CN105063007A - Method for adsorbing enterococcus by aid of attapulgite - Google Patents
Method for adsorbing enterococcus by aid of attapulgite Download PDFInfo
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- CN105063007A CN105063007A CN201510420815.XA CN201510420815A CN105063007A CN 105063007 A CN105063007 A CN 105063007A CN 201510420815 A CN201510420815 A CN 201510420815A CN 105063007 A CN105063007 A CN 105063007A
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- recessed soil
- lactococcus
- adsorption
- bacterium liquid
- recessed
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Abstract
The invention discloses a method for adsorbing enterococcus by the aid of attapulgite. The method includes pre-treating the preliminarily treated attapulgite by the aid of 1mol/L H<3>PO<4> buffer liquid; adsorbing enterococcus liquid by the aid of 0.2mol/L NaH<2>PO<4>-Na<2>HPO<4> buffer liquid with the pH (potential of hydrogen) of 6 at the temperature of 35 DEG C for 80min to realize the optima adsorption effects. Compared with the prior art, the method has the obvious advantages that the excellent adsorption effects can be realized, and the adsorption rate can reach 96% at least and is higher than existing adsorption rates; the method is simple, can be conveniently and quickly implemented and is suitable for industrial production.
Description
Technical field
The present invention relates to the preparation field of compound, particularly relate to a kind of recessed soil to the adsorption method of Lactococcus.
Background technology
20 middle of century, the success of antibiotic discovery and the synthesis of chemical antimicrobial substance, first comes into operation the mankind, is widely used as fodder additives subsequently, in animal health and husbandry sector, played vital role.But due to the life-time service of these additives and abuse, its drawback displays day by day; In order to overcome all drawbacks that antibiotic etc additive brings, the scientist of countries in the world makes great efforts to seek and develop the novel fodder additive had no side effect.And probiotics just with its green safety, have no side effect, the advantage of noresidue progressively becomes the main force of substitute antibiotics class additive.
Regulate body composition of gut flora because Lactococcus has, keep microecological balance, improve food digestion rate and biological value, reduce serum cholesterol, control intracellular toxin, suppress corrupt bacteria growing breeding in enteron aisle, etc. feature; We can attempt the microbiotic come with it in alternative feed.
But Lactococcus is very low by absorbed power after enteron aisle, its effect can not get embodying, and causes a large amount of waste.
Have the recessed soil of research and utilization to adsorb Lactococcus as carrier at present and prepare microbial preparation to increase Lactococcus by the specific absorption of enteron aisle, but the effective treatment process of a kind of system not yet proposes.
Summary of the invention
Goal of the invention: in order to overcome the defect existed in prior art, the present invention proposes a kind of advantages of good adsorption effect, the high recessed soil of adsorption rate is to the adsorption method of Lactococcus.
Technical scheme: in order to solve the problems of the technologies described above, the technical solution adopted in the present invention is:
Recessed soil, to an adsorption method for Lactococcus, comprises the following steps:
(1) pre-treatment of recessed soil: untreated recessed soil block is pulverized, use 200 mesh screen again, deionized water is added by the mass ratio of recessed soil and deionized water 1:30, after magnetic stirrer 24h, leave standstill 12h, get the recessed soil suspension in middle layer, add the deionized water of one times amount again, from whipping step, repeat 3 times, obtain the recessed soil of purifying, putting into refrigerator at the temperature of 0-10 DEG C saves backup;
(2) screening of Lactococcus: according to following steps, preparation → the substratum of MRS screening culture medium and the sterilizing of instrument → commercially available yoghourt gradient dilution → be down flat plate → coating → 37 DEG C constant temperature culture 24h → satisfactory flat board of observation bacterium colony number, picking colonies typical → gramstaining → microscopy → separation and purification → picking dominant strain carries out culture presevation → survey growth curve;
(3) making lactic acid coccus bacterium liquid: the bacterium liquid getting the 10mL logarithmic phase middle and later periods according to above-mentioned growth curve under 5000r/min condition centrifugal 10 minutes, abandoning supernatant, the physiological saline of 0.85% is added in precipitation, centrifuge washing twice, finally thalline is suspended in physiological saline, when the Lactococcus bacterium liquid needing the used time to be mixed with desired concn;
(4) adsorption treatment: with the H of 1mol/L
3pO
4recessed soil for subsequent use in damping fluid pre-treatment step (1) is the NaH of 6 at 0.2mol/L, pH
2pO
4-Na
2hPO
4mix in damping fluid, seal triangle bottleneck with cotton, be positioned over 35 DEG C, the shaking table of 150r/min vibrates, duration of oscillation is 80min, obtains the optimal adsorption effect of recessed soil to Lactococcus bacterium liquid.
