CN105950518A - Compound lactic acid bacterium micro-ecology liquid preparation for healthy dairy cow breeding - Google Patents

Compound lactic acid bacterium micro-ecology liquid preparation for healthy dairy cow breeding Download PDF

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CN105950518A
CN105950518A CN201610555996.1A CN201610555996A CN105950518A CN 105950518 A CN105950518 A CN 105950518A CN 201610555996 A CN201610555996 A CN 201610555996A CN 105950518 A CN105950518 A CN 105950518A
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lactobacillus plantarum
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包维臣
焦丽英
刘现伟
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Jinhua Yinhe Biological Science And Technology Co Ltd
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Abstract

The invention belongs to the field of feed probiotics preparation, and particularly relates to a compound lactic acid bacterium micro-ecology liquid preparation for healthy dairy cow breeding. The micro-biology liquid preparation is prepared from Lactobacillus plantarum SCI-01 and Lactobacillus plantarum SCI-02 through symbiotic coupling fermentation and covalent cross-linking embedding technology. The compound lactic acid bacterium micro-ecology liquid preparation can be well colonized on a gastrointestinal mucous membrane after being fed to dairy cows, pathogenic bacterium breeding is restrained, the intestinal effective microbial community is improved, the organism immunity is enhanced, the number of somatic cells can be effectively decreased, the recessive mastitis morbidity is decreased, and the milk yield is increased.

Description

A kind of compound lactobacillus Tiny ecosystem liquid preparation for milk cow health cultivation
Technical field:
The invention belongs to feeding micro-ecological preparation field, be specifically related to a kind of lactic acid bacteria compound microecological liquid preparation that can be used for milk cow health cultivation.
Background technology:
As far back as the seventies, more American-European developed countries develop novel microbial ecological agent in succession, and wish substitute antibiotics to a certain extent, and in succession limit or prohibit the use of antibiotic as feed additive.In recent years, vigorously advocating milk cow health cultivation with country, China's lactobacillus micro-ecological preparation is similarly obtained extensively application at China's feedstuff industry.
The patent of invention of Application No. 201310465699.4 discloses a kind of Chinese medicine microecological preparation and preparation method thereof, its composition comprises: probiotic bacteria, Chinese medicine composition and yeast cell wall polysaccharide, probiotic bacteria includes Bacillus coagulans, lactic acid bacteria, bacillus subtilis, Bacillus licheniformis, and Chinese medicine composition includes Herba Taraxaci, Flos Lonicerae, Semen Vaccariae, Fructus Forsythiae, Retinervus Luffae Fructus, adds and hang rattan.Chinese medicine composition of the present invention can improve probiotic active, strengthens the field planting ability of probiotic bacteria, and Chinese medicine composition can effectively prevent and treat the latent mammitis of milch cow simultaneously.
The Chinese invention patent public affairs of Patent No. ZL 201410396851.2 disclose a kind of preparation method preventing and treating bovine subclinical mastitis Traditional Chinese drug microecology additive, it is prepared as follows, by Folium Eucommiae, Folium Ginkgo, Folium Perillae, the Radix Pulsatillae, Echinacea, grass, Crusta Penaeus seu Panulirus, the Radix Astragali, Radix Glycyrrhizae micronizing, the intense fire that adds water boils, slow fire is incubated, cold filtration, collect filtrate, add white sugar to boil, cooling down to inoculating lactobacillus after room temperature, red tea fungus and yeast fermentation gained microbial ecological agent, gained microbial ecological agent is drunk to milch cow in proportion.
But domestic market microbial ecological agent product still suffers from many defects.Being mainly manifested in: microorganism resource is limited, lack the strain library of specialty, the major part prebiotic function of bacterial strain is poor；To use sporeformer, yeast, and really can use less with field planting lactic acid bacteria in intestinal；Product exists the most in solid form, and liquid type Tiny ecosystem product is less, liquid type microbial ecological agent unstable properties in the market, under normal temperature condition, viable count declines substantially (viable bacteria mortality rate ＞ 10% after typically storing 1 month), make product function play to be restricted, it is difficult to extensively apply.
