CN104774890B - One Bacillus species bacteriocin crude extract and its preparation method and application - Google Patents
One Bacillus species bacteriocin crude extract and its preparation method and application Download PDFInfo
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- CN104774890B CN104774890B CN201510174005.0A CN201510174005A CN104774890B CN 104774890 B CN104774890 B CN 104774890B CN 201510174005 A CN201510174005 A CN 201510174005A CN 104774890 B CN104774890 B CN 104774890B
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- 241000193830 Bacillus <bacterium> Species 0.000 title claims abstract description 93
- 108010062877 Bacteriocins Proteins 0.000 title claims abstract description 88
- 239000000287 crude extract Substances 0.000 title claims abstract description 69
- 238000002360 preparation method Methods 0.000 title claims abstract description 45
- 238000000855 fermentation Methods 0.000 claims abstract description 43
- 230000004151 fermentation Effects 0.000 claims abstract description 43
- 239000006228 supernatant Substances 0.000 claims abstract description 21
- 238000005119 centrifugation Methods 0.000 claims abstract description 20
- 239000007788 liquid Substances 0.000 claims abstract description 20
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims abstract description 19
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims abstract description 19
- 235000011130 ammonium sulphate Nutrition 0.000 claims abstract description 19
- 230000001376 precipitating effect Effects 0.000 claims abstract description 19
- 235000015099 wheat brans Nutrition 0.000 claims abstract description 17
- 239000001963 growth medium Substances 0.000 claims abstract description 16
- 241000592795 Paenibacillus sp. Species 0.000 claims abstract description 14
- 238000004090 dissolution Methods 0.000 claims abstract description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 34
- 230000009514 concussion Effects 0.000 claims description 13
- 238000000502 dialysis Methods 0.000 claims description 13
- 239000002054 inoculum Substances 0.000 claims description 7
- 238000004108 freeze drying Methods 0.000 claims description 6
- 235000019249 food preservative Nutrition 0.000 claims description 3
- 239000005452 food preservative Substances 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 2
- 238000000034 method Methods 0.000 abstract description 18
- 239000000047 product Substances 0.000 abstract description 14
- 235000013305 food Nutrition 0.000 abstract description 10
- 238000004519 manufacturing process Methods 0.000 abstract description 7
- 230000035755 proliferation Effects 0.000 abstract description 2
- 239000002994 raw material Substances 0.000 abstract description 2
- 239000012467 final product Substances 0.000 abstract 1
- 230000002401 inhibitory effect Effects 0.000 abstract 1
- 230000003385 bacteriostatic effect Effects 0.000 description 21
- 239000002609 medium Substances 0.000 description 19
- 239000012153 distilled water Substances 0.000 description 18
- 241000894006 Bacteria Species 0.000 description 16
- 230000001408 fungistatic effect Effects 0.000 description 16
- 241000191938 Micrococcus luteus Species 0.000 description 14
- 241000191967 Staphylococcus aureus Species 0.000 description 14
- 230000000844 anti-bacterial effect Effects 0.000 description 14
- 239000000243 solution Substances 0.000 description 13
- 238000001514 detection method Methods 0.000 description 11
- 230000001580 bacterial effect Effects 0.000 description 9
- 235000013580 sausages Nutrition 0.000 description 8
- 241000186781 Listeria Species 0.000 description 7
- 241000186779 Listeria monocytogenes Species 0.000 description 7
- 108010053775 Nisin Proteins 0.000 description 7
- 239000000463 material Substances 0.000 description 7
- NVNLLIYOARQCIX-MSHCCFNRSA-N Nisin Chemical compound N1C(=O)[C@@H](CC(C)C)NC(=O)C(=C)NC(=O)[C@@H]([C@H](C)CC)NC(=O)[C@@H](NC(=O)C(=C/C)/NC(=O)[C@H](N)[C@H](C)CC)CSC[C@@H]1C(=O)N[C@@H]1C(=O)N2CCC[C@@H]2C(=O)NCC(=O)N[C@@H](C(=O)N[C@H](CCCCN)C(=O)N[C@@H]2C(NCC(=O)N[C@H](C)C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCSC)C(=O)NCC(=O)N[C@H](CS[C@@H]2C)C(=O)N[C@H](CC(N)=O)C(=O)N[C@H](CCSC)C(=O)N[C@H](CCCCN)C(=O)N[C@@H]2C(N[C@H](C)C(=O)N[C@@H]3C(=O)N[C@@H](C(N[C@H](CC=4NC=NC=4)C(=O)N[C@H](CS[C@@H]3C)C(=O)N[C@H](CO)C(=O)N[C@H]([C@H](C)CC)C(=O)N[C@H](CC=3NC=NC=3)C(=O)N[C@H](C(C)C)C(=O)NC(=C)C(=O)N[C@H](CCCCN)C(O)=O)=O)CS[C@@H]2C)=O)=O)CS[C@@H]1C NVNLLIYOARQCIX-MSHCCFNRSA-N 0.000 description 6
