CN102559550A - Clostridium butyricum having diarrhea treating effect and application of clostridium butyricum as substitute of sodium butyrate - Google Patents
Clostridium butyricum having diarrhea treating effect and application of clostridium butyricum as substitute of sodium butyrate Download PDFInfo
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Abstract
The invention discloses a clostridium butyricum having a diarrhea treating effect. The strain is named as clostridium butyricum Cb-2 which is conserved in China Center for Type Culture Collection with the conservation number of CCTCC M2011384, in November 9th, 2011. A test proves that the clostridium butyricum disclosed by the invention has strong acid producing capability especially butyric acid producing capability; and the clostridium butyricum has obvious effects on diseases, such as intestinal tract flora infection, acute and chronic diarrhea, irritable bowel syndrome and non-ulcer dyspepsia, caused by various reasons, and has no toxicity and harm. Thus, the clostridium butyricum or clostridium butyricum powder disclosed by the invention is used as a raw material for preparing a micro-ecological preparation and used for replacing sodium butyrate to be applied; and when a specific application is carried out, a clostridium butyricum preparation can be singly used as an active component to be prepared into the micro-ecological preparation together with corn starch, or, the clostridium butyricum preparation is mixed with at least one of other bacterial preparations and the corn starch to prepare a composite micro-ecological preparation.
Description
Technical field
The present invention relates to that a strain has the clostridium butylicum of treatment diarrhoea effect and as the application of Sodium propanecarboxylate substitute.
Background technology
At present, along with improving constantly of constant development of economy and standard of living, people more and more recognize the basic meaning of " green ".But because the driving of economic interests, anti-main plain use has begun to spread unchecked, and particularly in feed, uses in a large number, the formation that causes normal microflora imbalance in the animal body and suppress organism immune response, and the serious superinfection that causes, even can cause death; Secondly, anti-main plain residual in the livestock and poultry body and in the food such as meat, egg, milk can not make its deactivation fully through heat treated, and edible for a long time this based food of people possibly cause chronic poisoning etc.In addition, modern medical service technology, agricultural chemicals, chemical fertilizer, hormone, isotropic substance and other heavy chemicals, and physical factor regular meeting directly or indirectly has a negative impact to intravital microecological balance, causes multiple disease.If in feed, use probiotics, will reduce antibiotic consumption greatly, solve the problem that microbiotic can't resolve, enhance productivity, advance to the direction of green agriculture.
Microecology is that the very fast pupil of development in recent years orders science, also be theory and practice combine science quite closely.Along with the progress of little ecological theory, it put into practice product one by one probiotics continue to bring out, and cause clinical medicine worker's great attention.Clostridium butylicum (Clostridium butyricum) has another name called Butylic acid bacteria, and it is a kind in the shuttle shape gemma Pseudomonas, is to go into closely to control doctor by Chiba, Japan medical university palace in 1933 at first to find and report, therefore is Clostridium Butyricum again.Nineteen thirty-five; Doctor Kingimiyairi isolates clostridium butylicum from people's ight soil and soil; Find subsequently to contain less lipid acid in the filtrate that its anaerobism cultivates; Have extremely strong whole intestines effect, it can suppress the pathogenic bacterium in the enteron aisle, the growth of probiotics such as bifidus bacillus and probiotic lactobacillus in the promotion enteron aisle.
Clostridium butylicum as the probiotics main characteristics is: (1) promotes the propagation and the growth of profitable strain (bifidus bacillus, milk-acid bacteria, bacillus faecalis etc.) in the enteron aisle; The growth and breeding that suppresses harmful bacterium such as staphylococcus, candidiasis, klebsiella, Bacillus proteus, Campylobacter, Pseudomonas aeruginosa, intestinal bacteria, dysentery bacterium, salmonella typhi in the enteron aisle and spoilage organism; Reduce the generation of amine, indoles material; And clostridium butylicum itself can reduce the generation of objectionable impuritiess such as ammonia, indoles, hydrogen sulfide because can not decomposing protein; (2) in enteron aisle, produce vitamin B complex, vitamin K, glycase, the good health care effect is arranged; (3) metabolite of clostridium butylicum---butyric acid is the main nutrient matter of intestinal epithelial tissue cell regeneration and reparation; (4) multiple antibiosis is have stronger tolerance, can not influence its biological action with microbiotic and time spent, and can reduce the sickness rate of pseudomembranous enteritis significantly; (5) clostridium butylicum is an anaerobism clostridium spp, does not receive hydrochloric acid in gastric juice, digestive ferment, bile acide etc. to influence its biological action in vivo, ability prolonged preservation under external room temperature; (6) clostridium butylicum is one of normal microflora in the human intestinal, and flora is disorderly in field planting of the harmful bacterium of prevention and invasion in enteron aisle, the correction enteron aisle.
At present, domestic research for butyric bacteria just began in nearly 2 years, did not also have manufacturer so far.Clostridium butyricum active bacteria formulation in the market is all from Japanese import, and price is higher.
Sodium propanecarboxylate has another name called the butanic acid sodium salt, and product shows white is to off-white color, and like the fine hair shape, but hygroscopic powder has the special cheese kind smell that becomes sour, and soluble in water, aqueous solution pH is alkalescence.Because of butyric acid has free property and volatile characteristics, be made into metastable sodium salt---Sodium propanecarboxylate in the fodder prodn.Sodium propanecarboxylate was to allow one of fodder additives kind of using in China, was listed in " fodder additives kind catalogue " by the Ministry of Agriculture in 2006.Application mode on animal produces mainly is in animal-feed, to add Sodium propanecarboxylate product (Ding Zhuansu, Jia Baoyu, intestines benefit are good for), plays effects such as intestinal mucosa nutrition agent, electrolyte balance regulator, gastrointestinal microecosystem regulator, composite souring agent, flavouring agent, phagostimulant.At present, domestic most of to young animal to the applied research of Sodium propanecarboxylate in herding is produced.But practical application finds that Sodium propanecarboxylate product effect is also unstable, analyze its reason have following some:
(1) feedstuff raw material has certain surge capability to Sodium propanecarboxylate: the surge capability of feedstuff raw material is the principal element that influences free acid amount in the stomach.Surge capability is big more, and the free acid that can adsorb in the stomach is just many more, and the free acid after this makes and searches for food in the stomach reduces, and stomach inner pH value raises, and then influences pepsic activity and proteinic digest and decompose.In general, the content of protein, calcium, phosphorus and mineral substance is high more, and the surge capability of feed is high more.
(2) the bad grasp of Sodium propanecarboxylate consumption: Sodium propanecarboxylate is as organic acid, and its liberation degree is little on the one hand, acidity a little less than, it is more much bigger than mineral acid to reach same souring ability addition, it is high to add cost.Sodium propanecarboxylate is as souring agent on the other hand, and the interpolation in the feed ration is direct, and the acid of interpolation by the alkali neutralization, loses acidification easily; Perhaps absorb under one's belt too fast, again can the reflectivity gastric acid inhibitory secretion, influence the normal development of stomach function; If can't arrive small intestine, then can not effectively reduce the pH value in the small intestine, can't reach the purpose that suppresses the harmful microorganism growth, promotes the probiotics growth.In addition, the age of the kind of feed diet, animal, body weight and feeding environment all can influence the usage quantity of Sodium propanecarboxylate.
(3) the storage transport condition is limited: the Sodium propanecarboxylate product is under routine storage and mode of transport; Be under low temperature and the sealed state stable; But can not mix preservation with incompatible material, dust, overheated article, strong oxidizer, the dilution caking take place in the storage process easily or cause feed to make moist.
(4) technical costs is high: the result of isotopic tagging method research SCFA (short chain fatty acid) absorption shows that the absorption of SCFA and metabolic capacity are very strong in the body colon, wherein again with butyric acid for the fastest.Because the butyric acid accretion rate is very fast, make that butyro-serum half-life only has only 6 minutes in the body.
