CN105950518B - Compound lactobacillus microecological liquid preparation for healthy breeding of dairy cows - Google Patents
Compound lactobacillus microecological liquid preparation for healthy breeding of dairy cows Download PDFInfo
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Abstract
The invention belongs to the field of feeding microecologics, and particularly relates to a lactobacillus composite microecological liquid preparation for healthy breeding of dairy cows. The microecological liquid preparation is prepared from a strain of Lactobacillus plantarum (Lactobacillus plantarum) SCI-01 and a strain of Lactobacillus plantarum (Lactobacillus plantarum) SCI-02 through symbiotic coupling fermentation and covalent crosslinking embedding technologies, and can be well colonized on gastrointestinal mucosa after being fed to dairy cows, inhibit pathogenic bacteria breeding, improve beneficial flora in intestinal tracts, enhance organism immunity, effectively reduce somatic cell count, reduce recessive mastitis morbidity and increase milk yield.
Description
The technical field is as follows:
The invention belongs to the field of feeding microecologics, and particularly relates to a lactobacillus composite microecological liquid preparation for healthy breeding of dairy cows.
background art:
as early as the 70 s, some developed countries in europe and the united states have been developing new probiotics and hoped to replace antibiotics to some extent and have been restricting or banning the use of antibiotics as feed additives. In recent years, along with the strong national advocated for the healthy breeding of dairy cows, the lactobacillus microecologics in China are also widely applied to the feed industry in China.
the invention patent with application number 201310465699.4 discloses a traditional Chinese medicine microecological preparation and a preparation method thereof, wherein the traditional Chinese medicine microecological preparation comprises the following components: probiotic bacteria, a traditional Chinese medicine composition and yeast cell wall polysaccharide, wherein the probiotic bacteria comprise bacillus coagulans, lactic acid bacteria, bacillus subtilis and bacillus licheniformis, and the traditional Chinese medicine composition comprises dandelion, honeysuckle, cowherb seed, fructus forsythiae, loofah sponge and fevervine herb. The traditional Chinese medicine composition can improve the activity of probiotics and strengthen the planting capacity of the probiotics, and meanwhile, the traditional Chinese medicine composition can effectively prevent and treat recessive mastitis of cows.
Chinese patent publication ZL 201410396851.2 discloses a preparation method of a traditional Chinese medicine microecological additive for preventing and treating cow recessive mastitis, which is prepared by micronizing folium Eucommiae, folium Ginkgo, Perillae herba, radix Pulsatillae, Echinacea purpurea, herba Humuli Scandentis, shrimp shell, radix astragali, and radix Glycyrrhizae, adding water, boiling with strong fire, keeping the temperature with slow fire, cooling, filtering, collecting filtrate, adding white sugar, boiling, cooling to room temperature, inoculating Lactobacillus, black tea fungus and yeast, fermenting to obtain microecological preparation, and proportionally adding the microecological preparation into cow for drinking.
but the microecological preparation products in the domestic market still have a plurality of defects. The main points are as follows: the strain resource is limited, a professional strain bank is lacked, and most strains have poor probiotic functions; uses bacillus and microzyme as main materials, and uses less lactic acid bacteria which can be really planted in the intestinal tract; the product mainly exists in a solid form, but the liquid microecological product is less, the performance of the liquid microecological preparation in the current market is unstable, the reduction of the number of viable bacteria is obvious under the normal temperature condition (the death rate of the viable bacteria is more than 10 percent after the product is generally stored for 1 month), the function exertion of the product is limited, and the wide application is difficult.
the healthy breeding of the dairy cows pursues high yield, stable yield, high quality, safety and maximized benefit, so that the intestinal health level of the dairy cows is important. Research shows that the quality of intestinal micro-ecological environment and the digestion function of the dairy cows become limiting factors for judging whether the dairy cows are healthy or not and improving the production performance. About 25% -30% of digestible substances and 50% of crude fiber in the forage are digested and absorbed in the intestinal tract of the cow, and microorganisms play an important role in the series of digestion processes. The microecological preparation can enhance the immunity of animal organisms, maintain the microecological balance of intestinal tracts, generate beneficial metabolites and the like, and has the advantages of no toxic or side effect, no residue, no environmental pollution and the like, thereby achieving the effects of reducing the number of somatic cells of milk, preventing and treating recessive mastitis of dairy cows and improving the yield of high-quality milk. Therefore, the microecological preparation has great potential and wide development prospect.