Milk cow health cultivation is all pursuing high yield, stable yields, high-quality, safety, maximizing the benefits, and therefore milch cow intestinal health level is again the most important thing.Research shows that the quality of milch cow intestine microenvironment and the height of digestive function become the restrictive factor that whether healthy milch cow and can production performance be improved.The digestible substance and 50% crude fibre that there are about 25%-30% in forage are digested and assimilated in milch cow rear intestinal, and in this series of digestion process, microorganism plays an important role.And microbial ecological agent can strengthen animal body immunity, maintain the several functions such as intestinal microecology balance, generation beneficial metabolic product, and have have no side effect, do not remain, the advantage such as free from environmental pollution, thus reduce milk somatic cell number, prevent and treat bovine subclinical mastitis and improve the effect of high-quality milk yield.Therefore microbial ecological agent has bigger potentiality and vast potential for future development.
Summary of the invention:
To achieve these goals, the present invention provides a kind of compound lactobacillus Tiny ecosystem liquid preparation for milk cow health cultivation, described Tiny ecosystem liquid preparation is made by a lactobacillus plantarum (Lactobacillus plantarum) SCI-01 and a lactobacillus plantarum (Lactobacillus plantarum) SCI-02 form through symbiosis coupled fermentation and covalent cross-linking embedding techniques, can be good at being colonizated in gastrointestinal tract mucous after feeding cow, pathogen grows in suppression, improve intestinal beneficial flora, enhancing human body immunity power, can effectively reduce somatic number, reduce latent mammitis sickness rate, increase milk yield.
Described Lactobacillus plantarum (Lactobacillus plantarum) SCI-01, this bacterial strain is preserved in (address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, China Committee for Culture Collection of Microorganisms's common micro-organisms center on the 24th in December in 2015, Institute of Microorganism, Academia Sinica, postcode 100101), deposit number CGMCC No.11930；This bacterial strain can be good at being colonizated in gastrointestinal tract mucous, and pathogen grows in suppression, improves intestinal beneficial flora, enhancing human body immunity power；
Described Lactobacillus plantarum (Lactobacillus plantarum) SCI-02, this bacterial strain is preserved in (address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, China Committee for Culture Collection of Microorganisms's common micro-organisms center on the 24th in December in 2015, Institute of Microorganism, Academia Sinica, postcode 100101), deposit number CGMCC No.11931；This bacterial strain can effective degradative fungi toxin, there is good acid resistance, energy for growth is strong, acid production speed is fast；Described SCI-02 bacterial strain by suppressing phytopathogenic fungi (aspergillus parasiticus, Aspergillus flavus, penicillium roqueforti, Fructus Lycopersici esculenti early epidemic, Fructus Melo withered, Fructus Mali pumilae anthrax, Fructus Melo phytophthora) and enteropathic antibacterial (escherichia coli, Mus hinder Salmonella, shigella flexneri, staphylococcus aureus, unit cell listeria spp) to screen, the natural promotion cow feeding quantity of material such as controlling pathogenic bacterium material and gamma-aminobutyric acid that presses down such as high yield phenyllactic acid；
Table 1 L.plantarum SCI-02 fungus inhibition test
Table 2 L.plantarum SCI-02 malignant bacteria inhibition test
The concrete processing technology of described Tiny ecosystem liquid preparation is as follows:
(1) actication of culture and cultivation: the Lactobacillus plantarum SCI-01 and Lactobacillus plantarum SCI-02 of freezen protective are inoculated in respectively in MRS fluid medium, cultivates 18-22h at temperature 37 DEG C, and such Secondary Culture obtains activated spawn 2-3 time；
Described MRS fluid medium composition is as follows: 10g peptone, 5g Carnis Bovis seu Bubali cream, 4g yeast leaching powder, 20g glucose, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g trisodium citrate, 1mL Tween 80,0.2g magnesium sulfate, 0.05g manganese sulfate add 1000mL distilled water, regulation pH to 6.5,121 DEG C of sterilizing 15min.
(2) symbiosis coupled fermentation: the Lactobacillus plantarum SCI-01 activated and Lactobacillus plantarum SCI-02 is inoculated in the fermentation tank of the above-mentioned MRS culture medium of sterilizing according to the ratio of each 5% (v/v) simultaneously, 37 DEG C of constant temperature culture, regulate initial pH6.5, after adding nertralizer control permanent pH5.0-5.9 fermentation 3-5 hour by stream, carry out natural fermentation again, when pH is less than 4.5, after adding nertralizer control pH to 5.0-5.9 fermentation 3-5 hour by stream, carry out natural fermentation again, so circulation, reaches 1 × 10 to viable count of lactobacillus10More than CFU/mL, terminates fermentation.
