CN107418924A - A kind of preparation method and applications of polar region beneficial bacteria member - Google Patents
A kind of preparation method and applications of polar region beneficial bacteria member Download PDFInfo
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- CN107418924A CN107418924A CN201710866203.2A CN201710866203A CN107418924A CN 107418924 A CN107418924 A CN 107418924A CN 201710866203 A CN201710866203 A CN 201710866203A CN 107418924 A CN107418924 A CN 107418924A
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Abstract
The invention discloses a kind of preparation method and applications of polar region beneficial bacteria member; its be by the excellent low temperature bacillus of upgrowth situation, low temperature lactobacillus and antibacterial peptide yeast respectively after cultivating, ferment, centrifuge; isolated thalline is added to containing being mixed in xylo-oligosaccharide, chitosan oligomer, beta glucan, trehalose, skimmed milk and ascorbic protective agent, then the polar region beneficial bacteria member is made through vacuum freeze drying.Gained polar region beneficial bacteria member can improve breeding water body environment, effectively remove ammonia nitrogen and nitrite in breeding water body, suppress the growth of harmful pathogenic bacteria in cultivation fish body, not only promote fish body growth, moreover it is possible to significantly improve aquatic seedling survival rate.
Description
Technical field
The invention belongs to feeding additive aquatic animal field, and in particular to the preparation method of a kind of polar region beneficial bacteria member and its should
With.
Background technology
With the sustainable development of domestic economy, the demand of culture fishery sustainable development is improved constantly, high density is supported
Grow pattern and abuse of antibiotics problem drastically influence the development of culture fishery, it is long-term conventional, cause breeding ecological environment
Imbalance, various diseases frequently occur.Furthermore antibiotic long term accumulation in aquatic livestock body, also human health is produced a kind of
Potential threat.Therefore, aquaculture model is changed, the substitute for finding antibiotic is imperative.
Polar region beneficial bacteria member is the product of new, alternative antibiotic, has a free of contamination feature of health, and long-term use can be with
Effectively change breeding water body microenvironment, regulation aquatic livestock gut flora balance, so as to ensure organism normal growth.Cause
This, has great economy and social effect.
Lactobacillus, gram-positive bacteria, aquatic livestock gut pH can be effectively reduced, so as to suppress other pathogenic bacteria
Growth and breeding, gut flora balance was both adjusted, has promoted body to digest and assimilate nutriment, and can improves the immunity energy of body
Power.Lactobacillus is generally acknowledged beneficial bacterium as animal and the dominant colony of human gastrointestinal tract.Lactobacillus is applied to aquaculture row
Industry is an effective way for solving the problems such as aquaculture thing survival rate is low.
Saccharomycete, containing abundant protein and vitamin, it is the excellent additive of aquatic feeds, has to aquatic livestock
Food calling, promote growth, improve immune effect.Antibacterial peptide has the bactericidal action of wide spectrum, is the important set of natural immune system
Into part, harmful pathogenic bacteria can be effectively killed, is not easy in-ductive drug -tolerance again, but natural antibacterial peptide content is few, it is impossible to be full
The needs of the sufficient mankind.The fusion egg formed by modifying fish antibacterial peptide hepcidin and amphibian antibacterial peptide maginin
White antibacterial peptide yeast is a kind of with antibacterial, immunologic function and the novel fodder additive built with genetic engineering.
Low temperature bacillus, gram-positive bacteria, thalline is shaft-like, majority motion, produces gemma.Low temperature bacillus is severe
Viability in environment is stronger, and with a variety of efficient enzymatic effects.High-quality low temperature bacillus can degrade rapidly
Various organic matters in breeding water body, including residual bait, the excreta of aquatic livestock, the residuum of dead organisms and bottom of pond mud
In organic matter, in addition, moreover it is possible to reduce the ammonia nitrogen and nitrite nitrogen in water body content, increase dissolved oxygen content, enter
And water quality is effectively improved, build good breeding ecological environment.By the low temperature with obvious bacteriostatic activity and low temperature enzymatic activity
It is an effective scheme for solving antibiotic pollution problem that bacillus, which is applied to aquaculture industry,.