- 239000012530 fluid Substances 0.000 description 6
- 239000004309 nisin Substances 0.000 description 6
- 235000010297 nisin Nutrition 0.000 description 6
- 235000015097 nutrients Nutrition 0.000 description 6
- 241000192125 Firmicutes Species 0.000 description 5
- 239000007864 aqueous solution Substances 0.000 description 5
- 229920002678 cellulose Polymers 0.000 description 4
- 239000001913 cellulose Substances 0.000 description 4
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 239000000654 additive Substances 0.000 description 3
- 230000000996 additive effect Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 2
- 241000726221 Gemma Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 239000000306 component Substances 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 239000012533 medium component Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 235000010288 sodium nitrite Nutrition 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000012549 training Methods 0.000 description 2
- NVNLLIYOARQCIX-GSJOZIGCSA-N 1414-45-5 Chemical compound N1C(=O)C(CC(C)C)NC(=O)C(=C)NC(=O)C(C(C)CC)NC(=O)C(NC(=O)C(=C/C)/NC(=O)C(N)C(C)CC)CSCC1C(=O)NC1C(=O)N2CCCC2C(=O)NCC(=O)NC(C(=O)NC(CCCCN)C(=O)NC2C(NCC(=O)NC(C)C(=O)NC(CC(C)C)C(=O)NC(CCSC)C(=O)NCC(=O)NC(CSC2C)C(=O)NC(CC(N)=O)C(=O)NC(CCSC)C(=O)NC(CCCCN)C(=O)NC2C(NC(C)C(=O)NC3C(=O)NC(C(NC(CC=4NC=NC=4)C(=O)NC(CSC3C)C(=O)NC(CO)C(=O)NC(C(C)CC)C(=O)NC(CC=3NC=NC=3)C(=O)NC(C(C)C)C(=O)NC(=C)C(=O)NC(CCCCN)C(O)=O)=O)CSC2C)=O)=O)CSC1C NVNLLIYOARQCIX-GSJOZIGCSA-N 0.000 description 1
- 244000207740 Lemna minor Species 0.000 description 1
- 235000006439 Lemna minor Nutrition 0.000 description 1
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 235000001855 Portulaca oleracea Nutrition 0.000 description 1
- 102000004245 Proteasome Endopeptidase Complex Human genes 0.000 description 1
- 108090000708 Proteasome Endopeptidase Complex Proteins 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 239000005864 Sulphur Substances 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 229940126678 chinese medicines Drugs 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
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- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000013376 functional food Nutrition 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 235000015110 jellies Nutrition 0.000 description 1
- 239000008274 jelly Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 235000013622 meat product Nutrition 0.000 description 1
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- 238000012986 modification Methods 0.000 description 1
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- 230000007935 neutral effect Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Food Preservation Except Freezing, Refrigeration, And Drying (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses Bacillus species bacteriocin crude extracts and its preparation method and application.Wherein the preparation method comprises the following steps: (1) being inoculated in series bacillus (Paenibacillus sp.) in the culture medium of culture series bacillus, it shakes fermented and cultured and obtains fermentation liquid, by gained fermentation liquid centrifuging and taking supernatant；(2) ammonium sulfate is added into gained supernatant, the precipitating being precipitated when ammonium sulfate saturation degree is 30~70% is collected by centrifugation, will dialyse, be freeze-dried after gained precipitating dissolution to obtain the final product.The raw material of series bacillus bacteriocin crude extract of the present invention is wheat bran, and source is wide, at low cost, and fermenting microbe is series bacillus single culture, and above-mentioned process advan is in the standardization of product quality and the cost control of industrial mass production.The bacteriocin crude extract is used directly for the production of food, reaches the proliferation for inhibiting spoilage organisms, extends the purpose of Food Shelf-life, reduce application cost.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to Bacillus species bacteriocin crude extract and preparation method thereof
And application.