(5) have distress: the animal oral mucosa is fine and soft, and feeling ability is strong, if with at a low price, the chemical grade Sodium propanecarboxylate of low-quality is during as phagostimulant; Expect to play the food calling effect healthy with keeping animal body; But often can cause that the animal mucous membrane burns, cause food consumption to descend, even food refusal.In addition, the issuable hazardous decomposition products of Sodium propanecarboxylate has carbon monoxide, carbonic acid gas, sodium oxide, supersensitivity and deleterious flue dust and gas, might cause also that cns suppresses, eye and allergic, digestive tube and respiratory tract stress.
In sum, seeking a kind of substitute of effective Sodium propanecarboxylate, is a great problem that solution is badly in need of in livestock industry and feed industry field.
Summary of the invention
To above-mentioned prior art, the invention provides the clostridium butylicum that a strain has the effect of treatment diarrhoea, and its application as the Sodium propanecarboxylate substitute is provided.
The present invention realizes through following technical scheme:
One strain has the clostridium butylicum of treatment diarrhoea effect, and this bacterial strain called after clostridium butylicum (Clostridium butyricum) Cb-2 is preserved in Chinese typical culture collection center on November 09th, 2011, and its deposit number is: CCTCC M2011384.
Being characterized as of said bacterial strain: be the Gram-positive genus bacillus of obligate anaerobic, its diameter is 0.6 μ m～1.2 μ m * 3.0 μ m～7.0 μ m, the blunt circle in two ends; Middle portion slightly expands, and bacterium is direct rod shape or bending is arranged slightly, and is single or paired; Short chain; Accidental have a thread thalline, and whole body flagellum can move.This bacterial strain can produce sour aerogenesis, and ability decomposing sucrose, glucose, fructose, SANMALT-S, melibiose, starch can not decompose melizitose, wood sugar; Can not make gelatine liquefication, reduction reindeer moss solidifies cow's milk; The cracked indigestion of grumeleuse, optimum growth temp are 37 ℃, and optimum growh pH value is 7.0.This bacterial strain has acidproof, anti-bile, resistance and high temperature resistance property; Has certain bacteriostasis simultaneously.
Through evidence, clostridium butylicum acid producing ability of the present invention is strong, especially produces the butyric acid ability.Through evidence, clostridium butylicum of the present invention has significant effect to diseases such as the intestinal microflora disorder due to a variety of causes, acute and chronic diarrhea, irritable bowel syndrome, non-ucler dyspepsias, and nontoxic.Therefore, can be raw material with clostridium butylicum of the present invention or clostridium butylicum pulvis, be used to prepare probiotics, be used for substituting Sodium propanecarboxylate and use.
Said clostridium butylicum pulvis obtains through cultivating above-mentioned clostridium butylicum, and its training method can be conventional training method.Its best cultivating and producing technical process is following:
(i) bacterial classification: select clostridium butylicum Clostridium butyricum Cb-2 for use, CCTCC M 2011384;
(ii) laboratory culture: lyophilized powder bacterial classification one ring of above-mentioned clostridium butylicum is inoculated on the substratum, and paraffin Vaseline (1: 1 v/v) is sealed up in aseptic technique, and 35～42 ℃ of following anaerobism are cultivated 24～38h;
(iii) prepare liquid seeds: adopt the 5000mL triangular flask, substratum loading amount 1000mL/5000mL triangular flask, 115 ℃ of sterilization 20min, the inoculation of cooling back, inoculum size 3%～8% (v/v), under 35～42 ℃ of conditions, anaerobism is cultivated 14～38h, is liquid seeds;
(iv) seeding tank fermentation: adopt the 50L seeding tank, the substratum loading amount is 30L; 115～121 ℃ of the temperature that disappears in fact, time 15～30min; Treat to inoculate when the substratum temperature is reduced to 40 ℃, inoculum size 3%～8% (v/v), tank pressure 0.05MPa, under 35～42 ℃ of conditions, anaerobism is cultivated 12～38h, changes fermentor cultivation over to;
(v) fermentor cultivation: adopt the 0.5T fermentor tank, the substratum loading amount is 350L; 115～121 ℃ of the temperature that disappears in fact, time 15～30min; Treat to inoculate when the substratum temperature is reduced to 40 ℃, inoculum size 3%～8% (v/v), tank pressure 0.05MPa, anaerobism is cultivated 16～32h;
(vi) after the fermentation ends, immediately that fermented liquid is centrifugal and clean with clear water, 2～3 times postlyophilizations are pulverized so repeatedly, are bacterium powder finished product; Or: after the fermentation ends, spraying drying promptly gets bacterium powder finished product immediately;
Above-mentioned steps (ii) (iii), (iv) described in culture medium prescription be: glucose 10g/L, Tryptones 5g/L, yeast extract paste 3g/L, beef extract 5g/L, lime carbonate 10g/L, during use, regulate the 20min that sterilizes under ℃ condition of pH to 6.5～7.5,115; (culture medium prescription v) is: glucose 10g/L, peptone 20g/L, yeast extract paste 15g/L, lime carbonate 10g/L, sal epsom 0.2g/L, potassium hydrogenphosphate 2g/L, manganous sulfate 0.2g/L during use, regulates pH to 6.5～7.5 to step.
Said clostridium butylicum preparation with the effect of treatment diarrhoea; Can be used to prepare probiotics; During concrete the application; The clostridium butylicum preparation can be processed probiotics as effective constituent (like fodder additives) and W-Gum separately, and perhaps: clostridium butylicum preparation and other at least a bacteria preparation and W-Gum are mixed and made into compound micro-ecological preparation.
Said other bacteria preparation is that milk-acid bacteria, sporeformer are or/and the conventional bacteria preparation of probiotic bacteriums such as bifidus bacillus; After composite, according to synergy is arranged, can strengthen the effect of product between each bacteria preparation; Promote the intestinal microecology balance, improve immunity of livestock and disease resistance, promote its healthy growth fast; Improve food conversion ratio, reduce the ight soil stink, solve problems such as antibiotic remains exceeds standard.
Clostridium butylicum of the present invention is as fodder additives of new generation, have " the acidifying enteron aisle, repair mucous membrane, to antimicrobial agent, be superior to Sodium propanecarboxylate " distinguishing feature, wide application prospect is arranged, will already produce profound significance to whole livestock industry and feed.
Description of drawings
Bacterial strain clostridium butylicum Cb-2 Clostridium butyricum Cb-2 provided by the invention; Be preserved in Chinese typical culture collection center on November 09th, 2011; Its deposit number is: CCTCC NO:M 2011384, the preservation address is: Chinese Wuhan Wuhan University.
Fig. 1 separates synoptic diagram for the clostridium butylicum plate streaking.
Fig. 2 is the Gram-stained Photomicrograph of clostridium butylicum (undyed part is a gemma in the thalline).
Fig. 3 is a clostridium butylicum acid resisting test synoptic diagram as a result.
Fig. 4 is the anti-bile test-results of a clostridium butylicum synoptic diagram.
Fig. 5 is a clostridium butylicum Cb-2 strain pH change curve.
Fig. 6 is healthy mice and diarrhoea model mice intestinal microflora comparison diagram.
Fig. 7 respectively organized flora comparison diagram in the enteron aisle on the 4th day for the treatment back.
Fig. 8 respectively organized flora comparison diagram in the enteron aisle on the 6th day for the treatment back.
Fig. 9 respectively organized flora comparison diagram in the enteron aisle on the 8th day for the treatment back.
Embodiment
Below in conjunction with embodiment the present invention is further described.
The isolation identification and the cultural characters of embodiment 11 bacterial classifications
1 materials and methods
1.1 the sample collecting Shandong Province Tai Ningyang healthy sow ight soil in ecological pasture.
1.2 substratum
Clostridium proliferated culture medium: Tryptones 5g/L, beef extract 5g/L, yeast extract paste 3g/L, glucose 10g/L, K
2HPO
42g/L, MgSO
40.2g/L, CaCO
310g/L, MnSO
40.2g/L; 7.0,121 ℃ of pH, the 15min sterilization.
