CN108815202A - A kind of chitosan microbial compound preparation and its preparation method and application - Google Patents
A kind of chitosan microbial compound preparation and its preparation method and application Download PDFInfo
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- CN108815202A CN108815202A CN201810741792.6A CN201810741792A CN108815202A CN 108815202 A CN108815202 A CN 108815202A CN 201810741792 A CN201810741792 A CN 201810741792A CN 108815202 A CN108815202 A CN 108815202A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/716—Glucans
- A61K31/722—Chitin, chitosan
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/30—Aerobic and anaerobic processes
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
- C02F2103/20—Nature of the water, waste water, sewage or sludge to be treated from animal husbandry
Abstract
The present invention relates to microbial preparation fields, a kind of chitosan microbial compound preparation is provided, including chitosan, Bacillus acidi lactici and composite bacillus, the composite bacillus includes bacillus licheniformis, bacillus subtilis and Paenibacillus polymyxa, living bacteria count 1.5~2.5 hundred million/ml, pH value are 6.8~7.0.In chitosan microbial compound preparation provided by the invention, chitosan is compounded with Bacillus acidi lactici, bacillus can effectively reduce ammonia nitrogen in breeding water body, phosphorus and nitrate content, so as to improve the water quality of breeding water body；Chitosan microbial compound preparation of the present invention can also effectively facilitate soft-shelled turtle growth, increase weight and improve at least 15% relative to the control group not added；Meanwhile having different degrees of raising for the sero-immunity protein content and oxidation resistance of soft-shelled turtle, to significantly improve the immunocompetence of soft-shelled turtle.
Description
Technical field
The present invention relates to microbial preparation fields, and in particular to a kind of chitosan microbial compound preparation and preparation method thereof
And application.
Background technique
Soft-shelled turtle also known as the soft-shelled turtle, foot fish, water fish, cuckold, soft-shelled turtle nourishing is abundant, contains protein, fat, calcium, iron, animal
Glue, keratin and multivitamin are rare invigorants.Soft-shelled turtle can also be used as medicine, and dorsal shield has nourishment for vitality, dissipating bind
The effect of calming the liver.Soft-shelled turtle is because of its fine and tender taste, and delicious flavour, nutritive value is high and is favored deeply by the majority of consumers.
Currently, soft-shelled turtle mainly uses artificial cultivating method, the young soft-shelled turtle of artificial breeding is then based on high-density breeding mode.
Under high-density breeding mode, for the density of greenhouse cultivation children soft-shelled turtle at 25~35 every square metre, the outer pool cultivates young soft-shelled turtle density one
As 3000 or more per acre.When cultivation density is high, residual baits nutriment and excreta and other suspensions such as a large amount of nitrogen, phosphorus in water body
Particulate matter resolves into the noxious materials such as hydrogen sulfide, ammonia nitrogen, nitrite by anaerobic bacteria, and water quality is caused to deteriorate quickly.In order to mitigate
Water quality deterioration and soft-shelled turtle disease occur, and farm, which generallys use, changes water measure and use bleaching powder disinfectant, antibiotic, nitro furan
The sterilization of the chemicals such as class of muttering, disinfection.
It is widely varied however, changing water and frequently easily causing water environment physicochemical property, often causes the stress reaction of soft-shelled turtle,
It is largely broken out with immunity degradation, disease rapidly so as to cause ingesting.Many drawbacks, medicine are brought using chemicals such as disinfectants
The residual of object causes antibody-resistant bacterium to be increasing, intestinal bacilli illness and uncontrollable disease.Some drugs directly launch water body,
One side polluted-water directly jeopardizes the environment of human survival, and on the other hand, residual of the drug in aquatic products passes through food chain
Into human body, allergic reaction and toxic effect are generated to the mankind, seriously affect the health of the mankind.Therefore, it is strong to carry out soft-shelled turtle ecology
Health cultivation prevents water quality deterioration and mitigates disease, and the most important thing is " to prevent ", followed by " controls ".
Summary of the invention
The present invention in order to solve the drug of water quality deterioration in soft-shelled turtle high-density breeding mode, soft-shelled turtle disease control to environment and
The unsafe problem of human body, breeding water body water quality can be effectively improved, improve soft-shelled turtle immunity and make its weight gain by providing one kind
Chitosan microbial compound preparation, safety and environmental protection.
