CN107338203B

CN107338203B - Enterococcus faecalis for feed, microecological preparation for preventing and treating heat stress of livestock and poultry and preparation method thereof

Info

Publication number: CN107338203B
Application number: CN201710559483.2A
Authority: CN
Inventors: 张炎达; 潘慧青; 杨荣平; 肖建设; 赵齐; 林贺桂
Original assignee: Fujian Brady Pharmaceutical Co ltd
Current assignee: Fujian Brady Pharmaceutical Co ltd
Priority date: 2017-07-11
Filing date: 2017-07-11
Publication date: 2020-02-21
Anticipated expiration: 2037-07-11
Also published as: CN107338203A

Abstract

The invention belongs to the field of Chinese herbal medicine microecological preparations, and discloses a microecological preparation for feeding enterococcus faecalis, preventing and treating heat stress of livestock and poultry and a preparation method thereof. The enterococcus faecalis BD-1 has a preservation number of CCTCC NO: m2017416. The substrate mass percentage of the microecological preparation product prepared by fermenting enterococcus faecalis BD-1 in the invention is as follows: 25-30% of green tea, 10-20% of green tea extraction residues, 10-15% of radix pseudostellariae fibrous roots, 10-15% of dried tangerine peels, 10-20% of hawthorn, 10-15% of denucleated dried jujubes and 0.05-0.1% of garlicin. The enterococcus faecalis BD-1 has good tea polyphenol and allicin resistance, and the radix pseudostellariae fibrous extract can remarkably promote the growth of the strain. The microecological preparation is beneficial to the storage of the effective components of the finished product, can obviously reduce the goods damage rate and prolong the shelf life.

Description

Enterococcus faecalis for feed, microecological preparation for preventing and treating heat stress of livestock and poultry and preparation method thereof

Technical Field

The invention belongs to the field of Chinese herbal medicine microecological preparations, and particularly relates to a microecological preparation for feeding enterococcus faecalis, preventing and treating heat stress of livestock and poultry and a preparation method thereof.

Background

The heat stress is a main factor influencing the growth performance of livestock in summer and is also a cause of various infectious diseases of the livestock. In the intensive cultivation today, with the increase of the breeding density and the trend of global warming, heat stress has become one of the important factors affecting the development of the cultivation industry. In the pig raising production, the loss caused by high temperature is prominent, the productivity is reduced, diseases are increased, the elimination rate and the death rate are increased, and the production cost is increased, so that the method becomes a great problem in the pig raising industry, particularly in a large-scale pig farm in summer. Similarly, poultry has abundant surface feathers, vigorous metabolism, no sweat glands and sensitivity to high temperature, gastrointestinal tracts are easy to suffer from intestinal injury due to ischemia in the heat stress process, the blood supply is reduced and the oxygen supply is reduced during ischemia, the oxygen intake and utilization capacity of the intestinal tracts is compensated and improved, so that epithelial cells of intestinal mucosa are damaged and edematous, cell membranes and intercellular junctions rupture cells and necrotize, the health of the poultry intestinal tracts is greatly influenced, the immunity of the intestinal tracts is easy to reduce, the internal environment of intestinal tracts is disturbed, the occurrence of other related diseases is promoted, and the production performance of the poultry is damaged.

At present, the approaches for preventing heat stress of livestock and poultry mainly focus on feeding management, addition of reducing agents (such as vitamins, tea polyphenol and the like), nutrition enhancement, spleen and stomach strengthening and intestinal probiotic supplementation. In the south livestock and poultry breeding industry, tea dust or tea residues are applied to the work of preventing heat stress of livestock and poultry, and researches find that tea polyphenol in the cheap tea dust or tea residues plays an important reductive antioxidation role. However, no high-efficiency fermentation type tea micro-ecological preparation product is reported in the market at present, the main reason is that tea fermentation probiotic strains capable of tolerating tea polyphenol are lacked, and even if the product is generally prepared, ideal application effects cannot be achieved due to small dissolution rate of the tea polyphenol and serious oxidation loss of the tea polyphenol. In addition, the existing fermentation type microecologics (with the water content of 10-40%) are mostly polluted by mould in the shelf life, and how to solve the problem is significant for improving the shelf life of finished products.

Disclosure of Invention

In order to overcome the problems that tea polyphenol-tolerant tea fermentation probiotic strains are lacked, the efficient fermentation type tea microecological preparation is difficult to prepare, and the shelf life of the finished microecological preparation is short in the prior art, the invention aims to provide the enterococcus faecalis for feeding, the microecological preparation for preventing and treating heat stress of livestock and poultry and the preparation method thereof.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a feed enterococcus faecalis (A)Enterococcus faecalis) BD-1, which the applicant has deposited in the China center for type culture Collection, at the address: wuhan city Wuchang Lojia mountain, preservation date: 7 months and 7 days in 2017; the preservation number is: CCTCC NO: m2017416.

