CN101554204B - Fermentation lactobacillus preparation - Google Patents
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Abstract
The invention provides a fermentation lactobacillus preparation which comprises the following components in parts by weight: 1-10 parts of strain liquid of fermentation lactobacillus CGMCC No.0843 and 30-150 parts of mineral carrier; the optimal composition contains 5-8 parts of strain liquid of fermentation lactobacillus CGMCC No.0843 and 60-100 parts of strain liquid carrier; and the concentration of the strain liquid is 10<7>-10<10>cfu/g. The preparation can be used for regulating the balance of microecology in the intestine of animals, promoting the growth of animals and improving the feed conversion rate at the sametime, thereby having the function of enhancing nonspecific immune function to prevent diseases; and meanwhile, by screening the prescriptions, the preparation has low cost and stable product quality.
Description
Technical field
The present invention relates to a kind of fermentation lactobacillus preparation, particularly a kind of preparation that contains fermentation lactobacillus CGMCC No.0843.
Background technology
Since finding that feeding antibiotic can be by influencing the field planting of enteric microorganism initial stage in host, thereby play after the somatotrophic effect, the antibiotic animal husbandry aspect that is applied in has produced huge economic benefit.But because antibiotic a large amount of uses, the negative effect of Chu Xianing also becomes increasingly conspicuous thereupon, and it is residual in animal product to show as pathogenic microorganism generation drug resistance and medicine, thereby human beings'health is produced potential harm.
In recent years, the clenbuterol hydrochloride poisoning in the rabid ox disease of Britain, Belgian dioxin incident and Hong Kong makes the consumer day by day pay close attention to the safety problem of animal product, and the increase of pathogenic bacteria of drug-resistant strain causes the difficulty of animal doctor and clinical medicine disease treatment.People are attributed to the interpolation of antibiotic at the medium-term and long-term subclinical therapeutic dose of feed naturally with the drug resistance of pathogen.This makes that adding probiotic in feed has caused the researcher and the producer's interest again.What bright clear grade (1986) significantly descends to Bacillus acidi lactici (Lactobacillus acidophilus) quantity in the diarrhoea intestine of young pigs of discovering of sick, strong intestine of young pigs flora, and coliform count significantly rises.Use the Bacillus acidi lactici probiotic can adjust micro ecology of gastrointestinal tract balance (Fuller etc., 1978), produce bacterial classification so Bacillus acidi lactici is a kind of up-and-coming probiotic.Totally 43 kinds of the safe microorganism kinds that U.S. FDA and AAFCO announced in 1989, wherein Bacillus acidi lactici accounts for 12 kinds, and The Ministry of Agriculture of the People's Republic of China, MOA announced in 12 kinds of microorganism fodders that allow to use in 1999 has 3 kinds to be Bacillus acidi lactici.In the U.S., the product of the industrial application of probiotic 57% contains Lactobacillus (Li Ling, 1995).The alctasin that the Kang Bai of Dalian Medical College etc. develop is a kind of drying agent of Bacillus acidi lactici, good stability between storage life, and every gram product contains Bacillus acidi lactici alive about 10
7Individual, piglet oral 3-4 gram every day can effectively be treated diarrhoea.
Application number is called the Chinese patent of Bacillus acidi lactici bacterial classification and isolation and selection method and purposes for the CN02159754.5 name, from swine alimentary canal, isolate four kinds of Bacillus acidi lacticis: lactobacillus gasseri (Lactobacillus gasseri), preserving number is CDMCC No.0840; Lactobacillus reuteri (Lactobacillus reuterii), preserving number is CGMCC No.0841; Lactobacillus acidophilus (Lactobacillus acidophilus), preserving number is CGMCCNo.0842; Fermentation lactobacillus (Lactobacillus fermentum), preserving number is CGMCC No.0843; A kind of compound additive also is provided simultaneously, by optimizing the combination of above-mentioned four kinds of Bacillus acidi lacticis, can better prevention post-weaning diarrhea in piglet and promotion growth.
But above-mentioned additive need comprise four kinds of Bacillus acidi lacticis, needs very high cultivation cost, and bacterial classification too much also makes unstable product quality.
In view of this, special proposition the present invention.
Summary of the invention
The object of the present invention is to provide a kind of fermentation lactobacillus preparation, said preparation can be used for regulating microecological balance in the animal intestine, thereby have the prophylactic effect of the non-specific immune function of enhancing, the while can also promote the growth of animal effect and improve food conversion ratio.
