CN108517308B - Micro-ecological microbial agent and preparation method and application thereof - Google Patents
Micro-ecological microbial agent and preparation method and application thereof Download PDFInfo
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- 239000003899 bactericide agent Substances 0.000 claims abstract description 52
- 240000006024 Lactobacillus plantarum Species 0.000 claims abstract description 50
- 235000013965 Lactobacillus plantarum Nutrition 0.000 claims abstract description 50
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
- C12N1/20—Bacteria; Culture media therefor
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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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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/125—Casei
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/169—Plantarum
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Abstract
The invention discloses a microecological bactericide and a preparation method thereof, wherein the microecological bactericide comprises the following steps: (a) respectively inoculating lactobacillus plantarum CGMCC No.0847 and lactobacillus casei CGMCC No.0828 into MRS culture medium for fermentation to obtain fermentation liquor; (b) centrifuging the obtained fermentation liquor, taking thalli precipitate, suspending the thalli by skim milk, freeze-drying, and dry-mixing and compounding to obtain the microbial inoculum. The application of the microecological bactericide in improving the feed utilization rate is also disclosed, and proved by verification, the microecological bactericide prepared by the invention can be mixed into the feed of dairy cows to obviously improve the feed utilization rate, and the mixed bactericide of lactobacillus plantarum CGMCC No.0847 and lactobacillus casei CGMCC No.0828 has the new application of improving the feed utilization rate, widens the microbial source of a feed degradation promoting preparation, and has good application prospect; the microecological bactericide has the advantages of long preservation period and good stability.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a microecological bactericide as well as a preparation method and application thereof.
Background
With the continuous adjustment of the animal husbandry production industry structure in China, the development of ruminants has gained great attention; the change of the resident dietary structure requires more beef, mutton and dairy products on the dining table. In recent 20 years, the degradation of grasslands is extremely serious due to the overload of livestock in grassland grasslands in China. In order to restore ecology and protect environment, the grassland animal husbandry in the areas of the middle and western parts of China is being reformed into the breeding industry, and the grazing livestock is changed into barn feeding and half barn feeding livestock. Sufficient forage resources are an important guarantee for the development of ruminant breeding industry and housing feeding livestock, China has a large number of agricultural and sideline products every year, wherein the yield of crops such as straws, alfalfa, oat grass and the like is large, and the forage resources are potential forage resources for ruminants.
Therefore, the problem of insufficient nutrition intake of ruminants can be solved by improving the utilization rate of the feed. There are two main ways to improve the utilization rate of feed, one is to weaken or destroy the ester bond between lignin and cellulose or hemicellulose by physically and chemically destroying the crystal structure of cellulose on plant cell wall. And secondly, the palatability of the coarse feed is changed, the food intake of animals is improved, and the circulation speed of chyme in the digestive tract is accelerated. Microorganisms are micro-organisms widely existing in the nature, most of the microorganisms have good probiotic effect in animal bodies, but reports on application of the microorganisms to improvement of feed utilization rate are relatively few, so that screening and obtaining the microorganisms with the effect of promoting feed degradation to prepare the micro-ecological microbial agent and applying the micro-ecological microbial agent to the dairy cow feed to improve the utilization rate of the micro-ecological microbial agent are urgent. These are problems that those skilled in the art are faced with.
Disclosure of Invention
Based on the technical problems, the invention provides a microecological bactericide and a preparation method thereof, and also provides application of the microecological bactericide in improving the utilization rate of dairy cow feed.
Specifically, the preparation method of the microecological bactericide is provided, and comprises the following steps: (a) respectively inoculating lactobacillus plantarum CGMCC No.0847 and lactobacillus casei CGMCC No.0828 into MRS culture medium for fermentation to obtain fermentation liquor; (b) centrifuging the obtained fermentation liquor, taking thalli precipitate, suspending the thalli by skim milk, freeze-drying, and dry-mixing and compounding to obtain the microbial inoculum.
Further, in the step (a), the inoculation amounts of the lactobacillus plantarum CGMCC No.0847 and the lactobacillus casei CGMCC No.0828 are both 7x106～3.5x107cfu/mL。
Further, in the step (a), the fermentation is anaerobic culture.
Further, in the step (a), the fermentation temperature is 26-42 ℃, and the fermentation time is 8-24 h.
Further, in the step (b), the centrifugation speed is 8000-12000 g, and the centrifugation time is 10-20 min.