Clostridium counting substratum: Tryptones 5g/L, beef extract 5g/L, yeast extract paste 3g/L, glucose 10g/L, K
2HPO
42g/L, MgSO
40.2g/L, CaCO
310g/L, MnSO
40.2g/L, agar 15g/L; 7.0,121 ℃ of pH, the 15min sterilization.
Clostridium isolation medium: in proliferated culture medium, add: taurocholate 5g/L, agar 15g/L transfers pH to 7.0, the 15min sterilization.
1.3 separation method
1) gets faecal samples 10g and be added in the 90mL sterilized water, put into 80 ℃ of water-bath 10min, kill non-sporeformer, put into proliferated culture medium then, cultivate 36h in 37 ℃.
2) gradient dilution nutrient solution is coated the clostridium isolation medium, puts into anaerobic jar, and (volume(tric)fraction is respectively 10% H2,10%CO to replace 3 gases
2And 80%N
2), and with platinum grain as catalyzer, 37 ℃ of anaerobism are cultivated 36h under the gaseous tension of 0.01MPa.
3) adopt replica-plating method to separate bacterial classification, carry out aerobic respectively and the anaerobism cultivation, choose the good bacterial strain of anaerobic growth, microscopy.
4) choosing bacterial strain that cultural characteristic, colonial morphology and microscopic morphology all meet the clostridium butylicum cultural characteristic carries out Physiology and biochemistry and identifies.
1.4 genus and crowd's preliminary evaluation
By " the correlation test that uncle Jie Shi Bacteria Identification handbook and " evaluation that bacterium belongs to is instructed " belong to the pure bacterial strain that is separated to; And crowd, the relevant physiological biochemical test of planting; Comprise respectively: gelatin liquification test, melibiose, melizitose, starch hydrolysis experiment and litmus milk tests.
1.5 acid producing ability and bacteriostasis are measured
Strain clostridium butylicum Cb-1 to new isolating clostridium butylicum Cb-2 and testing laboratory's preservation does acid producing ability and bacteriostasis mensuration respectively; Step is: two strain bacterium are inoculated in the clostridium proliferated culture medium respectively; In saline bottle, leave standstill cultivation; In 16h and 26h sampling, the centrifugal 10min of 4000rpm, supernatant is measured butyric acid and acetic acid content; The 16h sample carries out bacteriostatic test simultaneously, and cup-plate method is both bacteriostasis relatively.
2 results and discussion
2.1 the qualification result of bacterial classification
After 15 sample preparation cultivations, carry out the anaerobic and aerobic cultivation to xerox flat board, obtain 10 strains of strictly anaerobic bacterial strain, selectivity is cultivated the back microscopy and is obtained 1 strain of typical shuttle shape bacillus sp bacterial strain.Obtain bigger shaft-like, the inferior end of thalline after this bacterial strain is further purified and give birth to oval gemma bacterial strain, preliminary evaluation is a clostridium butylicum, and its diameter is 0.6 μ m～1.2 μ m * 3.0 μ m～7.0 μ m; The blunt circle in two ends, middle portion slightly expands, and bacterium is direct rod shape or bending is arranged slightly; Single or paired, short chain, accidental have a thread thalline; Whole body flagellum can move.Carry out physiological and biochemical test subsequently, find that this bacterial strain can produce sour aerogenesis, ability decomposing sucrose, glucose, fructose, SANMALT-S, starch, melibiose; Can not decompose melizitose, wood sugar, can not make gelatine liquefication, reduction reindeer moss; Solidify cow's milk, the cracked indigestion of grumeleuse (seeing table 1, Fig. 1 and Fig. 2), thus; The judgement isolated strains is a clostridium butylicum; Called after clostridium butylicum Clostridium butyricum Cb-2 is preserved in Chinese typical culture collection center on November 09th, 2011, and its deposit number is: CCTCC M 2011384.
The physiological and biochemical property of table 1 isolated strains
2.2 acid resisting test comparative result
Two strain bacterium are inoculated in the clostridium proliferated culture medium respectively, in saline bottle, leave standstill cultivation, and centrifugal at 16h and 26h sampling, supernatant is measured butyric acid and acetic acid content, and the result sees table 2.
The product butyric acid of table 2 liang strain clostridium butylicum, acetate ability are relatively
Test-results shows that former bacterial strain produces the acetate ability and is better than new bacterial strain slightly, is excellent with the latter then but produce the butyric acid ability.
2.3 bacteriostatic test comparative result
Two strain bacterium are inoculated in the clostridium proliferated culture medium respectively, in saline bottle, leave standstill cultivation, and the 16h sample carries out bacteriostatic test, compare the difference of the two bacteriostasis, and the result sees table 3.
The bacteriostasis of table 3 liang strain clostridium butylicum
Annotate: O1 is a chicken colibacillosis; C249 is a swine escherichia coli; K88 is the toxin producing intestinal bacteria; C79-20 fowl typhoid Salmonellas.
Bacteriostatic test is the result show; Two strain clostridium butylicums reach 14mm to the inhibition zone size of intestinal bacteria k88, streptococcus aureus, Sarcina lutea; Size to C249, O1, C79-20 inhibition zone does not wait at 12～14mm; Both do not have significant difference by bacteriostasis, and the bacteriostasis property of new isolated strains Cb-2 is a little more than former strain isolated Cb-1.
2 clostridium butylicum strain properties
The bacterial strain of selecting for use as feeding micro-ecological preparation must have certain characteristic, like the tolerance to the high bile environment of acidity, to restraining effect of harmful intestinal tract bacteria etc.The contriver at first studies some characteristics of this strain clostridium butylicum.
2.1 acid resisting test
The suspension 0.1mL that gets strain bacterium Cb-2 is injected in the anaerobism test tube that the pH that contains 2mL sterile phosphate damping fluid is respectively 1.0,2.0,3.0,4.0 (being transferred to final pH with hydrochloric acid).These anaerobism test tubes are put into 37 ℃ of biochemical incubators, and carry out live bacterial count respectively at 0h, 1h, 2h, 3h and 4h.The equal triplicate of each counting is averaged, and the result sees Fig. 3.
The acid resisting test result is illustrated under four pH, and through the acidproof experiment of 4h, viable count variation difference is little, and this result shows that further this bacterial strain has stronger acid resistance, can pass through stomach smoothly, arrives its effect of performance of enteron aisle position.
2.2 anti-bile test
The suspension 0.1mL that gets strain bacterium Cb-2 is injected into the drug concentration in bile that contains that contains 2mL respective liquid substratum and is respectively in 0%, 0.3%, 0.6%, 1.0% the anaerobism test tube.These anaerobism test tubes are put into 37 ℃ of biochemical incubators, and carry out live bacterial count respectively at 0h, 1.5h, 3h, 4.5h and 6h.The equal triplicate of each counting is averaged, and the result sees Fig. 4.
Clostridium butylicum Cb-2 is fine to the tolerance of these four drug concentration in bile, and the tolerance experiment through 6h has almost kept initial viable count.The result shows that this bacterial strain can tolerate gastrointestinal bile and keep greater activity.
2.3 high temperature resistant test
The 24h nutrient solution 10mL that gets clostridium butylicum Cb-2 places 90 ℃ of water bath processing in test tube, respectively at sampling and measuring viable count before the thermal treatment, when handling 5min and 10min, calculate survival rate, and the result sees table 4.
The high temperature resistant test-results of table 4
Test-results shows that survival rate is respectively more than 80% and 70% behind 90 ℃ of water bath processing 5min and 10min for clostridium butylicum Cb-2 bacterium liquid, and this point is promoted the meaning that particularly important is arranged to this bacterium as fodder additives.
2.4 bacteriostatic test
Analyze the full bacterium liquid of clostridium butylicum, thalline, meta-bolites nutrient solution and intestinal bacteria K88, C249, Q1, C79-20, streptococcus aureus, gamboge eight are folded the restraining effect of bacterium such as coccus again.Adopt the Oxford agar diffusion method to carry out bacteriostatic test, cultivate 8～10h for 37 ℃, measure and suppress the bacterium loop diameter.