Briefly, a kind of fermentation lactobacillus preparation provided by the invention is grouped into by the one-tenth of following weight portion; Strain liquid 1-10 part and mineral carrier 30-150 part of containing fermentation lactobacillus CGMCC No.0843; Preferred prescription is strain liquid 5-8 part and the strain liquid carrier 60-100 part that contains fermentation lactobacillus CGMCC No.0843; Strain liquid concentration is 107-1010cfu/g.
Wherein, described mineral carrier is one or more of calcium monohydrogen phosphate, calcium phosphate, calcium carbonate and zeolite, preferred zeolite.
Described mineral carrier is through micronization processes.
Described mineral carrier is through after the micronization processes, and 90% powder granularity is less than 100 μ m, and average grain diameter is less than 50 μ m, and described average grain diameter is 50% diameter of particle.
The strain liquid of described fermentation lactobacillus CGMCC No.0843 is the strain liquid through one time fermentation, secondary fermentation or three fermentation gained.
The carbon N/P ratio is 100: 5～10: 0.5～5 in the culture medium of described one time fermentation, secondary fermentation or three fermentations; Preferred 100: 10: 3.
Described culture medium is soya-bean cake powder, corn flour, molasses, and three's weight ratio is 20-50: 15-65 part: 10-30 part.
Described culture medium also comprises urea, and the weight ratio of itself and soya-bean cake powder is 10-30: 20-50.
Described culture medium also comprises phosphoric acid and/or soluble phosphate, and the weight ratio of itself and soya-bean cake powder is 10-30: 20-50.
It below is detailed description of the present invention.
Fermentation lactobacillus preparation of the present invention is made up of strain liquid that contains fermentation lactobacillus and mineral carrier.What wherein fermentation lactobacillus served as reasons that isolation and selection goes out in the healthy weanling pig mucous membrane of colon is preserved in the fermentation lactobacillus Lactobacillusfermentum at " China Committee for Culture Collection of Microorganisms common micro-organisms center " on December 2nd, 2002, and its preserving number is CGMCC No.0843.
Above-mentioned preserving number is the fermentation lactobacillus of CGMCC No.0843, its isolation and selection method can be called the Chinese patent of Bacillus acidi lactici bacterial classification and isolation and selection method and purposes for the CN02159754.5 name referring to application number, incorporates the full content of this patent into this literary composition in this mode by reference.The biological characteristics of this fermentation lactobacillus bacterial strain is as follows:
(1), the survival rate after pH2.0 handles 6 hours is 80%;
(2), heat-resisting survival rate is 45%;
(3), the storage tolerance survival rate is respectively 78%;
(4), reaching the highest growth concentration in back 24 hours in cultivation is 10
11Cfu/ml
Application number be CN02159754.5 patent disclosure contain the compound additive of fermentation lactobacillus CGMCC No.0843, lactobacillus gasseri (Lactobacillus gasseri) CDMCC No.0840, lactobacillus reuteri (Lactobacillusreuterii) CGMCC No.0841, Lactobacillus acidophilus (Lactobacillus acidophilus) CGMCC No.0842 combination; But above-mentioned additive need comprise four kinds of Bacillus acidi lacticis, needs very high cultivation cost, and bacterial classification too much also makes unstable product quality.Therefore, must improve, just can obtain ripe product above-mentioned additive.
The applicant finds in practice, adds mineral carriers such as calcium monohydrogen phosphate, calcium phosphate, calcium carbonate and zeolite in the strain liquid that contains fermentation lactobacillus CGMCC No.0843, even do not make up with other three kinds of bacterium; Still can effectively regulate microecological balance in the animal intestine, has the prophylactic effect of the non-specific immune function of enhancing, while can also promote the growth of animal effect and improve food conversion ratio, and effect is suitable with above-mentioned four kinds of bacterium combination, through experimental results demonstrate that the strain liquid concentration that contains fermentation lactobacillus CGMCC No.0843 is 10
7-10
10During cfu/g, above-mentioned best results.And to treating the coli-infection grice diarrhoea, promoting wean back piglet growth to have better effect.
Through studies show that; this is owing to contain the necessary most of mineral element of growth of animals or poultry in the mineral carrier; be not only the base stock of building animal skeleton as Ca, P element; also be the normal physiological activity of keeping the cell normal activity, keeping muscle and nerve, cushion blood and tissue fluid acid-base balance, finish the requisite base substance of various physiological functions.Therefore, in the strain liquid that contains fermentation lactobacillus CGMCC No.0843, add mineral carrier and can promote growth of animal, and can improve the quality of animal flesh, egg, milk.
