CN106591187A - Lactobacillus culture medium for production of animal feed and preparation and application of lactobacillus culture medium - Google Patents
Lactobacillus culture medium for production of animal feed and preparation and application of lactobacillus culture medium Download PDFInfo
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Abstract
The invention relates to the technical field of microorganisms, and particularly discloses a lactobacillus culture medium formula for production of animal feed. The lactobacillus culture medium formula for production of animal feed comprises 25-35 g/L of corn flour, 55-65 g/L of bean pulp, 10-18 g/L of molasses, 10-18 g/L of fructose, 0-2 g/L of K2HPO4.3H2O, 4-7 g/L of sodium acetate, 0-3 g/L of diammonium citrate, 0-0.5 g/L of MgSO4.7H2O, 0.2-0.5 g/L of MnSO4.H2O, 1-2 ml/L of tween-80 and 0.5-3 g/L of ascorbic acid. The lactobacillus culture medium formula is prepared with water, and the pH value of the lactobacillus culture medium formula is controlled to be 6.8-7.2. The invention further discloses a preparation method of the culture medium and a method for culturing lactobacillus by using the culture medium. While the costs for the raw materials of the culture medium is reduced, the viable count of the lactobacillus can be maintained or increased, and moreover, and the effect of inhibiting common pathogenic bacteria in aquaculture is good.
Description
Technical field
The present invention relates to microbial technology field, and in particular to a kind of animal feed production lactic acid bacteria culturing medium and its system
Standby and application.
Background technology
It is widely used in aquaculture using lactic acid bacteria as probiotic bacteria.As lactic acid bacteria can produce second in metabolic process
The acidic materials such as acid, lactic acid, reduce pH, scalable intestinal acid or alkali environment, promote digesting and assimilating for feedstuff, while lactic acid bacteria exists
The materials such as some antibacterial peptides, bacteriocin, somatomedin can be also produced in metabolic process, these products can suppress to a certain extent
The growth of harmful bacteria, improves the immunity of animal body.The multiplex lactic acid bacteria for treatment of culture fishery just waits aquatic biological normal in vain at present
See intestinal tract disease；Lactic acid bacteria is also a large amount of on poultry simultaneously uses, particularly in the fermentation of ablactational baby pig creep feed.
The multiplex MRS culture medium of lactic acid bacteria fermentation culture medium at this stage, the wherein price of carbon source and nitrogen source costly, be
Reduces cost in a large amount of production processes, has some patents at present and employs the materials such as bean cake, fish flour, Chinese medicine powder to improve lactic acid
The viable count of bacterium, effect have also obtained certain improvement really.But prior art means are also provided no advantage against in cost control,
And holding cannot be met or promote lactobacter growth and its inhibitory action to pathogen.
The content of the invention
In consideration of it, being necessary for the problems referred to above, there is provided a kind of to reduce the same of feeding lactobacillus culture medium raw material cost
When, lactobacter growth and its inhibitory action to pathogen can be effectively kept or promote, so as to be conducive to extending to extensive life
The animal feed production lactic acid bacteria culturing medium for producing and applying and its preparation and application.
For achieving the above object, the present invention takes technical scheme below：
The animal feed production lactic acid bacteria culturing medium of the present invention, its formula is：Semen Maydis powder 25-35g/L, bean cake 55-
65g/L, molasses 10-18g/L, Fructose 10-18g/L, K2HPO4·3H2O 0-2g/L, sodium acetate 4-7g/L, dibasic ammonium citrate 0-
3g/L、MgSO4·7H2O 0-0.5g/L、MnSO4·H2O 0.2-0.5g/L, tween 80 1-2ml/L, ascorbic acid 0.5-
3g/L, is prepared with water, and the pH value of lactic acid bacteria culturing medium is controlled in 6.8-7.2.
Used as preferred, animal feed production lactic acid bacteria culturing medium, its formula is：Semen Maydis powder 30g/L, bean cake 60g/L,
Molasses 15g/L, Fructose 15g/L, K2HPO4·3H2O 2g/L, sodium acetate 5g/L, dibasic ammonium citrate 2g/L, MgSO4·7H2O
0.5g/L、MnSO4·H2O 0.5g/L, tween 80 1ml/L, ascorbic acid 1g/L, are prepared with water.
