CN108102975A - One plant for the lactobacillus plantarum for the green forage that ferments and its production method of microbial inoculum - Google Patents
One plant for the lactobacillus plantarum for the green forage that ferments and its production method of microbial inoculum Download PDFInfo
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
- C12R2001/25—Lactobacillus plantarum
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
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- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23K50/00—Feeding-stuffs specially adapted for particular animals
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract
The invention discloses one plant for the lactobacillus plantarum for the green forage that ferments and its production method of microbial inoculum, wherein, the deposit number of the lactobacillus plantarum is CGMCC No.14616；The production method of microbial inoculum is：(1) by inoculation in seed culture medium, 36 DEG C~38 DEG C cultures are for 24 hours；(2) by the seed access fermentation medium of fermentation, 36 DEG C~38 DEG C cultures are for 24 hours；(3) zymotic fluid is centrifuged, is mixed into freeze drying protectant, be freeze-dried.The invention has the beneficial effects that：(1) lactobacillus plantarum can be effectively improved the Forage quality of ensilage, improve the sensory evaluation of ensilage, adjust the pH value of ensilage, control the ratio of ammoniacal nitrogen and total nitrogen, increase lactic acid content；(2) the production method fermentation time is short, and viable count is high, after fermentation for 24 hours viable count can reach 300,000,000,000/gram or more, it is lyophilized after viable count can reach 50,000,000,000/gram or more.
Description
Technical field
The present invention relates to the production methods of one plant of bacterial strain and its microbial inoculum, and in particular to one plant of plant for the green forage that ferments
The production method of object lactobacillus and its microbial inoculum, belongs to microbial technology field.
Background technology
Ensilage refers to：The green forage that moisture content is 65%~75% is after chopping, under conditions of airtight anoxia,
By the fermentation of anaerobism lactic acid bacteria, inhibit the breeding of various miscellaneous bacterias, and a kind of obtained roughage.
Ensilage smell acid perfume, soft and succulency, palatability be good, it is full of nutrition, beneficial to long-term preservation, be the excellent feeding of domestic animal
Expect source, be mainly used for feeding of ruminant animals, than fresh feed shelf-stable, nutritional ingredient is better than dry feed.
The content of the invention
First of the present invention be designed to provide one plant for the green forage that ferments, the feeding of ensilage can be effectively improved
With quality, improve the sensory evaluation of ensilage, the pH value for adjusting ensilage, the ratio of control ammoniacal nitrogen and total nitrogen, increase
The bacterial strain of lactic acid content.
Second object of the present invention is to provide the life containing above-mentioned bacterial strains, the microbial inoculum that fermentation time is short, viable count is high
Production method.
In order to realize above-mentioned first aim, the present invention adopts the following technical scheme that：
One plant of bacterial strain for the green forage that ferments, which is characterized in that Classification And Nomenclature is lactobacillus plantarum
(Lactobacillus plantarum), is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, protects
It is CGMCC No.14616 to hide number, and preservation date is on 09 15th, 2017.
In order to realize above-mentioned second target, the present invention adopts the following technical scheme that：
The production method of microbial inoculum containing foregoing bacterial strain, which is characterized in that comprise the following steps：
Step1：By inoculation in seed culture medium, 36 DEG C~38 DEG C cultures are for 24 hours；
Step2：The seed of fermentation is accessed in fermentation medium, control ph 6.0~8.0, fermentation temperature 36 DEG C~38
DEG C, rotating speed 210rpm~230rpm, ventilation ratio 1:0.4~1:0.6, tank pressure 0.04MPa~0.06MPa, culture is for 24 hours；
Step3：Gained zymotic fluid is centrifuged, by thalline：Freeze drying protectant mass ratio is 1:1~4 ratio is uniformly mixed；
Step4：Mixed thalline is placed in freeze drier and is freeze-dried to obtain bacterium powder.
Foregoing production method, which is characterized in that in Step1, foregoing seed culture medium is MRS culture mediums.
Foregoing production method, which is characterized in that in Step2, aforesaid fermentation culture medium is：By mass percentage,
Peptone 1%, beef extract 1%, yeast extract 0.5%, diammonium hydrogen citrate 0.2%, glucose 2%, lactose 1%, maltose
2%th, Tween 80 0.1%, sodium acetate 0.5%, dipotassium hydrogen phosphate 0.2%, magnesium sulfate 0.058%, manganese sulfate 0.025%, pH is certainly
So.
