CN107312730A - A kind of compound Chinese herb probiotics and its Quick production method based on solid state fermentation - Google Patents

A kind of compound Chinese herb probiotics and its Quick production method based on solid state fermentation Download PDF

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CN107312730A
CN107312730A CN201710559527.1A CN201710559527A CN107312730A CN 107312730 A CN107312730 A CN 107312730A CN 201710559527 A CN201710559527 A CN 201710559527A CN 107312730 A CN107312730 A CN 107312730A
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radix pseudostellariae
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张炎达
潘慧青
肖建设
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Fujian Brady Pharmaceutical Co ltd
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Abstract

The invention belongs to probiotics technology for producing field, a kind of compound Chinese herb probiotics and its Quick production method based on solid state fermentation are specifically disclosed.Including step：（1）Four kinds of fermentation strain activation；（2）Enterococcus faecalis BD 1 and clostridium butyricum RCM 2 mixes seed liquor A and prepared；（3）It is prepared by saccharomyces cerevisiae seed liquor B；（4）It is prepared by Young clostridium seed liquor C；（5）A, B, C proportioning prepare fermentation seed liquid；（6）Fermentation seed liquid is mixed with fermentation substrate matrix；（7）Fermentation.The method that the present invention is provided can substantially speed up the fermentation rate of solid matrix, it is easy to which the inner bag of fermented product is moulding, reduce the fermenting and producing time, beneficial to mass production, and mould contamination probability can be reduced, suitable for promoting the use of for packed solid fermentation operation.

Description

A kind of compound Chinese herb probiotics and its quick production based on solid state fermentation Method
Technical field
The invention belongs to probiotics technology for producing field, and in particular to a kind of compound Chinese herb probiotics And its Quick production method based on solid state fermentation.
Background technology
From last century, China's farming industry is developed rapidly, and it is first that the main livestock products total value of China occupy the world Position, and China's meat products total output is more than 100,000,000 tons, about 35,000,000 tons of birds, beasts and eggs amount, continues to hold a post or title the first place of world's total amount.Separately Outside, according to country, supply side is reformed in recent years, and national herding scale and its development will greatly drive the industry development, therefore, global From the point of view of, industrialization, scale, the development of standardization of China's animal husbandry will be promoted further, and China's livestock and poultry breeding industry will be in future Development potentiality it is huge, space is huge, significant.However, to there is abuse of antibiotics phenomenon extremely serious for China's aquaculture Phenomenon, in recent years country increased the management work of domestic livestock and poultry breeding industry abuse of antibiotics, Minister Agriculture of China is respectively at 2016 Year and 2017 put into effect the bulletin of part antibiotic disabling.Meanwhile, the cry that the domestic or even whole world is cultivated to nonreactive is increasing, Different antibiotic substitutes arise at the historic moment, and what current scientist had found probiotics, Chinese herbal medicine, antibacterial peptide etc. replaces anti-potentiality future Very big, compatibility product-probiotics of especially probiotics and Chinese herbal medicine is more quickly grown.Present scientific research is proved Probiotics can not only improve drug effect, nontoxic residue-free, moreover it is possible to which actively supplement beneficial bacteria of intestinal tract is beneficial to the reparation of gut flora And releveling, the collaboration of both pharmacology interworkings.
Currently, it is better with fermented type probiotics product in probiotics formulation, it is domestic increasing dynamic Protect enterprise to start to increase the production and selling of such preparation, but but govern entering for the industry always in the presence of two class general issues One step develops, and one is that probiotics fermenting speed needs longer time, document report up to now or enterprise practical life The fermentation time of solid probiotics is generally 2 weeks to 2 months from the point of view of production situation, and fermentation time length not only reduces goods Product shipment speed also increases cost, and can also increase the contamination probability of extraneous contamination bacterium with time growth；Two be Tiny ecosystem system Agent during the fermentation with there is a situation where living contaminants in shelf life, not only increase management work cost also significantly affect product Quality.
The content of the invention
Lack the tea leaf fermentation probiotics strain for being resistant to Tea Polyphenols in the prior art to overcome and be difficult to prepare efficient hair The short problem of ferment type tealeaves probiotics, probiotics finished product shelf life, it is an object of the invention to provide a kind of compound Chinese herbal microecological preparation and its Quick production method based on solid state fermentation.
To achieve the above object, the technical scheme that the present invention takes is as follows：
A kind of Quick production method of the compound Chinese herb probiotics based on solid state fermentation, step is as follows：
（1）, four kinds of fermentation strains activation：
Enterococcus faecalis（Enterococcus faecalis）BD-1 activation：By enterococcus faecalis BD-1 test tube slants strain in nothing Rinsed under collarium border with sterile saline, be then seeded into the MRS fluid nutrient mediums of the palpus extract 0.2 ~ 0.5% containing radix pseudostellariae In, 35 ~ 37 DEG C of 42 ~ 48h of static gas wave refrigerator；Wherein, the percentage of the radix pseudostellariae palpus extract is with quality percent by volume g/mL Meter（I.e. 100mL MRS fluid nutrient mediums must 0.2 ~ 0.5g of extract containing radix pseudostellariae）；Enterococcus faecalis BD-1 is new strains, applicant The bacterial strain is preserved in China typical culture collection center, address：Wuchang, wuhan Luo Jia Shan, preservation date：2017 7 The moon 7；Deposit number：CCTCC NO：M2017416；
Clostridium butyricum（Clostridium butyricum）RCM-2 activation：Clostridium butyricum RCM-2 test tube slants strain is existed Rinsed under gnotobasis with sterile saline, be then seeded into the MRS Liquid Cultures of the palpus extract 0.2 ~ 0.5% containing radix pseudostellariae In base, 35 ~ 37 DEG C of 42 ~ 48h of static gas wave refrigerator；Wherein, the percentage of the radix pseudostellariae palpus extract is with quality percent by volume g/ ML is counted（I.e. 100mL MRS fluid nutrient mediums must 0.2 ~ 0.5g of extract containing radix pseudostellariae）；Clostridium butyricum RCM-2 is new strains, Shen Ask someone that the bacterial strain is preserved in into China typical culture collection center, address：Wuchang, wuhan Luo Jia Shan, preservation date： On June 29th, 2017；Deposit number：CCTCC NO M2017396；
Saccharomyces cerevisiae（Saccharomyces cerevisiae）Activation：Commercialization edible saccharomyces cerevisiae powder is inoculated into liquid In body culture medium, 30 ~ 37 DEG C of 24 ~ 32h of static gas wave refrigerator；Viable count >=1.0 × 10 of saccharomyces cerevisiae powder8CFU/g；Fluid nutrient medium Constitute and be：Brewer's wort 40 ~ 60%, glucose 5 ~ 10%, radix pseudostellariae must extract 0.2 ~ 0.5% and surplus water, natural pH；Wherein, The percentage of brewer's wort with volume percentage, glucose and radix pseudostellariae must extract percentage in terms of g/mL（That is 100mL liquid Body culture medium is containing 40 ~ 60mL of brewer's wort, 5 ~ 10g of glucose, 0.2 ~ 0.5g of radix pseudostellariae palpus extract）；