Background technique
Bacteriocin is certain bacteriogenic substances with antibacterial activity, and main component is polypeptide, protein or albumen
Matter compound is proposed by Jacob in nineteen fifty-three earliest.In recent years, negative brought by the universal or even abuse of broad-spectrum antibiotic
Effect becomes increasingly conspicuous, and the research of new antibiotic substitute is extremely urgent.As the bacteriocin of protein substance, due to can quilt
Proteasome degradation, safety is very high, therefore receives more and more attention.Although the bacteriocin (Nisin) that exploitation obtains at present
Have been equipped with high anti-corrosion and antibacterial application value, but by the characteristic of Nisin itself (property is stablized under low ph condition, pH >
7.0 bacteriostatic activities are gradually lost) influence, the range of application receives great limitation, secondly, bacteriocin obtains at present
It must both be from chemical synthesis culture medium, such medium component multiplicity, formula is complicated, and process for preparation is cumbersome to have potential food peace
Full blast danger, furthermore, the cost for preparing bacteriocin is very high, bacteriocin source is relatively limited, the above problem largely upper limit
The development and utilization of bacteriocin is made.
Summary of the invention
Therefore, the technical problem to be solved in the present invention is to poor for existing bacteriocin stability, preparation cost is high, source
Deficiency, fermentation medium components multiplicity, formula is complicated, and process for preparation is cumbersome and has showing there are potential food safety risk
Shape provides Bacillus species bacteriocin crude extract and its preparation method and application.
The inventors discovered that series bacillus (Paenibacillus sp.) CGMCC No.8333 bacterial strain is through wheat bran culture
Gained supernatant is dialysed after being redissolved by the precipitating that ammonium sulfate precipitation, centrifugation obtain, is freeze-dried available tool after base fermentation
There is the series bacillus crude extract of strong bacteriostatic activity, so as to complete the present invention.
In order to solve the above technical problems, one of the technical solution that the present invention takes are as follows: a Bacillus species bacteriocin is thick
The preparation method of extract, the preparation method comprises the following steps:
(1) series bacillus (Paenibacillus sp.) is inoculated in the culture medium of culture series bacillus, concussion
Fermented and cultured obtains fermentation liquid, by gained fermentation liquid centrifuging and taking supernatant；
(2) ammonium sulfate is added into supernatant obtained by step (1), analysis when ammonium sulfate saturation degree is 30~70% is collected by centrifugation
Precipitating out, by gained precipitating dissolution after dialyse, freeze-drying to get.
Wherein series bacillus (Paenibacillus sp.) is inoculated in the culture of culture series bacillus by step (1)
In base, concussion fermented and cultured obtains fermentation liquid, by gained fermentation liquid centrifuging and taking supernatant.The wherein series bacillus
(Paenibacillus sp.) is the series bacillus of this field routine, preferably the class gemma bar of fermentation generation bacteriocin
Bacterium is more preferably series bacillus (Paenibacillus sp.), and deposit number is CGMCC No.8333.The class gemma
Bacillus is the prior art, and preparation method is this field customary preparation methods, or by obtaining from collection purchase.
Wherein the culture medium of the feeding series bacillus is the culture medium of this field routine, and the culture medium is preferably bran
Skin culture medium is more preferably wheat bran culture medium, and the preparation method of the bran mass is the preparation side of this field routine
Method.The formula of the bran mass preferably includes wheat bran and water, and wherein the content of wheat bran is preferably 1~5%, more preferably for
3%, surplus is water, and the percentage is mass percent.
Wherein the inoculum concentration of the series bacillus is preferably 1~5%, is more preferably 2~4%, is optimally 3%,
The percentage is percent by volume, and it is more preferably 28 DEG C that the fermentation temperature of the series bacillus, which is preferably 25~37 DEG C,
~35 DEG C, be most preferably 30 DEG C, and it is more preferably 60~78 hours, most preferably that the time of the fermentation, which is preferably 48~96 hours,
Ground is 72 hours, and it is more preferably 150~200rpm, most preferably that the speed of the concussion fermented and cultured, which is preferably 100~300rpm,
Ground is 180rpm.
Before the series bacillus CGMCC No.8333 is inoculated in culture medium by fermentation process of the present invention, compared with
Further include the steps that series bacillus CGMCC No.8333 activation obtains series bacillus seed goodly.The step is preferable
Ground the following steps are included: series bacillus CGMCC No.8333 of the present invention is seeded in TYC solid medium, 25~
30 DEG C are cultivated 18~28 hours up to series bacillus CGMCC No.8333 seed；By gained series bacillus CGMCC
The bacterium colony of No.8333 seed is dispersed in TYC fluid nutrient medium, then is inoculated with by the inoculum concentration of 2%~5% percent by volume
The shake culture in TYC fluid nutrient medium, shaking speed be 100~200rpm, cultivate 18-28 hours, by gained culture from
The heart discards supernatant, and after gained thallus is washed with sterile distilled water, is suspended with the sterile distilled water of former volume of culture and is used to get fermentation
Series bacillus seed.
