CN106191178A - Method for producing bacteriostatic active substance by using bacillus coagulans - Google Patents
Method for producing bacteriostatic active substance by using bacillus coagulans Download PDFInfo
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- CN106191178A CN106191178A CN201610724131.3A CN201610724131A CN106191178A CN 106191178 A CN106191178 A CN 106191178A CN 201610724131 A CN201610724131 A CN 201610724131A CN 106191178 A CN106191178 A CN 106191178A
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- bacillus coagulans
- culture medium
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- antibacterial
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- 241000193749 Bacillus coagulans Species 0.000 title claims abstract description 26
- 229940054340 bacillus coagulans Drugs 0.000 title claims abstract description 26
- 230000003385 bacteriostatic effect Effects 0.000 title abstract description 6
- 239000013543 active substance Substances 0.000 title abstract 3
- 238000004519 manufacturing process Methods 0.000 title abstract 2
- 239000001963 growth medium Substances 0.000 claims abstract description 25
- 241000894006 Bacteria Species 0.000 claims abstract description 20
- 230000000844 anti-bacterial effect Effects 0.000 claims description 31
- 239000000126 substance Substances 0.000 claims description 22
- 238000000034 method Methods 0.000 claims description 14
- 229920002472 Starch Polymers 0.000 claims description 11
- 239000008107 starch Substances 0.000 claims description 11
- 235000019698 starch Nutrition 0.000 claims description 11
- 229940041514 candida albicans extract Drugs 0.000 claims description 10
- 238000011081 inoculation Methods 0.000 claims description 10
- 239000012138 yeast extract Substances 0.000 claims description 10
- 239000001110 calcium chloride Substances 0.000 claims description 8
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 8
- 235000013373 food additive Nutrition 0.000 claims description 3
- 239000002778 food additive Substances 0.000 claims description 3
- 239000002054 inoculum Substances 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 2
- 230000005764 inhibitory process Effects 0.000 abstract description 17
- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 10
- 230000000694 effects Effects 0.000 abstract description 9
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 9
- FFRBMBIXVSCUFS-UHFFFAOYSA-N 2,4-dinitro-1-naphthol Chemical compound C1=CC=C2C(O)=C([N+]([O-])=O)C=C([N+]([O-])=O)C2=C1 FFRBMBIXVSCUFS-UHFFFAOYSA-N 0.000 abstract description 5
- 241000191940 Staphylococcus Species 0.000 abstract description 5
- 229960000723 ampicillin Drugs 0.000 abstract description 4
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 abstract description 4
- OOYGSFOGFJDDHP-KMCOLRRFSA-N kanamycin A sulfate Chemical compound OS(O)(=O)=O.O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N OOYGSFOGFJDDHP-KMCOLRRFSA-N 0.000 abstract description 4
- 229960002064 kanamycin sulfate Drugs 0.000 abstract description 4
- 239000005452 food preservative Substances 0.000 abstract description 3
- 235000019249 food preservative Nutrition 0.000 abstract description 3
- 229920001184 polypeptide Polymers 0.000 abstract description 3
- 230000002401 inhibitory effect Effects 0.000 abstract description 2
- 244000000010 microbial pathogen Species 0.000 abstract description 2
- 244000005700 microbiome Species 0.000 abstract description 2
- 239000003814 drug Substances 0.000 abstract 1
- 238000009629 microbiological culture Methods 0.000 abstract 1
- 239000003755 preservative agent Substances 0.000 abstract 1
- 238000000855 fermentation Methods 0.000 description 24
- 230000004151 fermentation Effects 0.000 description 24
- 239000007788 liquid Substances 0.000 description 13
- 239000002609 medium Substances 0.000 description 11
- 239000006228 supernatant Substances 0.000 description 10
- 241000896292 Odontothrips loti Species 0.000 description 8
- 241000191967 Staphylococcus aureus Species 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 229920001817 Agar Polymers 0.000 description 4
- 108010080698 Peptones Proteins 0.000 description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 4
- 239000008272 agar Substances 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 108091005804 Peptidases Proteins 0.000 description 3
- 239000001888 Peptone Substances 0.000 description 3
- 239000004365 Protease Substances 0.000 description 3
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 102000004142 Trypsin Human genes 0.000 description 3
- 108090000631 Trypsin Proteins 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 230000031700 light absorption Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 235000019319 peptone Nutrition 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000012588 trypsin Substances 0.000 description 3
