CN1177636A - Bacterial stain of bacillus subtilis and application thereof - Google Patents
Bacterial stain of bacillus subtilis and application thereof Download PDFInfo
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- CN1177636A CN1177636A CN97117032A CN97117032A CN1177636A CN 1177636 A CN1177636 A CN 1177636A CN 97117032 A CN97117032 A CN 97117032A CN 97117032 A CN97117032 A CN 97117032A CN 1177636 A CN1177636 A CN 1177636A
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present invention relates to a microbial stain. Said invented strain Bscillus subtilis BS-3 is defined as Bacillus subtilis, and is preserved in the General Microbe Centre of Chinese Microbial Strain Preservation Management Committee, its preservation number is CGMC No. 0321. Said strain can be used for curing dyspepsia, acute and chronic enteritis and diarrhea due to flora imbalance, and possesses obvious antagonism for enterorrhagia colibacillus (EHE. C. 0157). Said invented strain can be made into live strain dry powder and capsule preparation, etc..
Description
The present invention relates to a kind of microorganism strains.
China begins the fifties such as the faecalis preparation lactasinum of production for treating enteron aisle maldigestion and enteritis and yeast tablet etc.Because a large amount of widespread uses of microbiotic cause a large amount of appearance of endurance strain and flora imbalance diarrhoea to increase, this moment, the clinician more paid close attention to bionomic control to the eighties.Though use the preparation that the non-sporeformer of human body such as lactic acid, bifidus bacterium, faecalis etc. make for many years certain curative effect is arranged,, troubling the development of biologics industry always because the bacterium surviving rate in such preparation is low, preservation period short, curative effect is also unsatisfactory.Seek microorganism strains and microorganism medicine thereof safe, effective, long preservative period like this, become the direction that each scientist seeks.
The purpose of this invention is to provide a kind of bacillus subtilis BS-3 bacterial strain and application thereof, can treat because of the maldigestion that various factors causes, acute and chronic enteritis and flora imbalance diarrhoea with the medicament that this bacterial strain is made.Especially more effective to the treatment of bleeding type coli-infection enteritis.
The thalli morphology of bacillus subtilis BS-3 bacterial strain of the present invention (Bacillus Subtilis BS-3), observe with microscopy: this bacterial strain is shaft-like, and straight, single arrangement seldom becomes chain.Gram-positive (G+) size is 0.6～0.7 * 1.5-2.0um, the adnation flagellum, and the central spore ovalize, sporangiocyst does not expand, no PHB particle in the spore.This bacterial strain respectively through Chinese Academy of Sciences's microbe research in one's power Nat'l Pharmaceutical ﹠ Biological Products Control Institute be accredited as subtilis (Bacillus subtilis).And on September 3rd, 97 in the preservation of the center of China Committee for Culture Collection of Microorganisms's common micro-organisms, be numbered CGMCC.NO.0321.
Bacillus subtilis BS-3 bacterial strain of the present invention (Bacillus Subtilis BS-3) all can be cultivated on common nutrient broth and ordinary broth agar plate, under 18 ℃ of-45 ℃ of culture temperature, all can grow, the suitableeest growth temperature is 30-37 ℃, culture condition: pH value is 5-8, and the suitableeest growth pH value is 6.5-7.4.This bacterial strain is cultivated on common nutrient broth and nutrient broth agar flat board, can produce stronger amylase, proteolytic enzyme, bacitracin class antimicrobial substance in culturing process, and has the ability that suppresses and kill the blood group colon bacillus 0157.
Bacillus subtilis BS-3 bacterial strain of the present invention (Bacillus Subtilis BS-3) is to adopt following method separation and Culture:
1, the source of bacterial strain: the applicant has bought two bags of south from the market imitates the fresh milk that the dairy plant produces, place 4-10 ℃ of refrigerator, find when test next day that wherein one bag of milk takes place rotten, be that milk solidifies and has whey liquid to separate out, through this rotten milk is carried out preliminary bacteriology direct smear dyeing microscopic examination, find to have a kind of Gram-positive bacillus.
2, separation and Culture: above-mentioned rotten milk is inoculated in ordinary broth or plain agar substratum, adopts conventional method to turn out bacillus subtilis BS-3 bacterial strain of the present invention (Bacillus SubtilisBS-3).