Further, in the step (b), the concentration of the skim milk is 8 to 10% (w/v).
Further, in the step (b), the ratio of dry mixing and compounding of the lactobacillus plantarum and lactobacillus casei freeze-dried powder is as follows: 1: 9-9: 1 (w/w).
In a second aspect, a microecological microbial agent is also provided, which is prepared by any one of the preparation methods.
Furthermore, the viable bacteria number of the lactobacillus plantarum CGMCC No.0847 and the lactobacillus casei CGMCC No.0828 in the microecological bactericide is more than or equal to 6x108cfu/g。
In the third aspect, the application of the microecological bactericide in improving the utilization rate of the dairy cow feed is also provided, and the mass ratio of the added microecological bactericide to the dairy cow feed is 0.1-0.5%.
Further, the dairy cow feed is a Total Mixed Ration (TMR), alfalfa and oat grass.
Further, the utilization rate improvement effect is > 10%.
The technical scheme discloses the novel application of the mixed microbial inoculum of lactobacillus plantarum CGMCC No.0847 and lactobacillus casei CGMCC No.0828 in improving the feed utilization rate for the first time, widens the microbial source of the feed degradation promoting preparation, and has good application prospect. Meanwhile, the microecological bactericide has the advantages of preservation resistance and good stability.
Detailed Description
In order to more clearly illustrate the technical solution of the present invention, the technical solution of the present invention will be further illustrated with reference to the following specific embodiments:
in one embodiment, a method for preparing a microecological agent is provided, comprising the following steps: (a) respectively inoculating lactobacillus plantarum CGMCC No.0847 and lactobacillus casei CGMCC No.0828 into MRS culture medium for fermentation to obtain fermentation liquor; (b) centrifuging the obtained fermentation liquor, taking thalli precipitate, suspending the thalli by skim milk, freeze-drying, and dry-mixing and compounding to obtain the microbial inoculum.
Further, in the step (a), the inoculation amount of lactobacillus plantarum CGMCC No.0847 and lactobacillus casei CGMCC No.0828 is preferably 7x106～3.5x107cfu/mL; preferably 1.4x107～2.8x107cfu/mL, more preferably 2.1X107cfu/mL。
Further, in the step (a), the preferable culture method is anaerobic culture.
Further, in the step (a), the fermentation temperature is preferably 26 to 42 ℃; preferably 30 ℃ to 38 ℃; more preferably 34 deg.c.
Further, in the step (a), the preferable fermentation time is 8-24 h; preferably 12 to 20 hours; more preferably 16 h.
Further, in the step (b), the centrifugation speed is preferably 8000-12000 g, preferably 9000-11000 g, and more preferably 10000 g; the centrifugation time is preferably 10-20 min, more preferably 13-17 min, and still more preferably 15 min.
Further, in the step (b), the skim milk concentration is preferably 8 to 10% (w/v), more preferably 9% (w/v).
Further, in the step (b), the preferable dry-mixed ratio of lactobacillus plantarum and lactobacillus casei freeze-dried powder is as follows: 1:9 to 9:1(w/w), preferably 1:1 (w/w).
In combination with comparative example 1, it can be seen that when the preferred fermentation and compounding parameters are out of the range, the microecological microbial inoculum prepared by fermenting MRS culture medium with Lactobacillus plantarum CGMCC No.0847 and Lactobacillus casei CGMCC No.0828 has obviously reduced effect of improving the utilization rate of the cow feed. In the preferred range, the inoculation amount, the culture temperature, the fermentation time, the compounding ratio of the strains and the ratio of the microbial inoculum to the feed are mutually influenced, so that the improvement effect of the utilization rate of the dairy cow feed produced by the prepared microecological microbial inoculum is better.
In another embodiment, there is provided a microecological agent prepared by the process of any one of the above-described methods of preparation.
Furthermore, the viable bacteria number of the lactobacillus plantarum CGMCC No.0847 and the lactobacillus casei CGMCC No.0828 in the microecological bactericide is more than or equal to 6x108cfu/g。
In another specific embodiment, the application of the microecological microbial agent in the dairy cow feed to improve the utilization rate of the dairy cow feed is provided, and the mass ratio of the added microecological microbial agent to the dairy cow feed is 0.1-0.5%. The microecological microbial preparation is beneficial to the degradation of the milk cow feed in the rumen, and probiotics in the microecological microbial preparation are planted and grown on the gastrointestinal tissues of the cows, so that the fermentation function of the rumen can be obviously improved, and the utilization rate of the feed is improved.