Single beneficial bacteria member can not meet the requirement of a variety of cultivation objects and breeding environment, prepare compound polar region beneficial bacteria member
It is the effective way for solving the problems, such as aquaculture.
The content of the invention
It is an object of the invention to provide a kind of polar region beneficial bacteria member for aquaculture and its preparation method and application, both
Juvenile fish survival rate can be improved, and can improves cultivation water, has wide market prospects in terms of aquaculture.
To achieve the above object, the present invention adopts the following technical scheme that：
A kind of preparation method of polar region beneficial bacteria member for aquaculture：Comprise the following steps：
1）The culture of low temperature bacillus, low temperature lactobacillus and antibacterial peptide yeast：By low temperature bacillus, lactobacillus and resist
Bacterium peptide barms takes out from test tube slant culture medium, is inoculated into the conical flask equipped with fluid nutrient medium, then puts respectively
In shaking table, 24h is cultivated under the conditions of 20 DEG C, 150-250r/min；
2）By step 1）Low temperature bacillus bacterium solution, lactobacillus bacterium solution and antibacterial peptide yeast liquid in logarithmic phase press 1：1：
1 ratio, which is inoculated into fermentation medium, is fermented, and fermentation temperature is 20 DEG C, fermentation time 48-72h；
3）By step 2）Bacterial strain fermentation liquor in stationary phase centrifuges 20min in 10000r/min, with separating thallus and supernatant；
Gained thalline is added in protective agent jointly and stirred and evenly mixed, wherein thalline and protectant volume ratio are 1：3, then through vacuum refrigeration
Dry and the polar region beneficial bacteria member is made.
Step 1）Liquid Culture based formulas used in middle low temperature bacillus is：2- containing peptone in per 1000mL distilled water
8g, dusty yeast 0.5-3g；PH value 7.4-7.8, use the preceding 15-20min that sterilized in 121 DEG C；Fluid nutrient medium is used in lactobacillus
MRS culture mediums, fluid nutrient medium used in antibacterial peptide yeast are PDA culture medium, use the preceding 15-20min that sterilized in 121 DEG C.
Step 2）The inoculum concentration of middle lactobacillus bacterium solution, lactobacillus bacterium solution and antibacterial peptide yeast liquid is 2vol%, three
Fermentative medium formula used is：Soluble-containing starch 3-3.5g, peptone 10-12g, yeast extract 3- in per 1000mL distilled water
5g、KH2PO4 1.5-4g、KHPO4 2-4g、MgSO4 0.1-1g、ZnCl21-3g；PH value 7.4-7.6, gone out using preceding in 121 DEG C
Bacterium 15-20min.
Step 3）Described in protective agent by xylo-oligosaccharide, chitosan oligomer, beta glucan, trehalose, vitamin C, degreasing
Breast and water composition, can be alleviated or avoided the damage that freeze-drying is brought to bacterium, improve survival rate；Wherein, xylo-oligosaccharide and β-
The concentration of glucan is 1.5-2.5g/L, chitosan oligomer 0.3-0.6 wt%, and the concentration of trehalose is 3-6wt%, vitamin
C concentration is 0.08-0.12wt%, and the concentration of skimmed milk is 16-22wt%.
The polar region beneficial bacteria member can be used for aquaculture as feed addictive.
Low temperature lactobacillus has excellent digestive ferment ability, and under cryogenic, enzymatic productivity is not decreased obviously, and again may be used
Effectively to reduce fish body intestinal pH, decomposition of the fish body intestines and stomach to feed is promoted to absorb.The feed for adding low temperature lactobacillus can be with
The body weight of juvenile fish is dramatically increased, improves its abilities of digestive and absorption.
Antibacterial peptide yeast is a kind of beneficial bacterium extremely strong with antibacterial, immunologic function, can effectively suppress harmful pathogenic bacteria
Growth, improve fish body immunity.
Low temperature bacillus has the features such as superpower bacteriostatic activity, degraded organic ammonia nitrogen and nitrite, metabolizable life
Into multiple beneficial composition, such as a variety of organic acids, digestive ferment.Therefore it can effectively suppress harmful micro- life such as harmful bacteria, pathogen
The growth and breeding of thing, and the content of ammonia nitrogen and nitrite in water can be efficiently reduced, cultivation system dissolved oxygen rate is improved, and then
Improve water quality, improve breeding water body microenvironment.