Young clostridium（Clostridium ljungdahlii）Activation：The Young clostridium bacterium solution of cryopreservation tube preservation is disposable It is inoculated into the MRS liquid culture medium of the palpus extract 0.2 ~ 0.5% containing radix pseudostellariae（Fructose equivalent substitution glucose in culture medium） In, 35 ~ 37 DEG C of 42 ~ 48h of static gas wave refrigerator；Wherein, the percentage of the radix pseudostellariae palpus extract is with quality percent by volume g/mL Meter（I.e. 100mL MRS fluid nutrient mediums must 0.2 ~ 0.5g of extract containing radix pseudostellariae）；
（2）, prepare enterococcus faecalis BD-1 and clostridium butyricum RCM-2 mixed fermentation seed liquor, be calculated as seed liquor A：
Under gnotobasis, by step（1）Gained enterococcus faecalis BD-1 activating solution and clostridium butyricum RCM-2 activating solution press volume It is inoculated into than 1: 1 ~ 5: 1 composition admixture activation seed liquor, then by percent by volume 3 ~ 5% containing radix pseudostellariae palpus extract 0.2 ~ 0.5% MRS fluid nutrient mediums in, seed liquor A is made in 36 ~ 48h of static fermentation at 35 ~ 37 DEG C；Wherein, the radix pseudostellariae must extract Percentage in terms of quality percent by volume g/mL（I.e. 100mL MRS fluid nutrient mediums containing radix pseudostellariae must extract 0.2 ~ 0.5g）；
（3）, prepare saccharomyces cerevisiae seed liquor, be calculated as seed liquor B：
Under gnotobasis, by step（1）The activating solution of gained saccharomyces cerevisiae is inoculated into fluid nutrient medium by percent by volume 5 ~ 10% In, seed liquor B is made in 30 ~ 37 DEG C of 30 ~ 36h of static gas wave refrigerator；Fluid nutrient medium is constituted：Brewer's wort 40 ~ 60%, glucose 5 ~ 10%th, radix pseudostellariae palpus extract 0.2 ~ 0.5% and the water of surplus, natural pH；Wherein, the percentage of brewer's wort is with percent by volume The percentage of meter, glucose and radix pseudostellariae palpus extract is in terms of g/mL；
（4）, prepare Young clostridium seed liquor, be calculated as seed liquor C：
Under gnotobasis, by step（1）The activating solution of gained Young clostridium is inoculated into palpus containing radix pseudostellariae by percent by volume 3 ~ 7% In the MRS liquid culture medium of extract 0.2 ~ 0.5%, seed liquor C is made in 32 ~ 36h of static fermentation at 35 ~ 37 DEG C；Wherein, The percentage of the radix pseudostellariae palpus extract is in terms of quality percent by volume g/mL（I.e. 100mL MRS fluid nutrient mediums contain crown prince Ginseng must 0.2 ~ 0.5g of extract）；
（5）, seed liquor A, B, C proportioning prepare fermentation seed liquid：
By step（2）、（3）、（4）Gained seed liquor A, B, C are mixed into fermentation seed at A: B: C=1: 1: 1 ~ 5: 4: 3 by volume Liquid；
（6）, prepare fermentation substrate matrix：
In terms of mass fraction, the fermentation substrate matrix mixed after crushed by following raw materials constituted 1. or in constituting 2. and Into；
Composition is 1.：Radix pseudostellariae must 20 ~ 60 parts, 10 ~ 30 parts of dried orange peel, 10 ~ 30 parts of hawthorn, 10 ~ 30 parts of Medicated Leaven, 5 ~ 15 parts of green tea, 0.05 ~ 0.1 part of allicin；
Composition is 2.：20 ~ 30 parts of radix pseudostellariae, 20 ~ 25 parts of the Radix Astragali, 10 ~ 15 parts of Radix Codonopsis, 10 ~ 15 parts of Radix Isatidis, 10 ~ 15 parts of folium isatidis, 5 ~ 10 parts of honeysuckle, 10 ~ 15 parts of dried orange peel, 10 ~ 15 parts of RHIIZOMA DIOSCOREAE from Henan of China, 5 ~ 15 parts of green tea, 0.05 ~ 0.1 part of allicin；
（7）, fermentation：
By step（5）Gained fermentation seed liquid adds step（6）Gained fermentation substrate Medium Culture, is stirred and evenly mixed, and loads fermentation bag And seal, then fermentation bag is fermented 4 ~ 7d under the conditions of lucifuge in 32 ~ 37 DEG C, compound Chinese herb probiotics is made；Wherein, In terms of volume mass percentage L/kg, the percentage that fermentation seed liquid accounts for fermentation substrate matrix is 25 ~ 35%.
In the present invention, radix pseudostellariae must extract can be obtained by prior art, such as can refer to but be not limited to applicant in The patent of the preparation method of extract " a kind of radix pseudostellariae must " entitled filed in August in 2016 30 days（Application number 201610758305.8）Disclosed in method obtain.
Beneficial effect：
1st, enterococcus faecalis BD-1 is located away from peasant household of village under Zherong County in the present invention（Coordinate：119。51 ' 59.7 〞 E, 27。13′34.0〞 N）48h bacterium are cultivated in autotrophy healthy adult pig caecum mucous layer, gained enterococcus faecalis BD-1 safety non-toxics, 37 DEG C of MRS culture mediums Fall surface smooth bumps, periphery is neat, and colony colour canescence, 0.5 ~ 1.2mm of colony diameter, microscopy G+, no gemma, thalline is long About 0.5 ~ 1 μm of degree；Clostridium butyricum RCM-2 of the present invention is located away from peasant household of village under Zherong County（Coordinate：119。52 ' 04.1 〞 E, 27。13′ 32.7〞N）The caecum mucous membrane section of the healthy adult cock cultivated from circle mountain forest, gained clostridium butyricum RCM-2 safety non-toxics, 37 Culture 48h bacterium colonies surface smooth bumps in DEG C RCM culture mediums, periphery is neat, and colony colour milk yellow is opaque, and slightly acid is smelly Taste, 0.5 ~ 1.5cm of colony diameter, microscopy G+ have gemma, gemma oval, the life of gemma end or the life of secondary end, the thalline for spore of sprouting is in shuttle Shape, about 3.5 ~ 7.0 μm of thalline length；Enterococcus faecalis BD-1 and sour clostridium RCM-2 are with good resistance to Tea Polyphenols and allicin Ability, in addition, radix pseudostellariae palpus extract can remarkably promote the growth of two bacterial strains；
2nd, in fermentation process of the invention, saccharomyces cerevisiae can fast-growth first, produce a large amount of CO2Gas so that sealing hair Fast aeration makes residual air dilution not cracked ends check valve discharge in bag in the ferment bag short time, manufactures CO2Gaseous environment；Then Enterococcus faecalis BD-1 and clostridium butyricum RCM-2 being capable of cooperative fermentation, not stopping pregnancy acid and H2Gas so that fermentation bag is constantly inflated, And CO2Gas and H2Gas mixing；As substrate pH starts reduction, Young clostridium constantly grows, and treats organic carbohydrate in solid matrix During reduction（Other bacteria growings constantly slow down）Start constantly to accelerate to be beneficial to CO2With H2Gas produces organic acid and alcohol, further reduces close Gas content in fermentation bag is sealed, causes fermentation bag constantly to be inhaled flat and moulding, the profile of finished product preparation carries out suction bag machine suction with material Result is identical after bag, but the suction bag effect that method of the invention as time went on is caused is more excellent, and pole is beneficial to the storage of product, hair Organic acid, secondary vacuum and allicin in ferment finished product can also significantly reduce pollution, beneficial to transport, Shelf-life time；
3rd, the method that the present invention is provided can substantially speed up the fermentation rate of fermentation substrate matrix, it is easy to the interior fluid-pressure moulding of fermented product Shape, reduces the fermenting and producing time, beneficial to mass production, and mould contamination probability can be reduced, suitable for packed solid fermentation operation Promote the use of.
Brief description of the drawings
Fig. 1：Enterococcus faecalis BD-1 systematic evolution tree.
Fig. 2：Clostridium butyricum RCM-2 systematic evolution tree.
