CN107254424B

CN107254424B - Novel liquid composite microecological preparation for livestock and poultry and preparation method thereof

Info

Publication number: CN107254424B
Application number: CN201710569185.1A
Authority: CN
Inventors: 潘慧青; 张炎达; 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: 2023-02-28
Anticipated expiration: 2037-07-11
Also published as: CN107254424A

Abstract

The invention belongs to the technical field of production and preparation of microecologics, and particularly relates to a novel liquid composite microecologics for livestock and poultry and a preparation method thereof. The method comprises the following steps: (1) activating two fermentation strains; (2) preparing a fermentation seed liquid; (3) mixing and co-fermenting the two strains; (4) mixing liquid and solid; and (5) bottling. The method provided by the invention can accelerate the liquid fermentation rate, is beneficial to the preparation of high-concentration bacterial liquid, and the obtained microecological preparation can effectively prevent and treat diseases caused by staphylococcus aureus, micrococcus luteus and escherichia coli, supplement intestinal probiotics of livestock and poultry, and assist in preventing and treating heat stress syndrome of livestock and poultry; similarly, the method provided by the invention can also effectively build a new liquid preparation by in vitro compounding with the Chinese herbal medicine extract, is beneficial to diversified operation and has simple process; finally, the method does not need strict sterilization operation, obviously reduces the working difficulty, reduces the energy waste and saves the cost.

Description

Novel liquid composite microecological preparation for livestock and poultry and preparation method thereof

Technical Field

The invention belongs to the technical field of production and preparation of microecologics, and particularly relates to a novel liquid composite microecologics for livestock and poultry and a preparation method thereof.

Background

China is a big agricultural country, the development of animal husbandry occupies an important proportion, and according to the recent reform of the supply side of China, the scale and the development of livestock in China will greatly drive the development of industry, so that the industrialization, the scale and the standardization development of the animal husbandry in China will be further promoted globally, and the development potential of the livestock and poultry breeding in China during the 'thirteen-five' period in the future is huge, the space is huge, and the significance is great. However, the phenomenon of antibiotic abuse in the breeding industry of China is very serious, in recent years, the management work of antibiotic abuse in the livestock and poultry breeding industry of China has been increased, and the national ministry of agriculture announces that part of antibiotics are forbidden in 2016 and 2017 respectively. Meanwhile, the demand for antibiotic-free culture is increasing at home and even globally, different antibiotic substitutes come true, scientists find that probiotics prepared by fermentation are the most potential type of probiotics, and solid and liquid probiotics are developed rapidly. Scientific researches prove that the microecological preparation can improve the drug effect, has no toxicity or residue, can actively supplement intestinal probiotics, and is beneficial to the repair and rebalancing of intestinal flora.

At present, the effect of the fermentation type microecological preparation products is better in the dosage forms of the microecological preparation, and the fermentation type microecological preparation can be divided into three types of liquid microecological preparations, semi-solid microecological preparations and solid microecological preparations according to the dosage forms. The liquid microecological preparation utilizes the synergistic effect among microorganisms, exerts the combined action advantage of the groups, well improves the stability of thalli, keeps the bioactivity, plays an important role in regulating the physiological activity of animals, has simple preparation process, effectively reduces the cost and can generate great economic benefit. The prior data can be integrated to conclude that the liquid microecological preparation can be used in four modes of drinking water, oral taking, mixing and spraying, but the liquid microecological preparation is slightly more used in drinking water, oral taking and mixing. However, the current liquid microecological product has an urgent problem to solve: (1) strict sterilization is required before liquid fermentation, and the operation is complex and time-consuming; (2) the bottle expansion phenomenon appears on the bottled liquid microecological preparation product part, and the shelf life of the product is shortened; (3) the liquid fermentation lacks excellent synergistic fermentation mixed strains; (4) the fermentation accelerators in liquid fermentation are few in types, and the fermentation accelerators with homology of medicine and food are lacked.

Disclosure of Invention

In order to overcome the problems existing in the preparation of liquid microecologics in the prior art, the invention aims to provide a novel liquid composite microecologics for livestock and poultry and a preparation method thereof.

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

a preparation method of a novel liquid composite microecological preparation for livestock and poultry is characterized by comprising the following steps:

(1) Activation of two fermentation strains:

enterococcus faecalis: (Enterococcus faecalis) Activation of BD-1: washing enterococcus faecalis BD-1 test tube slant strains with sterile normal saline in an aseptic environment, then inoculating the washed enterococcus faecalis BD-1 test tube slant strains into an MRS liquid culture medium containing 0.2 to 0.5 percent of radix pseudostellariae fibrous extract, and statically culturing for 42 to 48h at the temperature of 35 to 37 ℃; wherein the percentage of the radix pseudostellariae fibrous extract is measured by mass volume percentage g/mL (namely 100mL MRS liquid culture medium contains 0.2 to 0.5g of radix pseudostellariae fibrous extract); enterococcus faecalis BD-1 is a new strain that applicant has deposited in the chinese collection of type cultures at the address: wuhan city Wuchang Lojia mountain, preservation date: 7 months and 7 days in 2017; the preservation number is as follows: CCTCC NO: m2017416;