Bacillus subtilis BS-3 bacterial strain of the present invention (Bacillus Subtilis BS-3) can be used as the application of preparation treatment maldigestion, acute and chronic enteritis and flora imbalance diarrhoea medicament.Also can be used as Medicines and Health Product, veterinary drug and fodder additives.
Bacillus subtilis BS-3 bacterial strain of the present invention (Bacillus Subtilis BS-3) can be made into the viable capsule preparation, can treat the maldigestion that causes because of various factors in the human body, acute and chronic enteritis and flora imbalance diarrhoea.The weight ratio of each component is in this capsule preparations:
The former bacterium powder of bacillus subtilis BS-3 bacterial strain is 1.5-2.5 part (parts by weight)
Vehicle is 96-100 part (parts by weight),
Wherein the viable count in the former bacterium powder of bacillus subtilis BS-3 bacterial strain is 〉=200 hundred million viable bacteria/grams.
Described vehicle is lactose and Zulkovsky starch, and wherein the weight ratio of lactose and Zulkovsky starch is:
Lactose 9-11 part (parts by weight)
Zulkovsky starch 87-89 part (parts by weight)
Also have, described former bacterium powder is made by 1: 3 weight ratio mixing drying by wet thallus and Carbon Dioxide calcium.
Bacillus subtilis BS-3 bacterial strain of the present invention (Bacillus Subtilis BS-3) can be made into dry powder formulations, treatment human or animal's maldigestion, acute and chronic enteritis, illnesss such as diarrhoea.The weight ratio of each component is in this powder preparation:
The former bacterium powder of bacillus subtilis BS-3 bacterial strain is 1.5-2.5 part (parts by weight)
Vehicle is 96-100 part (parts by weight)
Wherein the viable count in the former bacterium powder of bacillus subtilis BS-3 bacterial strain is 〉=200 hundred million viable bacteria/grams.
Described vehicle is lactose and Zulkovsky starch, and wherein the weight ratio of lactose and Zulkovsky starch is:
Lactose 9-11 part (parts by weight)
Zulkovsky starch 87-89 part (parts by weight)
Also have, described former bacterium powder is made by 1: 3 weight ratio mixing drying by wet thallus and Carbon Dioxide calcium.
Bacillus subtilis BS-3 bacterial strain of the present invention (Bacillus Subtilis BS-3) externally has tangible antagonistic action to enterohemorrhagic Escherichia coli (EHEC0157) through testing.Promptly showed inhibitory or killing effect in 24-48 hour.The 96 hours inhibitory or killing effects that extend to along with incubation time after 48 hours significantly strengthen.Its experimentation is as follows:
1, material therefor
(1) intestinal bacteria bacterial classification: enterohemorrhagic Escherichia coli 0157 (EHEC 0157), bacterium number: 88-2364.Provide by Inst. of Epidemiology and Microbiology, Chinese Academy of Preventive Medicine (.
(2) subtilis viable capsule of the present invention: specification 0.25 gram/grain (subtilis viable bacteria 〉=1.0 * 10
8Individual/gram)
(3) substratum:
A, broth medium
B, intestinal bacteria selective medium (EMB) dehydrated medium: every bottle 250 gram.Provide by Nat'l Pharmaceutical ﹠ Biological Products Control Institute.
C, 7%NaCl nutrient agar
2 methods:
(1) pilot study determines the logarithmic growth curve of EHEC0157 bacterium.
(2) preparation of EHEC0157 bacterium liquid:
Inoculation EHEC0157 bacterium is in the Erlenmeyer flask of 30 milliliters broth culture, and 15 hours (be the logarithmic phase of this bacterium this moment) cultivated in 37 ℃ of constant temperature water bath vibrations.With every milliliter of suitable EHEC0157 bacterium number in the standard bacterial turbidity pipe turbidimetric assay nutrient solution.To converse in the original fluid dilution of EHECO157 bacterium number with meat soup then is adjusted into every milliliter and contains 1 * 10 approximately
8Individual bacterium.
(3) preparation of subtilis viable capsule suspension:
Between aseptic technique, take by weighing capsule bacterium powder 2.0 grams, with 18 milliliters of meat soups.(every milliliter contains subtilis 1 * 10 approximately behind the mixing
8Individual bacterium).