The polar region beneficial bacteria member that low temperature bacillus, low temperature lactobacillus and antibacterial peptide yeast are mixed with by the present invention, can
To play feature synergy, from ecological angle, breeding water body ammonia-nitrogen content is reduced using microorganism, have it is natural,
It is nontoxic, have no side effect, be pollution-free, noresidue, safe and reliable superiority；Improve cultivation water using beneficial microbe again,
Suppress growth of pathogenic bacteria, reduce the incidence of common disease, promote fish body to digest and assimilate feed, so as to promote its growth,
Improve the survival rate of its seedling.
The beneficial effects of the present invention are：Gained polar region of the invention beneficial bacteria member can as aquaculture feed additive it is straight
Connect in input breeding water body, itself there is the features such as bacteriostasis rate is strong, and zymogenic rate is high, there is good improvement breeding water body micro-loop again
The effect in border, can reduce breeding water body ammonia-nitrogen content, and ammonia nitrogen clearance rate is reachable up to 60-68%, the clearance of nitrite
65-77%, and aquatic biological can be promoted to digest and assimilate nutriment, significantly improve the survival rate and rate of body weight gain of seedling.
Brief description of the drawings
Fig. 1 is antibacterial picture of the mixing supernatant of the present invention to vibrio parahemolyticus.
Fig. 2 be beneficial bacteria member in polar region of the present invention under different time to the ammonia nitrogen clearance rate change curve of breeding water body.
Fig. 3 be beneficial bacteria member in polar region of the present invention under different time to the nitrite clearance rate change curve of breeding water body.
Embodiment
In order that content of the present invention easily facilitates understanding, with reference to embodiment to of the present invention
Technical scheme is described further, but the present invention is not limited only to this.
Embodiment 1
A kind of polar region beneficial bacteria member for aquaculture, its preparation method comprise the following steps：
1）The culture of low temperature bacillus, low temperature lactobacillus and antibacterial peptide yeast：By low temperature bacillus, lactobacillus and resist
Bacterium peptide barms takes out from test tube slant culture medium, is inoculated into the conical flask equipped with fluid nutrient medium, then puts respectively
In shaking table, 24h is cultivated under the conditions of 20 DEG C, 150r/min；
2）By step 1）Low temperature bacillus bacterium solution, lactobacillus bacterium solution and antibacterial peptide yeast liquid in logarithmic phase press 1：1：
1 ratio, which is inoculated into fermentation medium, is fermented, and fermentation temperature is 20 DEG C, fermentation time 72h；
3）By step 2）Bacterial strain fermentation liquor in stationary phase centrifuges 20min in 10000r/min, with separating thallus and supernatant；
Gained thalline is added in the aqueous solution containing xylo-oligosaccharide and beta glucan jointly and mixed, adds chitosan oligomer, marine alga
Sugar, vitamin C, stir obtained suspension, is eventually adding skimmed milk as matrix, the pole is made through vacuum freeze drying
Ground beneficial bacteria member.
Step 1）Liquid Culture based formulas used in middle low temperature bacillus is：Contain peptone in per 1000mL distilled water
5g, dusty yeast 1g；PH value 7.6, use the preceding 20min that sterilized in 121 DEG C；Fluid nutrient medium used in lactobacillus is MRS culture mediums；It is anti-
Fluid nutrient medium used in bacterium peptide yeast is PDA culture medium, uses the preceding 20min that sterilized in 121 DEG C.
Step 2）The inoculum concentration of middle low temperature bacillus bacterium solution, lactobacillus bacterium solution and antibacterial peptide yeast liquid is
2vol%, fermentative medium formula used are：Soluble-containing starch 3g in per 1000mL distilled water, peptone 10g, yeast extract 3g,
KH2PO4 1.5g、KHPO4 2g、MgSO4 0.1g、ZnCl21g；PH value 7.6, use the preceding 15min that sterilized in 121 DEG C.
