CN101971920B - Porcine lactobacillus reuteri lyophilized preparation and preparation method thereof - Google Patents
Porcine lactobacillus reuteri lyophilized preparation and preparation method thereof Download PDFInfo
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- CN101971920B CN101971920B CN201010295637XA CN201010295637A CN101971920B CN 101971920 B CN101971920 B CN 101971920B CN 201010295637X A CN201010295637X A CN 201010295637XA CN 201010295637 A CN201010295637 A CN 201010295637A CN 101971920 B CN101971920 B CN 101971920B
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Abstract
The invention relates to a porcine lactobacillus reuteri lyophilized preparation and a preparation method thereof, belonging to the field of application of microbes. The porcine lactobacillus reuteri lyophilized preparation is characterized in that a protective agent in the lyophilized preparation comprises the following ingredients in percentage by weight: 10 percent of skimmed milk, 0.1 percent of sodium glutamate, 0.3 percent of cysteine, 10 percent of fucose, 8 percent of lactose, 10 percent of dextrine and the balance of water. Proved by experiments, the porcine lactobacillus reuteri lyophilized preparation of the invention can increase the viable lactobacillus content in nursery pig feces, can reduce the content of colon bacillus, and has the effect of improving the average daily gain and immunity of the nursery pigs. The lyophilized preparation prepared by the method of the invention has the viable bacteria count not less than 109cfu/g, and the viable bacteria survival rate is higher than 75 percent after the lyophilized preparation is stored for 90 days. The invention also provides an optimized culture medium for lactobacillus reuteri, and the viable bacteria count is six times higher than that that of the viable bacteria cultured on the conventional culture medium (Magnetic Resonance Spectrum (MRS) culture medium).
Description
Technical field
The present invention relates to microbe additive of feed stripped and preparation method thereof, particularly relate to a kind of viable bacteria lyophilized formulations that contains the Direct-fed that is formed by pig source lactobacillus reuteri and preparation method thereof.
Background technology
Exist a large amount of bacteriums in the humans and animals enteron aisle, about 30 belong to kind more than 500, have jointly consisted of normal intestinal flora.Existing positive relationship between these microorganisms and the host (commensalism, interpromoting relation in five elements and reciprocal phenomenon) also has negative sense relation (competition, partially harmful phenomenon), is jointly keeping microecological balance.Yet antibiotic is as a large amount of uses of feed addictive in recent years, but also killed the flora of normal symbiosis in the animal body when suppressing and killing pathogen, destroyed microecological balance.On the contrary, the effect of probio is to foster normal microorganism species, and the pathogen that flys up and down is adjusted to the ecological balance to disruption of ecological balance.Wherein, lactic acid bacteria is as dominant microflora in the chitling road, can regulating intestinal canal in the general character ratio of normal flora, keeping the balance of intestinal ecosystem.
Lactobacillus reuteri (Lactobacillus reuteri) belongs to obligate heterolactic fermentation bacterium, is the beneficial lactobacillus that is prevalent in the humans and animals enteron aisle, also is one of population important in the Lactobacillus.Studies show that the lactobacillus reuteri enteron aisle can be adjusted the gut flora balance, reduce the body cholesterol, regulate organism immune response etc.In addition, lactobacillus reuteri can also produce a kind of ablastins (reuterin) of uniqueness, can effectively suppress the multiple harmful bacterias such as large intestine dust Xi Shi, bacillus typhi murium, candida albicans, Aspergillus flavus, campylobacter jejuni and gemma clostridium, be considered to unique a kind of enteron aisle lactic acid bacteria culturers that can produce broad-spectrum antibacterial action.U.S. food and drug control association (FDA) are after classifying lactobacillus reuteri as safe microorganism fungus kind in 1989, and this bacterium is widely used.At present report that in the world maximum strain bacterial classifications is all lactobacillus reuteris of Sweden Biogaia AB company (Lactobacillus reuteri ATCC-55730), whole world consumption has surpassed 1,500,000,000 lactobacillus reuteri dosage, and not edible unsafe report.Japan has report to think in addition, more than 20 kind of probiotics fermention Ruzhong on the market, have only and contain the lactobacillus reuteri series products and can play inhibition to children caries, mainly be because this bacterium can suppress to be out of shape the growth and breeding of hammer (Streptococcus mutan), and Streptococcusmutan is the major microorganisms that causes carious tooth.China Ministry of Public Health ratified lactobacillus reuteri (Lactobacillusreuteri) for can be used for the probio bacterial classification of health food in 2003, domestic report about lactobacillus reuteri is mainly used in the human sour milk food at present, and the report negligible amounts.And as the lactobacillus reuteri of animal feeding rarely seen report especially, to such an extent as to this mainly is still not have systematic research because this bacterium identifies the product preparation processes such as application animal produces from separation, these effects limit this bacterial classification applying in animal produces.Therefore, according to the needs that herding is produced, by strict scientific experimentation, develop a kind of novel, practical lactobacillus preparation, have broad application prospects.