Foregoing production method, which is characterized in that in Step3, foregoing freeze drying protectant is：By weight, degreasing
Milk powder：Lactose：Sorbierite=2~8：1~4：1~2.
The invention has the beneficial effects that：
1st, deposit number provided by the invention is the lactobacillus plantarum (Lactobacillus of CGMCC No.14616
Plantarum), the Forage quality of ensilage can be effectively improved, improves the sensory evaluation of ensilage, adjusts ensilage
PH value, control the ratio of ammoniacal nitrogen and total nitrogen, increase lactic acid content.
2nd, the production method of microbial inoculum provided by the invention, fermentation time is short, and viable count is high, and viable count can be with after fermentation for 24 hours
Reach 300,000,000,000/gram or more, it is lyophilized after viable count can reach 50,000,000,000/gram or more.
Specific embodiment
Make specific introduce to the present invention below in conjunction with specific embodiment.
First, the separation and screening of bacterial strain
We will be collected in the ensilage sample in Zhucheng, and the method being separately cultured using dilution is coated on MRS culture mediums
On, culture is inverted in 37 DEG C of constant incubators, has then selected that growth is very fast, neat single one plant of bacterium colony.
Then we have carried out this plant of bacterium Molecular Identification using 16S rRNA sequence homology analysis technologies, and will obtain
Sequence compared in NCBI, comparison result is shown：The bacterial strain and lactobacillus plantarum that we select
The sequence similarity of (Lactobacillus plantarum) is 99%.
On 09 15th, 2017, we sent the bacterial strain to China Committee for Culture Collection of Microorganisms's common micro-organisms
Center (CGMCC) carries out preservation, preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, deposit number CGMCC
No.14616。
2nd, culture medium is prepared
1st, seed culture medium
Seed culture medium is MRS culture mediums：By mass percentage, peptone 1%, beef extract 1%, yeast extract 0.5%,
Diammonium hydrogen citrate 0.2%, glucose 2%, Tween 80 0.1%, sodium acetate 0.5%, dipotassium hydrogen phosphate 0.2%, magnesium sulfate
0.058%th, manganese sulfate 0.025%, agar 1.8%, pH are natural.
2nd, fermentation medium
Fermentation medium is new formula culture medium：By mass percentage, peptone 1%, beef extract 1%, yeast extract
0.5%th, diammonium hydrogen citrate 0.2%, glucose 2%, lactose 1%, maltose 2%, Tween 80 0.1%, sodium acetate 0.5%,
Dipotassium hydrogen phosphate 0.2%, magnesium sulfate 0.058%, manganese sulfate 0.025%, pH are natural.
Fermentation medium is that agar is eliminated on the basis of MRS culture mediums, is with the addition of lactose and maltose, nutrition is more
Fully, lactobacillus plantarum growth can faster, and yield also can higher.
3rd, microbial inoculum is produced
1st, seed liquor is obtained
By inoculation in seed culture medium, 36 DEG C~38 DEG C cultures are for 24 hours.
In the present embodiment, cultivation temperature is 37 DEG C.
2nd, fermented and cultured
The seed of fermentation is accessed in fermentation medium, control ph 6.0~8.0,36 DEG C~38 DEG C of fermentation temperature, rotating speed
210rpm~230rpm, ventilation ratio 1:0.4~1:0.6, tank pressure 0.04MPa~0.06MPa, culture is for 24 hours.
In the present embodiment, fermentation condition is：PH value 7.0,37 DEG C, rotating speed 220rpm of fermentation temperature, ventilation ratio 1:0.5,
Tank presses 0.05MPa.
3rd, freeze drying protectant is added in
(1) formula of freeze drying protectant is determined
Freeze drying protectant is：By weight, skimmed milk power：Lactose：Sorbierite=2~8：1~4：1~2.
| Skimmed milk power | Lactose | Sorbierite | |
| 1st group | 2 | 1 | 1 |
| 2nd group | 5 | 2 | 2 |
| 3rd group | 4 | 3 | 1 |
| 4th group | 6 | 4 | 1 |
| 5th group | 8 | 1 | 2 |
Gained zymotic fluid is centrifuged, by thalline：Freeze drying protectant mass ratio is 1:1 ratio is uniformly mixed, then will be mixed
Thalline after conjunction, which is placed in freeze drier, to be freeze-dried to obtain bacterium powder.