The invention content is as follows:
In order to achieve the purpose, the invention provides a composite Lactobacillus microecological liquid preparation for healthy breeding of dairy cows, which is prepared by carrying out symbiotic coupling fermentation and covalent crosslinking embedding technologies on a Lactobacillus plantarum (Lactobacillus plantarum) SCI-01 strain and a Lactobacillus plantarum (Lactobacillus plantarum) SCI-02 strain, and the microecological liquid preparation can be well planted in gastrointestinal mucosa after being fed to dairy cows to inhibit the breeding of pathogenic bacteria, improve beneficial intestinal flora and enhance the immunity of organisms, can effectively reduce the number of somatic cells, reduce the incidence of recessive mastitis and increase the milk yield.
The Lactobacillus plantarum SCI-01 is preserved in the general microbiological center of China Committee for culture Collection of microorganisms (the address: Beijing institute of microbiology, Japan institute of sciences, postal code 100101) at 12 months and 24 days in 2015 with the preservation number of CGMCC No. 11930; the strain can be well planted on gastrointestinal mucosa, inhibit pathogenic bacteria breeding, improve intestinal beneficial flora, and enhance organism immunity;
the Lactobacillus plantarum SCI-02 is preserved in the general microbiological center of China Committee for culture Collection of microorganisms (the address: Beijing institute of microbiology, Japan institute of sciences, postal code 100101) at 12 months and 24 days in 2015 with the preservation number of CGMCC No. 11931; the strain can effectively degrade mycotoxin, and has good acid resistance, strong growth capacity and high acid production speed; the SCI-02 strain is screened by inhibiting plant pathogenic fungi (aspergillus parasiticus, aspergillus flavus, penicillium roqueforti, tomato early blight, melon wilt, apple anthracnose and melon phytophthora melonis) and intestinal pathogenic bacteria (escherichia coli, salmonella typhimurium, shigella flexneri, staphylococcus aureus and listeria monocytogenes), and natural pathogenic bacteria inhibiting substances such as high-yield phenyllactic acid and substances promoting feed intake of dairy cows such as gamma aminobutyric acid and the like;
TABLE 1 L.plantarum SCI-02 fungus inhibition assay
TABLE 2 L.plantarum SCI-02 pathogenic bacteria inhibition assay
The specific preparation process of the microecological liquid preparation comprises the following steps:
(1) Strain activation and culture: respectively inoculating the lactobacillus plantarum SCI-01 and the lactobacillus plantarum SCI-02 which are frozen and preserved in an MRS liquid culture medium, culturing for 18-22h at the temperature of 37 ℃, and carrying out subculture for 2-3 times to obtain activated strains;
The MRS liquid culture medium comprises the following components: adding 10g of peptone, 5g of beef extract, 4g of yeast extract powder, 20g of glucose, 2g of dipotassium phosphate, 5g of sodium acetate, 2g of trisodium citrate, 1mL of Tween 80, 0.2g of magnesium sulfate and 0.05g of manganese sulfate into 1000mL of distilled water, adjusting the pH value to 6.5, and sterilizing at 121 ℃ for 15 min.
(2) symbiotic coupling fermentation: inoculating activated lactobacillus plantarum SCI-01 and lactobacillus plantarum SCI-02 into a fermentation tank of the sterilized MRS culture medium according to the proportion of 5% (v/v) respectively, culturing at the constant temperature of 37 ℃, adjusting the initial pH to 6.5, fermenting for 3-5 hours by controlling the constant pH to 5.0-5.9 through fed-in neutralizing agent, naturally fermenting again, controlling the pH to 5.0-5.9 through fed-in neutralizing agent when the pH is lower than 4.5, fermenting for 3-5 hours, naturally fermenting again, circulating in this way until the viable count of lactobacillus reaches more than 1 × 1010CFU/mL, and finishing the fermentation.