(4) experimental implementation step:
A, get two sterilization Erlenmeyer flasks, be respectively experiment bottle and control bottle.Add EHEC0157 bacterium liquid (1 * 10 in the experiment bottle
8Individual bacterium/milliliter) 9.0 milliliter.Add 9.0 milliliters of subtilis capsule suspensions (shaking up) again.Get 1.0ml behind the mixing, 10 times of serial dilutions to 10
-9, measure the subtilis number, as O hour subtilis viable bacteria radix, 37 ℃ of water-bath shaking culture.
Add EHEC0157 bacterium (1 * 10 in the control bottle
8Individual bacterium/milliliter) 9.0 milliliters, again with 9.0 milliliters of meat soups.Fully get 1.0 milliliters with 9.0 milliliters of meat soups behind the mixing, by 10 times of serial dilutions to 10
-9Each extent of dilution is respectively got 0.1 milliliter, and other goes on the EMB agar plate (double) after " L " rod is smeared, and puts in 37 ℃ of incubators and cultivates 24 hours, and counting is the large intestine colony number on the plate, with this as EHEC0157 basis number in O hour triangular flask.
B, bacterium number change to be observed
The bacterium number of EHEC0157 in respectively following different incubation times (0,24,48,72,96 hour) determination experiment group and the EHEC0157 control group.(CFU/ml)
3, experimental result
Table 1 subtilis active bacteria formulation in vitro the different incubation times of group as a result of antagonism EHEC0157 (hour) EHEC0157 bacterium number/ml (log
n)
There is significant difference live bacterial count (T check) P＜0.05 of 0 24 48 72 96 test group active bacteria formulation 8.3979 7.1760 7.2041 5.0000 4.7993+EHEC control groups, 8.3979 9.3979 9.1461 8.7634 8.5682 statistical analysis experimental group and control group
From table 1 result as can be seen 0 hour EHECO157 bacterium of test group to count logarithmic value be 8.3979, act on and promptly drop to 7.1760 after 24 hours, control group by 0 hour 8.3979, propagation is 9.3979.Effect to 96 hour, test group EHEC0157 bacterium number reduces to 4.7993, and control group only drops to 8.5682, there were significant differences in statistical study, illustrate that the subtilis active bacteria formulation promptly had antagonistic action external in 24 hours with the EHEC0157 effect, after 48 hours, this antagonistic action is further strengthened.
The made medicament of bacillus subtilis BS-3 bacterial strain of the present invention (Bacillus Subtilis BS-3) is treated the miniature pig of artificial experimental infection, and curative ratio is more than 80%, and is efficient 100%, and the treatment of natural occurrence piglet is efficient to reach 90%.Its experimentation is as follows:
1, material therefor:
(1) experimental infection miniature pig: attack in 20 miniature pigs of poison through toxigenicity intestinal bacteria (0149, K88), what diarrhoea took place in 48 hours has 18, the principal character of suffering from diarrhea of pigs: loose and watery stool, shapeless, minority is suffered from swine excrement and is water sample, and with bubble, cultivate through the ight soil sampling intestinal bacteria that suffer from pig to 18, suffer from pig for 18 and all isolate intestinal bacteria, and with the agglutination reaction that is positive of intestinal bacteria (0149) O antigens genotyping serum and K factor serum, prove that suffering from pig is intestinal bacteria (0149K88) strain infections.
(2) subtilis viable capsule: specification 0.25 gram/grain (containing viable bacteria 2.5 hundred million).
(3) chemical drug XIELITING: pulvis, 50 gram/bags, Taixing City, Jiangsu animal drug factory produces.
2, methods of treatment and dosage
Method: adopt bacterium powder or medicinal powder directly to irritate feeding through the oral cavity, control group only infects not administration to be observed synchronously.
Dosage: subtilis Capsules group: every each 0.5 gram bacterium powder (about 500,000,000 viable bacterias), every day 2 times, continuous irrigation was raised 3 days.
Chemical drug XIELITING group: every each 1.0 gram medicinal powder, every day 2 times, continuous irrigation was raised 3 days.
Curative effect index and criterion:
(1) cure: ight soil is shaped in the medication 3 days, food is normal, being full of animal spirits did not have recurrence in 7 days.