More preferred, the recessed native solid content chosen in described step (1) is 4mg/mL.
More preferred, in described step (2), commercially available yoghourt gradient dilution is from 10
-1to 10
-99 concentration gradients are divided to dilute.
More preferred, in described step (2), each gradient concentration of painting work repeats twice.
More preferred, the bacterium liquid OD value chosen in described step (2) is 1.3.
More preferred, the bacterium liquid CFU chosen in described step (2) is 1.68 × 10
10, 5.2g/L.
Beneficial effect: a kind of recessed soil provided by the invention to the adsorption method of Lactococcus, by the H of the recessed soil 1mol/L through pre-treatment
3pO
4damping fluid pre-treatment is the NaH of 6 in 0.2mol/L, pH
2pO
4-Na
2hPO
4damping fluid carries out absorption 80min in 35 DEG C to Lactococcus bacterium liquid can obtain optimal adsorption effect, compared with prior art has following obviously advantage: a) advantages of good adsorption effect: adsorption rate can reach more than 96%, breaches existing adsorption rate; B) method is simple, and simple operation, is applicable to suitability for industrialized production.
Accompanying drawing explanation
Fig. 1 is the adsorption isothermal line schematic diagram of the Lactococcus of one embodiment of the present invention;
Fig. 2 is that the differing temps of one embodiment of the present invention affects schematic diagram to recessed native kinetics of adsorption;
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail:
1) testing sequence:
(1) pre-treatment of recessed soil: choose the recessed soil that solid content is 4mg/mL, untreated recessed soil block is pulverized, use 200 mesh screen again, add deionized water, after magnetic stirrer 24h by the mass ratio of recessed soil and deionized water 1:30, leave standstill 12h, get the recessed soil suspension in middle layer, then add the deionized water of one times amount, from whipping step, repeat 3 times, obtain the recessed soil of purifying, putting into refrigerator at the temperature of 0-10 DEG C saves backup;
(2) screening of Lactococcus: according to following steps, preparation → the substratum of MRS screening culture medium and the sterilizing of instrument → commercially available yoghourt gradient dilution → be down flat plate → coating → 37 DEG C constant temperature culture 24h → satisfactory flat board of observation bacterium colony number, picking colonies typical → gramstaining → microscopy → separation and purification → picking dominant strain carries out culture presevation → survey growth curve; Wherein, commercially available yoghourt gradient dilution is from 10
-1to 10
-99 concentration gradients are divided to dilute; The each gradient concentration of painting work repeats twice to ensure accuracy, and the bacterium liquid OD value chosen is 1.3, CFU is 1.68 × 10
10, 5.2g/L;
(3) making lactic acid coccus bacterium liquid: the bacterium liquid getting the 10mL logarithmic phase middle and later periods according to above-mentioned growth curve under 5000r/min condition centrifugal 10 minutes, abandoning supernatant, the physiological saline of 0.85% is added in precipitation, centrifuge washing twice, finally thalline is suspended in physiological saline, when the Lactococcus bacterium liquid needing the used time to be mixed with desired concn;
(4) adsorption treatment: with the H of 1mol/L
3pO
4recessed soil for subsequent use in damping fluid pre-treatment step (1) is the NaH of 6 at 0.2mol/L, pH
2pO
4-Na
2hPO
4mix in damping fluid, seal triangle bottleneck with cotton, be positioned over 35 DEG C, the shaking table of 150r/min vibrates, duration of oscillation is 80min, and obtain the optimal adsorption effect of recessed soil to Lactococcus bacterium liquid, adsorption rate is 96.3%.
2) kinetics of adsorption and thermodynamic results research:
A) drafting of adsorption isothermal line
Select the Lactococcus bacterium liquid of several groups of different concns to carry out isothermal adsorption test, result is as shown in table 1, with quality of balance concentration p
eto equilibrium adsorption capacity q
emapping, obtains the adsorption isothermal line of recessed soil absorption Lactococcus, as shown in Figure 1.
The isothermal adsorption result of table 1 recessed soil absorption Lactococcus
As seen from Figure 1, along with the rising of temperature, recessed soil increases the adsorptive capacity of Lactococcus, and along with the increase of equilibrium concentration, equilibrium adsorption capacity first increases, and then tends towards stability.