The method for preparing the microecological preparation for preventing and treating the heat stress of the livestock and poultry by using the enterococcus faecalis BD-1 for feed comprises the following steps:

(1) activation of enterococcus faecalis BD-1:

washing slant strains of enterococcus faecalis BD-1 test tubes with sterile normal saline in a sterile environment, then inoculating the slant strains into an MRS liquid culture medium containing 0.2-0.5% of radix pseudostellariae fibrous extract, and statically culturing for 42-48 h at 35-37 ℃; wherein the percentage of the radix pseudostellariae fibrous extract is calculated by mass volume percentage g/mL (namely 100mL MRS liquid culture medium contains 0.2-0.5 g of radix pseudostellariae fibrous extract);

(2) preparing a enterococcus faecalis BD-1 fermentation seed solution:

inoculating 3-5% of the strain activation liquid obtained in the step (1) in a volume percentage into an MRS liquid culture medium containing 0.2-0.5% of radix pseudostellariae fibrous extract under an aseptic environment, and performing static fermentation for 42-48 h at 35-37 ℃ to obtain enterococcus faecalis BD-1 fermented seed liquid; wherein the percentage of the radix pseudostellariae fibrous extract is calculated by mass volume percentage g/mL (namely 100mL MRS liquid culture medium contains 0.2-0.5 g of radix pseudostellariae fibrous extract);

(3) preparing a fermentation substrate:

firstly, preparing raw materials according to mass percent: 25-30% of green tea, 10-20% of green tea extraction residues, 10-15% of radix pseudostellariae fibrous roots, 10-15% of dried tangerine peels, 10-20% of hawthorn fruits, 10-15% of denucleated dried jujubes and 0.05-0.1% of garlicin; then, crushing the raw materials except the allicin, and uniformly mixing the crushed raw material powder with the allicin to prepare a fermentation substrate;

(4) and fermenting:

adding the fermentation seed liquid obtained in the step (2) into the fermentation substrate obtained in the step (3), uniformly stirring, filling into a fermentation bag, sealing, and fermenting the fermentation bag for 14-21 days at 30-37 ℃ in a dark condition to obtain a microecological preparation; wherein the fermentation seed liquid accounts for 30-40% of the fermentation substrate matrix in terms of volume mass percent L/kg.

Preferably, in the step (3), the crushing particle size is preferably 60 to 80 meshes.

The microecological preparation for preventing and treating heat stress of livestock and poultry, which is prepared by the preparation method, is used for preventing and treating heat stress of livestock and poultry.

In the present invention, both the extraction residue of green tea (which refers to the residue left after extraction of green tea) and the fibrous root of pseudostellaria root extract can be obtained by the prior art, for example, the extraction method of green tea can refer to but is not limited to the prior water extraction method, and the fibrous root of pseudostellaria root can refer to but is not limited to the method disclosed in the patent (application No. 201610758305.8) entitled "a preparation method of fibrous root of pseudostellaria root extract" filed by the applicant at 2016, 8, 30.

Has the advantages that:

1. the enterococcus faecalis BD-1 for feed is separated from village peasant households (coordinate: 119) in Cudrania tricuspidata county^。51′59.7〞E，27^。13 '34.0'. N) of the black mucosa of the autotrophic healthy adult pig, the obtained enterococcus faecalis BD-1 is safe and nontoxic, the surface of a bacterial colony cultured in an MRS culture medium at 37 ℃ for 48 hours is smooth and convex, the periphery is neat, the color of the bacterial colony is grey white, the diameter of the bacterial colony is 0.5-1.2 mm, the diameter of the bacterial colony is G + detected by microscopy, no spore exists, and the length of the bacterial body is about 0.5-1 mu m. The enterococcus faecalis BD-1 grows well under anaerobic culture and aerobic culture, and the number of viable bacteria respectively reaches 2.82 multiplied by 10⁹CFU/mL、2.04×10⁹CFU/mL, the strain has good acid resistance, cholate resistance and high temperature resistance, and simultaneously has good tea polyphenol and allicin resistance, the concentration of the strain which can resist tea polyphenol and allicin is 1.0 percent and 0.1 percent respectively, the survival rate of the strain at the concentration is 70.26 percent and 70.43 percent respectively, and the survival rate can reach 53.72 percent and exceed 50 percent under the combined action of the tea polyphenol (2 percent) and the allicin (0.1 percent); in addition, the radix pseudostellariae fibrous extract can remarkably promote the growth of the strain; enterococcus faecalis BD-1 can remarkably inhibit the growth of pathogenic bacteria such as staphylococcus aureus, micrococcus luteus and Escherichia coli;