(3) The covalent crosslinking embedding technology comprises the following specific processes:
a. substances added in the covalent crosslinking embedding process except rennin and glutamine transaminase are subjected to microwave sterilization at 80-100 ℃ for 15-30 min;
b. Introducing nitrogen into the liquid after symbiotic coupling fermentation to replace air in the liquid, wherein the replacement time is 5 min;
c. Adding 30-40Kg of sucrose, 15-25Kg of molasses and 5-15Kg of yeast powder into every 1000L of fermentation liquor obtained in the step (2), keeping the rotating speed of a stirring paddle of a fermentation tank at 50-100r/min, and acting for 10-30min at the temperature of 37-42 ℃ to meet the nutritional requirement of maintaining microbial micro-metabolism;
d. Adding 0.3-1.0Kg of each of three antioxidants of L-cysteine salt, cystine and catalase into each 1000L of the treated fermentation liquor, maintaining the rotating speed of a stirring paddle of a fermentation tank at 60-100r/min, and acting for 20-30min at 37-42 ℃ to reduce or remove the damage to thalli caused by peroxide generated by microbial metabolism of the thalli in the storage process;
e. adding 3-10Kg of porous starch into each 1000L of the treated fermentation liquor, maintaining the rotating speed of a stirring paddle of the fermentation tank at 50-90r/min, acting for 30min at 37 ℃, ending the adsorption and embedding process, and adsorbing and embedding the thalli and the additives in the fermentation liquor in the process to form a relatively stable microenvironment;
f. Adding 5-20Kg of skim milk into every 1000L of the treated fermentation liquor, adjusting the pH to 5.0-5.9, keeping the constant temperature at 25-45 ℃, adjusting the flow of the treated fermentation liquor at the liquid inlet by an emulsifying machine to be 10-50L/min, adjusting the flow of the covalent crosslinking embedding mixed liquor to be 0.02-0.2L/min, carrying out covalent crosslinking reaction on the two, fully reacting the substances to form stable bacterial suspension after passing through the emulsifying machine, embedding the bacteria to form a very stable microenvironment, and finishing the covalent crosslinking embedding process to prepare the composite lactobacillus microecological liquid preparation. Thereby solving the problem that the death speed of live lactobacillus in the liquid preparation is higher;
The covalent crosslinking embedding mixed solution is an aqueous solution containing 0.5-1.0% (m/m) of rennin and 0.3-1.0% (m/m) of glutamine transaminase.
The application of the microecological preparation in preventing and improving cow mastitis comprises the following specific steps:
the feeding scheme comprises the following steps: feeding the composite microecological preparation 100mL each time 1 time in the morning and evening, uniformly spraying the microecological liquid preparation 100mL on the surface of the feed, uniformly mixing, feeding the cattle suffering from mastitis or health, and continuously feeding for 10 days.
has the advantages that:
the microecological preparation provided by the invention has the following positive effects:
(1) the composite micro-ecological liquid preparation can stably store the live bacteria in the fermentation liquor for 6 months at the normal temperature of 25 ℃, the survival rate of the live bacteria is more than 80 percent, and the stability of the product in the storage process is obviously improved.
(2) after the composite microecological preparation is uniformly mixed with the feed, the feed is used for feeding the dairy cattle with mastitis by 100mL each day, each cow is fed for 10 days in the morning and at night, the number of somatic cells can be effectively reduced, the reduction rate is 78.3%, and the milk quality is improved.
(3) After the composite microecological preparation is uniformly mixed with the feed, the feed is used for feeding healthy dairy cows with 100mL of the feed every day, each cow is fed 1 time in the morning and at the evening every day, and the feed is continuously fed for 10 days, so that the average daily milk yield of a single cow can be effectively increased by 1.6 Kg/day.