clostridium butyricum (C)Clostridium butyricum) Activation of RCM-2: washing a clostridium butyricum RCM-2 test tube slant strain with sterile normal saline in an aseptic environment, then inoculating the strain into an MRS liquid culture medium containing 0.2 to 0.5 percent of radix pseudostellariae fibrous extract, and statically culturing the strain for 42 to 48h at the temperature of 35 to 37 ℃; wherein the percentage of the radix pseudostellariae fibrous extract is calculated by mass volume percentage g/mL (namely, 100mL of MRS liquid culture medium contains 0.2 to 0.5g of the radix pseudostellariae fibrous extract); clostridium butyricum RCM-2 is a new strain that applicants have deposited in the chinese type culture collection, at address: wuhan city Wuchang Lojia mountain, preservation date: 2017, 6 and 29 months; the preservation number is: CCTCC NO M2017396;

(2) Respectively preparing fermented seed liquids of enterococcus faecalis BD-1 and clostridium butyricum RCM-2:

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

fermentation seed liquid of clostridium butyricum RCM-2: under an aseptic environment, inoculating the activated liquid of the clostridium butyricum RCM-2 obtained in the step (1) into an MRS liquid culture medium containing 0.2 to 0.5 mass percent of radix pseudostellariae fibrous extract according to the volume percent of 3~5%, and performing static fermentation for 42 to 48h at the temperature of 35 to 37 ℃; wherein the percentage of the radix pseudostellariae fibrous extract is measured by mass volume percentage g/mL (namely 100mL MRS liquid culture medium contains 0.2 to 0.5g of radix pseudostellariae fibrous extract);

(3) And mixing and co-fermenting the two strains:

firstly, injecting hot water with the temperature of 50 to 60 ℃ into a fermentation tank with an exhaust valve at the top, wherein the exhaust valve is in a completely closed state, then adding nutrient components, uniformly stirring, sealing the tank, adding tea polyphenol powder and allicin powder when the temperature of water in the fermentation tank is reduced to below 40 ℃, uniformly stirring, preserving the sealed tank for 30min to 1h, then adding a mixed seed liquid consisting of a fermentation seed liquid of enterococcus faecalis BD-1 and a fermentation seed liquid of clostridium butyricum RCM-2 according to the volume ratio of 1: 1~5: 1, sealing the tank by covering, performing static fermentation at the temperature of 32 to 37 ℃ for 6 to 8h, half opening the exhaust valve, then closing the exhaust valve when the fermentation is continued for 36 to 40h, and terminating the fermentation;

the nutritional ingredients comprise radix pseudostellariae fibrous extract, brown sugar, yeast extract, vitamin C, CMC and wort; the hot water-based mixed seed liquid comprises, by percentage, 0.2 to 0.5% of heterophylly falsestarwort root extract, 10 to 15% of brown sugar, 5~8% of yeast extract, 1~2% of vitamin C, 0.5 to 1.5% of CMC, 1~5% of wort, 0.1 to 0.25% of tea polyphenol powder, 0.01 to 0.025% of allicin powder and 5 to 10% of mixed seed liquid, wherein the percentages of the wort and the mixed seed liquid are volume ratio, and the rest is mass-volume ratio g/mL;

(4) And liquid-solid mixing:

the "liquid" refers to the fermentation liquid obtained in step (3), and the "solid" refers to the following solid (1) or solid (2):

the composition of the solid (1) was: the beverage comprises tea polyphenol powder, allicin powder and prebiotics, wherein the prebiotics are one or two of isomaltooligosaccharide and fructo-oligosaccharide, and comprise 0.25 to 1.5 percent of the tea polyphenol powder, 0.05 to 0.1 percent of the allicin powder and 1~5 percent of the prebiotics in percentage by mass of the total mass of the liquid-solid mixture;

the composition of the solid (2) is: tea polyphenol powder, garlicin powder and Chinese herbal medicine extract capable of improving immunity of livestock and poultry; 0.1 to 0.8 percent of tea polyphenol powder, 0.02 to 0.05 percent of allicin powder and 10 to 20 percent of Chinese herbal medicine extract by mass percentage of the total mass of the liquid-solid mixture;

(5) And (5) after the step (4) is finished, sealing the tank body again and preserving for 1 to 3 hours to obtain the novel liquid compound microecological preparation for the livestock and the poultry.

Preferably, the Chinese herbal medicine extract is prepared by performing alcohol extraction or/and water extraction on the following raw material medicines in parts by mass: 20 to 60 parts of radix pseudostellariae fibrous root, 10 to 30 parts of dried orange peel, 10 to 30 parts of hawthorn and 10 to 30 parts of medicated leaven.