Step 3）The concentration of middle xylo-oligosaccharide and beta glucan is 2.0g/L, the wt% of chitosan oligomer 0.5, trehalose
Concentration is 5wt%, and ascorbic concentration is 0.1wt%, and the concentration of skimmed milk is 20wt%.
Embodiment 2
A kind of polar region beneficial bacteria member for aquaculture, its preparation method comprise the following steps：
1）The culture of low temperature bacillus, low temperature lactobacillus and antibacterial peptide yeast：By low temperature bacillus, lactobacillus and resist
Bacterium peptide barms takes out from test tube slant culture medium, is inoculated into the conical flask equipped with fluid nutrient medium, then puts respectively
In shaking table, 24h is cultivated under the conditions of 20 DEG C, 200r/min；
2）By step 1）Low temperature bacillus bacterium solution, lactobacillus bacterium solution and antibacterial peptide yeast liquid in logarithmic phase press 1：1：
1 ratio, which is inoculated into fermentation medium, is fermented, and fermentation temperature is 20 DEG C, fermentation time 60h；
3）By step 2）Bacterial strain fermentation liquor in stationary phase centrifuges 20min in 10000r/min, with separating thallus and supernatant；
Gained thalline is added in the aqueous solution containing xylo-oligosaccharide and beta glucan jointly and mixed, adds chitosan oligomer, marine alga
Sugar, vitamin C, stir obtained suspension, is eventually adding skimmed milk as matrix, the pole is made through vacuum freeze drying
Ground beneficial bacteria member.
Step 1）Liquid Culture based formulas used in middle low temperature bacillus is：Contain peptone in per 1000mL distilled water
6g, dusty yeast 2g；PH value 7.4, use the preceding 18min that sterilized in 121 DEG C；Fluid nutrient medium used in lactobacillus is MRS culture mediums；It is anti-
Fluid nutrient medium used in bacterium peptide yeast is PDA culture medium, uses the preceding 18min that sterilized in 121 DEG C.
Step 2）The inoculum concentration of middle low temperature bacillus bacterium solution, lactobacillus bacterium solution and antibacterial peptide yeast liquid is
2vol%, fermentative medium formula used are：Soluble-containing starch 3.2g, peptone 11g, yeast extract in per 1000mL distilled water
4g、KH2PO4 2.5g、KHPO4 3g、MgSO4 0.6g、ZnCl22g；PH value 7.5, use the preceding 18min that sterilized in 121 DEG C.
Step 3）The concentration of middle xylo-oligosaccharide and beta glucan is 2.0g/L, the wt% of chitosan oligomer 0.5, trehalose
Concentration is 5wt%, and ascorbic concentration is 0.1wt%, and the concentration of skimmed milk is 20wt%.
Embodiment 3
A kind of polar region beneficial bacteria member for aquaculture, its preparation method comprise the following steps：
1）The culture of low temperature bacillus, low temperature lactobacillus and antibacterial peptide yeast：By low temperature bacillus, lactobacillus and resist
Bacterium peptide barms takes out from test tube slant culture medium, is inoculated into the conical flask equipped with fluid nutrient medium, then puts respectively
In shaking table, 24h is cultivated under the conditions of 20 DEG C, 150-250r/min；
2）By step 1）Low temperature bacillus bacterium solution, lactobacillus bacterium solution and antibacterial peptide yeast liquid in logarithmic phase press 1：1：
1 ratio, which is inoculated into fermentation medium, is fermented, and fermentation temperature is 20 DEG C, fermentation time 48h；
3）By step 2）Bacterial strain fermentation liquor in stationary phase centrifuges 20min in 10000r/min, with separating thallus and supernatant；
Gained thalline is added in the aqueous solution containing xylo-oligosaccharide and beta glucan jointly and mixed, adds chitosan oligomer, marine alga
Sugar, vitamin C, stir obtained suspension, is eventually adding skimmed milk as matrix, the pole is made through vacuum freeze drying
Ground beneficial bacteria member.