Embodiment
In following examples, Young clostridium is purchased from American Type Culture Collecti（ATCC 55380）；The radix pseudostellariae must be carried Thing method as disclosed in number of patent application is embodiment 1 in CN201610758305.8 is taken to be prepared；The formula of each culture medium For：
MRS fluid nutrient mediums (1L)：Peptone 10g, powdered beef 5g, glucose 20g, dusty yeast 4g, sodium acetate 5g, phosphoric acid hydrogen two Potassium 2g, magnesium sulfate 0.2g, Triammonium citrate 2g, manganese sulfate 0.05g, Tween 80 1mL, purified water 1L, pH value 6.2 ± 0.2；
MRS liquid culture medium (1L)：Difference with above-mentioned MRS fluid nutrient mediums is, fructose equivalent substitution in culture medium Glucose；
MRS agar plates or MRS slant mediums（1L）：Peptone 10g, powdered beef 5g, glucose 20g, dusty yeast 4g, acetic acid Sodium 5g, dipotassium hydrogen phosphate 2g, magnesium sulfate 0.2g, Triammonium citrate 2g, manganese sulfate 0.05g, Tween 80 1mL, agar 15g, purifying Water 1L, pH value 6.2 ± 0.2；
RCM agar plates or RCM slant mediums（1L）：Yeast extract 3g, beef extract 10g, tryptone 10g, glucose 5g, soluble starch 1g, sodium chloride 5g, sodium acetate trihydrate 3g, cysteine hydrochloride 0.15g, agar 15g, purified water 1L, PH value 6.8 ± 0.2, sterilizes standby；
Starch culture-medium（1L）：Beef extract 5g, peptone 5g, sodium chloride 0.5g, soluble starch 20g, agar 18g, purified water 1L, pH=7.2 ± 0.1, sterilize standby；
Wherein, the MRS fluid nutrient mediums containing Radix Pseudostellariae extract, Tea Polyphenols or allicin are each meant in above-mentioned MRS Liquid Cultures On the basis of based formulas, addition Radix Pseudostellariae extract, Tea Polyphenols or allicin are still further corresponded to；It is many containing Bromocresol purple, tea Each mean on the basis of above-mentioned MRS/RCM agar plates formula, still further correspond in the MRS/RCM agar plates of phenol or allicin Add Bromocresol purple, Tea Polyphenols or allicin.
Embodiment 1-- enterococcus faecalis BD-1 separation and identification
1st, enterococcus faecalis BD-1 separation, enrichment, purifying
Under aseptic technique, peasant household of village under Fujian Province Ningde City Zherong County is taken（Coordinate：119。51 ' 59.7 〞 E, 27。13′ 34.0〞N）Autotrophy healthy adult pig caecum mucous membrane section, scrapes mucous membrane mucus with the slide of sterilizing and is managed in EP, use sterile physiological salt Water dilution 102Times, take 0.1mL to be coated on the MRS agar plates containing Bromocresol purple (0.01%, g/mL), after coating MRS agar plates are placed in anaerobic culture box, 37 DEG C of culture 48h, select the bacterium colony line purifying of culture medium flavescence, choose mirror Gram-positive, nonspore-bearing bacterial strain are examined in containing bromocresol purple (0.01%, g/mL), Tea Polyphenols（0.01%, g/mL）With Allicin（0.01%, g/mL）MRS agar plates on secondary screening, it is final choose bacterium colony yellow color substantially, bacterium colony surface it is smooth convex Rise, periphery it is neat as object bacterial strain, Strain Designation is BD-1, and carries out slant preservation to strain with MRS slant mediums.
2nd, morphological observation
Observe bacterial strain BD-1 colony morphology characteristic, it is known that：Bacterium colony surface smooth bumps on MRS agar plates, periphery is neat, bacterium Fall color canescence, 0.5 ~ 1.2mm of colony diameter, microscopy G+, no gemma, about 0.5 ~ 1 μm of thalline diameter.
3rd, physiological and biochemical property
Bacterial strain BD-1 is identified by the biochemical identification experiment of lactic acid bacteria, motility, oxidase test etc. have been carried out respectively raw Change CHARACTERISTICS IDENTIFICATION, result of the test control《Primary Jie Shi Bacteria Identifications handbook》Carry out synthetic determination.It the results are shown in Table 1.
The physiology seriation index determining result of isolated strains shows to be consistent with lactic acid producing enterococcus strain result.
4th, the 16S rDNA sequencings of BD-1 bacterial strains are compared
Utilize bacterial genomes DNA extraction agent boxes（DP302-02, TIANGEN）Bacterial strain BD-1 STb gene is extracted, with bacterial strain BD-1 STb gene is template, primer P0 (5 '-AGAGTTTGATCCTGGCTCAG -3 ') and PC3 (5 ' - GGTTACCTTGTTACGACTT -3 ') guiding under PCR expand the 16S rDNA sequences of the bacterial strain, PCR reaction systems are：10 5.0 2.0 μ L, Primer 1 (20 μm of ol/L) of μ L, dNTP (10mmol/L) of × Buffer 1.0 μ L, Primer2 (20 μm of ol/L) 1.0 μ L, the μ L of template DNA 2.5, Taq enzyme (5.0U/ μ L) 0.5 μ L, ultra-pure water 38 μ L, total 50 μ L.PCR reaction conditions are：First 94 ℃5min；Then 94 DEG C of 1min, 55 DEG C of 1min, 72 DEG C of 1min, totally 30 circulations；Last 72 DEG C of extensions 8min.After reaction terminates, 1% agarose gel electrophoresis detection is carried out to pcr amplification product, marker is compared and obtains purpose band.With purchased from Axygen public affairs The Ago-Gel QIAquick Gel Extraction Kit of department reclaims and purifies the purpose band, send raw work bioengineering（Shanghai）Limited company Determined dna sequence is carried out, sequencing result is shown in SEQ ID No.1.
Measured sequence and the 16S rDNA sequences in Genbank are carried out into Blastn similarity analysis to be compared, as a result Bacterial strain BD-1 withEnterococcus faecium strain KCI1、Enterococcus lactis strain K12419C The homologys of 16S rDNA sequences reached 100%, therefore BD-1 is accredited as enterococcus faecalis, its systematic evolution tree such as Fig. 1.
With reference to above-mentioned morphology, physiological and biochemical property and sequencing and chadogram result, bacterial strain BD-1 of the present invention is identified simultaneously Classification And Nomenclature is enterococcus faecalis（Enterococcus faecalis）BD-1.
5th, Tea Polyphenols is to enterococcus faecalis BD-1 growths, pH influence
（1）Enterococcus faecalis BD-1 activation：Enterococcus faecalis BD-1 test tube slants strain is used into sterile physiological salt in an aseptic environment Water is rinsed, and is washed lower media surface lawn, is then seeded into MRS fluid nutrient mediums, 37 DEG C of static gas wave refrigerator 48h；
（2）By inoculum concentration 3%（Volume ratio）By step（1）Gained enterococcus faecalis BD-1 activating solution is seeded in anaerobic box to be contained The gradient of Tea Polyphenols is successively in the anaerobism serum bottle of MRS fluid nutrient mediums, in the MRS fluid nutrient mediums wherein in anaerobism serum bottle It is set to（g/mL）：0%th, 0.10%, 0.25%, 0.50%, 1.00%, zymotic fluid pH value and viable count, knot are determined when terminal is cultivated Fruit is shown in Table 2.
As seen from the results in Table 2：The concentration that enterococcus faecalis BD-1 of the present invention can tolerate Tea Polyphenols is 1.0%, the concentration hypothallus Survival rate is 70.26%.