Further, the dairy cow feed is a Total Mixed Ration (TMR), alfalfa and oat grass.
Further, the utilization rate improvement effect is > 10%.
The technical scheme discloses the novel application of the mixed microbial inoculum of lactobacillus plantarum CGMCC No.0847 and lactobacillus casei CGMCC No.0828 in improving the feed utilization rate for the first time, widens the microbial source of the feed degradation promoting preparation, and has good application prospect. Meanwhile, the microecological bactericide has the advantages of preservation resistance and good stability.
The following examples further illustrate the above embodiments, but do not therefore limit the invention within the scope of the examples described. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions. The reagents used in the examples, unless otherwise specified, were all analytical reagents and were purchased from the national pharmaceutical group. Other test instruments, reagents, and strains, unless otherwise specified, are commercially available.
Example 1
1. Materials and methods
(a) Preparation of seeds (fermentation strain):
lactobacillus plantarum CGMCC No. 0847: dissolving lyophilized powder of Lactobacillus plantarum CGMCC No.0847 (disclosed in Chinese patent with publication number CN 1467290A) in small amount of sterile distilled water, taking out a loop by using an inoculating loop, streaking on MRS solid culture medium (purchased from OXOID Co., UK), performing anaerobic culture at 37 deg.C for 24h, taking out a single colony by using an inoculating loop, placing the single colony in 10 mM MRS liquid culture medium (purchased from OXOID Co., UK), uniformly dispersing the colony in the liquid culture medium by using a vortex oscillator, performing anaerobic culture at 37 deg.C for 24h, inoculating the single colony in the MRS liquid culture medium by 2% (v/v) inoculation amount(purchased from OXOID Co., UK.) and anaerobically cultured at 37 ℃ for 24 hours, the culture was centrifuged at 15000rpm for 10 minutes, the supernatant was discarded, the cells were washed with sterile distilled water 2 times, and the cells were suspended in sterile distilled water of the original culture volume to obtain seeds for fermentation, the concentration of the seed solution was 7X108cfu/mL。
Lactobacillus casei CGMCC No. 0828: dissolving lyophilized powder of Lactobacillus casei CGMCC No.0828 (disclosed in Chinese patent with publication number CN 1566326A) in small amount of sterile distilled water, taking out a loop by inoculating loop, streaking on MRS solid culture medium (purchased from OXOID Co., UK), performing anaerobic culture at 37 deg.C for 24h, taking out single colony by inoculating loop, placing into 10 mM MRS liquid culture medium (purchased from OXOID Co., UK), uniformly dispersing the colony in the liquid culture medium by vortex oscillator, performing anaerobic culture at 37 deg.C for 24h, taking out, inoculated in MRS liquid medium (purchased from OXOID Co., UK) at an inoculum size of 2% (v/v), anaerobically cultured at 37 ℃ for 24h, the culture was centrifuged at 15000rpm for 10 minutes, the supernatant was discarded, the cells were washed with sterile distilled water 2 times, suspending with sterile distilled water of original culture volume to obtain seed for fermentation, wherein the bacterial concentration of the seed liquid is 7x 10.8cfu/mL。
(b) A method for measuring the effect of the microecological bactericide on improving the utilization rate of the dairy cow feed.
(1) Control group: directly feeding 10 adult cows (weight 550-600 kg) with permanent rumen fistula by using TMR feed three times a day (9: 00, 13: 00, 19: 00), freely drinking water, and continuously feeding for five days to obtain fistula cows of control group.
Experimental groups: the microecological microbial preparation is mixed with TMR feed at a ratio of 0.3% (w/w), fed to 10 adult cows with permanent rumen fistula in equal amount three times a day (9: 00, 13: 00, 19: 00), and fed with free water for five days to obtain the experimental group cow fistula.
(2) Weighing 10g of TMR, alfalfa and oat grass, respectively filling into three nylon bags, fixing on a plastic tube, and after feeding for 1h in the morning, putting the plastic tube into rumens of test cattle (a control group and an experimental group) through a fistula.
(3) After 24h, the feed in the nylon bag is taken out, the digestive juice is washed away, the nylon bag is placed in a blast drying oven to be dried to constant mass at 60 ℃, and the mass is recorded.