To solve the above-mentioned problems, the present invention provides following technical schemes：
The present invention provides a kind of chitosan microbial compound preparations, including chitosan, Bacillus acidi lactici and compound gemma bar
Bacterium, the composite bacillus include bacillus licheniformis, bacillus subtilis and Paenibacillus polymyxa, living bacteria count
1.5~2.5 hundred million/ml, pH value is 6.8~7.0.
Preferably, by weight, chitosan microbial compound preparation includes：5~10 parts of chitosan, living bacteria count
14~56 parts of composite bacillus of 7~14 parts of the Bacillus acidi lactici of 5.0 hundred million/ml or more and 2.0 hundred million/ml or more of living bacteria count.
Preferably, in the composite bacillus, bacillus licheniformis, bacillus subtilis and Paenibacillus polymyxa
Viable count ratio is 0.5~2:0.5~2:0.5~2.
It preferably, further include auxiliary material, the auxiliary material is selected from wheat bran and/or zeolite powder.
Preferably, the chitosan microbial compound preparation includes 30~35 parts of wheat bran and/or 15~20 parts of zeolite powder.
Preferably, the deacetylation of the chitosan is 85% or more, average molecular weight less than 100,000.
The present invention also provides the preparation method of chitosan microbial compound preparation described in above-mentioned technical proposal, including it is following
Step：
A, anaerobic fermentation is carried out to Bacillus acidi lactici with MRS fluid nutrient medium, obtains lactobacillus fermentation liquid；
B, with the beef-protein medium of improvement to bacillus subtilis, bacillus licheniformis and mostly viscous class gemma bar
Bacterium carries out mixed fermentation, obtains composite bacillus fermentation liquid；
By mass percentage, the beef-protein medium of the improvement includes beef extract 0.1~0.5%, albumen
Peptone 0.3~0.8%, sucrose 0.1~0.6%, sodium chloride 0.05~0.4%, dipotassium hydrogen phosphate 0.01~0.05%, biphosphate
The water of potassium 0.02~0.08% and surplus, pH are 6.8~7.2；
C, by lactobacillus fermentation liquid, composite bacillus fermentation liquid and chitosan mix, adjust pH value be 6.8~
7.0, obtain chitosan microbial compound preparation；
Out-of-order is successive between the step A and step B.
Preferably, when further including auxiliary material in chitosan microbial compound preparation, step C is replaced with：By auxiliary material and lactic acid
Bacillus fermentation liquid, composite bacillus fermentation liquid and chitosan mixing.
The present invention also provides chitosan microbial compound preparations described in preceding solution to improve breeding water body water quality
In application.
The present invention also provides chitosan microbial compound preparations described in preceding solution to improve soft-shelled turtle immunity, promote
Application into soft-shelled turtle growth.
Compared with prior art, technical solution provided by the invention has the following advantages that：
The present invention provides a kind of chitosan microbial compound preparations, including chitosan, Bacillus acidi lactici and compound gemma bar
Bacterium, the composite bacillus include bacillus licheniformis, bacillus subtilis and Paenibacillus polymyxa, living bacteria count
1.5~2.5 hundred million/ml, pH value is 6.8~7.0.In chitosan microbial compound preparation provided by the invention, chitosan and cream
Acidfast bacilli, bacillus compounding can effectively reduce ammonia nitrogen in breeding water body, phosphorus and nitrate content, so as to improve cultivation water
The water quality of body；Chitosan microbial compound preparation of the present invention can also effectively facilitate soft-shelled turtle growth, and weight gain is not relative to adding
The control group added improves at least 15%；Meanwhile it is different for the sero-immunity protein content and oxidation resistance of soft-shelled turtle
Degree improves, to significantly improve the immunocompetence of soft-shelled turtle.
In the present invention, it is compounded, can be played good with aerobic bacillus using the Bacillus acidi lactici of anaerobism
Complementary effect, Bacillus acidi lactici play the effect for improving animal intestinal tract and improving immunity, and it is organic that bacillus then plays degradation
Substance and the effect for inhibiting pathogen can generate and significantly change more fully with chitosan function synergic after mutual cooperation
Kind breeding water body water quality improves soft-shelled turtle immunity.
