CN109136156A - Solid composite bacteria agent and preparation method thereof and the application in officinal dendrobium stem plantation - Google Patents
Solid composite bacteria agent and preparation method thereof and the application in officinal dendrobium stem plantation Download PDFInfo
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Abstract
The invention discloses a kind of solid composite bacteria agent, which includes bacillus megaterium (Bacillus megaterium) NL-7 microbial inoculum, bacillus thuringiensis (Bacillus thuringiensis) NL-11 microbial inoculum and thermophilic carbon monoxide streptomycete (Streptomyces thermocarboxydus) NL-1 microbial inoculum.Also disclose the preparation method of above-mentioned composite bacteria agent and its application in officinal dendrobium stem plantation.The present invention has the advantages that increasing unit cultivated area dendrobium candidum stem weight after solid composite bacteria agent of the present invention is applied to dendrobium candidum, improving dendrobium polysaccharide content, the medical value of dendrobium candidum can be improved, there is good application and development prospect.
Description
Technical field
The invention belongs to microorganisms technical field, it is more particularly to a kind of solid composite bacteria agent and preparation method thereof and in iron
Application in skin dendrobe cultivation.
Background technique
Microbial bacterial agent after being manured into soil, passes through the quick of its specific bacterial strain as a kind of novel cultivation matrix modifying agent
Breeding can improve soil nutrient state of supply, play the potentiality of environment nutrient, and it is micro- to build a good soil for plant growth
Environment, reduce environmental pollution, improve crop yield and in terms of be of great significance.
Dendrobium candidum (Dendrobium officinale Kmiura et Migo) is orchid family Dendrobium Sw, is China
Traditional rare traditional Chinese medicine is listed in top grade in Shennong's Herbal and Compendium of Material Medica, and there is nourishing Yin and promoting production of body fluid, throat soothing to protect
Throat, warm stomach improving eyesight, kidney tonifying benefit power, the effect of promoting longevity.Modern pharmacological studies have shown that dendrobium candidum medical active power and more
Sugar amount height is related, and polysaccharide, which has, to be improved immunity, anti-aging, inhibits tumour, hypoglycemic, blood pressure lowering and other effects.Iron sheet stone
Dry measure used in former times growth scope is wide, but area is small, is substantially in spot distribution, likes warm, wet, shady and cool environment, limitation growth Yushan Hill
Ground moistens and in the original broad-leaf forest of closing, especially overhanging cliff, under face pool, have oblique fire sunlight reflection, the mountain ridge weight
Folded, ravines and guillies criss-cross microhabitat, is born on the rock for having humus to assemble or dry and decayed tree for being abound with moss with its aerial root.
Due to the high medical value of dendrobium candidum, wild resource is largely excavated, in addition its special growing environment,
Cause the wild resource of dendrobium candidum endangered.Dendrobium candidum is mainly artificial cultivation at present, but there are polysaccharide for artificial cultivation
Content is low, drug effect is not so good as the problems such as wild.It has been found that dendrobium candidum endogenetic fungus, which grows it, facilitation, moreover it is possible to
Improve its secondary metabolites content.But the microbial solid composite bacteria agent for directly acting on dendrobium officinale culture medium
Use, have not been reported yet.
Patent CN103087953A discloses bacillus megaterium (Bacillus megaterium) NL-7, deposit number
CCTCC NO:M 2012452, preservation date: on November 9th, 2012；Patent CN103087954A discloses Su Yun gold gemma bar
Bacterium (Bacillus thuringiensis) NL-11, deposit number CCTCC NO:M 2012453, preservation date: 2012 11
The moon 9；Patent CN103103151A discloses thermophilic carbon monoxide streptomycete (Streptomyces thermocarboxydus)
NL-1, deposit number CCTCC NO:M 2012460, preservation date: on November 19th, 2012.Above-mentioned bacterial strains are preserved in Chinese allusion quotation
Type culture collection, depositary institution address: Wuhan, China Wuhan University.Above-mentioned three kinds of bacterial strains are sieved from rock surface bacterium colony
Choosing obtains, and has very strong erosiveness to limestone, can promote the release of the leading ions such as Ca, Mg in rock, accelerates limestone
Cut out soil；Matrix capacity of sowing grass seeds by duster can be reduced, the impact resilience and shear resistance of matrix of sowing grass seeds by duster are enhanced.Wherein, bacillus megaterium NL-
7 and bacillus thuringiensis NL-11 also solves the potential of K, P；And data is shown, thermophilic carbon monoxide strepto- mushroom has degradation fine
Tie up disposition energy.But it there is no the report for being used in compounding three at present, also without the iron sheet being used for based on rock and bark
Report in dendrobe cultivation matrix.