Summary of the invention
The present invention is directed to the deficiency in above-mentioned field, a boar source lactobacillus reuteri lyophilized preparation is provided, can the Direct-fed animal.Can increase viable lactic acid bacteria content in the child care pig manure, reduce Escherichia coli content, and have the effect that improves child care pig average daily gain and immunity.
Pig source lactobacillus reuteri (Lactobacillus reuteri) lyophilized formulations; it is characterized in that the percentage by weight of protectant component and each component is in the lyophilized formulations: skimmed milk 10%, sodium glutamate 0.1%, cysteine 0.3%, trehalose 10%, lactose 8%, dextrin 10%, all the other are water.
Described pig source lactobacillus reuteri viable count 〉=10
9Cfu/g.
Described pig source lactobacillus reuteri derives from weanling pig caecum mucous membrane.
The preparation method of above-mentioned pig source lactobacillus reuteri lyophilized preparation the steps include: pig source lactobacillus reuteri fermented and enlarge to cultivate, and gets bacterium mud after the centrifugal zymotic fluid, and bacterium mud is joined in the described protective agent, and mixing, freeze-drying get final product.
Inoculum concentration was 1% during described fermentation expansion was cultivated, and fermentation temperature is 37 ℃, and pH is 6.4～6.5, and fermentation time is 24h.
Described fermentation enlarges the component of the culture medium that adopts in the cultivation and the percentage by weight of each component is: maltose 2%, casein peptone 1.5%, dusty yeast 0.5%, dipotassium hydrogen phosphate 0.4%, sodium acetate 0.7%, magnesium sulfate 0.058%, manganese sulfate 0.019%, tween 0.1%, beef extract 1%, diammonium hydrogen citrate 0.2%.
The application of above-mentioned lyophilized formulations in animal feed additive.
Described animal is domestic animal.
Described domestic animal is pig.
The present invention has obtained pig source lactobacillus reuteri in the separation of weanling pig caecum mucous membrane, test data shows that the lyophilized formulations that this pig source lactobacillus reuteri is made adds in the nursery-stage pig feed, can promote the growth of child care pig, improve its efficiency of feed utilization, reduce feedstuff-meat ratio and see Table 20; Can increase beneficial bacterium content in the child care chitling road, reduce harmful bacteria content, thereby keep the balance of chitling road micro-ecological environment, see Table 21; Having positive effect aspect protection liver, the enhancing immunity of organisms, see Table 22.
Based on the benefit of pig source lactobacillus reuteri in animal intestinal given birth to function; in order to make pig source lactobacillus reuteri in animal feed, obtain good application and to give full play to the effect of its probio; the present invention obtains a kind of frozen-dried protective agent prescription by a large amount of prescription screenings: 10% skimmed milk, 0.1% sodium glutamate, 0.3% cysteine, 8% lactose, 10% dextrin, 10% trehalose, all the other are water.This freeze drying protectant reaches 98.84% to the frozen-dried protective rate of pig source lactobacillus reuteri, sees Table 17; Make it after normal temperature is preserved 90d, the bacterium survival rate reaches 75.64%, sees Table 18.
The present invention also provides the preparation method of pig source lactobacillus reuteri lyophilized preparation, principal character is the pig source lactobacillus reuteri Optimal Medium that adopts optimization of the present invention to obtain, maltose 2%, casein peptone 1.5%, dusty yeast 0.5%, dipotassium hydrogen phosphate 0.4%, sodium acetate 0.7%, magnesium sulfate 0.058%, manganese sulfate 0.019%, tween 0.1%, beef extract 1%, diammonium hydrogen citrate 0.2% when increasing bacterium and prepare pig source lactobacillus reuteri.This culture medium is optimized at the MRS medium base, make it be suitable for pig source lactobacillus reuteri most, the viable count that is inoculated into the pig source lactobacillus reuteri of this culture medium is to be inoculated into simultaneously 6 times of pig source lactobacillus reuteri viable count on the MRS culture medium.