The research of the invention finds that for drop after bacillus subtilis, bacillus licheniformis and Paenibacillus polymyxa compounding
It solves organic matter and there is synergistic effect, more protease, amylase and chitinase can be generated after compounding, to improve pair
Organic matter quality capacity of decomposition.
Specific embodiment
The present invention provides a kind of chitosan microbial compound preparations, including chitosan, Bacillus acidi lactici and compound gemma bar
Bacterium, the composite bacillus include bacillus licheniformis, bacillus subtilis and Paenibacillus polymyxa, living bacteria count
1.5~2.5 hundred million/ml, pH value is 6.8~7.0.
In the present invention, the chitosan be preferably deacetylation in 85% or more, average molecular weight less than 100,000.At this
In invention, chitosan plays the role of improving soft-shelled turtle immunity, promotes soft-shelled turtle growth, reduces feed coefficient.The present invention is poly- to shell
The source of sugar is not particularly limited, using the commercial goods that can satisfy above-mentioned condition.
In the present invention, the Bacillus acidi lactici plays in chitosan microorganism formulation improves animal intestinal tract, improves body
The effect of immunity.Bacillus acidi lactici of the present invention is preferably lactobacillus acidophilus, specific bacterial strain of the present invention for Bacillus acidi lactici
It is not particularly limited, using known in the art, such as 393 bacterial strain of Bacillus acidi lactici ATCC.
In the present invention, the composite bacillus plays degradation of organic substances in chitosan microorganism formulation, inhibits disease
The effect of opportunistic pathogen.Composite bacillus of the present invention includes bacillus subtilis, bacillus licheniformis and mostly viscous class gemma
Bacillus, these three bacillus can significantly improve the degradation capability for organic substance after compounding, improve chitosan microorganism
Preparation is whole for the improvement of breeding water body water quality and the efficiency of feed utilization of soft-shelled turtle.In the present invention, the withered grass bud
Spore bacillus is preferably CICC20445；The bacillus licheniformis is preferably CICC 23584；The Paenibacillus polymyxa is preferred
For ATCC 842.
In the present invention, by weight, the chitosan microbial compound preparation preferably includes：Chitosan 5~10
Part, 7~14 parts of the Bacillus acidi lactici of 500,000,000/ml or more of viable count and the composite bacillus 14~56 of 2.0 hundred million/ml or more of viable count
Part；More preferably include：8 parts of chitosan, 10 parts of the Bacillus acidi lactici of 5.0 hundred million/ml or more of viable count and 2.0 hundred million/ml of viable count with
On 30 parts of composite bacillus.
It is currently preferred, in the composite bacillus, bacillus licheniformis, bacillus subtilis and mostly viscous class gemma
The viable count ratio of bacillus is 0.5~2:0.5~2:0.5~2；More preferably 1:1:1.
In the present invention, the chitosan microbial compound preparation preferably further includes auxiliary material, and the auxiliary material is selected from wheat bran
And/or zeolite powder.The present invention more preferably includes 30~35 parts of wheat bran, most preferably 32 parts；The present invention more preferably includes boiling
15~20 parts of mountain flour, most preferably 16 parts.
The purpose that the present invention adds auxiliary material is that various dosage forms are made in the compound of chitosan and microorganism.In the present invention
In, the dosage form of the chitosan microbial compound preparation includes but is not limited to pulvis, solution, suspending agent, emulsifiable concentrate or particle
Agent.The present invention can choose corresponding auxiliary material in order to which corresponding dosage form is made, and is prepared as using method well known in the art various
Dosage form.
The present invention also provides a kind of preparation methods of chitosan microbial compound preparation described in above-mentioned technical proposal, including
Following steps：
A, anaerobic fermentation is carried out to Bacillus acidi lactici with MRS fluid nutrient medium, obtains lactobacillus fermentation liquid；
B, with the beef-protein medium of improvement to bacillus subtilis, bacillus licheniformis and mostly viscous class gemma bar
Bacterium carries out mixed fermentation, obtains composite bacillus fermentation liquid；
According to mass percent meter, the beef-protein medium of the improvement includes beef extract 0.1~0.5%, egg
White peptone 0.3~0.8%, sucrose 0.1~0.6%, sodium chloride 0.05~0.4%, dipotassium hydrogen phosphate 0.01~0.05%, di(2-ethylhexyl)phosphate
The water of hydrogen potassium 0.02~0.08% and surplus, pH are 6.8~7.2；
C, by lactobacillus fermentation liquid, composite bacillus fermentation liquid and chitosan mix, adjust pH value be 6.8~
7.0, obtain chitosan microbial compound preparation；
Out-of-order is successive between the step A and step B.