(4) according to the invention, firstly, sucrose, molasses and yeast powder are added into the liquid after fermentation is finished so as to meet the nutritional requirement of maintaining microbial metabolism of the thalli; secondly, three antioxidants of L-cysteine salt, cystine and catalase are added to reduce or remove the damage to the thalli caused by peroxide generated by microbial metabolism of the thalli in the storage process; thirdly, adsorbing and embedding the thalli and the additives in the culture medium by using porous starch to form a relatively stable microenvironment; and fourthly, adding skim milk, carrying out covalent crosslinking reaction on the treated liquid, rennin and glutamine transaminage at the liquid inlet of an emulsifying machine, fully reacting the substances to form stable bacterial suspension after passing through the emulsifying machine, and embedding the bacteria to form a very stable microenvironment, so that the problem of high death rate of live lactobacillus in a liquid preparation is solved, and the stability of the product storage process is improved.
description of the drawings:
figure 1 effect of complex micro-ecological liquid preparation on average daily milk yield of dairy cows.
the specific implementation mode is as follows:
The present invention will be further illustrated with reference to the following examples; the following examples are illustrative only and do not limit the scope of the invention.
the experimental procedures in the following examples are conventional unless otherwise specified.
Example 1: micro-ecological liquid preparation and preparation method thereof
The microecological liquid preparation is prepared by carrying out symbiotic coupling fermentation and covalent crosslinking embedding technology on a strain of lactobacillus plantarum (SCI-01) and a strain of lactobacillus plantarum (SCI-02), and the preparation method comprises the following steps:
(1) strain activation and culture: respectively inoculating the lactobacillus plantarum SCI-01 and the lactobacillus plantarum SCI-02 which are frozen and preserved into an MRS liquid culture medium, culturing for 20h at the temperature of 37 ℃, and carrying out subculture for 3 times to obtain activated strains;
The MRS liquid culture medium comprises the following components: adding 10g of peptone, 5g of beef extract, 4g of yeast extract powder, 20g of glucose, 2g of dipotassium phosphate, 5g of sodium acetate, 2g of trisodium citrate, 1mL of Tween 80, 0.2g of magnesium sulfate and 0.05g of manganese sulfate into 1000mL of distilled water, adjusting the pH value to 6.5, and sterilizing at 121 ℃ for 15 min;
(2) Symbiotic coupling fermentation: inoculating activated lactobacillus plantarum SCI-01 and lactobacillus plantarum SCI-02 into a fermentation tank storing a sterilized MRS culture medium according to the proportion of 5% (V/V) respectively, culturing at the constant temperature of 37 ℃, controlling the pH to be 5.5 by feeding 25% (V/V) ammonia water as a neutralizing agent, fermenting for 3 hours at the constant pH value of 5.5, then performing natural fermentation, controlling the pH to be 5.5 by feeding 25% (V/V) ammonia water as a neutralizing agent when the pH is lower than 4.5, fermenting for 3 hours, performing natural fermentation again, and controlling for 20 hours in a circulating manner until the viable count of lactobacillus reaches more than 1 × 1010CFU/mL, and finishing the fermentation.
(3) The covalent crosslinking embedding technology comprises the following specific processes:
a. Substances added in the covalent crosslinking embedding process except rennin and glutamine transaminase are subjected to microwave sterilization at 90 ℃ for 20 min;
b. Introducing nitrogen into the liquid after symbiotic coupling fermentation to replace air in the liquid, wherein the replacement time is 5 min;
c. adding 35Kg of sucrose, 20Kg of molasses and 10Kg of yeast powder into every 1000L of the fermented liquid, maintaining the rotating speed of a stirring paddle of a fermentation tank at 80r/min, and acting for 20min at 37 ℃ to meet the nutritional requirement of maintaining microbial micro-metabolism;
d. adding 0.8Kg of each of three antioxidants of L-cysteine salt, cystine and catalase into every 1000L of fermentation liquor, maintaining the rotating speed of a stirring paddle of the fermentation tank at 80r/min, and acting for 25min at 37 ℃ to reduce or remove the damage to thalli caused by peroxide generated by thalli micro-metabolism in the storage process;
e. adding 8Kg of porous starch into every 1000L of fermentation broth, maintaining the rotation speed of the stirring paddle of the fermentation tank at 80r/min, acting for 30min at 37 ℃, and ending the adsorption embedding process;
f. Adding 10Kg of skim milk into every 1000L of fermentation liquor, adjusting the pH to 5.5, keeping the constant temperature at 37 ℃, adjusting the flow rate of the fermentation liquor after adjustment treatment at a liquid inlet by an emulsifying machine to be 25L/min, adjusting the flow rate of a covalent crosslinking embedding liquid (the embedding liquid is an aqueous solution containing 0.7% (m/m) rennin and 0.7% (m/m) glutamine transaminage) to be 0.1L/min, carrying out covalent crosslinking reaction on the covalent crosslinking embedding liquid in the emulsifying machine, fully reacting the substances after passing through the emulsifying machine to form stable bacterial suspension, embedding bacteria to form a very stable microenvironment, and finishing the covalent crosslinking embedding process to obtain the composite lactobacillus microecological liquid preparation.