(2) effective: doing well,improving or alleviate in the medication 3 days, ight soil is by rare retrogradation.
(3) invalid: medication after 3 days symptom do not improve or increase the weight of or dead.
Observation period: the experimental observation treatment phase is 7 days.
3, experimental result
(1) subtilis viable capsule treatment experimental infection colibacillus miniature pig diarrhoea result
Suffer from pig for 18 artificial experimental infection intestinal bacteria (0149K88), through two kinds of different pharmacological agenies, found that: 6 of subtilis viable capsule group treatments, cure 5, effective 1, efficient is 100%, 6 of chemical drug XIELITING group treatments, cure 4, effective 1, efficient is 83.3%; Untreated control group takes a turn for the better 1 naturally, in all the other 5 sick pigs 1 death is arranged, and 1 symptom of diarrhea increases the weight of in addition, the results are shown in Table 2.
The treatment result group experimental animal number of table 2 Chinese Small-sized pig experimental infection coliform diarrhea is cured number significant figure total effective rate %
(head) (head) % cures+effective subtilis 65 (83.3) 1 (16.6) 100 active bacteria formulation XIELITING 64 (66.6) 1 (16.6) 83.3 control groups 60 (0.0) 1 (16.6) 16.6
(2) the subtilis capsule for treating miniature pig experimental infection diarrhoea effect of different viable bacteria amounts relatively.
The subtilis capsule for treating miniature pig experimental infection diarrhoea effect of the different viable bacteria amounts of table 3 relatively.Group experimental animal number is cured number % significant figure % total effective rate % subtilis 55 (100) 0 (0) 10010 * 10
8Subtilis 55 (100) 0 (0) 1005 * 10
8Subtilis 54 (80) 1 (20%) 1002.5 * 10
8
As can be seen from Table 3 subtilis viable bacteria amount be 1,000,000,000 and 500,000,000 o'clock curative ratios be 100%, the viable bacteria amount is 2.5 hundred million o'clock, curative ratio is 80%, total effective rate is 100%.
(3) behind intestinal bacteria (0149 K88ac) experimental infection, through the change of 7 days several main floras of miniature pig enteron aisle of subtilis capsule for treating.
7 days several main floras of enteron aisle of table 4 subtilis capsule for treating experimental infection miniature pig
Measurement result (bacterium of every gram muck is counted the log value)
Group bacterial count kind
8.02 7.92 7.91 3.15 normal group 7.91 8.05 8.38 are organized in the treatment of intestinal bacteria faecalis Bacterium lacticum subtilis
Table 4 result shows that suffering from pig coliform count in subtilis viable capsule healing detection in 7 days ight soil is 8.02, and faecalis is 7.92, subtilis 3.15, and normal health miniature pig intestinal bacteria are 7.19.
The made medicament of bacillus subtilis BS-3 bacterial strain of the present invention (Bacillus subtilis BS-3) has the good preventing effect to newborn piglet diarrhoea, and its protection ratio is up to 90%, i.e. prevention effectively.Its experimentation is as follows:
Materials and methods
1, material therefor:
(1) pregnancy waits to produce the miniature pig sow: 2, China Agricultural University's miniature pig breeding field provides.
(2) subtilis viable capsule: specification 0.25 gram/grain (containing viable bacteria 2.5 hundred million).
2, method
Sow to be produced is divided into experimental group and control group, irritates immediately in 3-5 hour in experiment sows farrowing pig birth back and raise subtilis capsule bacterium powder, every day 2 times, each 1 (0.25 gram), for three days on end, not administration of control group is observed synchronously.
Observation index: routine number takes place in grice diarrhoea
Observation period: administration was observed to the 7th day in 3 days.
3, experimental result
Found that of using Bacillus subtilis viable capsule prevention newborn piglet diarrhea: experimental group has 1 example morbidity, and sickness rate is 10%, and death toll is 0; protection ratio is 90%, and non-prevention control group has 6 example morbidities, and sickness rate is 60%; wherein dead 3 examples, mortality ratio is 30%, the results are shown in Table 5.