Summary of the invention
Technical problem to be solved by the present invention lies in provide one kind to increase stem weight and the raising of dendrobium candidum
Solid composite bacteria agent of dendrobium polysaccharide content and preparation method thereof and the application in officinal dendrobium stem plantation.
The present invention is to solve above-mentioned technical problem by the following technical programs:
On the one hand, a kind of solid composite bacteria agent is provided, which includes: bacillus megaterium (Bacillus
Megaterium) NL-7 microbial inoculum, bacillus thuringiensis (Bacillus thuringiensis) NL-11 microbial inoculum and a thermophilic oxygen
Change carbochain mould (Streptomyces thermocarboxydus) NL-1 microbial inoculum.
Preferably, bacillus megaterium NL-7 microbial inoculum and bacillus thuringiensis NL-11 microbial inoculum viable count be all larger than 4 ×
108A/g, thermophilic carbon monoxide streptomycete NL-1 microbial inoculum viable count are greater than 1.5 × 108A/g.
Preferably, bacillus megaterium NL-7 microbial inoculum, bacillus thuringiensis NL-11 microbial inoculum and thermophilic carbon monoxide strepto-
The weight ratio of bacterium NL-1 microbial inoculum is 1:1:1.
On the other hand, the preparation method of above-mentioned solid composite bacteria agent is also provided, this method comprises: respectively by huge gemma bar
Bacterium NL-7 bacterial strain, bacillus thuringiensis NL-11 bacterial strain and thermophilic carbon monoxide streptomycete NL-1 strain inoculated are to activation culture
Base carries out activation culture；Bacterial strain after activation is seeded to seed culture medium culture respectively, obtains bacterial strain seed liquor；According to solid
The 3%~5% of body matrix weight is respectively by bacillus megaterium NL-7 bacterial strain seed liquor, bacillus thuringiensis NL-11 bacterial strain
It is cultivated in seed liquor and thermophilic carbon monoxide streptomycete NL-1 bacterial strain seed liquor access solid matrix, obtains bacillus megaterium
NL-7 microbial inoculum, bacillus thuringiensis NL-11 microbial inoculum and thermophilic carbon monoxide streptomycete NL-1 microbial inoculum；By bacillus megaterium
NL-7 microbial inoculum, bacillus thuringiensis NL-11 microbial inoculum and thermophilic carbon monoxide streptomycete NL-1 microbial inoculum are mixed according to weight ratio 1:1:1
It closes uniformly to get solid composite bacteria agent.
Preferably, bacterial strain is no less than in bacillus megaterium NL-7 and bacillus thuringiensis NL-11 bacterial strain seed liquor
1.5×109Cfu, thermophilic carbon monoxide streptomycete NL-1 bacterial strain seed liquor mycelium reach thick.
Preferably, bacillus megaterium NL-7 bacterial strain, bacillus thuringiensis NL-11 bacterial strain activation culture based component be
5g sucrose, 2g Na2HPO4、0.5g MgSO4·7H2O、0.005g FeCl3、0.5g CaCO3, 18g agar powder and 1000mL water；
The activation culture based component of thermophilic carbon monoxide streptomycete NL-1 bacterial strain is 1g KNO3, 20g soluble starch, 0.5g K2HPO4、
0.5g MgSO4·7H2O、0.5g NaCl、0.5gCaCO3、0.01g FeSO4, 18g agar powder and 1000mL water, every 300mL training
Support the potassium bichromate for being added that 1mL concentration is 3% in base.