(4) Feed utilization, i.e. rumen degradation rate (P):
p ═ feed mass before degradation-feed mass after degradation)/feed mass before degradation x 100
(5) The improvement effect of the feed utilization rate (T%):
T％＝(Pexperimental group-PControl group)/PControl group
2. Preparation of microecological bactericide and detection of effect of microecological bactericide on improvement of cow feed utilization rate
Inoculating lactobacillus plantarum CGMCC No.0847 and lactobacillus casei CGMCC No.0828 seeds in an inoculum size of 3% (v/v) in an MRS liquid culture medium in an aseptic manner, carrying out anaerobic culture at 37 ℃ for 16h to obtain fermentation liquid, respectively centrifuging 10000g of the two obtained fermentation liquids for 15min, discarding supernatant, taking a thallus precipitate, suspending thallus by 9% (w/v) skim milk, and freeze-drying to obtain lactobacillus plantarum CGMCC No.0847 and lactobacillus casei CGMCC No.0828 freeze-dried powder, wherein the ratio of 1:1(w/w) to obtain microecological bactericide A, wherein the viable count of Lactobacillus plantarum CGMCC No.0847 is 4.5x109cfu/g, the viable count of lactobacillus casei CGMCC No.0828 is 4.5x109cfu/g。
The effect of the microecological inoculant A on improving the utilization rate of the dairy cow feed is shown in Table 1:
TABLE 1 increasing effect of Microecological agent A on cow feed utilization
| TMR | Alfalfa | Herba Avenae Fatuae | |
| P control group | 38.1 | 34.2 | 32.7 |
| P experimental group | 47.4 | 41.5 | 38.5 |
| Increase T% | 24.3％ | 21.2％ | 17.8％ |
As can be seen from Table 1, the use ratio of the cow feed can be effectively improved by feeding the microecological agent A, and the use ratios of the TMR, the alfalfa and the oat grass are respectively improved by 24.3%, 21.2% and 17.8%.
Example 2
1. Materials and methods
The addition ratio of the microecological agent was 0.1% (w/w), and the same as in example 1 was repeated.
2. Preparation of microecological bactericide and detection of effect of microecological bactericide on improvement of cow feed utilization rate
Inoculating Lactobacillus plantarum CGMCC No.0847 and Lactobacillus casei CGMCC No.0828 seeds in an inoculation amount of 1% (v/v) in an MRS liquid culture medium in an aseptic manner, carrying out anaerobic culture at 40 ℃ for 20h to obtain fermentation liquid, respectively centrifuging 12000g of the two obtained fermentation liquids for 10min, discarding supernatant, taking a thallus precipitate, suspending thallus by using 10% (w/v) skim milk, and freeze-drying to obtain Lactobacillus plantarum CGMCC No.0847 and Lactobacillus casei CGMCC No.0828 freeze-dried powder, wherein the ratio of the freeze-dried powder to the lactobacillus plantarum CGMCC No.0828 seed is 9:1(w/w) to obtain microecological bactericide B, wherein the viable count of Lactobacillus plantarum CGMCC No.0847 is 6.3x109cfuThe viable count of the lactobacillus casei CGMCC No.0828 is 0.7x109cfu/g。
The effect of the microecological inoculant B on improving the utilization rate of the dairy cow feed is shown in the following table:
TABLE 2 improving effect of microecological agent B on cow feed utilization
| TMR | Alfalfa | Herba Avenae Fatuae | |
| P control group | 38.1 | 34.2 | 32.7 |
| P experimental group | 44.3 | 39.9 | 37.2 |
| Increase T% | 16.2％ | 16.6％ | 13.8％ |
As can be seen from Table 2, the use ratio of the cow feed can be effectively improved by feeding the microecological agent B, and the use ratios of the TMR, the alfalfa and the oat grass are respectively improved by 16.2%, 16.6% and 13.8%.
Example 3
1. Materials and methods
The addition ratio of the microecological agent was 0.5% (w/w), and the same as in example 1 was repeated.