The present invention carries out anaerobic fermentation to Bacillus acidi lactici with MRS fluid nutrient medium, when in the fermentation liquid for measuring Bacillus acidi lactici
Living bacteria count can terminate anaerobic fermentation when reaching 500,000,000/ml or more, obtain the lactic acid of 500,000,000/ml or more of living bacteria count
Bacillus fermentation liquid.
In the present invention, the anaerobic fermentation temperature is preferably 26~31 DEG C, and more preferably 28 DEG C；
The present invention is with the beef-protein medium improved to bacillus subtilis, bacillus licheniformis and mostly viscous class bud
Spore bacillus carries out mixed fermentation, can terminate to mix when measuring living bacteria count in mixed fermentation liquid and reaching 200,000,000/ml or more
Fermentation, obtains the composite bacillus fermentation liquid of effective 200,000,000/ml or more of viable bacteria.
In the present invention, the beef-protein medium of the improvement preferably includes beef extract 0.3%, peptone
0.5%, sucrose 0.3%, sodium chloride 0.2%, the water of dipotassium hydrogen phosphate 0.03%, potassium dihydrogen phosphate 0.05% and surplus, pH is
6.8~7.2.
In the present invention, the mixed fermentation carries out under aerobic condition, it is preferred that the oxyty of the mixed fermentation
Preferably 20~45%, more preferably 40%.In the present invention, the temperature of the mixed fermentation is preferably 27~31 DEG C, more excellent
It is selected as 29 DEG C.
Since Bacillus acidi lactici and bacillus are belonging respectively to anaerobic bacteria and aerobic bacteria, thus ferment respectively, the present invention
It is unlimited for the sequencing of anaerobic fermentation and mixed fermentation.
After lactobacillus fermentation liquid and composite bacillus fermentation liquid is prepared, the present invention is by both fermentation liquids and shell
Glycan mixing, adjusting pH value is 6.8~7.0, obtains chitosan microbial compound preparation.The pH value adjusting method is according to ability
Domain conventional technical means carries out, such as acid-base accommodation etc..
It is currently preferred, when the chitosan microbial compound preparation further includes auxiliary material, by auxiliary material and chitosan, cream
Acidfast bacilli fermentation liquid and composite bacillus fermentation liquid mix together.
The present invention also provides chitosan microbial compound preparations described in preceding solution to improve breeding water body water quality
In application.Specifically, chitosan microbial compound preparation of the present invention to be applied to the ammonia that can be effectively decomposed in water in water body
Nitrogen, phosphorus and nitrate improve the eutrophic state of breeding water body.Currently preferred, the chitosan microorganism is compound
Preparation is for when improving breeding water body water quality, mass concentration to be preferably 0.05~0.2%.
Chitosan microbial compound preparation of the present invention is for when improving breeding water body water quality, it directly to be shed in water
In body, splash again into water body after it can also being dissolved with water.In addition to this, the present invention can also be by the micro- life of chitosan
Object compound formulation feeds soft-shelled turtle after mixing with feed, can also achieve the effect that be effectively improved breeding water body water quality.
Soft-shelled turtle is improved in preparation the present invention also provides chitosan microbial compound preparation described in preceding solution to be immunized
Application in power, the soft-shelled turtle feed for promoting soft-shelled turtle to grow.Specifically, by chitosan microbial compound preparation of the present invention and first
Fish meal mixing is to get the soft-shelled turtle feed with raising soft-shelled turtle immunity and promotion soft-shelled turtle growth.
Currently preferred, the chitosan microbial compound preparation has raising soft-shelled turtle immunity and is promoting soft-shelled turtle raw
Mass fraction in long soft-shelled turtle feed is preferably 0.05~0.2%.