The preparation of the full bacterium liquid of clostridium butylicum, thalline, meta-bolites: with the centrifugal 30min of clostridium butylicum nutrient solution 3000rpm, get deposition, and clean 3 times, the thalline that obtains is diluted to original volume with saline water, process the clostridium butylicum thalline with saline water; Centrifugal resulting supernatant filters with sterilizing filter for the first time simultaneously, obtains the aseptic meta-bolites of clostridium butylicum.
The bacteriostasis of the full bacterium liquid of table 5 clostridium butylicum, thalline and meta-bolites relatively
Annotate: O1 is a chicken colibacillosis; C249 is a swine escherichia coli; K88 is the toxin producing intestinal bacteria; C79-20 fowl typhoid Salmonellas;-expression does not detect.
Test-results (seeing table 5) shows: the full bacterium liquid of butyric bacteria bacterium, meta-bolites reach 14mm to the inhibition zone size of k88 bacterium, streptococcus aureus, Sarcina lutea, and the size of C249, O1, C79-20 inhibition zone is not waited at 12～14mm; And the clostridium butylicum thalline is to the equal unrestraint effect of above-mentioned bacterium.This result shows that clostridium butylicum has the good restraining effect to pathogen enterobacteria, and rising inhibiting is clostridium butylicum and meta-bolites thereof.
2.5 produce the acid test
Carry out the fermentation of clostridium butylicum with the little saline bottle of 100mL, leave standstill and cultivate 36h, the pH value of different time sampling and measuring bacterium liquid, the result sees table 6 and Fig. 5.
The variation of pH value in the table 6 different time stage fermentation liquid
Test-results shows that the main metabolites of clostridium butylicum is exactly a butyric acid, and when thalline got into logarithmic phase, butyric acid promptly began to produce, produced simultaneously acetate in addition.The generation of butyric acid, acetate descends the pH value of fermented liquid rapidly, and during fermentation 27h, pH reaches Schwellenwert, and Schwellenwert is 4.56.
The optimization of 3 seed culture mediums
According to existing research, the contriver confirms that the initial medium of cultivation clostridium butylicum is the clostridium proliferated culture medium.Adopt deep liquid to leave standstill cultured method, the bacterium number is measured does not have the persimmon acid system to match with counting method of blood cell and Jiao's property.In order to improve Butylic acid bacteria thalline yield, this research substratum is formed as index with the viable count in the unit volume nutrient solution and culture condition such as initial pH, culture temperature are optimized.
3.1 the culture temperature of thalline
Clostridium butylicum Cb-2 is at 25 ℃, 28 ℃, 30 ℃, 32 ℃, 35 ℃, 37 ℃, 40 ℃ differing temps fermentation culture 36h, and after 80 ℃ of water-bath 15min of residual thalline inactivation treatment, the dull and stereotyped cultivation counted, and the result sees table 7.
Table 7 temperature is to the influence of bacterial concentration
Test-results shows that temperature has considerable influence to the growth of clostridium butylicum Cb-2, confirms that through testing the Cb-2 optimum growth temp is 37 ℃.
3.2 the cultivation pH value of thalline
Get the bacterium liquid 10mL of fermentation 36 h; The centrifugal 15min of 3000rpm abandons supernatant, and thalline is washed 3 times with saline water; Add saline water 5mL earlier; The pH value of regulating the bacteria suspension of clostridium butylicum Cb-2 is respectively 5.5,6.0,6.5,7.0,7.5,8.0, is settled to 10mL with saline water then, handles 2h in 37 ℃.Get bacteria suspension, on plate, do enumeration, the result sees table 8.
The different original ph of table 8 are to the influence of bacterial concentration
Different original ph, after clostridium butylicum Cb-2 cultivated, it is very remarkable that bacterial concentration changes difference; Test-results shows; In original ph is 7～8 o'clock, and bacterial concentration changes little, and the pH value is lower than 7 has bigger reduction; The pH value is too high also to be unfavorable for its growth, so confirms that it is 7.0 that clostridium butylicum Cb-2 cultivates ph optimum.
3.3 carbon, nitrogenous source proportioning test
Regulate carbon, nitrogenous source kind and addition,, thereby make cell concentration reach maximum for thalli growth provides enough nutrition.
3.3.1 different carbon sources are to the influence of cell concentration
Be carbon source with 1.0% glycerine, 1.0% glucose, 1.0% SANMALT-S, 1.0% starch, 1.0%D-fructose, 1.0% lactose, 1.0% sucrose respectively; 0.5% glucose in the replacement basic medium; Other components unchanged in the basic medium; Clostridium butylicum is carried out deep liquid leave standstill cultivation, cultivate 24h, the result sees table 9.
The different carbon sources of table 9 are to the influence of cell concentration
Test-results shows that best with 1.0% glucose carbon source culture effect, wherein starch and sucrose are as carbon source, and microscopy finds that the growth phase postpones.
3.3.2 the glucose addition is to the influence of cell concentration
With glucose is carbon source, cultivates with 0.3%, 0.5%, 1.0%, 1.2%, 1.5%5 consumption level respectively, and other composition remains unchanged in the basic medium.Cultivate 24h, the result sees table 10.
Table 10 glucose addition is to the influence of cell concentration
Test-results shows that 1.0%, 1.2%, 1.5% o'clock cell concentration of glucose addition is maximum, considers that from practicing thrift the cost angle glucose addition 1.0% is best.
3.3.3 dissimilar nitrogenous sources are to the influence of cell concentration
Since the protease activity of clostridium butylicum a little less than, can not utilize protein basically, therefore mainly select some solubility nitrogenous sources to compare, fixing other components unchanged in the initial medium, change nitrogenous source kind is respectively with (NH
4)
2SO
4, NaNO
3, Tryptones, yeast extract paste, Tryptones+yeast extract paste+beef extract, replace in the basic medium 1.0% beef extract, other components unchanged is cultivated 24h, the result sees table 11.
The dissimilar nitrogenous sources of table 11 are to the influence of cell concentration
Test-results shows that when Tryptones 1.0%, yeast extract paste 0.3%, beef extract 0.5%, cell concentration is the highest, therefore, confirms that nitrogenous source proportioning the best is Tryptones 1.0%, yeast extract paste 0.3%, beef extract 0.5%.
3.3.4 substratum carbon, nitrogenous source proportioning are confirmed
In single factor researchs such as above-mentioned carbon source, nitrogenous source, confirmed the concentration that carbon source and nitrogenous source are proper, but C/N ratio also there is very big influence to the growth of thalline.Through orthogonal test, press the primary and secondary of the big or small determinative of extreme difference, adopt L9 (3
4) carry out orthogonal test (gauge outfit design see table 12) and confirm suitable carbon-nitrogen ratio, test-results is seen table 13.
The design of table 12 gauge outfit
Table 13 orthogonal experimental design result
Can confirm that by above-mentioned test the seed minimum medium is: glucose 1.0%, yeast extract paste 0.3%, beef extract 0.5%, Tryptones 0.5%.
3.3.5 Different Ca CO
3Addition is to the influence of bacterial concentration
Adjust suitable carbon, the nitrogenous source proportioning can obtain higher cell concentration; Consider the acid of the big during the fermentation volume production of clostridium butylicum, it is very big to make that spoke falls in pH value, and pH is only in certain hour remains on right scope; Could promote the increase of bacterial concentration, add a certain amount of fermentation regulator CaCO
3Can neutralize the acid of part, the result sees table 14.
Table 14 Different Ca CO
3Addition is to the influence of bacterial concentration
Test-results shows, works as CaCO
3Addition be 1.0%, can obtain maximum total viable count, therefore, CaCO
3Concentration is chosen 1.0% and is optimum concn.
In sum, seed culture medium is confirmed as: glucose 1.0%, yeast extract paste 0.3%, beef extract 0.5%, Tryptones 0.5%, CaCO
31.0%.
