CN104762295A - Preparation method of acid-resistant salt-resistant facultative anaerobic bacillus - Google Patents
Preparation method of acid-resistant salt-resistant facultative anaerobic bacillus Download PDFInfo
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Abstract
A preparation method of acid-resistant salt-resistant facultative anaerobic bacillus includes preparation of protoplasts, wherein the protoplast of lactobacillus is prepared by enzymolysis to lactobacillus cells being activation-cultivated for 12 h with addition of lysozyme being 3.5 mg/mL in concentration under the pH of 7.0 at 37 DEG C for 45 min; and the protoplast of the bacillus is prepared by enzymolysis to bacillus cells being activation-cultivated for 6 h with addition of lysozyme being 2.0 mg/mL in concentration under the pH of 7.0 at 37 DEG C for 30 min. The protoplasts are respectively inactivated and the inactivated protoplasts of the lactobacillus and the bacillus are blended together. The method is simple in operation and is high in successful rate. The blended bacterial strain has an acid-resistant salt-resistant facultative anaerobic characteristic and is more excellent in use performances.
Description
Technical field
The present invention relates to a kind of Protoplast fusion technology, be specifically related to the preparation method of a kind of acidproof salt tolerant amphimicrobian genus bacillus.
Background technology
Current, the cultivation industry of China experiences transition and upgrade, and the agricultural breeding pattern that traditional middle and small scale is raised is progressively to the industrial aquaculture model transition of intensive mass-producing.Along with the transformation of aquaculture model, expose the key issue that some are urgently to be resolved hurrily.The feeding environment opposing seal of intensive culture, cultivation density is large, and animal immune ability and anti-disease reduced capability, to this, in cultivation, antibiotic use just highlights.In feeding process, microbiotic adds in a large number and result in the normal microflora imbalance of animal gastrointestinal tract, autogenous infection or superinfection; produce the drawbacks such as Resistant strain; antibiotic remains particularly in product can affect the health of the mankind by food chain, caused the problems such as a series of public safety problem, environmental protection problem, animal products quality.Due to antibiotic drug residue problem, China's animal cultivation products export is greatly affected.Along with the enforcement of socioeconomic development and national pollution-free food action plan, people's Consciousness of food security strengthens increasingly, more and more pays close attention to healthy and quality product, and the consumption of pollution-free food, green food worldwide becomes trend.Breeding production will experiencing by the huge historical change of scalar type to mass type.
Research both domestic and external shows that milk-acid bacteria etc. can produce lactic acid, acetic acid and secondary metabolite, regulates gastrointestinal bacterial flora balance in animal body, beneficial microorganism quantity is increased, enhances animal non-specific immune systems function; Bacillus micro-organism is owing to having stronger adverse-resistant characteristic, can secrete the digestive ferments such as highly active lipase, proteolytic enzyme and amylase, and above characteristic can improve the transformation efficiency of feed and diseases prevention, promotion animal digesting and assimilating and promoting growth performance and the probiotic effects preferably of animal nutritive substance.Therefore, the microorganism fodder being representative with milk-acid bacteria, genus bacillus etc. obtains a large amount of application in aquaculture, becomes the important impetus taking aquaculture transition and upgrade to.
At present, the microorganism used in cultivation field mainly comprises two types, and a class is the milk-acid bacteria of anaerobic type, and another kind of is the genus bacillus of aerobic.This two quasi-microorganism respectively has its relative merits in result of use: milk-acid bacteria can in the anoxic link of animal intestinal normal growth and play probiotic effects, but the heat-resisting effect of this bacterium is poor, acidproof salt resistance ability is also limited; Genus bacillus has the ability of secreting multiple digestive ferment, and has stronger resistance, but can not grow in anaerobism link, therefore limited to the probiotic effects of animal intestinal.
Therefore, be badly in need of a kind of novel microorganism fodder integrating milk-acid bacteria and the prebiotic advantage of genus bacillus of invention and maximize to make its probiotic effects, solve microorganism fodder in use Problems existing.
Summary of the invention
The present invention is directed to occurring in nature and there is amphimicrobian characteristic and to be applicable to the microbial population of animal rearing less, if be directly separated the feeding genus bacillus with amphimicrobian characteristic from physical environment, the defect that success ratio is extremely low, object is the preparation method providing a kind of acidproof salt tolerant amphimicrobian genus bacillus.
The present invention realizes especially by following technical scheme:
A preparation method for acidproof salt tolerant amphimicrobian genus bacillus, specifically comprises the following steps:
1) preparation of protoplastis: lactobacillus protoplast formation is: the lactobacillus cell getting activation culture 12h, adds the N,O-Diacetylmuramidase that concentration is 3.5mg/mL, enzymolysis 45min under the condition of pH7.0, temperature 37 DEG C; Protoplasts From Bacillus Subtilis And Bacillus Thuringiensis preparation condition is: the bacillus cell getting activation culture 6h, adds the N,O-Diacetylmuramidase that concentration is 2.0mg/mL, enzymolysis 30min under the condition of pH7.0, temperature 37 DEG C.
2) lactobacillus protoplastis and Protoplasts From Bacillus Subtilis And Bacillus Thuringiensis are carried out deactivation respectively;
3) lactobacillus after deactivation and the fusion of Protoplasts From Bacillus Subtilis And Bacillus Thuringiensis.
Described lactobacillus is screen the acidproof salt tolerant lactobacillus obtained; Described genus bacillus is screen the acidproof salt tolerant genus bacillus of product proteolytic enzyme obtained.