6th, allicin is to enterococcus faecalis BD-1 growths, pH influence
（1）Enterococcus faecalis BD-1 activation：With the step under the present embodiment the 5th（1）；
（2）By inoculum concentration 3%（Volume ratio）By step（1）Gained enterococcus faecalis BD-1 activating solution is seeded in anaerobic box to be contained The gradient of allicin is successively in the anaerobism serum bottle of MRS fluid nutrient mediums, in the MRS fluid nutrient mediums wherein in anaerobism serum bottle It is set to（g/mL）：0%th, 0.010%, 0.025%, 0.050%, 0.10%, determine zymotic fluid pH value and viable bacteria when terminal is cultivated Number, the results are shown in Table 3；
As seen from the results in Table 3：The concentration that enterococcus faecalis BD-1 of the present invention can tolerate allicin is 0.1%, concentration hypothallus survival Rate is 70.43%；
（3）Enterococcus faecalis is resistant to Tea Polyphenols and allicin growth contrast：
By inoculum concentration 3%（Volume ratio）Respectively by step（1）Gained enterococcus faecalis BD-1（It is calculated as A）Activating solution and control bacterium B, C, D activating solution are seeded in anaerobic box in the anaerobism serum bottle of the fluid nutrient medium containing MRS, wherein in anaerobism serum bottle In MRS fluid nutrient mediums 1. the gradient of Tea Polyphenols set gradually for（g/mL）：0%th, 1.00%, 2.00%, 2. the gradient of allicin according to It is secondary to be set to（g/mL）：0%th, 0.05%, 0.1%, 48h is continuously cultivated, control is used as by 0%+0%（Viable count is defined as X0）, pass through Formula：Thalline loss late=（X0-X）/ X0* 100% calculates enterococcus faecalis thalline loss late, the results are shown in Table 4.
As seen from the results in Table 4：In Tea Polyphenols（2%）And allicin（0.1%）Enterococcus faecalis BD-1 of the present invention under synergy Survival rate is up to 53.7%, beyond 50%, far above the similar bacterial strain in market.
7th, enterococcus faecalis bacteriostatic experiment
（1）Enterococcus faecalis BD-1 activation：With the step under the present embodiment the 5th（1）；
（2）The static fermentation of enterococcus faecalis BD-1 anaerobism in MRS fluid nutrient mediums：
By inoculum concentration 3%（Volume ratio）By step（1）Gained enterococcus faecalis BD-1 activating solution is seeded to containing MRS in anaerobic box In the anaerobism serum bottle of fluid nutrient medium, 37 DEG C of static gas wave refrigerator 48h take zymotic fluid to be detected using Odontothrips loti when fermentation termination To the fungistatic effect of staphylococcus aureus, micrococcus luteus and ETEC, using nonvaccinated MRS fluid nutrient mediums as Control；
（3）Enterococcus faecalis BD-1 aerobic fermentations in MRS fluid nutrient mediums：
By inoculum concentration（Volume ratio）3% by step（1）Gained enterococcus faecalis BD-1 activating solution access is equipped with 100 mL MRS liquid In 500 mL triangular flasks of culture medium, 37 DEG C, 48h is cultivated under 150rpm, takes zymotic fluid to utilize Odontothrips loti when terminal is cultivated The fungistatic effect to staphylococcus aureus, micrococcus luteus and ETEC is detected, with nonvaccinated MRS Liquid Cultures Base is control.
Antibacterial circle diameter size is shown in Table 5.
As seen from the results in Table 5：It is micro- that enterococcus faecalis BD-1 of the present invention can significantly inhibit pathogenic bacteria staphylococcus aureus, gamboge Coccus and the growth of ETEC, and anaerobic cultures are relatively better than aerobic culture.
Embodiment 2-- clostridium butyricums RCM-2 separation and identification
1st, clostridium butyricum RCM-2 separation, enrichment, purifying
Under aseptic technique, peasant household of village under Fujian Province Ningde City Zherong County is taken（Coordinate：119。52 ' 04.1 〞 E, 27。13′ 32.7〞N）The caecum mucous membrane section of the healthy adult cock cultivated from circle mountain forest, with the slide of sterilizing scrape mucous membrane mucus in EP is managed, and 10 are diluted with sterile saline2Times, 60 DEG C, 30min are then heated to, takes 0.1mL to be coated on and is indicated containing bromocresol purple On the RCM agar plates of agent (0.01%, g/mL), RCM agar plates are placed in anaerobic culture box after coating, 37 DEG C of cultures 48h, selects the bacterium colony line purifying of culture medium flavescence, choose microscopy Gram-positive, have the shaft-like bacterial strain of gemma in containing Bromocresol purple (0.01%, g/mL), Tea Polyphenols（0.01%, g/mL）And allicin（0.01%, g/mL）RCM agar plates on it is multiple Sieve, finally choose periphery of bacterial colonies yellow color substantially, bacterium colony surface smooth bumps, milk yellow is opaque, there is the conduct of acid smell Preparation bacterial strain, the maximum conduct pair of transparent circle is selected after preparation bacterial strain then is coated on into starch culture-medium, 37 DEG C of culture 48h again As bacterial strain, Strain Designation is RCM-2, and carries out slant preservation to strain with RCM slant mediums.
2nd, morphological observation
Observe bacterial strain RCM-2 colony morphology characteristic, it is known that：Bacterium colony surface smooth bumps on RCM agar plates, bacterium colony face yellow fraction Color is opaque, slightly acid smell, 0.5 ~ 1.5cm of colony diameter, microscopy G+, there is gemma, gemma oval, the life of gemma end or secondary end Raw, the thalline for spore of sprouting is in fusiform, about 3.5 ~ 7.0 μm of thalline length.
3rd, physiological and biochemical property
Reference《The outstanding Bacteria Identification handbook of uncle》Physiology and biochemistry identification is carried out to bacterial strain, respectively with catalase, amylase, nitrate also Former and sugar etc. carries out physiological and biochemical test, the results are shown in Table 6.
By its with《The outstanding Bacteria Identification handbook of uncle》Physiology and biochemistry with clostridium butyricum in common bacteria system identification handbook is special Property be compared, as a result find that bacterial strain RCM-2 is consistent substantially with the physio-biochemical characteristics of clostridium butyricum, therefore Preliminary Identification bacterial strain RCM-2 is clostridium butyricum.
4th, sequencing and chadogram
Utilize bacterial genomes DNA extraction agent boxes（DP302-02, TIANGEN）Bacterial strain RCM-2 STb gene is extracted, with bacterial strain RCM-2 STb gene is template, primer P0 (5 '-AGAGTTTGATCCTGGCTCAG -3 ') and PC3 (5 ' - GGTTACCTTGTTACGACTT -3 ') guiding under PCR expand the 16S rDNA sequences of the bacterial strain, PCR reaction systems are：10 5.0 2.0 μ L, Primer 1 (20 μm of ol/L) of μ L, dNTP (10mmol/L) of × Buffer 1.0 μ L, Primer2 (20 μm of ol/L) 1.0 μ L, the μ L of template DNA 2.5, Taq enzyme (5.0U/ μ L) 0.5 μ L, ultra-pure water 38 μ L, total 50 μ L.PCR reaction conditions are：First 94 ℃5min；Then 94 DEG C of 1min, 55 DEG C of 1min, 72 DEG C of 1min, totally 30 circulations；Last 72 DEG C of extensions 8min.After reaction terminates, 1% agarose gel electrophoresis detection is carried out to pcr amplification product, marker is compared and obtains purpose band.With purchased from Axygen public affairs The Ago-Gel QIAquick Gel Extraction Kit of department reclaims and purifies the purpose band, send raw work bioengineering（Shanghai）Limited company Determined dna sequence is carried out, sequencing result is shown in SEQ ID No.2.
Measured sequence and the 16S rDNA sequences in Genbank are carried out into Blastn similarity analysis to be compared, as a result Bacterial strain RCM-2 withClostridium butyricumThe homology of strain 1005-15098 16S rDNA sequences reaches 98%, therefore RCM-2 is accredited as clostridium butyricum, its systematic evolution tree such as Fig. 2.
With reference to above-mentioned morphology, physiological and biochemical property and sequencing and chadogram result, bacterial strain RCM-2 of the present invention is identified simultaneously Classification And Nomenclature is clostridium butyricum（Clostridium butyricum）RCM-2.