Table 5 subtilis viable capsule prevention newborn piglet diarrhea result.A group laboratory animal number case load 5% dead routine number % protect routine number % experimental group 10 1 (10) 0 (0) 90 control groups 10 6 (60) 3 (30)-
Bacillus subtilis BS-3 bacterial strain of the present invention (Bacillus subtilis BS-3) belongs to a kind of aerobic spore-forming bacteria, to human body and the useful nontoxic pair of effect of animal.The made preparation of this bacillus subtilis BS-3 bacterial strain is examined and determine through Nat'l Pharmaceutical ﹠ Biological Products Control Institute: do not find the abnormal response and the pathological change that are caused to animal by said preparation.Its probation redport is as follows:
Inspection product title: subtilis active bacteria formulation.
Specification: 0.25 gram/grain
Subacute toxicity test and overview: SPF level mouse is pressed 2.500g/kg body weight dosage, gavages subtilis viable bacteria continuous irrigation stomach 10 days every day.Experimental mice is not seen toxicity symptom and death.The mouse activity freely, hair color gloss, appetite is normal, mouthful the eye no secretory product, no symptoms of emesis, ight soil no abnormality seen, weight increase.
Blood test: red thin spore, white thin spore and hemoglobinometry result and control group mice no significant difference in the experiment mice blood.
Cut open inspection and pathological examination: experimental group and control group mice are through dissecting visual inspection, and the heart, liver, spleen, stomach, lung, kidney, intestines no abnormality seen are observed above-mentioned organ or tissue under the histopathology section mirror and also do not seen the pathology that causes because of said preparation.
Conclusion: do not find the abnormal response and the pathological change that cause to animal by said preparation.
Embodiment
(1) bacillus subtilis BS-3 bacterial strain (Bacillus subtilis BS-3) separates.
1, the source of sample: sample is aforementioned rotten bag milk.
2, isolation cultivation method:
(1) separatory substratum: adopt the ordinary broth liquid nutrient medium, pH value is 7.0-7.2; Or common nutrient broth agar substratum, pH value is 7.0-7.2, through 15 pounds of pressure, sterilizes and makes in 20 minutes.
(2) method:
Adopt the ordinary broth liquid culture method: the 0.1ml of the above-mentioned rotten bag milk of aseptic absorption, be inoculated in the ordinary broth and manage two, under 37 ℃ of conditions, cultivated 18-24 hour, observe the variation in the culture tube.
Adopt common nutrient broth agar medium therapy: with above-mentioned rotten bag milk one ring of the aseptic picking of platinum loop, line two separation of ordinary broth agar plate, under 37 ℃ of conditions, cultivated 18-24 hour, observe bacterial growth situation in the plate.
(3) separating resulting
In the ordinary broth culture tube, nutrient solution is little muddiness, and the culture tube liquid level forms complete one deck mycoderm, i.e. the microbial culture growth that is positive, and smear culture gramstaining is G
+Bacillus, the minority thalline has gemma, is positioned at thalline central authorities, ovalize.Be the bacillus subtilis BS-3 bacterial strain through calibrating.
On nutrient agar plate, it is positive that two flat boards are all cultivated, and the bacterium colony circle is irregular, and creamy is opaque, and colony diameter is 2-4mm, is G through gramstaining
+Bacillus, size are about in 0.6-0.7 * 1.5-2.0 hole um gemma living, are the bacillus subtilis BS-3 bacterial strain through calibrating.
(2) produce actication of culture and calibrating
With above-mentioned bacillus subtilis BS-3 inoculation in the common nutrient broth nutrient solution of 10ml in the pipe, under 35 ℃ of conditions, cultivated 18-24 hour, choose a ring line transferred species in nutrient agar plate with platinum loop then, under 35 ℃ of conditions, cultivated 18-24 hour, choose colonies typical, carry out the calibrating of morphology and characteristic, and the cultivation of going down to posterity.
(3) preparation of seed liquor
With the pure growth of assay approval, aseptic absorption 0.5ml is inoculated in the 10ml nutrient broth culturing bottle, cultivates 18-24 hour under 35 ℃ of conditions, carries out pure bacterium and bacterium shape flag check.
(4) produce substratum
Extractum carnis 5 gram, peptone 10 grams, glucose 10 grams, sodium-chlor 5 grams are added in the distilled water to 1000 milliliters, and after the said mixture heating for dissolving, accents pH value is 7.0-7.2, sterilizes 20 minutes under 15 pounds of pressure.