(3) described covalent cross-linking embedding techniques concrete technology is as follows:
A. in covalent cross-linking embedding process, institute's substance is in addition to Chymosin and paddy propionic acid amide. transaminase, and other is all through microwave disinfection, and microwave disinfection temperature is 80-100 DEG C, 15-30min；
B. the liquid after symbiosis coupled fermentation is passed through nitrogen and replaces air therein, time swap 5min；
C., step (2) gained every 1000L fermentation liquid adds 30-40Kg sucrose, 15-25Kg molasses and 5-15Kg yeast powder, and keep the rotating speed of fermentation tank stirring paddle 50-100r/min, under the conditions of 37-42 DEG C, effect 10-30min is with the nutritional need of the satisfied micro-metabolism of maintenance thalline；
D. to every 1000L fermentation liquid of above-mentioned process adds Cys salt, cystine and three kinds of each 0.3-1.0Kg of antioxidant of catalase, and keep the rotating speed of fermentation tank stirring paddle 60-100r/min, under the conditions of 37-42 DEG C, effect 20-30min reduces or removing micro-metabolism of thalline in storage produces peroxide and thalline is caused damage；
E. to every 1000L fermentation liquid of above-mentioned process adds 3-10Kg porous-starch, and keep the rotating speed of fermentation tank stirring paddle 50-90r/min, under the conditions of 37 DEG C, effect 30min, terminate absorption embedding process, this process carries out absorption embedding to the thalline in fermentation liquid and above-mentioned additive, forms metastable microenvironment；
F. to every 1000L fermentation liquid of above-mentioned process adds 5-20Kg skimmed milk, regulation pH to 5.0-5.9, keep constant temperature 25-45 DEG C, processing after fermentation flow quantity by mulser regulation at inlet is 10-50L/min, regulation covalent cross-linking embedding mixed liquor flow is 0.02-0.2L/min, covalent cross-linking reaction is there is in the two in mulser, and after mulser, these several materials fully react and form stable bacteria suspension, thalline embedding is defined highly stable microenvironment, terminate covalent cross-linking embedding process, prepare compound lactobacillus Tiny ecosystem liquid preparation.Thus solve viable lactic acid bacteria death rate problem faster in liquid preparation；
Described covalent cross-linking embedding mixed liquor is containing 0.5-1.0% (m/m) Chymosin and the aqueous solution of 0.3-1.0% (m/m) paddy propionic acid amide. transaminase.
Described microbial ecological agent is in prevention and improves the application in mammitis of cow, and concrete grammar is as follows:
Feed scheme: feed compound micro-ecological preparation morning and evening every day each 1 time, each 100mL, by 100mL Tiny ecosystem liquid preparation uniform sprinkling feed surface mix homogeneously, feed the cattle suffering from mastitis or health, feed 10 days continuously.
Beneficial effect:
The microbial ecological agent that the present invention provides has a following good effect:
(1) fermentation liquid viable bacteria stably can be stored 6 months under 25 DEG C of normal temperature conditions by compound microecological liquid preparation of the present invention, and Viable detection is more than 80%, significantly improves product storage stability.
(2) after compound micro-ecological preparation of the present invention is mixed homogeneously with feedstuff, feed the milch cow suffering from mastitis, feed 100mL every day, every cattle every day each 1 time sooner or later, feed continuously 10 days, can effectively reduce somatic number, reduction rate is 78.3%, improves milk quality.
(3) after compound micro-ecological preparation of the present invention is mixed homogeneously with feedstuff, the milch cow of Feeding of healthy, feed 100mL every day, every cattle every day each 1 time sooner or later, feed continuously 10 days, milch cow average single head daily yielding 1.6Kg/ days can be effectively increased.
(4) sucrose, molasses and yeast powder are added during the present invention first passes through the liquid to fermentation ends, with the nutritional need of the satisfied micro-metabolism of maintenance thalline；Second, add Cys salt, cystine and three kinds of antioxidants of catalase, reduce or remove the micro-metabolism of thalline in storage generation peroxide and thalline is caused damage；3rd, with porous-starch, thalline therein and above-mentioned additive are carried out absorption embedding, form metastable microenvironment；4th, add skimmed milk, at mulser inlet by process after liquid occur covalent cross-linking to react with Chymosin and paddy propionic acid amide. transaminase, and after mulser, these several materials fully react and form stable bacteria suspension, thalline embedding is defined highly stable microenvironment, thus solves viable lactic acid bacteria death rate problem faster in liquid preparation, improve product storage stability.
Accompanying drawing illustrates:
The effect of Fig. 1 compound microecological liquid preparation single head average to milch cow daily yielding.
Detailed description of the invention:
Below in conjunction with embodiment, the invention will be further described；Following embodiment is merely exemplary, and is not limiting as protection scope of the present invention.