2. the preparation method of the microecological preparation can be beneficial to dissolving out the effective components of the tea polyphenol, protect the tea polyphenol and reduce the loss of the tea polyphenol, can effectively improve the content of free tea polyphenol in a fermented finished product, can prolong the shelf life of the product, and can achieve the effect of well preventing mould pollution;

3. the enterococcus faecalis BD-1 is beneficial to restoration and rebalancing of the microecological internal environment of intestinal disorder, and the microecological preparation product prepared by using the enterococcus faecalis BD-1 has the effects of tonifying spleen and benefiting stomach, can be used for preventing and treating heat stress syndrome caused by intensive livestock and poultry breeding in summer, improving the livestock and poultry breeding production capacity in summer and reducing the occurrence of diseases, and is a potential heat stress-resistant ideal microecological fermentation preparation.

Drawings

FIG. 1: phylogenetic tree of enterococcus faecalis BD-1.

Detailed Description

In the following examples, the green tea extraction residue refers to a residue left after green tea is extracted by a conventional water extraction method; the radix pseudostellariae fibrous extract is prepared by the method disclosed in embodiment 1 in the patent application number of CN 201610758305.8; the formula of each culture medium is as follows:

MRS liquid medium (1L): 10g of peptone, 5g of beef powder, 20g of glucose, 4g of yeast powder, 5g of sodium acetate, 2g of dipotassium hydrogen phosphate, 0.2g of magnesium sulfate, 2g of triammonium citrate, 0.05g of manganese sulfate, 801 mL of tween, 1L of purified water and 6.2 +/-0.2 of pH value;

MRS agar plate or MRS slant medium (1L): 10g of peptone, 5g of beef powder, 20g of glucose, 4g of yeast powder, 5g of sodium acetate, 2g of dipotassium hydrogen phosphate, 0.2g of magnesium sulfate, 2g of triammonium citrate, 0.05g of manganese sulfate, 801 mL of tween, 15g of agar, 1L of purified water and pH value of 6.2 +/-0.2;

wherein, MRS liquid culture medium containing radix Pseudostellariae extract, tea polyphenol or allicin is prepared by adding radix Pseudostellariae extract, tea polyphenol or allicin on the basis of the above MRS liquid culture medium; the MRS agar plates containing bromcresol purple indicator and tea polyphenol or allicin are added with bromcresol purple indicator, tea polyphenol or allicin correspondingly on the basis of the formula of the MRS agar plates.

Example 1 isolation and identification of enterococcus faecalis BD-1

1. Separation, enrichment and purification of enterococcus faecalis BD-1

Under the aseptic operation condition, a village farmer (coordinate: 119) in Cudrania tricuspidata, Dendro, Fujian province is taken^。51′59.7〞E，27^。13' 34.0 "" N) of the cecal mucosa segment of an autotrophic healthy adult pig, scraping the mucosal mucus from the EP tube with a sterilized glass slide, diluting 10 with sterile physiological saline²Taking 0.1mL of the bacterial strain, coating the bacterial strain on an MRS agar plate containing a bromocresol purple indicator (0.01 percent, g/mL), placing the MRS agar plate in an anaerobic incubator after coating, culturing for 48 hours at 37 ℃, selecting bacterial colonies with a culture medium becoming yellow, streaking and purifying, selecting bacterial strains with gram-positive staining and no spores through microscopic examination, rescreening the bacterial strains on the MRS agar plate containing the bromocresol purple (0.01 percent, g/mL), tea polyphenol (0.01 percent, g/mL) and garlicin (0.01 percent, g/mL), and finally selecting bacterial strains with obvious yellow color, smooth and convex surfaces of the bacterial colonies, regular peripheries as target bacterial strains, wherein the bacterial strains are named as BD-1, and performing slant MRS preservation on the bacterial strains by using a slant culture medium.

2. Morphological observation

The morphological characteristics of the colony of the strain BD-1 were observed to show that: the surface of a bacterial colony on the MRS agar plate is smooth and convex, the periphery is neat, the color of the bacterial colony is grey white, the diameter of the bacterial colony is 0.5-1.2 mm, the diameter of the bacterial colony is G +, no spore exists, and the length of the thallus is about 0.5-1 mu m.

3. Physiological and biochemical characteristics

The strain BD-1 is identified according to the biochemical identification test of the lactic acid bacteria, the biochemical characteristics such as motility and oxidase tests are respectively identified, and the test result is comprehensively judged according to Bergey's bacteria identification handbook. The results are shown in Table 1.

Note: "+" indicates positive reaction; "-" indicates negative reaction.

The result of the physiological series index measurement of the isolated strain is consistent with the result of the enterococcus lactis strain.