The activation medium that described activation culture adopts is:
Lactobacillus: egg extractum carnis 10.0g, glucose 20.0g, white peptone 10.0g, yeast extract paste 5.0g, tween-80 1.0mL, K
2pHO
42.0g, sodium acetate 5.0g, Triammonium citrate 2.0g, MgSO
47H
2o 0.2g, MnSO
4h
2o 0.05g, distilled water is settled to 1L, pH 6.5,121 DEG C of sterilizing 20min.
Genus bacillus: extractum carnis 5.0g, glucose 10.0g, peptone 10.0g, yeast extract paste 5.0g, NaCl 5.0g, distilled water is settled to 1L, pH 7.2,121 DEG C of sterilizing 20min.
Described lytic enzyme SMM prepares, then is the sterile filters filtration sterilization of 0.22 μm with aperture, is distributed into the aliquot that single uses ,-20 DEG C of preservations.
Described deactivation condition is: lactobacillus protoplastis: 65 DEG C of thermal treatment 120min; Protoplasts From Bacillus Subtilis And Bacillus Thuringiensis: 15min is irradiated at 30W ultraviolet lamp 20cm place.
Described fusion conditions is 40%pH7.0 fusogen for selecting concentration, under 35 ~ 40 DEG C of conditions, merge 20 ~ 30min.
The preparation method of described fusogen is: 40g PEG, KH
2pO
40.27g, CaCl
20.11g, with the PBS damping fluid constant volume of pH7.0 to 100mL, 121 DEG C of sterilizing 20min, for subsequent use.
Beneficial effect of the present invention is: adopt the method for protoplast fusion artificially to build the amphimicrobian genus bacillus with acidproof salt-tolerant trait, the probability of success of this technological line far above random from environment separation screening there is the technological method of the amphimicrobian genus bacillus of acidproof salt-tolerant trait; Use protoplast fusion method to build and the fusant bacterial strain screening acquisition due to the excellent hereditary property (as acidproof salt tolerant, sporulation, anaerobic growth etc.) gathering former parent strain, there is more superior use properties.
Accompanying drawing explanation
Fig. 1 is the result that milk-acid bacteria tolerates survival rate under condition of different pH;
Fig. 2 is the cholate resistance characteristics of milk-acid bacteria;
The cholate resistance characteristics of genus bacillus when Fig. 3 is gallbladder salinity 0.2%;
The cholate resistance characteristics of genus bacillus when Fig. 4 is gallbladder salinity 0.5%;
Fig. 5 is the result that genus bacillus tolerates survival rate under condition of different pH;
Fig. 6 is the growth curve of lactobacillus A2 and genus bacillus E2
Fig. 7 is lysozyme concentration on the impact of lactobacillus A2 protoplast formation rate and regeneration rate;
Fig. 8 is lysozyme concentration on the impact of genus bacillus E2 protoplast formation rate and regeneration rate;
Fig. 9 is hydrolysis temperature on the impact of lactobacillus A2 protoplast formation rate and regeneration rate;
Figure 10 is hydrolysis temperature on the impact of genus bacillus E2 protoplast formation rate and regeneration rate;
Figure 11 is enzymolysis time on the impact of lactobacillus A2 protoplast formation rate and regeneration rate;
Figure 12 is enzymolysis time on the impact of genus bacillus E2 protoplast formation rate and regeneration rate
Figure 13 is the hot inactivation curves of lactobacillus A2 protoplasma;
Figure 14 is genus bacillus E2 protoplastis ultraviolet inactivation curve;
Figure 15 is the acidproof salt-tolerant trait analysis of fusant bacterial strain.
Embodiment
Below in conjunction with embodiment, the present invention is described further, the following stated, only to preferred embodiment of the present invention, not do other forms of restriction to the present invention, any those skilled in the art may utilize the technology contents of above-mentioned announcement to be changed to the Equivalent embodiments of equal change.Everyly do not depart from the present invention program's content, any simple modification done following examples according to technical spirit of the present invention or equivalent variations, all drop in protection scope of the present invention.
The separation screening of the acidproof salt tolerant feeding lactobacillus of embodiment 1
1.1 strain separating samples: the fermented feed of different sources, greenfeed, milk-product and leavened food
1.2 substratum
1) MRS liquid nutrient medium: peptone 1%, extractum carnis 1%, yeast extract paste 0.5%, glucose 2%, anhydrous sodium acetate 0.5%, tween 80 0.1%, dibasic ammonium citrate 0.2%, dipotassium hydrogen phosphate 0.2%, magnesium sulfate 0.00058%, manganous sulfate 0.00025%, pH 6.2-6.8,121 DEG C of sterilizing 20min;
2) MRS solid medium: add about 2% agar on liquid medium within basis, 121 DEG C of sterilizing 20min;
3) MRS solid medium calciferous: add 3% calcium carbonate on solid MRS medium base, 121 DEG C of sterilizing 20min;
4) semi-solid MRS substratum: the agar adding 3-6% on liquid medium within basis, 121 DEG C of sterilizing 20min;
5) tomato liquid nutrient medium: tomato juice 5%, yeast powder 0.5%, extractum carnis 1%, lactose 2%, glucose 0.2%, dipotassium hydrogen phosphate 0.2%, tween 80 0.1%, sodium acetate 0.5%, pH value 6.4,121 DEG C of sterilizing 20min;
6) the PY basic medium improved: peptone 0.5%, Tryptones 0.5%, yeast powder 1%, Calcium Chloride Powder Anhydrous 0.2%, magnesium sulfate heptahydrate 0.48%, dipotassium hydrogen phosphate 1%, potassium primary phosphate 1%, sodium bicarbonate 10%, sodium-chlor 2%, 121 DEG C of sterilizing 20min;
7) gelatin-based basal culture medium: gelatin 80%, peptone 1%, yeast extract paste 1%, glucose 0.1%, Calcium Chloride Powder Anhydrous 0.2%, magnesium sulfate heptahydrate 0.48%, dipotassium hydrogen phosphate 1%, potassium primary phosphate 1%, sodium bicarbonate 10%, sodium-chlor 2%, 115 DEG C of sterilizing 25min.