Experimental technique in following embodiment, if no special instructions, is conventional method.
Embodiment 1: a kind of Tiny ecosystem liquid preparation and preparation method thereof
Compound lactobacillus Tiny ecosystem liquid preparation, described Tiny ecosystem liquid preparation is made by a lactobacillus plantarum (Lactobacillus.plantarum) SCI-01 and a lactobacillus plantarum (Lactobacillus.plantarum) SCI-02 form through symbiosis coupled fermentation and covalent cross-linking embedding techniques, and concrete preparation method is as follows:
(1) actication of culture and cultivation: the Lactobacillus plantarum SCI-01 and Lactobacillus plantarum SCI-02 of freezen protective are inoculated in respectively in MRS fluid medium, cultivates 20h at temperature 37 DEG C, and such Secondary Culture obtains activated spawn 3 times；
MRS fluid medium composition is as follows: 10g peptone, 5g Carnis Bovis seu Bubali cream, 4g yeast leaching powder, 20g glucose, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g trisodium citrate, 1mL Tween 80,0.2g magnesium sulfate, 0.05g manganese sulfate add 1000mL distilled water, regulation pH to 6.5,121 DEG C of sterilizing 15min；
(2) symbiosis coupled fermentation: the Lactobacillus plantarum SCI-01 activated and Lactobacillus plantarum SCI-02 is inoculated into according to the ratio of each 5% (v/v) simultaneously and has in the fermentation tank of the MRS culture medium of sterilizing, 37 DEG C of constant temperature culture, initial pH 6.5, control after permanent pH5.5 ferments 3 hours by stream (V/V) ammonia that adds 25% as nertralizer, carry out natural fermentation again, when pH is less than 4.5, (V/V) ammonia controls pH to 5.5 fermentation 3h as nertralizer to add 25% by stream, carry out natural fermentation again, so loop control 20 hours, 1 × 10 is reached to viable count of lactobacillus10More than CFU/mL, terminates fermentation.
(3) described covalent cross-linking embedding techniques concrete technology is as follows:
A. in covalent cross-linking embedding process, institute's substance is in addition to Chymosin and paddy propionic acid amide. transaminase, and other is all through microwave disinfection, and microwave disinfection temperature is 90 DEG C, 20min；
B. the liquid after symbiosis coupled fermentation is passed through nitrogen and replaces air therein, time swap 5min；
C. every 1000L addition 35Kg sucrose, 20Kg molasses and 10Kg yeast powder in the liquid of fermentation ends, and keep the rotating speed of fermentation tank stirring paddle 80r/min, under the conditions of 37 DEG C, effect 20min is with satisfied maintenance bacterium The nutritional need of the micro-metabolism of body；
D. every 1000L fermentation liquid adds Cys salt, cystine and three kinds of each 0.8Kg of antioxidant of catalase, and keep the rotating speed of fermentation tank stirring paddle 80r/min, under the conditions of 37 DEG C, effect 25min reduces or removing micro-metabolism of thalline in storage produces peroxide and thalline is caused damage；
E. every 1000L fermentation liquid addition 8Kg porous-starch, and keep the rotating speed of fermentation tank stirring paddle 80r/min, under the conditions of 37 DEG C, act on 30min, terminate absorption embedding process；
F. every 1000L fermentation liquid adds 10Kg skimmed milk, regulation pH to 5.5, keep constant temperature 37 DEG C, processing after fermentation flow quantity by mulser regulation at inlet is 25L/min, regulation covalent cross-linking embedding liquid (described embedding liquid is containing 0.7% (m/m) Chymosin and the aqueous solution of 0.7% (m/m) paddy propionic acid amide. transaminase) flow is 0.1L/min, covalent cross-linking embedding liquid occurs covalent cross-linking to react in mulser, and after mulser, these several materials fully react and form stable bacteria suspension, thalline embedding is defined highly stable microenvironment, terminate covalent cross-linking embedding process, prepare compound lactobacillus Tiny ecosystem liquid preparation.