And mineral carrier can also play synergy with fermentation lactobacillus CGMCC No.0843, and effect is far longer than uses two kinds of materials respectively separately.Through research, find that this may be owing to following reason causes
At first, the mineral element in the mineral carrier can improve the activity of fermentation lactobacillus CGMCC No.0843, and fermentation lactobacillus is being measured under the constant situation, and the alimentary canal of animal is regulated the bigger effect of performance.
Secondly, mineral carrier can increase fermentation lactobacillus to gastral adhesive force.
Early stage studies show that adhesive force is the prerequisite of bacteria planting, is probiotic active main sign (Beachey, 1981; Servin, 2004).The higher bacterial strain of adhesive force is to host's health and production performance influence big (Majamaa etc., 1995; Shornikova etc., 1997; Kirjavainen etc. 1998; Ouwehand etc. 1999).Therefore, the size of sticking ability is to judge a major criterion of screening lactic acid bacteria strains.
Owing to can bring into play gastral regulating action after fermentation lactobacillus only can be attached on the alimentary canal surface, and through studies show that, mineral carrier can increase fermentation lactobacillus to gastral adhesive force, make more fermentation lactobacillus be attached on the alimentary canal surface, and then, reduce the breeding of the interior pathogen of alimentary canal and other harmful bacteria by the competition exclution effect.
In addition, mineral carriers such as calcium monohydrogen phosphate, calcium phosphate, calcium carbonate and zeolite can strengthen fermentation lactobacillus CGMCC No.0843 stability of formulation, particularly the stability under the room temperature condition.
Above-mentioned mineral carrier mainly is meant calcium monohydrogen phosphate, calcium phosphate, calcium carbonate and zeolite etc.Wherein calcium carbonate such as precipitated calcium carbonate, powdered whiting, oyster shell whiting etc. all can.Oyster shell whiting also contains several amino acids owing to not only contain calcium carbonate in the nacre of shell, therefore have better effect.
Mineral carrier can be one or more a mixture of above-mentioned several materials.But with the zeolite is best.Zeolite has kind more than 30, and more common have analcime, chabasie, scolecite, heulandite, sodalite, modenite, a foresite etc., all based on calcic, sodium.The general structural formula of zeolite is A (x/q) [(AlO
2) x (SiO
2) y] n (H
2O).Wherein: A is cations such as Ca, Na, K, Ba, Sr, and B is Al and Si, and q is the cationic compound valency, m is the cation number, and n is a water molecule number, and x is the Al atomicity, y is the Si atomicity, (y/x) usually between 1～5, (x+y) is tetrahedral number in the unit cell.
Usually contain the necessary very big part metals element of growth of animals or poultry in the zeolite, these element majorities exist with tradable ionic condition, the ammonia that organic feed is separated out in digestion process, hydrogen sulfide, the contour polar molecule of carbon dioxide, very easily with zeolite crystal in metal ion exchanged, force most of ion in the zeolite to be separated out and absorb for body; And the silicon in the zeolite, aluminium ion although content is a lot of, are formed the basic screen work of zeolite with the form of silicon-oxy tetrahedron or aluminum-oxygen tetrahedron, and Stability Analysis of Structures is difficult to decompose, and can't absorb.Owing to the result that the metal ion in ammonia, hydrogen sulfide, carbon dioxide isopolarity molecule and the zeolite exchanges mutually, greatly reduce the concentration of these polar molecules, improved the condition of work of stomach, protected the physiological function of stomach; And zeolite has absorbent properties efficiently, and some bacterium in the absorption stomach is arranged, and suppresses the possibility that some pathogen grows; Zeolite may also have the catalytic action of multiple biology enzyme in body, can promote the absorption of body to organic feed, improves the nutritive value of all feeds; And the result of zeolite adsorption ammonia, hydrogen sulfide, significantly reduced the foul smell of ight soil, reduced pollution, purified environment.
In order to make the better and fermentation lactobacillus of mineral carrier play synergy, preferably mineral carrier is carried out micronization processes.After the standard of micronization processes was micronization processes, 90% powder granularity was less than 100 μ m, and average grain diameter is less than 50 μ m, and described average grain diameter is 50% diameter of particle.
Micronization processes can increase the surface area of mineral carrier unit mass greatly, make mineral matter be more prone to be absorbed by the animal alimentary canal, micronizing can also increase the adsorbance of unit mass mineral carrier absorption fermentation lactobacillus CGMCC No.0843 strain liquid, both helped saving cost, can also improve the concentration of fermentation lactobacillus, increase the activity of fermentation lactobacillus, simultaneously, micronizing makes also that fermentation lactobacillus is easier to be attached on the alimentary canal surface, and then plays a role.