example 2A micro-ecological liquid preparation and a method for preparing the same
The microecological liquid preparation is prepared by carrying out symbiotic coupling fermentation and covalent crosslinking embedding technology on a strain of lactobacillus plantarum (SCI-01) and a strain of lactobacillus plantarum (SCI-02), and the preparation method comprises the following steps:
(1) Strain activation and culture: respectively inoculating the lactobacillus plantarum SCI-01 and the lactobacillus plantarum SCI-02 which are frozen and preserved into an MRS liquid culture medium, culturing for 18h at the temperature of 37 ℃, and carrying out subculture for 2 times to obtain activated strains;
the MRS liquid culture medium comprises the following components: adding 10g of peptone, 5g of beef extract, 4g of yeast extract powder, 20g of glucose, 2g of dipotassium phosphate, 5g of sodium acetate, 2g of trisodium citrate, 1mL of Tween 80, 0.2g of magnesium sulfate and 0.05g of manganese sulfate into 1000mL of distilled water, adjusting the pH value to 6.5, and sterilizing at 121 ℃ for 15 min.
(2) symbiotic coupling fermentation: inoculating activated lactobacillus plantarum SCI-01 and lactobacillus plantarum SCI-02 into a fermentation tank storing the sterilized MRS culture medium according to the proportion of 5% (V/V) respectively, culturing at the constant temperature of 37 ℃, adjusting the initial pH to 6.5, controlling the constant pH to 5.0 by adding 25% (V/V) ammonia water in a flowing manner, fermenting for 3 hours, naturally fermenting, controlling the pH to 5.0 by adding 25% (V/V) ammonia water in a flowing manner when the pH is lower than 4.5, fermenting for 3 hours, naturally fermenting again, circulating in the same manner until the viable count of lactobacillus reaches more than 1 × 1010CFU/mL, and finishing fermentation.
(3) The covalent crosslinking embedding technology comprises the following specific processes:
a. Substances added in the covalent crosslinking embedding process except rennin and glutamine transaminase are subjected to microwave sterilization at 80 ℃ for 15 min;
b. Introducing nitrogen into the liquid after symbiotic coupling fermentation to replace air in the liquid, wherein the replacement time is 5 min;
c. adding 30Kg of sucrose, 15Kg of molasses and 5Kg of yeast powder into every 1000L of fermentation broth, maintaining the rotation speed of a stirring paddle of a fermentation tank at 50r/min, and acting for 10min at 40 ℃ to meet the nutritional requirement of maintaining microbial micro-metabolism;
d. adding 0.3Kg of each of three antioxidants of L-cysteine salt, cystine and catalase into each 1000L of the treated fermentation liquor, keeping the rotating speed of a stirring paddle of a fermentation tank at 60r/min, and acting for 20min at 40 ℃ to reduce or remove the damage to thalli caused by peroxide generated by microbial metabolism of thalli in the storage process;
e. Adding 3Kg of porous starch into each 1000L of the treated fermentation liquor, maintaining the rotating speed of a stirring paddle of the fermentation tank at 50r/min, acting for 30min at 37 ℃, ending the adsorption and embedding process, and adsorbing and embedding the thalli and the additives in the fermentation liquor in the process to form a relatively stable microenvironment;
f. adding 5Kg of skim milk into every 1000L of the treated fermentation liquor, adjusting the pH to 5.0, keeping the constant temperature at 25 ℃, adjusting the flow rate of the treated fermentation liquor at the liquid inlet by an emulsifying machine to be 10L/min, adjusting the flow rate of the covalent crosslinking embedding mixed liquor to be 0.02L/min, carrying out covalent crosslinking reaction on the skim milk and the fermentation liquor in the emulsifying machine, fully reacting the substances to form stable bacterial suspension after passing through the emulsifying machine, embedding the bacteria to form a very stable microenvironment, and finishing the covalent crosslinking embedding process to prepare the composite lactobacillus microecological liquid preparation. Thereby solving the problem that the death speed of live lactobacillus in the liquid preparation is higher;
The covalent crosslinking embedding mixed solution is an aqueous solution containing 1.0% (m/m) of rennin and 0.3% (m/m) of glutamine transaminage.