Carry out Langmuir and Freundlich equation linear fit according to data in formula and table, result is as shown in table 2, table 3.
The Langmuir linear fit result of table 2 recessed soil absorption Lactococcus
The Freundlich linear fit result of table 3 recessed soil absorption Lactococcus
Contrast table 2 and table 3 known, the individual layer saturated extent of adsorption q of the theory deduction in table 2
mwith test gained saturated extent of adsorption q closely, thus in the temperature and concentration range of experimental study, recessed soil can meet Langmuir Tellurium determination preferably to the absorption of Lactococcus.
As shown in Table 2, temperature raises, individual layer saturated extent of adsorption q
mincrease, show that this absorption is endothermic process, namely heating up is conducive to the absorption of recessed soil to Lactococcus.Due to b>0, and there is 0<R
l=1/ (1+bp
0) <1, namely recessed soil is adsorbed as preferential absorption to Lactococcus.
B) result of Adsorption thermodynamics
Mapped to 1/T by lnb, regression equation is y=-0.001x+0.0027.Adsorption en-thalpy Δ H is can be calculated by straight slope.
By above formulae discovery adsorption thermodynamic parameter, the results are shown in Table 5.
The thermodynamic(al)function calculation result of table 4 recessed soil absorption Lactococcus
As can be seen from Table 4, under experiment condition, the adsorption enthalpy Δ H of recessed soil to Lactococcus is greater than zero, shows that absorption is endothermic process.Absorption is the coefficient result of motive power between adsorbate and sorbent material.In general, absorption is exothermic process, if do not have other factors to disturb, the impact of temperature on absorption is negative effect, and namely adsorptive capacity reduces with the rising of temperature.
Adsorption free energy is the embodiment of absorption impellent and the preferential property of absorption, can confirm that recessed soil is electrostatic attraction to the chief motivation that Lactococcus adsorbs by the value of adsorption free energy.
Total Entropy Changes of liquid-phase adsorption processes be adding of the absorption of solute and the desorb of solvent and, it depends on the power of solute and solvent and solid surface effect and the size of their molecular volumes.
C) result of kinetics of adsorption
By mixed solution being positioned over respectively 25 DEG C, 30 DEG C, 35 DEG C, the shaking table of 150r/min shakes, then every 10min sampling once, sample is centrifugal, get supernatant liquor, survey its OD value at 600nm wavelength place.Obtain result as shown in table 5.
The recessed soil of table 5 differing temps is to the adsorption kinetic data data of Lactococcus
Map according to table 5, obtain differing temps on the impact of recessed native kinetics of adsorption as shown in Figure 2.
As shown in Figure 2, temperature raises, and the adsorptive capacity of recessed soil to Lactococcus also increases, and all reaches adsorption equilibrium at 1 hours, but amplitude is little, and the impact of visible temperature on adsorptive capacity is little.Temperature is 298,303,308K time, the equilibrium adsorption capacity q of corresponding female soil to Lactococcus
e4.844 respectively, 4.916,4.968g/g.
Carry out linear fit by the data of pseudo-first-order and pseudo-second order kinetic equation his-and-hers watches 5 respectively, result is as shown in table 6, table 7.
The pseudo-first-order kinetics fitting result of table 6 temperature impact
The pseudo-second order kinetic fitting result of table 7 temperature impact
Contrast table 6 and table 7 can be found out, by the relation conefficient of pseudo-second order kinetic equation linear fit closer to 1, and Theoretical Equilibrium adsorptive capacity q
2with test gained equilibrium adsorption capacity q
emore close, thus with pseudo-second order kinetic equation, the absorption of recessed soil to Lactococcus can better be described.
To sum up, a kind of recessed soil of the present invention, to the adsorption method of Lactococcus, compared with prior art has following obviously advantage: a) advantages of good adsorption effect: adsorption rate can reach more than 96%, breaches existing adsorption rate; B) method is simple, and simple operation, is applicable to suitability for industrialized production.
Should be understood that, above embodiment is only not used in for illustration of the present invention and limits the scope of the invention, after having read the present invention, the amendment of those skilled in the art to the various equivalent form of value of the present invention has all fallen within the application's claims limited range.