Further, the extraction steps of the Chinese herbal medicine extract are as follows:

(i) Weighing the raw materials according to the formula, adding 75% ethanol, performing reflux extraction on the raw materials for 1~3 hours, cooling, separating residues and liquid to obtain residues and ethanol extract, adding water into the residues, decocting and extracting the residues for 1~2 times, and combining the ethanol extract and the water extract;

(ii) And (e) concentrating the combined extracting solution in the step (i) under reduced pressure to obtain thick paste with the relative density of 1.2 to 1.35 at the temperature of 28 ℃ so as to obtain the Chinese herbal medicine extract.

The novel liquid composite microecological preparation for livestock and poultry, which is prepared by the preparation method, is used for treating livestock and poultry.

In the present invention, the fibrous root extract of radix pseudostellariae can be obtained according to the prior art, such as but not limited to the method disclosed in the patent entitled "a method for preparing fibrous root extract of radix pseudostellariae" (application No. 201610758305.8) filed on 30/8/2016 by the applicant.

Has the beneficial effects that:

1. in the invention, enterococcus faecalis BD-1 is separated from village peasant households (coordinate: 119) in Cudrania tricuspidata county ^。 51′59.7〞E，27 ^。 13 '34.0N') autotrophic healthy adult pig cecal mucosa, the obtained enterococcus faecalis BD-1 is safe and nontoxic, the surface of a bacterial colony is smooth and convex after being cultured in an MRS culture medium at 37 ℃ for 48 hours, the periphery is neat, the color of the bacterial colony is off-white, the diameter of the bacterial colony is 0.5 to 1.2mm, G + is examined under a microscope, no spore exists, and the length of the bacterial body is about 0.5 to 1 mu m; the clostridium butyricum RCM-2 of the invention is separated from village farmers (coordinate: 119) in Cudrania tricuspidata county ^。 52′04.1〞E，27 ^。 13' (32.7 "" N) obtained from the cecal mucosa section of healthy adult roosters raised in the circled mountain forest landThe clostridium acidocaldarium RCM-2 is safe and nontoxic, the surface of a bacterial colony cultured in an RCM culture medium at 37 ℃ for 48 hours is smooth and convex, the periphery is neat, the color of the bacterial colony is milky yellow and opaque, the bacterial colony has slight acid odor, the diameter of the bacterial colony is 0.5 to 1.5cm, the bacterial colony is subjected to microscopic examination, G & lt + & gt has spores, a spore oval, and terminal or secondary spores, the thallus of the raw spores is fusiform, and the length of the thallus is about 3.5 to 7.0 mu m;

2. the fermentation strains enterococcus faecalis BD-1 and Clostridium butyricum RCM-2 are new strains with excellent tea polyphenol and allicin resistance, and in addition, the fermentation strains have better synergistic fermentation capacity, and the radix pseudostellariae fibrous extract provides a novel medicine-food homology fermentation promoter, so that the synergistic fermentation rate can be remarkably promoted, and the fermentation time can be saved; meanwhile, the fermentation strains enterococcus faecalis BD-1 and Clostridium butyricum RCM-2 can effectively prevent and treat diseases caused by staphylococcus aureus, micrococcus luteus and Escherichia coli, can supplement intestinal probiotics of livestock and poultry, are beneficial to repairing damaged intestinal micro-ecological environment, and can assist in preventing and treating heat stress syndrome of livestock and poultry;

3. the first stage of fermentation of the preparation method adopts normal hot water for normal temperature reduction, and the tea polyphenol powder with the percentage of 0.1 to 0.25 percent and the allicin powder with the percentage of 0.01 to 0.025 percent are added for inhibiting the growth of infectious microbes, rather than selecting the traditional high-temperature high-pressure sterilization (121 ℃ C., 20 to 30min), the strict sterilization operation is not needed, the working difficulty is obviously reduced, the energy consumption is saved, the time is saved, and the operation difficulty is simplified. Meanwhile, tea polyphenol and allicin are added again after fermentation, the stability of the tea polyphenol and the allicin can be remarkably promoted in an acidic fermentation liquid environment, the effective period of the tea polyphenol and the allicin can be prolonged, the bacteriostatic ability of the fermentation liquid can be synergistically enhanced, and the bottle expansion phenomenon of the liquid microecologics in the shelf life can be reduced; in addition, the tea polyphenol and the garlicin are also widely used as antioxidants and phagostimulants in livestock and poultry breeding, and can assist in preventing and treating the heat stress syndrome of the livestock and poultry; similarly, the method provided by the invention can also effectively build a new liquid microecological preparation by in vitro compounding with the Chinese herbal medicine extract, is beneficial to diversified operation and has simple process.

Drawings

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

FIG. 2: a phylogenetic tree of Clostridium butyricum RCM-2.