The enrichment culture of 1.3 milk-acid bacterias
Added in sterilized MRS liquid nutrient medium by often kind of sample according to 2% inoculum size, in the constant incubator being placed on 37 DEG C, quiet putting cultivates 24h, with enrichment milk-acid bacteria.
The separation and purification of 1.4 milk-acid bacterias and preservation
Get above 10mL bacterium liquid in 90mL stroke-physiological saline solution, shake up, then carry out 10 times of gradient dilutions with stroke-physiological saline solution 9mL.Get gradient 10-3 respectively, the 0.1mL bacterium liquid of 10-4,10-5,10-6,10-7,10-8 be put into sterilizing simultaneous temperature drop to 50-55 DEG C dress 15ml contain in the triangular flask of calcium carbonate MRS solid medium, fully shake up, pour into rapidly again in the culture dish of sterilizing, by the time the substratum inside culture dish solidifies, is placed upside down in the constant incubator of 37 DEG C and carries out cultivation 48h.Get the above bacterium colony having molten calcium circle, again carry out dilution purifying, the constant incubator being put in 37 DEG C carries out 48h cultivation.Repetition like this, basically identical to growing colonial morphology.By there being the molten calcium circle bacterium colony that colonial morphology is basically identical above simultaneously, lining on MRS solid slant culture base, be put in 37 DEG C of constant incubators and cultivate 24h, afterwards in 4 DEG C of preservations.
The qualification of 1.5 separating lactic acid bacterium
1.5.1 Morphological Identification
Observe separation screening bacterium colony out, carry out gramstaining observation, record thalli morphology.Result is as shown in table 1,2.
The colony characteristics result of table 1 isolated strains
Table 2 isolated strains morphological features result
1.5.2 the Physiology and biochemistry qualification of milk-acid bacteria
1) mobility test: the substratum using MRS, add the agar of 3-6% wherein, the bacterium screened with staight needle percutaneous puncture-inoculation, in semisolid medium, is put in 37 DEG C of constant incubators and carries out cultivation 24h, and whether the edge observed on the puncture line of inoculation is clear.
2) Catalase determination: by the microbionation that screens on solid MRS slant medium, cultivation 24h is carried out in 37 DEG C of constant incubators, get a ring and be grown on bacterium on solid MRS slant medium, be applied on clean slide glass, then add 3% superoxol thereon, observe and whether have bubble to produce.
3) KOH test: get a ring and be grown on bacterium on solid MRS slant medium, be applied on clean slide glass, then a 5%KOH solution is added thereon on clean slide glass, mixed with the transfering loop of sterilizing and stirred evenly, whether observe whether liquid retrogradation in short period of time, picking up transfering loop has wire drawing phenomenon.
4) gelatin liquification test: get a ring and be grown on bacterium on solid MRS slant medium, be inoculated in 37 DEG C of cultivations after gelatin-based basal culture medium, subzero treatment during observation, not connect the test tube of bacterium for control group, observes test tube and whether changes liquefaction.
5) carbohydrate fermentation produces the test of sour aerogenesis: in the PY basic medium of improvement, add 1% glucose, lactose, starch, sucrose respectively, packing test tube, be highly 4-5cm, inoculate after sterilizing, cultivate 48h for 37 DEG C, get the PY basic medium not adding carbohydrate to compare, use the instruction of BTB-MR reagent to produce sour degree, whether the Du Shi tubule put upside down is put in observation into simultaneously has bubble to produce.
6) detection of fermentation and acid: with glucose for fermentation test is carried out in sugared source in PY substratum, gets the kind of acid in the centrifugal rear liquid-mass spectrometric detection supernatant liquor of fermented liquid, to determine whether as lactic acid producing bacteria after 48h
Further biochemical identification shows (table 3 shown in): this 3 strain bacterium all can utilize glucose, lactose, starch and sucrose fermentation and acid, not aerogenesis, all not liquefy gelatin, non-decomposing H
2o
2, liquid not retrogradation, does not have wire drawing phenomenon, does not have mobility yet.Comprehensive morphological, biochemical test qualification result and milk-acid bacteria identification handbook, can infer that A1 ~ A24 all belongs to lactobacillus genus lactic acid bacteria.
Table 3 is separated the Physiology and biochemistry qualification result of bacterial classification
1.6 screenings with acid-resistant property milk-acid bacteria
The milk-acid bacteria of having identified picking from slant medium is placed in the MRS liquid nutrient medium of normal ph, is put in static gas wave refrigerator 24h in 37 DEG C of constant incubators.Afterwards according to 2% inoculum size, getting above bacterium liquid, to be placed on the pH value regulated with acidometer be in the MRS liquid nutrient medium of 2.0,3.0,4.0 respectively, and be placed in 37 DEG C of constant incubator quiescent culture 3h.Get bacterium liquid to dilute, live bacterial count, measure Viable detection, filter out the milk-acid bacteria with stronger acid-resistant property thus.Result as shown in Figure 1.
Acid resistance test result shows, in separating obtained 24 lactobacillus strains, have 11 strains to have relatively strong acid-resistant property, and after they retain 3h in the environment of pH2.0, the survival rate of bacterial strain is all more than 10%.