2 one kinds of Tiny ecosystem liquid preparations of embodiment and preparation method thereof
Compound lactobacillus Tiny ecosystem liquid preparation, described Tiny ecosystem liquid preparation is made by a lactobacillus plantarum (Lactobacillus.plantarum) SCI-01 and a lactobacillus plantarum (Lactobacillus.plantarum) SCI-02 form through symbiosis coupled fermentation and covalent cross-linking embedding techniques, and concrete preparation method is as follows:
(1) actication of culture and cultivation: the Lactobacillus plantarum SCI-01 and Lactobacillus plantarum SCI-02 of freezen protective are inoculated in respectively in MRS fluid medium, cultivates 18h at temperature 37 DEG C, and such Secondary Culture obtains activated spawn 2 times；
Described MRS fluid medium composition is as follows: 10g peptone, 5g Carnis Bovis seu Bubali cream, 4g yeast leaching powder, 20g glucose, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g trisodium citrate, 1mL Tween 80,0.2g magnesium sulfate, 0.05g manganese sulfate add 1000mL distilled water, regulation pH to 6.5,121 DEG C of sterilizing 15min.
(2) symbiosis coupled fermentation: the Lactobacillus plantarum SCI-01 activated and Lactobacillus plantarum SCI-02 is inoculated into according to the ratio of each 5% (v/v) simultaneously and has in the fermentation tank of the above-mentioned MRS culture medium of sterilizing, 37 DEG C of constant temperature culture, regulate initial pH6.5, control after permanent pH5.0 ferments 3 hours by stream (V/V) ammonia that adds 25%, carry out natural fermentation again, when pH is less than 4.5, control after pH to 5.0 ferments 3 hours by stream (V/V) ammonia that adds 25%, carry out natural fermentation again, so circulation, reaches 1 × 10 to viable count of lactobacillus10More than CFU/mL, terminates fermentation.
(3) described covalent cross-linking embedding techniques concrete technology is as follows:
A. in covalent cross-linking embedding process, institute's substance is in addition to Chymosin and paddy propionic acid amide. transaminase, and other is all through microwave disinfection, and microwave disinfection temperature is 80 DEG C, 15min；
B. the liquid after symbiosis coupled fermentation is passed through nitrogen and replaces air therein, time swap 5min；
C. addition 30Kg sucrose, 15Kg molasses and 5Kg yeast powder in every 1000L fermentation liquid, and keep the rotating speed of fermentation tank stirring paddle 50r/min, under the conditions of 40 DEG C, effect 10min is with the nutritional need of the satisfied micro-metabolism of maintenance thalline；
D. to every 1000L fermentation liquid of above-mentioned process adds Cys salt, cystine and three kinds of each 0.3Kg of antioxidant of catalase, and keep the rotating speed of fermentation tank stirring paddle 60r/min, under the conditions of 40 DEG C, effect 20min reduces or removing micro-metabolism of thalline in storage produces peroxide and thalline is caused damage；
E. to every 1000L fermentation liquid of above-mentioned process adds 3Kg porous-starch, and keep the rotating speed of fermentation tank stirring paddle 50r/min, under the conditions of 37 DEG C, effect 30min, terminate absorption embedding process, this process carries out absorption embedding to the thalline in fermentation liquid and above-mentioned additive, forms metastable microenvironment；
F. to every 1000L fermentation liquid of above-mentioned process adds 5Kg skimmed milk, regulation pH to 5.0, keep constant temperature 25 DEG C, processing after fermentation flow quantity by mulser regulation at inlet is 10L/min, regulation covalent cross-linking embedding mixed liquor flow is 0.02L/min, covalent cross-linking reaction is there is in the two in mulser, and after mulser, these several materials fully react and form stable bacteria suspension, thalline embedding is defined highly stable microenvironment, terminate covalent cross-linking embedding process, prepare compound lactobacillus Tiny ecosystem liquid preparation.Thus solve viable lactic acid bacteria death rate problem faster in liquid preparation；
Described covalent cross-linking embedding mixed liquor is containing 1.0% (m/m) Chymosin and the aqueous solution of 0.3% (m/m) paddy propionic acid amide. transaminase.
3 one kinds of Tiny ecosystem liquid preparations of embodiment and preparation method thereof
Compound lactobacillus Tiny ecosystem liquid preparation, described Tiny ecosystem liquid preparation is made by a lactobacillus plantarum (Lactobacillus.plantarum) SCI-01 and a lactobacillus plantarum (Lactobacillus.plantarum) SCI-02 form through symbiosis coupled fermentation and covalent cross-linking embedding techniques, and concrete preparation method is as follows:
(1) actication of culture and cultivation: the Lactobacillus plantarum SCI-01 and Lactobacillus plantarum SCI-02 of freezen protective are inoculated in respectively in MRS fluid medium, cultivates 22h at temperature 37 DEG C, and such Secondary Culture obtains activated spawn 3 times；
Described MRS fluid medium composition is as follows: 10g peptone, 5g Carnis Bovis seu Bubali cream, 4g yeast leaching powder, 20g glucose, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g trisodium citrate, 1mL Tween 80,0.2g magnesium sulfate, 0.05g manganese sulfate add 1000mL distilled water, regulation pH to 6.5,121 DEG C of sterilizing 15min.