Detailed Description

In the following examples, the radix pseudostellariae extract was prepared according to the method disclosed in example 1 of patent application No. 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 phosphate, 0.2g of magnesium sulfate, 2g of triammonium citrate, 0.05g of manganese sulfate, 80 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 phosphate, 0.2g of magnesium sulfate, 2g of triammonium citrate, 0.05g of manganese sulfate, 80 mL of tween, 15g of agar, 1L of purified water, and the pH value of the mixture is 6.2 +/-0.2;

RCM agar plate or RCM slant medium (1L): 3g of yeast extract, 10g of beef extract, 10g of tryptone, 5g of glucose, 1g of soluble starch, 5g of sodium chloride, 3g of sodium acetate trihydrate, 0.15g of cysteine hydrochloride, 15g of agar, 1L of purified water and 6.8 +/-0.2 of pH value, and sterilizing for later use;

starch medium (1L): 5g of beef extract, 5g of peptone, 0.5g of sodium chloride, 20g of soluble starch, 18g of agar, 1L of purified water, pH =7.2 +/-0.1, and sterilizing for later use;

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/RCM 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/RCM 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) autotrophic healthy adult porcine cecal mucosal segment, scraping mucosal mucus from EP tube with sterilized glass slide, diluting 10 with sterile physiological saline ² Taking 0.1mL of the bacterial colony, coating the bacterial colony 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 a bacterial colony with a yellowing culture medium, streaking and purifying, selecting bacterial strains with gram-positive staining and no spores through microscopic examination, and rescreening the bacterial colony on the MRS agar plate containing bromocresol purple (0.01 percent, g/mL), tea polyphenol (0.01 percent, g/mL) and allicin (0.01 percent, g/mL), finally selecting the bacterial colony with obvious yellow color, smooth and convex bacterial colony surface, taking the bacterial colony as an object bacterial strain with regular periphery, naming the bacterial strain as BD-1, and performing slant MRS preservation on the bacterial colony by using a slant culture medium.

2. Morphological observations

The morphological characteristics of the colony of the strain BD-1 are observed, and the following results are obtained: the surface of a bacterial colony on the MRS agar plate is smooth and convex, the periphery is neat, the bacterial colony is gray, the diameter of the bacterial colony is 0.5 to 1.2mm, the bacterial colony is G + examined by a microscope, no spore exists, and the length of the bacterial colony is about 0.5 to 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.

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 total DNA of the strain BD-1 by using a bacterial genome DNA extraction kit (DP 302-02, TIANGEN), and amplifying a 16S rDNA sequence of the strain by PCR under the guidance of primers P0 (5'-AGAGTTTGATCCTGGCTCAG-3') and 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 (10 mmol/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, and total 50 μ L. The PCR reaction conditions are as follows: firstly, 94 ℃ for 5min; then, 30 cycles of 94 ℃ for 1min,55 ℃ for 1min and 72 ℃ for 1min are performed; 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 band of interest was recovered and purified using an agarose gel recovery kit from Axygen, and sent to Biotechnology, inc. (Shanghai) for DNA sequencing, as shown in SEQ ID No.1.

The determined sequence was compared with the 16S rDNA sequence in Genbank for Blastn similarity analysis, resulting in the strains BD-1 andEnterococcus faecium strain KCI1、Enterococcus lactisthe 16S rDNA sequence of strain K12419C is 100% homologous, therefore 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。

5. Influence of tea polyphenols on growth and pH of enterococcus faecalis BD-1

(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) 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 to be (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 point of the culture are shown in Table 2.

From the results of table 2, it can be seen that: the concentration of the tolerant tea polyphenol of the enterococcus faecalis BD-1 is 1.0%, and the survival rate of thalli at the concentration is 70.26%.

6. Effect of allicin on growth and pH of enterococcus faecalis BD-1

(1) Activation of enterococcus faecalis BD-1: the same procedure as in step (1) of item 5 of this example;

(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 3;

from the results in Table 3, 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 the enterococcus faecalis BD-1 (counted as A) obtained in the step (1) and the activated liquid of the control bacterium B, C, D 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 (1) tea polyphenol in the MRS liquid culture medium in the anaerobic serum bottle is sequentially set as (g/mL): 0%, 1.00%, 2.00%, (2) gradient of allicin set to (g/mL): 0%, 0.05%, 0.1%, continuously culturing for 48h, and using 0% +0% as control (viable count is defined as X) ₀ ) By the formula: cell loss rate = (X) ₀ -X）/ X ₀ * The loss rate of the enterococcus faecalis cells was calculated at 100%, and the results are shown in Table 4.

From the results in Table 4, 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.

7. Enterococcus faecalis bacteriostasis experiment

(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 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 point of the culture, detecting the antibacterial effect on staphylococcus aureus, micrococcus luteus and Escherichia coli by 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 5.