In order to further illustrate the present invention, technical solution provided by the invention is retouched in detail below with reference to embodiment
It states, but they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
Prepare MRS fluid nutrient medium, with 10% inoculum concentration into MRS fluid nutrient medium inoculating lactobacillus (ATCC
393), anaerobic fermentation 90h under the conditions of 26 DEG C, obtains the lactobacillus fermentation liquid of 5.5 hundred million/ml of bacteria containing amount.
Prepare the beef-protein medium of improvement：Beef extract 0.3%, peptone 0.5%, sucrose 0.3%, sodium chloride
0.2%, dipotassium hydrogen phosphate 0.03%, potassium dihydrogen phosphate 0.05%, pH are the water of 6.8-7.2 and surplus, with 10% inoculum concentration
It is inoculated with bacillus subtilis (CICC 20445), bacillus licheniformis (CICC 23584) and Paenibacillus polymyxa respectively
(ATCC 842), mixed fermentation 75h under conditions of 27 DEG C, 20% oxyty obtain the compound of 5.2 hundred million/ml of bacteria containing amount
Fermentation of bacillus liquid.
Lactobacillus fermentation liquid 12kg, composite bacillus fermentation liquid 24kg, wheat bran 35kg, zeolite powder 20kg and shell are gathered
Sugared 10kg after mixing, is vacuum-packed to get chitosan microbial compound preparation.
Detected, be prepared chitosan microbial compound preparation pH value be 6.89, living bacteria count be 1.5 hundred million/
ml。
Embodiment 2
Prepare MRS fluid nutrient medium, with 10% inoculum concentration into MRS fluid nutrient medium inoculating lactobacillus (ATCC
393), anaerobic fermentation 90h under the conditions of 31 DEG C, obtains the lactobacillus fermentation liquid of 5.0 hundred million/ml of bacteria containing amount.
Prepare the beef-protein medium of improvement：Beef extract 0.3%, peptone 0.5%, sucrose 0.3%, sodium chloride
0.2%, the water of dipotassium hydrogen phosphate 0.03%, potassium dihydrogen phosphate 0.05% and surplus, pH is 6.8~7.2.With 10% inoculum concentration
It is inoculated with bacillus subtilis (CICC 20445), bacillus licheniformis (CICC 23584) and Paenibacillus polymyxa respectively
(ATCC 842), mixed fermentation 75h under conditions of 31 DEG C, 45%, obtains the composite bacillus of 2.6 hundred million/ml of bacteria containing amount
Fermentation liquid.
Lactobacillus fermentation liquid 8kg, composite bacillus fermentation liquid 32kg, wheat bran 30kg, zeolite powder 20kg and shell are gathered
Sugared 5kg after mixing, is vacuum-packed to get chitosan microbial compound preparation.
Detected, be prepared chitosan microbial compound preparation pH value be 6.92, living bacteria count be 2.0 hundred million/
ml。
Embodiment 3
Prepare MRS fluid nutrient medium, with 10% inoculum concentration into MRS fluid nutrient medium inoculating lactobacillus (ATCC
393), anaerobic fermentation 90h under the conditions of 28 DEG C, obtains the lactobacillus fermentation liquid of 5.0 hundred million/ml of bacteria containing amount.
Prepare the beef-protein medium of improvement：Beef extract 0.3%, peptone 0.5%, sucrose 0.3%, sodium chloride
0.2%, the water of dipotassium hydrogen phosphate 0.03%, potassium dihydrogen phosphate 0.05% and surplus, pH 6.8-7.2.With 10% inoculum concentration
It is inoculated with bacillus subtilis (CICC 20445), bacillus licheniformis (CICC 23584) and Paenibacillus polymyxa respectively
(ATCC 842), mixed fermentation 75h under conditions of 29 DEG C, 40%, obtains the composite bacillus of 2.0 hundred million/ml of bacteria containing amount
Fermentation liquid.
Lactobacillus fermentation liquid 10kg, composite bacillus fermentation liquid 28kg, wheat bran 31kg, zeolite powder 17kg and shell are gathered
Sugared 6kg after mixing, is vacuum-packed to get chitosan microbial compound preparation.