Because pig provided by the invention source lactobacillus reuteri lyophilized preparation, main feature is formula for lyophilized preparation, and lactobacillus reuteri wherein is the common probio of conventional animal enteron aisle, as all being found this bacterium in pig, the chicken intestines, therefore, this pig source lactobacillus reuteri lyophilized preparation can add in animal feed, and it is a kind of preferred version that the present invention is used for pig feed with this lyophilized formulations.
In sum; the freeze drying protectant of pig provided by the invention source lactobacillus reuteri lyophilized preparation; can provide good frozen-dried protective for lactobacillus reuteri; can obtain higher bacterium survival rate and storage stability; and production technology is simple; cost is reasonable, makes lyophilized formulations of the present invention be very suitable for being applied in the animal feed.
Specific implementation method
The preparation of embodiment 1 pig source lactobacillus reuteri lyophilized preparation
1.1 the separation of pig source Bacillus acidi lactici is identified
Piglet is cutd open extremely, and sterile working scraping caecum mucous membrane 1g places the triangular flask with bead that fills the 9ml SPSS, and fully concussion shakes up, and then draws 1.0ml bacterium liquid in the test tube that fills the 9.0ml SPSS, and this dilution factor is 10
-1, repeat above process, make successively 10
-2, 10
-3, 10
-4, 10
-5, 10
-6Bacterium liquid.Select 10
-4, 10
-5, 10
-6Three dilution factors are drawn 0.1ml bacterium liquid and are dripped respectively on the MRS culture medium flat plate, adopt 37 ℃ of candle jar methods to cultivate 24～48h, and pure culture is done in bacterium colony streak inoculation on the MRS culture medium that the picking form is different, and it is for subsequent use to be placed on 4 ℃ of Refrigerator stores.
Then the picking pure culture dyes, microscopy; Choose and carry out the biochemical tests such as catalase, nitrate reduction, gelatin liquefaction without the gemma Gram-positive bacillus, and the sugar alcohol fermentation test, the result is with " bacterial classification is determined in common bacteria system identification handbook, " lactic acid bacteria classification and experimental technique " contrast.Then deliver to Chinese industrial microorganism fungus kind preservation center and identify check.
Chinese industrial microorganism fungus kind preservation administrative center is that the qualification result of lactobacillus reuteri (Lactobacillusreuteri) is as follows to this Bacillus acidi lactici
Table 1 pig source lactobacillus reuteri physiological and biochemical property test report
Annotate: "+" expression 〉=90% bacterial strain is positive in the table, and "-" expression 〉=90% bacterial strain is negative, and " ND " represents undetermined.
1.2 pig source lactobacillus reuteri biological characteristics
1.2.1 acid resistance is measured
Behind actication of culture 2 times, adopt 5% inoculum concentration, be linked into respectively in the solution of pH=1.5, pH=2.0, pH=3, place 37 ℃ of insulations 0.5h, 1.5,2.5h, measure viable count, calculate survival rate.By the result as can be known, the lactobacillus reuteri thalline is along with the prolongation of the reduction of pH and temperature retention time is on a declining curve.Wherein, 2h is maximum to the thalline damage in the pH=1.5 insulation, and its survival rate only is 17.50%.
Table 2 pig source lactobacillus reuteri acid resistance is measured
1.2.2 bile tolerance performance measurement
Behind actication of culture 2 times, adopt 5% inoculum concentration, be linked into respectively and be incubated 1,3 in the liquid MRS culture medium that contains 0.10%, 0.20%, 0.30% pig cholate, 6h, get a certain amount of bacterium liquid and measure viable count, calculate survival rate.By the result as can be known, the pig gallbladder salinity is higher, and temperature retention time is longer, and the damage that the lactobacillus reuteri thalline is subject to is larger.Wherein, lactobacillus reuteri is when 0.30% pig cholate insulation 6h, and its survival rate only is 1.84%.
The lactobacillus reuteri bile tolerance performance measurement of table 3 pig source
1.3 pig source lactobacillus reuteri medium optimization
1.3.1 determining of basal medium
The preparation of lactic acid bacteria freeze drying preparation need to have good Optimal Medium, so that thalline is able to a large amount of propagation, and carries out the preparation of product with enough quantity.And the most basic process of Optimal Medium is exactly to determine first basal medium, and carries out based on this optimization of each composition.This test is with MRS culture medium (composition: peptone 10.0g, beef extract 10.0g, dusty yeast 5.0g, glucose 20.0g, anhydrous sodium acetate 5.0g, citric acid diamino 2.0g, Tween-80 1.0ml, dipotassium hydrogen phosphate 2.0g, magnesium sulfate 0.58g, manganese sulfate 0.19g, distilled water 1000ml, pH is 6.5) and TJA culture medium (composition: tomato juice 50ml, dusty yeast 5g, beef extract 10g, lactose 20g, glucose 2g, dipotassium hydrogen phosphate 2g, tween 1ml, sodium acetate 5g, distilled water 1000ml, pH are 6.8) cultivate the growth OD of this bacterium in these two kinds of culture mediums relatively as the basis of lactobacillus reuteri
600nmValue and pH value, thereby the best base basal culture medium of definite this bacterial strain.