Preferably, bacillus megaterium NL-7 bacterial strain, bacillus thuringiensis NL-11 bacterial strain seed culture based component be
5g sucrose, 2g Na2HPO4、0.5g MgSO4·7H2O、0.005g FeCl3、0.5g CaCO3With 1000mL water；A thermophilic oxidation
The seed culture based component of carbochain mould NL-1 bacterial strain is 1g KNO3, 20g soluble starch, 0.5g K2HPO4、0.5g
MgSO4·7H2O、0.5g NaCl、0.5g CaCO3、0.01g FeSO4With 1000mL water, 1mL is added in every 300mL culture medium
The potassium bichromate that concentration is 3%.
Preferably, solid matrix includes wheat bran, soybean powder and corn flour, and the weight ratio of wheat bran, soybean powder and corn flour is
3:1:1。
Preferably, the temperature of bacterial strain activation culture is 28 DEG C, bacillus megaterium NL-7 bacterial strain, bacillus thuringiensis
The activation culture time of NL-11 bacterial strain is 2~3 days；The activation culture time of thermophilic carbon monoxide streptomycete NL-1 bacterial strain be 4~
6 days；
The condition of culture of bacterial strain seed liquor culture is 28 DEG C, 180r/min shake culture 2~3 days；
The temperature that bacterial strain seed liquor is cultivated in solid matrix is 28 DEG C, and the time is 8~10 days.
Another aspect, also application of the offer solid composite bacteria agent in officinal dendrobium stem plantation.
Cultural method are as follows: peat soil, perlite, pine bark, sheep dung and rape cake are mixed, through overlay film compost fermentation 40~
After 60 days, fermentation material is sieved with the mesh screen of aperture about 2~3cm.The fermentation material for passing through mesh screen, it is standby as upper layer cultivation matrix
With；It with the stone that grade is 3~4cm is by volume that 2:3 is mixed by the fermentation material for not passing through mesh screen, as bottom cultivation base
Matter, it is spare.When cultivation of dendrobium officinale, upper layer cultivation matrix and solid composite bacteria agent 96:4 in mass ratio are mixed, cover iron sheet
Dendrobium nobile root.
Preferably, the mass ratio of peat soil, perlite, pine bark, sheep dung and rape cake is 10:10:60:3:2.
Preferably, upper layer cultivation matrix with a thickness of 2~3cm, bottom cultivation matrix with a thickness of 5~8cm.
The present invention has the advantage that solid composite bacteria agent of the invention compared with prior art, passes through specific bacterial strain kind
Class and proportion can increase unit cultivated area dendrobium candidum stem weight, improve dendrobium polysaccharide content, convenient to use, tool
There is good application and development prospect.
Specific embodiment
It elaborates below to the embodiment of the present invention, the present embodiment carries out under the premise of the technical scheme of the present invention
Implement, the detailed implementation method and specific operation process are given, but protection scope of the present invention is not limited to following implementation
Example.
Bacillus megaterium (Bacillus megaterium) NL-7 bacterial strain is disclosed in patent CN103087953A；Soviet Union
Cloud gold bacillus (Bacillus thuringiensis) NL-11 bacterial strain is disclosed in patent CN103087954A, and thermophilic one
Carbonoxide streptomycete (Streptomyces thermocarboxydus) NL-1 bacterial strain is disclosed in patent CN103103151A,
Above-mentioned bacterial strains are taken from the China typical culture collection center of Wuhan, China Wuhan University.
Embodiment 1 prepares solid composite bacteria agent:
The preparation of culture medium: the activation culture based component of bacillus thuringiensis NL-11, bacillus megaterium NL-7 are as follows:
5g sucrose, 2g Na2HPO4、0.5g MgSO4·7H2O、0.005g FeCl3、0.5g CaCO3, 18g agar powder and 1000mL water.