- 101800000263 Acidic protein Proteins 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 241000192125 Firmicutes Species 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- OHJMTUPIZMNBFR-UHFFFAOYSA-N biuret Chemical compound NC(=O)NC(N)=O OHJMTUPIZMNBFR-UHFFFAOYSA-N 0.000 description 2
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 2
- 238000009792 diffusion process Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 230000001408 fungistatic effect Effects 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 239000010413 mother solution Substances 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 108091005508 Acid proteases Proteins 0.000 description 1
- 108090000145 Bacillolysin Proteins 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000193755 Bacillus cereus Species 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 235000019733 Fish meal Nutrition 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108091005507 Neutral proteases Proteins 0.000 description 1
- 102000035092 Neutral proteases Human genes 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 1
- 240000000111 Saccharum officinarum Species 0.000 description 1
- 235000007201 Saccharum officinarum Nutrition 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 235000013877 carbamide Nutrition 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000004467 fishmeal Substances 0.000 description 1
- 235000001727 glucose Nutrition 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 238000009413 insulation Methods 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000010422 painting Methods 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- OQUKIQWCVTZJAF-UHFFFAOYSA-N phenol;sulfuric acid Chemical compound OS(O)(=O)=O.OC1=CC=CC=C1 OQUKIQWCVTZJAF-UHFFFAOYSA-N 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000001117 sulphuric acid Substances 0.000 description 1
- 235000011149 sulphuric acid Nutrition 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
- A23L3/3463—Organic compounds; Microorganisms; Enzymes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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Abstract
The invention discloses a method for producing bacteriostatic active substances by bacillus coagulans, belonging to the technical field of microorganisms. The invention adopts a bacillus coagulans strain which is preserved in China general microbiological culture collection center (CGMCC) and has the number of 1.949, the content of bacteriostatic polypeptide is improved from original 1.507mg/mL to 2.846mg/mL by adjusting a culture medium and culture conditions, golden yellow staphylococcus is taken as an indicator bacterium, and the inhibition effect is equivalent to the action effect of 0.5mg/mL ampicillin and 0.05mg/mL kanamycin sulfate. The bacillus coagulans can improve the capability of producing bacteriostatic active substances, and can be applied to food preservatives and preservatives or applied to the field of medicine and health care by utilizing the characteristic of inhibiting the growth of pathogenic microorganisms.
Description
Technical field
The present invention relates to a kind of method that Bacillus coagulans produces Substance, belong to microbial technology field.
Background technology
Bacillus coagulans is claimed lactic acid bacteria again, belongs to gram positive bacteria, amphimicrobian type, it is possible to produce breast
Acid, can produce Substance, suppresses some corrupt and pathogenic microorganism grows.Owing to it is harmless to human non-toxic, and not
Change the characteristic of food itself, can apply to field of food industry.In animal intestinal, pathogenic entero becteria can be had preferably
Restraint, can apply to medical health field.
Relevant research shows, condenses spore bacillus and can produce antibacterial substance, but owing to such product depends on fermentation bar
The control of part, and tunning complicated component, its content is relatively low, improve Bacillus coagulans Substance yield for
Preparing Substance, promoting it extensively to apply has great necessity.
Summary of the invention
First purpose of the present invention is the culture medium providing a kind of Bacillus coagulans to produce Substance, contains
Starch 8-12g/L, yeast extract 18-22g/L, CaCl214-16g/L, pH are 7.0.
In one embodiment of the invention, described culture medium contains soluble starch 10g/L, yeast extract 20g/L,
CaCl215g/L。
Second object of the present invention is a kind of method providing Bacillus coagulans to produce Substance, is by solidifying
Knot bacillus cereus is seeded in described culture medium cultivate 20～28h.
In one embodiment of the invention, described inoculation is to carry out with inoculum concentration 2～5mL bacterium solution/100mL culture medium
Inoculation.
In one embodiment of the invention, described cultivation is at 37 DEG C, 200～220rpm.
In one embodiment of the invention, described inoculation is inoculation Bacillus coagulans seed liquor；Described seed liquor
It is that 37 DEG C, 200～220r/min cultivate 8～20h acquisitions with single colony inoculation to LB culture medium.