Detected, be prepared chitosan microbial compound preparation pH value be 6.85, living bacteria count be 2.5 hundred million/
ml。
Embodiment 4
The organic matter degradation effect of composite bacillus is verified in this test
1, subjects：
Bacillus subtilis (CICC 20445), bacillus licheniformis (CICC 23584) and Paenibacillus polymyxa
(ATCC 842)；
Composite bacillus：Bacillus subtilis, bacillus licheniformis and the Paenibacillus polymyxa of equal viable counts compounding.
The protease to three kinds of bacillus individually and after compounding, amylase, chitinase yield detect respectively.
Degradation phosphorus effect of three kinds of bacillus individually and after compounding is detected.
2, detection method：
(1) protease activity determination
Protease activity determination culture medium：1.5% peptone, 1.0% sucrose, 0.045% magnesium sulfate, 0.03% chlorination
Sodium, 0.04% dipotassium hydrogen phosphate, pH7.0~7.5.
Bacillus subtilis, bacillus licheniformis, Paenibacillus polymyxa and three kinds of bacillus subtilises are pressed respectively
Meet according to equal proportion viable count and be inoculated into protease activity determination culture medium according to 10% volume, in 28~30 DEG C, 280rpm
Revolving speed shaking table culture 72h, takes supernatant to medium centrifugal 10min at 8000rpm after culture, using Folin phenol method
Measure the prolease activity in supernatant.
(2) amylase activity measures
Amylase activity measures culture medium：2.0% soluble starch, 0.5% peptone, 0.5% yeast extract, 0.08% chlorine
Change sodium, 0.06 potassium chloride, pH nature.
Bacillus subtilis, bacillus licheniformis, Paenibacillus polymyxa and three kinds of bacillus subtilises are pressed respectively
Meet according to equal proportion viable count and be inoculated into amylase activity measurement culture medium according to 10% volume, in 28~30 DEG C, 280rpm
Revolving speed shaking table culture 72h takes supernatant to medium centrifugal 10min at 8000rpm after culture, measures in supernatant
The method of amylase activity is referring to GB/8275-2009.
(3) chitinase activity measures
Chitinase activity measures culture medium：20% colloid chitin, 0.05% potassium dihydrogen phosphate, 0.05% phosphoric acid hydrogen two
Potassium, 0.05% sodium chloride, ferrous sulfate and manganese sulfate are micro, pH 6.0-6.5,
Bacillus subtilis, bacillus licheniformis, Paenibacillus polymyxa and three kinds of bacillus subtilises are pressed respectively
Meet according to equal proportion viable count and be inoculated into amylase activity measurement culture medium according to 10% volume, in 28~30 DEG C, 280rpm
Revolving speed shaking table culture 72h takes supernatant, chitin in supernatant to medium centrifugal 10min at 8000rpm after culture
Matter activity determination method is DNS (3,5- dinitrosalicylic acid).
(4) measurement of bacterium dissolving P capacity
Dissolving P capacity measures culture medium：1.0% glucose, 0.05% ammonium sulfate, 0.03% sodium chloride, 0.03% sulfuric acid
Magnesium, 0.15% tricalcium phosphate, 0.15% lecithin, manganese sulfate and ferrous sulfate are micro, pH 7.0~7.2.
Bacillus subtilis, bacillus licheniformis, Paenibacillus polymyxa and three kinds of bacillus subtilises are pressed respectively
Meet according to equal proportion viable count and be inoculated into amylase activity measurement culture medium according to 10% volume, in 28~30 DEG C, 280rpm
Revolving speed shaking table culture 72h takes supernatant to medium centrifugal 10min at 8000rpm after culture, measures in supernatant
The content of rapid available phosphorus uses molybdenum blue colorimetric method.
3, measurement result
1 three kinds of bacillus of table are applied alone compared with the effect of compounding
As can be seen from Table 1, under equivalent inoculation, protease, amylase and the chitinase of the mixing of three kinds of bacillus
Content is higher, has better degradation of organic substances matter effect, and synergistic effect is obvious.Show the present invention using three kinds of bacillus
Significantly more organic matter degradation effect can be obtained after compounding, hence for water quality purification and animal feed utilization efficiency all
It is significantly increased.