(2) symbiosis coupled fermentation: the Lactobacillus plantarum SCI-01 activated and Lactobacillus plantarum SCI-02 is inoculated in the fermentation tank of the above-mentioned MRS culture medium of sterilizing according to the ratio of each 5% (v/v) simultaneously, 37 DEG C of constant temperature culture, regulate initial pH6.5, control after permanent pH5.9 ferments 5 hours by stream (V/V) ammonia that adds 25%, carry out natural fermentation again, when pH is less than 4.5, control after pH to 5.9 ferments 5 hours by stream (V/V) ammonia that adds 25%, carry out natural fermentation again, so circulation, reaches 1 × 10 to viable count of lactobacillus10More than CFU/mL, terminates fermentation.
(3) described covalent cross-linking embedding techniques concrete technology is as follows:
A. in covalent cross-linking embedding process, institute's substance is in addition to Chymosin and paddy propionic acid amide. transaminase, and other is all through microwave disinfection, and microwave disinfection temperature is 100 DEG C, 30min；
B. the liquid after symbiosis coupled fermentation is passed through nitrogen and replaces air therein, time swap 5min；
C. addition 40Kg sucrose, 25Kg molasses and 15Kg yeast powder in every 1000L fermentation liquid, and keep the rotating speed of fermentation tank stirring paddle 100r/min, under the conditions of 42 DEG C, effect 30min is with the nutritional need of the satisfied micro-metabolism of maintenance thalline；
D. to every 1000L fermentation liquid of above-mentioned process adds Cys salt, cystine and three kinds of each 1.0Kg of antioxidant of catalase, and keep the rotating speed of fermentation tank stirring paddle 100r/min, under the conditions of 42 DEG C, effect 30min reduces or removing micro-metabolism of thalline in storage produces peroxide and thalline is caused damage；
E. to every 1000L fermentation liquid of above-mentioned process adds 10Kg porous-starch, and keep the rotating speed of fermentation tank stirring paddle 90r/min, under the conditions of 37 DEG C, effect 30min, terminate absorption embedding process, this process carries out absorption embedding to the thalline in fermentation liquid and above-mentioned additive, forms metastable microenvironment；
F. to every 1000L fermentation liquid of above-mentioned process adds 20Kg skimmed milk, regulation pH to 5.9, keep constant temperature 45 DEG C, processing after fermentation flow quantity by mulser regulation at inlet is 50L/min, regulation covalent cross-linking embedding mixed liquor flow is 0.2L/min, covalent cross-linking reaction is there is in the two in mulser, and after mulser, these several materials fully react and form stable bacteria suspension, thalline embedding is defined highly stable microenvironment, terminate covalent cross-linking embedding process, prepare compound lactobacillus Tiny ecosystem liquid preparation.Thus solve viable lactic acid bacteria death rate problem faster in liquid preparation；
Described covalent cross-linking embedding mixed liquor is containing 0.5% (m/m) Chymosin and the aqueous solution of 1.0% (m/m) paddy propionic acid amide. transaminase.
Embodiment 4: compound microecological liquid preparation storage at normal temperature stabilizing effect of the present invention
Tiny ecosystem liquid preparation embodiment 1 prepared at ambient temperature, is positioned in the sterile tube that sealing is good, respectively 0 day, 30 days, 60 days, 90 days, 120 days, 150 days and sampling in 180 days, carries out viable count inspection, and experimental data is shown in Table 3.
Table 3 compound microecological liquid preparation room temperature bin stability situation
From table 3 experimental result, being stored 180 days by this compound microecological liquid preparation under normal temperature condition, its viable count can reach 1.02 × 1010CFU/mL, its survival rate is 81.6%, illustrates that this compound microecological liquid preparation has good bin stability, is of very high actual application value.
Embodiment 5: compound micro-ecological preparation of the present invention is to improvement result somatic in Bovine Mastitis Milk.
Utilize embodiment 1 gained compound micro-ecological preparation pasture different with the Inner Mongol to Zhejiang, 20 cattle suffering from mastitis carry out somatic cell test, before feeding microbial ecological agent, utilize the somatic number in DHI detection method test Lac Bovis seu Bubali, feed microbial ecological agent every cattle respectively to feed 1 time every day, each 100mL, feed 10 days continuously, then utilize DHI detection method again to test the somatic number in Lac Bovis seu Bubali, compare feeding the somatic number before and after compound micro-ecological preparation.Result of the test is shown in Table 4.