The strain liquid that contains fermentation lactobacillus CGMCC No.0843 can prepare with any known method of prior art.As can in Rogosa meat soup, cultivating, its component comprises: 10g tryptone, 5g yeast soak powder, 10g glucose, 5g arabinose, 5g sucrose, 15g sodium acetate, 2g ammonium citrate, 6g potassium dihydrogen phosphate, 0.30g anhydrous magnesium sulfate, 0.12g manganese sulfate, 0.03g ferrous sulfate, 1g Tween-80,1000ml distilled water, and regulate pH to 5.4 with acetic acid.Or in the glucose nutrient broth, cultivate, its component is: peptone 10g, beef extract 5g, sodium chloride 5g, glucose 2.5g, agar 15g, distilled water 1000ml.
Strain liquid can also prepare with the method for industrial large scale fermentation.At first, after just being kept at the fermentation lactobacillus CGMCC No.0843 that locates resting state in sand pipe, the freeze drying pipe and inserting the test tube slant activation, enlarge step by step and cultivate and obtain some and the purebred process of quality through shaking bottle and seeding tank again.It is first procedure of fermenting and producing that the expansion of bacterial classification is cultivated, and this operation is referred to as the seed preparation again.Seed preparation not only will make thalline quantity increase, and the more important thing is that through seed preparation turns out and have high-quality seeding and use for fermenting and producing.
Next need fermentation lactobacillus CGMCC No.0843 is enlarged cultivation, obtain pure and strong culture, be i.e. acquisition flushes, the enough cultures of inoculation quantity.According to actual conditions, can select one grade fermemtation, second order fermentation or three grade fermemtation, this all is existing known technology.
With fermentation lactobacillus CGMCC No.0843 carry out one grade fermemtation, second order fermentation or even carry out three grade fermemtation, obtain strain liquid.Condition of culture to fermentation lactobacillus CGMCC No.0843 is studied, and finds in the culture medium of kefir milk acidfast bacilli or the nutrient solution that the carbon N/P ratio is 100: 5～10: 0.5～5 to be advisable; Preferred 100: 10: 3.
For fermentation lactobacillus CGMCC No.0843, glucides such as glucose, sucrose, maltose, lactose, dextrin, starch are optimum carbon source.Available molasses, soya-bean cake powder and corn flour are as carbon source in actual production, and three's weight ratio is 20-50: 15-65: 10-30, and soya-bean cake powder and corn flour can also provide nitrogenous source for fermentation lactobacillus.
Nitrogenous source can divide inorganic nitrogen-sourced and organic nitrogen source, inorganic nitrogen-sourcedly absorbs soon as ammoniacal liquor, nitrate, ammonium salt microorganism, is unfavorable for the long preservation of preparation.Preparation therefore of the present invention adopts organic nitrogen source.Organic nitrogen source commonly used such as groundnut meal, soya-bean cake powder, cottonseed meal, dusty yeast, wheat bran, fish meal, dried silkworm chrysalis meal, corn flour, peptone, urea, vinasse etc. all can adopt.For cost consideration, preferred soya-bean cake powder and corn flour, content is as mentioned above.Also optionally increase urea, the weight ratio of itself and soya-bean cake powder is 10-30: 20-50.
In addition, can also optionally increase the phosphorus source, the phosphorus source mainly is meant phosphoric acid and/or soluble phosphate, and soluble phosphate is commonly used sodium dihydrogen phosphate, sodium hydrogen phosphate etc.The weight ratio of itself and soya-bean cake powder is 10-30: 20-50.P elements can be used as the activator of enzyme in the microorganism growth process, the composition of physiological activator or the conditioning agent of physiological activity.
A kind of fermentation lactobacillus preparation provided by the invention can be used for regulating microecological balance in the animal intestine, thereby has the prophylactic effect of the non-specific immune function of enhancing, and the while can also promote the growth of animal effect and improve food conversion ratio.By the screening of prescription, make that preparation process of the present invention is simple simultaneously, cost is low, constant product quality, and less, the spawn activity of bacterial classification loss late loss is kept preferably during long preservation at normal temperatures.
The specific embodiment
Embodiment 1
Fermentation lactobacillus CGMCC No.0843 is inoculated in the glucose nutrient broth, and its component is: peptone 10g, beef extract 5g, sodium chloride 5g, glucose 2.5g, agar 15g, distilled water 1000ml, put common incubator 37 degree and cultivate after 18-24 hour, gradient dilution, obtaining concentration is 10
7-10
10The strain liquid of cfu/g.