Embodiment 5
This test improves water quality to chitosan microbial compound preparation and promotes growth to soft-shelled turtle, improves immune effect
Fruit is verified
1, material
The young soft-shelled turtle of China that soft-shelled turtle variety selection size is 50g is carried out in aquaculture pond, area is indoors as research material
15m2, depth of water 30-40cm Ni Dichi, the young soft-shelled turtle quantity of each cultivation China is 110, and aquaculture pond is equipped with two head inflator pumps and mends
It is oxygenated, dissolved oxygen is kept to be not less than 3.0mg/L.
Soft-shelled turtle feed：Young first special feed (soft-shelled turtle compound feed of Huzhou Hai Huang Biotechnology Co., Ltd production).
The chitosan microbial compound preparation prepared using embodiment 1.
2, test method
Chitosan microbial compound preparation is added to aquaculture pond using two ways：
Mode 1 is directly to splash, and three gradients are arranged, and is used according to water total volume 0.5 ‰, 1.0 ‰ and 2.0 ‰, often
It spilled once within 10 days：Chitosan microbial compound preparation first is diluted with water in a small amount of aquaculture pond, is uniformly splashed in the aquaculture pond water surface.
Mode 2 is to feed Shelled Turtle Trionyx Sinensis again after mixing with feed, and the dosage being added in soft-shelled turtle feed is 0.5 ‰, 1 ‰ and
2‰.It is fed according to weight 2%-3%, morning and afternoon is divided to feed twice.
Above-mentioned each processing sets the repetition of 3 aquaculture ponds, and raising samples after 2 months, investigation soft-shelled turtle gaining effect, in serum
Immune indexes and change of water quality etc..It is control (CK) without using microbial compound preparation.
3, test result
Body of fetching water after experiment measures corresponding water quality indicator, and water-quality determination measuring method is with reference to national water quality standard for fishery
(GB/11607-89).Soft-shelled turtle is taken to investigate weight, relatively front and back variation.Soft-shelled turtle obtains blood using decaptitating blood extracting method and makes
Standby serum, index of correlation biochemical measurement instrument or enzyme-linked immunoassay method measurement.
Influence of 2 different disposal of table to aquaculture pond water quality
Chitosan compound formulation provided by the invention has the water quality of breeding water body aobvious it can be seen from the data of table 2
Writing improves, and compares two kinds of processing modes, directly splashes and the effect of water correction is become apparent than being mixed into feeding in feed, sprinkle
Spill in mode NO in 0.5 ‰, 1.0 ‰ and 2.0 ‰ 3 concentration processing3 -、NO2 -、NH4+, total nitrogen and total phosphorus reduce than control respectively
67.98%, 330%, 47.21%, 44.15% and 53.79%；73.47%, 331%, 51.08%, 45.87% and
51.89%；68.84%, 331%, 40.88%, 44.52% and 63.29%.0.5 ‰, 1.0 ‰ and 2.0 ‰ 3 in feed mode
NO in a concentration processing3 -、NO2 -、NH4+, total nitrogen and total phosphorus reduce by 22.56% than control respectively, 328%, 15.86%,
17.18% and 22.15%；42.68%, 327%, 35.36%, 28.76% and 55.06%；43.29%, 327%,
33.95%, 31.77% and 56.96%.
Influence of 3 different disposal of table to the young soft-shelled turtle changes of body mass of China
Chitosan compound formulation provided by the invention has significant rush for the growth of soft-shelled turtle it can be seen from the data of table 3
Into effect, gaining effect has reached 8.0% or more.Meanwhile the gaining effect of mode 2 becomes apparent, i.e., answers chitosan
It closes preparation and soft-shelled turtle feed mixed feeding is more advantageous to and plays its effect for promoting soft-shelled turtle growth.
4 different disposal of table influences sero-immunity index etc.
Chitosan compound formulation provided by the invention is for total protein, white egg in serum it can be seen from the data of table 4
White, IgA, IgM and total antioxidant capacity are significantly increased, and show that chitosan compound formulation of the present invention can be mentioned effectively
The immune function of high soft-shelled turtle, and then soft-shelled turtle is improved to the resistance of disease.Two ways can significantly affect serum dependency number
According to comparing two ways, feed mode becomes apparent from than effect of directly splashing, 0.5 ‰, 1.0 ‰ and 2.0 ‰ 3 in feed mode
In concentration processing total protein, albumin, IgA, IgM and total antioxidant capacity improve 31.41% than control respectively, 15.70%,
4.51%, 8.52% and 10.73%；33.11%, 11.06%, 4.92%, 7.19% and 10.24%；36.96%,
11.89%, 5.48%, 7.81% and 25.95%.Total egg in 0.5 ‰, 1.0 ‰ and 2.0 ‰ 3 concentration processing in feed mode
White, albumin, IgA, IgM and total antioxidant capacity reduce by 27.03%, 0.93%, 2.85%, 3.03% and than control respectively
7.02%；33.64%, 8.92%, 3.18%, 4.16% and 16.49%；34.14%, 4.18%, 2.73%, 7.32% and
12.98%, from structure relatively from the point of view of, 2 method of application of mode is more preferably.