1.7 screenings with cholate resistance characteristics milk-acid bacteria
The above-mentioned milk-acid bacteria with stronger acid-resistant property filtered out picking from slant medium is out placed in the MRS liquid nutrient medium of normal condition, is put in static gas wave refrigerator 24h in 37 DEG C of constant incubators.Afterwards according to 2% inoculum size, get above bacterium liquid and be placed on respectively and added in the liquid MRS substratum of cholate 0.1%, 0.3%, 0.5%, 2%, and be placed in 37 DEG C of constant incubator static gas wave refrigerator 3h.Get bacterium liquid to dilute, live bacterial count, measure Viable detection, filter out the milk-acid bacteria with stronger salt-tolerant trait thus.Result as shown in Figure 2.
Salt tolerance result shows, in the above-mentioned acidproof milk-acid bacteria of 11 strain filtered out, have 4 strains to have relatively strong salt resistance ability, retain 3h in the environment of gallbladder salinity 0.2% after, the survival rate of bacterial strain is all more than 4%.Comparatively speaking, the cholate tolerance of bacterial strain 2 is the strongest, and its survival rate can reach about 11%.So far, obtain by experiment sieving the milk-acid bacteria A2 bacterial strain that a strain has stronger acidproof salt-tolerant trait.
The qualification of 1.8 milk-acid bacteria A2 bacterial strains
To be separated and the direct censorship of milk-acid bacteria A2 bacterial strain after purifying, and adopt 16SrDNA authentication method to carry out preliminary evaluation to this bacterial strain.Result shows, and milk-acid bacteria A2 bacterial strain is initially identified as Lactobacterium acidophilum.
The separation screening of the feeding genus bacillus of the acidproof salt tolerant of embodiment 2
1) strain separating sample: sewage, fresh animal enteron aisle, beans leavened food etc. near the fermented feed of different sources, greenfeed, dining room.
2) substratum
Liquid enrichment medium (%): yeast powder 0.5, peptone 1, NaCl 0.5, agar 1.5 ~ 2, pH7.0 ~ 7.2,121 DEG C of sterilizing 20min;
Primary dcreening operation substratum (%): skim-milk 4, sucrose 2, agar 1.5 ~ 2, natural pH, 105 DEG C of sterilizing 20min.
Seed culture medium (%): yeast powder 0.5, peptone 1, NaCl 0.5, pH7.0 ~ 7.2,121 DEG C of sterilizing 20min;
Multiple sieve and fermention medium (%): sucrose 4, peptone 2, wheat bran 3, dipotassium hydrogen phosphate 0.3, calcium carbonate 0.3, tween 80 0.1, pH 7.5 ~ 8.0,121 DEG C of sterilizing 20min;
Seed culture medium (%): yeast powder 0.5, peptone 1, NaCl 0.5, pH7.0 ~ 7.2,121 DEG C of sterilizing 20min;
Slant medium (%): yeast powder 0.5, peptone 1, NaCl 0.5, agar 1.5 ~ 2, pH7.0 ~ 7.2,121 DEG C of sterilizing 20min;
3) enrichment culture of genus bacillus
Get the sample 10g (or 10ml) after smashing to pieces, proceed in the triangular flask filling 90ml sterilized water and fully shake, then respectively get 2ml inoculation of suspension liquid in the triangular flask that 50mL enrichment medium is housed, sealing is placed on 37 DEG C, 24h is cultivated by the shaking table of 50r/min.
4) primary dcreening operation of proteolytic enzyme genus bacillus is produced
After enrichment culture liquid is hatched 20min in 85 DEG C of water-baths, by 10 times of gradient series dilutions, from gradient diluent, draw 0.2mL be respectively spread evenly across on primary dcreening operation substratum, be then placed in the lower 37 DEG C of constant temperature culture 24h of anaerobic environment.Select hydrolytic circle and the larger bacterium colony of colony diameter ratio from primary dcreening operation flat board, transferred 37 DEG C of constant temperature culture 24h on slant medium, test tube slant is placed in 4 DEG C of Refrigerator stores.
5) the multiple sieve (enzymatic production method) of proteolytic enzyme genus bacillus is produced
The bacterial strain that picking 2 ring primary dcreening operation obtains respectively is connected to the shaking table concussion being placed in 37 DEG C of 200r/min in 50mL seed culture medium and cultivates 24h; Then drawing 2mL seed culture fluid transfers in 50mL fermention medium, and 72h is cultivated in the shaking table concussion of 37 DEG C of 200r/min, after fermentation liquor is centrifugal, gets supernatant liquor 1mL and adopts Folin-phenol method to survey proteinase activity.Filter out genus bacillus 26 strain having and produce proteolytic enzyme ability thus.Result is as shown in table 4.
The shake flask fermentation that table 4 produces proteolytic enzyme genus bacillus sieves again
6) there is the screening of the Bacillus strain of cholate resistance characteristics
Product proteolytic enzyme Bacillus strain each picking one ring is inoculated in respectively 50mL seed culture medium and is placed in 37 DEG C of 200r/min shaking table activation 24h, draw respectively 2mL seed culture fluid transfer in gallbladder salinity be 0.2%, 0.5% and 1.0% 50mL seed culture medium, be placed in after 3h cultivated by 37 DEG C of 200r/min shaking tables, get bacterium liquid to dilute, live bacterial count, measure Viable detection, filter out the genus bacillus with stronger cholate resistance characteristics thus.Result as shown in Figure 3 and Figure 4.