The optimization of 4 clostridium butylicum fermentation tank culture medium and culture condition
Grope the fermentation culture conditions of clostridium butylicum, select best substratum and cultural method, for developing and develop the clostridium butylicum probiotics basic data is provided, and does some preliminary experimental studies.
4.1 confirming of fermention medium
If adopt the large-scale clostridium butylicum of producing of seed culture medium, cost is higher, and big production need be to being suitable for the fermention medium in the actual production process.The contriver is with reference to the existing substratum of company (glucose 2%, beef extract 1.5%, peptone 1.5%, CaCO
32%) is initial fermention medium, utilizes the 7L small fermentor, fermentation culture conditions is optimized with empirical method and statistical technique; Consider that the beef extract price is higher; The contriver replaces with Carnis Bovis seu Bubali cream in test the yeast extract paste of equivalent, has carried out orthogonal test, and the result sees table 15.
The orthogonal test of table 15 fermention medium
Press the primary and secondary of the big or small determinative of extreme difference, A
1B
2C
3D
1Be best of breed, final definite back glucose 1.0%, yeast extract paste 1.5%, peptone 2.0%, CaCO of optimizing
31.0%.With optimize before compare glucose and CaCO
3Each has reduced by 1 percentage point, and beef extract replaces with the cheaper yeast extract paste of price, and viable count does not only reduce, and provides cost savings.
4.2 inorganic salt and microelement match test
Suitable C/N ratio can improve the viable count of clostridium butylicum, and the gemma rate that how to improve thalline aborning is a most critical.Adjust in the substratum inorganic salt and microelement match to improve the gemma transformation efficiency at present.Potassium ion, mg ion, mn ion can promote gemma to form, so the contriver adds the K of different concns
2HPO
4, MgSO
4, MnSO
4And respectively the viable count in the nutrient solution is counted.
4.2.1K
2HPO
4Influence to cell concentration
Phosphorus is nucleic acid and proteinic necessary component, also is the component of ATP, and the growth of thalline is had material impact.Add the K of different concns
2HPO
4, and respectively the viable count in the nutrient solution is counted, the result sees table 16.
Table 16 K
2HPO
4Concentration is to the influence of bacterial concentration
Test-results shows that when the concentration of potassium ion was 0.2%, bacterium liquid viable count was maximum, therefore, confirms K in the bacterium liquid
2HPO
4The righttest interpolation concentration is 0.2%.
4.2.2MgSO
4Influence to cell concentration
Mg ion is the cofactor of many enzymes in the microbe body, and the growth of thalline is had material impact.Add the MgSO of different concns
4, and respectively the viable count in the nutrient solution is counted, the result sees table 17.
Table 17 MgSO
4Concentration is to the influence of bacterial concentration
Test-results shows that when the concentration of mg ion was 0.02%, bacterium liquid viable count was maximum, therefore, confirms MgSO in the bacterium liquid
4The righttest interpolation concentration is 0.02%.
4.2.3MnSO
4Influence to the bud cell concentration
Mn ion has significant promoter action to the growth of clostridium butylicum thalline and the formation of gemma, and the growth of thalline is had material impact.Add the MnSO of different concns
4, and respectively the viable count in the nutrient solution is counted, the result sees table 18.
Table 18 MnSO4 concentration is to the influence of bacterial concentration
Test-results shows that when the concentration of mn ion was 0.02%, bacterium liquid viable count was maximum, therefore, confirms MnSO in the bacterium liquid
4The righttest interpolation concentration is 0.02%.
4.2.4 orthogonal test is found out best proportioning
In single factor researchs such as above-mentioned potassium ion, mg ion and mn ion, confirmed K
2HPO
4, MgSO
4, MnSO
4And CaCO
3Proper concentration through orthogonal test, is pressed the primary and secondary of the big or small determinative of extreme difference, adopts L9 (3
4) carry out orthogonal test and confirm suitable carbon-nitrogen ratio, test-results is seen table 19.
Table 19 inorganic salt orthogonal experiments
Be that the inorganic salt proportioning is: MgSO
40.02%, K
2HPO
40.2%, MnSO
40.02%, CaCO
31.0%.Through above test, the contriver has confirmed K
2HPO
4, MgSO
4, MnSO
4,CaCO
3Concentration is respectively 0.2%, 0.02%, 0.02%, 1.0%.
4.3 the selection of inoculum size
The inoculum size size has certain influence to cultivation results; Big inoculum size can impel thalline after inoculation, to recover in the short period to increase; Shorten the time that cell concentration peaks; But inoculum size is crossed the meta-bolites that the more seed culture phase is brought in conference into, and makes the thalline early growth too fast, and final bacterium number is descended.The too small growing microorganism that then makes of inoculum size is slow, and incubation time prolongs.Test has adopted 1%, 2%, 5%, 8%, 10%, 12%6 inoculum size to cultivate respectively, cultivates 24h, and the result sees table 20.
Table 20 clostridium butylicum inoculum size is to the influence of cell concentration
Test-results shows that microscopy is found when cultivating 24h, and 10% inoculum size group gets into stationary phase, but cell concentration is not the highest; 1%, 2% inoculum size group still is in exponential phase, and it is slow to grow, and therefore 5% is optimum inoculation amount.
4.4 clostridium butyricum to grow and pH value change curve
According to above research, confirm that the substratum after butyric acid acid bacterium is optimized is glucose 1.0%, yeast extract paste 1.5%, peptone 2.0%, CaCO to clostridium butylicum substratum and culture condition
31.0%, K
2HPO
40.2%, MgSO
40.02%, MnSO
40.02%, pH7.0, culture temperature is 37 ℃, inoculum size is 5%.According to above culture condition, measure pH value change curve and bacterial concentration and gemma conversion situation in the 33h, the result sees table 21.
Clostridium butyricum to grow and pH value change in the table 21 different fermentations stage fermentation liquid
Test-results shows that during incubation time 18h, viable count is bigger, but the gemma transformation efficiency is on the low side.When cultivating 30h, viable count descends a little, and the gemma rate reaches 90%, and expulsion rate reaches 40～60%, and for avoiding the long gemma diauxic growth that occurs of fermentation time, the contriver is decided to be best incubation time with 30h.
4.5, the preparation of pilot scale scale-up and bacterium powder
The 70L fermentor tank is selected in the pilot scale amplification for use, fermentor tank volume 100L, and coefficient 0.6, inoculum size 5%, 37 ℃ of leavening temperatures, the initial pH of fermention medium is controlled at 7.0.Carry out pilot plant test to optimize good culture medium prescription; And measure living weight, pH value and butyric acid and the acetic acid content of the clostridium butylicum Cb-2 of fermented liquid at the different fermentations time sampling; Microscopy is observed the growth and the gemma formation situation of clostridium butylicum simultaneously, and the result sees table 22,23,24.
The living weight of clostridium butylicum in the table 22 different fermentations stage bacterium liquid
The variation of pH value in the table 23 different fermentations stage bacterium liquid
The mensuration of butyric acid, acetic acid content in the table 24 different fermentations stage bacterium liquid
Fermented liquid is when 15h, and microscopy finds, clostridium butylicum has got into logarithmic growth latter stage, and thalline rolls up; Thalline partly forms gemma when 18h; Thalline gemma rate reaches more than 90% during 24h, and maturing rate reaches 50%, fermentation ends and stop the fermentation.Living weight reaches the highest (7.9mg/mL) in the fermented liquid when 18h, and as time passes, living weight descends on the contrary, maybe be relevant with the minimizing of thalline behind the thalline formation this moment gemma; The pH value reaches Schwellenwert 6.18 at 15h, prolongs in time and gos up; The content of butyric acid and acetate increases very fast at 12～15h, this with this moment thalline breed relevantly in a large number, then raise again during later stage 21h, then maybe with CaCO in this moment fermented liquid
3Utilization reach maximum and can't continue to neutralize with it relevant.
Test-results shows that the 70L expanding test is comparatively similar with the fermentation process results of 7L jar, but difference is also arranged: clostridium butylicum 18h in the jar of 70L has promptly formed gemma, when 24h, stops fermentation, has shortened 8h than 7L canister fermentation time; The fermented liquid viable count is up to 1.8 * 10 than higher
9Cfu/mL has improved 1.5 times than 7L jar.