Behind actication of culture, be inoculated in respectively and minute be equipped with in MRS and the TJA fluid nutrient medium, place 37 ℃ of cultivations, respectively 12,15,18,21,24,27h measures OD
600nmValue and pH value are determined the best base basal culture medium.It the results are shown in Table 4, Fig. 3.By the result as can be known, the lactobacillus reuteri OD that in the MRS culture medium, grows
600nmBe worth higher, produce acid can be better.Therefore selecting the MRS culture medium is basal medium, then each composition in the culture medium is optimized.
The growth OD of table 4 lactobacillus reuteri under two kinds of culture medium condition
600nmValue and pH value
Annotate: data are mean+SD in the table.
1.3.2 sugar, peptone class material determines in the lactobacillus reuteri culture medium of pig source
In culture medium, carbohydrate provides carbon source and energy substance for bacterial growth, and peptone then is one of composition the most frequently used when preparing culture medium, and it can provide bacterial growth to breed needed nitrogenous source.Therefore, carbohydrate and peptone class material are that growth and breeding has great importance for bacterium.Studies show that different bacterial classifications is selectively to the utilization of these two kinds of materials.Therefore, this test has been carried out the comparative test of different carbohydrates and peptone class material to lactobacillus reuteri, further optimizes for culture medium to lay the foundation.
Test changes carbohydrate and peptone with other components unchanged in the MRS culture medium, respectively different carbohydrates and peptone class is made up in twos, makes 16 kinds of different culture mediums.Activated spawn is inoculated in the different culture media by 1% inoculum concentration, measures viable count, determine best carbohydrate and peptone class.By the result as can be known, the 16th group of (maltose+casein peptone) effect is best, and its viable count can reach 1.33 * 10
8Cfu/ml.
Table 5 pig source lactobacillus reuteri viable count situation in different culture media
| Group | Culture medium carbon/nitrogenous source composition | Viable count (* 10 7cfu/ml) |
| 1 | Glucose+peptone | 3.10 |
| 2 | Glucose+tryptone | 8.95 |
| 3 | Glucose+soy peptone | 11.1 |
| 4 | Glucose+casein peptone | 4.20 |
| 5 | Lactose+peptone | 4.80 |
| 6 | Lactose+tryptone | 8.15 |
| 7 | Lactose+soy peptone | 6.50 |
| 8 | Lactose+casein peptone | 3.35 |
| 9 | Sucrose+peptone | 1.01 |
| 10 | Sucrose+tryptone | 7.8 |
| 11 | Sucrose+soy peptone | 6.10 |
| 12 | Sucrose+casein peptone | 4.45 |
| 13 | Maltose+peptone | 2.35 |
| 14 | Maltose+tryptone | 8.95 |
| 15 | Maltose+soy peptone | 5.30 |
| 16 | Maltose+casein peptone | 13.3 |
1.3.3 growth factor, inorganic salts and buffer salt determines in the culture medium
According to 1.3.2 result, determine that lactobacillus reuteri carbohydrate and peptone class are respectively maltose and casein peptone.Then adopt Orthogonal Experiment and Design (L
9(3
4) growth factor (A), inorganic salts (B) and buffer salt (C) optimal components and content are optimized.Its experimental design sees Table 6, table 8, and result of the test sees Table 7, table 9.By the result as can be known, the lactobacillus reuteri optimum growh factor, inorganic salts and buffering salt composition and content are respectively 0.5% dusty yeast, 0.7% sodium acetate, 0.4% potassium dihydrogen phosphate.