By weight, will contain 30 parts of mixing of 1 part of strain liquid, calcium monohydrogen phosphate of fermentation lactobacillus CGMCC No.0843, promptly obtain fermentation lactobacillus preparation.
Embodiment 2
Fermentation lactobacillus CGMCC No.0843 is inoculated in the Rogosa meat soup cultivates, its component comprises: 10g tryptone, 5g yeast soak powder, 10g glucose, 5g arabinose, 5g sucrose, 15g sodium acetate, 2g ammonium citrate, 6g potassium dihydrogen phosphate, 0.30g anhydrous magnesium sulfate, 0.12g manganese sulfate, 0.03g ferrous sulfate, 1g Tween-80,1000ml distilled water, and regulate pH to 5.4 with acetic acid; Put common incubator 37 degree and cultivate after 18-24 hour, gradient dilution, obtaining concentration is 10
7-10
10The strain liquid of cfu/g.
By weight, will contain 150 parts of mixing of 10 parts of strain liquids, calcium phosphate of fermentation lactobacillus CGMCC No.0843, calcium phosphate carried out micronization processes in advance, promptly obtained fermentation lactobacillus preparation.
Embodiment 3
Place fermentation tank to carry out one grade fermemtation with cultivating the fermentation lactobacillus CGMCC No.0843 that obtains among the embodiment 1, cultivate after 18-24 hour, gradient dilution, obtaining concentration is 10
7-10
10The strain liquid of cfu/g.The fermentation tank culture medium component is 20 parts in soya-bean cake powder, 15 parts of corn flour, 10 parts in molasses, and other adds suitable quantity of water.The carbon N/P ratio is 100: 5: 0.5 in the culture medium.
By weight, to contain 20 parts of mixing of 80 parts of 5 parts of strain liquids, precipitated calcium carbonates, calcium phosphate of fermentation lactobacillus CGMCC No.0843, precipitated calcium carbonate, calcium phosphate all pass through micronization processes in advance, and (90% powder granularity is less than 100 μ m, average grain diameter is less than 50 μ m, described average grain diameter is 50% diameter of particle), promptly obtain fermentation lactobacillus preparation.
Embodiment 4
Place seeding tank to carry out one grade fermemtation with cultivating the fermentation lactobacillus CGMCC No.0843 that obtains among the embodiment 1, place the production jar to carry out second order fermentation then, gradient dilution, obtaining concentration is 10
7-10
10The strain liquid of cfu/g.The fermentation tank culture medium component is 50 parts in soya-bean cake powder, 65 parts of corn flour, 30 parts in molasses, 30 parts in urea, 30 parts of mixing of sodium dihydrogen phosphate, and other adds suitable quantity of water.The carbon N/P ratio is 100: 10: 5 in the culture medium.
By weight, to contain 60 parts of mixing of 8 parts of strain liquids, zeolite of fermentation lactobacillus CGMCC No.0843, zeolite passes through micronization processes in advance, and (90% powder granularity is less than 100 μ m, average grain diameter is less than 50 μ m, described average grain diameter is 50% diameter of particle), promptly obtain fermentation lactobacillus preparation.
Embodiment 5
Place seeding tank to carry out fermenting twice continuously with cultivating the fermentation lactobacillus CGMCC No.0843 that obtains among the embodiment 2, place the production jar to carry out three grade fermemtation then, gradient dilution, obtaining concentration is 10
7-10
10The strain liquid of cfu/g.The fermentation tank culture medium component is 30 parts of groundnut meals, 55 parts of peptones, 20 parts in molasses, 25 parts in urea, 20 parts of phosphoric acid, and the carbon N/P ratio is 100: 7: 2.
By weight, to contain 20 parts of mixing of 50 parts in 6 parts of strain liquids, zeolite, Bake powder of fermentation lactobacillus CGMCC No.0843, zeolite and Bake powder pass through micronization processes in advance, and (90% powder granularity is less than 100 μ m, average grain diameter is less than 50 μ m, described average grain diameter is 50% diameter of particle), promptly obtain fermentation lactobacillus preparation.
Embodiment 6
Contain fermentation lactobacillus CGMCC No.0843 strain liquid preparation method with embodiment 5.
By weight, to contain 20 parts of mixing of 40 parts in 7 parts of strain liquids, zeolite, powdered whiting of fermentation lactobacillus CGMCC No.0843, zeolite and powdered whiting pass through micronization processes in advance, and (90% powder granularity is less than 100 μ m, average grain diameter is less than 50 μ m, described average grain diameter is 50% diameter of particle), promptly obtain fermentation lactobacillus preparation.