2. Preparation of microecological bactericide and detection of effect of microecological bactericide on improvement of cow feed utilization rate
Inoculating Lactobacillus plantarum CGMCC No.0847 and Lactobacillus casei CGMCC No.0828 seeds in an inoculum size of 5% (v/v) in an MRS liquid culture medium in an aseptic manner, carrying out anaerobic culture at 32 ℃ for 8h to obtain fermentation liquid, respectively centrifuging 8000g of the two obtained fermentation liquids for 20min, discarding supernatant, taking a thallus precipitate, suspending thallus by using 8% (w/v) skim milk, and freeze-drying to obtain the lactobacillus plantarum CGMCC No.0847 and lactobacillus casei CGMCC No.0828 freeze-dried powders, wherein the ratio of the freeze-dried powders is 1: 9(w/w) to obtain microecological bactericide C, wherein the viable count of Lactobacillus plantarum CGMCC No.0847 is 0.6x109cfu/g, the viable count of lactobacillus casei CGMCC No.0828 is 5.4x109cfu/g。
The effect of the microecological inoculant C on improving the utilization rate of the dairy cow feed is shown in the following table:
TABLE 3 improving effect of microecological inoculant C on cow feed utilization
| TMR | Alfalfa | Herba Avenae Fatuae | |
| P control group | 38.1 | 34.2 | 32.7 |
| P experimental group | 43.5 | 39.3 | 37 |
| Increase T% | 14.3％ | 14.9％ | 13.1％ |
As can be seen from Table 3, the use ratio of the cow feed can be effectively improved by feeding the microecological bactericide C, and the use ratios of the TMR, the alfalfa and the oat grass are respectively improved by 14.3%, 14.9% and 13.1%.
Example 4
1. Materials and methods
The addition ratio of the microecological agent was 0.2% (w/w), and the same as in example 1 was repeated.
2. Preparation of microecological bactericide and detection of effect of microecological bactericide on improvement of cow feed utilization rate
Inoculating Lactobacillus plantarum CGMCC No.0847 and Lactobacillus casei CGMCC No.0828 seeds in an inoculum size of 2% (v/v) in an MRS liquid culture medium in an aseptic manner, carrying out anaerobic culture at 42 ℃ for 24h to obtain fermentation liquid, respectively centrifuging 9000g of the two obtained fermentation liquids for 18min, discarding supernatant, taking a thallus precipitate, suspending the thallus by 9.5% (w/v) skim milk, and freeze-drying to obtain Lactobacillus plantarum CGMCC No.0847 and Lactobacillus casei 0828 freeze-dried powder with the ratio of CGMCC No. 3: 7(w/w) to obtain microecological bactericide D, wherein the viable count of Lactobacillus plantarum CGMCC No.0847 is 0.9x109cfu/g, the viable count of lactobacillus casei CGMCC No.0828 is 2.1x109cfu/g。
The effect of the microecological bactericide D on the utilization rate of the dairy cow feed is shown in the following table:
TABLE 4 improving effect of microecological agent D on cow feed utilization
| TMR | Alfalfa | Herba Avenae Fatuae | |
| P control group | 38.1 | 34.2 | 32.7 |
| P experimental group | 44.0 | 39.3 | 37.8 |
| Increase T% | 15.6％ | 14.8％ | 15.5％ |
As can be seen from Table 4, the use ratio of the cow feed can be effectively improved by feeding the microecological agent D, and the use ratios of the TMR, the alfalfa and the oat grass are respectively improved by 15.6%, 14.8% and 15.5%.
Example 5
1. Materials and methods
The addition ratio of the microecological agent was 0.4% (w/w), and the same as in example 1 was repeated.
2. Preparation of microecological bactericide and detection of effect of microecological bactericide on improvement of cow feed utilization rate
Inoculating lactobacillus plantarum CGMCC No.0847 and lactobacillus casei CGMCC No.0828 seeds in an inoculum size of 4% (v/v) in an MRS liquid culture medium in an aseptic manner, carrying out anaerobic culture at 26 ℃ for 12h to obtain fermentation liquor, respectively centrifuging 11000g of the two obtained fermentation liquors for 12min, discarding supernatant, taking a thallus precipitate, suspending thallus by 8.5% (w/v) skim milk, and freeze-drying to obtain lactobacillus plantarum CGMCC No.0847 and lactobacillus casei 0828 freeze-dried powder in a CGMCC No. 6: 4(w/w) to obtain microecological bactericide E, wherein the viable count of Lactobacillus plantarum CGMCC No.0847 is 2.4x109cfu/g, the viable count of lactobacillus casei CGMCC No.0828 is 1.6x109cfu/g。
The effect of the microecological inoculant E on improving the utilization rate of the dairy cow feed is shown in the following table:
TABLE 5 increasing effect of microecological agent E on cow feed utilization
| TMR | Alfalfa | Herba Avenae Fatuae | |
| P control group | 38.1 | 34.2 | 32.7 |
| P experimental group | 44.7 | 40.4 | 38.2 |
| Increase T% | 17.3％ | 18.1％ | 16.9％ |
As can be seen from Table 5, the use ratio of the cow feed can be effectively improved by feeding the microecological agent E, and the use ratios of the TMR, the alfalfa and the oat grass are respectively improved by 17.3%, 18.1% and 16.9%.