As preferred, component in formula：Semen Maydis powder 30g/L, bean cake 60g/L, molasses 15g/L, Fructose 15g/L.
Used as preferred, the protein content of bean cake is more than 38%, and the carbohydrate content of Semen Maydis powder is not less than 40%.
The preparation of above-mentioned lactic acid bacteria culturing medium, step are as follows：
1) bean cake is weighed, plus 10-12 times of distilled water, pulping, with six layers of filtered through gauze, filtrate is stand-by；
2) Semen Maydis powder is weighed, plus 9-11 times of distilled water, to boil to sticky, with six layers of filtered through gauze, cool, filtrate is stand-by；
3) L-AA is weighed, is dissolved in appropriate distilled water, filtration sterilization, filtrate are stand-by；
4) molasses, Fructose, K are weighed respectively2HPO4·3H2O, sodium acetate, dibasic ammonium citrate, MgSO4·7H2O、MnSO4·
H2O, tween 80, are dissolved in appropriate distilled water, add 1), 2) in solution, plus appropriate distilled water mixes, sterilizing；
5), after by solution sterilization in 4), 50 DEG C or less are cooled to, add 3) obtained by filtrate, mix.
Using the method for above-mentioned culture medium culturing lactic acid bacteria, specially：
Extracting lactic acid bacterium carries out three rides, culture activation in MRS flat boards；On flat board, picking single bacterium colony is inoculated in MRS trainings
After cultivating in foster base, it is inoculated in MRS culture medium after culture with the inoculum concentration of 1-2%, then this is inoculated in the inoculum concentration of 2-5%
Cultivate in the culture medium of invention.
Beneficial effects of the present invention are：
The culture medium of the present invention adopts Semen Maydis powder and bean cake as culture media nitrogen source, and the carbon source component to culture medium is carried out
Optimization, while culture medium raw material cost is reduced, can keep or improve the viable count of lactic acid bacteria, and for Aquatic product is supported
The inhibition for growing common pathogen (such as vibrio parahaemolytious) also plays facilitation.
Description of the drawings
Fungistatic effect figures of the Fig. 1 for the culture medium culturing lactic acid bacteria of embodiment 1, in figure, " new culture medium " is the present embodiment
Culture medium；
Fungistatic effect figures of the Fig. 2 for the culture medium culturing lactic acid bacteria of embodiment 2, in figure, " new culture medium " is the present embodiment
Culture medium；
Fungistatic effect figures of the Fig. 3 for the culture medium culturing lactic acid bacteria of embodiment 3, in figure, " new culture medium " is the present embodiment
Culture medium；
Fungistatic effect figures of the Fig. 4 for the culture medium culturing lactic acid bacteria of embodiment 4, in figure, " new culture medium " is the present embodiment
Culture medium.
Specific embodiment
To make the object, technical solutions and advantages of the present invention clearer, below in conjunction with the embodiment of the present invention, to this
Bright technical scheme is made further clearly and completely to describe.It should be noted that described embodiment is only the present invention one
Section Example, rather than the embodiment of whole.Based on the embodiment in the present invention, those of ordinary skill in the art are not doing
The every other embodiment obtained under the premise of going out creative work, belongs to the scope of protection of the invention.
Embodiment 1
The preparation of above-mentioned lactic acid bacteria fermentation culture medium, step are as follows：
1) 60g bean cake, plus 500ml distilled water are taken, pulping, with six layers of filtered through gauze, filtrate is stand-by；
2) 30g Semen Maydis powder, plus 300ml distilled water are taken, is boiled to sticky (about 20min), with six layers of filtered through gauze, is put
Cool, filtrate is stand-by；
3) L-AA 1g is weighed, is dissolved in 10ml distilled water, filtration sterilization, filtrate are stand-by；
4) molasses 15g, Fructose 15g, K are weighed2HPO4·3H2O 2g, sodium acetate 5g, dibasic ammonium citrate 2g, MgSO4·7H2O
0.5g、MnSO4·H2O 0.5g, tween 80 1ml, are dissolved in 200ml distilled water, add 1), 2) obtained by filtrate, add distillation
Water is settled to 990ml, mixes, and 121 DEG C sterilize 15 minutes；
5), after by solution sterilization in 4), 50 DEG C are cooled to, add 3) obtained by filtrate, mix.