Step 1）Liquid Culture based formulas used in middle low temperature bacillus is：Contain peptone in per 1000mL distilled water
8g, dusty yeast 3g；PH value 7.8, use the preceding 20min that sterilized in 121 DEG C；Fluid nutrient medium used in lactobacillus is MRS culture mediums；It is anti-
Fluid nutrient medium used in bacterium peptide yeast is PDA culture medium, uses the preceding 20min that sterilized in 121 DEG C.Contain egg in per 1000mL distilled water
White peptone 2-5g, dusty yeast 0.5-1.5g；PH value 7.4-7.8
Step 2）The inoculum concentration of middle low temperature bacillus bacterium solution, lactobacillus bacterium solution and antibacterial peptide yeast liquid is 2vol%, institute
It is with fermentative medium formula：Soluble-containing starch 3.5g, peptone 12g, yeast extract 5g, KH in per 1000mL distilled water2PO4
4g、KHPO4 4g、MgSO4 1g、ZnCl23g；PH value 7.6, use the preceding 20min that sterilized in 121 DEG C.
Step 3）The concentration of middle xylo-oligosaccharide and beta glucan is 2.0g/L, the wt% of chitosan oligomer 0.5, trehalose
Concentration is 5wt%, and ascorbic concentration is 0.1wt%, and the concentration of skimmed milk is 20wt%.
Beneficial bacteria element performance test in polar region in embodiment 1
Test 1：The step 2 of embodiment 1）Bacteriostatic experiment of the zymotic fluid obtained after bacterial strain compounding to vibrio parahaemolytious
Bacteriostatic experiment of the zymotic fluid to vibrio parahaemolytious is done with Kirby-Bauer methods, 1 is sterilized water, and 2 is mould for 0.5mg/mL chlorine
Element, 3 be blank nutrient solution, and 4 be zymotic fluid.As seen from Figure 1, supernatant has inhibitory action to vibrio parahaemolytious.
Test 2：Polar region beneficial bacteria member removes ammonia nitrogen experiment
Ammonia-nitrogen content, the alkaline solution and the light red palm fibre of ammonia reaction generation of mercuric iodixde and KI are determined using reagent colorimetric method
Coloring agent state compound, its colourity is directly proportional to ammonia-nitrogen content, absorbance can be determined in the range of 410 ~ 425nm of wavelength, and calculate
Go out content.
Figure it is seen that polar region beneficial bacteria member reaches as high as 68% to the clearance rate of ammonia nitrogen in breeding water body.
Test 3：Polar region beneficial bacteria member removes nitrites test
Diazo coupling Spectrophotometric Determination of Nitrite content, during pH ＜ 1.7, water nitrite and P-aminobenzene-sulfonamide
Diazotising, then with hydrochloric acid N-（1- naphthalenes）- ethylenediamine produces coupling reaction, generates the azo dyes of aubergine, colorimetric assay.From figure
3 as can be seen that polar region beneficial bacteria member is up to 77% to the clearance rate of breeding water body nitrite.
Test 3：Polar region beneficial bacteria member stability test
After polar region beneficial bacteria member is preserved into 0 month, 1 month, 2 months, 3 months under the conditions of lucifuge at 4 DEG C, detect respectively therein
The content of low temperature bacillus, low temperature lactobacillus and antibacterial peptide yeast, as a result such as table 1.
The stability test of table 1
From table 1, beneficial bacteria member in polar region of the present invention has long-time stability under 4 DEG C of environment.
Test 4：The culture experiment of Cynoglossus semilaevis
Cynoglossus semilaevis seedling is divided into two groups, every group of 100 tails, polar region beneficial bacteria made from one of which addition inventive embodiments 1 is first,
To be fed 1% polar region beneficial bacteria member is mixed with chow diet, another set is fed only with conventional feed, after raising 8 weeks, sky
Abdomen 24h is measured, and calculates the rate of body weight gain of Cynoglossus semilaevis, and its computational methods is as follows：
Rate of body weight gain（%）=（W2-W1）/W1×100%
Wherein W1、W2Respectively Cynoglossus semilaevis initial weight and end weight.As a result such as table 2.
The cynoglossus semilaevis cultivation of table 2 is tested
From table 2, the Cynoglossus semilaevis average growth rate that mixed with chow diet group of 1% feed addictive is 68.49%, survival rate
For 95%, all higher than chow diet nursing group Cynoglossus semilaevis average growth rate 55.29% and survival rate 78%.