Control group：It is not added with freeze drying protectant.
After testing, the corresponding viable count of each group bacterium powder is as follows：
| Viable count | |
| 1st group | 150300000000/gram |
| 2nd group | 149600000000/gram |
| 3rd group | 154000000000/gram |
| 4th group | 151000000000/gram |
| 5th group | 150800000000/gram |
| Control group | 76000000000/gram |
As seen from the above table, skimmed milk power：Lactose：Sorbierite is 2~8：1~4：1~2 in the range of this, can be preferable
Thalline is protected, wherein, skimmed milk power：Lactose：Sorbierite is by weight=4：3：During 1 mixing, protecting effect is best.
(2) dosage of freeze drying protectant is determined
Gained zymotic fluid is centrifuged, by thalline：Freeze drying protectant (skimmed milk power：Lactose：Sorbierite is by weight=4：3：
1) mass ratio is 1:1~4 ratio is uniformly mixed, and then mixed thalline is placed in freeze drier and is freeze-dried
Bacterium powder is obtained, after testing, the corresponding viable count of each group bacterium powder is as follows：
| Thalline：Freeze drying protectant | Viable count | |
| 1st group | 1:1 | 154000000000/gram |
| 2nd group | 1:2 | 106000000000/gram |
| 3rd group | 1:3 | 76000000000/gram |
| 4th group | 1:4 | 64000000000/gram |
As seen from the above table, thalline：Freeze drying protectant is 1:1~4 in the range of this, can preferably protect thalline, considers
To the use cost of freeze drying protectant, thalline：Freeze drying protectant=1:1 is optimal operational version.
(3) thalline is mixed with freeze drying protectant
In the present embodiment, by thalline and freeze drying protectant in mass ratio 1:1 ratio is uniformly mixed, wherein, it freezes and protects
Shield agent uses skimmed milk power：Lactose：Sorbierite is by weight=4：3：1 mixing.
4th, it is freeze-dried
Mixed thalline is placed in freeze drier and is freeze-dried to obtain bacterium powder.
4th, viable count measures
Sample 1：Thalline is obtained after zymotic fluid centrifugation, therefrom gathers 10g as sample 1.
Sample 2：Bacterium powder is obtained after freeze-drying, therefrom gathers 10g as sample 2.
Sample 1 and sample 2 are diluted are separately cultured respectively, is coated on MRS culture mediums, culture carries out viable bacteria for 24 hours
Fall to count.
| Viable count | |
| Sample 1 | 380000000000/gram |
| Sample 2 | 160000000000/gram |
As seen from the above table, the viable count of two samples is all higher.
5th, ferment green forage
Two tons of maize straws that are fresh, crushing is taken to spray the above-mentioned bacterium powders of 1kg into matrix as matrix, be then placed in
In green forage fermentation vat, 45d is sealed by fermentation.
After fermentation, feed is stirred evenly, 100g is taken to carry out sample identification.
1st, sensory evaluation
| Color | Smell | Tart flavour | Structure |
| Yellow green | Fragrant tart flavour | It is dense | It moistens, is close |
As can be seen from the above table, deposit number provided by the invention is the lactobacillus plantarum of CGMCC No.14616
(Lactobacillus plantarum) can improve the sensory evaluation of ensilage, make ensilage condition, appearance more preferable.
2nd, Forage quality
Randomly choose the normal milk production of 10 adults from cattle farm, and weight in 500kg, daily yielding more than 20kg's
Cow in milk, wherein, 5 are used as experimental group to feed above-mentioned fermented feed, and 5 feed original feed as a control group.