Lactic acid bacteria culture and test are carried out using above-mentioned culture medium, specially：
1.1 experiment strain subjects：Lactobacillus salivarius, Lactobacillus plantarum, lactobacillus rhamnosuss, enterococcus faecalis.It is main next
Source：This laboratory separates the strain for obtaining from aquatic animal intestinal, and in the glycerol tube preservation of own -80 DEG C of laboratory.
Indicator bacteria used by 1.2 experiments：Vibrio parahaemolytious ATCC 17802.Main source：Buy in Huan Kai Reagent Companies.
1.3 experimental program：Above-mentioned four strains of lactic acid bacteria is carried out into three ride activation, 37 DEG C of cultures with MRS culture medium first
24h；Then in MRS culture medium 2ml EP pipes, 37 DEG C are cultivated 18h to picking single bacterium colony；MRS trainings are connected to 2% inoculum concentration again
Foster base (100ml centrifuge tubes), 37 DEG C of culture 18h；The culture medium that the present embodiment is prepared into is inoculated in 3% inoculum concentration finally
In, 37 DEG C of cultures, different time points are sampled, and determine fungistatic effect, and determine 48h viable counts.
1.4 experimental result：A.48h Lactobacillus salivarius, Lactobacillus plantarum, lactobacillus rhamnosuss, enterococcus faecalis viable count divide
Wei (unit CFU/ml, similarly hereinafter)：4.10×109、4.5×109、4.7×109、4.3×109, as shown in table 1；B. antibacterial effect
Fruit is as shown in Figure 1.
Embodiment 2
1) culture medium prescription of the present embodiment is as follows：Semen Maydis powder 27g/L, bean cake 55g/L, molasses 12g/L, Fructose 12g/L,
K2HPO4·3H2O 2g/L, sodium acetate 5g/L, dibasic ammonium citrate 2g/L, MgSO4·7H2O 0.5g/L、MnSO4·H2O 0.5g/
L, tween 80 1ml/L, ascorbic acid 1g/L, are prepared with water.Compound method is same as Example 1.
1.1 experiment strain subjects：It is same as Example 1.
Indicator bacteria used by 1.2 experiments：It is same as Example 1.
1.3 experimental program：The present embodiment with the difference of embodiment 1 is：This enforcement is inoculated in 3% inoculum concentration finally
Example is prepared in the culture medium of gained, and 37 DEG C of cultures, different time points are sampled, and determine fungistatic effect, and determine 48h viable bacterias
Number.
1.4 experimental result：A.48h Lactobacillus salivarius, Lactobacillus plantarum, lactobacillus rhamnosuss, enterococcus faecalis viable count divide
It is not：4.35×109、4.7×109、4.5×109、4.3×109, as shown in table 1；B. fungistatic effect is as shown in Figure 2.
Embodiment 3
The culture medium prescription of the present embodiment is as follows：Semen Maydis powder 35g/L, bean cake 65g/L, molasses 18g/L, Fructose 18g/L,
K2HPO4·3H2O 2g/L, sodium acetate 5g/L, dibasic ammonium citrate 2g/L, MgSO4·7H2O 0.5g/L、MnSO4·H2O 0.5g/
L, tween 80 1ml/L, ascorbic acid 1g/L, are prepared with water.Compound method is same as Example 1.
1.1 experiment strain subjects：It is same as Example 1.
Indicator bacteria used by 1.2 experiments：It is same as Example 1.
1.3 experimental program：The present embodiment with the difference of embodiment 1 is：This enforcement is inoculated in 3% inoculum concentration finally
In the culture medium that example is prepared, 37 DEG C of cultures, different time points are sampled, and determine fungistatic effect, and determine 48h viable bacterias
Number.