Example 3A micro-ecological liquid preparation and method for preparing the same
the microecological liquid preparation is prepared by carrying out symbiotic coupling fermentation and covalent crosslinking embedding technology on a strain of lactobacillus plantarum (SCI-01) and a strain of lactobacillus plantarum (SCI-02), and the preparation method comprises the following steps:
(1) strain activation and culture: respectively inoculating the lactobacillus plantarum SCI-01 and the lactobacillus plantarum SCI-02 which are frozen and preserved into an MRS liquid culture medium, culturing for 22h at the temperature of 37 ℃, and carrying out subculture for 3 times to obtain activated strains;
The MRS liquid culture medium comprises the following components: adding 10g of peptone, 5g of beef extract, 4g of yeast extract powder, 20g of glucose, 2g of dipotassium phosphate, 5g of sodium acetate, 2g of trisodium citrate, 1mL of Tween 80, 0.2g of magnesium sulfate and 0.05g of manganese sulfate into 1000mL of distilled water, adjusting the pH value to 6.5, and sterilizing at 121 ℃ for 15 min.
(2) symbiotic coupling fermentation: inoculating activated lactobacillus plantarum SCI-01 and lactobacillus plantarum SCI-02 into a sterilized fermentation tank of the MRS culture medium according to the proportion of 5% (V/V) respectively, culturing at a constant temperature of 37 ℃, adjusting the initial pH to 6.5, fermenting for 5 hours by controlling the constant pH to 5.9 through feeding 25% (V/V) ammonia water, then performing natural fermentation, and when the pH is lower than 4.5, controlling the pH to 5.9 through feeding 25% (V/V) ammonia water, fermenting for 5 hours, then performing natural fermentation, and circulating in this way until the viable count of lactobacillus reaches more than 1 × 1010CFU/mL, and finishing the fermentation.
(3) The covalent crosslinking embedding technology comprises the following specific processes:
a. substances added in the covalent crosslinking embedding process except rennin and glutamine transaminase are subjected to microwave sterilization at 100 ℃ for 30 min;
b. introducing nitrogen into the liquid after symbiotic coupling fermentation to replace air in the liquid, wherein the replacement time is 5 min;
c. adding 40Kg of sucrose, 25Kg of molasses and 15Kg of yeast powder into every 1000L of fermentation broth, maintaining the rotation speed of a stirring paddle of a fermentation tank at 100r/min, and acting for 30min at 42 ℃ to meet the nutritional requirement of maintaining microbial micro-metabolism;
d. adding 1.0Kg of each of three antioxidants of L-cysteine salt, cystine and catalase into each 1000L of the treated fermentation liquor, maintaining the rotating speed of a stirring paddle of a fermentation tank at 100r/min, and acting for 30min at 42 ℃ to reduce or remove the damage to thalli caused by peroxide generated by microbial metabolism of thalli in the storage process;
e. Adding 10Kg of porous starch into each 1000L of the treated fermentation liquor, maintaining the rotating speed of a stirring paddle of the fermentation tank at 90r/min, acting for 30min at 37 ℃, ending the adsorption and embedding process, and adsorbing and embedding the thalli and the additives in the fermentation liquor in the process to form a relatively stable microenvironment;
f. Adding 20Kg of skim milk into every 1000L of the treated fermentation liquor, adjusting the pH to 5.9, keeping the constant temperature at 45 ℃, adjusting the flow rate of the treated fermentation liquor at the liquid inlet by an emulsifying machine to be 50L/min, adjusting the flow rate of the covalent crosslinking embedding mixed liquor to be 0.2L/min, carrying out covalent crosslinking reaction on the skim milk and the fermentation liquor in the emulsifying machine, fully reacting the substances to form stable bacterial suspension after passing through the emulsifying machine, embedding the bacteria to form a very stable microenvironment, and finishing the covalent crosslinking embedding process to prepare the composite lactobacillus microecological liquid preparation. Thereby solving the problem that the death speed of live lactobacillus in the liquid preparation is higher;
The covalent crosslinking embedding mixed solution is an aqueous solution containing 0.5% (m/m) of rennin and 1.0% (m/m) of glutamine transaminage.