4. 16S rDNA sequencing alignment of BD-1 strains

Extracting the total DNA of the strain BD-1 by using a bacterial genome DNA extraction kit (DP 302-02, TIANGEN), and carrying out PCR amplification on a 16S rDNA sequence of the strain under the guidance of a primer P0 (5'-AGAGTTTGATCCTGGCTCAG-3') and a PC3 (5'-GGTTACCTTGTTACGACTT-3') by using the total DNA of the strain BD-1 as a template, wherein the PCR reaction system is as follows: 10 XBuffer 5.0 μ L, dNTP (10mmol/L)2.0 μ L, Primer 1(20 μmol/L)1.0 μ L, Primer2(20 μmol/L)1.0 μ L, template DNA 2.5 μ L, Taq enzyme (5.0U/μ L)0.5 μ L, ultrapure water 38 μ L, total 50 μ L. The PCR reaction conditions are as follows: firstly, 94 ℃ for 5 min; then 30 cycles of 94 ℃ for 1min, 55 ℃ for 1min and 72 ℃ for 1 min; finally, extension is carried out for 8min at 72 ℃. After the reaction is finished, carrying out 1% agarose gel electrophoresis detection on the PCR amplification product, and comparing with a marker to obtain a target band. The target band was recovered and purified using an agarose gel recovery kit from Axygen, and sent to Biotechnology engineering (Shanghai) Co., Ltd for DNA sequencing, and the sequencing results are shown in SEQ ID No. 1.

The determined sequence was compared with the 16S rDNA sequence in Genbank for Blast similarity analysis, resulting in strain BD-1 andEnterococcus faeciumstrain KCI1、Enterococcus lactisthe 16S rDNA sequence of strain K12419C showed 100% homology, and BD-1 was identified as enterococcus faecalis and its phylogenetic tree is shown in FIG. 1.

The strain BD-1 is identified and classified and named as enterococcus faecalis by combining the morphological, physiological and biochemical characteristics and sequencing and evolutionary tree results (Enterococcus faecalis）BD-1。

Example 2 anaerobic and aerobic fermentation of enterococcus faecalis BD-1 in MRS liquid Medium

(1) Activation of enterococcus faecalis BD-1: washing slant strain of enterococcus faecalis BD-1 test tube with sterile normal saline in sterile environment, washing off thallus Porphyrae on the surface of culture medium, inoculating into MRS liquid culture medium, and static culturing at 37 deg.C for 48 hr;

(2) anaerobic static fermentation of enterococcus faecalis BD-1 in MRS liquid culture medium:

inoculating the activating solution of the enterococcus faecalis BD-1 obtained in the step (1) into an anaerobic serum bottle containing an MRS liquid culture medium in an anaerobic box according to the inoculation amount of 3 percent (volume ratio), statically culturing at 37 ℃ for 48 hours, and measuring the viable count and the pH value of the fermentation liquor at the end point of the culture;

(3) aerobic fermentation of enterococcus faecalis BD-1 in MRS liquid medium:

inoculating the activating solution of the enterococcus faecalis BD-1 obtained in the step (1) into a 500 mL triangular flask filled with 100mL MRS liquid culture medium according to the inoculation amount of 3% (volume ratio), culturing at 37 ℃ and 150rpm for 48h, and measuring the viable count and pH value of the fermentation liquid at the culture endpoint.

The results are shown in Table 2.

Note △ pH = pH-pH₀，pH₀The initial pH value for the inoculation culture represents the acid production degree in the reaction growth process.

Example 3 Effect of radix Pseudostellariae Leptoradix Pseudostellariae extract on growth and pH of enterococcus faecalis BD-1

(1) Activation of enterococcus faecalis BD-1: same as example 2, step (1);

(2) inoculating the activating solution of the enterococcus faecalis BD-1 obtained in the step (1) into an anaerobic serum bottle containing an MRS liquid culture medium in an anaerobic box according to the inoculation amount of 3% (volume ratio), wherein the gradient of the radix pseudostellariae fibrous extract in the MRS liquid culture medium in the anaerobic serum bottle is sequentially set as (g/mL): 0%, 0.10%, 0.25%, 0.5%, static culturing at 37 deg.C for 48h, measuring pH value and viable count of the fermentation broth at the end point of the culturing, and the results are shown in Table 3;

(3) inoculating the activating solution of the enterococcus faecalis BD-1 obtained in the step (1) into a 500 mL triangular flask filled with 100mL MRS liquid culture medium according to the inoculation amount of 3% (volume ratio), wherein the gradients of the radix pseudostellariae fibrous extracts in the MRS liquid culture medium in the flask are sequentially set as (g/mL): 0%, 0.10%, 0.25%, 0.5%, culturing at 37 deg.C and 150rpm for 48h, and determining pH value and viable count of the fermentation broth at the end point of the culture, the results are shown in Table 4;

as is clear from the results in tables 3 to 4: the radix pseudostellariae fibrous extract can obviously promote the growth of the enterococcus faecalis BD-1.