Comparative example 1
This experiment is to measure the influence of preparation of the present invention to piglet growth performance.
1. materials and methods
The test group lactobacillus preparation is embodiment 4 gained preparations.Bacillus acidi lactici adds up to 9.0 * 10 in the preparation of making
8CFU/g.
Control group lactobacillus preparation preparation method is with embodiment 4, but wherein strain liquid carrier zeolite changes the degreasing powdered rice hulls into, and the powdered rice hulls stable in properties can not cause extra influence to the piglet state.Bacillus acidi lactici adds up to 9.0 * 10 in the preparation of making
8CFU/g.
1.1 experimental animal
Select 36 28 ± 2d weanling pigs, body weight 7.65 ± 1.09kg, by completely random block design animal experiment, test divides 2 processing, and each handles 6 repetitions, each repeats 3 piglets, high-rise online flat supporting, the hurdle circle is of a size of 2.0 * 2.0m, and every circle is a repetition, each handles 3 circle boars, 3 circle sows.
1.2 test grouping
It is two processing that test is divided into, and handling 1 is test group, and basal diet adds weight ratio 0.1% test group lactobacillus preparation; Handling 2 is control group, and basal diet adds weight ratio 0.1% control group lactobacillus preparation; Each arrangement of handling sees Table 1.The composition and the nutritional labeling of basal diet see Table 2.
1.3 feeding and management
The duration of test feeding piglet is in fully closed child care piglet house, and the temperature in the house remains on 24～27 ℃.Free choice feeding, each hurdle circle are equipped with the duck-beak type water fountain and freely drink water for piglet.1% premix autogamy of basal diet does not contain any antibiotic.The immunity of piglet is undertaken by the immune programme for children of pig routine veterinary infectious disease, the strict health and epidemic prevention system of carrying out of feeding piglet control measures.
Table 1 experimental design
The basal diet of table 2 growth test is formed and trophic level
Annotate: 1. crude protein, lysine, methionine, cystine, threonine, calcium and phosphorus are measured value.
2. the per kilogram daily ration provides: vitamin A, 11,000IU; Vitamin D
3, 1503IU; Vitamin E, 44.1IU; Vitamin K, 4.0mg; Riboflavin, 5.22mg; Pantothenic acid, 20.0mg; Nicotinic acid, 26.0mg; Cobastab
12, 0.01mg; Manganese, 35.0mg; Iron, 100.0mg; Zinc, 90.0mg; Copper, 16.5mg; Iodine, 0.30mg; Selenium, 0.30mg.
1.4 sample is collected and is handled
Test claims the piglet individual weight when beginning, 7d, 14d and 21d, record daily gain and feed feed intake weekly.
1.5 data statistics
(SPSSInc., USA) statistics is handled in the check of the independent sample t of statistical software to test all The data SPSS12.0.
2. result and discussion
2.1 fermentation lactobacillus preparation to weaned piglet after the influence of growth performance
Table 3 compares the influence of fermentation lactobacillus preparation to the weanling pig growth performance with antibiotic
Annotate: the table intermediate value is a mean+SD.
Can go out from table 3, wean back 1wk and 2wk add piglet average daily gain (ADG) comparison of test group fermentation lactobacillus group and you can well imagine high 27.6% and 31.7% according to component, significant difference (P＜0.05), average daily ingestion amount (ADFI) comparison you can well imagine high 17.9% and 28.5% according to component, significant difference (P＜0.05), wean back 2wks adds the piglet average daily gain of fermentation lactobacillus group and improves 30.2% than control group, daily ingestion amount improves 23.9% than control group, and difference is (P＜0.01) extremely significantly.
Carry out same experiment with other embodiment and also obtained identical conclusion.
Comparative example 2
This experiment purpose is to study the influence of embodiment 5 fermentation lactobacillus preparations to the coli-infection grice diarrhoea.
1. materials and methods
Lactobacillus preparation contains fermentation lactobacillus 9.0 * 10
8CFU/g.