From the results in Table 5, 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 2 isolation and identification of Clostridium butyricum RCM-2

1. Separation, enrichment and purification of clostridium butyricum RCM-2

Get fortune under aseptic operation conditionJian province Ning De city, cudrania tricuspidata county village peasant household (coordinate: 119) ^。 52′04.1〞E，27 ^。 13'32.7 "" N) mucosal sections of the cecum of healthy adult roosters bred from circinate arena, mucosal mucus was scraped from EP tubes with sterilized slides, diluted 10 with sterile physiological saline ² Heating to 60 ℃ for 30min, taking 0.1mL of the bacterial colony coated with a bromocresol purple indicator (0.01 percent, g/mL) on an RCM agar plate, placing the RCM agar plate in an anaerobic incubator after coating, culturing for 48h at 37 ℃, selecting a bacterial colony with a yellowing culture medium, streaking and purifying, selecting a rod-shaped bacterial strain with gram-positive staining and spores for microscopic examination, re-screening on the RCM agar plate containing bromocresol purple (0.01 percent, g/mL), tea polyphenol (0.01 percent, g/mL) and garlicin (0.01 percent, g/mL), finally selecting a bacterial colony with obvious yellow color, smooth and convex surface, milky yellow and non-transparent and acid odor as a preparation bacterial strain, then coating the preparation bacterial strain on a starch culture medium, culturing for 48h at 37 ℃, selecting a strain with the largest transparent circle as a target bacterial strain, using the bacterial strain RCM-2 as a slant culture medium, and preserving the bacterial strain by using the RCM slant culture medium.

2. Morphological observation

The colony morphology characteristics of the strain RCM-2 are observed, and the following can be known: the surface of a colony on the RCM agar plate is smooth and convex, the colony is milky yellow and opaque and has slight acid odor, the diameter of the colony is 0.5 to 1.5cm, the colony is G +, spores, spore oval, spore end-growth or sub-end-growth, the thallus of the spore is fusiform, and the length of the thallus is about 3.5 to 7.0 mu m.

3. Physiological and biochemical characteristics

The bacterial strains are subjected to physiological and biochemical identification according to Bergey's Manual of bacteria identification, and physiological and biochemical tests are respectively carried out by catalase, amylase, nitrate reduction, sugar and the like, and the results are shown in Table 6.

The strain RCM-2 is compared with the physiological and biochemical characteristics of clostridium butyricum in Bergey's Manual of identification of bacteria and the manual of identification of common bacteria systems, and the result shows that the strain RCM-2 basically accords with the physiological and biochemical characteristics of clostridium butyricum, so that the strain RCM-2 is preliminarily identified to be the clostridium butyricum.

4. Sequencing and evolutionary trees

Extracting total DNA of the strain RCM-2 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 RCM-2 as a template, wherein the PCR reaction system is as follows: 10 XBuffer 5.0 μ L, dNTP (10 mmol/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, and total 50 μ L. The PCR reaction conditions were: firstly, 94 ℃ for 5min; then, 30 cycles of 94 ℃ for 1min,55 ℃ for 1min and 72 ℃ for 1min are performed; 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.2.

The measured sequence was compared with the 16S rDNA sequence in Genbank by Blastn similarity analysis, and as a result, the strain RCM-2 had 98% homology with the 16S rDNA sequence of Clostridium butyricum strain 1005-15098, so that RCM-2 was identified as Clostridium butyricum, and its phylogenetic tree is shown in FIG. 2.

The strain RCM-2 is identified and classified as clostridium butyricum by combining the morphological, physiological and biochemical characteristics and sequencing and evolutionary tree resultsClostridium butyricum）RCM-2。

5. Influence of tea polyphenols on growth and pH of Clostridium butyricum RCM-2

(1) Activation of Clostridium butyricum RCM-2: washing the clostridium butyricum RCM-2 test tube slant strain with sterile normal saline in a sterile environment, washing down the lawn on the surface of a culture medium, then inoculating the lawn into an MRS liquid culture medium, and statically culturing for 48 hours at 37 ℃;

(2) Inoculating the activated liquid of clostridium butyricum RCM-2 obtained in the step (1) into an anaerobic serum bottle containing MRS liquid culture medium in an anaerobic box according to the inoculation amount of 3 percent (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 point of the culture are shown in Table 7.

From the results of Table 7, it can be seen that: the concentration of tea polyphenol which can be tolerated by clostridium butyricum RCM-2 is 1.0%, and the survival rate of thalli at the concentration is 87.3%.

6. Effect of allicin on growth and pH of Clostridium butyricum RCM-2

(1) Activation of Clostridium butyricum RCM-2: the same procedure as in step (1) of item 5 of this example;

(2) Inoculating the activated liquid of clostridium butyricum RCM-2 obtained in the step (1) into an anaerobic serum bottle containing 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 8;

from the results in Table 8, it can be seen that: the concentration of the clostridium butyricum RCM-2 which can tolerate allicin is 0.1 percent, and the survival rate of thalli at the concentration is 85.5 percent;

(3) Clostridium butyricum tolerates tea polyphenols and allicin growth comparisons:

inoculating the activated liquid of clostridium butyricum RCM-2 (counted as A) obtained in the step (1) into an anaerobic serum bottle containing MRS liquid culture medium in an anaerobic box according to the inoculation amount of 3 percent (volume ratio), wherein the gradient of (1) tea polyphenol in the MRS liquid culture medium in the anaerobic serum bottle is sequentially set as (g/mL): 0%, 1.00%, 2.00%, (2) gradient of allicin set to (g/mL): 0%, 0.05%, 0.1%, continuously culturing for 48h, using 0% +0% as control (alive)The number of bacteria is defined as X ₀ ) By the formula: cell loss rate = (X) ₀ -X）/ X ₀ * The loss rate of Clostridium butyricum cells was calculated at 100%, and the results are shown in Table 9.

