing for 30min after the preparation;

(2) Fully crushing corn stalks by using a crusher, uniformly spreading the crushed corn stalks in a fermentation tank, layering and spraying a probiotic aqueous solution into the corn stalks, and rapidly covering and compacting each layer of probiotic aqueous solution; after the probiotics water solution is sprayed, the fermentation tank is sealed immediately, and the fermentation time is 100 days.

8. The use according to claim 7, wherein 100 g of lactobacillus plantarum JYLP-375 lyophilized powder silage maize straw is 20 tons.