Table 4 feeds before and after compound micro-ecological preparation somatic number situation of change (unit: ten thousand/mL) in Lac Bovis seu Bubali
By table 4 data analysis, in 20 test cattle, being decreased obviously occurs in the somatic number of 18 cattle, and only 2 cattle are to no effect.If being more than 500,000/mL by somatic number is clinic mastitis, then result of the test illustrates: wherein 8 cattle are cured, and 10 cattle effects are obvious.Before 10 cattle tests, average body cell number is 882.9 ten thousand/mL, and after test, average body cell number is 191.2 ten thousand/mL, have dropped 78.3%, shows that feeding compound micro-ecological preparation can be effectively improved mammitis of cow, reduces the somatic cell in Lac Bovis seu Bubali.
Embodiment 6: the effect of compound microecological liquid preparation of the present invention single head average to milch cow daily yielding.
20 the healthy cattle utilizing embodiment 1 gained compound microecological liquid preparation Inner Mongolia pasture are tested, feed microbial ecological agent every cattle respectively to feed 1 time every day, 100mL every time, feed 10 days continuously, then the weight of giving milk of every day is recorded, result shows that the average single head daily yielding of 20 cow heads substantially increases, and result of the test is shown in Fig. 1, illustrates that this compound micro-ecological preparation can be effectively increased milk production of cow.
By Fig. 1 data analysis, 20 cow heads are after 7 days feed compound microecological liquid preparation, milch cow average single head daily yielding was increased to 31.1Kg/ days by initial 29.5Kg/ days, increase 1.6Kg/ days, thereafter within 3 days, average single head daily yielding maintains about 31Kg/ days, illustrates that this compound micro-ecological preparation has remarkable result in terms of having increase milk production of cow.
By Fig. 1 data analysis, 20 cow heads are after 7 days feed compound microecological liquid preparation, milch cow average single head daily yielding was increased to 31.1Kg/ days by initial 29.5Kg/ days, increase 1.6Kg/ days, thereafter within 3 days, average single head daily yielding maintains about 31Kg/ days, illustrates that this compound micro-ecological preparation has remarkable result in terms of having increase milk production of cow.

Claims (8)

1. a compound lactobacillus Tiny ecosystem liquid preparation, it is characterised in that described Tiny ecosystem liquid preparation is by Lactobacillus plantarum (Lactobacillus plantarum) SCI-01 and Lactobacillus plantarum (Lactobacillus plantarum) SCI-02 is through symbiosis Coupled fermentation and covalent cross-linking embedding are made；Described Lactobacillus plantarum (Lactobacillus plantarum) SCI-01 deposit number CGMCC No.11930；Told Lactobacillus plantarum (Lactobacillus plantarum) SCI-02 deposit number CGMCC No.11931。
2. the preparation method of Tiny ecosystem liquid preparation described in claim 1, it is characterised in that specific as follows:
(1) actication of culture and cultivation: the Lactobacillus plantarum SCI-01 and Lactobacillus plantarum SCI-02 of freezen protective are inoculated respectively In MRS fluid medium, cultivating 18-22h at temperature 37 DEG C, such Secondary Culture obtains activated spawn 2-3 time；
(2) symbiosis coupled fermentation: by the Lactobacillus plantarum SCI-01 activated and Lactobacillus plantarum SCI-02 according to each 5% ratio Example is inoculated in MRS culture medium simultaneously, 37 DEG C of constant temperature culture, initial pH 6.5, adds nertralizer by stream and controls permanent pH5.0-5.9 After fermenting 3-5 hour, then carry out natural fermentation, when pH is less than 4.5, adds nertralizer by stream and control pH to 5.0-5.9, After fermenting 3-5 hour, then carry out natural fermentation, so circulate, reach 1 × 10 to viable count of lactobacillus10More than CFU/mL, Terminate fermentation；
(3) covalent cross-linking embedding:
A. in covalent cross-linking embedding process, institute's substance is in addition to Chymosin and paddy propionic acid amide. transaminase, and other all kills through microwave Bacterium, microwave disinfection temperature is 80-100 DEG C, 15-30min；
B. the liquid after symbiosis coupled fermentation is passed through nitrogen and replaces air therein, time swap 5min；
C. in the fermentation liquid of step (2) fermentation ends, every 1000L adds 30-40Kg sucrose, 15-25Kg molasses and 5-15Kg Yeast powder, and keep the rotating speed of fermentation tank stirring paddle 50-100r/min, under the conditions of 37-42 DEG C, act on 10-30min；
D. addition antioxidant 0.9-3.0Kg in every 1000L fermentation liquid, and keep the rotating speed of fermentation tank stirring paddle 60-100r/min, Under the conditions of 37-42 DEG C, act on 20-30min；
E. addition 3-10Kg porous-starch in every 1000L fermentation liquid, and keep the rotating speed of fermentation tank stirring paddle 50-90r/min, Under the conditions of 37 DEG C, act on 30min；
F. every 1000L fermentation liquid adds 5-20Kg skimmed milk, regulate pH to 5.0-5.9, keep constant temperature 25-45 DEG C, by breast It is 10-50L/min that the regulation at inlet of change machine processes after fermentation flow quantity, and regulation covalent cross-linking embedding mixed liquor flow is 0.02-0.2L/min, the two occurs covalent cross-linking to react in mulser.