In one embodiment of the invention, described Bacillus coagulans is CGMCC 1.949.
Third object of the present invention is to provide the Substance that described method produces.
Fourth object of the present invention is to provide described Substance in terms of preparation food additive, antibacterial
Application.
In one embodiment of the invention, described food additive includes food preservative, antistaling agent.
Beneficial effect: by culture medium and training systern so that Bacillus coagulans 1.949 bacterial strain produces antibacterial many
Peptide content is risen to 2.846mg/mL by original 1.507mg/mL, using golden yellow staphylococcus as indicator bacteria, measures inhibition zone
Diameter, its inhibition is suitable with the kanamycin sulfate action effect of the ampicillin of 0.5mg/mL and 0.05mg/mL.
It is laid good basis for food preservative and field of health care products in the future.
Accompanying drawing explanation
Fig. 1 is Bacillus coagulans inhibition zone size in LB culture medium；
Fig. 2 is that Bacillus coagulans is produced the impact of antibacterial substance by carbon source；
Fig. 3 is that Bacillus coagulans is produced the impact of antibacterial substance by nitrogen source；
Fig. 4 is that bacterial strain is produced the impact of antibacterial substance by inorganic salt；
Fig. 5 is Bacillus coagulans fungistatic effect figure (being from left to right followed successively by embodiment 5-7) under different culture media
Fig. 6 be different protease bacterial strain is produced the activity influence of antibacterial substance (1, acidic protein ferment treatment, 2, neutral protein
Ferment treatment, 3, trypsin treatment).
Detailed description of the invention
Culture medium:
LB culture medium (g/L): yeast powder 5, NaCl 10, peptone 10, pH7.0
LB solid medium (g/L): yeast powder 5, NaCl 10, peptone 10, agar 1.5-2, pH 7.0
Seed culture: picking list colony inoculation to LB culture medium, cultivates 8～20h for 37 DEG C 200～220r/min.
Fermentation condition: inoculum concentration 2～5mL bacterium solution/100mL fermentation medium, condition of culture 37 DEG C, 200～220r/min,
Fermentation 24h.
Improvement Odontothrips loti: the Solid agar culture of falling 5ml (volume ratio is that 1.5%-2% agar is dissolved in deionized water) in
Bottom culture dish, placing Oxford cup, after LB solid medium temperature is reduced to 40 DEG C, (every 100mL trains to be added to indicator bacteria
Support base and add indicator bacteria 2mL), shake up and prepare solid medium, after it solidifies, take out Oxford cup with aseptic nipper, every hole adds
Enter 100 μ L and be centrifuged after fermentation supernatant.Put into 4 DEG C of refrigerator diffusion 1h, place into 37 DEG C of incubator growth 24h.
Inhibition zone measurement: outer ring antibacterial circle diameter and inner ring Oxford cup diameter (8cm) difference are final antibacterial circle diameter.
The mensuration of reducing sugar: use DNS method, first draw the standard curve of glucose, by the dilutest for fermented liquid supernatant liquid
After releasing, take 2ml and accurately add 1.5mlDNS reagent boiling water bath 5min, place and treat that it cools down under room temperature, be settled to ultra-pure water
25ml, measures its light absorption value at 520nm.According to the standard curve drawn before, calculate the reducing sugar total amount of fermentation liquid.
The mensuration of protein content: Coomassie Brilliant Blue, first draws standard curve, is suitably diluted by fermented liquid supernatant liquid,
Take the Coomassie brilliant blue that the sample 1ml after dilution adds 4ml, room temperature reaction two minutes, at 595nm, measure absorbance.According to painting
The standard curve of system, calculates protein content in fermentation liquid.
The mensuration of total sugar: Phenol sulfuric acid procedure, first draws the standard curve of glucose, draws the fermentation liquid after suitably dilution
2mL, adding 1mL mass fraction is the phenol solution of 6%, then adds 5mL concentrated sulphuric acid lentamente, shakes up, at room temperature the most anti-
Answer 20min, survey and at 490nm, survey light absorption value.Total sugar content in fermentation liquid is calculated according to the standard curve drawn in advance.
Determining content of peptides: using biuret method, take supernatant 1ml and accurately add the biuret reagent of 4ml, mixing is shaken
React 30min after even at room temperature, measure its light absorption value at 540nm.Content of peptides in fermentation liquid is calculated according to standard curve.