(5) inoculation and cultivation
Aforementioned seed liquor is seeded in the fermentor tank that above-mentioned nutrient solution is housed in 1-3% ratio (volume ratio) behind assay approval, cultivates 48-72 hour under 35 ℃ of conditions, checks the ratio that gemma forms.
(6) collect thalline
To cultivate overdue culture, the spore rate of seeking survival reaches more than 90%, through pure bacterium test, confirms no living contaminants again.Divide rotating speed centrifugal 20 minutes with above-mentioned culture with 4000rpm/, abandon supernatant, collect wet thallus.
(7) the former bacterium powder of preparation
With above-mentioned wet thallus and Carbon Dioxide calcium in 1: 3 ratio (weight ratio) be mixed into wet bacterium powder, dry under 70-80 ℃ of condition, and to grind to form dry powder be former bacterium powder.The viable count of this former bacterium powder is controlled to be 〉=20,000,000,000 viable bacteria/grams.
(8) breading
Above-mentioned former bacterium powder and sterilization are sieved the lactose, Zulkovsky starch of (sieve aperture is 80 orders) by following weight ratio mixing: 2 parts in former bacterium powder, 10 parts of lactose, 88 parts of Zulkovsky starches, and fully stir evenly, the viable count of wherein above-mentioned bacterium powder mixture be controlled to be 〉=and 1 * 10
8Individual viable bacteria/gram.And be packaged into bag by 0.25 gram/bag or 50 gram/bags, make the viable bacteria dry powder formulations of bacillus subtilis BS-3 bacterial strain.
(9) canned capsule
Above-mentioned bacterium powder mixture also can be made the viable capsule preparation, the bacterium powder mixture is placed in the capsule canning machine canned, and pressing plate, packing are made the viable capsule preparation of bacillus subtilis BS-3 bacterial strain, and wherein every capsules is 0.25 gram/grain.
In sum, bacillus subtilis BS-3 bacterial strain provided by the present invention can be made into the preparation of viable bacteria dry powder formulations, viable capsule preparation or other form, can treat the maldigestion, acute and chronic enteritis and the flora imbalance diarrhoea that cause because of various factors of people, animal.Especially more effective to the treatment of Enterohemorrhagic E.coli infective enteritis.
Claims (5)
1, a kind of bacillus subtilis BS-3 bacterial strain (Bacillus Subtilis BS-3), its deposit number at China Committee for Culture Collection of Microorganisms common micro-organisms center is CGMCC.NO.0321.
2, the described bacillus subtilis BS-3 bacterial strain of claim 1 (Bacillus Subtilis BS-3) is as the application of preparation treatment maldigestion, acute and chronic enteritis and flora imbalance diarrhoea medicament.
3, the application of bacillus subtilis BS-3 bacterial strain according to claim 2 (Bacillus SubtilisBS-3), it is characterized in that: this medicament is a capsule preparations, and the weight ratio of each component is in this capsule preparations:
The former bacterium powder of bacillus subtilis BS-3 bacterial strain is 1.5-2.5 part (parts by weight)
Vehicle is 96-100 part (parts by weight),
Wherein the viable count in the former bacterium powder of bacillus subtilis BS-3 bacterial strain is 〉=200 hundred million viable bacteria/grams.
4, the application of bacillus subtilis BS-3 bacterial strain according to claim 2 (Bacillus SubtilisBS-3), it is characterized in that: this medicament is a powder preparation, and the weight ratio of each component is in this powder preparation:
The former bacterium powder of bacillus subtilis BS-3 bacterial strain is 1.5-2.5 part (parts by weight)
Vehicle is 96-100 part (parts by weight)
Wherein the viable count in the former bacterium powder of bacillus subtilis BS-3 bacterial strain is 〉=200 hundred million viable bacteria/grams.
5, according to the application of claim 3 or 4 described bacillus subtilis BS-3 bacterial strains (BacillusSubtilis BS-3), it is characterized in that: described vehicle is lactose and Zulkovsky starch, and wherein the weight ratio of lactose and Zulkovsky starch is:
Lactose 9-11 part (parts by weight)
Zulkovsky starch 87-89 part (parts by weight)
Also have, described former bacterium powder is made by 1: 3 weight ratio mixing drying by wet thallus and Carbon Dioxide calcium.
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