From the results of Table 9, it can be seen that: under the combined action of tea polyphenol (1%) and allicin (0.05%), the survival rate of the clostridium butyricum RCM-2 can reach 88.5%, is over 50%, and is far higher than that of the similar strains in the market.

7. Clostridium butyricum bacteriostatic test

(1) Activation of Clostridium butyricum RCM-2: the same as step (1) in item 5 of this embodiment;

(2) Inoculating the activated liquid of the clostridium butyricum RCM-2 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 for 48h at 37 ℃, taking a fermentation liquid at the end of fermentation, 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 reference; the diameter of the zone is shown in Table 10.

From the results in Table 10, it can be seen that: the clostridium butyricum RCM-2 can obviously inhibit the growth of pathogenic bacteria staphylococcus aureus, micrococcus luteus and escherichia coli.

Example 3 symbiotic growth of enterococcus faecalis BD-1 and Clostridium butyricum RCM-2

(1) Activation of the Strain

Activation of enterococcus faecalis BD-1: washing test tube slant strain of enterococcus faecalis BD-1 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;

activation of Clostridium butyricum RCM-2: washing test tube slant strain of Clostridium butyricum RCM-2 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) Then inoculating the activated liquid of enterococcus faecalis BD-1, the activated liquid of Clostridium butyricum RCM-2 and the activated liquid compounded by the two according to the volume ratio of 1:1 into a 200mL anaerobic serum bottle of an MRS liquid culture medium respectively 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 anaerobic serum bottle are sequentially set as (mass ratio): 0 percent and 0.25 percent, static culture is carried out for 48 hours at 37 ℃, the viable count of the fermentation liquor is measured when the culture is finished, and in addition, the spore rate of the clostridium butyricum RCM-2 is measured; the results are shown in Table 11.

As can be seen from Table 11: the radix pseudostellariae fibrous extract can remarkably promote the symbiotic growth of enterococcus faecalis BD-1 and clostridium butyricum RCM-2, and the symbiotic promotion degree of the two strains is larger than the sum of the number of the two single strains under the same concentration; in addition, the spore rate of RCM-2 can be promoted at a concentration of 0.25%, and the spore rate of symbiotic culture is further improved.

Example 4 preparation of a Microecological preparation

The preparation steps are as follows:

(1) Activation of two fermentation strains:

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; wherein the percentage of the radix pseudostellariae fibrous extract is calculated by mass volume percentage g/mL;

washing slant strain of Clostridium butyricum RCM-2 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; wherein the percentage of the radix pseudostellariae fibrous extract is calculated by mass volume percentage g/mL;

(2) Respectively preparing fermentation seed liquid of enterococcus faecalis BD-1 and clostridium butyricum RCM-2:

fermentation seed liquid of enterococcus faecalis BD-1: under an aseptic environment, inoculating the activated liquid of enterococcus faecalis BD-1 obtained in the step (1) into a 1L anaerobic serum bottle filled with MRS liquid culture medium containing 0.25% of radix pseudostellariae fibrous extract according to the volume percentage of 3%, and performing static fermentation at 37 ℃ for 36h to obtain a fermented seed liquid of enterococcus faecalis BD-1; wherein the percentage of the radix pseudostellariae fibrous extract is calculated by mass volume percentage g/mL;

fermentation seed liquid of clostridium butyricum RCM-2: inoculating 3 percent by volume of the clostridium butyricum RCM-2 activation solution obtained in the step (1) into a 1L anaerobic serum bottle filled with MRS liquid culture medium containing 0.25 percent of radix pseudostellariae fibrous extract under the aseptic environment, and performing static fermentation for 36h at 37 ℃ to obtain clostridium butyricum RCM-2 fermentation seed solution; wherein the percentage of the radix pseudostellariae fibrous extract is calculated by mass volume percentage g/mL;