Above-mentioned result of the test shows, compared with conventional feed is fed, using beneficial bacteria member in polar region of the present invention to Cynoglossus semilaevis
Cultivation shows preferable effect.
The foregoing is only presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, it should all belong to the covering scope of the present invention.
Claims (6)
- A kind of 1. preparation method of polar region beneficial bacteria member applied to aquaculture, it is characterised in that：Comprise the following steps：1）The culture of low temperature bacillus, low temperature lactobacillus and antibacterial peptide yeast：By low temperature bacillus, low temperature lactobacillus with And antibacterial peptide yeast takes out from test tube slant culture medium, is inoculated into respectively in fluid nutrient medium, shaking table temperature setting is 20 DEG C, Rotating speed 150-250r/min, cultivate 24h；2）By step 1）Low temperature bacillus bacterium solution, lactobacillus bacterium solution and antibacterial peptide yeast in logarithmic phase press 1：1：1 Ratio is inoculated into fermentation medium together is fermented, and fermentation temperature is 20 DEG C, fermentation time 48-72h；3）By step 2）Bacterial strain fermentation liquor in stationary phase, 20min is centrifuged in 10000r/min, with separating thallus and supernatant Liquid；Gained thalline is added by xylo-oligosaccharide, chitosan oligomer, beta glucan, trehalose, skimmed milk, vitamin C and water jointly Stirred and evenly mixed in the protective agent of composition, wherein thalline and protectant volume ratio are 1：3, then it is made described through vacuum freeze drying Polar region beneficial bacteria member.
- 2. the preparation method of polar region beneficial bacteria member according to claim 1, it is characterised in that：Step 1）Middle low temperature bacillus institute Liquid Culture based formulas is：2-8g containing peptone, dusty yeast 0.5-3g in per 1000mL distilled water；PH value 7.4-7.8；It is low Warm lactobacillus used medium is MRS culture mediums；Antibacterial peptide yeast used medium is PDA culture medium.
- 3. the preparation method of polar region beneficial bacteria member according to claim 1, it is characterised in that：Step 2）Middle fermented and cultured basigamy Fang Wei：Soluble-containing starch 3-3.5g, peptone 10-12g, yeast extract 3-5g, KH in per 1000mL distilled water2PO4 1.5-4g、 KHPO4 2-4g、MgSO4 0.1-1g、ZnCl21-3g；PH value 7.4-7.6.
- 4. the preparation method of polar region beneficial bacteria member according to claim 1, it is characterised in that：Step 3）Described in it is protectant Component content is that xylo-oligosaccharide and beta glucan distinguish 1.5-2.5g/L, chitosan oligomer 0.3-0.6 wt%, trehalose 3- 6wt%, skimmed milk 16-22wt%, vitamin C 0.08-0.12wt%.
- 5. one kind beneficial bacteria member in polar region as made from any methods describeds of claim 1-4.
- A kind of 6. application of the member of polar region beneficial bacteria as claimed in claim 5, it is characterised in that：It is applied to aquatic products as feed addictive Cultivation.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109022317A (en) * | 2018-08-15 | 2018-12-18 | 河南金百合生物科技股份有限公司 | A kind of preparation method of clostridium butyricum bacterium powder |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105907674A (en) * | 2016-05-04 | 2016-08-31 | 福州大学 | Preparation method and application of composite microbial ecological agent |
| CN106957808A (en) * | 2017-05-12 | 2017-07-18 | 福州大学 | A kind of probiotics for cynoglossus semilaevis cultivation |
-
2017
- 2017-09-22 CN CN201710866203.2A patent/CN107418924A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105907674A (en) * | 2016-05-04 | 2016-08-31 | 福州大学 | Preparation method and application of composite microbial ecological agent |
| CN106957808A (en) * | 2017-05-12 | 2017-07-18 | 福州大学 | A kind of probiotics for cynoglossus semilaevis cultivation |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109022317A (en) * | 2018-08-15 | 2018-12-18 | 河南金百合生物科技股份有限公司 | A kind of preparation method of clostridium butyricum bacterium powder |
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