Table 6 lactobacillus reuteri growth factor, inorganic salts and buffering salt component are determined orthogonal test factor level table
Table 7 lactobacillus reuteri growth factor, inorganic salts and buffering salt component are determined orthogonal experiments
Table 8 lactobacillus reuteri growth factor, inorganic salts and buffering salt content orthogonal test factor level table
| Level | Dusty yeast (A) | Dipotassium hydrogen phosphate (B) | Sodium acetate (C) |
| 1 | 0.5％ | 0.2％ | 0.7％ |
| 2 | 0.75％ | 0.4％ | 0.5％ |
| 3 | 1.0％ | 0.6％ | 0.3％ |
The table 9 growth of lactobacillus reuteri factor, inorganic salts and buffering salt content orthogonal experiments
1.3.4 determining of carbohydrate in the culture medium, peptone class, the best adding proportion of inulin
According to above result, determine that maltose is best carbohydrate, casein peptone is the optimum protein peptone, adopts Orthogonal Experiment and Design (L
9(3
4)), determine optimum addition.Wherein 3 levels of A factor (maltose) are respectively: 1%, 2%, 3%; 3 levels of B factor (casein peptone) are respectively: 1%, 1.5%, 2%; 3 levels of C factor (inulin) are respectively: 1%, 2%, 3%.Its experimental design and the results are shown in Table 10, table 11.By the result as can be known, best carbohydrate is 2% maltose in the lactobacillus reuteri culture medium of pig source, optimum protein peptone 1.5% casein peptone, and best inulin adding proportion is 3%.Therefore, pig source lactobacillus reuteri through the optimal medium after improveing is: 2% maltose+1.5% casein peptone+dusty yeast 0.5%+ potassium dihydrogen phosphate 0.4%+ sodium acetate 0.7%+0.058% magnesium sulfate+0.019% manganese sulfate+3% inulin.
Carbohydrate, peptone class, the best adding proportion orthogonal test table of inulin in table 10 culture medium
| Level | Maltose (A) | Casein peptone (B) | Inulin (C) |
| 1 | 1％ | 1％ | 1％ |
| 2 | 2％ | 1.5％ | 2％ |
| 3 | 3％ | 2％ | 3％ |
Carbohydrate, peptone class, the best adding proportion orthogonal experiments of inulin in table 11 culture medium
1.3.5 the viable count of improved culture medium and MRS culture medium relatively
With after the bacterium inoculation activation respectively the inoculum concentration with 1% be inoculated in improved culture medium and the MRS culture medium, cultivate 24h for 37 ℃, get respectively a certain amount of bacterium liquid and measure viable count, result such as table 12, as shown in Table 12, the enriching effect of improved culture medium is significantly better than the MRS culture medium, and viable count can reach 3.03 * 10
8Cfu/ml improves about 6 times than MRS culture medium.
Table 12 lactobacillus reuteri is the viable count situation in two kinds of culture mediums
| Culture medium | Viable count (cfu/ml) |
| Improved culture medium | 3.03×10 8 |
| The MRS culture medium | 5.82×10 7 |
1.4 the preparation of pig source lactobacillus reuteri lyophilized preparation
1.4.1 the primary dcreening operation of freeze drying protectant
Behind the lactobacillus reuteri fermented and cultured 24h of pig source, collect the centrifugal 10min of zymotic fluid 6000rpm and get bacterium mud, add the protective agent of various combination, make the bacteria suspension identical with centrifugal primary fermentation liquid concentration.Protective agent adopts Orthogonal Experiment and Design (L
9(3
4)) at first optimize the consumption of skimmed milk (A), sodium glutamate (B), cysteine (C), trehalose (D), factor level sees Table 13, and result of the test sees Table 14.Effect was best when by the result as can be known, the primary dcreening operation result of lactobacillus reuteri freeze drying protectant was 10% skimmed milk, 0.1% sodium glutamate, 0.3% cysteine.
Table 13 lactobacillus reuteri protective agent primary dcreening operation Orthogonal Experiment and Design table
| Level | Skimmed milk (A) | Sodium glutamate (B) | Cysteine (C) |
| 1 | 10％ | 0.1％ | 0.1％ |
| 2 | 12％ | 0.2％ | 0.3％ |
| 3 | 14％ | 0.3％ | 0.5％ |
Table 14 lactobacillus reuteri protective agent primary dcreening operation orthogonal experiments
1.4.2 the multiple sieve of freeze drying protectant
According to the primary dcreening operation result, adopt Orthogonal Experiment and Design (L
9(3
4)) consumption of lactose (A), dextrin sodium (B), trehalose (C) is optimized, the experimental design table sees 15, result of the test sees Table 16.By the result as can be known, lactobacillus reuteri best freeze drying protectant in pig source is 10% skimmed milk, 0.1% sodium glutamate, 0.3% cysteine, 8% lactose, 10% dextrin, 10% trehalose.