5th, Tea Polyphenols is to clostridium butyricum RCM-2 growths, pH influence
（1）Clostridium butyricum RCM-2 activation：Clostridium butyricum RCM-2 test tube slants strain is used into sterile physiological in an aseptic environment Normal saline washing, washes lower media surface lawn, is then seeded into MRS fluid nutrient mediums, 37 DEG C of static gas wave refrigerator 48h；
（2）By inoculum concentration 3%（Volume ratio）By step（1）Gained clostridium butyricum RCM-2 activating solution is seeded in anaerobic box to be contained The gradient of Tea Polyphenols is successively in the anaerobism serum bottle of MRS fluid nutrient mediums, in the MRS fluid nutrient mediums wherein in anaerobism serum bottle It is set to（g/mL）：0%th, 0.10%, 0.25%, 0.50%, 1.00%, zymotic fluid pH value and viable count, knot are determined when terminal is cultivated Fruit is shown in Table 7.
As seen from the results in Table 7：The concentration that clostridium butyricum RCM-2 of the present invention can tolerate Tea Polyphenols is bacterium under 1.0%, the concentration Body survival rate is 87.3%.
6th, allicin is to clostridium butyricum RCM-2 growths, pH influence
（1）Clostridium butyricum RCM-2 activation：With the step under the present embodiment the 5th（1）；
（2）By inoculum concentration 3%（Volume ratio）By step（1）Gained clostridium butyricum RCM-2 activating solution is seeded in anaerobic box to be contained The gradient of allicin is successively in the anaerobism serum bottle of MRS fluid nutrient mediums, in the MRS fluid nutrient mediums wherein in anaerobism serum bottle It is set to（g/mL）：0%th, 0.010%, 0.025%, 0.050%, 0.10%, determine zymotic fluid pH value and viable bacteria when terminal is cultivated Number, the results are shown in Table 8；
As seen from the results in Table 8：The concentration that clostridium butyricum RCM-2 of the present invention can tolerate allicin is 0.1%, and the concentration hypothallus is deposited Motility rate is 85.5%；
（3）Clostridium butyricum is resistant to Tea Polyphenols and allicin growth contrast：
By inoculum concentration 3%（Volume ratio）By step（1）Gained clostridium butyricum RCM-2（It is calculated as A）Activating solution be inoculated with anaerobic box Into the anaerobism serum bottle of the fluid nutrient medium containing MRS, the 1. ladder of Tea Polyphenols in the MRS fluid nutrient mediums wherein in anaerobism serum bottle Degree set gradually for（g/mL）：0%th, 1.00%, 2.00%, 2. the gradient of allicin set gradually for（g/mL）：0%、0.05%、 0.1%, 48h is continuously cultivated, control is used as by 0%+0%（Viable count is defined as X0）, pass through formula：Thalline loss late=（X0-X）/ X0* 100% calculates clostridium butyricum thalline loss late, the results are shown in Table 9.
As seen from the results in Table 9：In Tea Polyphenols（1%）And allicin（0.05%）Clostridium butyricum RCM- of the present invention under synergy 2 survival rates are up to 88.5%, beyond 50%, far above the similar bacterial strain in market.
7th, clostridium butyricum bacteriostatic experiment
（1）Clostridium butyricum RCM-2 activation：With the step under the present embodiment the 5th（1）；
（2）By inoculum concentration 3%（Volume ratio）By step（1）Gained clostridium butyricum RCM-2 activating solution is seeded in anaerobic box to be contained In the anaerobism serum bottle of MRS fluid nutrient mediums, 37 DEG C of static gas wave refrigerator 48h take zymotic fluid to utilize Odontothrips loti when fermentation termination The fungistatic effect to staphylococcus aureus, micrococcus luteus and ETEC is detected, with nonvaccinated MRS Liquid Cultures Base is control；Antibacterial circle diameter size is shown in Table 10.
As seen from the results in Table 10：Clostridium butyricum RCM-2 of the present invention can significantly inhibit pathogenic bacteria staphylococcus aureus, M. luteus Bacterium and the growth of ETEC.
Embodiment 3-- radix pseudostellariaes must growth promotion experiment of the extract to fermentation strain
（1）, four kinds of fermentation strains activation：
Enterococcus faecalis BD-1 activation：Enterococcus faecalis BD-1 test tube slant strain is used into sterile saline in an aseptic environment Rinse, wash lower media surface lawn, be then seeded into MRS fluid nutrient mediums, 37 DEG C of static gas wave refrigerator 48h；
Clostridium butyricum RCM-2 activation：Clostridium butyricum RCM-2 test tube slant strain is used into sterile physiological salt in an aseptic environment Water is rinsed, and is washed lower media surface lawn, is then seeded into MRS fluid nutrient mediums, 37 DEG C of static gas wave refrigerator 48h；
The activation of saccharomyces cerevisiae：Commercialization edible saccharomyces cerevisiae powder is inoculated into fluid nutrient medium, 32 DEG C of static gas wave refrigerators 32h；Viable count >=1.0 × 10 of saccharomyces cerevisiae powder8CFU/g；Fluid nutrient medium is constituted：Brewer's wort 45%, glucose 6% and remaining The water of amount, natural pH, wherein, the percentage of brewer's wort is with volume percentage, and the percentage of glucose is in terms of g/mL；
The activation of Young clostridium：The Young clostridium bacterium solution of cryopreservation tube preservation is disposably inoculated into MRS liquid culture medium, 37 DEG C of static gas wave refrigerator 48h；
（2）Growth promotion is tested：
By inoculum concentration 3%（Volume ratio）By step（1）Gained enterococcus faecalis BD-1 activating solution is seeded to containing MRS in anaerobic box In the anaerobism serum bottle of fluid nutrient medium, the ladder of radix pseudostellariae palpus extract in the MRS fluid nutrient mediums wherein in anaerobism serum bottle Degree set gradually for（g/mL）：0%th, 0.10%, 0.25%, 37 DEG C of static gas wave refrigerator 48h, zymotic fluid pH value is determined when terminal is cultivated And viable count；
By inoculum concentration 3%（Volume ratio）By step（1）Gained clostridium butyricum RCM-2 activating solution is seeded to containing MRS in anaerobic box The anaerobism serum bottle of fluid nutrient medium（Top is externally connected with sensitive gas pressure meter apparatus）In, wherein in anaerobism serum bottle In MRS fluid nutrient mediums radix pseudostellariae must extract gradient set gradually for（g/mL）：0%th, 0.10%, 0.25%, 37 DEG C of static trainings 48h is supported, zymotic fluid pH value, viable count and terminal pressure value are determined when terminal is cultivated；
By inoculum concentration 5%（Volume ratio）By step（1）The activating solution of gained saccharomyces cerevisiae is inoculated into the anaerobism blood containing fluid nutrient medium Clear bottle（Top is externally connected with sensitive gas pressure meter apparatus）In, 32 DEG C of static gas wave refrigerator 32h determine zymotic fluid when terminal is cultivated PH value, viable count and terminal pressure value；Fluid nutrient medium is constituted：Brewer's wort 45%, glucose 6%, radix pseudostellariae must extract and The water of surplus, natural pH, wherein, the percentage of brewer's wort with volume percentage, glucose and radix pseudostellariae must extract hundred Divide ratio in terms of g/mL；In fluid nutrient medium in anaerobism serum bottle radix pseudostellariae must extract gradient set gradually for：0%、 0.10%、0.25%；
By inoculum concentration 5%（Volume ratio）By step（1）The activating solution of gained Young clostridium is inoculated into MRS liquid culture medium, Wherein in MRS liquid culture medium radix pseudostellariae must extract gradient set gradually for（g/mL）：0%th, 0.10%, 0.25%, 37 Static fermentation 32h at DEG C, zymotic fluid pH value and viable count are determined when terminal is cultivated.
Viable count the results are shown in Table 11, and terminal pressure increment rate the results are shown in Table 12.
From table 11-12：Radix pseudostellariae must extract can remarkably promote the enterococcus faecalis BD-1 of the present invention, clostridium butyricum RCM-2, Saccharomyces cerevisiae and the growth of Young clostridium；0.1 ~ 0.25% radix pseudostellariae must extract not only notable RCM-2, saccharomyces cerevisiae simultaneously Growth, be also obviously improved the fermentation gas ability of RCM-2, saccharomyces cerevisiae, therefore 0.1% ~ 0.25% radix pseudostellariae must extract Addition promote thalli growth and rapid aerogenesis and then accelerate sealing and fermenting bag exhaust speed when will be for the fermentation of later stage host solid Rate provides robust techniques reference.