Wherein step (2) is that ammonium sulfate is added into supernatant obtained by step (1), and it is 30 that ammonium sulfate saturation degree, which is collected by centrifugation,
The precipitating being precipitated when~70%, by gained precipitating dissolution after dialyse, freeze-drying to get.
Wherein the ammonium sulfate saturation degree is preferably 30~70%, is more preferably 40~60%, the speed of centrifugation is preferable
Ground is 8,000~15,000g, is more preferably 10,000~12,000g, and the time of centrifugation is preferably 5~15 minutes, more preferably
It is 8~10 minutes, it is more preferably 5 DEG C~7 DEG C that the temperature of centrifugation, which is preferably 4 DEG C~8 DEG C, the dialysis membrane used of dialysing
Molecular cut off be preferably 800Da~1200Da, be more preferably 1000Da, dialysis time is preferably 1~3 day, more preferably
Ground be 2 days, wherein the preferred solvents of the dissolution be water, be more preferably distilled water.Preferably further include in dialysis procedure
Water step is changed, the number for changing water is preferably 2~4 times/day.When the condition of the centrifugation or the technical parameter value for condition of dialysing
When the present invention is claimed except range, the bacteriostatic activity of gained series bacillus bacteriocin crude extract can be made to generate significant drop
It is low.
Wherein the freeze-drying is the freeze drying technology of this field routine, as long as can remove solvent, is obtained with antibacterial
Active series bacillus bacteriocin crude extract.
In order to solve the above technical problems, the two of the technical solution that the present invention takes are as follows: preparation method institute as described herein
Obtain series bacillus bacteriocin crude extract.
Technical characteristic described in each step is preferred in the preparation method of series bacillus bacteriocin crude extract of the present invention
Range and corresponding technology contents in preparation method described above are completely the same, specifically refer to techniques described above content, this
Place repeats no more.
In order to solve the above technical problems, the three of the technical solution that the present invention takes are as follows: series bacillus as described herein
Bacteriocin crude extract is preparing the application in functional food.
Series bacillus bacteriocin crude extract of the present invention can be widely applied to prepare various food preservatives, function
In property food or functional feed.The application is preferably preparing the application in food preservative.
On the basis of common knowledge of the art, above-mentioned each optimum condition, can any combination to get each preferable reality of the present invention
Example.
The reagents and materials used in the present invention are commercially available.
The positive effect of the present invention is that:
1, the present invention solves the narrow status in current bacteriocin source, and series bacillus is applied to bacterium in a creative way
The preparation process of element.
2, the preparation of current bacteriocin all relies on chemical synthesis culture medium, and prepares bacteriocin with bran mass
Research had not been reported, and the present invention is applied to the system of bacteriocin using bran mass as a kind of natural fermentation medium for the first time
It is standby, thus its product has higher foodsafety.
3, the raw material of the present invention for being used to prepare bacteriocin crude extract is wheat bran, and source is wide, at low cost, fermenting microbe
For series bacillus single culture, said combination is conducive to the standardization of product quality and the cost control of industrial mass production
System.
4, wheat bran is a kind of byproduct of wheat processing, is the bacteriocin of base-material fermentation preparation than routine side using it therefore
The chemical synthesis culture medium that method uses has higher foodsafety.
5, more stable using bacteriocin crude extract property prepared by the present invention compared with conventional bacteria element Nisin, especially exist
Bacteriostatic activity is significantly higher than Nisin under neutrallty condition, therefore, has application value more preferably than Nisin.
6, the production of food is used directly for using bacteriocin crude extract prepared by the present invention, to reach inhibition corruption
The proliferation of bacterium extends the purpose of Food Shelf-life, thereby reduces the cost of application.
Specific embodiment
The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to the reality
It applies among a range.In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or according to quotient
The selection of product specification.Heretofore described " room temperature " refers to the temperature for the operation room tested, generally 15~25 DEG C, real
It is analytical reagents if applying reagent used in example not plus illustrating, buys from Chinese medicines group.