3. the preparation method of Tiny ecosystem liquid preparation as claimed in claim 2, it is characterised in that described MRS fluid medium Form as follows: 10g peptone, 5g Carnis Bovis seu Bubali cream, 4g yeast leaching powder, 20g glucose, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g trisodium citrate, 1mL Tween 80,0.2g magnesium sulfate, 0.05g manganese sulfate add 1000mL distilled water, and regulation pH is extremely 6.5,121 DEG C of sterilizing 15min.
4. the preparation method of Tiny ecosystem liquid preparation as claimed in claim 2, it is characterised in that described nertralizer is that volume ratio is The ammonia of 25%.
5. the preparation method of Tiny ecosystem liquid preparation as claimed in claim 2, it is characterised in that described antioxidant is L-half Cystine salt, cystine and catalase, addition is each 0.5Kg of 1000L fermentation liquid.
6. the preparation method of Tiny ecosystem liquid preparation as claimed in claim 2, it is characterised in that described covalent cross-linking embedding mixing Liquid is containing 0.5-1.0% Chymosin and the aqueous solution of 0.3-1.0% paddy propionic acid amide. transaminase.
7. compound lactobacillus Tiny ecosystem liquid preparation described in claim 1 is in the application of field of fodder.
8. compound lactobacillus Tiny ecosystem liquid preparation described in claim 7 is in the application of field of fodder, it is characterised in that specifically side Method is as follows: feeding compound micro-ecological preparation morning and evening every day each 1 time, each 100mL, by equal for 100mL Tiny ecosystem liquid preparation Even sprinkling feed surface mix homogeneously, feed the cattle suffering from mastitis or health, feed 10 days continuously.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108220193A (en) * 2018-01-08 2018-06-29 金华银河生物科技有限公司 One plant of bifidobacterium lactis, a kind of bifidobacterium lactis freeze-dried powder and preparation method thereof
CN108504648A (en) * 2018-03-19 2018-09-07 金华银河生物科技有限公司 A kind of covalent embedding method and lactobacillus preparation of lactic acid bacteria
CN113040270A (en) * 2021-04-29 2021-06-29 西南科技大学 Preparation method of microecological preparation for pigs

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105748534A (en) * 2016-04-29 2016-07-13 内蒙古和美科盛生物技术有限公司 Composite lactobacillus nipple cleaning solution for improving dairy cow nipple micro-ecosystem

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105748534A (en) * 2016-04-29 2016-07-13 内蒙古和美科盛生物技术有限公司 Composite lactobacillus nipple cleaning solution for improving dairy cow nipple micro-ecosystem

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
王颉等: "《食品加工工艺学》", 30 September 2006, 中国农业科学技术出版社 *
郭建林: "益生菌Lactobacillus casei Zhang包埋稳定性研究", 《中国优秀硕士学位论文全文数据库工程科技I辑》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108220193A (en) * 2018-01-08 2018-06-29 金华银河生物科技有限公司 One plant of bifidobacterium lactis, a kind of bifidobacterium lactis freeze-dried powder and preparation method thereof
CN108220193B (en) * 2018-01-08 2021-01-15 金华银河生物科技有限公司 Bifidobacterium lactis, bifidobacterium lactis freeze-dried powder and preparation method thereof
CN108504648A (en) * 2018-03-19 2018-09-07 金华银河生物科技有限公司 A kind of covalent embedding method and lactobacillus preparation of lactic acid bacteria
CN113040270A (en) * 2021-04-29 2021-06-29 西南科技大学 Preparation method of microecological preparation for pigs

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