Embodiment 1
With LB culture medium as fermentation medium, use improvement Odontothrips loti, using golden yellow race staphylococcus as indicator bacteria,
It produces inhibition zone as it is shown in figure 1, after measured, and antibacterial circle diameter meansigma methods is 18.3mm.Content of peptides in fermentation liquid is carried out
Measuring, result shows, antibacterial content of peptides is 1.507mg/mL.
Embodiment 2
Using LB culture medium as initial medium, add mannitol, glucose, sugarcane with the addition of 2g/100mL respectively
Sugar, soluble starch, use improvement Odontothrips loti, using staphylococcus aureus as indicator bacteria, measure antibacterial circle diameter, wherein
Record inhibition zone after adding the culture medium fermentation of soluble starch and be 13mm to the maximum, result such as Fig. 2.
Embodiment 3
Using LB culture medium as initial medium, respectively with the addition addition peptone of 1g/100mL, fish meal protein peptone,
Yeast extract, carbamide；Use improvement Odontothrips loti, using staphylococcus aureus as indicator bacteria, measure antibacterial circle diameter, wherein
Record inhibition zone after adding the culture medium fermentation of yeast extract and be 12mm to the maximum, result such as Fig. 3.
Embodiment 4
Using LB culture medium as initial medium, add CaCl with 1g/100mL respectively2, KCl, NaCl, use Crossbred Cattle
Tianjin agar diffusion method, using golden yellow race staphylococcus as indicator bacteria, measures antibacterial circle diameter, wherein adds CaCl2Culture medium fermentation
After record inhibition zone and be 10mm to the maximum, result such as Fig. 4.
Embodiment 5
With soluble starch 10g/L, yeast extract 15g/L, CaCl215g/L prepares fermentation medium, regulates initial pH
Being 7, at 37 DEG C, 220r/min, ferment 24h, uses improvement Odontothrips loti, using staphylococcus aureus as indicator bacteria, measures
Antibacterial circle diameter 22mm (as shown in Figure 5).
Embodiment 6
Embodiment is with embodiment 5, and its difference is, changing yeast extract addition is 20g/L, measures antibacterial circle diameter
24mm.(as shown in Figure 5)
Embodiment 7
Embodiment is with embodiment 5, and its difference is, changing soluble starch addition is 5g/L, measures inhibition zone straight
Footpath 20mm.(as shown in Figure 5)
Embodiment 8
Embodiment is with embodiment 5, and its difference is, changing yeast extract addition is 15g/L, measures antibacterial circle diameter
19mm。
Embodiment 9
Embodiment is with embodiment 5, and its difference is, changes CaCl2Addition is 5g/L, measures antibacterial circle diameter
13mm。
Embodiment 10
Embodiment is with embodiment 5, and its difference is, changing soluble starch addition is 15g/L, measures inhibition zone straight
Footpath 14mm.
Embodiment 11
Embodiment is with embodiment 5, and its difference is, regulating initial pH is 6, uses improvement Odontothrips loti, with golden yellow
Staphylococcus, as indicator bacteria, has no that transparent circle occurs.
Embodiment 12
Embodiment is with embodiment 11, and its difference is, changing initial medium pH is 8, uses improvement Odontothrips loti, with
Staphylococcus aureus, as indicator bacteria, has no that transparent circle occurs.
Embodiment 13
With starch 10g/L, yeast extract 20g/L, CaCl215g/L prepares fermentation medium, and regulating initial pH is 7,
37 DEG C, 220r/min, ferment 20h, uses improvement Odontothrips loti, using staphylococcus aureus as indicator bacteria, measures inhibition zone
A diameter of 19mm.
Embodiment 14
Embodiment is with embodiment 13, and its difference is, change fermentation time is 22h, measures antibacterial circle diameter 22mm.
Embodiment 15
Embodiment is with embodiment 13, and its difference is, change fermentation time is 26h, measures antibacterial circle diameter 24mm.
Embodiment 16
Embodiment is with embodiment 13, and its difference is, change fermentation time is 28h, along with fermentation time increases, antibacterial
Material has the inactivation that is partly decomposed.Measuring antibacterial circle diameter after fermentation 28h is 20mm.