Cholate resistance test result shows, genus bacillus generally has stronger cholate tolerance.Above-mentioned filter out 26 strains produce proteolytic enzyme genus bacillus except indivedual bacterial strain except (bacterial strain unlisted in figure) all show very strong cholate tolerance, even in the environment of gallbladder salinity 0.5% 37 DEG C reservation 3h still have more than 40% survival rate.
7) there is the screening of the Bacillus strain of acid-resistant property
21 strains of above-mentioned screening gained had the genus bacillus slant activation of producing proteolytic enzyme and stronger cholate resistance characteristics, picking one ring inclined-plane bacterial strain is connected to the shaking table being placed in 37 DEG C of 200r/min in 50mL seed culture medium and cultivates activation 24h respectively, then draw 2mL seed culture fluid transfer respectively in pH be 2.0,3.0 and 4.0 50mL seed culture medium, be placed in after 3h cultivated by 37 DEG C of 200r/min shaking tables, get bacterium liquid to dilute, live bacterial count, measure Viable detection, filter out the Bacillus strain with stronger acid-resistant property thus.Result as shown in Figure 5.
Acid resistance test result shows, have in the Bacillus strain of salt-tolerant trait at above-mentioned separating obtained 21, only have 11 strains to have relatively strong acid-resistant property, retain 3h in the environment of pH3.0 after, bacterial strain still has the survival rate of about 10%.In these bacterial strains, the genus bacillus E2 deriving from ooze has the strongest acid-fast ability, also has the survival rate of 20% after it retains 3h in the environment of pH2.0.So far, separation obtains the genus bacillus E2 bacterial strain with stronger acidproof salt-tolerant trait.
8) qualification of genus bacillus E2
To be separated and the direct censorship of genus bacillus E2 bacterial strain after purifying, and adopt 16SrDNA authentication method to carry out preliminary evaluation to this bacterial strain.Result shows, and genus bacillus E2 bacterial strain is initially identified as Bacillus licheniformis.
Embodiment 3
1) parent strain is merged
Acidproof salt tolerant lactobacillus A2; Produce proteolytic enzyme acidproof salt tolerant genus bacillus E2.
2) substratum and reagent
Lactobacillus A2 activation medium: egg extractum carnis 10.0g, glucose 20.0g, white peptone 10.0g, yeast extract paste 5.0g, tween-80 1.0mL, K
2pHO
42.0g, sodium acetate 5.0g, Triammonium citrate 2.0g, MgSO
47H
2o 0.2g, MnSO
4h
2o 0.05g, distilled water is settled to 1L, pH 6.5,121 DEG C of sterilizing 20min.
Genus bacillus E2 activation medium: extractum carnis 5.0g, glucose 10.0g, peptone 10.0g, yeast extract paste 5.0g, NaCl 5.0g, distilled water is settled to 1L, pH 7.2,121 DEG C of sterilizing 20min.
Regeneration culture medium: extractum carnis 5.0g, glucose 10.0g, peptone 10.0g, yeast extract paste 5.0g, sucrose 171.2g, hydrolyzed casein 0.1g, NaCl5.0g, MgCl
21.9g, CaCl
21.7g, agar 15g, distilled water is settled to 1L, 121 DEG C of sterilizing 20min.
Hypertonic solution (SMM): sucrose 171.2g, MgCl
21.9g, maleic acid 23.2g, distilled water is settled to 1L, pH6.5, and 121 DEG C of sterilizing 20min are for subsequent use.
N,O-Diacetylmuramidase liquid: prepare with SMM, then be the sterile filters filtration sterilization of 0.22 μm with aperture, be distributed into the aliquot that single uses ,-20 DEG C of preservations.
Phosphoric acid buffer (PBS): first liquid: KH
2pO
41.361g, 100mL distilled water; Second liquid: Na
2hPO
41.78g, 100mL distilled water, 7mL first liquid+6mL second liquid (pH 7.0), 121 DEG C of sterilizing 20min are for subsequent use.
Physiological saline: NaCl 0.9g, distilled water is settled to 100mL, and 121 DEG C of sterilizing 20min are for subsequent use.
3) mensuration of lactobacillus A2 and genus bacillus E2 growth curve
Respectively bacterial classification A2 and E2 is inoculated into activation medium and 37 DEG C of cultivations, getting pure growth every 2h and measure absorbance at wavelength 600nm place, take incubation time as X-coordinate, and absorbancy is ordinate zou mapping, determines the mid log phase of two bacterial strains thus.Result as shown in Figure 6.
Mid log phase about the 12h after inoculation of lactobacillus A2 is determined by test; Mid log phase about the 6h after inoculation of genus bacillus E2.
4) preparation of protoplast suspension
The cell bacterium liquid getting 10mL mid log phase respectively, in the centrifugal 10min of 6000r/min, is abandoned supernatant, is washed 2 times with phosphoric acid buffer, centrifugal, abandons collecting cell after supernatant.Respectively enzymolysis is carried out to lactobacillus A2 and genus bacillus E2 with N,O-Diacetylmuramidase, make protoplast pellet at the centrifugal 10min of 4000r/min respectively after enzymolysis, abandon supernatant.Wash 2 times with phosphoric acid buffer, then protoplastis is suspended in 5mL high osmotic buffer (SMM).
Protoplast formation rate=A/B × 100%=(B-C)/B × 100%;
Protoplast calli=(D-C)/A × 100%;
In formula: A-protoplastis number;
B-without the total count of ferment treatment;
Remaining total count after C-after ferment treatment (the bacterium liquid after ferment treatment is suspended in stroke-physiological saline solution, make protoplastis because of osmotic pressure cracking dead.Then sample in regenerated solids substratum, cultivate 24 ~ 36h for 37 DEG C and calculate colony number, obtain the total count after ferment treatment.)