Example 4 Effect of tea polyphenols on growth and pH of enterococcus faecalis BD-1

(1) Activation of enterococcus faecalis BD-1: same as example 2, step (1);

(2) inoculating the activated liquid of the enterococcus faecalis BD-1 obtained in the step (1) into an anaerobic serum bottle containing an MRS liquid culture medium in an anaerobic box according to the inoculation amount of 3% (volume ratio), wherein the gradient of tea polyphenol in the MRS liquid culture medium in the anaerobic serum bottle is sequentially set as (g/mL): 0%, 0.10%, 0.25%, 0.50%, 1.00%, and the results of measuring the pH value and viable count of the fermentation broth at the end of the incubation are shown in Table 5.

From the results in Table 5, it can be seen that: the concentration of the tea polyphenol which can be tolerated by the enterococcus faecalis BD-1 is 1.0%, and the survival rate of thalli under the concentration is 70.26%.

Example 5 Effect of allicin on growth and pH of enterococcus faecalis BD-1

(1) Activation of enterococcus faecalis BD-1: same as example 2, step (1);

(2) inoculating the activated liquid of the enterococcus faecalis BD-1 obtained in the step (1) into an anaerobic serum bottle containing an MRS liquid culture medium in an anaerobic box according to the inoculation amount of 3 percent (volume ratio), wherein the gradient of allicin in the MRS liquid culture medium in the anaerobic serum bottle is sequentially set as (g/mL): 0%, 0.010%, 0.025%, 0.050%, 0.10%, and the results of measuring the pH value and viable count of the fermentation broth at the end point of the culture are shown in Table 6;

from the results in Table 6, it can be seen that: the concentration of the tolerant allicin of the enterococcus faecalis BD-1 is 0.1 percent, and the survival rate of thalli under the concentration is 70.43 percent;

(3) growth comparison of enterococcus faecalis resistant to tea polyphenols and allicin:

respectively inoculating the activated liquid of enterococcus faecalis BD-1 (counted as A) and the activated liquid of control bacterium B, C, D obtained in the step (1) into an anaerobic serum bottle containing an MRS liquid culture medium in an anaerobic box according to the inoculation amount of 3 percent (volume ratio), wherein the gradients of ① tea polyphenol in the MRS liquid culture medium in the anaerobic serum bottle are sequentially set to be (g/mL): 0%, 1.00%, 2.00%, and ② allicin are sequentially set to be (g/mL): 0%, 0.05%, and 0.1%, continuously culturing for 48h, and taking 0% +0% as a control (the number of viable bacteria is defined as X)₀) By the formula: cell loss rate = (X)₀-X）/ X₀The loss rate of enterococcus faecalis cells was calculated as 100%, and the results are shown in table 7.

Note: a is enterococcus faecalis BD-1, B, C, D purchased from enterococcus faecalis for feeding of Zhengzhou Europe Becker Biotech limited, Zhengzhou Beddi Biotech limited and Shanghai Bansen Biotech limited, respectively. Among them, the activation process of B, C, D strain was the same as A.

From the results in Table 7, it can be seen that: under the combined action of tea polyphenol (2%) and allicin (0.1%), the survival rate of enterococcus faecalis BD-1 can reach 53.7%, which exceeds 50%, and is far higher than that of similar strains in the market.

Example 6 enterococcus faecalis bacteriostasis test

(1) Activation of enterococcus faecalis BD-1: same as example 2, step (1);

inoculating the activating solution of the enterococcus faecalis BD-1 obtained in the step (1) into an anaerobic serum bottle containing an MRS liquid culture medium according to the inoculation amount of 3% (volume ratio), statically culturing at 37 ℃ for 48h, taking a fermentation liquid at the fermentation end point, detecting the bacteriostatic effect of the fermentation liquid on staphylococcus aureus, micrococcus luteus and Escherichia coli by using an Oxford cup method, and taking the non-inoculated MRS liquid culture medium as a reference;

(3) aerobic fermentation of enterococcus faecalis BD-1 in MRS liquid medium:

and (2) inoculating the activating solution of the enterococcus faecalis BD-1 obtained in the step (1) into a 500 mL triangular flask filled with 100mL of MRS liquid culture medium according to the inoculation amount (volume ratio) of 3%, culturing at 37 ℃ and 150rpm for 48h, taking the fermentation liquid at the end of the culture, detecting the bacteriostatic effect on staphylococcus aureus, micrococcus luteus and Escherichia coli by using an Oxford cup method, and taking the non-inoculated MRS liquid culture medium as a control.

The diameter of the zone of inhibition is shown in Table 8.