1.1 experimental animal and feeding and management
Select the health Du * length * big commodity hybridization piglet of 12 28 ± 2d wean, body weight is 7.65 ± 1.10kg.Be divided into 2 groups at random, single cage is raised in metabolic cage, and the room temperature of respiratory chamber is controlled at 25 ℃～27 ℃, 6 piglets of processed group oral liquid lactic acid bacillus preparation of drinking water every day, drink into that the sum of probio is about 107 every day, 6 piglet drinking public water supplies of control group in contrast.Test mixed-culture medium (108) oral challenge of 8d with 20mL e. coli k99, K88 and 987P, the colibacillary of three kinds of serotypes is in equal proportions in this mixed liquor, one twice-daily, be total to 1d, attacking poison back pig only still freely drinks running water and searches for food, mensuration is attacked poison back diarrhoea disease time and diarrhoea index, experimental period 14d.Attacking poison back 7d morning, vena cava anterior blood sampling 10mL, then each group pig is all butchered, piglet dead before the off-test is butchered immediately, an aseptic stomach, duodenum, jejunum, ileum, caecum and each 10cm of colon of rounding, with the ligation of surgical thread two ends, be placed in-80 ℃ of refrigerators, be used to measure the wherein microorganism species of content and mucous membrane.
1.2 test daily ration
The test piglet basal diet of searching for food does not contain any antibiotic, prepares with reference to the requirement that NRC (1998) recommends.Comparative example 1 table 2 is seen in the basal diet configuration.
1.3 diarrhoea index
The diarrhoea index is that the diarrhoea head multiply by corresponding diarrhoea score, and the diarrhoea score is calculated according to the diarrhoea standards of grading of table 4.Diarrhea rate is that the inferior piglet number divided by every day of the total head of the interior piglet of suffering from diarrhoea of 7d multiply by 7 after attacking poison.
Table 4 diarrhoea standards of grading
1.4 statistical analysis
SPSS12.0 is adopted in the statistical analysis of data, and (SPSSInc., USA) statistical analysis is carried out in the independent sample t-check of software.
2 results and discussion
Result of the test sees Table 5, and the test group diarrhea disease percentage descends 69.1% than control group, and the diarrhoea index has descended 66.Control group promptly had first piglet to suffer from diarrhoea in back second day attacking poison, and the Bacillus acidi lactici group until attack poison afterwards 4d just find first grice diarrhoea.In China, many experimental studies have proved the diarrhoea that probiotic can effectively be prevented and treated suckling pig and weanling pig, and some effect is better than antibiotic or recombinant vaccine (Bai Lin and Li Xuewei, 2002; Jiang Yuwen etc., 2000; Nie's practice etc., 1999; Ma Xiuhai and Feng Jiaxi, 1996).The Bacillus acidi lactici of the lactic acid producing rhzomorph that contains in beneficial lactogenesis acidfast bacilli preparation has a good Chinese People's Anti-Japanese Military and Political College enterobacteria characteristic external, according to have in Hoefling (1989) the report newborn piglet diarrhoea 26% since enterotoxigenic Escherichia coli cause.The test group piglet was being attacked poison preceding 7 days, the oral always drinking-water that contains Bacillus acidi lactici, and Bacillus acidi lactici exists in a large number at intestines and stomach chyme and mucous membrane, and at specific intestines and stomach microhabitat performance prebiotic effect.Piglet is subjected to Escherichia coli when attacking poison; because Bacillus acidi lactici can suppress colibacillary growth and breeding; thereby protected body to avoid the grievous injury that a large amount of breedings of Escherichia coli cause effectively, reduced the order of severity of diarrhea disease percentage and diarrhoea, postponed the diarrhoea disease time simultaneously.
Table 5 fermentation lactobacillus preparation is to the influence of coli-infection diarrhea of weaned piglets
Carry out same experiment with other embodiment and also obtained identical conclusion.
Comparative example 3
This experimental example is the external adhesive force test of the casing slime of different carriers Bacillus acidi lactici.
1, material
With test group and control group in the comparative example 1.
2, method
The test of the adhesive force of casing slime is carried out with reference to the method for (2003) such as Conway etc. (1990) and Gusils.It is 0.5mg/ml that mucus HEPES-Hanks buffer solution is diluted to protein concentration.The supernatant of getting 200ul is the goldbeater's skin tissue fixedly, preserves 24 hours under 4 ℃ of conditions.Organize in contrast with bovine serum albumin.Mucus and BSA (bovine serum albumin) remove floating preteins twice with the HEPES-Hanks buffer solution flushing of 250 μ l.The lactic acid bacteria that adds 150 μ l marks in each hole activates 1 hour down at 37 ℃ then.Before sticking bacterium, dull and stereotyped with 250 μ l HEPES-Hanks buffer solution for cleaning 3 times, stick bacterium with 250 μ l cell pyrolysis liquids (the 0.1mol1-1 sodium hydroxide solution of 1%SDS) cracking 1 hour under 60 ℃ of water area conditions.Each bacterial strain all has the relatively suction of bacterium energy of control group, and the preparation procedure of control group is with top the same except cleaning.The sample in each hole is collected together with after two milliliters of scintillation solutions mix and is spent the night.The radioactivity of sample is calculated with scintillation counter.The adhesive force index is the ratio of the radiation power and the control group hole radiation power in test group hole.Three kinds of aaerosol solution MRS broth bouillons, Bacillus acidi lactici culture supernatant 4.0 and PBS are used to the mensuration of four kinds of different lactic acid bacterias to dissimilar enteron aisle mucus adhesive forces.Each mark bacterium all suspends once with original bacteria liquid again in the culture supernatant processed group.