Effect example 1Stability of microecological bactericide for improving utilization rate of dairy cow feed under refrigeration condition
The microecologics A, B, C, D and E prepared in examples 1 to 5 were stored under refrigeration (8 ℃) for 0, 60, 120, and 180 days and then taken out, and the effect of each sample on the improvement of the cow feed (TMR) utilization rate was measured, respectively, and the results are shown in Table 6.
TABLE 6 stability to Dairy cow feed (TMR) utilization improvement under Cold storage conditions
As can be seen from Table 6, all tested microecologics maintained the same level of improvement effect on the cow feed (TMR) utilization rate after being stored for 180 days at refrigeration (8 ℃), and had better stability.
Comparative example 1
The inoculum size, culture temperature, fermentation time, compounding ratio of the strains and the ratio of the microbial inoculum to the feed in example 1 were adjusted one by one to obtain a group of microecological microbial agents prepared by the following different methods, and the improvement effect of each group of microbial agents on the feed utilization rate of the dairy cows was determined by referring to the method described in example 1, with the results shown in table 7.
TABLE 7 microecological agents prepared by different methods improve the effect on the improvement of the utilization rate of the cow feed
Referring to the results shown in table 7, it can be seen that when the inoculation amount, the culture temperature, the fermentation time, the compounding ratio of the strain and the ratio of the microbial inoculum to the feed in the preparation method of the microecological microbial agent are adjusted to be out of the preferred range, the prepared microbial agent can still improve the utilization rate of the feed for the dairy cows, but the effect is obviously reduced.
Comparative example 2
The effect of improving the feed utilization of cows by microecologics prepared from lactobacillus plantarum CGMCC No.0847 and lactobacillus casei CGMCC No.0828, lactobacillus plantarum ATCC14917 and lactobacillus casei ATCC393 (purchased from ATCC) was compared with the method described in reference example 1, and the specific operations were as follows:
1. materials and methods
(a) Preparation of seeds (fermentation strain):
lactobacillus plantarum CGMCC No.0847, lactobacillus casei CGMCC No.0828, lactobacillus plantarum ATCC14917, lactobacillus casei ATCC 393: dissolving lyophilized powder of Lactobacillus plantarum CGMCC No.0847, Lactobacillus casei CGMCC No.0828, Lactobacillus plantarum ATCC14917 and Lactobacillus casei ATCC393 in a small amount of sterile distilled water, taking out a loop by using an inoculating loop, marking on MRS solid culture medium (purchased from OXOID Co., UK), carrying out anaerobic culture at 37 ℃ for 24h, taking out a single colony by using the inoculating loop, putting the single colony into 10mL of MRS liquid culture medium (purchased from OXOID Co., UK), uniformly dispersing the colony in the liquid culture medium by using a vortex oscillator, carrying out anaerobic culture at 37 ℃ for 24h, taking out, inoculated in MRS liquid medium (purchased from OXOID Co., UK) at an inoculum size of 2% (v/v), anaerobically cultured at 37 ℃ for 24h, the culture was centrifuged at 15000rpm for 10 minutes, the supernatant was discarded, the cells were washed with sterile distilled water 2 times, suspending with sterile distilled water of original culture volume to obtain seed for fermentation, wherein the bacterial concentration of the seed liquid is 7x 10.8cfu/mL(CGMCC No.0847)、7x108cfu/mL(CGMCC No.0828)、6x108cfu/mL(ATCC 14917)、5x108cfu/mL(ATCC 393)。
(b) The determination method of the effect of the microecological bactericides prepared by the lactobacillus plantarum CGMCC No.0847 and the lactobacillus casei CGMCC No.0828 and the microecological bactericides prepared by the lactobacillus plantarum ATCC14917 and the lactobacillus casei ATCC393 on the improvement of the feed utilization rate of the dairy cows comprises the following steps: the same as in example 1.