1.4 experimental result：A.48h Lactobacillus salivarius, Lactobacillus plantarum, lactobacillus rhamnosuss, enterococcus faecalis viable count divide
It is not：4.2×109、4.8×109、4.9×109、4.4×109, as shown in table 1；B. fungistatic effect is as shown in Figure 3.
Embodiment 4
The culture medium prescription of the present embodiment is as follows：Tryptone 10.0g, Carnis Bovis seu Bubali cream 10.0g, yeast extract 5.0g, sugar
Sweet 15g/L, Fructose 15g/L, K2HPO4·3H2O 2g/L, sodium acetate 5g/L, dibasic ammonium citrate 2g/L, MgSO4·7H2O
0.5g/L、MnSO4·H2O 0.5g/L, tween 80 1ml/L, are routinely prepared with water.
1.1 experiment strain subjects：It is same as Example 1.
Indicator bacteria used by 1.2 experiments：It is same as Example 1.
1.3 experimental program：The present embodiment with the difference of embodiment 1 is：This enforcement is inoculated in 3% inoculum concentration finally
In the culture medium that example is prepared, 37 DEG C of cultures, different time points are sampled, and determine fungistatic effect, and determine 48h viable bacterias
Number.
1.4 experimental result：A.48h Lactobacillus salivarius, Lactobacillus plantarum, lactobacillus rhamnosuss, enterococcus faecalis viable count divide
It is not：3.6×109、3.5×109、4.2×109、4.0×109, as shown in table 1；B. fungistatic effect is as shown in Figure 4.
Reference examples 1
Reference examples are conventional MRS culture medium, and Ju Ti Pei Fang is as follows：Tryptone 10.0g, Carnis Bovis seu Bubali cream 10.0g, yeast are carried
Take thing 5.0g, glucose 20.0g, K2HPO4·3H2O 2g/L, sodium acetate 5g/L, dibasic ammonium citrate 2g/L, MgSO4·7H2O
0.5g/L、MnSO4·H2O 0.5g/L, tween 80 1ml/L, are prepared with water.
1.1 experiment strain subjects：It is same as Example 1.
Indicator bacteria used by 1.2 experiments：It is same as Example 1.
1.3 experimental program：The present embodiment with the difference of embodiment 1 is：This enforcement is inoculated in 3% inoculum concentration finally
In the culture medium that example is prepared, 37 DEG C of cultures, different time points are sampled, and determine fungistatic effect, and determine 48h viable bacterias
Number.
1.4 experimental result：A.48h Lactobacillus salivarius, Lactobacillus plantarum, lactobacillus rhamnosuss, enterococcus faecalis viable count divide
It is not：3.3×109、3.2×109、3.9×109、3.6×109, as shown in table 1；B. the MRS that fungistatic effect is shown in Fig. 1-Fig. 4
Curve.
1 each embodiment 48h viable count of lactobacillus (CFU/ml) of table
| Lactobacillus salivarius | Lactobacillus plantarum | Lactobacillus rhamnosuss | Enterococcus faecalis | |
| Embodiment 1 | 4.10×109 | 4.5×109 | 4.7×109 | 4.3×109 |
| Embodiment 2 | 4.35×109 | 4.7×109 | 4.5×109 | 4.3×109 |
| Embodiment 3 | 4.2×109 | 4.8×109 | 4.9×109 | 4.4×109 |
| Embodiment 4 | 3.6×109 | 3.5×109 | 4.2×109 | 4.0×109 |
| Reference examples 1 | 3.3×109 | 3.2×109 | 3.9×109 | 3.6×109 |
As can be seen from Table 1, in testing, 48h viable counts are above using control embodiments of the invention 1-3 formula
The MRS culture medium culturings of example 1.
In embodiment 4, when using tryptone 10.0g, Carnis Bovis seu Bubali cream 10.0g, yeast extract 5.0g substitute Semen Maydis powder and
Bean cake, and without ascorbic acid under, be compared with MRS culture medium, only change carbon source, nitrogen source does not make any adjustments, sees
Whether its result changes.The viable count of test result embodiment 4 is compared embodiment 1-3 and has been declined, but slightly better than adopts
MRS culture medium culturings are adopted in reference examples 1；
Can be seen that from Fig. 1-Fig. 4 the fungistatic effect of embodiment of the present invention 1-4 is close to MRS in even above reference examples 1
The fungistatic effect of culture medium culturing.