Table 15 lactobacillus reuteri protective agent sieves the Orthogonal Experiment and Design table again
| Level | Lactose (A) | Dextrin (B) | Trehalose (C) |
| 1 | 4％ | 8％ | 8％ |
| 2 | 6％ | 10％ | 10％ |
| 3 | 8％ | 12％ | 12％ |
Table 16 lactobacillus reuteri protective agent sieves orthogonal experiments again
1.4.3 the middle trial production of pig source lactobacillus reuteri lyophilized preparation
Pig source lactobacillus reuteri adopts the 50L fermentation tank to enlarge and cultivates, the fermentation tank running parameter is respectively: fermentation jar temperature is 37 ℃, culture medium adopts the MRS culture medium, inoculum concentration 1%, liquid amount is 50%, and rotating speed is 100r/min, initial pH6.4～6.5, keep from start to finish tank pressure at 0.05MPa, fermentation period is 24h.After the fermentation ends, get an amount of bacterium liquid and measure viable count.Measuring viable count is 7.30 * 10
8Cfu/ml.
1.4.4 the preparation of pig source lactobacillus reuteri lyophilized formulations
Collecting zymotic fluid adopts the centrifugal 10min of 6000rpm to get bacterium mud; join in the protective agent; protectant percentage by weight is: 10% skimmed milk+0.1% sodium glutamate+0.3% cysteine+10% trehalose+8% lactose+10% dextrin; all the other are water, make the bacteria suspension identical with centrifugal primary fermentation liquid concentration.Bacteria suspension is poured in the dry materials dish of freeze dryer, vacuumized and begin to carry out freeze-drying.Namely get the lactobacillus reuteri lyophilized product in pig source after the freeze-drying.Wherein produce record sheet as a trial and see Table 17.As shown in Table 17, the lactobacillus reuteri viable count is 2.00 * 10 after the freeze-drying
9Cfu/g, the frozen-dried protective rate reaches 98.84%.
Frozen-dried protective rate (%)=(after the freeze-drying after the outstanding bacterium powder viable count * freeze-drying of bacterium bacterium powder total amount)/(before the freeze-drying before bacteria suspension viable count * freeze-drying bacteria suspension volume) * 100
Table 17 lactobacillus reuteri lyophilized preparation pilot scale record of production table
1.4.5 pig source lactobacillus reuteri lyophilized preparation bin stability is measured
With lactobacillus reuteri lyophilized preparation with packaging of aluminium foil bag after, normal temperature is got the bacterium powder after preserving 30d, 60d, 90d, carry out gradient dilution with SPSS after, adopt the MRS culture medium to measure viable count.The results are shown in Table 18.As shown in Table 18, after lactobacillus reuteri lyophilized formulations normal temperature was preserved 90d, viable count still can reach 1.52 * 10
9Cfu/g.
Table 18 lactobacillus reuteri lyophilized preparation shelf characteric is measured
| Holding time (d) | Viable count (cfu/ml) | Survival rate (%) |
| 0 | 2.00×10 9 | 100 |
| 30 | 1.87×10 9 | 93.52 |
| 60 | 1.67×10 9 | 83.33 |
| 90 | 1.52×10 9 | 75.64 |
1.4.6 the security performance of pig source lactobacillus reuteri lyophilized preparation is measured
Pig source lactobacillus reuteri lyophilized preparation is delivered to medicinesafety supervision and inspection center of the Ministry of Agriculture (Beijing) carried out safety examination, through rat, its mouse oral acute toxicity test, the result proves: pig source lactobacillus reuteri lyophilized formulations is for rat, its mouse oral LD
50Greater than the 5000mg/kg body weight.Illustrate that pig source lactobacillus reuteri lyophilized preparation is safe.
The effect of embodiment 2 pig source lactobacillus reuteri lyophilized preparations in the child care pig produces
2.1 experimental animal and grouping
Choose 64 of the child care pigs that age in days is close, body weight is suitable (Du * length * Dasanyuan hybridized pig), be divided at random 4 groups, establish two repetitions for every group, each repeats 8.1 group is control group, the basal diet+antibiotic of feeding (every 100kg complete diet pellet adds colistin 0.98g, Bacitracin Zinc 4g, roxarsone 5g), 2,3,4 groups of lactobacillus reuteris that add 250g, 500g, 750g at every 100kg complete diet pellet.Experimental period is 30d.Test is carried out in closed pig house, the cement flooring, and the stable breeding of net bed, free choice feeding provides sufficient cleaning drinking-water with Duckbill-type drinking trough.Immunity, expelling parasite and sterilization are undertaken by the pig farm rules.