(3) And mixing and co-fermenting the two strains:

firstly, injecting hot water with the temperature of 55 ℃ into a fermentation tank with an exhaust valve at the top, wherein the exhaust valve is in a completely closed state, then adding nutrient components, stirring for 5min at 90rpm, sealing the tank, adding tea polyphenol powder and allicin powder when the water temperature in the fermentation tank is reduced to 37 ℃, uniformly stirring, preserving in the sealed tank for 45min, then adding a mixed seed solution consisting of a fermentation seed solution of enterococcus faecalis BD-1 and a fermentation seed solution of clostridium butyricum RCM-2 according to the volume ratio of 1:1, sealing by a cover, carrying out static fermentation at 37 ℃ for 6h, semi-opening the exhaust valve, then closing the exhaust valve when continuing to ferment for 38h, and terminating the fermentation;

the nutritional ingredients comprise radix pseudostellariae fibrous extract, brown sugar, yeast extract, vitamin C, CMC and wort; the hot water-soluble health-care beverage comprises, by weight, 0.25% of radix pseudostellariae fibrous extract, 15% of brown sugar, 8% of yeast extract, 2% of vitamin C, 1.0% of CMC, 3% of wort, 0.15% of tea polyphenol powder, 0.02% of garlicin powder and 7% of mixed seed liquid, wherein the wort and the mixed seed liquid are in percentage by volume, and the balance is in mass-to-volume ratio of g/mL;

(4) Mixing liquid and solid, uniformly mixing and stirring at the rotating speed of 100rpm for 20min:

the liquid refers to the fermentation liquid obtained in the step (3), and the solid refers to tea polyphenol powder, allicin powder and prebiotics, wherein the prebiotics are isomaltooligosaccharide, and comprise 0.5% of tea polyphenol powder, 0.1% of allicin powder and 3% of prebiotics in percentage by mass of the total mass of the liquid-solid mixture;

(5) And (4) after the step (4) is finished, sealing the tank body again and preserving for 2 hours to obtain the novel liquid compound microecological preparation for the livestock and the poultry, wherein the novel liquid compound microecological preparation can be weighed and bottled for subpackaging, the bottle adopts a 250mL brown plastic bottle, and the liquid volume in each bottle is 200mL.

Respectively taking 10 bottles, equivalently dividing into 2 groups A and B, and respectively detecting the rate of mixed bacteria and the diameter of an inhibition zone of pathogenic bacteria (staphylococcus aureus, micrococcus luteus and escherichia coli) by opening a cover and sampling in the field of the group A; and the group B is stored in an air-conditioning temperature-control room at the temperature of 35 ℃ for 6 months, and then the infectious microbe rate, the diameter of the inhibition zone of pathogenic bacteria (staphylococcus aureus, micrococcus luteus and escherichia coli) and the bottle expansion rate are respectively detected. The results are shown in Table 12.

As can be seen from table 12: the liquid microecologics prepared by the embodiment has no bottle expansion phenomenon, and the shelf life of the liquid microecologics is prolonged.

The product prepared in the embodiment is used as an experimental medicament, a nutrient liquid culture medium is activated by staphylococcus aureus, micrococcus luteus and escherichia coli = 1:1 respectively (three pathogenic bacteria of staphylococcus aureus, micrococcus luteus and escherichia coli are respectively and independently subjected to nutrient liquid culture medium activation, the activated bacterial liquid is added into a triangular medicine bottle containing 100mL of nutrient liquid culture medium according to the volume ratio of 3% to be cultured in a medicine bottle at 100rpm for 24h to obtain bacterial liquid, and finally the pathogenic bacteria compound liquid is prepared according to the volume ratio of 1: 1), wherein 1L of the nutrient liquid culture medium comprises 10g of peptone, 3g of beef extract powder, 5g of sodium chloride and final pH of 7.3 +/-0.2) and is continuously poured into healthy chickens for 2d to cause diseases caused by artificial infection of the test chickens to construct model animals, wherein the total number of the model chickens is 200, the model chickens are divided into two groups, one group is treated by using the product of the embodiment, and the other group is not treated (a control group). The application of the product of the embodiment adopts feeding mode for treatment, three times a day, 2 mL/patient each time, free drinking, feeding and moving, and 3 days of treatment. After three days, the cure rate (cure means that the clinical symptoms of the chickens completely disappear and the mental status completely returns to normal; cure rate is the percentage of the number of cured chickens in the total number of the group of chickens), the death rate (death rate is the percentage of the number of dead chickens in the total number of the group of chickens), the condition of fresh excrements of the chickens are checked, and the results are shown in the following table 13.

EXAMPLE 5 preparation of a Microecological formulation

The difference from example 4 is that: in the step (4), the solid refers to tea polyphenol powder, allicin powder and Chinese herbal medicine extract capable of improving the immunity of livestock and poultry, and the tea polyphenol powder accounts for 0.20 percent, the allicin powder accounts for 0.04 percent and the Chinese herbal medicine extract accounts for 15 percent of the total mass of the liquid-solid mixture; the Chinese herbal medicine extract is extracted from the following raw material medicines in proportion: 40 parts of radix pseudostellariae fibrous roots, 20 parts of dried orange peels, 20 parts of hawthorns and 20 parts of medicated leavens; the extraction steps are as follows:

(i) Weighing the raw materials according to a formula, adding 75% ethanol, performing reflux extraction for 2 hours, cooling, separating residue and liquid to obtain residue and ethanol extract, decocting the residue in water for 2 times, and mixing the ethanol extract and the water extract;

(ii) And (ii) concentrating the combined extracting solution obtained in the step (i) under reduced pressure to obtain thick paste with the relative density of 1.2 at 28 ℃ so as to obtain the Chinese herbal medicine extract.