Adopt spray-drying process that fermented liquid is carried out drying, dry back bacterium powder counting, the result is 3.34 * 10
9Cfu/g.Bacterium powder viable count is not high, and analyzing reason possibly be that the clostridium butylicum thalline is bigger, in spray-drying process, can't bear cf-and the death of nearly 12000rpm.The contriver attempts carrying out lyophilize (earlier with 3000rpm centrifugal after spraying drying again) to fermented liquid, and surviving rate has only about 40%, and cost is too high.Therefore the last handling process of fermented liquid also needs further to explore.
4.6 preparation clostridium butylicum bacterium powder
The concrete steps of said preparation clostridium butylicum bacterium powder are following
(i) bacterial classification: select clostridium butylicum Clostridium butyricum Cb-2 for use, CCTCC M 2011384;
(ii) laboratory culture: lyophilized powder bacterial classification one ring of above-mentioned clostridium butylicum is inoculated on the substratum, and paraffin Vaseline (1: 1 v/v) is sealed up in aseptic technique, and 37 ℃ of following anaerobism are cultivated 36h;
(iii) prepare liquid seeds: adopt the 5000mL triangular flask, substratum loading amount 1000mL/5000mL triangular flask, 115 ℃ of sterilization 20min, the inoculation of cooling back, inoculum size 5% (v/v), under 37 ℃ of conditions, anaerobism is cultivated 24h, is liquid seeds;
(iv) seeding tank fermentation: adopt the 50L seeding tank, the substratum loading amount is 30L; 121 ℃ of the temperature that disappears in fact, time 30min; Treat to inoculate when the substratum temperature is reduced to 40 ℃, inoculum size 5% (v/v), tank pressure 0.05MPa, under 37 ℃ of conditions, anaerobism is cultivated 30h, changes fermentor cultivation over to;
(v) fermentor cultivation: adopt the 0.5T fermentor tank, the substratum loading amount is 350L; 121 ℃ of the temperature that disappears in fact, time 30min; Treat to inoculate when the substratum temperature is reduced to 40 ℃, inoculum size 5% (v/v), tank pressure 0.05MPa, anaerobism is cultivated 24h;
(vi) after the fermentation ends, immediately that fermented liquid is centrifugal and clean with clear water, 3 times postlyophilizations are pulverized so repeatedly, are bacterium powder finished product; Or: after the fermentation ends, spraying drying promptly gets bacterium powder finished product immediately;
Above-mentioned steps (ii) (iii), (iv) described in culture medium prescription be: glucose 10g/L, Tryptones 5g/L, yeast extract paste 3g/L, beef extract 5g/L, lime carbonate 10g/L, during use, regulate pH to 7.0,20min sterilizes under 115 ℃ of conditions; (culture medium prescription v) is: glucose 10g/L, peptone 20g/L, yeast extract paste 15g/L, lime carbonate 10g/L, sal epsom 0.2g/L, potassium hydrogenphosphate 2g/L, manganous sulfate 0.2g/L during use, regulates pH to 7.0 to step.
Purpose and meaning
Weaning syndrome of piglets is the healthy major issue of cultivating of restriction piglet always, and present conventional means is after wean, to add organic acid in the daily ration.Sodium propanecarboxylate is the metastable sodium salt of butyric acid, at the treatment grice diarrhoea, improve the gi tract function, improve and to bring into play important effect aspect the breeding performonce fo animals.
Clostridium butylicum is a kind of probiotic bacterium in the animal intestinal; The existing report of its research as microbial forage additive; Simultaneously because this bacterium can form gemma; Environment has very strong resistibility to external world, and strong stress resistance, high temperature high voltage resistant reach multiple antibiosis is have resistance, therefore becomes the focus of current research and application.
Research Sodium propanecarboxylate and clostridium butylicum substitute Sodium propanecarboxylate Application feasibility on piglet produces to clostridium butylicum and verify the influence of weanling pig production performance.The difference of research clostridium butylicum is added the influence of level to weanling pig food consumption, average daily gain, feed efficiency, diarrhea rate, for its application in actual production provides theoretical foundation and guidance.
1 materials and methods
1.1 test materials
16 of wean ternary piglets, parity and body weight are close and be in a good state of health.Prepare clostridium butylicum Cb-2 pulvis according to 4.6 method among the embodiment 1, promptly clostridium butylicum Cb-2 obtains fermented liquid through fermentative processing, adds 6% (weight percentage) W-Gum (trade name: W-Gum then; Article number: 6920420601219; Manufacturer: process clostridium butylicum bacterium powder through spraying drying Shandong Jin Cheng limited-liability company), viable count is 2.0 * 10
8Cfu/g, swinery adopts fermentation bed.The basis grain ration is the piglet fermentation bed complete diet pellet of our company oneself preparation, fills a prescription like table 25 unit: gram.
Table 25 conventional feed prescription
1.2 test period, place
Test in late April, 2010 to late May, come ecologically raising pigs factory of Ningyang of company in the Bora profit, the time is 35 days.
1.3 selection and the grouping of test piglet
The practical situation of consideration company, the contriver has selected 16 piglets that are in a good state of health for use from the original piglet of pig factory, close according to body weight; Male and female half and half principle is assigned to 4 treatment group at random; Every group 4,1. group is Sodium propanecarboxylate control group, the Sodium propanecarboxylate of the basal diet of feeding+0.15%; 2., 3., 4. test group is the clostridium butylicum group, the basal diet of feeding+clostridium butylicum Cb-2, and addition is respectively 0.1%, 0.5%, 1.0%, and viable count is respectively 1.0 * 10
5Cfu/g, 5.0 * 10
5Cfu/g, 1.0 * 10
6Cfu/g.
1.4 feeding and management
Pig house adopts fermentation bed, and the environmental health condition is good, and free choice feeding is freely drunk water.
1.5 testing index
Take off data comprises just starting weight, heavy, the average mouthful weightening finish in end, feed consumption, feedstuff-meat ratio and diarrhea rate.During on-test, every piglet is claimed initial body weight morning on an empty stomach, and off-test is weighed morning next day on an empty stomach, in order to calculate piglet average daily gain (ADG); Write down each charging capacity of each treatment group and surplus material amount respectively; In order to calculate the average food consumption (ADFI) of piglet; Calculate feed conversion rate according to piglet average weight gain and daily ingestion amount; Record suffer from diarrhoea every day piglet a number of times and calculate the diarrhea rate of each treatment group piglet, total piglet number * 100% in diarrhea rate (%)=diarrhoea piglet number/processing.
2 test-results
Experimental stage is respectively organized body weight, day weight gain, feedstuff-meat ratio and the diarrhea rate of piglet, sees table 26.
Table 26 Sodium propanecarboxylate, clostridium butylicum are to the influence of piglet production performance
Test-results shows, adds the test group of clostridium butylicum Cb-2 and compares with the control group that adds Sodium propanecarboxylate, and day weight gain is best to add 0.5% clostridium butylicum Cb-2 effect; Feedstuff-meat ratio serves as better with 0.5% clostridium butylicum Cb-2 group; All test pig are not all suffered from diarrhoea at duration of test.
3 analyze discussion
This test-results shows; Sodium propanecarboxylate group and each dose groups of clostridium butylicum Cb-2 all have apparent preferably effect to day weight gain and the feedstuff-meat ratio aspect of pig; This is consistent with the data of literatures report; Explain that clostridium butylicum and Sodium propanecarboxylate have good promoter action to improving the GI micro-ecological environment of piglet, thereby guarantee that piglet has good digestion and utilising efficiency to feed, obtained feeding effect preferably.Especially the alternative Sodium propanecarboxylate test of clostridium butylicum Cb-2 shows that clostridium butylicum Cb-2 can substitute Sodium propanecarboxylate fully, really plays " acidifying enteron aisle, reparation mucous membrane; To antimicrobial agent, be superior to Sodium propanecarboxylate " effect.