The difference of bacillus thuringiensis NL-11, the seed culture based component of bacillus megaterium NL-7 and activation culture based component are only
It is not add agar powder in seed culture medium.The activation culture based component of thermophilic carbon monoxide streptomycete NL-1 are as follows: 1g
KNO3, 20g soluble starch, 0.5gK2HPO4、0.5g MgSO4·7H2O、0.5g NaCl、0.5g CaCO3、0.01g
FeSO4, 18g agar powder and 1000mL water, it is 3% potassium bichromate that 1mL concentration, which is added, in every 300mL culture medium.A thermophilic oxidation
The seed culture based component of carbochain mould NL-1 and the difference of activation culture based component, which are only that in seed culture medium, does not add fine jade
Cosmetics.For above-mentioned culture medium through 121 DEG C, 30min sterilizing is spare.
Bacterial strain activation: the bacillus thuringiensis NL-11 deposited that goes bail for, bacillus megaterium NL-7 bacterial strain are coated on equipped with it
In the culture dish of activation medium, cultivated 2~3 days in 28 DEG C, it is spare.The thermophilic carbon monoxide streptomycete NL-1 deposited that goes bail for is coated with
In the culture dish equipped with its activation medium, cultivated 4~6 days in 28 DEG C, it is spare.
Seed liquor preparation: bacillus thuringiensis NL-11, the bacillus megaterium of above-mentioned activation are taken with transfer needle respectively
The pure bacterium of NL-7, thermophilic carbon monoxide streptomycete NL-1 are inoculated in the 250mL triangle equipped with the respective seed culture medium of 50mL respectively
In bottle, 28 DEG C, 180r/min shake culture 2~3 days, bacterial strain seed liquor is obtained.Wherein, bacillus megaterium NL-7 and Su Yunjin
Bacterial strain number is no less than 1.5 × 10 in bacillus NL-11 bacterial strain seed liquor9cfu；Thermophilic carbon monoxide streptomycete NL-1 bacterial strain kind
Sub- liquid mycelium reaches thick.
Solid matrix pretreatment: it dries, smash to pieces, mix after 3Kg wheat bran, 1Kg soybean powder, 1kg corn flour are cooked.Inoculation
First 121 DEG C, 30min sterilizing.It is dried after sterilizing in sterile ventilation, prevents from agglomerating.
The preparation of solid fungicide: according to the amount of solid matrix weight 3%~5%, respectively by the huge gemma of above-mentioned preparation
The seed liquor of bacillus NL-7 bacterium, bacillus thuringiensis NL-11 bacterium and thermophilic carbon monoxide streptomycete NL-1 bacterium is inoculated into above-mentioned
It is mixed in solid matrix, is 60% or so with the water content that distilled water adjusts mixture, the disposable paper after being loaded into sterilizing
Matter cutlery box is wrapped up with 4 layers of sterilizing yarn, is placed in 28 DEG C of cultures and stirs, stir under gnotobasis at interval of 2 days on superclean bench
It mixes, the disposable paper cutlery box after reloading sterilizing is wrapped up with 4 layers of sterilizing yarn, is placed in 28 DEG C of cultures, is obtained after 8~10 days
Obtain bacillus megaterium NL-7 microbial inoculum, bacillus thuringiensis NL-11 microbial inoculum and thermophilic carbon monoxide streptomycete NL-1 microbial inoculum.Its
In, bacillus megaterium NL-7 microbial inoculum and bacillus thuringiensis NL-11 microbial inoculum viable count are greater than 4 × 108A/g, a thermophilic oxygen
Change carbochain mould NL-1 microbial inoculum viable count and is greater than 1.5 × 108A/g.
The preparation of solid composite bacteria agent: by bacillus megaterium NL-7 microbial inoculum, bacillus thuringiensis NL-11 microbial inoculum and thermophilic
Hot carbon monoxide streptomycete NL-1 microbial inoculum is uniformly mixed according to weight ratio 1:1:1, that is, prepares solid composite bacteria agent.It is obtained
Solid composite bacteria agent is brown, micro- stink pulvis.
Application of the 2 solid composite bacteria agent of embodiment in officinal dendrobium stem plantation
Peat soil 10Kg, perlite 10Kg, pine bark 60Kg, sheep dung 3Kg, rape cake 2Kg are mixed, sent out through overlay film compost
Ferment after 40~60 days, is sieved with the mesh screen of aperture about 2~3cm.The fermentation material of mesh screen is passed through as covering dendrobium candidum root
Upper layer cultivation matrix, cladding thickness be 2~3cm；To not pass through the fermentation materials such as the coarse bark of mesh screen and grade be 3~
The stone of 4cm is layered on bottom as bottom cultivation matrix, with a thickness of 5~8cm after 2:3 is mixed by volume.