3, result
Table 6 has been listed Bacillus acidi lactici to isolate the index that sticks of mucus from different small intestine sections.Being contrast to sticking of BSA.
Table 6 different carriers Bacillus acidi lactici is to different intestinal mucosa adhesive force indexes
To annotate: each numeric representation least square is poor ± standard error; *: expression is remarkable with the poor heteropole of BSA.
As seen, mineral carrier can strengthen Lactic Acid Bacilus Adhesion power index significantly.
Carry out same experiment with other embodiment and also obtained identical conclusion.
Comparative example 4:
1, experiment purpose:
Find that different strain liquid carrier adsorbs long term storage stability influence under the prepared preparation comparison room temperature of the present invention
2, experimental technique
Adopt the preparation of embodiment 2 methods to contain fermentation lactobacillus CGMCC No.0843 strain liquid, concentration is 10
10Cfu/g.The strain liquid carrier is respectively zeolite powder, precipitated calcium carbonate powder, phosphoric acid calcium powder, calcium monohydrogen phosphate powder, degreasing powdered rice hulls, wood dust, vinasse powder and milled powders of cornstalk.
Measure spawn activity at once after being made into preparation, placed for 1,6,12,18 week respectively under the normal temperature then after, measure spawn activity more respectively; Calculate and place the percentage that the back spawn activity accounts for the preceding spawn activity of placement.
3, experimental result
As shown in table 7
Table 7 different strain liquid carrier adsorbs the Journal of Sex Research steady in a long-term of prepared preparation
As seen preparation of the present invention is more stable in mineral carrier.
Claims (1)
1. a fermentation lactobacillus preparation is characterized in that, is grouped into by the one-tenth of following weight portion;
The strain liquid 5-8 part that contains fermentation lactobacillus CGMCC No.0843; With
Mineral carrier 60-100 part;
Described strain liquid concentration is 10
7-10
10Cfu/g; Described mineral carrier is one or more of calcium monohydrogen phosphate, calcium phosphate, calcium carbonate and zeolite; Described mineral carrier is through micronization processes; Described mineral carrier is through after the micronization processes, and 90% powder granularity is less than 100 μ m, and average grain diameter is less than 50 μ m, and described average grain diameter is 50% diameter of particle; The strain liquid of described fermentation lactobacillus CGMCC No.0843 is the strain liquid through one time fermentation, secondary fermentation or three fermentation gained; The carbon N/P ratio is 100: 5～10: 0.5～5 in the culture medium of described one time fermentation, secondary fermentation or three fermentations; Described nutrient media components is soya-bean cake powder, corn flour, molasses, urea, phosphoric acid and/or soluble phosphate, and the weight ratio of each component is 20-50: 15-65: 10-30: 10-30: 10-30.
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| CN102212476B (en) * | 2011-03-07 | 2013-04-03 | 杭州向天歌生物科技有限公司 | Method for cleanly and efficiently producing microorganism bactericide |
| CN102212495B (en) * | 2011-04-15 | 2013-04-10 | 北京龙科方舟生物工程技术有限公司 | Lactobacillus acidophilus and application thereof |
| CN102226157B (en) * | 2011-05-06 | 2013-01-09 | 北京龙科方舟生物工程技术有限公司 | Lactobacillus fermentum and application thereof |
| CN102178110B (en) * | 2011-05-12 | 2012-10-17 | 比利美英伟营养饲料（深圳）有限公司 | Premix feed for improving reproductive performance of boar |
| CN102304489B (en) * | 2011-09-23 | 2013-06-12 | 北京龙科方舟生物工程技术有限公司 | Lactobacillus reuteri strain and application thereof |
| CN106191044A (en) * | 2016-08-05 | 2016-12-07 | 福建农林大学 | A kind of gene engineering method improving lactein plnK yield |
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| CN1511945A (en) * | 2002-12-30 | 2004-07-14 | 中国农业大学 | Lactic bacillus strain and its separating and breeding method and use |
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| CN1511945A (en) * | 2002-12-30 | 2004-07-14 | 中国农业大学 | Lactic bacillus strain and its separating and breeding method and use |
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