Total count on D-regeneration culture medium.
1. lysozyme concentration is on the impact of protoplast formation rate and regeneration rate
Process lactobacillus A2 and genus bacillus E2 regular hour with the N,O-Diacetylmuramidase of different concns in 37 DEG C respectively, measure the rate of formation of protoplastis; Get the protoplastis parent of preparation respectively in stroke-physiological saline solution, make ten times of serial dilutions, get 0.1mL sample liquid respectively and mix in regeneration culture medium and be down flat plate, at 37 DEG C, cultivate 24h, measure the regeneration rate of protoplastis.Then be X-coordinate with lysozyme concentration, the rate of formation of protoplastis and regeneration rate are that ordinate zou is mapped.Result as shown in Figure 7,8.
Determined by test: best lysozyme concentration prepared by lactobacillus A2 protoplasma is 3.5mg/mL; Best lysozyme concentration prepared by genus bacillus E2 protoplastis is 2mg/mL.
2. the rate of formation of hydrolysis temperature on protoplastis and the impact of regeneration rate
After adding appropriate N,O-Diacetylmuramidase respectively in two bacteria culture fluids, the water-bath being placed in differing temps respectively processes, and take enzymolysis time as X-coordinate, and the rate of formation of protoplastis and regeneration rate are that ordinate zou is mapped.
As shown in Figures 9 and 10, lactobacillus A2 and the suitable hydrolysis temperature of genus bacillus E2 protoplastis preparation is determined at about 37 DEG C by test.
3. the rate of formation of enzymolysis time on protoplastis and the impact of regeneration rate
Two bacteria culture fluids are respectively with the rate of formation and the regeneration rate thereof that measure protoplastis after N,O-Diacetylmuramidase process different time.Take enzymolysis time as X-coordinate, the rate of formation of protoplastis and regeneration rate are that ordinate zou is mapped.
As shown in FIG. 11 and 12, the enzymolysis time determining the preparation of lactobacillus A2 protoplastis suitable by test is 45min; The enzymolysis time that the preparation of genus bacillus E2 protoplastis is suitable for is 30min.
Obtaining lactobacillus A2 protoplast formation according to above-mentioned test is: get the lactobacillus A2 cell that activation culture is about 12h, add the N,O-Diacetylmuramidase that concentration is 3.5mg/mL, enzymolysis 45min under the condition of pH7.0, temperature 37 DEG C; Genus bacillus E2 protoplast formation is: get the genus bacillus E2 cell that activation culture is about 6h, add the N,O-Diacetylmuramidase that concentration is 2.0mg/mL, enzymolysis 30min under the condition of pH7.0, temperature 37 DEG C.
Embodiment 4 lactobacillus A2 and genus bacillus E2 protoplast fusion build acidproof salt tolerant amphimicrobian genus bacillus
1) protoplastis preparation
Aforesaid method is adopted to prepare the protoplastis of lactobacillus A2 and genus bacillus E2.
2) substratum and reagent
Protoplast regeneration substratum: extractum carnis 5.0g, glucose 10.0g, peptone 10.0g, yeast extract paste 5.0g, sucrose 171.2g, hydrolyzed casein 0.1g, NaCl 5.0g, MgCl
21.9g, CaCl
21.7g, agar 15g, distilled water is settled to 1L, 121 DEG C of sterilizing 20min.
Fusogen: 40g PEG, KH
2pO
40.27g, CaCl
20.11g, is settled to 100mL with the PBS damping fluid of pH7.0, and 121 DEG C of sterilizing 20min are for subsequent use.
3) protoplasma body heat deactivation
Get the lactobacillus A2 protoplastis bacteria suspension prepared, regulate bacteria concentration at 106/mL, be placed in temperature 65 DEG C of constant water bath box and be incubated, interrupted oscillation.Sample at set intervals, dilution spread regenerated plate, to contrast without heat-inactivated protoplast suspension, 37 DEG C of constant temperature temperature cultivate 3d-5d.Calculate inactivation ratio according to the following formula.Take heat treatment time as X-coordinate, inactivation ratio is that ordinate zou draws inactivation curves, as shown in figure 13.
The condition being determined lactobacillus A2 protoplasma complete inactivation by test is: 65 DEG C of thermal treatment 120min.
Get the genus bacillus E2 protoplast suspension for preparing and be diluted to 106/mL, getting 5mL and be placed in the sterilizing plate that diameter is 9cm, irradiate in 30W ultraviolet lamp 20cm place and stir with magnetic stir bar.Sample at set intervals, dilution spread protoplastis is dull and stereotyped, with the protoplast suspension without ultraviolet inactivation for contrast, and 37 DEG C of heat insulating culture 3d-5d.Calculating inactivation ratio, take ultraviolet irradiation time as X-coordinate, and inactivation ratio is that ordinate zou draws inactivation curves, as shown in figure 14.
The condition being determined genus bacillus E2 protoplastis complete inactivation by test is: 15min is irradiated at 30W ultraviolet lamp 20cm place.
4) fusion of the lactobacillus A2 after deactivation and genus bacillus E2 protoplastis
By the lactobacillus A2 after deactivation and genus bacillus E2 protoplastis balanced mix, centrifugally remove supernatant liquor, add the fusogen of preheating, 37 DEG C of insulations are after several minutes, centrifugal segregation fusogen.With coating in regenerated plate after hypertonic solution dilution, compare with the mixed bacteria liquid of non-deactivation, 37 DEG C of lucifuges cultivate 3d ~ 5d.The bacterium colony that regeneration culture medium is formed is fusant, calculates fusion rate according to following formula:
Fusion rate=A/B × 100%
Total number of bacterial colony on rear regenerated plate is merged in A---deactivation, individual/mL;
B---the total number of bacterial colony of protoplastis on regenerated plate before deactivation, individual/mL.