Average daily milk production is measured daily, and METHOD FOR CONTINUOUS DETERMINATION 7 days, measurement result is as follows：
| Experimental group | Control group | |
| 1st day | 23kg | 20kg |
| 2nd day | 26kg | 24kg |
| 3rd day | 29kg | 22kg |
| 4th day | 30kg | 21kg |
| 5th day | 29kg | 23kg |
| 6th day | 29kg | 25kg |
| 7th day | 30kg | 24kg |
As can be seen from the above table, deposit number provided by the invention is the lactobacillus plantarum of CGMCC No.14616
(Lactobacillus plantarum) can be effectively improved the Forage quality of ensilage, make ensilage more nutritious, and
And it is more easy to be absorbed.
3rd, pH value, ammoniacal nitrogen and total nitrogen ratio, lactic acid content
| PH value | Ammoniacal nitrogen and total nitrogen ratio | Lactic acid content |
| 4.1 | <5% | 1.3% |
As can be seen from the above table, deposit number provided by the invention is the lactobacillus plantarum of CGMCC No.14616
(Lactobacillus plantarum) can not only adjust the pH value of (reduction) ensilage, be not easy ensilage miscellaneous
Bacterium pollutes, and effectively protects ensilage, and can control the ratio of ammoniacal nitrogen and total nitrogen (lower), makes ensilage albumen
Matter decomposition is less, effectively protects the nutrition of ensilage, while can also increase lactic acid content, and lactic acid content is evaluation ensiling
The reliability index of feed quality containing more lactic acid in good ensilage, can improve mouthfeel and nutrient absorption.
It should be noted that the invention is not limited in any way for above-described embodiment, it is all to use equivalent substitution or equivalent change
The technical solution that the mode changed is obtained, all falls in protection scope of the present invention.
Claims (5)
1. one plant of bacterial strain for the green forage that ferments, which is characterized in that Classification And Nomenclature is lactobacillus plantarum
(Lactobacillus plantarum), is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, protects
It is CGMCC No.14616 to hide number, and preservation date is on 09 15th, 2017.
2. the production method of the microbial inoculum containing bacterial strain described in claim 1, which is characterized in that comprise the following steps：
Step1：By inoculation in seed culture medium, 36 DEG C~38 DEG C cultures are for 24 hours；
Step2：By in the seed access fermentation medium of fermentation, control ph 6.0~8.0,36 DEG C~38 DEG C of fermentation temperature turns
Fast 210rpm~230rpm, ventilation ratio 1:0.4~1:0.6, tank pressure 0.04MPa~0.06MPa, culture is for 24 hours；
Step3：Gained zymotic fluid is centrifuged, by thalline：Freeze drying protectant mass ratio is 1:1~4 ratio is uniformly mixed；
Step4：Mixed thalline is placed in freeze drier and is freeze-dried to obtain bacterium powder.
3. production method according to claim 2, which is characterized in that in Step1, the seed culture medium is trained for MRS
Support base.
4. production method according to claim 2, which is characterized in that in Step2, the fermentation medium is：By matter
Measure percentages, peptone 1%, beef extract 1%, yeast extract 0.5%, diammonium hydrogen citrate 0.2%, glucose 2%, lactose
1%th, maltose 2%, Tween 80 0.1%, sodium acetate 0.5%, dipotassium hydrogen phosphate 0.2%, magnesium sulfate 0.058%, manganese sulfate
0.025%, pH are natural.
5. production method according to claim 2, which is characterized in that in Step3, the freeze drying protectant is：By weight
Amount is than meter, skimmed milk power：Lactose：Sorbierite=2~8：1~4：1~2.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109355229A (en) * | 2018-11-22 | 2019-02-19 | 中国科学院青岛生物能源与过程研究所 | A kind of lactobacillus plantarum and its application in the preparation of Chinese silvergrass ensilage |
| CN112725183A (en) * | 2020-12-30 | 2021-04-30 | 杭州娃哈哈科技有限公司 | Method for improving freeze drying survival rate of probiotics |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109355229A (en) * | 2018-11-22 | 2019-02-19 | 中国科学院青岛生物能源与过程研究所 | A kind of lactobacillus plantarum and its application in the preparation of Chinese silvergrass ensilage |
| CN112725183A (en) * | 2020-12-30 | 2021-04-30 | 杭州娃哈哈科技有限公司 | Method for improving freeze drying survival rate of probiotics |
| CN112725183B (en) * | 2020-12-30 | 2023-04-28 | 杭州娃哈哈科技有限公司 | Method for improving freeze-drying survival rate of probiotics |
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