In conclusion chitosan microbial compound preparation provided by the invention tool be significantly improved breeding water body water quality and
Promote soft-shelled turtle growth, improve its immunity.It is more bright for reducing breeding water body Effects of Water Quality by the way of being exerted into water body
It is aobvious, chitosan microbial compound preparation is more excellent to the effect of soft-shelled turtle growth and raising immunocompetence is promoted.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of chitosan microbial compound preparation, including chitosan, Bacillus acidi lactici and composite bacillus, the compound gemma
Bacillus includes bacillus licheniformis, bacillus subtilis and Paenibacillus polymyxa, living bacteria count 1.5~2.5 hundred million/ml,
PH value is 6.8~7.0.
2. chitosan microbial compound preparation according to claim 1, which is characterized in that by weight, including：Chitosan
5~10 parts, 7~14 parts of the Bacillus acidi lactici of 5.0 hundred million/ml or more of living bacteria count and 2.0 hundred million/ml or more of living bacteria count it is compound
14~56 parts of bacillus.
3. chitosan microbial compound preparation according to claim 1 or 2, which is characterized in that the composite bacillus
In, the viable count ratio of bacillus licheniformis, bacillus subtilis and Paenibacillus polymyxa is 0.5~2:0.5~2:0.5~2.
4. chitosan microbial compound preparation according to claim 1 or 2, which is characterized in that it further include auxiliary material, it is described auxiliary
Material is selected from wheat bran and/or zeolite powder.
5. chitosan microbial compound preparation according to claim 4, which is characterized in that the chitosan microorganism is compound
Preparation includes 30~35 parts of wheat bran and/or 15~20 parts of zeolite powder.
6. chitosan microbial compound preparation according to claim 1, which is characterized in that the deacetylation of the chitosan
It is 85% or more, average molecular weight less than 100,000.
7. the preparation method of chitosan microbial compound preparation, includes the following steps described in claim 1~6 any one：
A, anaerobic fermentation is carried out to Bacillus acidi lactici with MRS fluid nutrient medium, obtains lactobacillus fermentation liquid；
B, with the beef-protein medium of improvement to bacillus subtilis, bacillus licheniformis and Paenibacillus polymyxa into
Row mixed fermentation obtains composite bacillus fermentation liquid；
By mass percentage, the beef-protein medium of the improvement includes beef extract 0.1~0.5%, peptone 0.3
~0.8%, sucrose 0.1~0.6%, sodium chloride 0.05~0.4%, dipotassium hydrogen phosphate 0.01~0.05%, potassium dihydrogen phosphate
0.02~0.08% and surplus water, pH be 6.8~7.2；
C, lactobacillus fermentation liquid, composite bacillus fermentation liquid and chitosan are mixed, adjusting pH value is 6.8~7.0, is obtained
To chitosan microbial compound preparation；
Out-of-order is successive between the step A and step B.
8. the preparation method of chitosan microbial compound preparation according to claim 7, which is characterized in that when the micro- life of chitosan
When further including auxiliary material in object compound formulation, step C is replaced with：Auxiliary material and lactobacillus fermentation liquid, composite bacillus are fermented
Liquid and chitosan mixing.
9. chitosan microbial compound preparation described in claim 1~6 any one is improving the application in breeding water body water quality.
10. chitosan microbial compound preparation described in claim 1~6 any one is improving soft-shelled turtle immunity, is promoting soft-shelled turtle
Application in growth.
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| CN117413924A (en) * | 2023-11-02 | 2024-01-19 | 北京衍微科技有限公司 | Composition with antibacterial and fermentation promoting effects and application thereof |
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