Purpose: observe the diarrhea of mouse that clostridium butylicum Cb-2 causes penbritin and the influence of intestinal microflora, estimate clostridium butylicum Cb-2 in the effect of regulating aspect the intestinal microflora balance.
Method: adopt penbritin to irritate the method for stomach, make the diarrhea of mouse model; Modeling success back is irritated clothes various dose clostridium butylicum Cb-2 and is treated, and observes the ight soil situation of different treatment times and detects the quantity of bifidus bacillus, probiotic lactobacillus, enterobacteria, faecalis and bacterioide.
1 materials and methods
1.1 experimental animal: kunming mice, male, body weight 18～22g is available from the Tai Bang institute of biological products.
1.2 medicine: penbritin, available from health ground grace bio-pharmaceuticals limited-liability company.
1.3 substratum: BDS, LBS, eosin methylene blue agar (EMB), TPY, sodium azide-Viola crystallina-Vitamin C2 agar
1.4 test strain: prepare clostridium butylicum Cb-2 pulvis (1.0 * 10 according to 4.6 method in the embodiment example 1
8Cfu/mL), promptly clostridium butylicum Cb-2 obtains fermented liquid through fermentative processing, adds 6% (weight percentage) W-Gum (trade name: W-Gum then; Article number: 6920420601219; Manufacturer: process clostridium butylicum bacterium powder through spraying drying Shandong Jin Cheng limited-liability company),, be mixed with viable count with sterile saline and be respectively 1.0 * 10
4Cfu/mL, 1.0 * 10
5Cfu/mL, 1.0 * 10
6The liquid preparation of cfu/mL concentration refrigerates subsequent use.
1.5 TP
1.5.1 diarrhea of mouse model
The molding environment: mouse cage fully cleans back shop 5～6 layers of toilet paper (being convenient to visual inspection ight soil situation), and 2 mouse of every cage are pasted the grouping mark on the cage, and mouse goes into behind the cage to change every day toilet paper.
Molding medication: prepare molding and calculate with penbritin concentration 22.4g/kg body weight.Calculate by each every mouse stomach amount 0.5mL, every day 2 times, continuous 5 days, capacity penbritin powder is dissolved in SPSS, fully after the vibration dissolving, put 4 ℃ of refrigerators and preserve subsequent use.
Model preparation: tried mouse and carry out after all flexibility raises, fasting 12h (freely drinking water) before modeling presses the 22.4g/kg body weight and prepares capacity penbritin physiological salt soln; Mouse stomach 0.5mL/ time. only; Every day 2 times, continuous 5 days, until causing the diarrhea of mouse symptom to take place.
1.5.2 test is divided into groups
The normal microflora group: 15, normal health mouse, blank.
The diarrhoea model group: 15, carry out the intestinal microflora analysis after the modeling immediately, whether verification model is successfully set up.
Natural recovering group: 15, do not do any treatment after the modeling success and handle, to observe continuously 7 days, record symptom and flora change.
The saline water group: 15, every the mouse in modeling success back is irritated stomach saline water 0.5mL at every turn, every day 2 times, continuous 4 days, observes the 7th day.
Clostridium butylicum group: high (1.0 * 10
6Cfu/mL), in (1.0 * 10
5Cfu/mL), low by (1.0 * 10
4Cfu/mL) three dose groups, 15 every group, every the mouse in modeling success back is irritated stomach clostridium butylicum solution 0.5mL at every turn, every day 2 times, continuous 4 days, observes to the 8th day.
1.5.3 symptomology is observed: penbritin solution is observed the medication reaction after irritating stomach immediately, notes observing movable performance of mouse and ight soil situation later every day; Movable performance of observed and recorded mouse and ight soil changing conditions during the treatment.
1.5.4 intestinal microflora analysis:
Get the fresh excreta of 5 mouse before the modeling and do the flora analysis, the aseptic stool in mice of taking in modeling success back is carried out live bacterial count, and with the comparative analysis of normal microflora analysis bank; With each dose groups treatment the 4th day (D4), the 6th day (D6) in back of clostridium butylicum, the 8th day (D8); From each group, getting 5 mouse cuts open extremely; The aseptic cecal content of getting, the back 10 times of dilutions of weighing are counted wherein enterobacteria, faecalis, probiotic lactobacillus, bacterioide and bifidus bacillus respectively.Natural recovering group and the sampling of saline water group are with the treatment group.
1.5.5 statistical procedures: the normal intestinal flora analytical results is converted into the log value.Mean relatively adopts the T check between group, and all data are carried out statistical procedures with statistics SPSS10.0 software.
2 results and analysis
2.1 symptom of diarrhea relatively
Before the treatment, each is organized mouse and all presents watery stool diarrhoea in various degree, treats back 3 days, and the middle and high dose groups of clostridium butylicum has half diarrhea of mouse symptom to take a turn for the better, and low dose group and saline water group effect are poor slightly; Treated back 5 days, each dose groups does not almost have watery stool, and the saline water group also has 2 diarrhoea; Most of mouse has recovered normal ight soil after 7 days, and the saline water group has 1 continuation just rare, and concrete outcome is seen table 27.
Each treatment group test mice symptom of diarrhea of table 27 is replied situation
2.2 penbritin is to the influence of mouse intestinal flora, the result sees table 28 and Fig. 6.
Table 28 normal mouse and model mice intestinal microflora average ratio
Annotate: O1 is a flora before the moulding; O2 is the saline water group; T1 is the diarrhoea model group.
Test-results shows, saline water group O2 with modeling before in the mouse O1 intestines normal microflora compare, quantity does not have considerable change, difference is not remarkable.After the penbritin modeling, the quantity of 5 kinds of leading floras obviously changes in the enteron aisle, compares with the corresponding flora quantity of normal mouse, and enterobacteria, bacterioide, probiotic lactobacillus, bifidus bacillus and faecalis amount reduce sharply; Exist significant difference (p＜0.05) between model group and the normal group mouse intestinal flora, explain that the microbial population balance is destroyed in the model mice enteron aisle, dysbacteriosis occurs.
2.3 the different treatment of clostridium butylicum fate intestinal microflora changes: like table 29, table 30, table 31, Fig. 7, Fig. 8 and shown in Figure 9.
Table 29 is respectively organized mouse and is handled the 4th day intestinal flora quantitative analysis results in back
Annotate: O2 is the saline water group; O3 is the diarrhoea natural recovering group; T2 is 10
4The Cb-2 group; T3 is 10
5The Cb-2 group; T4 is 10
6The Cb-2 group;-representative does not detect.
Table 30 is respectively organized mouse and is handled the 6th day intestinal flora quantitative analysis results in back
Annotate: O2 is the saline water group; O3 is the diarrhoea natural recovering group; T2 is 10
4The Cb-2 group; T3 is 10
5The Cb-2 group; T4 is 10
6The Cb-2 group;-representative does not detect.
Table 31 is respectively organized mouse and is handled the 8th day intestinal flora quantitative analysis results in back
Annotate: O2 is the saline water group; O3 is the diarrhoea natural recovering group; T2 is 10
4The Cb-2 group; T3 is 10
5The Cb-2 group; T4 is 10
6The Cb-2 group;-representative does not detect.
Test-results shows, recovers O3 group, saline water group modeling success back naturally the 4th, 6,8 day, and faecalis, probiotic lactobacillus, bifidus bacillus quantity levels are risen progressively gradually, but normal healthy mice intestinal physiology bacterium level is on the low side.Each dose groups treatment back of clostridium butylicum the 4th day, probiotic lactobacillus and bifidus bacillus increase very obvious, and bacterioide, enterobacteria and faecalis increase obviously; Treatment back the 6th day, bifidus bacillus increases obviously, and other bacterium are not obvious; Each flora then was tending towards normal value basically in the 8th day.