The preparation of 1 series bacillus bacteriocin crude extract of embodiment and the detection of bacteriostatic activity
1, materials and methods
The preparation of seed (fermenting microbe): by series bacillus (Paenibacillus sp.) (series bacillus
Deposit number be CGMCC No.8333, the source of the bacterial strain refers to the Chinese patent of Publication No. CN103740618A) jelly
Dry powder is dissolved with a small amount of sterile distilled water, is taken a ring to line TYC solid medium with oese and (is bought from OXOID Co., English
State), 30 DEG C of aerobic culture 48h take out, and are put into 10mL TYC fluid nutrient medium with oese picking single colonie and (buy from OXOID
Co., Britain), bacterium colony is dispersed in fluid nutrient medium with vortex oscillator, 30 DEG C, 180rpm shake culture takes for 24 hours
Out, it is inoculated in TYC fluid nutrient medium (buying from OXOID Co., Britain) with 2% (percent by volume) inoculum concentration, 30 DEG C,
180rpm shake culture for 24 hours after, culture 15,000rpm be centrifuged 10 minutes, discard supernatant, thallus washs 2 with sterile distilled water
After secondary, suspended with the sterile distilled water of former volume of culture, obtain the seed of fermentation.
Indicator strain: staphylococcus aureus (Staphylococcus aureus) CGMCC 1.879, micrococcus luteus
(Micrococcus luteus) CGMCC 1.1848, single increasing listeria spp (Listeria monocytogenes) CGMCC
1.9136, bacterial strain indicated above is all bought from CGMCC.
Indicate the preparation of bacterium solution: by staphylococcus aureus (Staphylococcus aureus), micrococcus luteus
(Micrococcus luteus), single freeze-dried powder for increasing listeria spp (Listeria monocytogenes) are used a small amount of sterile
Distilled water dissolution takes a ring to line LB solid medium (buying from OXOID Co., Britain), 30 DEG C of aerobic trainings with oese
It supports 48h to take out, is put into 10mL LB liquid medium (buying from OXOID Co., Britain) with oese picking single colonie, uses
Bacterium colony is dispersed in fluid nutrient medium by vortex oscillator, 30 DEG C, 180rpm shake culture take out for 24 hours, with 2% (volume
Percentage) inoculum concentration is inoculated in LB liquid medium (purchase from OXOID Co., Britain), 30 DEG C, 180rpm shake culture 20h
Afterwards to get corresponding instruction bacterium solution.
It indicates the preparation of plate: instruction bacterium solution prepared by the above method being diluted, makes its bacterial concentration be about
107Cfu/mL draws diluted instruction bacterium solution with the ratio of volume ratio 1:150 and injects in 45 DEG C of sterile LB solid mediums, fills
Divide rapid inverted plate after mixing, after plate solidification and surface moisture evaporation completely.
The detection method of bacteriostatic activity: 20 μ L samples to be tested are added dropwise on instruction plate with dibbling method, are placed in 30 DEG C of cultures
Antibacterial circle diameter is measured and recorded after 20h.
Wheat bran purchase from Shandong Heze (Taobao's trade name: the Yellow River beach peasant household other, http: //
Item.taobao.com/item.htm? spm=a230r.1.14.34.6aY7qR&id=23590856555&ns=1&
Abbucket=5#detail).The preparation of fermentation medium: wheat bran and distilled water are uniformly mixed in required ratio, 118
DEG C sterilizing 15min, be cooled to room temperature to get sterile fermentation medium.
2, the preparation of series bacillus bacteriocin crude extract
Series bacillus (Paenibacillus sp.) CGMCC No.8333 bacterial strain is inoculated with 5% (percent by volume)
Amount is inoculated in sterile fermentation medium, 25 DEG C, 100rpm concussion fermented and cultured 48h acquisition fermentation liquid, by above-mentioned fermentation liquid 4
DEG C, 8,000g centrifugation 15min take supernatant, ammonium sulfate are slowly added into supernatant, 8,000g is centrifuged 15min and collects sulfuric acid again
The precipitating that ammonium saturation degree is precipitated when being 30~60%, the precipitating are saturating for the bag filter of 1000Da with interception after being redissolved with distilled water
Analysis 1 day, is changed water 4 times daily, and dialysis is completed solution in back pkt. and is freeze-dried to get bacteriocin crude extract A.Wherein, fermented and cultured
The formula of base are as follows: wheat bran 5%, surplus are water, and the percentage is mass percent.
3, the detection of series bacillus bacteriocin crude extract bacteriostatic activity
Bacteriocin crude extract A is configured to the aqueous solution of 50mg/mL with sterile distilled water dissolution, measures its bacteriostatic activity.
As a result as shown in the table:
The fungistatic effect of 1 series bacillus bacteriocin crude extract of table
As can be seen from Table 1, series bacillus bacteriocin crude extract A acts on staphylococcus aureus
(Staphylococcus aureus), micrococcus luteus (Micrococcus luteus), single increasing listeria spp
The antibacterial circle diameter that (Listeria monocytogenes) is generated is respectively 13mm, 16mm and 13mm, it can be seen that, the present invention
Gained series bacillus bacteriocin crude extract A has significant fungistatic effect to above-mentioned gram-positive bacteria.