2.2 test diet design
For raising daily ration with reference to China's " swine rearing standard " preparation, Diet Formula and trophic level see Table 12.
Table 19 basal diet prescription and trophic level
| Daily ration forms | w/％ | Trophic level | |
| Corn | 59 | Digestible energy (MC/kg) | 3.16 |
| Dregs of beans | 26 | Thick protein (%) | 18.9 |
| Imported fish meal | 3 | Calcium (%) | 0.82 |
| Wheat bran | 8 | Available phosphorus (%) | 0.42 |
| Premix | 4 | Salt (%) | 0.48 |
| Lysine (%) | 1.023 | ||
| Methionine (%) | 0.365 |
Annotate: every kilogram of daily ration provides: retinol1 3500IU; Vitamin D
32850IU; Vitamin E 64mg; Vitamin K
32.4mg; Cobastab
12.4mg; Cobastab
26.6mg; Cobastab
63mg; Cobastab
120.045mg; Niacinamide 25.5mg; Pantothenic acid 13.5mg; Biotin 0.12mg; Copper 12mg; Iron 150mg; Zinc 155mg; Manganese 55mg; Iodine 5mg; Selenium 0.3mg.
2.3 method
When on-test and end, individual empty stomach is weighed; Observe the swinery growing state every day, record food consumption situation, record grice diarrhoea situation; After the off-test, calculate daily gain, feed intake and feedstuff-meat ratio, take fresh excrement sample for every group, measure lactic acid bacteria and Escherichia coli viable count situation in the excrement, and from 4 piglets of every group of random choose, vena cava anterior blood sampling 5mL measures glutamic-pyruvic transaminase (ALT), immunoglobulin A (IgA) content.Data statistics adopts SPSS 17.0 statistical softwares to carry out one-way analysis of variance, and the result represents with mean+SD.
2.4 results and analysis
2.4.1 pig source lactobacillus reuteri lyophilized preparation is on the impact of child care pig production performance
As shown in Table 20,3,4 groups are added the daily gain that all can significantly improve the child care pig behind the lactobacillus reuteri lyophilized preparation, and the contrast group has improved 2.06% and 13.92 respectively.This shows, when the lactobacillus reuteri lyophilized formulations of doses in the child care swine rations, can promote the growth of child care pig, improve its efficiency of feed utilization, reduce feedstuff-meat ratio.
Table 20 lactobacillus reuteri is on the impact of the average daily gain of child care pig and feedstuff-meat ratio
| Group | Daily gain | Feedstuff-meat ratio |
| 1 | 390.625±13.258 ab | 2.496 |
| 2 | 365.556±29.856 a | 2.531 |
| 3 | 398.661±51.981 ab | 2.495 |
| 4 | 445.000±10.589 b | 2.168 |
Annotate: with the different significant differences that represent of shoulder mark, the different expression of capitalization difference is (P＜0.01) extremely significantly, the different expression of lowercase significant difference (P＜0.05).
2.4.2 pig source lactobacillus reuteri lyophilized preparation is on lactic acid bacteria in the child care pig manure and colibacillary impact
By the result as can be known, all can increase lactic acid bacteria content in the excrement after 3 test group are added lactobacillus reuteri lyophilized preparation, reduce Escherichia coli content, wherein, it is best to test 3 groups of effects.This shows that the lactobacillus reuteri preparation can increase beneficial bacterium content in the child care chitling road, reduce harmful bacteria content, thereby keep the balance of chitling road micro-ecological environment.
Lactic acid bacteria and Escherichia coli viable count (log in the table 21 child care pig manure
10Cfu/g)
| Group | Lactic acid bacteria | Escherichia coli |
| 1 | 8.097±0.025 A | 7.638±0.021 A |
| 2 | 8.437±0.078 B | 7.755±0.043 A |
| 3 | 8.476±0.020 B | 6.639±0.228 B |
| 4 | 8.728±0.040 C | 6.830±0.013 A |
Annotate: with the different significant differences that represent of shoulder mark, the different expression of capitalization difference is (P＜0.01) extremely significantly, the different expression of lowercase significant difference (P＜0.05).