1. PEG concentration and pH value are on the impact of protoplast fusion rate
In protoplastis mixed solution, add the PEG of different concns and different pH, after 27 DEG C of fusion treatment 20min, get 0.1mL and be down flat plate on regenerated solids substratum, count colony number after 37 DEG C of cultivation 3 ~ 5d, calculate fusion rate, as shown in table 5.
Table 5 PEG concentration and pH value are on the impact of protoplast fusion rate
The optimum concn determining fusogen PEG by above-mentioned test is 40%, pH7.0.
2. fusion temperature and time of fusion are on the impact of protoplast fusion rate
Carry out fusion treatment under being placed in differing temps after adding fusogen in protoplastis mixed solution respectively, take out 0.1mL at regular intervals and be down flat plate on regenerated solids substratum, count colony number after 37 DEG C of cultivation 3 ~ 5d, calculate fusion rate, as shown in table 6.
Table 6 fusion temperature and time of fusion are on the impact of protoplast fusion rate
Best fusion temperature is determined and the time is respectively: temperature 35 ~ 40 DEG C, time of fusion 20 ~ 30min by above-mentioned test.
The lactobacillus A2 protoplastis of deactivation and the best fusion conditions of genus bacillus E2 protoplastis is obtained: select fusogen (40g PEG, KH according to above-mentioned series of experiments
2pO
40.27g, CaCl
20.11g, with the PBS damping fluid constant volume of pH7.0 to 100mL), control PEG concentration is 40%, pH7.0, at 35 ~ 40 DEG C of fusion treatment 20 ~ 30min.With this understanding, protoplast fusion rate can reach 7 × 10
-6.
The screening of embodiment 5 acidproof salt tolerant amphimicrobian genus bacillus, qualification and character analysis
Get 0.1mL merge after Protoplast suspension coating regenerated plate, 37 DEG C cultivate 3 ~ 5d, picking fusant bacterium colony from regenerated plate and transfer test tube slant preserve.
1) preparation of fusant
Single bacterium colony that regeneration culture medium grows is selected and is transferred on test tube slant, cryopreservation after cultivating 1 ~ 2d in 37 DEG C.From multiple different regenerated plate picking 10 fusant list bacterium colonies altogether, number consecutively is RHae-1 ~ RHae-10.
2) substratum
Basal liquid medium: peptone 10g, extractum carnis 5g, glucose 10g, NaCl 5g, MgSO
47H
2o 2.4g, distilled water is settled to 1L, pH 6.5,121 DEG C of sterilizing 20min, for subsequent use.
Solid medium: peptone 10g, extractum carnis 5g, glucose 10g, NaCl 5g, MgSO
47H
2o 2.4g, agar 15g, distilled water is settled to 1L, pH 6.5,121 DEG C of sterilizing 20min, for subsequent use.
Fermention medium: peptone 10.0g, yeast extract paste 5.0g, glucose 20.0g, NaCl1.0g, MgSO
47H
2o 0.2g, K
2hPO
40.3g, distilled water is settled to 1L, pH 6.5,121 DEG C of sterilizing 20min, for subsequent use.
3) screening of fusant and qualification
1. morphological observation and gemma microscopy
From 10 fusant bacterial strains that regeneration culture medium is picked out, after smear, dyeing, microscopy is observed, and found that in these 10 fusant bacterial strains and only have RHae-4 not have gemma, all the other bacterial strains all have gemma.
2. the detection of fusant bacterial strain gemma production rate
10 the fusant bacterial strains preserved are inoculated in the triangular flask that 50mL liquid nutrient medium is housed respectively, in 37 DEG C, after 200r/min shaking culture 48h, respectively get 2mL nutrient solution after 10 times of serial dilutions, to get 0.1mL bacterium liquid respectively coat solid medium flat board, the colony number (total viable count) after 12 ~ 24 on calculating flat board is cultivated in 37 DEG C, then residue bacterium liquid is placed in 80 DEG C of water-bath thermal treatment 15min to kill non-sporeformer, getting 0.1mL bacterium liquid more respectively coats on flat board, 24 ~ 48h is cultivated in 37 DEG C of thermostat containers, observe and record the colony number (sporeformer sum) on flat board.Gemma production rate is calculated by following formula:
Gemma production rate=(gemma number/total viable count) × 100%.
Table 7 fusant bacterial strain gemma production rate
As shown in table 7, the fusant (except RHae-4) picked out all has the ability forming gemma, and the sporulation ability of some fusant, even close to former parent genus bacillus E2 bacterial strain, illustrates that they remain the hereditary property of parent genus bacillus E2 bacterial strain.
3. the anaerobic growth of fusant bacterial strain and product acid activity analysis
Pick out 9 fusant bacterial strains (except RHae-4) are seeded in fermention medium respectively, after 37 DEG C of standing anaerobic condition bottom fermentations cultivate 24h, measure fermented liquid pH value, and measuring bacterium liquid optical density(OD) in the condition of 600nm.Determine anaerobic growth and the acid producing ability of bacterial strain thus.Result is as shown in table 8.
The anaerobic growth of table 8 fusant bacterial strain and product acid activity
As shown in table 8, in 9 fusant bacterial strains of test, except other bacterial strain of RHae-6 all can under the condition of anaerobism normal growth and synthesizing lactic acid, its growth and lactic acid metabolism ability and parent strain lactobacillus A2 substantially quite, describe the hereditary property that fusant bacterial strain remains parent strain lactobacillus A2 anaerobic growth and metabolism substantially.