The planting density of dendrobium candidum be every square meter 10 × 10 clumps, 3~5 plants every clump.Cultivation in First Year April in spring, on
Solid composite bacteria agent prepared by layer cultivation matrix and embodiment 1 is mixed by weight 96:4 to be used.Setting control matrix group and sky
White control group, control matrix group is only that with the difference of above-mentioned solid composite bacteria agent group, is added in the cultivation matrix of dendrobium candidum upper layer
Equivalent compares matrix, compares preparation of the processing with solid fungicide of matrix, and equivalent is added in solid matrix and does not access bacterial strain
The seed culture medium of corresponding three kinds of bacterial strains；Blank control group and the difference of above-mentioned cultivation matrix are only that upper layer cultivation matrix is not
Any processing is done, i.e., does not add solid composite bacteria agent or control matrix.Second year is added into upper layer cultivation matrix April again
The solid composite bacteria agent of layer cultivation matrix weight 2% (do not appoint by the control matrix of control matrix group addition equivalent, blank control group
Where reason), other management measures are consistent, and every kind of processing repeats multiple samples are arranged.
Acquiring dendrobium candidum second year December, raw aerial part stem (divide for sampling by the similar region of selection growing way for the year
Analysis), it is placed in 60 DEG C of baking oven drying and causes constant weight, weigh its stem weight respectively；Determination of polysaccharide is according to 2015 editions " Chinese Pharmacopoeias "
" dendrobium candidum " Methods in Determination of Polysaccaride Content measures in (one) medicinal material and medicine materical crude slice.
Unit cultivated area stem reflects dendrobium candidum medicinal effects yield again, seen from table 1, at solid composite bacteria agent
Stem is higher by 14.5%, 15.5% than control matrix group and blank control group respectively again after reason, compares matrix group and blank control
There was no significant difference for the stem weight of group.
Dendrobium polysaccharide is the medicinal main component of dendrobium candidum, reflects the quality of dendrobium candidum, 2015 editions " middle traditional Chinese medicines
Allusion quotation " (one) regulation dendrobium polysaccharide must not be less than 25.0%.As shown in Table 1, dendrobium polysaccharide in stem after solid composite bacteria agent is handled
Average content is 38.1%, improves 22.1% and 23.3%, control matrix group and sky than control matrix group and blank control group
There was no significant difference for the dendrobium polysaccharide content of white control group.
Influence of the 1 solid composite bacteria agent of table to dendrobium candidum stem weight and dendrobium polysaccharide content
| Processing | Stem weight (g/m2) | Dendrobium polysaccharide (%) |
| Solid composite bacteria agent group | 119.4±11.3a | 38.1±4.3a |
| Compare matrix group | 104.3±9.9b | 31.2±4.8b |
| Blank control group | 103.4±9.4b | 30.9±4.7b |
Note: numerical value is mean ± standard deviation in table, is existed in 0.05 level between the different alphabetical expression processing of same column significant
Difference.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (8)
1. a kind of solid composite bacteria agent, which is characterized in that the solid composite bacteria agent includes: bacillus megaterium (Bacillus
Megaterium) NL-7 microbial inoculum, bacillus thuringiensis (Bacillus thuringiensis) NL-11 microbial inoculum and a thermophilic oxygen
Change carbochain mould (Streptomyces thermocarboxydus) NL-1 microbial inoculum.
2. solid composite bacteria agent according to claim 1, which is characterized in that the bacillus megaterium NL-7 microbial inoculum and Soviet Union
Cloud gold bacillus NL-11 microbial inoculum viable count is all larger than 4 × 108A/g, the thermophilic carbon monoxide streptomycete NL-1 microbial inoculum are living
Bacterium number is greater than 1.5 × 108A/g.
3. solid composite bacteria agent according to claim 1, which is characterized in that the bacillus megaterium NL-7 microbial inoculum, Soviet Union
The weight ratio of cloud gold bacillus NL-11 microbial inoculum and thermophilic carbon monoxide streptomycete NL-1 microbial inoculum is 1:1:1.