Embodiment 4-- enterococcus faecalis BD-1 and clostridium butyricum RCM-2 symbiosis are long
（1）The activation of bacterial strain
Enterococcus faecalis BD-1 activation：Enterococcus faecalis BD-1 test tube slant strain is used into sterile saline in an aseptic environment Rinse, wash lower media surface lawn, be then seeded into MRS fluid nutrient mediums, 37 DEG C of static gas wave refrigerator 48h；
Clostridium butyricum RCM-2 activation：Clostridium butyricum RCM-2 test tube slant strain is used into sterile physiological salt in an aseptic environment Water is rinsed, and is washed lower media surface lawn, is then seeded into MRS fluid nutrient mediums, 37 DEG C of static gas wave refrigerator 48h；
（2）Then by enterococcus faecalis BD-1 activating solution, clostridium butyricum RCM-2 activating solution and both by volume 1:1 answers The activating solution of conjunction is respectively according to inoculum concentration 3%（Volume ratio）It is inoculated in 200 mL anaerobism serum bottles of MRS fluid nutrient mediums, its In MRS fluid nutrient mediums in middle anaerobism serum bottle radix pseudostellariae must extract gradient set gradually for（Mass ratio）：0%、 0.25%, 37 DEG C of static gas wave refrigerator 48h, determine zymotic fluid viable count when terminal is cultivated, clostridium butyricum RCM-2 bud are determined in addition Spore rate；It the results are shown in Table 13.
As shown in Table 13：Radix pseudostellariae palpus extract can remarkably promote enterococcus faecalis BD-1 and clostridium butyricum RCM-2 symbiosis It is long, and promote degree to be more than two plants of single strain quantity sums two plants of bacterium symbiosis under comparable sodium；In addition, under 0.25% concentration RCM-2 gemma rate can be respectively facilitated, the gemma rate of symbiosis culture is further improved.
Embodiment 5-- safety testings
（1）, four kinds of fermentation strains activation：
Enterococcus faecalis BD-1 activation：Enterococcus faecalis BD-1 test tube slant strain is used into sterile saline in an aseptic environment Rinse, in the MRS fluid nutrient mediums for being then seeded into the palpus extract 0.25% containing radix pseudostellariae, 37 DEG C of static gas wave refrigerator 48h；Wherein, The percentage of the radix pseudostellariae palpus extract is in terms of quality percent by volume g/mL；
Clostridium butyricum RCM-2 activation：Clostridium butyricum RCM-2 test tube slant strain is used into sterile physiological salt in an aseptic environment Water is rinsed, in the MRS fluid nutrient mediums for being then seeded into the palpus extract 0.25% containing radix pseudostellariae, 37 DEG C of static gas wave refrigerator 48h；Wherein, The percentage of the radix pseudostellariae palpus extract is in terms of quality percent by volume g/mL；
The activation of saccharomyces cerevisiae：Commercialization edible saccharomyces cerevisiae powder is inoculated into fluid nutrient medium, 32 DEG C of static gas wave refrigerators 32h；Viable count >=1.0 × 10 of saccharomyces cerevisiae powder8CFU/g；Fluid nutrient medium is constituted：Brewer's wort 45%, glucose 6%, too Son ginseng must extract 0.25% and surplus water, natural pH, wherein, the percentage of brewer's wort with volume percentage, glucose and The percentage of radix pseudostellariae palpus extract is in terms of g/mL；
The activation of Young clostridium：The Young clostridium bacterium solution of cryopreservation tube preservation is disposably inoculated into must extract containing radix pseudostellariae In 0.25% MRS liquid culture medium, 37 DEG C of static gas wave refrigerator 48h；Wherein, the radix pseudostellariae must extract percentage with Quality percent by volume g/mL is counted；
（2）, prepare enterococcus faecalis BD-1 and clostridium butyricum RCM-2 mixed fermentation seed liquor, be calculated as seed liquor A：
Under gnotobasis, by step（1）Gained enterococcus faecalis BD-1 activating solution and clostridium butyricum RCM-2 activating solution press volume Than 1:1 composition admixture activation seed liquor, then by percent by volume 3% be inoculated into containing radix pseudostellariae must extract 0.25% MRS liquid In culture medium, seed liquor A is made in static fermentation 36h at 37 DEG C；Wherein, the percentage of the radix pseudostellariae palpus extract is with quality Percent by volume g/mL is counted；
（3）, prepare saccharomyces cerevisiae seed liquor, be calculated as seed liquor B：
Under gnotobasis, by step（1）The activating solution of gained saccharomyces cerevisiae is inoculated into fluid nutrient medium by percent by volume 8%, 32 DEG C of static gas wave refrigerator 32h, are made seed liquor B；Fluid nutrient medium is constituted：Brewer's wort 45%, glucose 6%, radix pseudostellariae must be extracted The water of thing 0.25% and surplus, natural pH, wherein, the percentage of brewer's wort must be carried with volume percentage, glucose and radix pseudostellariae The percentage of thing is taken in terms of g/mL；
（4）, prepare Young clostridium seed liquor, be calculated as seed liquor C：
Under gnotobasis, by step（1）The activating solution of gained Young clostridium is inoculated into by percent by volume 5% and must carried containing radix pseudostellariae In the MRS liquid culture medium for taking thing 0.25%, seed liquor C is made in static fermentation 32h at 37 DEG C；Wherein, the radix pseudostellariae The percentage of palpus extract is in terms of quality percent by volume g/mL；
（5）, safety testing
Experimental animal：KM kind small white mouses, SPF grades, 18 ~ 22g of body weight, male and female half and half are purchased from institute of Chinese materia medica of Chongqing City.
Test bacterium solution：With volume basis, A: B: C=1: 1: 1 and 5: 4: 3 two kind of concentration cooperation fermentation seed liquid.
Experiment：Choose healthy mice 10, male and female half and half, average weight：It is female to be（21.90±0.5）G, hero is（22.00 ±0.5）g.Mouse fasting（It can't help water）After 12h, with experiment bacterium solution gastric infusion, 3 times a day equivalent administration, accumulated dose, which is administered, is 1.5mL.Mouse is normally raised in cleaning grade receptacle, room temperature after administration（21±0.5）DEG C, relative humidity 45% ~ 60%, observation is dynamic The activity of thing, fur, diet, excrement whether there is abnormal change, and observe whether there is poisoning symptom and death, Continuous Observation three weeks, knot Fruit is shown in Table 14.
Understand：Fermentation strain composition safety non-toxic of the present invention.
The preparation of embodiment 6-- probioticses
The preparation of probiotics of the present invention, step is as follows：
Step（1）-（4）：Respectively be the same as Example 5 the step of（1）-（4）It is identical；
（5）, seed liquor A, B, C proportioning prepare fermentation seed liquid：
By step（2）、（3）、（4）Gained seed liquor A, B, C are mixed into fermentation seed liquid at A: B: C=1: 1: 1 by volume；
（6）, prepare fermentation substrate matrix：
In terms of mass fraction, the fermentation substrate matrix is mixed after crushed by following raw materials：Radix pseudostellariae must 25 parts, dried orange peel 20 parts, 20 parts of hawthorn, 20 parts of Medicated Leaven, 15 parts of green tea, 0.06 part of allicin；
（7）, fermentation：
First by step（6）The fermentation substrate matrix of preparation is dosed in mixing plant（Equipment uses utility model patent equipment, The patent No.：201620053478.5）, 5min is premixed, mixing rotating speed is 100 rpm, by step（5）Gained fermentation seed liquid is added Added in equipment, flow velocity is 30mL/s, and stirring, speed of agitator is 100 rpm, and the retention time is 20min, wherein, with volume Mass percent L/kg is counted, and the percentages that fermentation seed liquid accounts for fermentation substrate matrix are 35%；Rapidly will mixing after stirring Material is successively weighed and packed, and fermentation bag is using utility model patent bag（The patent No.：201620018088.4）It is interior, pack close It is honored as a queen and bag pressing discharge additional air is paved into bag face rapidly, check valve upward, and is placed in fermenting vehicle successively（Utility model is special Profit, the patent No.：201620981592.4）It is interior, then fermentation plant is promoted, fermentation plant requires constant temperature（35℃）And lucifuge, in hair Fermentation bag will be arranged after stacking in ferment workshop, start fermentation.