The preparation of 2 series bacillus bacteriocin crude extract of embodiment and the detection of bacteriostatic activity
1, materials and methods: with embodiment 1.
2, the preparation of series bacillus bacteriocin crude extract
Series bacillus (Paenibacillus sp.) CGMCC No.8333 bacterial strain is inoculated with 1% (percent by volume)
Amount is inoculated in sterile fermentation medium, 37 DEG C, 300rpm concussion fermented and cultured 96h acquisition fermentation liquid, by above-mentioned fermentation liquid 8
DEG C, 10,000g centrifugation 10min take supernatant, ammonium sulfate are slowly added into supernatant, 10,000g is centrifuged 10min and collects sulphur again
The precipitating that sour ammonium saturation degree is precipitated when being 50~70%, the precipitating use interception for the bag filter of 1000Da after redissolving with distilled water
Dialysis 1 day, is changed water 4 times daily, and dialysis is completed solution in back pkt. and is freeze-dried to get bacteriocin crude extract B.Wherein, fermentation training
Support the formula of base are as follows: wheat bran 1%, surplus are water, and the percentage is mass percent.
3, the detection of series bacillus bacteriocin crude extract bacteriostatic activity
Bacteriocin crude extract B is configured to the aqueous solution of 50mg/mL with sterile distilled water dissolution, measures its bacteriostatic activity.
As a result as shown in the table:
The fungistatic effect of 2 series bacillus bacteriocin crude extract of table
As can be seen from Table 2, series bacillus bacteriocin crude extract B acts on staphylococcus aureus
(Staphylococcus aureus), micrococcus luteus (Micrococcus luteus), single increasing listeria spp
The antibacterial circle diameter that (Listeria monocytogenes) is generated is respectively 9mm, 12mm and 10mm, it can be seen that, the present invention
Gained series bacillus bacteriocin crude extract B has significant fungistatic effect to above-mentioned gram-positive bacteria.
The preparation of 3 series bacillus bacteriocin crude extract of embodiment and the detection of bacteriostatic activity
1, materials and methods: with embodiment 1.
2, the preparation of series bacillus bacteriocin crude extract
Series bacillus (Paenibacillus sp.) CGMCC No.8333 bacterial strain is inoculated with 2% (percent by volume)
Amount is inoculated in sterile culture medium, 30 DEG C, 180rpm concussion fermented and cultured 72h obtain fermentation liquid, by above-mentioned fermentation liquid, 4 DEG C,
15,000g centrifugation 5min take supernatant, ammonium sulfate are slowly added into supernatant, 15,000g is centrifuged 5min and collects ammonium sulfate again
The precipitating that saturation degree is precipitated when being 40~60%, the precipitating are dialysed after being redissolved with distilled water with interception for the bag filter of 1000Da
It 2 days, changes daily water 3 times, dialysis is completed solution in back pkt. and is freeze-dried to get bacteriocin crude extract C.Wherein, fermentation medium
Formula are as follows: wheat bran 3%, surplus are water, the percentage be mass percent.
3, the detection of series bacillus bacteriocin crude extract bacteriostatic activity
Bacteriocin crude extract C is configured to the aqueous solution of 50mg/mL with sterile distilled water dissolution, measures its bacteriostatic activity.
As a result as shown in the table:
The fungistatic effect of 3 series bacillus bacteriocin crude extract of table
As can be seen from Table 3, series bacillus bacteriocin crude extract C acts on staphylococcus aureus
(Staphylococcus aureus), micrococcus luteus (Micrococcus luteus), single increasing listeria spp
The antibacterial circle diameter that (Listeria monocytogenes) is generated is respectively 15mm, 20mm and 16mm, it can be seen that, the present invention
Gained series bacillus bacteriocin crude extract C has significant fungistatic effect to above-mentioned gram-positive bacteria.
The preparation of 4 series bacillus bacteriocin crude extract of embodiment and the detection of bacteriostatic activity
1, materials and methods: with embodiment 1.