Claims (6)
1. recessed soil is to an adsorption method for Lactococcus, it is characterized in that comprising the following steps:
(1) pre-treatment of recessed soil: untreated recessed soil block is pulverized, use 200 mesh screen again, deionized water is added by the mass ratio of recessed soil and deionized water 1:30, after magnetic stirrer 24h, leave standstill 12h, get the recessed soil suspension in middle layer, add the deionized water of one times amount again, from whipping step, repeat 3 times, obtain the recessed soil of purifying, putting into refrigerator at the temperature of 0-10 DEG C saves backup;
(2) screening of Lactococcus: according to following steps, preparation → the substratum of MRS screening culture medium and the sterilizing of instrument → commercially available yoghourt gradient dilution → be down flat plate → coating → 37 DEG C constant temperature culture 24h → satisfactory flat board of observation bacterium colony number, picking colonies typical → gramstaining → microscopy → separation and purification → picking dominant strain carries out culture presevation → survey growth curve;
(3) making lactic acid coccus bacterium liquid: the bacterium liquid getting the 10mL logarithmic phase middle and later periods according to above-mentioned growth curve under 5000r/min condition centrifugal 10 minutes, abandoning supernatant, the physiological saline of 0.85% is added in precipitation, centrifuge washing twice, finally thalline is suspended in physiological saline, when the Lactococcus bacterium liquid needing the used time to be mixed with desired concn;
(4) adsorption treatment: with the H of 1mol/L
3pO
4recessed soil for subsequent use in damping fluid pre-treatment step (1) is the NaH of 6 at 0.2mol/L, pH
2pO
4-Na
2hPO
4mix in damping fluid, seal triangle bottleneck with cotton, be positioned over 35 DEG C, the shaking table of 150r/min vibrates, duration of oscillation is 80min, obtains the optimal adsorption effect of recessed soil to Lactococcus bacterium liquid.
2. recessed soil according to claim 1 is to the adsorption method of Lactococcus, it is characterized in that: the recessed native solid content chosen in described step (1) is 4mg/mL.
3. recessed soil according to claim 1 is to the adsorption method of Lactococcus, it is characterized in that: in described step (2), commercially available yoghourt gradient dilution is from 10
-1to 10
-99 concentration gradients are divided to dilute.
4. recessed soil according to claim 1 is to the adsorption method of Lactococcus, it is characterized in that: in described step (2), each gradient concentration of painting work repeats twice.
5. recessed soil according to claim 1 is to the adsorption method of Lactococcus, it is characterized in that: the bacterium liquid OD value chosen in described step (2) is 1.3.
6. recessed soil according to claim 1 is to the adsorption method of Lactococcus, it is characterized in that: the bacterium liquid CFU chosen in described step (2) is 1.68 × 10
10, 5.2g/L.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114288985A (en) * | 2021-12-29 | 2022-04-08 | 浙江工业大学 | Black talc adsorption material and preparation method and application thereof |
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| CN104012754A (en) * | 2014-06-10 | 2014-09-03 | 寿阳华阳农牧有限公司 | Feed rich in lactococcus lactis and preparation method thereof |
| CN104772107A (en) * | 2015-03-23 | 2015-07-15 | 广州优克日用品有限公司 | Modified attapulgite material, preparation method and application thereof |
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2015
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Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1648237A (en) * | 2004-12-15 | 2005-08-03 | 江南大学 | Process for preparing high active powder composite green storage inoculating agent and its carrier adsorption agent |
| CN101919879A (en) * | 2010-08-06 | 2010-12-22 | 淮阴工学院 | Method for preparing probiotics preparation by taking attapulgite as carrier |
| CN102212476A (en) * | 2011-03-07 | 2011-10-12 | 杭州向天歌生物科技有限公司 | Method for cleanly and efficiently producing microorganism bactericide |
| CN103045581A (en) * | 2012-12-21 | 2013-04-17 | 淮阴工学院 | Method for immobilizing myxococcus fulvus coated by chitosan loaded with attapulgite |
| CN103271244A (en) * | 2013-05-13 | 2013-09-04 | 仝荣 | Preparation method of pig feed zymophyte additive |
| CN104012754A (en) * | 2014-06-10 | 2014-09-03 | 寿阳华阳农牧有限公司 | Feed rich in lactococcus lactis and preparation method thereof |
| CN104772107A (en) * | 2015-03-23 | 2015-07-15 | 广州优克日用品有限公司 | Modified attapulgite material, preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114288985A (en) * | 2021-12-29 | 2022-04-08 | 浙江工业大学 | Black talc adsorption material and preparation method and application thereof |
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