Compare the preparation of probiotics：By commercially available mixed fermenting agent（It is limited purchased from Zhengzhou Ou Kebaike biotechnologys Company, trade name：Bai Kesheng；Mixed fermenting agent composition is bacillus subtilis, lactic acid bacteria, saccharomycete, total viable count >=1.0 ×109CFU/g）As control strain, prepare control fermentation seed liquor according to the activation of product description method and replace the present embodiment Fermentation seed liquid, then to be fermented with identical fermentation substrate matrix and condition of the invention.
Suction bag is started using fermentation bag and it is moulding when as fermentation ends time point, then sampling and respectively with total viable count, end Point pH value, the moulding intensity of fermentation bag suction bag, bacterial contamination rate are test index, and random 5 bags mix and sample.Wherein two classes are sent out 30 bags of ferment, per packed amount 10kg.
It the results are shown in Table 15.
The preparation of embodiment 7 -- probiotics
It is with the difference of embodiment 6：Step（5）In, A: B: C=5: 4: 3；Step（6）In, fermentation substrate matrix group Turn into：20 parts of radix pseudostellariae, 20 parts of the Radix Astragali, 10 parts of Radix Codonopsis, 10 parts of Radix Isatidis, 10 parts of folium isatidis, 5 parts of honeysuckle, 10 parts of dried orange peel, bosom 10 parts of Chinese yam, 5 parts of green tea, 0.06 part of allicin.
It the results are shown in Table 16.
From table 15-16：Probiotics of the present invention has shorter fermentation period, nearly shortens half the time, and arrive hair Probiotics of the present invention has higher total viable count, lower matrix pH value, stronger fermentation bag suction bag modeling during ferment terminal Shape intensity and lower mould contamination rate.
The application examples of embodiment 8-- finished product probioticses
（1）Influence of the probiotics product of the present invention of embodiment 6 to meat chicken growth performance
Experimental program：120 1 age in days AA broiler chickens are randomly divided into 2 groups, every group of 3 repetitions are each to repeat 20.Control Group feeding basal diet, test group adds 0.2wt% on the basis of basal diet（In terms of the mass percent for accounting for basal diet） The probiotics product of the present invention of embodiment 6, basal diet is corn-soybean meal powder-material, young with reference to NRC (1994) meat Chicken trophic level and Chinese feed nutrient table are formulated.Experiment broiler chicken uses cage, each repeats individually feed, freely Feeding, drinking-water, daily management are routinely carried out with immune programme for children.Culturing time is set as 6 weeks.Raised in 4 weekends and 6 weekends early It is preceding to be weighed, daily gain and feed-weight ratio are calculated respectively, the results are shown in Table 17.
（2）The probiotics product of the present invention of embodiment 7 is to the influence to piglet growth performance
Experimental program：The DLY ternary weanling pig of 60 first 21 ages in days and body weight about 20kg is randomly divided into 2 groups, every group 3 Repeat, it is each to repeat 10.Control group fed basal diet, test group adds 0.25wt% respectively on the basis of basal diet The probiotics product of the present invention of embodiment 7, basal diet formula：The wt% of corn 56.00, dregs of beans 30.00wt%, wheat bran 10.00wt%, premix 4.00wt%（Every kilogram of premix 15000IU containing vitamin A, vitamin D3 8000IU, vitamin E 60IU、VK 2.5IU）.Test pig carries out group feeding, fully feeding, and daily 4 times, free water, experimental period 40d, management is normal. Weightening and feed conversion rate are determined during off-test, 18 are the results are shown in Table.
From table 17-18：Compared to control group, finished product probiotics group of the present invention can remarkably promote growth of animals or poultry, And significantly reduce feedstuff-meat ratio.
SEQUENCE LISTING
<110>Fujian Bei Di pharmaceutcal corporation, Ltds
<120>A kind of compound Chinese herb probiotics and its Quick production method based on solid state fermentation
<130>
<160> 2
<170> PatentIn version 3.4
<210> 1
<211> 1461
<212> DNA
<213>It is unknown
<400> 1
cgcaggcgcg tactatacat gcagtcgtac gcttcttttt ccaccggagc ttgctccacc 60
ggaaaaagag gagtggcgaa cgggtgagta acacgtgggt aacctgccca tcagaagggg 120
ataacacttg gaaacaggtg ctaataccgt ataacaatcg aaaccgcatg gttttgattt 180
gaaaggcgct ttcgggtgtc gctgatggat ggacccgcgg tgcattagct agttggtgag 240
gtaacggctc accaaggcca cgatgcatag ccgacctgag agggtgatcg gccacattgg 300
gactgagaca cggcccaaac tcctacggga ggcagcagta gggaatcttc ggcaatggac 360
gaaagtctga ccgagcaacg ccgcgtgagt gaagaaggtt ttcggatcgt aaaactctgt 420
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gccacggcta actacgtgcc agcagccgcg gtaatacgta ggtggcaagc gttgtccgga 540
tttattgggc gtaaagcgag cgcaggcggt ttcttaagtc tgatgtgaaa gcccccggct 600
caaccgggga gggtcattgg aaactgggag acttgagtgc agaagaggag agtggaattc 660
catgtgtagc ggtgaaatgc gtagatatat ggaggaacac cagtggcgaa ggcggctctc 720
tggtctgtaa ctgacgctga ggctcgaaag cgtggggagc aaacaggatt agataccctg 780
gtagtccacg ccgtaaacga tgagtgctaa gtgttggagg gtttccgccc ttcagtgctg 840
cagctaacgc attaagcact ccgcctgggg agtacgaccg caaggttgaa actcaaagga 900
attgacgggg gcccgcacaa gcggtggagc atgtggttta attcgaagca acgcgaagaa 960
ccttaccagg tcttgacatc ctttgaccac tctagagata gagcttcccc ttcgggggca 1020
aagtgacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc 1080
gcaacgagcg caacccttat tgttagttgc catcattcag ttgggcactc tagcaagact 1140
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gggctacaca cgtgctacaa tgggaagtac aacgagttgc gaagtcgcga ggctaagcta 1260
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atcgctagta atcgcggatc agcacgccgc ggtgaatacg ttcccgggcc ttgtacacac 1380
cgcccgtcac accacgagag tttgtaacac ccgaagtcgg tgaggtaacc tttttggagc 1440
cagccgccta agtgattgat t 1461
<210> 2
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<212> DNA
<213>It is unknown
<400> 2
ggcagtggcg gcatcttacc atgcagtcga gcgatgaagc tccttcggga gtggattagc 60
ggcggacggg tgagtaacac gtgggtaacc tgcctcatag aggggaatag cctttcgaaa 120
ggaagattaa taccgcataa gattgtagta ccgcatggta cagcaattaa aggagtaatc 180
cgctatgaga tggacccgcg tcgcattagc tagttggtga ggtaacggct caccaaggcg 240
acgatgcgta gccgacctga gagggtgatc ggccacattg ggactgagac acggcccaga 300
ctcctacggg aggcagcagt ggggaatatt gcacaatggg ggaaaccctg atgcagcaac 360
gccgcgtgag tgatgacggt cttcggattg taaagctctg tctttaggga cgataatgac 420
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gcaagcgttg tccggattta ctgggcgtaa agggagcgta ggtggatatt taagtgggat 540
gtgaaatacc cgggcttaac ctgggtgctg cattccaaac tggatatcta gagtgcagga 600