2, the preparation of series bacillus bacteriocin crude extract
Series bacillus (Paenibacillus sp.) CGMCC No.8333 bacterial strain is inoculated with 4% (percent by volume)
Amount is inoculated in sterile culture medium, 28 DEG C, 150rpm concussion fermented and cultured 60h obtain fermentation liquid, by 5 DEG C of above-mentioned fermentation liquid,
12,000g centrifugation 8min take supernatant, ammonium sulfate are slowly added into supernatant, 15,000g is centrifuged 5min and collects ammonium sulfate again
The precipitating that saturation degree is precipitated when being 30~70%, the precipitating are dialysed after being redissolved with distilled water with interception for the bag filter of 1000Da
It 3 days, changes daily water 3 times, dialysis is completed solution in back pkt. and is freeze-dried to get bacteriocin crude extract.Wherein, fermentation medium
Formula are as follows: wheat bran 3%, surplus are water, and the percentage is mass percent.
3, the detection of series bacillus bacteriocin crude extract bacteriostatic activity
Gained series bacillus bacteriocin crude extract is configured to the aqueous solution of 50mg/mL with sterile distilled water dissolution, is surveyed
Its fixed bacteriostatic activity.The antibacterial result of gained are as follows: to staphylococcus aureus (Staphylococcus aureus), M. luteus
Bacterium (Micrococcus luteus), single antibacterial circle diameter for increasing listeria spp (Listeria monocytogenes) and generating
11mm, 14mm and 12mm respectively, it can be seen that, the resulting bacteriocin crude extract of the present invention has above-mentioned gram-positive bacteria aobvious
The fungistatic effect of work.
The preparation of 5 series bacillus bacteriocin crude extract of embodiment and the detection of bacteriostatic activity
1, materials and methods: with embodiment 1.
2, the preparation of series bacillus bacteriocin crude extract
Series bacillus (Paenibacillus sp.) CGMCC No.8333 bacterial strain is inoculated with 4% (percent by volume)
Amount is inoculated in sterile culture medium, 35 DEG C, 200rpm concussion fermented and cultured 78h obtain fermentation liquid, by 7 DEG C of above-mentioned fermentation liquid,
12,000g centrifugation 8min take supernatant, ammonium sulfate are slowly added into supernatant, 15,000g is centrifuged 5min and collects ammonium sulfate again
The precipitating that saturation degree is precipitated when being 30~70%, the precipitating are dialysed after being redissolved with distilled water with interception for the bag filter of 1000Da
It 3 days, changes daily water 3 times, dialysis is completed solution in back pkt. and is freeze-dried to get bacteriocin crude extract.Wherein, fermentation medium
Formula are as follows: wheat bran 3%, surplus are water, and the percentage is mass percent.
3, the detection of series bacillus bacteriocin crude extract bacteriostatic activity
Gained series bacillus bacteriocin crude extract is configured to the aqueous solution of 50mg/mL with sterile distilled water dissolution, is surveyed
Its fixed bacteriostatic activity.The antibacterial result of gained are as follows: to staphylococcus aureus (Staphylococcus aureus), M. luteus
Bacterium (Micrococcus luteus), single antibacterial circle diameter for increasing listeria spp (Listeria monocytogenes) and generating
11mm, 13mm and 10mm respectively, it can be seen that, the resulting bacteriocin crude extract of the present invention has above-mentioned gram-positive bacteria aobvious
The fungistatic effect of work.
Comparative example 1
By the inoculum concentration in embodiment 3, cultivation temperature, fermentation time, the speed for concussion of fermenting, wheat bran concentration, ammonium sulfate
Saturation degree and dialysis time are adjusted one by one, obtain the bacteriocin crude extract of following set of distinct methods preparation.Each group
After series bacillus bacteriocin crude extract is reduced to the volume before freeze-drying, shown in the fungistatic effect following table of measurement.
The fungistatic effect of 4 distinct methods of table preparation gained series bacillus bacteriocin crude extract
It can be concluded that, it will be connect in the preparation method of the series bacillus bacteriocin crude extract from result shown in table 4
Kind amount, cultivation temperature, fermentation time, the speed for concussion of fermenting, wheat bran concentration, ammonium sulfate saturation degree and dialysis time are adjusted to
When except the present invention, the fungistatic effect of gained series bacillus bacteriocin crude extract is significantly reduced.
Comparative example 2
Using the series bacillus bacteriocin crude extract C of 3 the method for embodiment preparation as sample to be tested, while selecting current
The conventional general woods of bacteriocin product Nysa (Nisaplin is bought from Danisco A/S BJ Rep Office) is control in the market, and the general woods of Nysa is
The antibacterial product of traditional commercialization, core effective component therein be nisin Nisin, in addition there are sodium chloride etc. its
His composition.Series bacillus bacteriocin crude extract C and the general woods of Nysa the 0.2M phosphate for being dissolved separately in pH=3,5,7,9 are delayed
It rushes in solution, obtains sample to be tested group S-3, S-5, S-7, S-9 and control group N-3, N-5, N-7, N-9 that concentration is 50mg/mL,
The fungistatic effect of each group sample is as shown in the table.