3 analyses and discussion
3.1 irritate the clothes mouse with penbritin, make test intestine dysbacteriosis disease model, be intended to verify the pharmacodynamic action of the clostridium butylicum Cb-2 of various dose group to the mouse intestine dysbacteriosis.Test-results shows, irritates stomach after 5 days with penbritin, during mouse has occurred, severe diarrhoea; The intestinal microflora quantitative analysis shows; The quantity of the normal dominant microflora of model mice is far below the normal health mouse; This symptom shows that there is serious intestine dysbacteriosis disease in mouse, expression modeling success, and model mice can be used as the objective basis of estimating the clostridium butylicum treatment.
3.2 see that from result of treatment before the clostridium butylicum preparation for treating, severe diarrhoea is 100%, treatment back the 4th day, 10
5Cb-2 group curative ratio is 30%, 10
6Cb-2 group curative ratio is 40%; Treatment back the 6th day, 10
5Cb-2 group curative ratio is 70%, 10
6Cb-2 group curative ratio is 80%; Treatment back the 8th day then curative ratio reaches 100%; 10
4Cb-2 group curative effect is better than natural recovering group.Can find out that clostridium butylicum has good result of treatment, 10 to diarrhea of mouse
5Cb-2 group, 10
6The Cb-2 group has fast and significantly effect to correcting intestine dysbacteriosis property diarrhoea, exists certain dependency with dosage.
3.3 see from the intestinal flora quantitative analysis; In the week after undressed two groups of control group modelings success, five kinds of normal intestinal microflora quantity are gone up gradually, explain that intestinal flora has the self-healing tendency; But still normal healthy mice intestinal microflora quantity levels is on the low side; Explain that alteration of intestinal flora is auxiliary in no extraneous factor, recover normally slower at short notice, will cause multiple influence to body like this.Five kinds of advantage physiology bacterium propagation levels are higher than control group after clostridium butylicum is treated, and explain that clostridium butylicum recovers intestinal physiology property bacterium quantity and weight of structure has rapidly and active influence.It should be noted that; The quantity increasing degree of the main dominant microflora bifid of enteron aisle bar, probiotic lactobacillus is higher than other bacterium; Adjust and replenish outside the normal microflora of enteron aisle road from the decapacitation of an aspect explanation clostridium butylicum; Can also with probiotics symbiosis in the enteron aisles such as bifidus bacillus and probiotic lactobacillus, and impel its growth and breeding.
In sum, clostridium butylicum of the present invention has following effect: the acidifying enteron aisle promotes the small intestine digestion and absorption function; Repair mucous membrane, keep the epithelial normal morphology of intestinal mucosa, raise immunity; To antimicrobial agent, as the energy derive of intestinal cell; Safeguard beneficial microbe colony in the gi tract.During application, can earlier the clostridium butylicum nutrient solution be processed pre-mixture, Preblend, and then mix the back with other component of mixed feed and use; Also can be directly and the mixed feed each component mix the back and use; Both can be applicable to feed factory, also can directly apply to plant.
It is shown in table 32 to add consumption:
The usage quantity explanation of table 32 clostridium butylicum
| Animal varieties and period | Recommend addition (%) |
| Piglet (2 weeks of back of wean～wean just) | 0.5 |
| Growing pig (body weight<=30kg) | 0.2～0.5 |
| Fryer (0～21 day) | 0.2～0.5 |
| Laying hen (brood time) | 0.2～0.5 |
Annotate: press clostridium butyricum active bacteria number >=100,000,000/gram, the kg/t mixed feed
The substitute that clostridium butylicum of the present invention can be used as Sodium propanecarboxylate is used for livestock industry and feed industry.
Claims (9)
1. a strain has the clostridium butylicum of treatment diarrhoea effect; It is characterized in that: this bacterial strain called after clostridium butylicum Clostridium butyricum Cb-2; Be preserved in Chinese typical culture collection center on November 09th, 2011, its deposit number is: CCTCC M 2011384.
2. the clostridium butylicum with the effect of treatment diarrhoea according to claim 1, it is characterized in that: be the Gram-positive genus bacillus of obligate anaerobic, its diameter is 0.6 μ m～1.2 μ m * 3.0 μ m～7.0 μ m; The blunt circle in two ends, middle portion slightly expands, and bacterium is direct rod shape or bending is arranged slightly; Single or paired, short chain, accidental have a thread thalline; Whole body flagellum can move; This bacterial strain can produce sour aerogenesis, and ability decomposing sucrose, glucose, fructose, SANMALT-S, melibiose, starch can not decompose melizitose, wood sugar; Can not make gelatine liquefication, reduction reindeer moss solidifies cow's milk; The cracked indigestion of grumeleuse, optimum growth temp are 37 ℃, and optimum growh pH value is 7.0.
3. the described application of clostridium butylicum in the preparation probiotics of claim 1 with the effect of treatment diarrhoea.
4. the described application of claim 1 with clostridium butylicum of treatment diarrhoea effect as the Sodium propanecarboxylate substitute.
5. the clostridium butylicum preparation with the effect of treatment diarrhoea is characterized in that: be to obtain through cultivating the described clostridium butylicum with the effect of treatment diarrhoea of claim 1.
6. the described preparation method with the clostridium butylicum preparation of treating the diarrhoea effect of claim 5 is characterized in that, concrete culturing step is following:
(i) bacterial classification: select clostridium butylicum Clostridium butyricum Cb-2 for use, CCTCC M 2011384;
(ii) laboratory culture: lyophilized powder bacterial classification one ring of above-mentioned clostridium butylicum is inoculated on the substratum, and paraffin Vaseline is sealed up in aseptic technique, and 35～42 ℃ of following anaerobism are cultivated 24～38h;
(iii) prepare liquid seeds: adopt the 5000mL triangular flask, substratum loading amount 1000mL/5000mL triangular flask, 115 ℃ of sterilization 20min, the inoculation of cooling back, inoculum size 3%～8%, under 35～42 ℃ of conditions, anaerobism is cultivated 14～38h, is liquid seeds;
(iv) seeding tank fermentation: adopt the 50L seeding tank, the substratum loading amount is 30L; 115～121 ℃ of the temperature that disappears in fact, time 15～30min; Treat to inoculate when the substratum temperature is reduced to 40 ℃, inoculum size 3%～8%, tank pressure 0.05MPa, under 35～42 ℃ of conditions, anaerobism is cultivated 12～38h, changes fermentor cultivation over to;
(v) fermentor cultivation: adopt the 0.5T fermentor tank, the substratum loading amount is 350L; 115～121 ℃ of the temperature that disappears in fact, time 15～30min treats to inoculate when the substratum temperature is reduced to 40 ℃, inoculum size 3%～8%, tank pressure 0.05MPa, anaerobism is cultivated 16～32h,
(vi) after the fermentation ends, immediately that fermented liquid is centrifugal and clean with clear water, 2～3 times postlyophilizations are pulverized so repeatedly, are bacterium powder finished product; Or: after the fermentation ends, spraying drying promptly gets bacterium powder finished product immediately;
Above-mentioned steps (ii) (iii), (iv) described in culture medium prescription be: glucose 10g/L, Tryptones 5g/L, yeast extract paste 3g/L, beef extract 5g/L, lime carbonate 10g/L, during use, regulate the 20min that sterilizes under ℃ condition of pH to 6.5～7.5,115; (culture medium prescription v) is: glucose 10g/L, peptone 20g/L, yeast extract paste 15g/L, lime carbonate 10g/L, sal epsom 0.2g/L, potassium hydrogenphosphate 2g/L, manganous sulfate 0.2g/L during use, regulates pH to 6.5～7.5 to step.
7. the described application of clostridium butylicum preparation in the preparation probiotics of claim 5 with the effect of treatment diarrhoea.
8. application according to claim 7; It is characterized in that: during application; The clostridium butylicum preparation is processed probiotics as effective constituent and W-Gum separately, or: clostridium butylicum preparation and other at least a bacteria preparation and W-Gum are mixed and made into compound micro-ecological preparation.
9. application according to claim 8 is characterized in that: said other bacteria preparation is that milk-acid bacteria, sporeformer are or/and the conventional bacteria preparation of bifidus bacillus.
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