4. the preparation method of solid composite bacteria agent described in any one of claims 1 to 3, which is characterized in that the preparation side
Method includes: respectively by bacillus megaterium NL-7 bacterial strain, bacillus thuringiensis NL-11 bacterial strain and thermophilic carbon monoxide streptomycete
NL-1 strain inoculated carries out activation culture to activation medium；Bacterial strain after activation is seeded to seed culture medium culture respectively,
Obtain bacterial strain seed liquor；According to solid matrix weight 3%~5% respectively by bacillus megaterium NL-7 bacterial strain seed liquor, Soviet Union
Cloud gold bacillus NL-11 bacterial strain seed liquor and thermophilic carbon monoxide streptomycete NL-1 bacterial strain seed liquor access the solid matrix
Middle culture obtains bacillus NL-7 microbial inoculum, bacillus thuringiensis NL-11 microbial inoculum and thermophilic carbon monoxide streptomycete NL-1 bacterium
Agent；By the bacillus megaterium NL-7 microbial inoculum, bacillus thuringiensis NL-11 microbial inoculum and thermophilic carbon monoxide streptomycete NL-1
Microbial inoculum is uniformly mixed according to weight ratio 1:1:1 to get solid composite bacteria agent.
5. the preparation method of solid composite bacteria agent according to claim 4, which is characterized in that the bacillus megaterium
Bacterial strain is no less than 1.5 × 10 in NL-7 and bacillus thuringiensis NL-11 bacterial strain seed liquor9Cfu, the thermophilic carbon monoxide
Streptomycete NL-1 bacterial strain seed liquor mycelium reaches thick.
6. the preparation method of solid composite bacteria agent according to claim 4, which is characterized in that the bacillus megaterium
NL-7 bacterial strain, bacillus thuringiensis NL-11 bacterial strain activation culture based component be 5g sucrose, 2g Na2HPO4、0.5g
MgSO4·7H2O、0.005g FeCl3、0.5g CaCO3, 18g agar powder and 1000mL water；The thermophilic carbon monoxide streptomycete
The activation culture based component of NL-1 bacterial strain is 1g KNO3, 20g soluble starch, 0.5g K2HPO4、0.5g MgSO4·7H2O、
0.5g NaCl、0.5g CaCO3、0.01g FeSO4, 18g agar powder and 1000mL water, it is dense that 1mL is added in every 300mL culture medium
The potassium bichromate that degree is 3%；
The bacillus megaterium NL-7 bacterial strain, bacillus thuringiensis NL-11 bacterial strain seed culture based component be 5g sucrose,
2g Na2HPO4、0.5g MgSO4·7H2O、0.005g FeCl3、0.5g CaCO3With 1000mL water；The thermophilic carbon monoxide
The seed culture based component of streptomycete NL-1 bacterial strain is 1g KNO3, 20g soluble starch, 0.5g K2HPO4、0.5g MgSO4·
7H2O、0.5g NaCl、0.5g CaCO3、0.01g FeSO4With 1000mL water, 1mL concentration is added in every 300mL culture medium is
3% potassium bichromate；
The solid matrix includes wheat bran, soybean powder and corn flour, and the weight ratio of wheat bran, soybean powder and corn flour is 3:1:1.
7. the preparation method of solid composite bacteria agent according to claim 4, which is characterized in that the bacterial strain activation culture
Temperature be 28 DEG C, the bacillus megaterium NL-7 bacterial strain, bacillus thuringiensis NL-11 bacterial strain the activation culture time be 2
~3 days；The activation culture time of the thermophilic carbon monoxide streptomycete NL-1 bacterial strain is 4~6 days；
The condition of culture of the bacterial strain seed liquor culture is 28 DEG C, 180r/min shake culture 2~3 days；
The temperature that the bacterial strain seed liquor is cultivated in solid matrix is 28 DEG C, and the time is 8~10 days.
8. application of the solid composite bacteria agent in officinal dendrobium stem plantation described in any one of claims 1 to 3.
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