Table 5 is compared with the fungistatic effect of Conventional bacteria element product
Table 5 shows conventional bacterium cellulose product from series bacillus bacteriocin crude extract prepared by the present invention in different pH
Under the conditions of fungistatic effect, statistics indicate that, under the conditions of pH of the general woods of Nysa more than neutral or neutrality, bacteriostatic activity starts to lose
It loses, and series bacillus bacteriocin crude extract prepared by the present invention still remains stable antibacterial energy under the same conditions
Power, it can be seen that, series bacillus bacteriocin crude extract prepared by the present invention has more excellent antimicrobial stability, which pushes away
Having turned over existing bacterium cellulose product can be only applied to the limitation of the food processing field under meta-acid environment, therefore, present invention preparation
The applicable food processing range of series bacillus bacteriocin crude extract will be wider than existing bacterium cellulose product.
Effect example 1
By the sausage of traditional handicraft production, (production method is quoted from following documents: Yuan Qiu duckweed nisin is in meat products
In application [J] food industry science and technology, 1998 (4): 27-28.), wherein only add sodium nitrite in the sausage of control group 1,
Additive amount is 0.15g/kg, and sodium nitrite and the general woods of Conventional bacteria cellulose product Nysa, additive amount point are added in the sausage of control group 2
Not Wei 0.04g/kg and 0.4g/kg, be added to nitrite in the sausage of sample sets and bacteriocin that embodiment 3 prepares be thick
Extract C, additive amount are respectively 0.04g/kg and 0.3g/kg, measure total plate count after the completion of each sausage sample making, as a result as follows
Shown in table.
The fungistatic effect of series bacillus bacteriocin crude extract in 6 sausage maker skill of table
As shown in Table 6, in the manufacture craft of sausage, series bacillus bacteriocin prepared by the addition present invention is slightly mentioned
Object, the fungistatic effect and existing bacteriocin product in finished product are similar, and the color of sausage does not change significantly, by
This is as it can be seen that with the alternative existing bacteriocin product completely of series bacillus bacteriocin crude extract prepared by the present invention various anti-
Rotten, antibacterial etc. application.
It should be understood that those skilled in the art can make the present invention various after having read above content of the invention
Change or modification, these equivalent forms also fall within the scope of the appended claims of the present application.
Claims (5)
1. the preparation method of a Bacillus species bacteriocin crude extract, which is characterized in that the preparation method includes following step
It is rapid:
(1) series bacillus (Paenibacillus sp.) is inoculated in the culture medium of culture series bacillus, concussion fermentation
Culture obtains fermentation liquid, by gained fermentation liquid centrifuging and taking supernatant；
(2) ammonium sulfate is added into supernatant obtained by step (1), is collected by centrifugation and is precipitated when ammonium sulfate saturation degree is 30~70%
Precipitating, by gained precipitating dissolution after dialyse, freeze-drying to get；
Wherein, the deposit number of step (1) described series bacillus (Paenibacillus sp.) is CGMCC No.8333；
The formula of the culture medium of step (1) the culture series bacillus is bran mass, the formula of the bran mass
Are as follows: wheat bran 1~5%, surplus are water, and the percentage is mass percent；
The inoculum concentration of step (1) described series bacillus is 1~5%, and the percentage is percent by volume, the temperature of the fermentation
Degree is 25 DEG C~37 DEG C, and the speed of the concussion is 100~300rpm, and the time of the fermentation is 48~96 hours；
The molecular cut off of step (2) dialysis membrane used of dialysing is 800Da~1200Da, and dialysis time is 1~3 day；
The speed of step (2) described centrifugation is 8,000~15,000g, and the time of centrifugation is 5~15 minutes, and the temperature of centrifugation is 4
DEG C~8 DEG C.
2. preparation method as described in claim 1, which is characterized in that the inoculum concentration of step (1) described series bacillus be 2~
4%, the percentage is percent by volume, and the temperature of the fermentation is 28 DEG C~35 DEG C, the speed of the concussion is 150~
200rpm, the time of the fermentation are 60~78 hours.
3. preparation method as described in claim 1, which is characterized in that the speed of step (2) described centrifugation is 10,000~12,
000g, the time of centrifugation are 8~10 minutes, and the temperature of centrifugation is 5 DEG C~7 DEG C.
4. series bacillus bacteriocin crude extract obtained by one kind preparation method as described in any one of claims 1 to 3.
5. series bacillus bacteriocin crude extract as claimed in claim 4 is preparing the application in food preservative.
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