Note: the unit is mm, and the control group has no inhibition zone.

From the results in Table 8, it can be seen that: the enterococcus faecalis BD-1 can obviously inhibit the growth of pathogenic bacteria staphylococcus aureus, micrococcus luteus and Escherichia coli, and the anaerobic culture is stronger than the aerobic culture.

EXAMPLE 7 acid, bile salt and high temperature resistant test of enterococcus faecalis BD-1

(1) Activation of enterococcus faecalis BD-1: same as example 2, step (1);

(2) acid resistance test: inoculating the activating solution of the enterococcus faecalis BD-1 obtained in the step (1) into an anaerobic serum bottle containing an MRS liquid culture medium in an anaerobic box according to the inoculation amount of 3 percent (volume ratio), and statically culturing for 32 hours at 37 ℃ to serve as acid-resistant strain liquid; then adjusting the initial pH value of the MRS liquid culture medium by using 1M hydrochloric acid solution, setting the initial pH value gradient of the MRS liquid culture medium to be 2.0, 3.0, 4.0, 5.0, 6.0 and 7.0 respectively, sterilizing at 115 ℃ for 20min, and cooling to room temperature; then inoculating the acid-resistant strain liquid into MRS liquid culture medium with various gradients according to the inoculation amount of 3 percent (volume ratio), uniformly mixing, standing at 37 ℃ for 48h, and respectively measuring when finishingThe mixture solution at each pH gradient was diluted to 1mL with 25-fold physiological saline, and the OD was measured using 25-fold dilution of the non-inoculated MRS liquid medium at each gradient with physiological saline as a control₆₀₀The value is obtained. The results are shown in Table 9;

(3) bile salt resistance test: inoculating the activating solution of the enterococcus faecalis BD-1 obtained in the step (1) into an anaerobic serum bottle containing an MRS liquid culture medium in an anaerobic box according to the inoculation amount of 3 percent (volume ratio), and statically culturing for 32 hours at 37 ℃ to obtain cholate-resistant bacteria liquid; then adding bile salt into the MRS liquid culture medium, setting the initial bile salt gradient (g/mL) of the MRS liquid culture medium to be 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1.0% and 2.0% respectively, then taking 9mL of the solution, transferring the solution into a test tube, covering a test tube plug, sterilizing at 115 ℃ for 20min, and cooling to room temperature; then adding 1mL of cholate-resistant strain liquid into a test tube of 9mL of MRS liquid culture medium in an aseptic environment, uniformly mixing and sealing, standing at 37 ℃ for 2h, and simultaneously measuring 1mL of acid-resistant strain liquid into a test tube containing 9mL of MRS liquid culture medium without cholate addition, wherein the method is the same as the above and is used as a reference; respectively measuring 1mL of mixed solution of each cholate gradient and the control group at 2h, and carrying out the measurement for 10 h⁸Diluting with normal saline, spreading on MRS agar plate with pH of 6.2 + -0.2, culturing at 37 deg.C for 48 hr, counting, and calculating survival rate; survival rate = (number of viable control group-number of viable test group)/number of viable control group 100%. The results are shown in Table 10;

(4) high temperature resistance test:

inoculating the activating solution of the enterococcus faecalis BD-1 obtained in the step (1) into an anaerobic serum bottle containing an MRS liquid culture medium in an anaerobic box according to the inoculation amount of 3% (volume ratio), and performing static culture at 37 ℃ for 32 hours to obtain a high-temperature-resistant strain solution; then transferring 4.5mL into 5mL EP tube, sealing with cover, standing in hot water bath at 37 deg.C, 40 deg.C, 50 deg.C, 60 deg.C and 70 deg.C for 0.5 hr, wherein 37 deg.C is used as control, and most preferablyThen respectively taking 1mL of the high-temperature resistant strain liquid treated at different temperatures, and carrying out 10⁸Diluting with normal saline, spreading on MRS agar plate with pH of 6.2 + -0.2, culturing at 37 deg.C for 48 hr, counting, and calculating survival rate. Survival rate = (number of viable bacteria in control group-number of viable bacteria in test group)/number of viable bacteria in control group 100%; the results are shown in Table 11;

from the results of tables 9 to 11, it can be seen that: the enterococcus faecalis BD-1 has good acid resistance, alkali resistance, cholate resistance and high temperature resistance.

Example 8 safety test

Test animals: KM mice, SPF grade, 18-22g weight, half male and half female, purchased from the institute of Chinese medicine, Chongqing.