To sum up, under same culture conditions, with Semen Maydis powder and bean cake as nitrogen source, with Fructose and molasses as carbon source, not only may be used
The growth performance of various lactobacillus to reduce production cost, can also be kept or be improved, and is kept or is increased to secondary haemolysis arc
The fungistatic effect of bacterium.
Embodiment described above only expresses the several embodiments of the present invention, and its description is more concrete and detailed, but and
Therefore the restriction to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that for one of ordinary skill in the art
For, without departing from the inventive concept of the premise, some deformations and improvement can also be made, these belong to the guarantor of the present invention
Shield scope.Therefore, the protection domain of patent of the present invention should be defined by claims.
Claims (8)
1. a kind of animal feed production lactic acid bacteria culturing medium, it is characterised in that formula is：Semen Maydis powder 25-35g/L, bean cake 55-
65g/L, molasses 10-18g/L, Fructose 10-18g/L, K2HPO4·3H2O 0-2g/L, sodium acetate 4-7g/L, dibasic ammonium citrate 0-
3g/L、MgSO4·7H2O 0-0.5g/L、MnSO4·H2O 0.2-0.5g/L, tween 80 1-2ml/L, ascorbic acid 0.5-
3g/L, is prepared with water, and the pH value of lactic acid bacteria culturing medium is controlled in 6.8-7.2.
2. animal feed production lactic acid bacteria culturing medium according to claim 1, it is characterised in that component in formula：It is beautiful
Rice flour 30g/L, bean cake 60g/L, molasses 15g/L, Fructose 15g/L.
3. animal feed production lactic acid bacteria culturing medium according to claim 1, it is characterised in that formula is：Semen Maydis powder
30g/L, bean cake 60g/L, molasses 15g/L, Fructose 15g/L, K2HPO4·3H2O 2g/L, sodium acetate 5g/L, dibasic ammonium citrate 2g/
L、MgSO4·7H2O 0.5g/L、MnSO4·H2O 0.5g/L, tween 80 1ml/L, ascorbic acid 1g/L, are prepared with water.
4. the animal feed production lactic acid bacteria culturing medium according to claim 1-3 any one, it is characterised in that bean cake
Protein content be more than 38%, the carbohydrate content of Semen Maydis powder is not less than 40%.
5. the preparation method of the animal feed production lactic acid bacteria culturing medium described in a kind of claim 1-4 any one, which is special
Levy and be, step is as follows：
1) bean cake is weighed, plus 10-12 times of distilled water, pulping, with six layers of filtered through gauze, filtrate is stand-by；
2) Semen Maydis powder is weighed, plus 9-11 times of distilled water, to boil to sticky, with six layers of filtered through gauze, cool, filtrate is stand-by；
3) L-AA is weighed, is dissolved in appropriate distilled water, filtration sterilization, filtrate are stand-by；
4) molasses, Fructose, K are weighed respectively2HPO4·3H2O, sodium acetate, dibasic ammonium citrate, MgSO4·7H2O、MnSO4·H2O, tell
Temperature -80, is dissolved in appropriate distilled water, add 1), 2) obtained by filtrate, plus appropriate distilled water mixes, sterilizing；
5), after by solution sterilization in 4), 50 DEG C or less are cooled to, add 3) obtained by filtrate, mix.
6. the culture medium of the method preparation gained of the culture medium or claim 5 described in claim 1-4 any one is being sent out
Application in ferment lactic acid bacteria.
7. a kind of method of culture lactic acid bacteria, it is characterised in that extracting lactic acid bacterium carries out three rides in MRS flat boards, culture activation；
On flat board, picking single bacterium colony is inoculated in MRS culture medium after culture, is inoculated in MRS culture medium with the inoculum concentration of 1-2% and is trained
After supporting, then the culture medium described in claim 1-4 any one is inoculated in or using claim 5 with the inoculum concentration of 2-5%
Method prepare gained culture medium in cultivate.
8. application of the lactic acid bacteria that will be gone according to right obtained by 7 method in production animal feed.
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