2.4.3 pig source lactobacillus reuteri lyophilized preparation is on the impact of child care pig blood index
Glutamic-pyruvic transaminase (ALT) is that the most responsive liver function of animal detects one of index, and the activity rising degree of this enzyme can reflect the degree of hepatocellular injury.Immunoglobulin A (IgA) is immunoglobulin (Ig) main in the exocrine secretion, plays a crucial role aspect the invasion of opposing pathogenic microorganism at protection enteron aisle, respiratory tract, urogenital tract, mammary gland and eyes.In this test, test 3,4 groups of child care pig serum alt content and all be lower than control group, and the lactobacillus reuteri lyophilized preparation that adds 3 levels all can improve the content of IgA in the child care pig serum, wherein, especially best to test 3 groups of effects.Therefore proved also that lactobacillus reuteri lyophilized preparation has positive effect aspect protection liver, the enhancing immunity of organisms.
Table 22 lactobacillus reuteri is on the impact of child care pig immunocompetence
| Group | ALT(U/L | IgA(mg/ml) |
| 1 | 54.50±3.79 | 1.033±0.106 ab |
| 2 | 57.00±6.48 | 1.161±0.017 ab |
| 3 | 51.75±4.27 | 1.408±0.241 b |
| 4 | 46.00±5.03 | 1.345±0.368 ab |
Annotate: with the different significant differences that represent of shoulder mark, the different expression of capitalization difference is (P＜0.01) extremely significantly, the different expression of lowercase significant difference (P＜0.05).
Claims (9)
1. pig source lactobacillus reuteri (Lactobacillus reuteri) lyophilized formulations; the protective agent prescription that it is characterized in that the lyophilized formulations employing is as follows: the percentage by weight of protectant component and each component is: skimmed milk 10%, sodium glutamate 0.1%, cysteine 0.3%, trehalose 10%, lactose 8%, dextrin 10%, all the other are water.
2. lyophilized formulations according to claim 1, described pig source lactobacillus reuteri viable count 〉=10
9Cfu/g.
3. lyophilized formulations according to claim 1, described pig source lactobacillus reuteri derives from weanling pig caecum mucous membrane.
4. the preparation method of the arbitrary described pig of claim 1～3 source lactobacillus reuteri lyophilized preparation; the steps include: pig source lactobacillus reuteri fermented and enlarge to cultivate; get bacterium mud after the centrifugal zymotic fluid, bacterium mud is joined in the described protective agent, mixing, freeze-drying get final product.
5. preparation method according to claim 4, described fermentation enlarge cultivate in inoculum concentration be 1%, fermentation temperature is 37 ℃, pH is 6.4～6.5, fermentation time is 24h.
6. preparation method according to claim 4, described fermentation enlarges the component of the culture medium that adopts in cultivating and the percentage by weight of each component is: maltose 2%, casein peptone 1.5%, dusty yeast 0.5%, dipotassium hydrogen phosphate 0.4%, sodium acetate 0.7%, magnesium sulfate 0.058%, manganese sulfate 0.019%, tween 0.1%, beef extract 1%, diammonium hydrogen citrate 0.2%.
7. the application of lactobacillus reuteri agent lyophilized formulations in the arbitrary described pig of claim 1～3 source in animal feed additive.
8. application according to claim 7, described animal are domestic animal.
9. application according to claim 8, described domestic animal are pig.
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| CN102660477B (en) * | 2012-05-10 | 2013-06-19 | 北京市农林科学院 | Lactobacillus brevis, freeze-dried powder of Lactobacillus brevis and application of freeze-dried powder |
| CN102726637A (en) * | 2012-07-04 | 2012-10-17 | 北京大北农科技集团股份有限公司 | Application of lactobacillus johnsonii in lowering pH value of intestinal tract of weaning piglet |
| CN104605139A (en) * | 2015-01-08 | 2015-05-13 | 河北农业大学 | Bacillus subtilis-acanthopanax senticosus polysaccharide synbiotics, preparation method thereof and application of bacillus subtilis-acanthopanax senticosus polysaccharide synbiotics as feed additive |
| CN105274032B (en) * | 2015-11-13 | 2019-01-11 | 江南大学 | A kind of antagonism campylobacter jejuni and the lactobacillus reuteri for inhibiting its flaA gene expression |
| CN105950496A (en) * | 2016-02-04 | 2016-09-21 | 曲靖市千村农牧科技有限公司 | Lactobacillus agilis and application thereof |
| CN107043722A (en) * | 2017-04-13 | 2017-08-15 | 东北农业大学 | One plant of pig source lactobacillus reuteri and its solid-state probiotics of preparation |
| CN110065688B (en) * | 2019-04-22 | 2021-08-13 | 刘坚 | Production and packaging method of pig manure-derived biofertilizer |
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