example 4: the composite micro-ecological liquid preparation has the effect of stability in normal temperature storage
the microecological liquid preparation prepared in example 1 was placed in a sterile tube with good sealing property at room temperature, and sampled at 0 day, 30 days, 60 days, 90 days, 120 days, 150 days and 180 days, respectively, for viable cell number test, and the experimental data are shown in table 3.
TABLE 3 storage stability at room temperature of the complex microecological liquid formulations
the experimental results in table 3 show that the composite microecological liquid preparation has viable count of 1.02 × 1010CFU/mL and survival rate of 81.6% when stored at normal temperature for 180 days, which indicates that the composite microecological liquid preparation has good storage stability and high practical application value.
Example 5: the composite microecological preparation has the effect of improving somatic cells in mastitis milk.
the composite microecologics prepared in example 1 are used for carrying out somatic cell tests on 20 cows with mastitis in different pastures of Zhejiang and inner Mongolia, the number of somatic cells in the cow milk is tested by a DHI (denaturing high integrity) detection method before the microecologics are fed, the fed microecologics are fed for 10 days by feeding each cow 1 time in the morning and evening and 100mL each time, then the number of somatic cells in the cow milk is tested again by the DHI detection method, and the number of somatic cells before and after the composite microecologics are fed is compared. The test results are shown in Table 4.
TABLE 4 number of somatic cells in milk before and after feeding the composite microecological preparation (Unit: ten thousand/mL)
As can be seen from the data analysis in Table 4, the somatic cell count of 18 out of 20 test cattle was significantly decreased, and only 2 cattle had no effect. If the number of somatic cells is more than 50 ten thousand/mL, the clinical mastitis is determined, and the test result indicates that: wherein 8 cattle are cured, and 10 cattle have obvious effect. The average somatic cell number of 10 cattle before test is 882.9 ten thousand/mL, and the average somatic cell number after test is 191.2 ten thousand/mL, which is reduced by 78.3%, which indicates that the compound microecological preparation fed by the cattle feed can effectively improve the mastitis of the cows and reduce the somatic cells in the milk.
example 6: the composite microecological liquid preparation has the effect of average daily milk yield of a single cow.
the composite microecological liquid preparation obtained in the example 1 is used for testing 20 healthy cows in an inner Mongolia pasture, each cow fed with the microecological preparation is fed for 1 time in the morning and evening every day, each time is 100mL, the continuous feeding is carried out for 10 days, then the daily milk production weight is recorded, the result shows that the average daily milk production of a single cow of 20 cows is obviously increased, and the test result is shown in figure 1, which shows that the composite microecological preparation can effectively increase the milk production of cows.
as can be seen from the data analysis in FIG. 1, after 20 cows are fed with the composite microecological liquid preparation for 7 days, the average daily milk yield of one cow is increased from the initial 29.5 Kg/day to 31.1 Kg/day by 1.6 Kg/day, and then the average daily milk yield of one cow on a 3-balance scale is maintained at about 31 Kg/day, which indicates that the composite microecological preparation has a significant effect in increasing the milk yield of cows.
As can be seen from the data analysis in FIG. 1, after 20 cows are fed with the composite microecological liquid preparation for 7 days, the average daily milk yield of one cow is increased from the initial 29.5 Kg/day to 31.1 Kg/day by 1.6 Kg/day, and then the average daily milk yield of one cow on a 3-balance scale is maintained at about 31 Kg/day, which indicates that the composite microecological preparation has a significant effect in increasing the milk yield of cows.