Respectively taking 10 bottles, equivalently dividing into 2 groups A and B, and respectively detecting the rate of mixed bacteria and the diameter of an inhibition zone of pathogenic bacteria (staphylococcus aureus, micrococcus luteus and escherichia coli) by opening a cover and sampling in the field of the group A; and the group B is stored in an air-conditioning temperature-control room at the temperature of 35 ℃ for 6 months, and then the infectious microbe rate, the diameter of the inhibition zone of pathogenic bacteria (staphylococcus aureus, micrococcus luteus and escherichia coli) and the bottle expansion rate are respectively detected. The results are shown in Table 14.

As can be seen from table 14: the liquid microecologics prepared by the embodiment has no bottle expansion phenomenon, and the shelf life of the liquid microecologics is prolonged.

The product prepared in the embodiment is taken as an experimental medicament, and a healthy chicken model building animal is continuously fed with a staphylococcus aureus, micrococcus luteus and escherichia coli = 1:1 pathogenic bacteria compound liquid (the method is the same as the embodiment) for 2d so that the experimental chicken is infected by human diseases to build the model animal, wherein the total number of the model chicken is 200, the model chicken is divided into two groups, one group is treated by the product of the embodiment, and the other group is controlled by Chinese herbal medicine extract. The product and the Chinese herbal medicine extract of the embodiment are used for treatment by feeding, the dosage of the product and the Chinese herbal medicine extract is respectively 2 mL/piece and 0.3 g/piece three times a day, the drinking, the ingestion and the movement are free, and the treatment lasts for 7 days. After 3 days of the test, the cure rate (cure means that the clinical symptoms of the chickens are completely disappeared and the mental status is completely recovered, the cure rate is the percentage of the cured chickens in the total number of the group of chickens), the death rate (the death rate is the percentage of the dead chickens in the total number of the group of chickens) and the condition of fresh excrements of the chickens are checked. The weight of each surviving test chicken on day 1 and day 7 was also recorded and the average gain in weight (g/chicken) was calculated from the average weights of the chickens on day 1 and day 7, and the results are shown in Table 15 below.

SEQUENCE LISTING

<110> Fujian Beidi pharmaceutical Co Ltd

<120> novel liquid composite microecological preparation for livestock and poultry and preparation method thereof

<130>

<160> 2

<170> PatentIn version 3.4

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Claims

1. A preparation method of a novel liquid composite microecological preparation for livestock and poultry is characterized by comprising the following steps:

(1) Activation of two fermentation strains:

enterococcus faecalis: (Enterococcus faecalis) Activation of BD-1: washing enterococcus faecalis BD-1 test tube slant strains with sterile normal saline in an aseptic environment, then inoculating the washed enterococcus faecalis BD-1 test tube slant strains into an MRS liquid culture medium containing 0.2 to 0.5 percent of radix pseudostellariae fibrous extract, and statically culturing for 42 to 48h at the temperature of 35 to 37 ℃; wherein the percentage of the radix pseudostellariae fibrous extract is calculated by mass volume percentage g/mL; enterococcus faecalis BD-1 is a new strain with a preservation number of CCTCC NO: m2017416;

clostridium butyricum (C)Clostridium butyricum) Activation of RCM-2: butyric acidThe strain of the test tube slant of the clostridium RCM-2 is washed by sterile normal saline under the sterile environment, and then is inoculated into an MRS liquid culture medium containing 0.2 to 0.5 percent of radix pseudostellariae fibrous extract, and is statically cultured for 42 to 48h at the temperature of 35 to 37 ℃; wherein the percentage of the radix pseudostellariae fibrous extract is calculated by mass volume percentage g/mL; the clostridium butyricum RCM-2 is a new strain with the preservation number of CCTCC NO M2017396;

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

fermentation seed liquid of clostridium butyricum RCM-2: under an aseptic environment, inoculating the activated liquid of the clostridium butyricum RCM-2 obtained in the step (1) into an MRS liquid culture medium containing 0.2 to 0.5 mass percent of radix pseudostellariae fibrous extract according to the volume percent of 3~5%, and performing static fermentation for 42 to 48h at the temperature of 35 to 37 ℃; wherein the percentage of the radix pseudostellariae fibrous extract is calculated by mass volume percentage g/mL;

(3) And mixing and co-fermenting the two strains:

(4) And liquid-solid mixing:

2. The method of claim 1, wherein: the Chinese herbal medicine extract is prepared by extracting the following raw material medicines in parts by mass: 20 to 60 parts of radix pseudostellariae fibrous root, 10 to 30 parts of dried orange peel, 10 to 30 parts of hawthorn and 10 to 30 parts of medicated leaven;

the extraction steps of the Chinese herbal medicine extract are as follows:

3. A novel liquid complex microecological preparation for livestock and poultry prepared by the preparation method according to claim 1 or 2.