4. the acidproof salt-tolerant trait analysis of fusant bacterial strain
Pick out 10 fusants (removing RHae-4 and Rhae-6) bacterial strain picking from slant medium is transferred in basal liquid medium, quiescent culture 24h in 37 DEG C of constant incubators.Afterwards with 2% inoculum size, get cultured bacterium liquid and be transferred to the basal liquid medium of pH2.0 and be added with in the basal liquid medium of 0.5% cholate, be placed in 37 DEG C of constant incubator quiescent culture 3h.Get bacterium liquid to dilute, live bacterial count, measure Viable detection, result as shown in figure 15.
Test-results shows, and the acidproof salt-tolerant trait between the fusant bacterial strain picked out has larger difference, also illustrate that their inherited character there are differences.The bacterial strain in these 8 fusant bacterial strains with stronger acid-resistant property (acidproof survival rate is greater than 20%) has RHae-2, RHae-3 and RHae-5; The bacterial strain with stronger salt-tolerant trait (salt tolerant survival rate is greater than 20%) has RHae-3, RHae-5RHae-9 and RHae-10; Fusant bacterial strain RHae-3 and RHae-5 has the dual resistance characteristics of stronger acidproof salt tolerant.So far, fusant bacterial strain RHae-3 and RHae-5 with excellent acidproof salt-tolerant trait is obtained by protoplast fusion and screening.
5. the stability analysis of fusant bacterial strain
Fusant bacterial strain RHae-3 with RHae-5 is carried out continuously 5 " single bacterium colony is separated and preservation inclined-plane of transferring " Secondary Culture.Get the 5th slant culture bacterial strain, activate and measure its corresponding gemma production rate, anaerobic condition growth and acid producing ability, acidproof and salt resistance ability.
Result shows, fusant bacterial strain RHae-3 and RHae-5 after 5 times go down to posterity is not lost in the inherited character obtained in fusion, the acid of its distinctive sporulation, anaerobic growth and product and acidproof salt-tolerant trait and first-generation fusant bacterial strain there was no significant difference, illustrate that the fusant bacterial strain of screening acquisition has good genetic stability thus.
Claims (6)
1. a preparation method for acidproof salt tolerant amphimicrobian genus bacillus, is characterized in that: comprise the following steps:
1) preparation of protoplastis:
Acidproof salt tolerant lactobacillus protoplast formation is: the lactobacillus cell getting activation culture 12h, adds the N,O-Diacetylmuramidase that concentration is 3.5mg/mL, enzymolysis 45min under the condition of pH7.0, temperature 37 DEG C;
Producing proteolytic enzyme acidproof salt tolerant Protoplasts From Bacillus Subtilis And Bacillus Thuringiensis preparation condition is: the bacillus cell getting activation culture 6h, adds the N,O-Diacetylmuramidase that concentration is 2.0mg/mL, enzymolysis 30min under the condition of pH7.0, temperature 37 DEG C;
2) lactobacillus protoplastis and Protoplasts From Bacillus Subtilis And Bacillus Thuringiensis are carried out deactivation respectively;
3) lactobacillus after deactivation and Protoplasts From Bacillus Subtilis And Bacillus Thuringiensis utilize fusogen to merge.
2. the preparation method of a kind of acidproof salt tolerant amphimicrobian genus bacillus according to claim 1, is characterized in that: the activation medium that described activation culture adopts is:
Lactobacillus: egg extractum carnis 10.0g, glucose 20.0g, white peptone 10.0g, yeast extract paste 5.0g, tween-80 1.0mL, K
2pHO
42.0g, sodium acetate 5.0g, Triammonium citrate 2.0g, MgSO
47H
2o 0.2g, MnSO
4h
2o 0.05g, distilled water is settled to 1L, pH 6.5,121 DEG C of sterilizing 20min;
Genus bacillus: extractum carnis 5.0g, glucose 10.0g, peptone 10.0g, yeast extract paste 5.0g, NaCl 5.0g, distilled water is settled to 1L, pH 7.2,121 DEG C of sterilizing 20min.
3. the preparation method of a kind of acidproof salt tolerant amphimicrobian genus bacillus according to claim 1, it is characterized in that: described lytic enzyme is for prepare with SMM, be the sterile filters filtration sterilization of 0.22 μm again with aperture, be distributed into the aliquot that single uses ,-20 DEG C of preservations.
4. the preparation method of a kind of acidproof salt tolerant amphimicrobian genus bacillus according to claim 1, is characterized in that: described deactivation condition is: lactobacillus protoplastis: 65 DEG C of thermal treatment 120min; Protoplasts From Bacillus Subtilis And Bacillus Thuringiensis: 15min is irradiated at 30W ultraviolet lamp 20cm place.
5. the preparation method of a kind of acidproof salt tolerant amphimicrobian genus bacillus according to claim 1, is characterized in that: described fusion conditions is 40%pH7.0 fusogen for selecting concentration, under 35 ~ 40 DEG C of conditions, merge 20 ~ 30min.
6. the preparation method of a kind of acidproof salt tolerant amphimicrobian genus bacillus according to claim 5, is characterized in that: the preparation method of described fusogen is: 40g PEG, KH
2pO
40.27g, CaCl
20.11g, with the PBS damping fluid constant volume of pH7.0 to 100mL, 121 DEG C of sterilizing 20min, for subsequent use.
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| CN105685479A (en) * | 2016-03-09 | 2016-06-22 | 广东海洋大学 | Preparation method of fermented soybean meal |
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