Claims (6)
1. the composite Lactobacillus microecological liquid preparation is characterized by being prepared by carrying out symbiotic coupling fermentation on Lactobacillus plantarum (SCI-01) and Lactobacillus plantarum (SCI-02) and covalently crosslinking embedding mixed liquid for covalent crosslinking and embedding; the preservation number of the Lactobacillus plantarum (Lactobacillus plantarum) SCI-01 is CGMCC No. 11930; the preservation number of the Lactobacillus plantarum (Lactobacillus plantarum) SCI-02 is CGMCC No. 11931;
The covalent cross-linking embedding mixed solution is an aqueous solution containing 0.5-1.0% of rennin and 0.3-1.0% of glutamine transaminase.
2. the method for preparing the microecological liquid preparation according to claim 1, which comprises the following steps:
(1) strain activation and culture: respectively inoculating the lactobacillus plantarum SCI-01 and the lactobacillus plantarum SCI-02 which are frozen and preserved in an MRS liquid culture medium, culturing for 18-22h at the temperature of 37 ℃, and carrying out subculture for 2-3 times to obtain activated strains;
(2) symbiotic coupling fermentation: inoculating activated lactobacillus plantarum SCI-01 and lactobacillus plantarum SCI-02 to an MRS culture medium at the same time according to the proportion of 5 percent of each, culturing at the constant temperature of 37 ℃, controlling the initial pH to be 6.5, controlling the constant pH to be 5.0-5.9 by feeding neutralizing agent, fermenting for 3-5 hours, then performing natural fermentation, controlling the pH to be 5.0-5.9 by feeding neutralizing agent when the pH is lower than 4.5, fermenting for 3-5 hours, then performing natural fermentation, circulating in the same way until the viable count of lactobacillus reaches more than 1 multiplied by 1010CFU/mL, and finishing the fermentation;
(3) covalent crosslinking embedding:
a. substances added in the covalent crosslinking embedding process except rennin and glutamine transaminase are subjected to microwave sterilization at 80-100 ℃ for 15-30 min;
b. introducing nitrogen into the liquid after symbiotic coupling fermentation to replace air in the liquid, wherein the replacement time is 5 min;
c. Adding 30-40Kg of sucrose, 15-25Kg of molasses and 5-15Kg of yeast powder into every 1000L of the fermentation broth after the fermentation in the step (2), maintaining the rotation speed of a stirring paddle of a fermentation tank at 50-100r/min, and acting for 10-30min at the temperature of 37-42 ℃;
d. adding antioxidant 0.9-3.0Kg into every 1000L fermentation broth, maintaining the rotation speed of the stirring paddle of the fermentation tank at 60-100r/min, and acting at 37-42 deg.C for 20-30 min;
e. Adding 3-10Kg of porous starch into every 1000L of fermentation broth, maintaining the rotation speed of the stirring paddle of the fermentation tank at 50-90r/min, and acting at 37 deg.C for 30 min;
f. adding 5-20Kg of skim milk into every 1000L of fermentation liquor, adjusting pH to 5.0-5.9, keeping constant temperature at 25-45 deg.C, adjusting the flow rate of the fermentation liquor at the inlet of the emulsifying machine to 10-50L/min, adjusting the flow rate of the covalent crosslinking embedding mixed liquor to 0.02-0.2L/min, and allowing the two to undergo covalent crosslinking reaction in the emulsifying machine.
3. The method for preparing a micro-ecological liquid preparation according to claim 2, wherein the MRS liquid medium consists of: adding 10g of peptone, 5g of beef extract, 4g of yeast extract powder, 20g of glucose, 2g of dipotassium phosphate, 5g of sodium acetate, 2g of trisodium citrate, 1mL of Tween 80, 0.2g of magnesium sulfate and 0.05g of manganese sulfate into 1000mL of distilled water, adjusting the pH value to 6.5, and sterilizing at 121 ℃ for 15 min.
4. the method for preparing a microecological liquid preparation according to claim 2, wherein the neutralizing agent is 25% by volume of aqueous ammonia.
5. The method for preparing a microecological liquid preparation according to claim 2, wherein the antioxidant comprises L-cysteine salt, cystine and catalase in an amount of 0.5Kg for each 1000L of fermentation broth.
6. the use of the complex lactic acid bacteria micro-ecological liquid preparation of claim 1 in the field of preparing feed.
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