And (3) testing: selecting 10 healthy mice, each half of the mice in male and female, and weighing the mice in average: female (21.90 +/-0.5) g and male (22.00 +/-0.5) g; after the mice were fasted (without water deprivation) for 12h, a suspension of enterococcus faecalis BD-1 (approximately 5.0X 10)⁸CFU/mL), and the total dose of administration is 1.5mL after 3 times of equal dose administration in one day; wherein the preparation process of the suspension of enterococcus faecalis BD-1 comprises the following steps:

(1) activation of enterococcus faecalis BD-1: washing slant strain of enterococcus faecalis BD-1 test tube with sterile normal saline in sterile environment, inoculating into MRS liquid culture medium, and static culturing at 37 deg.C for 48 hr;

(2) and (3) expanding culture: inoculating the activating solution of the enterococcus faecalis BD-1 obtained in the step (1) into an anaerobic serum bottle containing an MRS liquid culture medium in an anaerobic box according to the inoculation amount of 5% (volume ratio), and statically culturing for 48h at 37 ℃;

(3) diluting: and (3) measuring the total viable count of the culture solution obtained in the step (2), and diluting the culture solution to a required concentration according to the total viable count and sterile normal saline.

After administration, the mice are normally raised in a clean-grade raising room at room temperature (21 +/-0.5) DEG C and with the relative humidity of 45-60%, the animals are observed to have abnormal changes in activity, fur, diet and excrement, toxic symptoms and death are observed, and the results are shown in table 12 after three weeks of continuous observation.

From the results in Table 12, it can be seen that: the enterococcus faecalis BD-1 is safe and nontoxic.

EXAMPLE 9 preparation of a Microecological formulation

The preparation steps are as follows:

(1) activation of enterococcus faecalis BD-1:

washing slant strain of enterococcus faecalis BD-1 test tube with sterile normal saline in sterile environment, washing off thallus Porphyrae on the surface of culture medium, inoculating into 500 mL anaerobic serum bottle containing MRS liquid culture medium containing radix Pseudostellariae fibrous extract 0.25%, and static culturing at 37 deg.C for 48 hr; the percentage of the radix pseudostellariae fibrous extract is calculated by mass volume percentage g/mL (namely 100mLMRS liquid culture medium contains 0.25g of radix pseudostellariae fibrous extract);

(2) preparing a enterococcus faecalis BD-1 fermentation seed solution:

under an aseptic environment, inoculating the activated liquid of the enterococcus faecalis BD-1 obtained in the step (1) into a fermentation tank filled with an MRS liquid culture medium containing 0.25% of radix pseudostellariae fibrous extract according to the volume percentage of 5%, and performing static fermentation for 48h at 37 ℃ to obtain an enterococcus faecalis BD-1 fermented seed liquid; the percentage of the radix pseudostellariae fibrous extract is calculated by mass volume percentage g/mL (namely 100mL MRS liquid culture medium contains 0.25g of radix pseudostellariae fibrous extract);

(3) preparing a fermentation substrate:

firstly, preparing raw materials according to mass percent: 25% of green tea, 15% of green tea extraction residue, 14.94% of radix pseudostellariae fibrous root, 15% of dried orange peel, 15% of hawthorn, 15% of denucleated dried jujube and 0.06% of garlicin; then, the raw materials except the allicin are firstly crushed, the particle size is 80 meshes, and then the crushed raw material powder is uniformly mixed with the allicin by using the authorized utility model patent equipment (patent number: 201620106247.6) to prepare a fermentation substrate;

(4) and fermenting:

firstly, feeding the fermentation substrate prepared in the step (3) into stirring equipment (the equipment adopts utility model patent equipment, the patent number is 201620053478.5), then adding the fermentation seed liquid obtained in the step (2) into the equipment, stirring, wherein the stirring speed is 100 rpm, and the holding time is 25min, wherein the fermentation seed liquid accounts for 35% of the fermentation substrate in terms of volume mass percent L/kg; after the mixture is uniformly stirred, the mixture is quickly filled into a fermentation bag (the fermentation bag adopts a utility model patent bag, the patent number is 201620018088.4), the mixture is sealed, after the split charging is finished, the fermentation bag is directly transported to a fermentation workshop, the fermentation workshop requires constant temperature (35 ℃) and light shielding, after the fermentation bags are arranged and stacked in the fermentation workshop, the fermentation time is 21d, and the microecological preparation is prepared.

Preparation of control microecologics: using B, C, D strains of the same type in the market as control strains, adopting the same method and conditions as the enterococcus faecalis BD-1 to carry out activation and preparation of fermentation seed liquid, and then carrying out fermentation for 21d by using the same fermentation substrate.

Respectively detecting 3 indexes of viable count of thalli, polyphenol content of free tea and mould pollution rate of the microecological preparation of the invention and a control finished product; and then the finished product of the microecological preparation is stored on a shelf for one year, and the survival rate of thalli, the polyphenol content of free tea and the mould pollution rate of the finished product are detected again by 3 indexes. The results are shown in Table 13.