Embodiment 17
Bacillus coagulans strain fermented liquid embodiment 6 obtained is at 4 DEG C, and 12000r/min is centrifuged 5min, to supernatant
Reducing sugar in liquid, total sugar, protein, content of peptides is measured.
Result shows, in the active antibacterial material that Bacillus coagulans produces, containing total sugar 6.515mg/mL, reducing sugar
0.908mg/mL, polypeptide 2.846mg/mL, protein 0.624mg/mL.
Embodiment 18
The detection Substance sensitivity to different protease, bacterial strain supernatant acid protease, neutral protein
Enzyme, trypsin acts on respectively under corresponding optimal condition, and the bacteriostatic activity before and after then acting on is analysed and compared, first handle
Three kinds of protease pH6.5, it is 1mg/mL mother solution that the phosphate buffer of 3mmol/L is made into mass concentration, is separately added into fermentation
In supernatant, the optimum pH of regulation pH value to three kinds of enzymes, using the most enzyme-added fermented supernatant fluid as blank, 37 DEG C of water-baths
Insulation 2h, adjusts each enzymolysis solution pH value to 6.5 by the NaOH solution of the HCl solution of 1mol/L or 1mol/L after cooling, then with
Staphylococcus aureus is that indicator bacteria carries out improveing Oxford cup measuring antibacterial circle diameter size.As shown in Figure 6, find antibacterial
Material is antibacterial substance still retentive activity in acidic protein ferment treatment supernatant, and neutral protease, live after trypsin treatment
Property disappear.Tentatively judge that antibacterial substance activity is the most relevant with the proteins and peptides of the inside.
Use ampicillin and kanamycin sulfate to carry out control experiment, choose two kinds of antibiosis that concentration is 50mg/mL
Element, as mother solution, dilutes original 10,10 respectively-2, 10-3,10-4,10-5,10-6Times, using staphylococcus aureus as instruction
Bacterium carries out bacteriostatic experiment, and the fermented supernatant fluid inhibition zone size obtained with embodiment 7 compares, and measures discovery through inhibition zone and contains
The inhibition zone effect of the fermentation liquid (corresponding 24mm inhibition zone) of the antibacterial polypeptide of 2.846mg/mL and equal inhibition zone size
The ampicillin of 0.5mg/mL and the kanamycin sulfate action effect of 0.05mg/mL are suitable.
Embodiment 19
Choose gram positive bacteria staphylococcus aureus, bacillus subtilis；Choose gram negative bacteria escherichia coli,
Pseudomonas aeruginosa；Choosing a kind of Mycophyta saccharomyces cerevisiae makes dense all control of its bacterium carry out condensing spore bar at 9lg (CFU/mL)
The antimicrobial spectrum experiment of bacterium antibacterial substance.Find that gram positive bacteria is had the most antibacterial by this bacillus coagulans inhibiting product
Effect (is shown in Table 1), to saccharomyces cerevisiae without obvious bacteriostasis.
Table 1 Bacillus coagulans 1.949 fungistatic effect to different microorganisms
Claims (9)
1. the culture medium of a Bacillus coagulans product Substance, it is characterised in that containing soluble starch 8-12g/
L, yeast extract 18-22g/L, CaCl214-16g/L, pH are 7.0.
Culture medium the most according to claim 1, it is characterised in that containing soluble starch 10 according to claim 1
Culture medium, it is characterised in that containing soluble starch 10g/L, yeast extract 20g/L, CaCl215g/L。
3. the method that a Bacillus coagulans produces Substance, it is characterised in that Bacillus coagulans is seeded to power
Profit requires the culture medium described in 1, cultivates 20～28h.
Method the most according to claim 3, it is characterised in that described inoculation is with 2～5mL bacterium solution/100mL culture medium
Inoculum concentration is inoculated.
Method the most according to claim 3, it is characterised in that described cultivation is at 37 DEG C, rotating speed 200-220r min-1。
Method the most according to claim 3, it is characterised in that described inoculation is inoculation Bacillus coagulans seed liquor；Institute
Stating seed liquor is that 37 DEG C, 200～220r/min cultivate 8～20h acquisitions with single colony inoculation to LB culture medium.
Method the most according to claim 3, it is characterised in that described Bacillus coagulans is CGMCC 1.949.
8. the Substance that the arbitrary described method of claim 3-7 produces.
9. the application in terms of preparation food additive, antibacterial of the Substance described in claim 8.
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