2. Preparation of each group of microecological bactericide and detection of effect of each group of microecological bactericide on improvement of cow feed utilization rate
Inoculating each strain seed in MRS liquid culture medium with 3% (v/v) of inoculum size in a sterile manner, carrying out anaerobic culture at 37 ℃ for 16h to obtain fermentation liquor, respectively centrifuging 10000g of the two obtained fermentation liquor for 15min, discarding supernatant, taking a strain precipitate, suspending the strain by 9% (w/v) skim milk, and freeze-drying the obtained lactobacillus plantarum CGMCC No.0847 and lactobacillus casei CGMCC No.0828 freeze-dried powder in a ratio of 1:1(w/w) to obtain the microecological bactericide S, wherein the viable count of the lactobacillus plantarum CGMCC No.0847 is 4.5x109cfu/g, the viable count of lactobacillus casei CGMCC No.0828 is 4.5x109cfu/g. The lyophilized powder of lactobacillus plantarum ATCC14917, lactobacillus casei ATCC393 were also mixed in a ratio of 1:1(w/w) to obtain microecological bactericide T1, wherein the viable count of Lactobacillus plantarum ATCC14917 is 3.9x109cfu/g, viable count of Lactobacillus casei ATCC393 3.0x109cfu/g. The lactobacillus plantarum CGMCC No.0847 freeze-dried powder is directly used as a microecological bactericide T2, wherein the viable count of the lactobacillus plantarum CGMCC No.0847 is 9.0x109cfu/g. The lactobacillus casei CGMCC No.0828 freeze-dried powder is directly used as a microecological bactericide T3, wherein the viable count of the lactobacillus casei CGMCC No.0828 is 9.0x109cfu/g。
The effect of each group of microecological agents on the improvement of the feed utilization rate of dairy cows is shown in table 8.
TABLE 8 improvement of the efficiency of cow feed utilization by microecological agents prepared with different strains
As can be seen from Table 8, the microecological bactericide prepared from other conventional strains basically has no capability of improving the utilization rate of the cow feed, but the microecological bactericide prepared from a single bacterium of Lactobacillus plantarum CGMCC No.0847 and Lactobacillus casei CGMCC No.0828 has a poor (less than 10%) improvement effect on the cow feed, but the microecological bactericide prepared by compounding two bacteria of Lactobacillus plantarum CGMCC No.0847 and Lactobacillus casei CGMCC No.0828 can obviously promote the degradation of the cow feed in rumen, and finally achieves the purpose of improving the utilization rate of the feed.
The microecological bactericide provided by the invention, and the preparation method and the application thereof are described in detail above. The principles and embodiments of the present invention are explained herein using specific examples, which are presented only to assist in understanding the method and its core concepts. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.
Claims (4)
1. The application of the microecological bactericide is characterized in that the microecological bactericide is added into the dairy cow feed to improve the utilization rate of the microecological bactericide, and the mass ratio of the added microecological bactericide to the dairy cow feed is 0.1-0.5%;
the preparation method of the microecological bactericide comprises the following steps:
(a) respectively inoculating lactobacillus plantarum CGMCC No.0847 and lactobacillus casei CGMCC No.0828 into MRS culture medium for fermentation to obtain fermentation liquor;
(b) centrifuging the obtained fermentation liquor, taking thalli precipitate, suspending the thalli by skim milk, and performing freeze drying and dry mixing compounding to obtain the microbial inoculum;
the inoculation amounts of the lactobacillus plantarum CGMCC No.0847 and the lactobacillus casei CGMCC No.0828 are both 7x106～3.5x107cfu/mL；
The fermentation is anaerobic culture;
the fermentation temperature is 26-42 ℃, and the fermentation time is 8-24 h.
2. The use of the microecological agent according to claim 1, wherein in the step (b), the centrifugation speed is 8000-12000 g, and the centrifugation time is 10-20 min.
3. The use of the microecological agent according to claim 1, wherein the concentration of the skim milk in step (b) is 8-10%.
4. The use of the microecological agent according to claim 1, wherein in step (b), the dry-blended mixture of the lactobacillus plantarum and lactobacillus casei lyophilized powder comprises the following mass ratios: 1: 9-9: 1.
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| CN1566326A (en) * | 2003-06-20 | 2005-01-19 | 上海光明乳业股份有限公司 | Lactobacillus casei LC2W strain and its application in treatment of high blood pressure |
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| CN1566326A (en) * | 2003-06-20 | 2005-01-19 | 上海光明乳业股份有限公司 | Lactobacillus casei LC2W strain and its application in treatment of high blood pressure |
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