As can be seen from table 13: compared with similar strains in other markets, the enterococcus faecalis BD-1 has high viable count, obviously increases the content of free tea polyphenol of finished products, prolongs the shelf life of the products, and can achieve the effect of preventing mould pollution.

Example 10 Effect of finished Microecological formulations on piglet growth Performance

The finished microecological preparation prepared by using enterococcus faecalis BD-1 in example 9 as a research object was tested in 2016 for 7 months and 1 day to 8 months and 1 day (summer), 60 healthy 30-day-old ternary miscellaneous weaned piglets (Du x Long x big) were selected as test animals and randomly divided into three groups, wherein each group was repeatedly divided into five groups, ① control group, namely basic diet group, ② basic diet + the finished microecological preparation of the present invention (added in an amount of 5% of the basic diet by mass ratio), ③ basic diet + antibiotics (mucobacill sulfate, purity of 10% and added in an amount of 0.05% of the basic diet by mass ratio).

The composition of the basal diet is shown in table 14.

The results are shown in Table 15.

As can be seen from table 15: compared with a control group and an antibiotic group, the finished product microecological preparation group has the advantages of obvious weight increment, increased feed utilization rate, reduced bacterial diarrhea and mortality and guarantee of healthy growth of piglets.

Example 11 Effect of finished Microecological formulations on the production Performance of egg-laying hens

In example 9, finished microecologics prepared from enterococcus faecalis BD-1 of the present invention were used as study subjects, and tested in 2016 years from 7 months 1 day to 8 months 1 day (summer), 300 healthy 28-week-old Hailan brown laying hens with similar laying rate and an average laying rate of 85% or more of the whole group and with uniform and healthy body weight were randomly divided into three groups, and 100 of each group were repeated, wherein ① control group, i.e., basal diet group, ② basal diet + the finished microecologics of the present invention (added in mass ratio of 3%) group, ③ basal diet + antibiotics (mucobacteriocin sulfate, purity of 10%, added in mass ratio of 0.05%) of basal diet, during the test period, the laying hens were raised in the hen house, the free food consumption was increased to > 30 ℃, the humidity of 60-90%, free-drip water was provided to the hen house for free water, the management was conducted in the house in accordance with the daily management manner, the daily morning, the daily free food consumption was calculated, the daily free-radical-free-feed consumption was calculated, and the total free-radical-free-enzyme-free-feed production rate (SOD) was calculated, and the total free-feed-free-feed-free-feed-.

The composition of the basal ration is shown in table 16.

The results are shown in Table 17.

As can be seen from table 17: compared with a control group and an antibiotic group, the finished product microecological preparation group has the advantages of obviously improving the anti-oxidation heat stress resistance of the laying hens, increasing the feed utilization rate, reducing bacterial diarrhea and the death rate, and ensuring the healthy production performance of the laying hens.

SEQUENCE LISTING

<110> Fujian Beidi pharmaceutical Co Ltd

<120> enterococcus faecalis for feed, microecological preparation for preventing and treating heat stress of livestock and poultry and preparation method thereof

<130>

<160>1

<170>PatentIn version 3.4

<210>1

<211>1461

<212>DNA

<213> unknown

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Claims

1. A feed enterococcus faecalis (A)Enterococcus faecalis) BD-1, characterized by: the preservation number is CCTCC NO: m2017416.

2. The method for preparing the microecological preparation for preventing and treating the heat stress of the livestock and poultry by using the enterococcus faecalis BD-1 for feeding according to claim 1, which is characterized by comprising the following steps of:

(1) activation of enterococcus faecalis BD-1:

washing slant strains of enterococcus faecalis BD-1 test tubes with sterile normal saline in a sterile environment, then inoculating the slant strains into an MRS liquid culture medium containing 0.2-0.5% of radix pseudostellariae fibrous extract, and statically culturing for 42-48 h at 35-37 ℃; wherein the percentage of the radix pseudostellariae fibrous extract is calculated by mass volume percentage g/mL;

(2) preparing a enterococcus faecalis BD-1 fermentation seed solution:

inoculating 3-5% of the strain activation liquid obtained in the step (1) in a volume percentage into an MRS liquid culture medium containing 0.2-0.5% of radix pseudostellariae fibrous extract under an aseptic environment, and performing static fermentation for 42-48 h at 35-37 ℃ to obtain enterococcus faecalis BD-1 fermented seed liquid; wherein the percentage of the radix pseudostellariae fibrous extract is calculated by mass volume percentage g/mL;

(3) preparing a fermentation substrate:

(4) and fermenting:

3. The method of claim 2, wherein: in the step (3), the crushing granularity is 60-80 meshes.

4. A microecological preparation for preventing and treating heat stress of livestock and poultry, which is prepared by the preparation method according to claim 2 or 3.