gaggaaagga gaattcctag tgtagcggtg aaatgcgtag agattaggaa gaataccagt 660
ggcgaaggcg cctttctgga ctgtaactga cactgaggct cgaaagcgtg gggagcaaac 720
aggattagat accctggtag tccacgccgt aaacgatgaa tactaggtgt aggggttgtc 780
atgacctctg tgccgccgct aacgcattaa gtattccgcc tggggagtac ggtcgcaaga 840
ttaaaactca aaggaattga cgggggcccg cacaagcagc ggagcatgtg gtttaattag 900
aagcaacgag aagcacctta cctagacttg acatctcctg aatttctgtg taatggagga 960
agccatttcg gtcgcaggaa cacaggtggc gcatggttgt tgtcagctcg tgtcgtgaga 1020
tgttgggtta agtcccgcaa cgagcgcacc ctttattttt agttgctccc atttagttga 1080
gcactctagc gagactcccg gggttaaccg ggaggaaggt ggggatgacg tcaaatcctc 1140
atccccctta tgtatagggc cacacacgtg ctacaatggt cggtacaatg agacgcaccc 1200
tcgcgagagt gagcaaaact ataaaaccga tctcatttcg gattgtaggc tgaatctcgc 1260
ccacaaaaag ctggagtttc tagtactcgc gactcaaaat gtcgcggaga atacgttccc 1320
gcgccttgca cacacccccc ctcaccccat gagagttcgc aatacccaaa gttcgtgagc 1380
taacctcaag gaggcagcga cctaagtagt agcgt 1415

Claims (2)

1. a kind of Quick production method of the compound Chinese herb probiotics based on solid state fermentation, it is characterised in that step is as follows：
（1）, four kinds of fermentation strains activation：
Enterococcus faecalis（Enterococcus faecalis）BD-1 activation：By enterococcus faecalis BD-1 test tube slants strain in nothing Rinsed under collarium border with sterile saline, be then seeded into the MRS fluid nutrient mediums of the palpus extract 0.2 ~ 0.5% containing radix pseudostellariae In, 35 ~ 37 DEG C of 42 ~ 48h of static gas wave refrigerator；Wherein, the percentage of the radix pseudostellariae palpus extract is with quality percent by volume g/mL Meter；Enterococcus faecalis BD-1 is new strains, and deposit number is CCTCC NO：M2017416；
Clostridium butyricum（Clostridium butyricum）RCM-2 activation：Clostridium butyricum RCM-2 test tube slants strain is existed Rinsed under gnotobasis with sterile saline, be then seeded into the MRS Liquid Cultures of the palpus extract 0.2 ~ 0.5% containing radix pseudostellariae In base, 35 ~ 37 DEG C of 42 ~ 48h of static gas wave refrigerator；Wherein, the percentage of the radix pseudostellariae palpus extract is with quality percent by volume g/ ML is counted；Clostridium butyricum RCM-2 is new strains, and deposit number is CCTCC NO M2017396；
Saccharomyces cerevisiae（Saccharomyces cerevisiae）Activation：Commercialization edible saccharomyces cerevisiae powder is inoculated into liquid In body culture medium, 30 ~ 37 DEG C of 24 ~ 32h of static gas wave refrigerator；Fluid nutrient medium is constituted：Brewer's wort 40 ~ 60%, glucose 5 ~ 10%, too The water of son ginseng palpus extract 0.2 ~ 0.5% and surplus, natural pH；Wherein, the percentage of brewer's wort is with volume percentage, grape The percentage of sugar and radix pseudostellariae palpus extract is in terms of g/mL；
Young clostridium（Clostridium ljungdahlii）Activation：The Young clostridium bacterium solution of cryopreservation tube preservation is disposable In the MRS liquid culture medium for being inoculated into the palpus extract 0.2 ~ 0.5% containing radix pseudostellariae, 35 ~ 37 DEG C of 42 ~ 48h of static gas wave refrigerator；Its In, the percentage of the radix pseudostellariae palpus extract is in terms of quality percent by volume g/mL；
（2）, prepare enterococcus faecalis BD-1 and clostridium butyricum RCM-2 mixed fermentation seed liquor, be calculated as seed liquor A：
Under gnotobasis, by step（1）Gained enterococcus faecalis BD-1 activating solution and clostridium butyricum RCM-2 activating solution press volume It is inoculated into than 1: 1 ~ 5: 1 composition admixture activation seed liquor, then by percent by volume 3 ~ 5% containing radix pseudostellariae palpus extract 0.2 ~ 0.5% MRS fluid nutrient mediums in, seed liquor A is made in 36 ~ 48h of static fermentation at 35 ~ 37 DEG C；Wherein, the radix pseudostellariae must extract Percentage in terms of quality percent by volume g/mL；
（3）, prepare saccharomyces cerevisiae seed liquor, be calculated as seed liquor B：
Under gnotobasis, by step（1）The activating solution of gained saccharomyces cerevisiae is inoculated into fluid nutrient medium by percent by volume 5 ~ 10% In, seed liquor B is made in 30 ~ 37 DEG C of 30 ~ 36h of static gas wave refrigerator；Fluid nutrient medium is constituted：Brewer's wort 40 ~ 60%, glucose 5 ~ 10%th, radix pseudostellariae palpus extract 0.2 ~ 0.5% and the water of surplus, natural pH；Wherein, the percentage of brewer's wort is with percent by volume The percentage of meter, glucose and radix pseudostellariae palpus extract is in terms of g/mL；
（4）, prepare Young clostridium seed liquor, be calculated as seed liquor C：
Under gnotobasis, by step（1）The activating solution of gained Young clostridium is inoculated into palpus containing radix pseudostellariae by percent by volume 3 ~ 7% In the MRS liquid culture medium of extract 0.2 ~ 0.5%, seed liquor C is made in 32 ~ 36h of static fermentation at 35 ~ 37 DEG C；Wherein, The percentage of the radix pseudostellariae palpus extract is in terms of quality percent by volume g/mL；
（5）, seed liquor A, B, C proportioning prepare fermentation seed liquid：
By step（2）、（3）、（4）Gained seed liquor A, B, C are mixed into fermentation seed at A: B: C=1: 1: 1 ~ 5: 4: 3 by volume Liquid；
（6）, prepare fermentation substrate matrix：
In terms of mass fraction, the fermentation substrate matrix mixed after crushed by following raw materials constituted 1. or in constituting 2. and Into；
Composition is 1.：Radix pseudostellariae must 20 ~ 60 parts, 10 ~ 30 parts of dried orange peel, 10 ~ 30 parts of hawthorn, 10 ~ 30 parts of Medicated Leaven, 5 ~ 15 parts of green tea, 0.05 ~ 0.1 part of allicin；
Composition is 2.：20 ~ 30 parts of radix pseudostellariae, 20 ~ 25 parts of the Radix Astragali, 10 ~ 15 parts of Radix Codonopsis, 10 ~ 15 parts of Radix Isatidis, 10 ~ 15 parts of folium isatidis, 5 ~ 10 parts of honeysuckle, 10 ~ 15 parts of dried orange peel, 10 ~ 15 parts of RHIIZOMA DIOSCOREAE from Henan of China, 5 ~ 15 parts of green tea, 0.05 ~ 0.1 part of allicin；
（7）, fermentation：
By step（5）Gained fermentation seed liquid adds step（6）Gained fermentation substrate Medium Culture, is stirred and evenly mixed, and loads fermentation bag And seal, then fermentation bag is fermented 4 ~ 7d under the conditions of lucifuge in 32 ~ 37 DEG C, compound Chinese herb probiotics is made；Wherein, In terms of volume mass percentage L/kg, the percentage that fermentation seed liquid accounts for fermentation substrate matrix is 25 ~ 35%.
2. a kind of compound Chinese herb probiotics prepared using preparation method as claimed in claim 1.
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